Professional Documents
Culture Documents
Autinmunidad
Autinmunidad
Autinmunidad
Series Editor
John M. Walker
School of Life Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK
Second Edition
Edited by
Andras Perl
Departments of Medicine, Microbiology and Immunology, Biochemistry and Molecular Biology,
State University of New York, Upstate Medical University, College of Medicine, Syracuse, NY, USA
Editor
Andras Perl
Departments of Medicine
Microbiology and Immunology
Biochemistry and Molecular Biology
State University of New York
Upstate Medical University
College of Medicine
Syracuse, NY, USA
To my parents, Ibolya and Miklos, and family, Katalin, Annmarie, Marcel, and Daniel, for
their inspiration, support, and love in my pursuit of medicine and science.
Preface
The first edition of Autoimmunity: Methods and Protocols, published in 2005, has been
initiated to provide a ready-to-use guide to establish and interrogate human and animal
models of autoimmune diseases. The second edition contains several updated chapters and
many new ones. Due to continued refinement of and widespread access to transgenic tech-
nology, perhaps hundreds of new animal models have been developed that exhibit features
of autoimmune disease. Alternatively, the increasing resolution of whole genome typing
oligonucleotide chips and full genome sequencing identified many new pathways that can
lead to autoimmunity.
The first chapter, Pathogenesis and Spectrum of Autoimmunity, discusses major hypoth-
eses driving this most tantalizing area of research since the concept of autoimmunity has
been proposed by Paul Ehrlich in 1900. Considering the great diversity and ever-changing
spectrum of autoimmunity, it has not been possible to include models and experimental
protocols for each known clinical disorder. Rather, several chapters have been devoted to
the most prevalent and complex diseases, such as systemic lupus erythematosus (SLE),
rheumatoid arthritis (RA), insulin-dependent diabetes mellitus (IDDM), scleroderma or
progressive systemic sclerosis (PSS), and multiple sclerosis (MS). The chapters are contrib-
uted by laboratories actively using the presented models. Each chapter contains an intro-
ductory section that discusses relevance of the model for a particular disease and autoimmunity
in general.
The first set of eight chapters contains methods and protocols used to assess immuno-
logical and biochemical pathways of diseases pathogenesis in human samples. Chapters in
this section focus on methods to identify susceptibility genes, intercellular signaling via
cytokines, intracellular signaling through the T-cell receptor and signal processing via pro-
tein kinases, identification and enumeration of autoantigen-specific T cells and autoanti-
bodies, dysregulation of apoptosis and its role in modification of self antigens, and epigenetic
control of gene expression via DNA methylation. The second section, Chapters 923, con-
tains protocols to establish, to investigate, and to treat inflammatory arthritis, experimental
allergic encephalomyelitis (EAE), IDDM, scleroderma, and uveitis in animal models. The
methods focus on assessment of genetic, immunological, and biochemical parameters
underlying spontaneous or exogenous antigen-induced diseases. Although the individual
protocols focus on a specific disease, they can be adapted to investigate additional signaling
pathways or pathogenic autoantigens.
This book does not replace laboratory manuals with recipes for standard cell and molec-
ular biology and immunology techniques, such as cell culture, gene cloning, sequencing,
and amplification by polymerase chain reaction, vector design for generation of transgenic
and knockout animals, flow cytometry, fluorescence microscopy, electrophoresis, and gene
vii
viii Preface
and protein microarray and sequencing methods. Although these general methods are not
described in detail, they are appropriately referenced in each section. With both my col-
leagues in the field and newcomers in mind, step-by-step protocols and detailed trouble-
shooting guides supplement all chapters.
I am thankful to Professor John Walker for inviting me to organize the 2nd edition and
Dr. Paul Phillips for continued encouragement and support. I am grateful to the distin-
guished authors for their time, expertise, and devotion that made this book possible. If the
reader feels that a particularly relevant disease or model is missing, I should be held respon-
sible. Refining and extracting new meaning of old models and developing new ones is a
constantly ongoing process. Therefore, we invite our readers to approach the authors with
questions and comments or offer new models and protocols for a future volume of this
endeavor.
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii
Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xi
ix
x Contents
xi
xii Contributors
AMR H. SAWALHA University of Michigan and the Ann Arbor Veterans Affairs
Hospital, Ann Arbor, MI, USA
R. HAL SCOFIELD Arthritis and Clinical Immunology Program, Oklahoma Medical
Research Foundation, Oklahoma City, OK, USA; Department of Medicine,
College of Medicine, University of Oklahoma Health Sciences Center,
Oklahoma City, OK, USA; Department of Veterans Affairs Medical Center,
Oklahoma City, OK, USA
DAVID W. SCOTT Department of Medicine, Uniformed Services University
of the Health Sciences, Bethesda, MD, USA
BENJAMIN M. SEGAL Department of Neurology, University of Michigan
Multiple Sclerosis Center and Holtom-Garrett Program in Neuroimmunology,
University of Michigan, Ann Arbor, MI, USA
PHYLLIS B. SILVER Laboratory of Immunology, National Eye Institute, NIH,
Bethesda, MD, USA
KATERYNA SOLOVIOVA Department of Pathology, Uniformed Services University
of Health Sciences, Bethesda, MD, USA
TIFFANY TELARICO Departments of Medicine, Microbiology and Immunology,
Biochemistry and Molecular Biology, College of Medicine, State University
of New York, Upstate Medical University, Syracuse, NY, USA
GEORGE C. TSOKOS Division of Rheumatology, Department of Medicine,
Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
KOICHI TSUNEYAMA Department of Pathology, University of Toyama, Toyama, Japan
CHARLES S. VIA Department of Pathology, Uniformed Services University of Health
Sciences, Bethesda, MD, USA
HONGYANG WANG Department of Medicine and Microbiology and Center
of Immunity, Inflammation and Regenerative Medicine, University of Virginia
School of Medicine, Charlottesville, VA, USA
CHUNGWEN WEI Division of Allergy, Immunology, and Rheumatology,
Department of Medicine, University of Rochester Medical Center, Rochester,
NY, USA
GUO XIANG YANG Division of Rheumatology, Allergy and Clinical Immunology,
School of Medicine, University of California, Davis, CA, USA
RAYMOND YUNG Ann Arbor Veterans Affairs Hospital, University of Michigan,
Ann Arbor, MI, USA
AI-HONG ZHANG Department of Medicine, Uniformed Services University
of the Health Sciences, Baltimore, MD, USA
Chapter 1
Abstract
The immune system specifically recognizes and eliminates foreign antigens and, thus, protects integrity of
the host. During maturation of the immune system, tolerance mechanisms develop that prevent or inhibit
potentially harmful reactivities to self-antigens. Autoreactive B and T cells that are generated during
immune responses are eliminated by apoptosis in the thymus, lymph nodes, or peripheral circulation or
actively suppressed by regulatory T cells. However, autoreactive cells may survive due to failure of apopto-
sis or molecular mimicry, i.e., presentation and recognition of cryptic epitopes of self-antigens, or aberrant
lymphokine production. Preservation of the host requires the development of immune responses to for-
eign antigen and tolerance to self-antigens. Autoimmunity results from a breakdown of tolerance to self-
antigens through an interplay of genetic and environmental factors.
One of the basic functions of the immune system is to specifically recognize and eliminate foreign
antigens and, thus, protect integrity of the host. Through rearrangements and somatic mutations of vari-
ous gene segments encoding T and B cell receptors and antibody molecules, the immune system acquires
tremendous diversity. During maturation of the immune system, recognition of self-antigens plays an
important role in shaping the repertoires of immune receptors. Tolerance mechanisms develop that pre-
vent or inhibit potentially harmful reactivities to self-antigens. These self-defense mechanisms are mediated
on the levels of central and peripheral tolerance, i.e., autoreactive T cells are either eliminated by apoptosis
in the thymus, lymph nodes, or peripheral circulation or actively suppressed by regulatory T cells. Likewise,
autoreactive B cells are eliminated in the bone marrow or peripheral lymphoid organs. However, immune
responses triggered by foreign antigens may be sustained by molecular mimicry, i.e., presentation and
recognition of cryptic epitopes of self-antigens. Further downstream, execution of immune responses
depends on the functioning of intracellular signaling networks and the cooperation of many cell types com-
municating via surface receptors, cytokines, chemokines, and antibody molecules. Therefore, autoimmu-
nity represents the end result of the breakdown of one or multiple basic mechanisms of immune tolerance
(Table 1).
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_1, Springer Science+Business Media New York 2012
1
2
A. Perl
Table 1
Exogenous and endogenous factors involved in autoimmunity
Table 2
Systemic of autoimmune diseases
Table 3
Organ-specific autoimmune diseases
inflammatory bowel diseases extend to fewer tissues, the gut, the joints,
and the eye. Organ-specific diseases include insulin-dependent dia-
betes mellitus, multiple sclerosis, uveitis, thyroiditis, pernicious
anemia, autoimmune hemolytic anemia, thrombocytopenia, hepa-
titis, primary biliary cirrhosis, pemphigus, pemphigoid, and vitiligo
(Table 3). Individual patients may have more than one autoimmune
disorder, concurrently, and subsequently.
While the causes of autoimmune diseases have not been clearly
defined, independent lines of evidence have implicated environ-
mental factors and genetic determinants of the host (8). As shown
in Table 1, polymorphisms of HLA molecules that regulate antigen
presentation (9), complement deficiency states (10), inactivation
of the transcription factor FoxP3 (4) have been identified as
inherited factors influencing disease susceptibility. Concordance
1 Pathogenesis and Spectrum of Autoimmunity 5
rates for autoimmune diseases, such as SLE, IDDM, RA, and MS,
are approximately 25 % in monozygotic twins. The latest approaches
and successes to map lupus susceptibility genes are described in
Chapter 2. Alternatively, the discordance rate may be as high as
70 % among monozygotic twins (11), suggesting a significant role
for exogenous agents.
The concept of autoimmunity, designated as horror autotoxi-
cus, was first proposed by Paul Ehrlich in 1900 (12). The clonal
selection theory and specific elimination of self-reactive forbid-
den clones, as means of preventing autoimmunity, were hypoth-
esized by McFarlane Burnet (12). The first organ-specific
autoimmune disease, experimental thyroiditis, was described by
Noel Rose and Ernest Witebsky in 1956 (13). Rose and Vladutiu
recognized the influence of the major histocompatibility (MHC)
gene locus on development of autoimmunity. Similar to the
consequences of microbial infections, autoimmune diseases are
characterized by polyclonal T cell expansions and antibody pro-
duction, suggesting antigen-driven process (1, 14). During the
past century, tremendous efforts have been made to identify self-
antigens and infectious agents capable of inducing autoimmunity
in humans and animal models. Such studies led to the discovery of
disease-specific autoantigens that have become instrumental in
clinical diagnosis (15). As examples, antinuclear antibodies, rheu-
matoid factor, cyclic citrullinated peptide antibodies, proteinase
3-specific cytoplasmic anti-neutrophil cytoplasmic antibodies
(cANCAs), anti-glomerular basement membrane antibodies, thy-
roid-stimulating hormone receptor, thyroid peroxidase, and thyro-
globulin antibodies are routinely used to establish diagnosis of
SLE, RA, GPA, Goodpasture syndrome, Graves disease, and
Hashimotos thyroiditis, respectively. Various antigens from tissues
targeted by organ-specific autoreactivities have been used to gener-
ate useful animal models. Joint cartilage-derived antigens, such as
collagen (16) and proteoglycan (17), can induce inflammatory
arthritis in mice. Myelin-derived antigens, myelin basic protein,
myelin oligodendrocyte glycoprotein, proteolipid protein (Chapters
18 and 19), as well as mimicking viral antigens can trigger enceph-
alomyelitis resembling MS (Chapter 19). Certain animal strains
spontaneously develop insulin-dependent diabetes mellitus
(IDDM) characterized by autoreactivities to pancreas-derived
antigens, such as insulin (Chapter 17) or glutamic acid
decarboxylase (18). These animal models provide new information
on pathogenesis of autoimmunity: identify and confirm relevant
autoantigens, and delineate critical checkpoints of signaling net-
works that can be targeted for therapeutic interventions. As an
example, tumor necrosis factor (TNF) antagonists reduced
severity of collagen-induced arthritis in animal models and became
a major breakthrough in treatment of RA (19). Along the same
line, TNF has been shown to protect against lupus (20) and
6 A. Perl
References
1. Oldstone MBA (1987) Molecular mimicry lupus erythematosus. Arthritis Rheum 25:
and autoimmune disease. Cell 50:819820 12711277
2. Nagata S (2010) Apoptosis and autoimmune 8. Perl A (2010) Systems biology of lupus: map-
diseases. Ann N Y Acad Sci 1209:1016 ping the impact of genomic and environmental
3. Waterman PM, Cambier JC (2010) The factors on gene expression signatures, cellular
conundrum of inhibitory signaling by ITAM- signaling, metabolic pathways, hormonal and
containing immunoreceptors: potential molec- cytokine imbalance, and selecting targets for
ular mechanisms. FEBS Lett 584:48784882 treatment. Autoimmunity 43:3247
4. Sakaguchi S (2011) Regulatory T cells: history 9. Silverstein AM, Rose NR (1997) On the mys-
and perspective. Methods Mol Biol 707:317 tique of the immunological self [review] [91
5. Silverman GJ (2011) Regulatory natural refs]. Immunol Rev 159:197206, discussion
autoantibodies to apoptotic cells: pallbearers 10. Agnello V (1986) Lupus diseases associated
and protectors. Arthritis Rheum 63:597602 with hereditary and acquired deficiencies of
6. Arnett FC, Edworthy SM, Bloch DA, McShane complement. Springer Semin Immunpathol
DJ, Fries JF, Cooper NS, Healey LA, Kaplan 9:183219
SR, Liang MH, Luthra HS (1988) The 11. Arnett FC, Reveille JD (1992) Genetics of sys-
American Rheumatism Association 1987 temic lupus erythematosus. Rheum Dis Clin
revised criteria for the classification of rheuma- North Am 18:865892
toid arthritis. Arthritis Rheum 31:315324 12. Ehrlich P (1900) On immunity with special
7. Tan EM, Cohen AS, Fries JF, Masi AT, reference to cell life. Proc R Soc Lond (Biol)
McShane DJ, Rothfield NF, Schaller JG, Talal 66:428448
N, Winchester RJ (1982) The 1982 revised 13. Rose NR, Witebsky E (1956) Studies on organ
criteria for the classification of systemic specificity. V. Changes in the thyroid glands of
8 A. Perl
rabbits following active immunization with rabbit lymphoproliferative syndrome type II. Cell
thyroid extracts. J Immunol 76:417427 98:4758
14. Steinberg AD, Gourley MF, Klinman DM, 26. Cohen PL, Eisenberg RA (1991) Lpr and gld:
Tsokos GC, Scott DE, Krieg AM (1991) Single gene models of systemic autoimmunity
Systemic lupus erythematosus. Ann Intern and lymphoproliferative disease. Annu Rev
Med 115:548559 Immunol 9:243269
15. Samter M, Talmage DW, Frank MM, Austen 27. Clerici M, Fusi ML, Caputo D, Guerini FR,
KF, Claman HN (1988) Immunological dis- Trabattoni D, Salvaggio A, Cazzullo CL,
eases. Little Brown, Boston, MA Arienti D, Villa ML, Urnovitz HB, Ferrante P
16. Brand DD, Kang AH, Rosloniec EF (2004) (1999) Immune responses to antigens of
The mouse model of collagen-induced arthri- human endogenous retroviruses in patients
tis. In: Perl A (ed) Autoimmunity: methods with acute or stable multiple sclerosis.
and protocols, 102nd edn. Humana, Totowa, J Neuroimmunol 99:173182
NJ, pp 295312 28. Georgescu L, Vakkalanka RK, Elkon KB, Crow
17. Abu-Hamad S, Sivan S, Shoshan-Barmatz V MK (1997) Interleukin-10 promotes activa-
(2006) The expression level of the voltage- tion-induced cell death of SLE lymphocytes
dependent anion channel controls life and mediated by Fas ligand. J Clin Invest 100:
death of the cell. PNAS 103:57875792 26222633
18. Liu E, Yu L, Moriyama H, Eisenbarth GS 29. Wang JH, Pappas D, De Jager PL, Pelletier D,
(2004) Animal models of insulin-dependent de Bakker PI, Kappos L, Polman CH, Chibnik
diabetes, 102 edn. In: Perl A (ed) Humana, LB, Hafler DA, Matthews PM, Hauser SL,
Totowa, NJ. pp 195212 Baranzini SE, Oksenberg JR (2011) Modeling
19. Slavin A, Kelly-Modis L, Labadia M, Ryan K, the cumulative genetic risk for multiple
Brown ML (2010) Pathogenic mechanisms sclerosis from genome-wide association data.
and experimental models of multiple sclerosis. Genome Med 3:3
Autoimmunity 43:504513 30. Steck AK, Rewers MJ (2011) Genetics of type
20. Mageed RA, Isenberg DA (2002) Tumour 1 diabetes. Clin Chem 57:176185
necrosis factor alpha in systemic lupus erythe- 31. Crow MK, Kirou KA (2004) Interferon-alpha
matosus and anti-DNA autoantibody produc- in systemic lupus erythematosus. [Review] [74
tion [review] [45 refs]. Lupus 11:850855 refs]. Curr Opin Rheumatol 16:541547
21. Wang J, Asensio VC, Campbell IL (2002) 32. Perl A (1999) Mechanisms of viral pathogen-
Cytokines and chemokines as mediators of pro- esis in rheumatic diseases (Invited Review).
tection and injury in the central nervous system Ann Rheum Dis 58:454461
assessed in transgenic mice. [Review] [146 refs]. 33. Lai Z-W, Hanczko R, Bonilla E, Caza TN,
Curr Top Microbiol Immunol 265:2348 Clair B, Bartos A, Miklossy G, Jimah J, Doherty E,
22. Ghezzi P, Mennini T (2001) Tumor necrosis Tily H, Francis L, Garcia R, Dawood M, Yu J,
factor and motoneuronal degeneration: Ramos I, Coman I, Faraone SV, Phillips PE,
an open problem [Review] [51 refs]. Perl A (2012) N-acetylcysteine reduces disease
Neuroimmunomodulation 9:178182 activity by blocking mTOR in T cells of lupus
23. Fisher GH, Rosenberg FJ, Straus SE, Dale JK, patients. Arth Rheum Accepted Article,
Middleton LA, Lin AY, Strober W, Lenardo DOI 10.1002/art.34502
MJ, Puck JM (1995) Dominant interfering 34. Fernandez D, Bonilla E, Mirza N, Perl A
Fas gene mutations impair apoptosis in a (2006) Rapamycin reduces disease activity and
human autoimmune lymphoproliferative syn- normalizes T-cell activation-induced calcium
drome. Cell 81:935946 fluxing in patients with systemic lupus erythe-
24. Drappa J, Vaishnaw AK, Sullivan KE, Chu J-L, matosus. Arthritis Rheum 54:29832988
Elkon KB (1996) Fas gene mutations in the 35. Warner LM, Adams LM, Sehgal SN (1994)
Canale-Smith syndrome, an inherited lym- Rapamycin prolongs survival and arrests
phoproliferative disorder associated with auto- pathophysiologic changes in murine systemic
immunity. N Engl J Med 335:16431649 lupus erythematosus. Arthritis Rheum 37:
25. Wang J, Zheng L, Lobito A, Chan FK, Dale J, 289297
Sneller M, Yao X, Puck JM, Straus SE, Lenardo 36. Wu T, Qin X, Kurepa Z, Kumar KR, Liu K,
MJ (1999) (1999) Inherited human Caspase Kanta H, Zhou XJ, Satterthwaite AB, Davis
10 mutations underlie defective lymphocyte LS, Mohan C (2007) Shared signaling net-
and dendritic cell apoptosis in autoimmune works active in B cells isolated from genetically
1 Pathogenesis and Spectrum of Autoimmunity 9
distinct mouse models of lupus [Article]. 42. Reap EA, Roof K, Maynor K, Borrero M,
J Clin Invest 117:21862196 Booker J, Cohen PL (1997) Radiation and
37. Esposito M, Ruffini F, Bellone M, Gagliani N, stress-induced apoptosis: a role for Fas/Fas
Battaglia M, Martino G, Furlan R (2010) ligand interactions. Proc Natl Acad Sci U S A
Rapamycin inhibits relapsing experimental 94:57505755
autoimmune encephalomyelitis by both 43. Vladutiu AO, Rose NR (1971) Autoimmune
effector and regulatory T cells modulation. murine thyroiditis relation to histocompatibil-
J Neuroimmunol 220:5263 ity (H-2) type. Science 174:11371139
38. Donia M, Mangano K, Amoroso A, Mazzarino 44. Lockshin MD (2002) Sex ratio and rheumatic
MC, Imbesi R, Castrogiovanni P, Coco M, disease: excerpts from an Institute of Medicine
Meroni P, Nicoletti F (2009) Treatment with report [Review] [30 refs]. Lupus 11:
rapamycin ameliorates clinical and histological 662666
signs of protracted relapsing experimental 45. Llorente L, Zou W, Levy Y, Richaud-Patin Y,
allergic encephalomyelitis in Dark Agouti rats Wijdenes J, Alcocer-Varela J, Morel-Fourrier
and induces expansion of peripheral B, Brouet JC, Alarcon-Segovia D, Galanaud P
CD4 + CD25 + Foxp3+ regulatory T cells. (1995) Role of interleukin 10 in the B
J Autoimmun 33:135140 lymphocyte hyperactivity and autoantibody
39. Tian J, Lehmann PV, Kaufman DL (1994) T production of human systemic lupus erythe-
cell cross-reactivity between Coxsackievirus matosus. J Exp Med 181:839844
and glutamate decarboxylase is associated with 46. Gergely PJ, Niland B, Gonchoroff N, Pullmann
a murine diabetes susceptibility allele. J Exp R Jr, Phillips PE, Perl A (2002) Persistent
Med 180:19791984 mitochondrial hyperpolarization, increased
40. Baum H, Davies H, Peakman M (1996) reactive oxygen intermediate production, and
Molecular mimicry in the MHC: hidden clues cytoplasmic alkalinization characterize altered
to autoimmunity? Immunol Today 17:6470 IL-10 signaling in patients with systemic lupus
41. Richardson BC, Strahler JR, Pivirotto TS, erythematosus. J Immunol 169:10921101
Quddus J, Bayliss GE, Gross LA, ORourke KS, 47. Wong HK, Kammer GM, Dennis G, Tsokos
Powers D, Hanash SM, Johnson MA (1992) GC (1999) Abnormal NF-kappa B activity in
Phenotypic and functional similarities between T lymphocytes from patients with systemic
5-azacytidine-treated T cells and a T-cell subset lupus erythematosus is associated with
in patients with active systemic lupus erythema- decreased p65-RelA protein expression.
tosus. Arthritis Rheum 35:647662 J Immunol 163:16821689
Chapter 2
Abstract
Genome-wide association studies have identified many dozen genetic intervals that harbor single-nucleotide
polymorphisms (SNPs) showing statistical association with systemic lupus erythematosus. Despite the
wealth of data produced, there are limitations of these studies. The causal alleles at a given locus are not
identified; only SNP is strong linkage disequilibrium with the putative causative alleles. In order to address
identification of the causative SNPs for lupus susceptibility genes, we have initiated a candidate gene study
for which more than 40 investigators have contributed patient and control samples. In addition, these
investigators have designated SNPs to be placed on a custom array. In this way fine mapping of genetic
association findings can occur in order to identify causal alleles. These efforts have thus far benefitted
greatly from comparisons of different ethnicities. Work on about ten previously identified associations has
been published using this resource. Genome-wide association studies cannot identify rare SNPs or muta-
tions, which may impart greater relative risks than common variants. Much of the genetics of lupus may be
from rare variants or mutations. In order to approach this aspect of lupus genetics, next-generation
sequencing has begun in which all exons will be sequenced in controls and patients. This effort can also be
used to identify causal alleles from association intervals not yet otherwise identified.
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_2, Springer Science+Business Media New York 2012
11
12 R.H. Scofield and K.M. Kaufman
2. Methods
2.1. Genome-Wide In the past several years genome-wide genetic association studies
Association Studies (GWAS) have been performed in several large collections of SLE
patients and controls. These genome-wide studies of up to 500,000
SNPs have identified at least 30 and perhaps up to 50 genetic asso-
ciations for SLE (21, 22). A review of this rapidly changing field in
2009 found 20 confirmed results with p values exceeding the
genome-wide significance level of less than 1 108 (23). There are
now replication studies, including in non-white cohorts (2428).
As discussed below, study of these findings across racial/ethnic
groups may be informative, especially in regard to identifying the
causative alleles (27, 28). However, it is of note that all the GWAS
in SLE have been in either European-derived or Asian subjects.
And, while the causative, functional alleles that impart the risk
of SLE were not likely identified in genome-wide association stud-
ies, there are common themes among the genes and the gene
products thus far implicated. For example, B lymphocyte activa-
tion, apoptosis, or the interferon signaling pathway genes can be
identified as commonly represented among those genes thus far
implicated (23, 29). Interestingly, only the IRF5 association is seen
in all four populations that have been currently studied. Many of
the associations cross population boundaries but not always.
Perhaps this is due to population-specific genetic risks for lupus.
Alternatively, these results may be due to the fact that the func-
tional polymorphisms have not been identified. That is, the pres-
ently identified alleles are only in linkage disequilibrium with the
actual causative allele, and this linkage differs between human pop-
ulation groups. As more of the functional polymorphisms are
identified our ability to fully examine the associations across popu-
lation boundaries will be increased.
The genome-wide association study in which our research
group participated was a collaborative effort formed and supported
by the Alliance for Lupus Research that was known as the
International Consortium for Systemic Lupus Erythematosus
Genetics (SLEGEN). For this study, 317,501 SNPs were typed for
alleles in 720 American women of European ancestry with SLE
along with 2,337 racially matched controls. As a replication cohort,
we studied consistently associated SNPs in two additional indepen-
dent sample sets of 1,846 SLE-affected women and 1,825 con-
trols. There was strong genetic association with the HLA region
on chromosome 6p21 as well as the previously confirmed associa-
tion at chromosome 7p32 at the IRF5 gene. In addition, there was
evidence of association at genome-wide statistical significance with
replication at 16p11.2 (ITGAM), 11p15.5 (KIAA1542), 3p14.3
(PXK), and 1q25.1 (rs10798269).
16 R.H. Scofield and K.M. Kaufman
2.2. Large Lupus In order to confirm, consolidate, and extend the findings from the
Association Study 2 various SLE GWASs, we initiated candidate gene study that, similar
to SLEGEN, would be a collaborative effort of many investigators
interested in the genetics of SLE. Collaborators agreed to contrib-
ute DNA samples on at least 50 SLE patients and in return those
contributors could select SNPs to be included in the custom array.
Additional SNPs were included based on the financial contribution
of each collaborator to the project. For production of the SNP typ-
ing array, this project utilized the Illumina Infinium genotyping
assay. Initially, 33,788 custom SNPs were submitted by the 42 col-
laborators (see Table 1). After manufacturing, 32,217 SNPs passed
Illumina Quality control measurements (95.3 %). A total of 3,241
SNPs were removed due to an allele call rate below 90 %. An addi-
tional 3,445 SNPs were removed due to a major allele frequency
less than 1 %. Thus, 27,276 SNPs passed quality control proce-
dure, and were used for analysis. In addition, approximately 3,000
ancestrally informative SNPs were included. Each collaborator was
supplied with the results of the SNPs that he or she had included
in the study as well as any information that others had selected any
of these same SNPs for typing.
For a typical Large Lupus Association Study 2 (LLAS2), more
than 4,000 lupus patients with about 4,000 controls of European
descent, about 1,500 African-American lupus patients with more
than 1,800 African-American controls, about 1,000 Hispanic
patients with approximately 350 Hispanic controls enriched for
Amerindian-European admixture, and about 1,300 Asian lupus
patients with an equal number of Asian controls can be included.
Thus, this study uses samples from multiple racial and ethnic
groups. All SLE cases meet four or more of the 1997 ACR revised
criteria for the classification of SLE (11). Samples from these
patients were provided from multiple sites to the Oklahoma
Medical Research Foundation (OMRF). Each recruiting site had
Institutional Review Board (IRB) approval to recruit subjects and
the overall study was approved by OMRFs IRB.
2 Mapping Susceptibility Gene in Systemic Lupus Erythematosus 17
Table 1
Collaborators and their role in the LLAS2 study
Collaborator Role
Table 1
(continued)
Collaborator Role
Anne Stevens SamplesC, AA, As, H
Betty Tsao SamplesC, AA, As, H
Tim Vyse SamplesC
John Harley Study design; samplesC, AA, H, Am
B Freedman SamplesAA
Stuart Glenn Clinical data organization and quality
control
C = Caucasians, AA = African-Americans, H = Hispanic, As = Asians, Am = Amerindian
Table 2
Papers published or in press as a result of LLAS2
2.3. Next-Generation Genome-wide association studies are technically feasible and pro-
Sequencing ductive as demonstrated by the several that have been performed
in an uncommon disease such as SLE with identification of about
30 risk alleles. Nevertheless, there are significant drawbacks and
20 R.H. Scofield and K.M. Kaufman
3. Conclusion
References
1. Hochberg MC (1997) The epidemiology of 8. Manderson AP, Botto M, Walport MJ (2004)
systemic lupus erythematosus. In: Wallace DJ, The role of complement in the development of
Hahn BH (eds) Dubois lupus erythematosus. systemic lupus erythematosus. Annu Rev
Williams and Wilkins, Baltimore, MD, pp Immunol 22:43156
4965 9. Wu YL, Yang Y, Chung EK, Zhou B, Kitzmiller
2. Lawrence RC, Helmick CG, Arnett FC, Deyo KJ, Savelli SL, Nagaraja HN, Birmingham DJ,
RA, Felson DT, Giannini EH, Heyse SP, Tsao BP, Rovin BH, Hebert LA, Yu CY (2008)
Hirsch R, Hochberg MC, Hunder GG, Liang Phenotypes, genotypes and disease susceptibil-
MH, Pillemer SR, Steen VD, Wolfe F (1998) ity associated with gene copy number varia-
Estimates of the prevalence of arthritis and tions: complement C4 CNVs in European
selected musculoskeletal disorders in the American healthy subjects and those with sys-
United States. Arthritis Rheum 41:778799 temic lupus erythematosus. Cytogenet
3. Petri M (1998) The effect of race on incidence Genome Res 123:13141
and clinical course in systemic lupus erythema- 10. Aggarwal R, Sestak AL, DSousa A, Dillon SP,
tosus: the Hopkins Lupus Cohort. J Am Med Namjou B, Scofield RH (2010) Complete
Womens Assoc 53:916 complement deficiency in a large cohort of
4. Borchers AT, Naguwa SM, Shoenfeld Y, familial systemic lupus erythematosus. Lupus
Gershwin ME (2010) The geoepidemiology of 19:5257
systemic lupus erythematosus. Autoimmun 11. Hochberg MC (1997) Updating the American
Rev 9:A277287 College of Rheumatology revised criteria for
5. Vyse TJ, Todd JA (1996) Genetic analysis of the classification of systemic lupus erythemato-
autoimmune disease. Cell 85:311318 sus. Arthritis Rheum 40:1725
6. Deapen D, Escalante A, Weinrib L, Horwitz 12. Schur PH (1995) Genetics of systemic lupus
D, Bachman B, Roy-Burman P, Walker A, erythematosus. Lupus 4:425437
Mack TM (1992) A revised estimate of twin 13. Harley JB, Reichlin M, Arnett FC, Alexander
concordance in systemic lupus erythematosus. EL, Bias WB, Provost TT (1986) Gene inter-
Arthritis Rheum 35:311318 action at HLA-DQ enhances autoantibody
7. Harley IT, Kaufman KM, Langefeld CD, production in primary Sjogrens syndrome.
Harley JB, Kelly JA (2009) Genetic suscepti- Science 232:11451147
bility to SLE: new insights from fine mapping 14. Scofield RH, Frank MB, Neas BR, Horowitz
and genome-wide association studies. Nat Rev RM, Hardgrave KL, Fujisaku A, McArthur R,
Genet 10:285290 Harley JB (1994) Cooperative association of
22 R.H. Scofield and K.M. Kaufman
T cell beta receptor and HLA-DQ alleles in the PXK, KIAA1542 and other loci. Nat Genet
production of anti-Ro in systemic lupus ery- 40:204210
thematosus. Clin Immunol Immunopathol 22. Hom G, Graham RR, Modrek B, Taylor KE,
72:335341 Ortmann W, Garnier S, Lee AT, Chung SA,
15. Moser KL, Neas BR, Salmon JE, Yu H, Gray- Ferreira RC, Pant PV, Ballinger DG, Kosoy R,
McGuire C, Asundi N, Bruner GR, Fox J, Demirci FY, Kamboh MI, Kao AH, Tian C,
Kelly J, Henshall S, Bacino D, Dietz M, Hogue Gunnarsson I, Bengtsson AA, Rantapaa-
R, Koelsch G, Nightingale L, Shaver T, Abdou Dahlqvist S, Petri M, Manzi S, Seldin MF,
NI, Albert DA, Carson C, Petri M, Treadwell Ronnblom L, Syvanen AC, Criswell LA,
EL, James JA, Harley JB (1998) Genome scan Gregersen PK, Behrens TW (2008) Association
of human systemic lupus erythematosus: evi- of systemic lupus erythematosus with C8orf13-
dence for linkage on chromosome 1q in BLK and ITGAM-ITGAX. N Engl J Med
African-American pedigrees. Proc Natl Acad 358:900909
Sci U S A 95:1486914874 23. Graham RR, Hom G, Ortmann W, Behrens
16. Tsao BP et al (1997) Evidence for linkage of a TW (2009) Review of recent genome-wide
candidate chromosome 1 region to human sys- association scans in lupus. J Intern Med
temic lupus erythematosus. J Clin Invest 265:680688
99:725731 24. Suarez-Gestal M, Calaza M, Endreffy E,
17. Forabosco P et al (2006) Meta-analysis of Pullmann R, Ordi-Ros J, Domenico Sebastiani
genome-wide linkage studies of systemic lupus G, Ruzickova S, Jose Santos M, Papasteriades
erythematosus. Genes Immun 7:609614 C, Marchini M, Skopouli FN, Suarez A, Blanco
FJ, DAlfonso S, Bijl M, Carreira P, Witte T,
18. Lee YH, Nath SK (2005) Systemic lupus ery- Migliaresi S, Gomez-Reino JJ, Gonzalez A,
thematosus susceptibility loci defined by European Consortium of SLE DNA Collections
genome scan meta-analysis. Hum Genet (2009) Replication of recently identified sys-
118:434443 temic lupus erythematosus genetic associa-
19. Karlson EW, Sanchez-Guerrero J, Wright EA, tions: a case-control study. Arthritis Res Ther
Lew RA, Daltroy LH, Katz JN, Liang MH 11:R69
(1995) A connective tissue disease screening 25. Gateva V, Sandling JK, Hom G, Taylor KE,
questionnaire for population studies. Ann Chung SA, Sun X, Ortmann W, Kosoy R,
Epidemiol 5:297302 Ferreira RC, Nordmark G, Gunnarsson I,
20. Rasmussen A, Sevier S, Kelly J, Glenn S, Aberle Svenungsson E, Padyukov L, Sturfelt G,
T, Cooney CM, Grether A, Goodmon E, Ning Jonsen A, Bengtsson AA, Rantapaa-Dahlqvist
J, Tesiram J, Morrisey J, Powe T, Drexel M, S, Baechler EC, Brown EE, Alarcon GS,
Daniel W, Namjou B, Ojwang JO, Nguyen Edberg JC, Ramsey-Goldman R, McGwin G
KM, Cavett JW, Te JL, James JA, Scofield RH, Jr, Reveille JD, Vila LM, Kimberly RP, Manzi
Moser KL, Gilkeson GS, Kamen DL, Carson S, Petri MA, Lee A, Gregersen PK, Seldin MF,
C, MdC B, Punaro MG, Quintero-del-Rio Ronnblom L, Criswell LA, Syvanen AC,
AL, Karp DR, Wallace DJ, Weisman M, Merrill Behrens TW, Graham RR (2009) A large-scale
J, Rivera R, Petri MA, Albert DA, Espinoza replication study identifies TNIP1, PRDM1,
LR, Utset TO, Shaver TS, Arthur E, Anaya JAZF1, UHRF1BP1 and IL10 as risk loci for
JM, Bruner GR, Harley JB (2010) The lupus systemic lupus erythematosus. Nat Genet
family registry and repository. Rheumatology 41:12281233
(Oxford) 50:4759 26. Han S, Kim-Howard X, Deshmukh H,
21. Harley JB, Alarcon-Riquelme ME, Criswell Kamatani Y, Viswanathan P, Guthridge JM,
LA, Jacob CO, Kimberly RP, Moser KL, Tsao Thomas K, Kaufman KM, Ojwang J, Rojas-
BP, Vyse TJ, Langefeld CD, Nath SK, Villarraga A, Baca V, Orozco L, Rhodes B,
Guthridge JM, Cobb BL, Mirel DB, Marion Choi CB, Gregersen PK, Merrill JT, James JA,
MC, Williams AH, Divers J, Wang W, Frank Gaffney PM, Moser KL, Jacob CO, Kimberly
SG, Namjou B, Gabriel SB, Lee AT, Gregersen RP, Harley JB, Bae SC, Anaya JM, Alarcon-
PK, Behrens TW, Taylor KE, Fernando M, Riquelme ME, Matsuda K, Vyse TJ, Nath SK
Zidovetzki R, Gaffney PM, Edberg JC, Rioux (2009) Evaluation of imputation-based asso-
JD, Ojwang JO, James JA, Merrill JT, Gilkeson ciation in and around the integrin-alpha-M
GS, Seldin MF, Yin H, Baechler EC, Li QZ, (ITGAM) gene and replication of robust asso-
Wakeland EK, Bruner GR, Kaufman KM, Kelly ciation between a non-synonymous functional
JA (2008) Genome-wide association scan in variant within ITGAM and systemic lupus
women with systemic lupus erythematosus erythematosus (SLE). Hum Mol Genet
identifies susceptibility variants in ITGAM, 18:11711180
2 Mapping Susceptibility Gene in Systemic Lupus Erythematosus 23
27. Ito I, Kawasaki A, Ito S, Hayashi T, Goto D, Vila LM, Vyse TJ, Yu C-Y, Guthridge JM,
Matsumoto I, Tsutsumi A, Hom G, Graham Bruner GR, Langefeld CD, Montgomery C,
RR, Takasaki Y, Hashimoto H, Ohashi J, Harley JB, Scofield RH, Gaffney PM, Moser
Behrens TW, Sumida T, Tsuchiya N (2009) KL (2011) Identification of a systemic lupus
Replication of the association between the erythematosus susceptibility locus at 11p13
C8orf13-BLK region and systemic lupus ery- between PDHX and CD44 in a multi-ethnic
thematosus in a Japanese population. Arthritis study. Am J Hum Genet 88:8391
Rheum 60:553558 35. Adrianto I, Wen F, Templeton A, Wiley G,
28. Molineros JE, Kim-Howard X, Deshmukh H, King JB, Lessard CJ, Bates JS, Hu Y, Kelly JA,
Jacob CO, Harley JB, Nath SK (2009) Kaufman KM, Guthridge JM, Alarcon-
Admixture in Hispanic Americans: its impact Riquelme ME for the BIOLUPUS and
on ITGAM association and implications for GENLES Networks, Anaya JM, Bae SC, Bang
admixture mapping in SLE. Genes Immun SY, Boackle SA, Brown EE, Petri MA, Gallant
10:539545 C, Ramsey-Goldman R, Reveille JD, Vila LM,
29. Perl A (2009) Emerging new pathways of Criswell LA, Edberg JC, Freedman BI,
pathogenesis and targets for treatment in sys- Gregersen PK, Gilkeson GS, Jacob CO, James
temic lupus erythematosus and Sjogrens syn- JA, Kamen DL, Kimberly RP, Martin J, Merrill
drome. Curr Opin Rheumatol 21:443447 JT, Niewold TB, Park SY, Pons-Estel BA,
30. Kariuki SN, Franek BS, Kumar AA, Arrington Scofield RH, Stevens AM, Tsao BP, Vyse TJ,
J, Mikolaitis RA, Utset TO, Jolly M, Crow Langefeld CD, Harley JB, Moser KL, Webb
MK, Skol AD, Niewold TB (2010) Trait- CF, Humphrey MB, Montgomery CG,
stratified genome-wide association study Gaffney PM (2011) Association of a functional
identifies novel and diverse genetic associations variant downstream of TNFAIP3 with systemic
with serologic and cytokine phenotypes in sys- lupus erythematosus. Nat Genet 43:253258
temic lupus erythematosus. Arthritis Res Ther 36. Manolio TA, Collins FS, Cox NJ, Goldstein
12:R151 DB, Hindorff LA, Hunter DJ, McCarthy MI,
31. Taylor KE, Chung SA, Graham RR, Ortmann Ramos EM, Cardon LR, Chakravarti A, Cho
WA, Lee AT et al (2011) Risk alleles for sys- JH, Guttmacher AE, Kong A, Kruglyak L,
temic lupus erythematosus in a large case-con- Mardis E, Rotimi CN, Slatkin M, Valle D,
trol collection and associations with clinical Whittemore AS, Boehnke M, Clark AG,
subphenotypes. PLoS Genet 7:e1001311 Eichler EE, Gibson G, Haines JL, Mackay TF,
McCarroll SA, Visscher PM (2009) Finding
32. Hughes T, Kim X, Kelly JA, Kaufman KM,
the missing heritability of complex diseases.
Langefeld CD, Ziegler J, Kimberly RP, Edberg
Nature 461:747753
JC, Ramsey-Goldman R, Petri M, Reveille JD,
Brown EE, Vila LM, James JA, Gilkeson GS, 37. Cirulli ET, Goldstein DB (2010) Uncovering
Moser KL, Gaffney PM, Merrill JT, Vyse TJ, the roles of rare variants in common disease
Alarcon-Riquelme ME, Nath SK, Harley JB, through whole-genome sequencing. Nat Rev
Sawalha AH (2011) Fine mapping and trans- Genet 11:415425
ethnicity genotyping in IL2/IL21 establish 38. Bronson PG, Goldstein BA, Ramsay PP,
the genetic association between IL21 and sys- Beckman KM, Noble JA, Lane JA, Sledin MF,
temic lupus erythematosus. Arthritis Rheum Kelly JA, Harley JB, Moser KL, Gaffney PM,
63:16891697 Behrens TW, Criswell LA, Barcellos LF (2011)
33. Scofield RH, Bruner GR, Kelly JA, Kilpatrick The rs4774 CIITA missense variant is associ-
J, Bacino D, Nath SK, Harley JB (2003) ated with risk of systemic lupus erythematosus.
Thrombocytopenia identifies a severe familial Genes Immun 12:667671
phenotype of systemic lupus erythematosus 39. Namjou B, Kothari PH, Kelly JA, Glenn SB,
and reveals genetic linkages at 1q22 and Ojwang JO, Adler A, Alarcon-Riquelme ME,
11p13. Blood 101:9927 Gallant CJ, Boackle SA, Criswell LA, Kimberly
34. Lessard CJ, Adrianto I, Kelly JA, Kaufman RP, Brown E, Edberg J, Stevens AM, Jacob
KM, Grundahl KM, Adler A, Williams AH, CO, Tsao BP, Gilkeson GS, Kamen DL, Merrill
Gallant C, Alarcn-Riquelme ME for the JT, Petri M, Goldman RR, Vila LM, Anaya
BIOLUPUS and GENLES Networks, Anaya JM, Niewold TB, Martin J, Pons-Estel BA,
J-M, Bae S-C, Boackle SA, Brown EE, Chang Sabio JM, Callejas JL, Vyse TJ, Bae SC, Perrino
D-M, Criswell LA, Edberg JC, Freedman BI, FW, Freedman BI, Scofield RH, Moser KL,
Gregersen PK, Gilkeson GS, Jacob CO, James Gaffney PM, James JA, Langefeld CD,
JA Kamen DL, Kimberly RP, Martin J, Merrill Kaufman KM, Harley JB, Atkinson JP (2011)
JT, Niewold TB, Park S-Y, Petri MA, Pons- Evaluation of the TREX1 gene in a large
Estel BA, Rosalind Ramsey-Goldman R, multi-ancestral lupus cohort. Genes Immun
Reveille JD, Song YW, Stevens AM, Tsao BP, 12:2709
24 R.H. Scofield and K.M. Kaufman
40. Sanchez E, Webb RD, Rasmussen A, Kelly JA, 42. Zhao J, Wu H, Khosravi M, Cui H, Qian X,
Riba L, Kaufman KM, Torre IG, Moctezuma Kelly JA, Kaufman KM, Langefeld CD,
JF, Maradiaga-Cecea MA, Cardiel-Rios MH, Williams AH, Comeau ME, Ziegler JT, Marion
Acevedo E, Cucho-Venegas M, Garcia MA, MC, Adler A, Glenn SB, Alarcon-Riquelme
Gamron S, Pons-Estel BA, Vasconcelos C, ME, Pons-Estel BA, Harley JB, Bae SC, Bang
Martin J, Tusi-Luna T, Harley JB, Richardson SY, Cho SK, Jacob CO, Vyse TJ, Niewold TB,
B, Sawalha AH, Alarcn-Riquelme ME (2011) Gaffney PM, Moser KL, Kimberly RP, Edberg
Genetically determined Amerindian ancestry JC, Brown EE, Petri MA, Ramsey-Goldman
correlates with increased frequency of risk R, Vila LM, Reveille JD, James JA, Gilkeson
alleles for systemic lupus erythematosus. GS, Kamen DL, Freedman BI, Anaya JM,
Arthritis Rheum 62:37223729 Merrill JT, Criswell LA, Scofield RH, Stevens
41. Webb R, Kelly JA, Somers EC, Hughes T, AM, Boackle SA, Guthridge JM, Chang DM,
Kaufman KM, Sanchez E, Nath SK, Bruner G, Song YW, Park JA, Lee EY, Grossman JM,
Alarcon-Riquelme ME, Gilkeson GS, Kamen Hahn BH, Goodship T, Cantor RM, Yu CY,
DL, Richardson BC, Harley JB, Sawalha AH Shen N, Tsao BP (2011) Association of genetic
(2011) Early disease onset is associated with a variants in complement factor H and factor
more severe phenotype in lupus patients and is H-related genes with systemic lupus
predicted by a higher genetic risk for lupus. erythematosus susceptibility. PLoS Genet
Ann Rheum Dis 70:151156 7(5):e1002079
Chapter 3
Abstract
Abnormal expression of key signaling molecules and defective functions of T lymphocytes play a significant
role in the pathogenesis of systemic lupus erythematosus (SLE). T cell receptor (TCR/CD3)-mediated
stimulation of SLE T cells show increased protein tyrosine phosphorylation of cellular proteins with faster
kinetics, heightened calcium flux response, and decreased IL-2 production. The molecular mechanisms of
T cell signaling abnormalities in SLE T cells are complex. Current research has been directed towards
investigating various factors that contribute to abnormal tyrosine phosphorylation, intracellular calcium
response, and cytokine production. Central to this dysfunction is the aberrant expression and function of
the TCR/CD3 chain. Latest developments suggest multiple explanations are involved, including altered
receptor structure, supramolecular assembly, modulation of membrane clustering, aberrant cellular
distribution, and pre-compartmentalization with lipid-rafts. The methods and protocols described here
pertaining to T cell signaling abnormalities in SLE T cells are optimized in many ways and are derived by
the combined task and continuous efforts of many researchers in the lab over a long period of time. These
simplified protocols can be readily applied to study T cell signaling abnormalities in SLE to identify the
genetic, molecular, and biochemical factors contributing to aberrant immune cell function and unravel
the pathophysiology of SLE.
Key words: T cell isolation, Cell signaling, Calcium response, Tyrosine phosphorylation,
Immunoblotting, Cell proliferation, Cytokines, Autoimmune disease, Systemic lupus erythematosus,
T cell receptor, CD3
1. Introduction
1.1. TCR Signaling One of the earliest steps in the signal transduction after TCR/CD3
engagement is the phosphorylation of the tyrosine residues within
the three immunoreceptor tyrosine-based activation motifs
(ITAMs) of the chain by Lck and Fyn, leading to the association
and activation of ZAP-70 (14). Once activated, Fyn, Lck, and
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_3, Springer Science+Business Media New York 2012
25
26 V.R. Moulton et al.
1.2. Applications Signaling studies have several applications in SLE and other auto-
immune disorders. The precise pathologic mechanisms behind
abnormal T cell functions in SLE remain incompletely understood.
Identification of underlying genetic, molecular, and biochemical
mechanisms that is responsible aberrant T cell signaling will con-
tribute to our understanding of the pathogenesis of SLE as well as
provide novel targets for future pharmacological intervention.
1.3. Study Design Normal T cells or cells from patients with other autoimmune
diseases such as rheumatoid arthritis, Sjogrens syndrome, anti-
phospholipid syndrome and dermatomyositis can be used as
3 Methods and Protocols to Study T Cell Signaling Abnormalities 27
2. Materials
2.3.2. Cross-linking Goat anti-mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
Antibody
2.3.3. Isotype Controls 1. Mouse IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
2. Rabbit IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
3. Goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA).
4. Human IgG (Sigma Aldrich, St. Louis, MO) used for blocking
antibodies.
2.4. Kits 1. Magnetic column separation (MACS) Pan human T cell isola-
tion kit (Miltenyi Biotech, Auburn, CA).
2. RosetteSep-human T cell enrichment Cocktail (StemCell
Technologies Inc., Vancouver BC, Canada).
3. Quantikine human IL-2 Immunoasssay kit (R&D Systems Inc,
Minneapolis, MN).
4. Protein assay kit (BioRad, Hercules, CA).
5. RNeasy Mini kit (Qiagen, Valencia, CA).
2.5. Electrophoresis, 1. Novex precast gels 412 % BisTris NuPageTM gels (Invitrogen,
Western Transfer, Carlsbad, CA).
Immunoblotting, and 2. Whatman filter papers (Fisher Scientific, Suwanee, GA).
Immunopreciptation
3. PVDF membrane, Immobilon-P, Pore size: 0.45 m (Sigma
Reagents Aldrich, St. Louis, MO).
4. NuPageTM-reducing agent (10) (Invitrogen Corporation,
Carlsbad, CA).
5. NuPageTM-transfer buffer (20) (Invitrogen Corporation,
Carlsbad, CA).
6. NuPageTM-LDS (laureal dodecyl sulfate) sample buffer (4)
(Invitrogen, Carlsbad, CA).
7. SeeBlue Plus 2 protein ladder (Invitrogen, Carlsbad, CA).
8. Protein A/G plus-agarose-2 ml 50 % slurry (Santa Cruz
Biotechnology, Santa Cruz, CA).
9. Phenyl phosphate disodium salt (Sigma Aldrich, St. Louis, MO).
10. Kodak X-ray film (Sigma Aldrich, St. Louis, MO).
11. Tween-20 (Sigma Aldrich, St. Louis, MO).
12. Triton-X-100 (Fisher Scientific, Suwanee, GA).
2.6. Flow Cytometry 1. FACS tubes, 12 75 mm polypropylene test tubes without cap
and Fluorescence (Fisher Scientific, Suwanee, GA).
Microscopy Reagents 2. Polylysine-coated Poly Prep slides (Sigma Aldrich).
30 V.R. Moulton et al.
11. Power supplies, BioRad Power Pac 300 (Bio-Rad, Hercules, CA).
12. Light microscope, Olympus (OPELCO, Dulles, VA).
13. Dounce homogenizer (Fisher Scientific).
14. Beckman ultracentrifuge and SW41 rotor (Palo Alto, CA).
15. PCR machine, T3 thermocycler (Biometra, Heidelberg,
Germany).
16. Lightcycler 480 real-time PCR instrument (Roche,
Germany).
17. Confocal microscope, Bio-Rad Radiance 2100 (Bio-Rad; Laser
Sharp 2000 software).
18. Olympus model IX70 fluorescence microscope (OPELCO,
Dulles, VA).
19. Nucleoporator (Lonza, Cologne, Germany).
20. Sonicator (Fisher Scientific).
21. Nanodrop 2000 spectrophotometer (Thermo Scientific,
Wilmington, DE).
3. Methods
3.1. Isolation The blood should be collected in heparin green top blood collec-
and Cell Culture tion tubes and kept at room temperature (see Note 1). Peripheral
of T Lymphocytes blood mononuclear cells (PBMCs) are isolated by density gradient
centrifugation over Ficoll-Hypaque and the whole process is done
3.1.1. Separation
at room temperature in a sterile hood.
of Peripheral Blood
Mononuclear Cells 1. Aliquot blood into 50 ml conical disposable centrifuge tubes,
10 ml per tube.
2. Add 30 ml RPMI 1640 medium with Pen-Strep, cap, and mix
by inverting.
3. Carefully underlay the sample with 10 ml Lymphoprep.
4. Centrifuge in a Sorvall table top centrifuge at 650g for 45 min.
Turn off brake during centrifugation to prevent mixing of
the density gradient layers during deceleration process.
5. Remove the interface of white ring or buffy coat containing
PBMCs and transfer into new 50 ml conical tubes.
6. Dilute the PBMCs with 3 volumes of RPMI-1640 with Pen-
Strep, mix, and centrifuge at 370g for 5 min.
7. Discard the supernatant and resuspend the cells in appropriate
volume of RPMI-1640.
8. Count the cells using a hemocytometer and light microscope by
diluting 10 l of cell suspension into 200 l trypan blue. The
32 V.R. Moulton et al.
cells should be counted from all four corner squares and the
total number obtained should be more than 200 for accurate
results. Total number of cells = cell count/4 20 104 cells/ml.
Note: If the PBMCs are contaminated with red blood cells (RBCs)
and the sample appears red it can be hemolyzed using ACK lysis
buffer (0.15 M NH4Cl, 1 mM KHCO3, and 0.1 mM EDTA).
PBMCs should be maintained in RPMI containing Pen-Strep and
10 % FBS at 37 C in a 5 % CO2 incubator. If going directly on to
T cell isolation, the serum can be omitted from the medium.
3.1.2. Isolation of T Magnetic separation and RosetteSep methods routinely yield >97 %
Lymphocytes from PBMCs CD3-positive T lymphocytes and have distinct advantages.
Rosetting using sheep RBCs is inexpensive but is less preferred due
to less pure yields (see Note 2).
Magnetic Separation Using T cells can be isolated by negative depletion by magnetic separation
MicroBeads using a kit from Miltenyi Biotech. This procedure is simple and
involves incubation of the PBMCs with a cocktail of hapten-
conjugated antibodies that bind to non-T lymphocytes followed
by removal of the antibody bound non-T lymphocyte by a magnetic
bead coated with secondary anti-hapten antibody. The non-T
lymphocytes bound to magnetic beads are retained in a column
under the influence of a powerful magnet. The flow-through
containing purified T cells are used for the studies. Although the
manufacturers protocol suggests that PBMCs may be stored in
the refrigerator overnight in PBS containing 2 mM EDTA supple-
mented with 10 % autologous serum after the last washing step
before separation using MACS column, we found that storing SLE
PBMCs under these conditions sometimes results in large clumps
that fail to resuspend and later clog the separation column.
Overnight incubation of SLE PBMCs also leads to decreased
viability of the cells and cell lysis.
1. Count the PBMCs and wash them in 10 ml MACS buffer
(1 PBS, 1 % FBS or 0.5 % BSA, 2 mM EDTA).
2. Remove the supernatant very carefully and resuspend the cells
in 80 l (per 107 cells) ice cold MACS buffer.
3. Add 20 l hapten-antibody cocktail per 107 cells, mix well, and
incubate at 6 C for 10 min.
4. Wash two times with MACS buffer (20 times the labeling
volume).
5. Resuspend the cells in 80 l MACS buffer and add 20 l MACS
anti-hapten MicroBeads per 107 cells, mix well, and incubate at
6 C for 15 min.
6. Wash the cells one time as in step 4 and resuspend in 500 l
MACS buffer.
3 Methods and Protocols to Study T Cell Signaling Abnormalities 33
7. Set up the MACS column in between the magnet and wash the
column with 3 ml of MACS buffer.
8. Load the cells to the column and collect the flow-through as
well as washing with 10 ml of MACS buffer.
9. Centrifuge the T-lymphocytes at 200g for 5 min in Sorvall
table-top centrifuge and discard the supernatant.
10. Resupend the cells in appropriate volume of RPMI-1640
containing 10 % FBS and Pen-Strep.
RosetteSep T lymphocytes can be directly isolated from the SLE blood samples
using this protocol. The method of T cell isolation by RosetteSep
is essentially based on cross-linking the non-T lymphocytes with
antibodies coated to latex beads that separate with RBCs in Ficoll-
Hypaque density gradient centrifugation. This negative selection
protocol takes less time than MACS separation. However, the yield
of purified T lymphocytes is slightly lower than MACS isolation
from PBMCs.
1. Aliquot blood into 50 ml conical disposable centrifuge tubes,
10 ml per tube.
2. Add 250 l StemCell T cell isolation antibody to each tube.
Mix well using a 25 ml pipette and incubate at room tempera-
ture for 30 min.
3. Add equal volume of PBS with 10 % FBS; mix by gentle
pipetting so as not to disturb the already-formed rosettes.
4. Underlay the sample with 10 ml of Lymphoprep and centrifuge
at 1000g for 30 min at room temperature, with the brake
turned off to prevent layers from mixing during deceleration.
5. Carefully collect the white interphase layer of cell and transfer
to fresh tubes.
6. Wash cells by adding 10 ml of PBS with 10 % FBS, followed by
centrifugation for 10 min at 650g (brake may be turned on
again).
7. Repeat wash step.
8. Resuspend the purified T cells in 1 ml of 10 % FBS plus Pen-
Strep. Count cells as per Subheading 3.1.1.
9. If not being used immediately, T cells may stored in liquid
nitrogen as cell suspension in 90 % FBS with 10 % DMSO. Cell
density should not exceed 106 cells/ml. Cells should be frozen
first at 80 C for 24 h before transfer to liquid nitrogen tank.
3.1.3. Cell Culture 1. Prepare culture media with RPMI-1640 containing 10 % FBS
of T Lymphocytes and Pen-Strep.
2. Add 1 g/ml phytohemagglutinin.
34 V.R. Moulton et al.
3.2. Analysis of TCR/ Intracellular calcium flux response is measured by flow cytometry
CD3-Mediated after labeling the cells with INDO-1 (Molecular Probes, Eugene,
Signaling in OR) (11). INDO-1 loaded cells are analyzed using an Epics Altra
Lymphocytes (Coulter Beckmann, Hialeah, FL) flow cytometer equipped with a
high power dual wavelength (365 and 488 nm) argon laser and
3.2.1. Measurement UV source. In each run, the cells are first run unstimulated to
of TCR/CD3-Mediated record the baseline fluorescence ratio, which represents the resting
Intracellular Calcium [Ca2+]i levels. After 40 s either antibody OKT3 or other activating
Response in SLE T Cells antibodies or the isotype control mIgG2a, is added to the tube
followed by cross-linking with the goat anti-mouse IgG at 130 s
and the calcium response is recorded for 10 min. The mean
fluorescence ratio calculated by the software MultiTime (version 3,
Phoenix Flow Systems, San Diego, CA) is directly proportional to
the free cytosolic Ca2+.
It is very important to perform the calcium analysis as soon as
possible after blood collection to be representative of signaling
in vivo. Isolated T lymphocytes (5 106/ml) should be recovered
in RPMI 1640 containing 10 % FBS and Pen-Strep at 37 C in 5 %
CO2 for 46 h for an optimal response and should not be
maintained more than 16 h. Longer periods of incubation of the
lymphocytes in culture medium may decrease the effective calcium
response qualitatively and quantitatively. When comparing the
intracellular calcium response of SLE T cells to normal T cells,
experiments should be done simultaneously on the same day to
avoid instrumental variations.
1. Prepare 100 ml of RPMI-1640 medium containing 1 % FBS,
10 mM HEPES, pH 7.4, and Pen-Strep and warm to 37 C.
2. Take 1 ml of SLE T cells maintained in RPMI-1640
(1 106 cells/ml) in a flow cytometry tube. Add 1 l INDO-1
(add 50 l DMSO to one vial of 50 g INDO-1, keep pro-
tected from light) and incubate at 37 C for 1 h.
3. Dilute 200 l INDO-1 loaded T lymphocytes into 2 ml in pre-
warmed culture medium and load onto EPICs Altra flow
cytometer.
3 Methods and Protocols to Study T Cell Signaling Abnormalities 35
Fig. 1. TCR/CD3-induced intracellular calcium response in SLE T cells. 1 106 SLE T cells
were resuspended in 1 ml pre-warmed culture medium and loaded with 1 L (1 g/ml in
DMSO) INDO-1 (Molecular Probes, Eugene OR) for 60 min at 37 C. INDO-1 loaded cells
were diluted ten times (0.22 ml) with pre-warmed filtered RPMI containing 1 % FBS.
After establishing the baseline fluorescence ratio which represents the resting [Ca2+]i lev-
els for 40 s, the cells were stimulated with either OKT3 (2.5 g/ml), or the isotype control
mIgG2a followed by 10 g/ml of goat anti-mouse Ab at 130 s. The intracellular calcium
response was continuously monitored for 10 min with intermittent shaking.
36 V.R. Moulton et al.
Protein Assay Protein concentrations can be measured with any of the standard
commercially-available kits such as the Bradford assay systems from
BioRad (Hercules, CA) or Sigma (St. Louis, MO). Normal T lympho-
cytes yield approximately 25 g/l protein if five million cells are
lysed in 100 l lysis buffer. Jurkat T lymphoma cell lines yield
approximately 20 g/l protein if five million cells are lysed in
150 l lysis buffer. Generally, electrophoresis of 1520 g of the
protein sample is required for easy detection by immunoblotting.
3 Methods and Protocols to Study T Cell Signaling Abnormalities 37
Protein Separation by Depending on the molecular weight of the protein under investigation,
Polyacrylamide Gel different gel types can be used (see Note 3). Upper molecular
Electrophoresis weights above 100 kDa can be better resolved with a 6 % gel,
50100 kDa can be better resolved with a 10 % gel and to visualize
lower molecular weight proteins a 12 % gel can be used. Sometimes
a gradient gel such as a 412 % BisTris gel may be necessary to see
the whole spectrum of proteins. The Invitrogen-Novex pre-cast
gel is set up after removing the tape and comb in the electrophore-
sis apparatus according to manufacturers instructions and both
inner and outer chambers are filled with SDS running buffer. The
samples are denatured in SDS loading buffer and reduced using
DTT or 2-mercaptoethanol (12).
1. Pipette 1015 g protein lysate from different samples in a
1.5 ml microfuge tube.
2. Add equal volume of 4 loading buffer (Invitrogen-Novex,
Carlsbad, CA) containing bromophenol blue, SDS (sodium
dodecyl sulfate) and 2 mercaptoethanol. The total volume of
the sample is limited by the size of the well, and in general
should not exceed 2530 l.
3. Boil for 5 min in a water bath and cool to room temperature.
4. Assemble the pre-cast gel in the electrophoresis apparatus
according to manufacturers instructions.
5. Fill inner and outer chambers with running buffer. Gently flush
the wells with running buffer using a pipette.
6. Load samples carefully into each well.
7. Load protein ladder (SeeBlue Protein Plus, Invitrogen).
8. Run gel for 1 h at constant voltage of 200 V until the dye front
reaches the bottom of the gel. Remove the gel and continue
with staining or transfer to membrane.
Note: Care should be taken to avoid spillage of samples into adja-
cent wells. The voltage should be reduced if smiling of the gel
occurs.
3.2.3. Kinetics of Tyrosine The kinetics of early tyrosine phosphorylation of cellular proteins
Phosphorylation of Cellular can be studied by activation of SLE T cells for 0, 1, 2, and 3 min,
Proteins and analysis of tyrosine phosphorylated proteins as mentioned
above. The kinetics of phosphorylation of cellular protein sub-
strates in faster in SLE T cells compared normal T cells (Fig. 2).
3.2.4. Western Blot 1. After ECL detection, wash membrane with TBS-T for 5 min
Stripping and Reprobing before incubating with Immunopure stripping buffer (Pierce
Chemical Co., Rockford, IL) for 90 min at room temperature.
2. Wash membrane three times with TBS-T as described in
Subheading Immunoblotting.
3. The membrane can then be reprobed for another protein by
following the same protocol as in Subheading Immunoblotting.
Membranes can be stripped and re-probed at least 34 times,
but sensitivity will diminish significantly with each stripping.
3.2.6. Estimation of Lipid The functionally important lipid raft-associated form of the
Raft-Associated TCR- TCR-chain is quantitated by treating the SLE T cells with cho-
Chain lesterol-solubilizing agent methyl--cyclodextrin, which disrupts
lipid rafts. The increase in the amount of TCR-chain in the
detergent-soluble fraction after treatment corresponds to the lipid
raft-associated form of TCR-chain (see Note 4). Application of
this method has shown that in contrast to normal controls, the
TCR-chain is more segregated within lipid rafts in SLE T cells
(7, 13) (Fig. 3a). The membrane-bound linker for activation of T
cells (LAT) is used to demonstrate co-localization of the TCR
within lipid rafts.
1. Pipette 5 106 T cells into two microfuge tubes and resuspend
in 1 ml of RPMI-1640 without serum.
2. T cells are treated with or without 30 mM methyl--
cyclodextrin (Sigma Aldrich) for 30 min at room temperature.
3. Lyse cells in 1 % NP-40 lysis buffer as described in Subheading
Cell Activation and Lysis and centrifuge at 18,000g for
10 min at 4 C.
3 Methods and Protocols to Study T Cell Signaling Abnormalities 41
Fig. 3. Analysis of the membrane lipid raft-associated and actin cytoskeleton associated
forms of the TCR-chain in SLE T cells. (a) SLE T cells were treated with the lipid raft-
disrupting agent methyl--cyclodextrin for 30 min. The cells were lysed, and the
detergent-soluble and detergent-insoluble fractions were collected by centrifugation,
separated by SDS-PAGE, transferred, and blotted with the TCR-chain C-terminal mono-
clonal antibody (mAb). On treatment with methyl--cyclodextrin, the increased level of the
p16 and p21 23-kDa forms of the TCR-chain in the detergent-soluble fraction represent
lipid raft-associated TCR-chain. Densitometric analysis showed that, in SLE T cells, the lipid
raft-associated TCR-chain is increased compared to normal donors. (b) Sucrose density
gradient analysis showing distribution of LAT in raft and nonraft fractions; 100 106 cells
were lysed with a lysis buffer containing 1 % Brij58 and fractionated by discontinuous
sucrose gradient. Immunoblot analysis of LAT was performed with an equal volume of cell
lysate from each fraction. Raft and nonraft fractions were as indicated.
3.2.7. Lipid Raft Isolation Lipid raft and associated proteins can be isolated from T cells using
density gradient centrifugation in an ultracentrifuge (14, 15). This
allows more comprehensive evaluation of the lipid raft components.
1. T cells (25 million) are pelleted and lysed in 1 ml of 1 % Brij58
or 0.2 % Triton X-100 lysis buffer (0.2 % Triton X-100 or 1 %
Brij58 in MBS buffer [25 mM MES, 150 mM NaCl, pH 6.5],
supplemented with 1 mM sodium orthovanadate, 2 mM
EDTA, 1 mM PMSF, and 1 g/ml aprotinin). The lysis buf-
fer is prepared as follows for 200 ml stock solution: 2 g 1 %
Brij58 or 400 l 0.2 % Triton X-100 400, 1.0665 g 25 mM
MES, 1.743 g 150 mM NaCl. Freshly add 800 l 2 mM
EDTA of 0.5 M, 1 mM Na3VO4, 1 mM PMSF, 1 g/ml
aprotinin.
42 V.R. Moulton et al.
3.2.8. Cell Proliferation SLE T cells do not proliferate as well as cells from normal con-
Assay trols. This is measured most commonly by assaying thymidine
incorporation using radioactive-labeled thymidine. This protocol
also uses immobilized stimulating antibodies, obviating need for a
cross-linker.
1. Pre-coat a 96-well culture plate with 100 l per well of anti-
CD3 antibody (OKT3, 110 g/ml) and anti-CD28 antibody
(12.5 g/ml), diluted in PBS. Wrap plate in plastic wrap and
3 Methods and Protocols to Study T Cell Signaling Abnormalities 43
3.2.9. Cytokine Expression 1. Plate 5 106 SLE T cells in 1 ml medium in a 24 well culture
plate.
2. Activate cells by adding 10 g/ml anti-CD3 antibody plus
2.5 g/ml anti-CD28 antibody for 24 or 48 h.
3. Transfer cell suspension to a microfuge tube and centrifuged at
18,000g for 2 min at 4 C.
4. Harvest supernatants and use immediately or freeze at 80 C.
5. Measure IL-2 concentration using 100 l of the culture super-
natant in triplicates by Quantikine ELISA kit (R&D systems,
Minneapolis, MN). A standard curve should always be estab-
lished for reliability and reproducibility.
6. The levels of IFN- and IL-4 or other cytokines in culture
supernatants can be measured similarly using other commer-
cially available kits.
7. Read the absorbance of the color reactions at 450 nm in an
ELISA microplate reader (Bio-Rad, Hercules, CA).
3.3.1. Reverse 1. Lyse 5 106 cells in 350 l RLT buffer supplied with Qiagen
Transcriptase Polymerase RNeasy minikit (Qiagen Inc.). If not used immediately, the
Chain Reaction sample can be frozen at 80 C for several months.
Isolation of mRNA 2. Homogenize the samples by centrifugation at 18,000g for
2 min in a Qiashredder.
3. Add 350 l of 70 % ethanol to the homogenized lysate and mix
well by pipetting.
4. Apply 700 l of the sample to RNeasy minicolumn placed in a
2-ml collection tube and spin at 10,000g for 15 s. Discard the
filtrate, reload the remaining sample, and spin as above.
44 V.R. Moulton et al.
3.3.2. Real-Time PCR 1. The reaction is set up in a 20 l volume in a 96-well plate using
the SYBR green master mix from Roche (Germany).
2. Add 10 l of SYBR Green Master mix, 6 l PCR grade water,
1 l each forward and reverse primers, and 2 l of single-
stranded cDNA.
3. Pipette the sample onto a 96-well plate, seal plate with sealing
foil, and centrifuge at 1,500g for 2 min.
4. Load plate onto a Roche Lightcycler 480 instrument.
5. Run the PCR under the following conditions: denaturation at
94 C for 1 min, followed by 40 cycles of amplification: 94 C
for 15 s, 67 C annealing for 15 s, 72 C extension for 30 s,
followed by 1 cycle of melting curve at 95 C for 15 s, 65 C
for 2 min, and 97 C continuous and a final cooling step at
37 C.
6. Analyze data based on the fluorescence cycle threshold (Ct)
differences between target genes compared to housekeeping
genes using the Ct relative quantification method. If abso-
lute quantification is desired, run serial dilutions of cDNA to
generate a standard curve and calculate absolute values for
unknown genes.
7. The reaction products can also be run on agarose gel electro-
phoresis to verify for specific products.
3.3.3. mRNA Stability T cells can be treated with 10 g/ml actinomycin D to arrest RNA
Assay synthesis and can be incubated for different times. At the end of
each period, the total RNA is isolated, and RT-PCR or real-time
PCR is carried out as described above. The decrease in the level of
TCR-chain mRNA at different times compared to zero time gives
an indication of mRNA stability.
Cross-linking of the Cells 1. Take 1 106 T cells. Activate the cells as preferred.
and Preparation of Cell 2. Add 1/37 volume of 37 % formaldehyde to the cells and mix
Extract immediately. Close the cap and incubate the cells 20 min at
37 C.
3. Add 12 volumes of ice-cold PBS to cells to stop the reaction.
Spin down the cells at 650g for 5 min.
4. Wash cells twice with ice-cold PBS.
5. Lyse the cells in an Eppendorf tube at room temperature in
lysis buffer (50 mM TrisHCl, 10 mM EDTA, 1 % SDS at pH
8.1; just before use, add 1 mM PMSF, 1 g/ml aprotinin, and
1 g/ml leupeptin) at 1 106 cells/200 l. Incubate on ice for
10 min.
6. Sonicate samples on ice five times, with the duration of each
burst 10 s at continuous output and with a 30 s pause between
each burst.
7. Spin cells at 300g for 15 min at 4 C. Transfer the supernatant
to another tube and discard the pellet.
8. Freeze the sample at 80 C or proceed with immuno-
precipitation.
Chromatin Immuno- 1. Thaw the frozen sample and add 1.8 ml of ChIP dilution buf-
precipitation fer (0.01 % SDS, 1.1 % Triton X-100, 1.2 mM EDTA, 16.7 mM
TrisHCl at pH 8.1, 167 mM NaCl; just before use, add the
protease inhibitors).
2. To reduce nonspecific background, preclear the 2 ml diluted
sample with 80 l of salmon sperm DNA-blocked protein A/G
Sepharose bead (50 % slurry). Incubate the samples 1 h at
4 C, shaking in a nutator.
3. Pellet the protein A/G agarose by centrifugation at 18,000g or
top speed for 30 s and transfer the supernatant to a new tube.
4. Add TCR-chain transcription factor, Elf-1 antibody
(612 g/2 ml) and incubate overnight with shaking at 4 C.
For a negative control, use a no antibody or isotype control
antibody immunoprecipitation by incubating the supernatant
fraction with 60 l PBS or with isotype control antibody.
3 Methods and Protocols to Study T Cell Signaling Abnormalities 49
3.3.7. ChIP to Detect the The ChIP protocol can also be exploited to analyze the level of
Binding of Phosphorylated phosphorylated transcription factor bound to promoter DNA by
Transcription Factors first immunoprecipitation with anti-phosphotyrosine antibody and
then with the antibody of interest.
1. Immunoprecipitate the cell lysates with anti-phosphotyrosine
antibody 4 G10 (12 l per 2 ml) or anti-phosphoserine and
anti-phosphothreonine for 1 h and capture with 80 l (50 %
slurry) of protein A/G Sepharose.
2. Elute the anti-phosphotyrosine immunoprecipitates from the
protein A/G Sepharose beads by incubating with 10 mM phe-
nyl phosphate in lysis buffer containing 1 % NP-40 at 4 C for
15 min.
3. Reprecipitate phosphotyrosine-immunoprecipitated samples
with the antibody of interest, and the ChIP assay is continued
as described above.
3.4.1. Flow Cytometry The technique can be applied to molecules that have extracellular
domains, and fluorescent-conjugated antibodies directed to the
Cell Surface Staining
extracellular domain are readily available. For each assay, a sample
with an isotype control of the primary antibody is required to
determine the background binding. Before adding the antibodies,
the nonspecific Fc receptor binding should be blocked using
52 V.R. Moulton et al.
Intracellular Staining Flow cytometry using permeabilized cells can be applied to the
TCR-chain or other signaling molecules that do not have sub-
stantial extracellular domains or to detect cytokine expression.
Flow cytometry using permeabilized cells will provide an estimate
of the total level of expression of the TCR-chain. For each assay,
a sample with an isotype control of the primary antibody is required
to determine the background binding. Before adding the antibod-
ies, the nonspecific Fc receptor binding should be blocked using
excess human IgG. It is important to precool the centrifuge and to
perform the experiment at 4 C, except for permeabilization with
detergents, which is done at room temperature. When discarding
3 Methods and Protocols to Study T Cell Signaling Abnormalities 53
3.4.3. Confocal Microscopy Confocal microscopy can be used to determine the distribution of
molecules such as TCR-chain in three dimensions in SLE T cells
before and after activation (Fig. 5b). The microscope will take sec-
tioned images of the stained cells, and the overall distribution of
the TCR-chain can be analyzed. Also, there is better resolution of
the specimen by confocal microscopy because the laser is focused
on a single plane, as opposed to the diffuse direction of the laser
beam in fluorescence microscopy. An isotype control is required
for both nonactivated and activated samples.
1. Make two circles on a polylysine-coated slide using a pap pen
and add 1 million cells in 100 l RPMI-1640 containing
1 % FBS.
2. Place the slides in a box and keep the box at room temperature
for 1 h to adhere the cells.
3. Activate the cells by OKT3 or anti-CD3 IgM antibody at 37 C
for the time required. Clustering requires a short time activa-
tion of 23 min, whereas capping requires activation for
10 min.
4. Remove the medium by suction and fix the cells in 3.7 %
paraformaldehyde (dissolve 3.7 % paraformaldehyde in approx.
50 ml PBS, increase the pH using NaOH to dissolve, and read-
just the final pH to 7.0 using 6 N HCl and make up the volume
to 100 ml) for 30 min. Alternatively, the cells can also be
activated in a microfuge tube and then adhered to the
polylysine-coated glass slides.
5. Wash with 200 l PBS.
6. Permeabilize using 100 l permeabilization buffer, add 50 g
of human IgG to block Fc receptor binding, and incubate at
room temperature for 15 min.
56 V.R. Moulton et al.
4. Notes
are: forward 5 AGC CTC TGC CTC CCA GCC TCT TTC
TGA G 3 (3562 sense bps) and reverse 5 TCA GTG GCT
GAG AAG AGT GAA CCG GGT TG 3 (669641 antisense
bps according to numbering of Weissman et. al. (24)) (Fig. 6a).
Primers for amplifying the TCR-chain promoter are as fol-
lows: forward 5 CCA TCG AGA ACT TGT ATT TG 3, 307
to 288 sense bps; and reverse 5 GCC CTA CCT GTA ATC
GG 3, +129 to +113 antisense bps according to the number-
ing of Rellahan et al. (18) (Fig. 6b). The primers for amplify-
ing the TCR-chain 3 untranslated region are as follows:
forward 5 CAG CCA GGG GAT TTC CAC CAC TCA AAG
3 (567592 sense base pairs); and reverse 5 CCC TAG TAC
ATT GAC GGG TTT TTC CTG 3 (14721443 antisense
bps) (Fig. 6c). The -actin primers used in these experiments
are as follows: forward 5 CAT GGG TCA GAA GGA TTC
CT 3; and reverse 5 AGC TGG TAG CTC TTC TCC A 3.
3 Methods and Protocols to Study T Cell Signaling Abnormalities 59
References
1. Irving BA, Chan AC, Weiss A (1993) Functional patients with systemic lupus erythematosus.
characterization of a signal transducing motif Scand J Immunol 15:475482
present in the T cell antigen receptor zeta 14. Darlington PJ, Baroja ML, Chau TA, Siu E, Ling
chain. J Exp Med 177:10931103 V, Carreno BM, Madrenas J (2002) Surface cyto-
2. Zenner G, Vorherr T, Mustelin T, Burn P toxic T lymphocyte-associated antigen 4 partitions
(1996) Differential and multiple binding of within lipid rafts and relocates to the immunologi-
signal transducing molecules to the ITAMs of cal synapse under conditions of inhibition of T cell
the TCR-zeta chain. J Cell Biochem activation. J Exp Med 195:13371347
63:94103 15. Xavier R, Brennan T, Li Q, McCormack C,
3. Kersh EN, Shaw AS, Allen PM (1998) Fidelity Seed B (1998) Membrane compartmentation
of T cell activation through multistep T cell is required for efficient T cell activation.
receptor zeta phosphorylation. Science (N Y) Immunity 8:723732
281:572575 16. Herndon TM, Juang YT, Solomou EE,
4. Kersh EN, Kersh GJ, Allen PM (1999) Partially Rothwell SW, Gourley MF, Tsokos GC (2002)
phosphorylated T cell receptor zeta molecules Direct transfer of p65 into T lymphocytes from
can inhibit T cell activation. J Exp Med systemic lupus erythematosus patients leads to
190:16271636 increased levels of interleukin-2 promoter activ-
5. Weiss A, Littman DR (1994) Signal transduc- ity. Clin Immunol 103:145153
tion by lymphocyte antigen receptors. Cell 17. Nambiar MP, Fisher CU, Kumar A, Tsokos CG,
76:263274 Warke VG, Tsokos GC (2003) Forced expres-
6. Wange RL, Samelson LE (1996) Complex sion of the Fc receptor gamma-chain renders
complexes: signaling at the TCR. Immunity human T cells hyperresponsive to TCR/CD3
5:197205 stimulation. J Immunol 170:28712876
7. Nambiar MP, Enyedy EJ, Fisher CU, Krishnan 18. Rellahan BL, Jensen JP, Howcroft TK, Singer
S, Warke VG, Gilliland WR, Oglesby RJ, Tsokos DS, Bonvini E, Weissman AM (1998) Elf-1
GC (2002) Abnormal expression of various regulates basal expression from the T cell
molecular forms and distribution of T cell recep- antigen receptor zeta-chain gene promoter.
tor zeta chain in patients with systemic lupus J Immunol 160:27942801
erythematosus. Arthritis Rheum 46:163174 19. Valensin S, Paccani SR, Ulivieri C, Mercati D,
8. Takeuchi T, Tsuzaka K, Pang M, Amano K, Pacini S, Patrussi L, Hirst T, Lupetti P, Baldari
Koide J, Abe T (1998) TCR zeta chain lacking CT (2002) F-actin dynamics control segrega-
exon 7 in two patients with systemic lupus ery- tion of the TCR signaling cascade to clustered
thematosus. Int Immunol 10:911921 lipid rafts. Eur J Immunol 32:435446
9. Moulton VR, Tsokos GC (2010) Alternative 20. Nambiar MP, Fisher CU, Enyedy EJ, Warke VG,
splicing factor/splicing factor 2 regulates the Krishnan S, Tsokos GC (2000) Heat stress down-
expression of the zeta subunit of the human T regulates TCR zeta chain expression in human T
cell receptor-associated CD3 complex. J Biol lymphocytes. J Cell Biochem 79:416426
Chem 285:1249012496 21. Caplan S, Baniyash M (1996) Normal T cells
10. Moulton VR, Kyttaris VC, Juang YT, express two T cell antigen receptor popula-
Chowdhury B, Tsokos GC (2008) The RNA- tions, one of which is linked to the cytoskeleton
stabilizing protein HuR regulates the expres- via zeta chain and displays a unique activation-
sion of zeta chain of the human T cell dependent phosphorylation pattern. J Biol
receptor-associated CD3 complex. J Biol Chem Chem 271:2070520712
283:2003720044 22. Rozdzial MM, Malissen B, Finkel TH (1995)
11. Liossis SN, Ding XZ, Dennis GJ, Tsokos GC Tyrosine-phosphorylated T cell receptor zeta
(1998) Altered pattern of TCR/CD3-mediated chain associates with the actin cytoskeleton
protein-tyrosyl phosphorylation in T cells from upon activation of mature T lymphocytes.
patients with systemic lupus erythematosus. Immunity 3:623633
Deficient expression of the T cell receptor zeta 23. Rozdzial MM, Pleiman CM, Cambier JC,
chain. J Clin Invest 101:14481457 Finkel TH (1998) pp 56Lck mediates TCR
12. Laemmli UK (1970) Cleavage of structural zeta-chain binding to the microfilament
proteins during the assembly of the head of cytoskeleton. J Immunol 161:54915499
bacteriophage T4. Nature 227:680685 24. Weissman AM, Baniyash M, Hou D, Samelson
13. Abe T, Toguchi T, Takeuchi T, Kiyotaki M, LE, Burgess WH, Klausner RD (1988) Molecular
Homma M (1981) Mitogenic responses to cloning of the zeta chain of the T cell antigen
lipopolysaccharide by B lymphocytes from receptor. Science (N Y) 239:10181021
Chapter 4
Abstract
Systemic lupus erythematosus (SLE) is characterized by abnormal activation and cell death signaling within
the immune system. Activation, proliferation, or death of cells of the immune system is dependent on
controlled reactive oxygen intermediates (ROI) production and ATP synthesis in mitochondria. The mito-
chondrial transmembrane potential (ym) reflects the energy stored in the electrochemical gradient across
the inner mitochondrial membrane which, in turn, is used by F0F1-ATPase to convert ADP to ATP during
oxidative phosphorylation. Mitochondrial hyperpolarization (MHP) and transient ATP depletion repre-
sent early and reversible steps in T cell activation and apoptosis. By contrast, T lymphocytes of patients
with SLE exhibit elevated ym, i.e., persistent mitochondrial hyperpolarization (MHP), cytoplasmic alka-
linization, increased ROI production, as well as diminished levels of intracellular glutathione and ATP.
Increased production of nitric oxide has been identified as a cause of MHP and increased mitochondrial
biogenesis. Oxidative stress affects signaling through the T cell receptor as well as activity of redox-
sensitive caspases. ATP depletion causes diminished activation-induced apoptosis and sensitizes lupus
T cells to necrosis. Activation of the mammalian target of rapamycin (mTOR) has recently emerged as a
key sensor of MHP and mediator of enhanced Ca2+ flux in lupus T cells.
Key words: Systemic lupus erythematosus, Mitochondrial hyperpolarization, Reactive oxygen inter-
mediates, Cytoplasmic alkalinization, Caspases, Glutathione depletion, ATP depletion, Apoptosis,
Necrosis, mTOR
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_4, Springer Science+Business Media New York 2012
61
62 A. Perl et al.
1.1. Mitochondrial Both cell proliferation and apoptosis are energy-dependent pro-
Checkpoints of T Cell cesses. Energy in the form of ATP is provided through glycolysis
Activation and and oxidative phosphorylation. The mitochondrion, the site of
Apoptosis oxidative phosphorylation, has long been identified as a source of
energy and cell survival (8). The synthesis of ATP is driven by an
electrochemical gradient across the inner mitochondrial membrane
maintained by an electron transport chain and the membrane
potential (negative inside and positive outside). A small fraction of
electrons react directly with oxygen and form reactive oxygen
intermediates (ROI). Disruption of the mitochondrial membrane
potential has been proposed as the point of no return in apoptotic
signaling (911). Mitochondrial membrane permeability is subject
to regulation by an oxidationreduction equilibrium of ROI, pyri-
dine nucleotides (NADH/NAD + NADPH/NADP) and GSH
levels (12). Regeneration of GSH by glutathione reductase from
its oxidized form, GSSG, depends on NADPH produced by the
pentose phosphate pathway (PPP) (13). ROI levels and ym are
regulated by the supply of reducing equivalents from PPP (14, 15).
While ROI have been considered as toxic by-products of aerobic
existence, evidence is now accumulating that controlled levels of
ROI modulate various aspects of cellular function and are neces-
sary for signal-transduction pathways, including those mediating
T cell activation and apoptosis (16).
Increased production of ROI was demonstrated in TNF
(1719) and Fas-mediated cell death (9, 14, 2023). Disruption of
the mitochondrial membrane potential (ym) has been proposed
as the point of no return in apoptotic signaling (911). Interestingly,
elevation of ym, mitochondrial hyperpolarization (MHP), and
ROI production precede phosphatidylserine (PS) externalization
and a disruption of ym in Fas- (15) and H2O2-induced apoptosis
of Jurkat human leukemia T cells and normal human peripheral
blood lymphocytes (24). These observations were extended to p53
(25), tumor necrosis factor a (26), staurosporin (27), camptothe-
cin (28), and nitric oxide-induced apoptosis (29). Elevation of ym
is independent from activation of caspases and represents an early
event in apoptosis (15, 25). Pretreatment with caspase inhibitors,
DEVD, Z-VAD, and Boc-Asp, completely abrogated Fas-induced
PS externalization, indicating that activation of caspase-3, caspase-8,
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 63
1.2. Pharmacological MHP predisposes for increased ROI production (38). Oxidative
Targeting stress affects activity of transcription factors AP-1 and NF-kB
of Mitochondrial (44, 45), and, further downstream, may lead to the skewed
Dysfunction in SLE expression of IL-2, TNF, and IL-10 (46). Increased spontaneous
apoptosis of lymphocytes has been linked to increased IL-10 pro-
duction, release of Fas ligand, and overexpression of Fas receptor
in SLE (47). Since increased ROI levels confer sensitivity to H2O2,
NO, TNF, and Fas-induced cell death (14, 15), elevated baseline
Table 1
Signaling abnormalities of T cell death in patients with SLE
Fig. 1. (continued) space (34). Phosphorylation of BAD by mitochondria-anchored PKA results in anti-apoptotic sequestration
of BAD into the cytosol (85). Signaling through cell death receptors, such as Fas (15), CD3/CD28 co-stimulation (36, 37),
ROS (24), NO (29), as well as lymphokines, IL-3, IL-10, IFN-g, and TGF-b1 influence ym, ATP synthesis and susceptibility
to apoptosis (37). MHP and mitochondrial biogenesis is mediated via production of NO by eNOS or nNOS (39) and
up-regulation of transcription factors PGC-1a, Tfam, and ALAS (86). NO production by eNOS may be compartmentalized to
the T cell synapse (87). NO causes transient MHP via reversible inhibition of complex IV/cytochrome c oxidase (29) and
persistent MHP via S-nitrosylation of complex I of the ETC in a state of GSH depletion (40). mTOR senses ym (41), interacts
with the small GTPase HRES-1/Rab4 (43), and regulates Ca2+ release (42).
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 65
Fig. 1. Overview of mitochondrial redox and metabolic checkpoints of T cell activation and apoptosis signals. Antigen
binding-initiated signaling through the T cell receptor complex/CD3 and the CD28 co-stimulatory molecule activate phso-
phatidylinositol 3-kinase (PI3K) and protein tyrosine kinases (PTK). Increased cytosolic Ca2+ concentration activates the
serine/threonine phosphatase calcineurin which dephosphorylates the NFAT. Dephosphorylated NFAT can translocate to the
nucleus where it promotes transcription of IL-2 in concert with AP-1, NF6B, and Oct-1. Ca2+ flux into mitochondria increases
production of ROS and NF-6B activation (7981). Mitochondrial membrane integrity is maintained by a balance of mem-
brane-stabilizing bcl-2 and bcl-XL and pore-inducing bax and bad (34) as well as the metabolic capacity to synthesize
reducing equivalents, NADPH, GSH, and TRX. Controlled increase of ROS levels activates NF-6B and promotes cell growth.
Excess ROS production and disruption of ym lead to activation-induced cell death executed by caspase 3 (digesting vitally
important proteins PARP, 70K U1RNP, lamin, and actin) and caspase 3-dependent DNase (CAD, causing nuclear DNA frag-
mentation). Cleavage by caspase 3 is thought to expose cryptic epitomes and cause autoantigenicity of self antigens (82).
Activity of redox-sensitive transcription factors NF-6B, p53, AP-1, and Sp1 is regulated through release from inhibitor
complexes and conformational changes in their active sites. Intracellular antioxidants reduced glutathione (GSH) and thi-
oredoxin (TRX-DT) are regenerated at the expense of NADPH supplied primarily through metabolism of glucose via the
pentose phosphate pathway (PPP) (83). Among PPP products, ribose 5-phosphate is required for nucleotide and DNA syn-
thesis and support cell growth, C3C7 sugars influence mitochondrial function and ROS production, inositol and ADP-ribose
serve as precursors for second messengers, inositol phosphates and cADP-ribose, respectively. Dehydroascorbate (DHA) is
imported through GLUT1. DHA is metabolized through the PPP, thereby enhancing GSH levels. DHA also increases surface
expression of Fas-R (84). Glutathione reductase and TRX reductase synthesize GSH and TRX-DT at the expense of NADPH.
Formulation of the PPP and its efficiency to provide NADPH is dependent on the expression of G6PD and TAL (14, 15). ym
is controlled by intracellular GSH/NADH/NADPH levels, integrity of the permeability transition pore complex largely com-
prised of adenine nucleotide translocator (ANT, inner membrane), voltage-dependent anion channel (VDAC, outer mem-
brane), and translocation and dimerization of pro- and anti-apoptotic bcl-2 family members in the intermembrane
66 A. Perl et al.
ym, ROI production, and pHi may have key roles in altered
activation and death of lupus T cells. Although MHP was not
affected, IL-10 antibody or IL-12 normalized ROI production
and intracellular alkalinization in lupus PBL (37). Therefore, IL-10
antagonists may partially correct signaling dysfunction in lupus.
Recent studies showed diminished GSH/GSSG ratios in the
kidneys of 8-month-old vs. 4-month-old (NZB NZW) F1 mice;
treatment with N-acetylcysteine (NAC), a precursor of GSH and
stimulator of its de novo biosynthesis, prevented the decline of
GSH/GSSG ratios, reduced autoantibody production and devel-
opment of glomerulopnephritis (GN) and prolonged the survival
of (NZB NZW) F1 mice (48). Oral NAC has been used to treat
oxidative stress in patients with idiopathic pulmonary fibrosis (IPF)
(49). In a 1-year study of IPF patients treated with prednisone and
azathioprine, addition of NAC (3 600 mg/day) improved vital
capacity and reduced myelotoxicity in comparison to placebo.
Therefore, prospective clinical studies appear justified to assess
whether NAC treatment can reverse GSH depletion, correct T cell
signaling defects and provide clinical benefit to patients with
lupus.
NO production is a particularly interesting target because it
provides a link between seemingly dissociated features of T cell
activation and mitochondrial function. NO induces MHP and
mitochondrial biogenesis, increases Ca2+ in the cytosol and mito-
chondria of normal T cells, and recapitulates the enhanced CD3/
CD28-induced Ca2+ fluxing of lupus T cells (50). NO contributes
to the development of GN in the MRL/lpr lupus mouse model
(51). Inactivation of iNOS does not block the development of
lupus (52), suggesting a role for eNOS and nNOS isoforms
expressed in T cells. However, given the widespread expression of
these isoforms in vascular smooth muscle and brain, it will be nec-
essary to develop T-cell-specific approaches for inhibiting NOS to
avoid potentially deleterious side effects.
2. Materials
24. Nigericin (Sigma, St. Louis, MO; diluted from a stock solution
of 500 mg/ml in ethanol).
25. Deproteinizing buffer for glutathione (GSH) assay: 70 % per-
chloric acid and 15 mM bathophenanthrolinedisulfonic acid
(BPDS, Sigma).
26. g-Glutamyl glutamate (g-Glu-Glu, Sigma), internal standard
for GSH assay.
27. After repeated freezing and thawing, samples were centrifuged
at 15,000 g for 3 min. 50 ml of 100 mM mono-iodo-acetic
acid in 0.2 mM m-cresol purple was added to 500 ml superna-
tant. Samples were neutralized by addition of 480 ml of 2 M
KOH and 2.4 M KHCO3 and incubated in the dark at room
temperature for 10 min. Then, 1 ml of 1 % fluoro-dinitro-
benzene was added and the samples were incubated in the dark
at 4 C overnight. After centrifugation and filtering, 100 ml of
supernatants were injected into the HPLC Model 2690 (Waters
Alliance System, Milford, MA) equipped with a Model 996
photodiode array detector and Spherisorb NH2 column
(4.6 250 mm; 10 mm; Waters).
28. 7-Amino-4-trifluoromethyl-coumarin (AFC, Sigma).
29. Caspase substrate peptides: DEVD-AFC, Z-IETD-AFC, where
Z represents a benzyloxycarbonyl group; caspase inhibitor
peptides Z-Val-Ala-Asp(Ome).fmk (Z-VAD), Boc-Asp.fmk
(Boc-Asp) as well as non-caspase cysteine protease inhibitor,
Z-Phe-Ala.fmk (Z-FA) can be obtained from Enzyme Systems
Products (Livermore, CA).
30. Caspase assay buffer: 250 mM sucrose, 20 mM HEPESKOH
pH 7.5, 50 mM KCl, 2.5 mM MgCl2, 1 mM dithiothreitol.
31. Versene (Life Technologies).
32. Concanavalin A (Con A, Sigma).
33. Goat anti-mouse IgG (ICN, Aurora OH).
34. Tritiated thymidine, 3HTdR (ICN).
35. CH-11 IgM monoclonal antibody to Fas/Apo-1/CD95
(Upstate Biotechnology, Saranac Lake, NY).
36. Complete RPMI medium: RPMI 1640 supplemented with
10 % fetal calf serum, 2 mM L-glutamine, 100 IU/ml penicil-
lin, 100 mg/ml gentamicin, and 10 mg/ml amphotericin B.
37. Plastic tissue culture dishes (Becton Dickinson, Franklin
Lake, NJ).
38. Phenol, chloroform, isoamyl alcohol, proteinase K, agarose
(all RNase and DNase-free. molecular biology grade, from
Sigma).
39. Spectrophotometer.
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 69
40. Luminometer.
41. Carbonyl cyanide m-chlorophenylhydrazone (mClCCP,
Sigma).
42. Folin & Ciocalteus Phenol Reagent Solution (Sigma).
43. 4 mm diameter 0.45 mm polypropylene filter (Whatman,
Mainstead, England).
44. Monoclonal antibody to poly(ADP-ribose) polymerase (PARP)
C-2-10 (53).
45. Monoclonal antibody 5F7 directed to C-terminal amino acids
176-460 of human FLICE/Mch5/caspase-8 (Panvera,
Madison, WI).
46. Monoclonal antibody 31A1067 directed to caspase 3 (Gene
Therapy Systems, San Diego, CA).
47. Monoclonal antibody C4 directed to human b actin
(Boehringer, Indianapolis, IN).
48. Biotinylated secondary antibodies and horseradish peroxidase-
conjugated avidin (Jackson Laboratories, West Grove, PA).
49. 4-Chloronaphthol (Sigma).
50. Enhanced chemiluminescence detection kit (Western Lightning
Chemiluminescence Reagent Plus, PerkinElmer Life Sciences,
Boston, MA).
51. Kodak Image Station 440CF equipped with Kodak 1D
Image Analysis Software (Eastman Kodak Company,
Rochester, NY).
3. Methods
3.1.2. Separation 1. Precoat Petri dishes with autologous serum for 30 min
of Monocytes and at 37 C.
Peripheral Blood 2. Add 5 ml of PBMC (maximum 5 106/ml) to serum-pre-
Lymphocytes treated dishes and incubate for 1 h at 37 C.
3. Remove nonadherent cells by washing three times with 5 ml of
warm (37 C) complete RPMI medium.
4. To obtain a monocyte-enriched cell fraction wash dishes vigor-
ously with warm medium.
5. Add 4 ml of ice-cold 0.05 % Versene and 1 ml autologous
serum to each dish for 15 min at room temperature.
6. Scrape off loosely adherent monocytes with a rubber police-
man under inverse microscopic control.
7. The monocyte-depleted fraction of peripheral blood lympho-
cytes (PBL) should contain less than 2 % monocytes, while the
monocyte-enriched fraction should contain 9095 % mono-
cytes by staining with CD14 monoclonal antibody.
3.1.3. Cell Culture, Human PBL undergo apoptosis in response to repetitive activation
Activation, and Viability through the T cell receptor, i.e., CD3/CD28 co-stimulation
Assays resulting in activation-induced cell death (AICD) (36, 37), cross-
linking of cell surface death receptors such as Fas/Apo-1/CD95
(15) or elevation of intracellular ROI levels after treatment with
H2O2 (24, 36). Monocytes/macrophages remove apoptotic bod-
ies via phagocytosis, therefore processing of cell death signals by
lymphocytes is best evaluated using PBL (see Note 2).
CD3/CD28 Co-stimulation 1. Precoat 10 cm diameter plastic Petri dishes with 100 mg/ml
of PBL goat anti-mouse IgG (diluted in PBS) for 2 h at 37 C.
2. Wash plates with PBS, add OKT3 monoclonal antibody (1 mg/
ml), and incubate for 1 h at 37 C.
3. Add PBL (106 cells/ml in complete RPMI medium).
4. For CD28 co-stimulation add 500 ng/ml mAb CD28.2 and
incubate cells at 37 C for the desired period of time.
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 71
Treatment with H2O2 1. Prepare fresh 10 mM H2O2 in PBS from 30 % stock solution.
2. Seed PBL at 2 106 cells/ml in complete RPMI medium.
3. Add an equal volume of complete RPMI medium without
(control) or with 100 mM H2O2.
4. Incubate cells at 37 C for the desired period of time.
Monitoring of Cell Death Apoptosis is monitored by observing cell shrinkage while counting
trypan blue-stained cells, DNA fragmentation using agarose gel
electrophoresis and quantified by flow cytometry after concurrent
staining with fluorescein-conjugated annexin V (annexin V-FITC,
R&D Systems, Minneapolis, MN; FL-1) and propidium iodide
(PI, FL-2) as earlier described (14, 15, 55, 56). Staining with phy-
coerythrin-conjugated annexin V (annexin V-PE, R&D Systems)
was used to monitor PS externalization (FL-2) in parallel with
measurement of ROI levels and ym (see below). Apoptosis rates
are expressed as percentage of annexin V-positive/PI-negative
cells. Necrosis is assessed by observing cellular and nuclear swell-
ing. Swollen nuclei of necrotic cells can be observed by staining
with propidium iodide (PI, 50 mg/ml). Necrotic cells are enumer-
ated by direct PI staining using flow cytometry and fluorescence
microscopy. Necrosis rates are expressed as percentage of PI-positive
population within annexin-positive cells (36) (see Note 4).
DNA Fragmentation Assay
1. Wash cells 3 in PBS using screw-capped 15 ml polypropylene
tubes.
2. Resuspend up to 5 107 cells in 100 mM NaCl, 10 mM
TrisHCL pH 8.0, and 1 mM EDTA.
3. Add 250 ml of 10 % SDS.
72 A. Perl et al.
Specific Protocol
1. Following apoptosis assay, wash cells two times in 5 mM
HEPES-buffered saline (HBS, containing 0.9 % NaCl)
pH 7.4.
2. Resuspend 2 105 cells in 200 ml of annexin binding buffer if
concurrently stained with Annexin V-FITC or Annexin V-PE
matching oxidation-sensitive dyes emitting FL-2 (HE) or FL-1
fluorescence (DCF, R123), respectively. Alternatively, 2 105
cells can be resuspended in 200 ml of 5 mM HBS. This
buffer, lacking Ca2+, does not allow concurrent staining with
Annexin V.
3. Subsequently, aliquots of cell suspensions are stained with
several potentiometric dyes in parallel.
(a) Add 200 ml of dye solution containing 0.1 mM DHR (exci-
tation: 488 nm, emission: 530 nm recorded in FL-1). This
dye can be added in combination with Annexin V-PE
emitting FL-2 fluorescence. Staining is done in the dark at
room temperature for 2 min, timed with a stopwatch, fol-
lowed by running on the flow cytometer.
(b) Add 200 ml of dye solution containing 1 mM DCFH-DA
(excitation: 488 nm, emission: 525 nm recorded in FL-1).
This dye can be added in combination with Annexin V-PE
emitting FL-2 fluorescence. Staining is done in the dark at
room temperature for 15 min, timed with a stopwatch,
followed by running on the flow cytometer.
(c) Add 200 ml of dye solution containing 1 mM HE (excita-
tion: 488 nm, emission: 605 nm recorded in FL-2). This
dye can be added in combination with Annexin V-FITC
emitting FL-1 fluorescence. Staining is done in the dark at
room temperature for 15 min, timed with a stopwatch,
followed by running on the flow cytometer.
4. For each sample, measurements are carried out on 10,000
cells.
3.4. Intracellular Intracellular pH measurements are carried out with flow cytometry
pH Measurement using the pH-sensitive dye carboxy SNARF-1-acetoxymethyl ester
acetate (SNARF-1) as described by Wieder et al. (62). SNARF-1
enters cells passively as a nonpolar ester. It is then hydrolyzed by
intracellular esterases into a polar compound unable to leave mem-
brane-intact cells. The emission spectrum of SNARF-1 undergoes
a pH-dependent wavelength shift. The ratio of fluorescence inten-
sities emitted at two different wavelengths (FL2:580 nm and
FL3:650 nm) is used for determination of pH.
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 75
3.5. Measurement T cell activation and apoptosis require the energy provided by
of Intracellular ATP ATP (63). Intracellular ATP concentration is a key switch in the
and ADP Levels cells decision to die via apoptosis or necrosis (64) and, therefore,
depletion of ATP may be responsible for defective apoptosis and a
predisposition to necrosis in patients with SLE (36). Intracellular
ATP levels can be determined with great sensitivity and specificity
using the luciferinluciferase method (65). Addition of luciferin
and firefly luciferase to ATP-containing biological sample results in
light emission. The luminometric ATP assay is based on the firefly
luciferase reaction:
ATP + D-Luciferin + O2 AMP + pyrophosphate
+ oxyluciferin + CO2 + light.
The quantum efficiency is very high resulting in almost one
photon per ATP molecule consumed in the reaction. The light is
measured in luminometer. Under assay conditions of constant
luciferase activity, intensity of the emitted light is proportional to
the ATP concentration. The assay is calibrated by the addition of a
known amount of ATP.
3.5.1. Collection of Cells 1. Collect 5 106 PBL by centrifugation at 300 g for 10 min and
for ATP Assay wash once in PBS.
2. Resuspend cell pellet in 50 ml of PBS and mix with equal vol-
umes of 2.5 % trichloroacetic acid. Such extracts can be stored
at 20 C.
3. Measure the total protein content of each sample using the
Lowry assay (66).
76 A. Perl et al.
Assay Procedure 1. Add 100 ml of sample (sample + buffer = 100 ml) per tube.
2. Add 1.0 ml of Lowry stock reagent to each tube.
3. Incubate 30 min at room temperature.
4. Add 100 ml of Folins reagent to each tube.
5. Incubate 30 min at room temperature.
6. Read in a spectrophotometer at 595 nm.
3.5.3. ATP Assay The ATP contents of PBLs from patients with SLE and control
donors were assayed in parallel.
The bioluminescence assay was performed using an ATP deter-
mination kit (A22066, Invitrogen, Carslbad, CA) according to the
manufacturers instructions. Utilizing a Synergy 2 Multi-Mode
Microplate Reader (BioTek, Winooski, VT), equipped with auto-
matic reagent dispensers; in all-white, flat-bottom, 96-well plates
(Cat. No 7571, Thermo Scientific, Rochester, NY), ATP standard
Table 2
Preparation of protein standard solution for Lowry assay
Standard solution protein amount (mg) Standard solution volume(ml) Buffer volume (ml)
0 0 100
10 10 90
20 20 80
30 30 70
50 50 50
75 75 25
100 100 0
The standard calibration solution is dissolved at a concentration of 1 mg/ml in a buffer similar to the biological sample
of unknown protein content, such as PBS
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 77
Assessment of ATP/ADP ADP can be measured by its conversion to ATP, using the ApoGlow
Ratio and ADP Levels kit (Lumitech, Nottingham, UK). The produced ATP is then
detected by the above luciferinluciferase method.
1. Dispense 100 ml of standard reaction solution with experimental
sample as described in step 8a in ATP Assay Protocol into 96
well plates.
2. Load plate into 96-well plate reader and record luminescence:
reading A.
3. After 10 min lag period, add 20 ml of ADP-converting reagent
to each well using multichannel pipettor or autodispenser built
into luminometer if available.
4. Take a 1 s integrated reading: reading B. If autoinjector is
unavailable, reading B should be taken before addition of
ADP-converting reagent.
5. After 5 min incubation allowing conversion of ADP to ATP
take a final 1 s integrated reading: reading C.
6. ADP:ATP ratio is calculated as follows: (C B)/A.
3.6. HPLC Assay Reduced (GSH) and oxidized glutathione (GSSG) as well as other
of Glutathione Levels intermediates of GSH metabolism can be concurrently measured
by reverse phase ion-exchange high-performance liquid chroma-
tography (HPLC) using UV detection at 365 nm (68).
We use a two-step derivatization procedure: (1) S-carbo-
xymethylation of the reduced SH groups with iodoacetic acid to
prevent their oxidation and (2) N-dinitrophenylation with
1-fluoro,2,4-dinitrobenzene to allow UV detection (Fig. 2).
1. Wash 2 107 PBL once in 5 ml of PBS and store cell pellet
at 80 C until assay.
2. Resuspend cell pellet in 250 ml of H2O. Use 10 ml of cell
suspension to measure protein content as described in
Subheading 3.5.2.
3. Add 50 ml of 70 % perchloric acid, 25 ml of 15 mM BPDS to
deproteinize sample as well as 25 ml of g-Glu-Glu as internal
standard.
1. 2.
1-fluoro-2,4-
iodoacetic acid dinitrobenze
10 min, room 4 C,
temperature, in dark, overnight, in
pH=8-9 dark
3.7. Caspase Enzyme Activation of the caspase enzyme cascade is a hallmark of apoptosis.
Assays Caspase-3 is a key effector of all apoptosis pathways, amplifying the
signal from initiator caspases (such as caspase-8) and indicating a
final commitment to cellular disassembly. In addition to cleaving
other caspases in the enzyme cascade, caspase-3 has been shown to
cleave poly(ADP-ribose) polymerase (PARP), DNA-dependent
protein kinase, protein kinase C, the 70 kDa component of U1
snRNP, and actin (see Note 6).
1. Induce apoptosis in cells by desired method. Remember to
incubate a concurrent control culture without induction.
Include cells treated with caspase inhibitor DEVD-CHO as
negative control (15).
2. After washing cells once in PBS, pellet 106 cells per experimental
sample at 400 g for 5 min. Cell pellet can be stored at 80 C
until measurement.
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 81
3.8. Measurement The method described here explains (a) how to permeabilize PBL
of Mitochondrial (70); (b) and measure the activity of the mitochondrial ETC
Electron Transport (71, 72).
Chain Activity 1. Mannitol (Sigma).
3.8.1. Reagents 2. KCl (Fisher).
3. MgCl (J.T. Baker, Phillipsburg, NJ).
4. K2PO4 (USB).
5. Digitonin (Sigma).
6. Bovine serum albumin fraction V, heat shock, fatty-acid free
(BSA; Roche).
7. ADP (Sigma).
8. Sodium pyruvate (Sigma).
9. Malic acid (Sigma).
10. Rotenone (Sigma).
11. Succinic acid (Fisher).
12. ATP (Sigma).
13. Antimycin A (Sigma).
14. L-Ascorbic acid (Sigma).
15. N,N,N ,N -Tetramethyl-p-phenylenediamine (TMPD; Sigma).
16. Oxygraph system (Hansatech Instruments, Norfolk,
England).
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 83
3.8.2. Permeabilizing PBL 1. Wash PBL in PBS twice this is to ensure the cells are as clean as
possible if they are not truly clean it can affect how well the
cells are permeabilized.
2. Resuspend at a concentration of 5 106/ml in respiration
buffer (RB) containing 0.3 M mannitol, 10 mM KCl, 5 mM
MgCl, 10 mM K2PO4 pH adjusted to 7.4.
3. Set aside 300 ml of the cell suspension for whole cell respiration
to determine the baseline respiration rate of your cells.
4. Add digitonin to a final concentration of 60 mg/ml, as an
example, add 21 ml of a 2 mg/ml stock solution to 700 ml of
cell suspension; digitonin purification is described in 3.9.4.
5. Incubate for 1 min at room temperature.
6. Add 5 volumes (e.g., 5 21 ml = 105 ml, for 700 ml of cell
suspension) of RB containing 1 mg/ml of BSA (made fresh
that day) to stop the permeablization of the cell membranes.
7. Spin down cells at 500 g for 10 min.
8. Resuspend the pellet at 5 106 cells/ml in RB containing
1 mg/ml BSA and 0.5 mM ADP (made fresh that day).
9. Let the cells rest at room temperature for 5 min.
Testing Complex I (a) Place 300 ml of cell suspension into the oxygraph chamber.
(b) Collect a baseline reading.
(c) Add pyruvate and malate to final concentrations of 8 mM and
0.2 mM, respectively, to measure complex I activity.
Testing Complexes IIIV (a) Inhibit complex I by adding Rotenone to a final concentration
(If Possible Done of 3 mM.
in Separate Chamber) (b) Complex II is measured with the addition of 10 mM final
concentration succinate and 130 mM ATP.
(c) Inhibit complex II with a final concentration of 1 mM anti-
mycin A.
(d) Complexes III and IV are measured through the addition of
ascorbate and TMPD to final concentrations of 10 and 0.2 mM
respectively.
4. Notes
References
1. Elkon KB (1994) Apoptosis in SLEtoo little 13. Mayes PA (1993) The pentose phosphate path-
or too much? [Review] [45 refs]. Clin Exp way & other pathways of hexose metabolism.
Rheumatol 12:553559 In: Murray RK, Granner DK, Mayes PA,
2. Perl A, Banki K (1999) Molecular mimicry, Rodwell VW (eds) Harpers biochemistry, 23rd
altered apoptosis, and immunomodulation as edn. Appleton & Lange, Norwalk, CT, pp
mechanisms of viral pathogenesis in systemic 201211
lupus erythematosus. In: Kammer GM, Tsokos 14. Banki K, Hutter E, Colombo E, Gonchoroff
GC (eds) Lupus: molecular and cellular patho- NJ, Perl A (1996) Glutathione levels and sensi-
genesis, 1st edn. Humana, Totowa, NJ, pp tivity to apoptosis are regulated by changes in
4364 transaldolase expression. J Biol Chem
3. Cohen JJ, Duke RC, Fadok VA, Sellins KS 271:3299433001
(1992) Apoptosis and programmed cell death 15. Banki K, Hutter E, Gonchoroff N, Perl A
in immunity. Annu Rev Immunol 10:267293 (1999) Elevation of mitochondrial transmem-
4. Thompson CB (1995) Apoptosis in the patho- brane potential and reactive oxygen intermedi-
genesis and treatment of disease. Science ate levels are early events and occur
267:14561462 independently from activation of caspases in
Fas signaling. J Immunol 162:14661479
5. Emlen W, Niebur JA, Kadera R (1994)
16. Perl A, Gergely P Jr, Puskas F, Banki K (2002)
Accelerated in vitro apoptosis of lymphocytes
Metabolic switches of T-cell activation and
from patients with systemic lupus erythemato-
apoptosis. Antiox Redox Signal 4(5):427443
sus. J Immunol 152:36853692
17. Meier B, Radeke HH, Selle S, Younes M, Sies
6. Casciola-Rosen LA, Anhalt G, Rosen A (1994) H, Resch K et al (1989) Human fibroblasts
Autoantigens targeted in systemic lupus ery- release reactive oxygen species in response to
thematosus are clustered in two populations of interleukin-1 or tumor necrosis factor-a.
surface structures on apoptotic keratinocytes. J Biochem J 263:539545
Exp Med 179:13171330
18. Hennet T, Richter C, Peterhans E (1993)
7. Kovacs B, Vassilopoulos D, Vogelgesang SA, Tumor necrosis factor-a induces superoxide
Tsokos GC (1996) Defective CD3-mediated anion generation in mitochondria of L929
cell death in activated T cells from patients with cells. Biochem J 289:587592
systemic lupus erythematosus: role of decreased
19. Schulze-Osthoff K, Krammer PH, Droge W
intracellular TNF-alpha. Clin Immunol
(1994) Divergent signaling via APO-1/Fas and
Immunopathol 81:293302
the TNFreceptor, two homologous molecules
8. Skulachev VP (1999) Mitochondrial physiol- involved in physiological cell death. EMBO J
ogy and pathology; concepts of programmed 13:45874596
death of organelles, cells and organisms. Mol 20. Gergely P, Nekam K, Lang I, Kalmar L,
Aspects Med 20:139140 Gonzalez-Cabello R, Perl A (1984)
9. Xiang J, Chao DT, Korsmeyer SJ (1996) BAX- Ketoconazole in vitro inhibits mitogen-induced
induced cell death may not require interleukin blastogenesis, antibody-dependent cellular
1b-converting enzyme-like proteases. Proc cytotoxicity, natural killer activity and random
Natl Acad Sci U S A 93:1455914563 migration of human leukocytes.
10. Susin SA, Zamzami N, Castedo M, Daugas E, Immunopharmacology 7:167170
Wang H-G, Geley S et al (1997) The central 21. Williams MS, Henkart PA (1996) Role of reac-
executioner of apoptosis: multiple connections tive oxygen intermediates in TCR-induced
between protease activation and mitochondria death of T cell blasts and hybridomas.
in Fas/Apo-1/CD95- and ceramide-induced J Immunol 157:23952402
apoptosis. J Exp Med 186:2537 22. Gulbins E, Brenner B, Schlottmann K, Welsch
11. Vander Heiden M, Chandel NS, Williamson J, Heinle H, Koppenhoefer UL et al (1996)
EK, Schumaker PT, Thompson CB (1997) Fas-induced programmed cell death is medi-
Bcl-XL regulates the membrane potential and ated by a Ras-regulated O2-synthesis.
volume homeostasis of mitochondria. Cell Immunology 89:205212
91:627637 23. Um HD, Orenstein JM, Wahl SM (1996) Fas
12. Constantini P, Chernyak BV, Petronilli V, mediates apoptosis in human monocytes by a
Bernardi P (1996) Modulation of the mitochon- reactive oxygen intermediate dependent path-
drial permeability transition pore by pyridine way. J Immunol 156:34693477
nucleotides and dithiol oxidation at two sep- 24. Puskas F, Gergely P, Banki K, Perl A (2000)
arate sites. J Biol Chem 271:67466751 Stimulation of the pentose phosphate pathway
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 87
the female NZB NZW F1 mouse model of 58. Tanner MK, Wellhausen SR, Klein JB (1993)
systemic lupus erythematosus (SLE). Lupus Flow cytometric analysis of altered mononu-
10(4):258265 clear cell transmembrane potential induced by
49. Demedts M, Behr J, Buhl R, Costabel U, cyclosporin. Cytometry 14:5969
Dekhuijzen R, Jansen HM et al (2005) High- 59. Smiley ST, Reers M, Mottola-Hartshorn C,
dose acetylcysteine in idiopathic pulmonary Lin M, Chen A, Smith TW et al (1991)
fibrosis [see comment]. N Engl J Med Intracellular heterogeneity in mitochondrial
353:22292242 membrane potentials revealed by a J-aggregate-
50. Nagy G, Barcza M, Gonchoroff N, Phillips PE, forming cation JC-1. Proc Natl Acad Sci U S A
Perl A (2004) Nitric oxide-dependent mito- 88:36713675
chondrial biogenesis generates Ca2+ signaling 60. Cossarizza A, Franceschi C, Monti D, Salvioli
profile of lupus T cells. J Immunol S, Bellesia E, Rivabene R et al (1995) Protective
173(6):36763683 effect of N-acetylcysteine in tumor necrosis
51. Weinberg JB, Granger DL, Pisetsky DS, Seldin factor-a-induced apoptosis in U937 cells: The
MF, Misukonis MA, Mason SN et al (1994) role of mitochondria. Exp Eye Res
The role of nitric oxide in the pathogenesis of 220:232240
spontaneous murine autoimmune disease: 61. Royall JA, Ischiropoulos H (1993) Evaluation
Increased nitric oxide production and nitric of 2,7-dichlorofluorescein and dihydrorhod-
oxide synthase expression in MRL-lpr/lpr amine 123 as fluorescent probes for intracellu-
mice, and reduction of spontaneous glomeru- lar H2O2 in cultured endothelial cells. Arch
lonephritis and arthritis by orally administered Biochem Biophys 302:348355
NG-monomethyl-L-arginine. J Exp Med 62. Wieder ED, Hang H, Fox MH (1993)
179:651660 Measurement of intracellular pH using flow
52. Gilkeson GS, Mudgett JS, Seldin MF, Ruiz P, cytometry with carboxy-SNARF-1. Cytometry
Alexander AA, Misukonis MA et al (1997) 14:916921
Clinical and serologic manifestations of auto- 63. Fiers W, Beyaert R, Declercq W, Vandenabeele
immune disease in MRL-lpr/lpr mice lacking P (1999) More than one way to die: apoptosis,
nitric oxide synthase type 2. J Exp Med necrosis and reactive oxygen damage. Oncogene
186:365373 18:77197730
53. Lamarre D, Talbot B, De Murcia G, LaPlante 64. Leist M, Single B, Castoldi AF, Kuhnle S,
C, LeDuc Y, Mazen A et al (1988) Structural Nicotera P (1997) Intracellular adenosine
and functional analysis of poly(ADP-ribose) triphosphate (ATP) concentration: a switch in
polymerase: an immunological study. Biochim the decision between apoptosis and necrosis. J
Biophys Acta 950:147160 Exp Med 185:14811486
54. Colombo E, Banki K, Tatum AH, Daucher J, 65. Lundin A (2000) Use of firefly luciferase in
Ferrante P, Murray RS et al (1997) Comparative ATP-related assays of biomass, enzymes, and
analysis of antibody and cell-mediated autoim- metabolites. Methods Enzymol 305:346370
munity to transaldolase and myelin basic pro- 66. Lowry OH, Rosebrough NJ, Farr AL, Randall
tein in patients with multiple sclerosis. J Clin RJ (1951) Protein measurement with the Folin
Invest 99:12381250 phenol reagent. J Biol Chem 193:265275
55. Vermes I, Haanen C, Steffens-Nakken H, 67. Lundin A (1994) ATP extractants neutralized
Reutelingsperger C (1995) A novel assay for by cyclodextrins. In: Campbell AK, Kricka LJ,
apoptosis. Flow cytometric detection of phos- Stanley PE (eds) Biolumimescence and chemi-
phatidylserine expression on early apoptotic luminescence. Wiley, Chichester, pp 399402
cells using fluorescein labelled Annexin V. J 68. Fariss MW, Reed DJ (1987) High-performance
Immunol Methods 184:3951 liquid chromatography of thiols and disulfides:
56. Martin SJ, Reutelingsperger CPM, McGahon dinitrophenol derivatives. Methods Enzymol
AJ, Rader JA, van Schie CAA, LaFace DM et al 143:101109
(1995) Early redistribution of plasma mem- 69. Towbin HH, Staehelin T, Gordon J (1979)
brane phosphatidylserine is a general feature of Electrophoretic transfer of proteins from poly-
apoptosis regardless of the initiating stimulus: acrylamide gels to nitrocellulose sheets: proce-
inhibition by overexpression of bcl-2 and Abl. J dure and some applications. Proc Natl Acad Sci
Exp Med 182:15451556 U S A 76:43504354
57. Petit PX, OConnor JE, Grunwald D, Brown 70. Rustin P, Parfait B, Chretien D, Bourgeron T,
SC (1990) Analysis of the membrane potential Djouadi F, Bastin J et al (1996) Fluxes of nico-
of rat- and mouse-liver mitochondria by flow tinamide adenine dinucleotides through mito-
cytometry and possible applications. Eur J chondrial membranes in human cultured cells.
Biochem 194:389397 J Biol Chem 271(25):1478514790
4 Assessment of Mitochondrial Dysfunction in Lymphocytes of Patients 89
71. Barrientos A (2002) In vivo and in organello interactions [Review] [50 refs]. Immunol Res
assessment of OXPHOS activities. Methods 13:234243
26(4):307316 80. Los M, Schenk H, Hexel K, Baeuerle PA,
72. Rustin P, Chretien D, Bourgeron T, Gerard B, Droge W, Schulze-Osthoff K (1995) IL-2
Rotig A, Saudubray JM et al (1994) Biochemical gene expression and NF-kappa B activation
and molecular investigations in respiratory through CD28 requires reactive oxygen pro-
chain deficiencies. [Review] [25 refs]. Clin duction by 5-lipoxygenase. EMBO J
Chim Acta 228(1):3551 14:37313740
73. Nicoletti I, Migliorati G, Pagliacci MC, 81. Tatla S, Woodhead V, Foreman JC, Chain BM
Grignani F, Riccardi C (1991) A rapid and sim- (1999) The role of reactive oxygen species in
ple method for measuring thymocyte apoptosis triggering proliferation and IL-2 secretion in T
by propidium iodide staining and flow cytom- cells. Free Radic Biol Med 26:1424
etry. J Immunol Methods 139:271279 82. Casciola-Rosen LA, Miller DK, Anhalt GJ,
74. Nicholson DW, Ali A, Thornberry NA, Rosen A (1994) Specific cleavage of the 70 kDa
Vaillancourt JP, Ding CK, Gallant M et al protein component of the U1 small nuclear
(1995) Identification and inhibition of ICE/ ribonucleoprotein is a characteristic biochemi-
CED-3 protease necessary for mammalian cal feature of apoptotic cell death. J Biol Chem
apoptosis. Nature 376:3743 269:3075730760
75. Armstrong RC, Aja T, Xiang J, Gaur S, Krebs 83. Perl A, Banki K (2000) Genetic and metabolic
JF, Hoang K et al (1996) Fas-induced activa- control of the mitochondrial transmembrane
tion of the cell death-related protease CPP32 is potential and reactive oxygen intermediate pro-
inhibited by bcl-2 and by ICE family protease duction in HIV disease. Antioxid Redox Signal
inhibitors. J Biol Chem 271:1685016855 2:551573
76. Lorenz HM, Grunke M, Hieronymus T, 84. Puskas F, Gergely PJ, Niland B, Banki K, Perl A
Herrmann M, Kuhnel A, Manger B (1997) In (2002) Differential regulation of hydrogen
vitro apoptosis and expression of apoptosis- peroxide and Fas-dependent apoptosis path-
related molecules in lymphocytes from patients ways by dehydroascorbate, the oxidized form
with systemic lupus erythematosus and other of vitamin C. Antioxid Redox Signal
autoimmune diseases. Arthritis Rheum 4(3):358369
40:306317 85. Harada H, Becknell B, Wilm M, Mann M,
77. Eisner MD, Amory J, Mullaney B, Tierney L Huang LJ, Taylor SS et al (1999)
Jr, Browner WS (1996) Necrotizing lymph- Phosphorylation and inactivation of BAD by
adenitis associated with systemic lupus erythe- mitochondria-anchored protein kinase A. Mol
matosus. Semin Arthritis Rheum 26:477482 Cell 3:413422
78. Llorente L, Zou W, Levy Y, Richaud-Patin Y, 86. Nisoli E, Carruba MO (2006) Nitric oxide and
Wijdenes J, Alcocer-Varela J et al (1995) Role mitochondrial biogenesis. J Cell Sci
of interleukin 10 in the B lymphocyte hyperac- 119(14):28552862
tivity and autoantibody production of human 87. Ibiza S, Victor VM, Bosca I, Ortega A,
systemic lupus erythematosus. J Exp Med Urzainqui A, OConnor JE et al (2006)
181:839844 Endothelial nitric oxide synthase regulates T
79. Green JM, Thompson CB (1994) Modulation cell receptor signaling at the immunological
of T cell proliferative response by accessory cell synapse. Immunity 24(6):753765
Chapter 5
Abstract
Abnormalities in T cell signal transduction underlie pathology in systemic lupus erythematosus. Lupus T
cells are more sensitive to stimulation, yet have reduced expression of T cell antigen receptor (TCR) at the
surface. The amount of TCR expressed at the surface of a T cell directly determines the ability of a T cell
to become activated. The endocytic recycling machinery regulates transport of T cell receptors to the
plasma membrane, internalization of surface receptors, and recycling to the cell surface, which determines
the ability of a T cell to become activated. Increased recycling of CD3 and CD4 receptors occurs in lupus
T cells, and could represent a mechanism by which T cells are sensitized to stimulation. This chapter
explains methods used to investigate endocytic recycling of the TCR, CD4, and CD8 co-receptors in
peripheral blood lymphocytes, T cells, and in splenocytes from lupus-prone murine models. The assays
described will allow the study of surface receptor turnover in live untouched lymphocytes by flow
cytometry.
Key words: Receptor recycling, Early endosome, T cell antigen receptor, CD4, Mammalian target of
rapamycin, Systemic lupus erythematosus
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_5, Springer Science+Business Media New York 2012
91
92 T. Telarico and A. Perl
1.1. Metabolic Control SLE T cells are metabolically active and have increased baseline
of the Endocytic nitric oxide (NO) production (10). NO production at the immu-
Pathway in SLE T Cells nological synapse (IS) is required to sustain the signal post
TCR ligation by MHCIIpeptide complex and maintain T cell
activation (11). NO overproduction leads to elevation of the
mitochondrial transmembrane potential (inducing mitochondrial
hyperpolarization, MHP) and increases mitochondrial biogenesis
(12). MHP increases production of reactive oxygen intermediates
(ROIs) and reduces ATP production, which predisposes chroni-
cally stimulated cells to undergo necrosis (4, 13).
The mammalian target of rapamycin (mTOR) is activated in
response to NO, and further enhances NO production through
activation of YY1 and PGC1, factors required for transcription of
nitric oxide synthases (14). Inhibition of mTORC1 activity by
rapamycin reduces NO production through inducing proteosomal
degradation of inducible nitric oxide synthase (iNOS) in mac-
rophages (15). mTOR senses MHP through association with the
voltage-dependent anion channel-1 (VDAC1). mTOR traffics to
mitochondria, through formation of a complex at the mitochon-
drial outer membrane with Bcl-xL and VDAC1 (16). mTOR activ-
ity has been directly implicated in promoting mitochondrial
function, as treatment with rapamycin reduces mitochondrial
transmembrane potential, decreases oxygen consumption, and
reduces ATP synthesis (17).
Target of rapamycin (TOR) proteins were identified as a
component of isolated endocytic patches, suggesting a role for
mTOR in endocytic trafficking (18). mTOR overexpression in
SLE T cells promotes expression of the small GTPase HRES-1/
Rab4, a regulator of endocytic recycling (19). Treatment with
rapamycin reduces HRES-1/Rab4 overexpression in SLE T cells
(19). mTOR and HRES-1/Rab4 colocalize on early endosomes
(19). The endocytic recycling machinery feeds back to regulate
mTOR activity through its intracellular location (20). Rab pro-
teins were found to be indispensible for signaling through mTOR
complex I (mTORC1) (20), and can serve as regulators of
mTORC1 activity (21).
5 The Role of Endocytic Recycling in Autoimmunity 93
APC
MHC-II-peptide
complex
CD4 CD3
complex
eNOS
FcRI Syk
Lck
NO NOSIP P- PLC
Endocytic
recycling
HRES -1/Rab4
CD4,CD3,
Dy m NOSIP
EE
Lysosome
CRAC
NFAT
PP2a
IL-2
ER
Elf-1
CD3
Nucleus
T cell
Fig. 1. HRES-1/Rab4 overexpression in SLE T cells occurs downstream of NO-dependent mTOR activation (19). NO is
overproduced in SLE T cells, resulting in MHP, mitochondrial biogenesis, and transcription of mTOR via activation of PGC1
(12, 14). HRES-1/Rab4 feeds back to increase NO production via degradation of NOSIP, a negative regulator of eNOS
(unpublished data). HRES-1/Rab4 overexpression reorganizes the IS through promoting CD3 and CD4 receptor recycling
and lysosomal degradation, reducing expression of CD4 and CD3 (19). CD3 is functionally replaced with FcRI, which
interacts with Syk, resulting in activation of PLC and enhanced intracytoplasmic Ca2+ fluxing upon T cell activation (35).
APC, antigen-presenting cell; CRAC, Ca2+ release-activated calcium channel; EE, early endosome; ER, endoplasmic reticu-
lum; MHP, mitochondrial hyperpolarization; mTOR, mammalian target of rapamycin; NFAT, nuclear factor of activated
T cells; NOSIP, nitric oxide synthase interacting protein; eNOS, endothelial nitric oxide synthase; ROI, reactive oxygen inter-
mediates. Colorized lettering in red represents downregulation in SLE T cells; green lettering represents upregulation in
SLE T cells.
5 The Role of Endocytic Recycling in Autoimmunity 95
130
120 120
120
110 110
110
100 100
100
90 90 90
0 30 60 90 120 150 0 30 60 90 120 150 0 30 60 90 120 150
Time (minutes) Time (minutes) Time (minutes)
Fig. 2. SLE T cells exhibit increased recycling of CD3 and CD4, but not CD8 receptors. T cells were negatively isolated from
seven SLE patients and seven healthy controls, then stained on ice with APC-Cy7-conjugated antibody to CD3, Alexa
647-conjugated antibody to CD4, and PE-conjugated antibody to CD8 for 30 min, and then washed three times in 4 C
RPMI 1640 medium. T cells were resuspended in pre-warmed 37 C medium and incubated at 37 C for 2.5 h, removing
samples every 30 min and placing them on ice. At the end of sample collection, the cells were restained with CD3, CD4,
and CD8 antibodies on ice for 30 min, then washed three times with ice-cold RPMI complete medium, and analyzed by flow
cytometry. Recycling was measured by the increase in mean fluorescence intensity (MFI) at each time point over the time
0 sample.
96 T. Telarico and A. Perl
2. Materials
3. Methods
3.1.2. Separation 1. Pre-coat 10 cm Petri dishes with 750 l autologous serum and
of Peripheral Blood incubate plates at 37 C CO2 tissue culture incubator for
Lymphocytes from PBMC 30 min.
2. Add 515 ml of PBMC (12 106 cells/ml) onto coated dishes
and incubate at 37 C for 1 h.
After incubation period, remove peripheral blood lym-
phocytes (PBLs) into a sterile conical tube. Wash plate gently
three times with 5 ml PBS and add these washes into tube (see
Note 4).
3. Centrifuge PBL at 1,300 rpm for 10 min at 8 C.
4. Resuspend PBL in RPMI 1640 complete medium at
5 106 cells/ml.
5. Monocyte-depleted PBL should contain <2 % monocytes. This
can be verified by flow cytometry after staining with a fluorescent
conjugated anti-human CD11b antibody.
3.1.3. Negative Isolation Adapted from Invitrogen Dynabeads untouched human T cell kit
of T Lymphocytes manufacturers protocol (catalog #113.44D).
1. Aliquot 20 106 PBMC or PBL into a 15 ml conical tube and
pellet cells by centrifugation at 1,300 rpm for 10 min at 8 C.
2. Resuspend cells in 10 ml T cell isolation buffer (0.5 g BSA,
2 mM EDTA, in autoclaved PBS). Wash cells by centrifugation
at 1,300 rpm at 8 C for 10 min.
3. Resuspend cell pellet in 200 l T cell isolation buffer. Add
40 l heat-inactivated and filter-sterilized FBS. Add 40 l of
antibody mix from Invitrogen Dynabeads untouched human T
cell kit and mix by inverting tubes. The antibody mix contains
IgG antibodies against human CD14, CD16, HLA class II
DR/DP, CD56, and CD235a antigens.
4. Incubate cells on ice for 20 min.
5. After incubation period, add 10 ml of T cell isolation buffer to
cells to wash off non-bound antibody. Centrifuge at 1,300 rpm
at 4 C for 10 min.
6. Aspirate off isolation buffer and resuspend pellet in 200 l T
cell isolation buffer.
7. Wash 1 ml Dynabeads per sample by placing in DynaMag-15
magnet for 2 min and resuspending in an equal volume of T
cell isolation buffer. Add 1 ml of negative isolation Dynabeads
per sample.
8. Incubate cells for 20 min at room temperature with low-speed
rotation.
9. After incubation period, place tubes in DynaMag-15 magnet
for 2 min.
100 T. Telarico and A. Perl
3.3. Recycling Assay 1. Aliquot 3 106 PBL or isolated T cells in a 600 l volume into
sterile 5 ml tubes. Aliquot five tubes containing 1 106 PBL in
3.3.1. Recycling Assays
a 200 l volume to use as single color compensation controls
on Human PBL
(see Note 5).
and Isolated T Cells
2. Add 50 g/ml cycloheximide to samples to inhibit synthesis of
new surface receptors.
3. Stain cells on ice with APC Cy7-conjugated antibody to CD3,
Alexa Fluor 647-conjugated antibody to CD4, PE-conjugated
antibody to CD8, and PE Cy7 antibody conjugated to CD19
(for PBL only) using 2 g per sample. Add 0.5 g antibody per
5 The Role of Endocytic Recycling in Autoimmunity 101
3.4. Western Blotting 1. To determine differences in total CD3, CD4, and CD8 surface
for Surface Receptor receptor expression, lyse 2 106 cells in 50 l RIPA lysis buffer
Expression (150 mM sodium chloride, 1 % Non-Idet-40, 0.5 % sodium
deoxycholate, 0.1 % SDS, 50 mM Tris pH 8.0, 1 mM PMSF,
1 g/ml aprotinin, 1 g/ml pepstatin, 1 g/ml leupeptin,
1 mM NaF, 1 mM sodium orthovanadate, 0.1 mM sodium
molybdate, 10 mM sodium pyrophosphate) (see Noe 1).
2. Place cells on ice after lysis, and use 2 l lysate to measure pro-
tein concentration by Bradford assay (43). For Bradford assay,
prepare a set of standard concentrations of BSA from 0 to
12 g in Bradford reagent (0.15 M NaCl, Coomassie brilliant
blue G-250) and measure absorbance in spectrophotometer at
595 nanometers to create a standard curve (40). Add 2 l of
5 The Role of Endocytic Recycling in Autoimmunity 103
4. Notes
References
1. Perl A, Fernandez DR, Telarico T, Doherty E, 8. Fernandez D, Bonilla E, Mirza N, Niland B,
Francis L, Phillips PE (2009) T-cell and B-cell Perl A (2006) Rapamycin reduces disease activ-
signaling biomarkers and treatment targets in ity and normalizes activation-induced calcium
lupus. Curr Opin Rheumatol 21(5):454464, fluxing in patients with systemic lupus erythe-
http://www.ncbi.nlm.nih.gov/pubmed/ matosus. Arthritis Rheum 54(9):29832988,
19550330 http://www.ncbi.nlm.nih.gov/pubmed/
2. Banchereau J, Pascual V, Palucka AK (2004) 16947529
Autoimmunity through cytokine-induced den- 9. Solomou EE, Juang YT, Gourley MF, Kammer
dritic cell activation. Immunity 20(5):539550, GM, Tsokos GC (2001) Molecular basis of
http://www.ncbi.nlm.nih.gov/pubmed/ deficient IL-2 production in T cells from
15142523 patients with systemic lupus erythematosus.
3. Emlen W, Niebur J, Kadera R (1994) J Immunol 166(6):42164222, http://www.
Accelerated in vitro apoptosis of lymphocytes ncbi.nlm.nih.gov/pubmed/11238674
from patients with systemic lupus erythemato- 10. Nagy G, Barcza M, Gonchoroff N, Phillips PE,
sus. J Immunol 152(7):36853692, http:// Perl A (2004) Nitric oxide-dependent mito-
www.ncbi.nlm.nih.gov/pubmed/8144943 chondrial biogenesis generates Ca2+ signaling
4. Fernandez D, Perl A (2009) Metabolic control profile of lupus T cells. J Immunol 173(6):
of T cell activation and death in SLE. 36763683, http://www.ncbi.nlm.nih.gov/
Autoimmun Rev 8(3):184189, http://www. pubmed/15356113
ncbi.nlm.nih.gov/pubmed/18722557 11. Ibiza S, Victor VM, Bosca I, Ortega A,
5. Ma L, Chan KW, Trendell-Smith NJ, Wu A, Urzainqui A, OConnor JE, Sanchez-Madrid
Tian L, Lam AC, Chan AK, Lo CK, Chik S, Ko F, Espluques JV, Serrador JM (2006)
KH, To CK, Kam SK, Xs L, Yang CH, Leung Endothelial nitric oxide synthase regulates T
SY, Ng MH, Stott DI, MacPherson GG, Huang cell receptor signaling at the immunological
FP (2005) Systemic autoimmune disease synapse. Immunity 24(6):753765, http://
induced by dendritic cells that have captured www.ncbi.nlm.nih.gov/pubmed/16782031
necrotic but not apoptotic cells in susceptible 12. Nagy G, Koncz A, Fernandez D, Perl A (2007)
mouse strains. Eur J Immunol 35(11): Nitric oxide, mitochondrial hyperpolarization,
33643375, http://www.ncbi.nlm.nih.gov/ and T cell activation. Free Radic Biol Med
pubmed/16224814 42(11):16251631, http://www.ncbi.nlm.nih.
6. Blasini AM, Alonzo E, Chacon R, Riera R, gov/pubmed/17462531
Stekman IL, Rodriguez MA (1998) Abnormal 13. Leist M, Single B, Castoldi AF, Kuhnle S,
pattern of tyrosine phosphorylation in unstim- Nicotera P (1997) Intracellular adenosine
ulated peripheral blood T lymphocytes from triphosphate concentration: a switch in the
patients with systemic lupus erythematosus. decision between apoptosis and necrosis. J Exp
Lupus 7(8):515523, http://www.ncbi.nlm. Med 185(8):14811486, http://www.ncbi.
nih.gov/pubmed/9863892 nlm.nih.gov/pubmed/9126928
7. Karonitsch T, Feierl E, Steiner CW, Dalwigk K, 14. Cunningham JT, Rodgers JT, Arlow DH,
Korb A, Binder N, Rapp A, Steiner G, Vazquez F, Mootha VK, Puiqserver P (2007)
Scheinecker C, Smolen J, Aringer M (2009) mTOR controls mitochondrial oxidative func-
Activation of the interferon-gamma signaling tion through a YY1-PGC1alpha transcriptional
pathway in systemic lupus erythematosus complex. Nature 450(7170):736740, http://
peripheral blood mononuclear cells. Arthritis www.ncbi.nlm.nih.gov/pubmed/18046414
Rheum 60(5):14631471, http://www.ncbi. 15. Jin HK, Ahn SH, Yoon JW, Park JW, Lee EK,
nlm.nih.gov/pubmed/19404947 Yoo JS, Lee JC, Choi WS, Han JW (2009)
106 T. Telarico and A. Perl
Rapamycin down-regulates inducible nitric required for autophagy. Journal of Cell Biology,
oxide synthase by inducing proteasomal degra- 305-321. http://www.ncbi.nlm.nih.gov/
dation. Biol Pharm Bull 32(6):988992, pubmed/19364919
http://www.ncbi.nlm.nih.gov/pubmed/ 25. Nambiar MP, Enyedy EJ, Fisher CU, Krishnan
19483303 S, Warke VG, Gilliland WR, Oglesby RJ, Tsokos
16. Ramanathan A, Schreiber SL (2009) Direct GC (2002) Abnormal expression of various
control of mitochondrial function by mTOR. molecular forms and distribution of T cell
Proc Natl Acad Sci 106(52):2222922232, receptor zeta chain in patients with systemic
http://www.ncbi.nlm.nih.gov/pubmed/ lupus erythematosus. Arthritis Rheum
20080789 46(1):163174, http://www.ncbi.nlm.nih.
17. Schieke SM, Phillips D, McCoy JP Jr, Aponte gov/pubmed/11817588
AM, Shen RF, Balaban RS, Finkel T (2006) 26. Nambiar MP, Enyedy EJ, Warke VG, Krishnan
The mammalian target of rapamycin (mTOR) S, Dennis G, Kammer GM, Tsokos GC (2001)
pathway regulates mitochondrial oxygen con- Polymorphisms/mutations of TCR-zeta-chain
sumption and oxidative capacity. J Biol Chem promoter and 3 untranslated region and selec-
281(37):2764327652, http://www.ncbi.nlm. tive expression of TCR zeta-chain with an
nih.gov/pubmed/16847060 alternatively spliced 3 untranslated region in
18. Michelot A, Costanzo M, Sarkeshik A, Boone patients with systemic lupus erythematosus.
C, Yates JR III, Drubin DG (2010) J Autoimmun 16(2):133142, http://www.
Reconstitution and protein composition analy- ncbi.nlm.nih.gov/pubmed/11247639
sis of endocytic actin patches. Curr Biol 27. Juang YT, Wang Y, Jiang G, Peng HB, Ergin S,
20:18901899, http://www.ncbi.nlm.nih. Finnell M, Magilavy A, Kyttaris VC, Tsokos
gov/pubmed/21035341 GC (2008) PP2A dephosphorylates Elf-1 and
19. Fernandez DR, Telarico T, Bonilla E, Li Q, determines the expression of CD3 zeta and
Banerjee S, Middleton FA, Phillips PE, Crow FcRgamma in human systemi lupus erythema-
MK, Oess S, Muller-Esterl W, Perl A (2009) tosus T cells. J Immunol 181(5):36583664,
Activation of mammalian target of rapamycin http://www.ncbi.nlm.nih.gov/pubmed/
controls the loss of TCR zeta in lupus T cells 18714041
through HRES-1/Rab4-regulated lysosomal 28. Takeuchi T, Tsuzaka K, Pang M, Amano K,
degradation. J Immunol 182(4):20632073, Koide J, Abe T (1998) TCR zeta chain lacking
http://www.ncbi.nlm.nih.gov/pubmed/ exon 7 in two patients with systemic lupus ery-
19201859 thematosus. Int Immunol 10(7):911921,
20. Flinn RJ, Yan Y, Goswami S, Parker PJ, Backer http://www.ncbi.nlm.nih.gov/pubmed/
JM (2010) The late endosome is essential for 9701029
mTORC1 signaling. Mol Biol Cell 21(5): 29. Krishnan S, Kiang JG, Fisher CU, Nambiar
833841, http://www.ncbi.nlm.nih.gov/ MP, Nguyen HT, Kyttaris VC, Chowdhury B,
pubmed/20053679 Rus V, Tsokos GC (2005) Increased caspase-3
21. Li L, Kim E, Yuan H, Inoki K, Goraksha-Hicks expression and activity contribute to reduced
P, Schiesher RL, Neufeld TP, Guan KL (2010) CD3zeta expression in systemic lupus erythe-
Regulation of mTORC1 by the Rab and Arf matosus T cells. J Immunol 175(5):
GTPases. J Biol Chem 285(26):1970519711, 34173423, http://www.ncbi.nlm.nih.gov/
http://www.ncbi.nlm.nih.gov/pubmed/ pubmed/16116236
20457610 30. Valitutti S, Muller S, Salio M, Lanzavecchia A
22. Huang H, Jeon MS, Liao L, Yang C, Elly C, (1997) Degradation of T cell receptor (TCR)-
Yates JR 3rd, Liu YC (2010) K33-linked polyu- CD3zeta complexes after antigenic stimulation.
biquination of T cell receptor zeta regulates J Exp Med 185(10):18591864, http://www.
proteolysis-independent T cell signaling. ncbi.nlm.nih.gov/pubmed/9151711
Immunity 33(1):6070, http://www.ncbi.nlm. 31. Holst J, Wang H, Eder KD, Workman CJ, Boyd
nih.gov/pubmed/20637659 KL, Baguet Z, Singh H, Forbes SK, Chruscinski
23. Cormont M, Meton I, Mari M, Monzo P, A, Smeyne R, van Oers NS, Utz PJ, Vignali DA
Keslair F, Gaskin C, McGraw TE, Le Marchand- (2008) Scalable signaling mediated by T cell
Brustel Y (2003) CD2AP/CMS regulates antigen receptor-CD3 ITAMs ensures effective
endosome morphology and traffic to the degra- negative selection and precents autoimmunity.
dative pathway through its interaction with Nat Immunol 9(6):658666, http://www.ncbi.
Rab4 and c-Cbl. Traffic 4(2):97112, http:// nlm.nih.gov/pubmed/18469818
www.ncbi.nlm.nih.gov/pubmed/12559036 32. Nambiar MP, Fisher CU, Warke VG, Krishnan
24. Razi M, Chan EY, Tooze SA (2009). Early S, Mitchell JP, Delaney N, Tsokos GC (2003)
endosomes and endosomal coatomer are Reconstitution of deficient T cell receptor zeta
5 The Role of Endocytic Recycling in Autoimmunity 107
chain restores T cell signaling and augments T 38. Krangel MS (1987) Endocytosis and recycling
cell receptor/CD3-induced interleukin-2 pro- of the T3 T-cell receptor complex. J Exp Med
duction in patients with systemic lupus erythe- 165(4):11411159, http://www.ncbi.nlm.
matosus. Arthritis Rheum 48(7):19481955, nih.gov/pubmed/3104527
http://www.ncbi.nlm.nih.gov/pubmed/ 39. Liu H, Rhodes M, Wiest DL, Vignali DAA
12847689 (2000) On the dynamics of TCR:CD3 com-
33. Howard FD, Rodewald HR, Kinet JP, Reinherz plex cell surface expression and downmodula-
EL (1990) CD3zeta subunit can substitute for tion. Immunity 13:665675, http://www.
the gamma subunit of Fc epsilon receptor type ncbi.nlm.nih.gov/pubmed/11114379
I in assembly and functional expression of the 40. Kovacs B, Vassilopoulous D, Vogelgesang SA,
high-affinity IgE receptor: evidence for inter- Tsokos GC (1996) Defective CD3-mediated
receptor complementation. Proc Natl Acad Sci cell death in activated T cells from patients
87(18):70157019, http://www.ncbi.nlm.nih. with systemic lupus erythematosus: role of
gov/pubmed/1698288 decreased intracellular TNF-alpha. Clin
34. Enyedy EJ, Nambiar MP, Liossis SN, Dennis Immunol Immunopathol 81(3):293302,
G, Kammer GM, Tsokos GC (2001) Fc epsilon http://www.ncbi.nlm.nih.gov/pubmed/
receptor type I gamma chain replaces the 8938108
deficient T cell receptor zeta chain in T cells of 41. Patino-Lopez G, Dong X, Ben-Aissa K, Bernot
patients with systemic lupus erythematosus. KM, Itoh T, Fukuda M, Kruhlak MJ, Samelson
Arthritis Rheum 44(5):11141121, http:// LE, Shaw S (2008) Rab35 and its GAP EPI64C
www.ncbi.nlm.nih.gov/pubmed/11352243 in T cells regulate receptor recycling and immu-
35. Krishnan S, Farber DL, Tsokos GC (2003) T cell nological synapse formation. J Biol Chem
rewiring in differentiation and disease. J Immunol 283(26):1832318330, http://www.ncbi.
171(7):33253331, http://www.ncbi.nlm.nih. nlm.nih.gov/pubmed/18450757
gov/pubmed/14500623 42. Batista A, Millan J, Mittelbrunn-M Sanchez-
36. Ueda H, Howson JM, Esposito L, Heward J, Madrid F, Alonso MA (2004) Recruitment of
Snook H, Chamberlain G, Rainbow DB, transferrin receptor to immunological synapse
Hunter KM, Smith AN, Di Genova G, Herr in response to TCR engagement. J Immunol
MH, Dahlman I, Payne F, Smyth D, Lowe C, 172(11):67096714, http://www.ncbi.nlm.
Twells RC, Howlett S, Healy B, Nutland S, nih.gov/pubmed/15153487
Rance HE, Everett V, Smink LJ, Lam AC, 43. Bradford MM (1976) Rapid and sensitive
Cordell HJ, Walker NM, Bordin C, Hulme J, method for the quantitation of microgram
Motzo C, Cucca F, Hess JF, Metzker ML, quantities of protein utilizing the principle of
Rogers J, Gregory S, Allahabadia A, protein-dye binding. Anal Biochem 72:
Nithiyananthan R, Tuomilehto-Wolf E, 248254, http://www.ncbi.nlm.nih.gov/
Tuomilehto J, Bingley P, Gillespie KM, Undlien pubmed/942051
DE, Rnningen KS, Guja C, Ionescu-Trgovi te 44. Alcover A, Alarcon B (2000) Internalization
C, Savage DA, Maxwell AP, Carson DJ, and intracellular fate of TCR-CD3 complexes.
Patterson CC, Franklyn JA, Clayton DG, Crit Rev Immunol 20(4):325346, http://
Peterson LB, Wicker LS, Todd JA, Gough SC www.ncbi.nlm.nih.gov/pubmed/11100805
(2003) Association of the T-cell regulatory
gene CTLA4 with susceptibility to autoim- 45. Minami Y, Samelson LE, Klausner RD (1987)
mune disease. Nature 423(6939):506511, Internalization and cycling of the T cell antigen
http://www.ncbi.nlm.nih.gov/pubmed/ receptor. J Biol Chem 262(27):1334213347,
12724780 http://www.ncbi.nlm.nih.gov/pubmed/
3498715
37. D Oro U, Munitic I, Chacko G, Karpova T,
McNally J, Ashwell JD (2002) Regulation of 46. Smythe E, Redelmeier TE, Schmid SL (1992)
constitutive TCR internalization by the zeta Receptor-mediated endocytosis in semiintact
chain. J Immunol 169:62696278, http:// cells. Methods Enzymol 219:223234, http://
www.ncbi.nlm.nih.gov/pubmed/12444133 www.ncbi.nlm.nih.gov/pubmed/1487996
Chapter 6
Abstract
B lymphocyte involvement in systemic lupus erythematosus has been recognized for several decades,
mainly in the context of autoantibody production. Both mouse and human studies reveal that different
types of antibody responses, as well as antibody-independent effector functions can be ascribed to distinct
subpopulations (subsets) of circulating B cells. Characterizing human B cell subsets can advance the field
of autoimmunity even further by establishing B cell signatures associated with disease severity, progression,
and response-to-treatment. For this purpose, we have developed specialized B cell reagent panels for mul-
tiparameter flow cytometry, and combine their use with advanced bioinformatics strategies that together
will likely be advantageous for improving the characterization, prognosis, and for possibly improving treat-
ment regimens of chronic inflammatory diseases such as lupus.
Key words: B lymphocyte, Autoantibody, 9G4, Transitional B cell, Mature-nave B cell, Memory B cell,
Antibody-secreting cell, B cell subset, Systemic lupus erythematosus, Principal component analysis
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_6, Springer Science+Business Media New York 2012
109
110 D.A. Kaminski et al.
1.2. B Cell Subsets In adult bone marrow, nascent B cells begin expressing cell-surface
in SLE BCR. In a transitional stage, the BCR+ B cell enters the periph-
eral circulation and differentiates into a mature-nave cell that is
competent for responding to BCR engagement by antigen. In
addition to transitional and mature-nave B cells, human blood
contains 34 additional core B cell subsets with characteristics of
antigen exposure (Ig somatic hypermutation, Ig class switching,
and/or markers associated with Ig secretion). Such B cells that are
neither nave nor secreting antibody are generally regarded as
memory in most nomenclatures (31) (Fig. 1b, c), which are dis-
cussed below. All of these basic core CD19+ populations can be
identified using flow cytometry by various combinations of IgD
BCR (more nave) and CD27 (more differentiated) surface expres-
sion (Fig. 1c) (31). Further subsetting these core populations using
additional markers is a highly advantageous way to characterize
human B cells (31).
1.2.1. Transitional The CD10 marker on human BCR B cell progenitors continues to
and Mature-Nave B Cells be expressed on immature B cells that have acquired the first class
of BCR, IgM, as well as on IgM+ transitional B cells coexpressing
an IgD BCR with an identical V region. In healthy subjects, the
frequency of autoreactive B cells is reduced when the BCR reaches
the cell surface (6), and then again when CD10 is lost (7). At each
step, more of these B cells are self-reactive in SLE patients com-
pared with healthy subjects (7, 8). Despite breaching these toler-
ance checkpoints, SLE patients have decreased numbers of nave
B cells contributing to overall reduced numbers of total CD19+
B cells (13, 22, 32, 33). The cause and significance of this reduc-
tion is poorly understood.
Given the critical involvement of the transitional-to-mature
conversion in determining the SLE B cell repertoire, it is advanta-
geous to define such cells and their subsets in clearer detail. This
goal can be achieved using multiparameter flow cytometry that can
a
112
SSC-A
SSC-A
SSC-W
100K 100K 100K 100K
FSC-W
2
10
CD3-Pacific Blue
50K 50K 50K 50K 10.3
42.2 0
0 0 0 0
0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 50K 100K 150K 200K 250K 0 0
D.A. Kaminski et al.
b
5 3.25
10 0.656
CD19+
populations in Bm3+4 Bm2
B-mature (Bm) 104
nomenclature
Bm2
103
74.2
0
CD38-PerCP-Cy5.5
Bm5 Bm1
14.2 7.72
0 102 103 104 105
IgD-FITC
c CD27+
SwMe NSM CD19+ gate CD19+ IgD- gate
0 0 102
CD27-Qdot605
CD24-PE-Alexa610
N
0
5.24 80.9
0 2 3 4 5 3 4 5 4
10 10 10 10 0 10 10 10 0 103 10 105
DN
IgD-FITC CD38-PerCP-Cy5.5 CD38-PerCP-Cy5.5
IgD-CD27+ IgD-CD27- IgD+CD27-
d
100 100 100 100 100
80 80 80 80 80
60 60 60 60 60
% of Max
40 40 40 40 40
20 20 20 20 20
0 0 0 0 0
0 103 104 105 0102 103 104 105 0 103 104 105 0 102 103 104 105 0 102 103 104 105
B220-PE-Cy7 CD24-PE-Alexa610 CD21-PE-Cy5 CD95-APC CXCR3-PE
6
Cell number
5 500 20 2.67
5 50
CD27-Qdot605
102
0
0 0 0 0 0
0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105 0 103 104 105
9G4-Alexa680 9G4-Alexa680
Fig. 1. Healthy human PB B cell subsets and phenotypic characterization of memory B cells. (a) Sequential gating strategy for (left to right) lymphocytes, singlets based on FSC,
singlets based on SSC, viable cells that do not take up amine-reactive Aqua-fluorescent dye (Live/Dead-negative), CD3CD19+ B cells. (b) The Bm (mature B cell) nomenclature,
first developed to classify tonsil B cells by IgD and CD38 expression, is commonly used in PBL analysis. This approach fails to separate IgD+CD27+ nonswitched memory cells, which
fall into the Bm1 and Bm2 gates together with nave cells (Bm1 and Bm2). Transitional 1 and 2 (T1/2) are included with Bm2 (pre-GC). Furthermore, isotype-switched CD27+ cells
are not distinguished from CD27 memory cells within the Bm5 population (IgDCD38+/). (c) The more commonly used IgD/CD27 classification scheme identifies four core B cell
subsets in human peripheral blood: IgD+CD27 nave B cells (N), IgD+CD27+ nonswitched memory B cells (NSM), IgDCD27+ switched memory cells (CD27+ SwMe), and IgDCD27
double-negative B cells (DN). In this scheme, transitional T1/2 colocalize with nave cells in the CD27 vs IgD plot, but can be identified based on their CD24++CD38++ phenotype
(middle panel). Likewise, plasmablasts (PB) are embedded in the IgDCD27+ fraction and can be accurately identified by first gating on IgD fraction followed by gating on the
Multiparameter Flow Cytometry and Bioanalytics
CD27++CD38++ region as depicted in the third panel. (d) The differential expression patterns of the surface markers in the IgDCD27+ (blue histograms) and IgDCD27 (green histo-
grams) memory B cells were overlaid on the IgD+CD27 nave B cells (filled red histograms). (e) Most autoreactive 9G4+ B cells are substantially CD27 and thus underrepresented
within the CD27+ memory compartment as depicted in the bivariate plot of the total CD19+ B cells (right). Histograms further demonstrate that among the core subsets (gated as in
113
c), the transitional and nave B cells exhibit the highest fraction of 9G4+ cells.
114 D.A. Kaminski et al.
1.2.2. Germinal Center Antigen engagement of the BCR on a mature-nave B cell can
B Cells stimulate activation and differentiation along any of several path-
ways that, ideally, help eliminate an invading organism and protect
the host. One of these pathways is the germinal center (GC) reac-
tion in secondary lymphoid tissue follicles. Typically, GC reactions
require T cell help to promote B cell proliferation, BCR class
switching from IgM/IgD to IgG, IgA, or IgE, and also Ig V region
gene somatic hypermutation that changes the affinity of the
encoded BCR for its cognate antigen (36). Somatic mutations in
autoantibodies contribute to their self-reactivity (11, 37, 38).
Antigen-driven selection results in clonal expansion of mainly the
highest affinity B cells. In normal individuals with intact peripheral
tolerance, these B cells are ideally only reactive with antigens from
invading organisms. The surviving cells can differentiate into anti-
body-secreting plasma cells or into memory B cells (each discussed
below) that are ready for a rapid response to a second encounter
with an invading organism.
In healthy human tonsil, non-self-reactive B cells are evenly
distributed among the B cell populations, but self-reactive 9G4+ B
6 Multiparameter Flow Cytometry and Bioanalytics 115
1.2.3. CD27+ Memory B cell memory (specific and rapid recall responses to previously
B Cells encountered antigens) is often inferred from cells restimulated
in vitro and from the ability to transfer such memory to nave
experimental animals (4143). In humans, most of this activity is
attributed to CD27+ B cells. Compared with CD27 (mostly
mature-nave and transitional) B cells, CD27+ B cells are slightly
larger (34, 44), are more proliferative in vitro (4547) and in vivo
(48), more efficiently stimulate allogeneic CD4 T cell proliferation
(46), and more readily differentiate into antibody-secreting cells
(47). GC, memory, and in vitro-activated nave B cells can all be
CD27+. However, as GC are generally confined to lymphoid tis-
sues, and few circulating CD27+ B cells show evidence of on-going
proliferation (34), GC and recently activated B cells unlikely
account for a significant portion of the CD27+ B cell pool, at least
in healthy humans. Additionally, CD27+ B cells are barely detect-
able in natal cord blood, but proportionally increase with age and
cumulative antigen exposure (44, 4850). The few CD27+ B cells
in the cord blood lack Ig V somatic hypermutations and have been
attributed to a newly described innate-like human B1 B cell
equivalent (51).
Adult CD27+ B cells either (1) express an IgD/IgM BCR
[IgM memory or nonswitched memory (NSM) B cells] or (2) have
lost expression of these markers after the irreversible process of
antibody class switching (switched memory B cells). CD27+ B cells
are ~1030 % each IgM+D+, IgG+, or IgA+ (34, 5254). It is
unknown whether these memory B cell subsets derive from com-
mon or from distinct differentiation pathways, or even if such
pathways are common to all cells in a given pool. Thus, the switched
memory B cell pool may contain cells that class-switched prior to
and also those that class-switched after acquiring CD27. Similarly,
the NSM pool may contain B cells that will remain NSM together
with intermediates that will eventually class-switch. Preliminary
analysis using high-parameter flow cytometry combined with auto-
mated analysis examining all parameters simultaneously (55) sug-
gests that most NSM cells have more in common with other
compartments than they have with each other (unpublished).
However, most studies to-date have compared all NSM with all
switched memory B cells using conventional manual gating meth-
ods. Briefly, both compartments have somatically hypermutated Ig
116 D.A. Kaminski et al.
1.2.6. Evolving SLE B Cell We have thus far discussed the tendency of SLE patients to have
Signature proportional increases in CD19+IgDCD27 DN B cells (which
may become the largest switched memory subset) and in plas-
mablasts, but decreased proportions of NSM B cells. Previous
studies and our own observations suggest that including additional
cell-surface markers will provide additional advantages in further
characterizing these populations in lupus. As previously reported
(31), CD27+ resting memory cells in healthy subjects are predomi-
nantly B220 (Fig. 1d) as the expression of this marker is typically
lost during germinal center differentiation. By contrast, B220
expression predominates in IgDCD27 cells. This subset is char-
acterized in SLE by the downregulation of CD24, a marker typi-
cally expressed by most PBL B cells in general and memory B cells
in particular (see Fig. 1d for healthy subject example). Consequently,
expanded fractions of B220+CD24 cells within the IgDCD27
compartment are prominent in lupus patients. Loss of CD21 and
upregulation of CD95 have been independently associated with
memory B cell activation (83, 84). Accordingly, an indication of
memory B cell activation in SLE can be found in both SwMe and
DN cells in which the CD21 and CD95+ fractions are greatly
expanded in lupus compared to the healthy controls. By contrast,
CXCR3 expression (suggesting migratory potential of activated
cells to nonlymphoid, systemic inflamed tissues) is concentrated in
6 Multiparameter Flow Cytometry and Bioanalytics 119
2. Materials
Primary analysis
Draw
conclusions
Independently
validate with new
Develop sorting experiments patient samples
for functional analysis
3. Methods
3.1. Sample 1. Record the original volume of blood in the heparinized blood-
Preparation collection tube before proceeding. Centrifuge with no brake at
400 g for 15 min.
3.1.1. Isolation of PBMC
2. Remove plasma from the top of the sample, leaving ~0.2 mL
of plasma behind. If needed, reserve the plasma at 4 C.
3. Collect the white blood cells (WBC) from the interface (ini-
tially between the plasma and the middle layer of Ficoll; red
blood cells will be in the bottom layer) plus ~0.2 mL above
and below. Transfer to a clean 50-mL centrifuge tube.
4. Dilute the collected WBC up to the original blood-volume
with PBS.
5. Add 15 mL Ficoll-Paque into a separate, clean 50-mL conical
tube.
6. Hold the tube as close to horizontal as possible and slowly
layer the diluted WBC sample onto the Ficoll-Paque, being
careful not to mix layers.
7. Centrifuge at 400 g for 35 min at 20 C with brake off.
8. Using a Pasteur pipette and avoiding Ficoll-Paque, collect the
mononuclear cell layer at the interface (buffy coat), and trans-
fer to a clean 15-mL conical tube.
9. Fill tube to 15 mL with cold PBS or RPMI, cap the tube, and
mix by inverting. Centrifuge for 10 min at 400 g at 4 C with
high brake.
10. Discard supernatant and resuspend pellet in 10 mL sterile PBS
or RPMI. Centrifuge again.
11. Gently dislodge the pellet and repeat steps 9 and 10.
12. Remove supernatant and resuspend in FACS buffer.
13. Count the cells by Trypan blue exclusion.
14. For cells to be stained fresh, transfer 107 cells per sample for
each multicolor panel into separate FACS tubes. Remaining
cells can be frozen.
3.1.2. Freezing 1. Pellet cells reserved for freezing, discard supernatant, and
and Thawing resuspend in cold freezing medium at 107/mL. Densities less
than 5 106/mL will reduce cell recovery.
Freezing Cells
2. Immediately freeze at 80 C for 2448 h and then transfer to
permanent storage, such as a liquid N2 freezer (optimal
180 C).
Thawing Cells 1. Before retrieving cells from frozen storage, warm FACS buffer
to 37 C.
2. Remove the vial of cells from the freezer and hold in a 37 C
water bath while continuously shaking and monitoring the
6 Multiparameter Flow Cytometry and Bioanalytics 123
3.1.3. Staining Cells with 1. Set up twelve, 1.5-mL microfuge tubes and dispense two drops
the 9G4 Memory B Cell of Simply Cellular Compensation Standard beads into each
Panel tube.
Stain Compensation 2. Reserve one tube as the unstained control. To each remaining
Controls tube, add 0.22 g of one of the other antibodies. Gently
vortex.
3. Incubate on ice for 30 min in the dark. [Note: for the Alexa680
channel, it is a 2-step staining: First with biotin-CD3 and then
with SAv-Alexa680. Stain 30 min for each step with a wash in
between.]
4. Wash the beads once with 1 mL FACS buffer. Pellet the beads
by centrifugation in a microcentrifuge at 900 g for 5 min.
Resuspend the beads in 200 L of 0.5 % formaldehyde, and
transfer to separate 5-mL FACS tubes.
5. For the Aqua compensation control, gently vortex ArC bead
components. Add one drop of Component A (reactive beads)
to a clean microfuge tube. Allow beads to sit at room tempera-
ture for at least 5 min. Add 1 L Aqua L/D stain directly to
the droplet of the reactive beads and incubate for 30 min.
6. Transfer to a FACS tube with the addition of 3 mL FACS buf-
fer. Centrifuge at 300 g for 5 min. Add 500 L FACS buffer
to the tube, plus one drop of ArC (negative beads) to the
tube.
Stain Blood-Cell Samples 1. Prepare antibody cocktails using FACS buffer in the presence
(3-Step Staining) of NMS and NRS (1:20 dilution each). Prepare a cocktail of
the fluorescent and biotinylated antibodies sufficient for stain-
ing the number of cell samples (100 L per sample). Prepare
also separate 1-sample mixtures of the same cocktail omitting
one reagent per cocktail for the fluorescence-minus-one
controls.
2. Pellet the cells reserved for staining at 300 g for 10 min at
4 C. Resuspend each pellet with 100 L of the appropriate
antibody cocktails. Incubate on ice for 30 min in the dark.
3. Wash the cells once with 2.5 mL FACS buffer.
4. Resuspend the cells with 100 L SAv-A680 (at 1:500 dilution)
on ice for 30 min in the dark.
5. Wash the cells once with 2.5 mL PBS (no BSA).
6. Incubate the cells in 1 mL PBS (no BSA) containing aqua-
fluorescent reactive dye (1:1,000) on ice for 30 min in the
dark.
124 D.A. Kaminski et al.
3.2. Primary Analysis Another integral element needed for consistent and reproducible
flow results is a standardized strategy for flow data analysis. A ratio-
nalized gating strategy can significantly reduce user-to-user vari-
ability to ensure consistent interpretation of the data among all
operators (e.g., Fig. 1). The following protocol assumes basic
knowledge of FlowJo software. Tutorials on getting started with
FlowJo are available at the TreeStar Web site (http://www.flowjo.
com/home/basictutorial.html) and can also be arranged with
TreeStar personnel.
3.3. Secondary Once primary analysis (gating) has been performed and cross-
Analysis checked, the resulting data can be analyzed in numerous ways,
depending on the experimental question. Considerations include
whether to report the data as mean fluorescent intensity of a par-
ticular fluorescent parameter, the percentage of cells within a par-
ent population, as a percentage of all B cells, or as the absolute
cell number per volume of blood. For cell-number calculations, it
is necessary to acquire a clinical blood count (CBC) from an
6 Multiparameter Flow Cytometry and Bioanalytics 125
aliquot of whole blood in which total white blood cells and lym-
phocytes are measured. These procedures can be requested from
clinical staff or performed by the analyst using conventional or
automated methods, such as a Sysmex XE-2100 instrument.
Multiparameter flow analysis invariably generates large quantities
of data that need to be managed efficiently in order to derive pro-
ductive output. For large-scale studies with associated clinical
information, it is recommended that the data be structured and
housed in a relational database to support flexible interrogation of
the data.
In some circumstances, many reagents in the panel are used for
narrowing down a particular subset of interest, which is then ana-
lyzed for various characteristics and by standard statistical tests with
conventional graphing techniques. In other circumstances, a more
global profiling approach can be used in which all of the data are
considered together to obtain a system-wide view of immune cell
populations. Profiling is useful for class-discovery in which natural
groupings of samples can be sought (85, 86). Furthermore, it may
be possible to relate those groupings to sets of samples with differ-
ent disease states, disease activities, or clinical manifestations.
Univariate approaches on individual subsets fail to reveal how col-
lections of subsets and their relative distributions might contribute
to such sample groupings.
3.3.1. Calculating Cell 1. Calculate B cell numbers with the following formula in which
Numbers both WBC/mL and % lymphocytes are taken from the CBC,
and % B cells among lymphocytes is taken from gating
analysis:
WBC %lymphocytes % CD19+ CD3 # B cells
= .
mL 100 100 mL blood
2. Calculate the core subset (e.g., switched memory) numbers:
# Bcells %IgD CD27 + # switchedmemoryB cells
= .
mL 100 mL blood
3. Calculate finer subpopulations (e.g., in which CD95+ was mea-
sured as a per cent of the switched memory parent):
# switched memoryB cells %CD95+
mL 100
#CD95+ switchedmemoryB cells
= .
mL blood
3.3.2. Clustering
The following represent a generic workflow that can be performed
and Principal-Component
by several software packages or by technical computing platforms.
Analysis
This protocol assumes basic knowledge of Matlab software
(Mathworks, Natick MA; http://www.mathworks.com/
products/matlab/).
126 D.A. Kaminski et al.
4. Notes
a
c
% Variance Explained
100
HC
80 8 SLE
60
40 6
20
0 4
1 2 3 4 5 6 7 8 9 10
Principal component
Principal Component 2
b 2
40 B cell Subsets Ordered by PC1 Loadings
-2
-4
-6
-8
-0.3 0 0.3 -8 -6 -4 -2 0 2 4 6 8
Loading
Principal Component 1
Fig. 3. PCA applied to summarized human B cell phenotyping data. Twenty PBMC samples were selected from our lupus
cross-sectional study (in preparation) as follows: ten healthy controls were selected at random. Ten flaring lupus patients
were chosen, including two severely flaring patients, three moderately flaring patients and five additional mildly flaring
patients that were selected randomly from a larger pool of available samples. Primary analysis (gating as per Fig. 1) of these
samples resulted in 40 cell subsets (the original variables). (a) Pareto plot showing the contribution to the overall variance
by the first ten (out of 40) principal components (PCs). The line indicates the cumulative contribution of the PCs to the overall
variance of the data from the samples. Most of the variance is captured by the first few components. (b) Contributions
(loadings) of each original variable to the first principal component. Each horizontal line corresponds to one of the original
cell subsets. Similar plots can be generated for other principal components as well. The loading (x axis) is the coefficient of
an original variable for the principal component (with values from 1 to 1); larger coefficients indicate a larger contribution
of that subset to the principal component in question. (c) Score plot of 40 B cell subsets reduced to the first two principal
components, representing an informative summary of the phenotypic profiles. Frequency data were first log-transformed
and then normalized. Open circles are healthy controls (HC) and asterisks are flaring lupus patients (SLE). Note how the two
groups of samples segregate based on their phenotypic profiles (mainly along Principal Component 2, y axis).
Acknowledgments
We thank John Jung and Ravi Misra for reading the manuscript
and members of the Sanz lab for help and advice. Supported by
NIH R01 AI049660-01A1 and U19 Autoimmunity Center of
Excellence AI56390 to I.S.
References
1. Pons-Estel GJ et al (2008) Understanding the 14. Erdei A et al (2009) Expression and role of
epidemiology and progression of systemic lupus CR1 and CR2 on B and T lymphocytes under
erythematosis. Semin Arthritis Rheum 39: physiological and autoimmune conditions. Mol
257268 Immunol 46:27672773
2. Borchers AT et al (2010) The geoepidemiol- 15. Casciola-Rosen LA, Anhalt G, Rosen A (1994)
ogy of systemic lupus erythematosus. Autoantigens targeted in systemic lupus ery-
Autoimmun Rev 9:A277A287 thematosus are clustered in two populations of
3. Bertsias GK, Salmon JE, Boumpas DT (2010) surface structures on apoptotic keratinocytes.
Therapeutic opportunities in systemic lupus ery- J Exp Med 179:13171330
thematosus: state of the art and prospects for the 16. Schutters K, Reutelingsperger C (2010)
new decade. Ann Rheum Dis 69:16031611 Phosphatidylserine targeting for diagnosis and
4. Arbuckle MR et al (2003) Development of treatment of human diseases. Apoptosis 15:
autoantibodies before the clinical onset of sys- 10721082
temic lupus erythematosus. N Engl J Med 349: 17. Pugh-Bernard AE et al (2001) Regulation of
15261533 inherently autoreactive VH4-34 B cells in the
5. Anolik JH et al (2009) Insights into the het- maintenance of human B cell tolerance. J Clin
erogeneity of human B cells: diverse functions, Invest 108:10611070
roles in autoimmunity, and use as therapeutic 18. Isenberg D et al (1993) Identification of the
targets. Immunol Res (2009) 45:144-158 9G4 idiotope in systemic lupus erythematosus.
6. Wardemann H et al (2003) Predominant Br J Rheumatol 32:876882
autoantibody production by early human B cell 19. van Vollenhoven RF et al (1999) VH4-34
precursors. Science 301:13741377 encoded antibodies in systemic lupus erythe-
7. Yurasov S et al (2005) Defective B cell tolerance matosus: a specific diagnostic marker that
checkpoints in systemic lupus erythematosus. correlates with clinical disease characteristics.
J Exp Med 201:703711 J Rheumatol 26:17271733
8. Yurasov S et al (2006) Persistent expression of 20. Bhat NM et al (2002) VH4-34 encoded anti-
autoantibodies in SLE patients in remission. body in systemic lupus erythematosus: effect of
J Exp Med 203:22552261 isotype. J Rheumatol 29:21142121
9. Yurasov S et al (2005) B-cell tolerance check- 21. Sanz I, Lee FE (2010) B cells as therapeutic tar-
points in healthy humans and patients with sys- gets in SLE. Nat Rev Rheumatol 6:326337
temic lupus erythematosus. Ann N Y Acad Sci 22. Anolik JH et al (2004) Rituximab improves
1062:165174 peripheral B cell abnormalities in human sys-
10. Yurasov S, Nussenzweig MC (2007) Regulation temic lupus erythematosus. Arthritis Rheum
of autoreactive antibodies. Curr Opin 50:35803590
Rheumatol 19:421426 23. Calero I, Sanz I (2010) Targeting B cells for
11. Tiller T et al (2007) Autoreactivity in human the treatment of SLE: the beginning of the end
IgG+ memory B cells. Immunity 26:205213 or the end of the beginning? Discov Med 10:
12. Sherer Y et al (2004) Autoantibody explosion 416424
in systemic lupus erythematosus: more than 24. Shlomchik MJ et al (1994) The role of B cells
100 different antibodies found in SLE patients. in lpr/lpr-induced autoimmunity. J Exp Med
Semin Arthritis Rheum 34:501537 180:12951306
13. Huang W et al (2002) The effect of anti-CD40 25. Chan OT et al (1999) A novel mouse with B
ligand antibody on B cells in human systemic cells but lacking serum antibody reveals an
lupus erythematosus. Arthritis Rheum 46: antibody-independent role for B cells in murine
15541562 lupus. J Exp Med 189:16391648
132 D.A. Kaminski et al.
26. Ahuja A et al (2007) Depletion of B cells in 43. Anderson SM, Tomayko MM, Shlomchik MJ
murine lupus: efficacy and resistance. J Immunol (2006) Intrinsic properties of human and
179:33513361 murine memory B cells. Immunol Rev 211:
27. Looney RJ et al (2004) B cell depletion as a 280294
novel treatment for systemic lupus erythemato- 44. Agematsu K et al (1997) B cell subpopulations
sus: a phase I/II dose-escalation trial of ritux- separated by CD27 and crucial collaboration of
imab. Arthritis Rheum 50:25802589 CD27+ B cells and helper T cells in immuno-
28. Navarra SV et al (2001) Efficacy and safety of globulin production. Eur J Immunol 27:
belimumab in patients with active systemic 20732079
lupus erythematosus: a randomised, placebo- 45. Tangye SG et al (2003) Intrinsic differences in
controlled, phase 3 trial. Lancet 377:721731 the proliferation of naive and memory human
29. Chan O, Shlomchik MJ (1998) A new role for B cells as a mechanism for enhanced secondary
B cells in systemic autoimmunity: B cells pro- immune responses. J Immunol 170:686694
mote spontaneous T cell activation in MRL- 46. Good KL, Avery DT, Tangye SG (2009)
lpr/lpr mice. J Immunol 160:5159 Resting human memory B cells are intrinsically
30. Anolik JH et al (2007) Delayed memory B cell programmed for enhanced survival and respon-
recovery in peripheral blood and lymphoid tissue siveness to diverse stimuli compared to naive
in systemic lupus erythematosus after B cell deple- B cells. J Immunol 182:890901
tion therapy. Arthritis Rheum 56:30443056 47. Tangye SG, Avery DT, Hodgkin PD (2003)
31. Sanz I et al (2008) Phenotypic and functional A division-linked mechanism for the rapid gen-
heterogeneity of human memory B cells. Semin eration of Ig-secreting cells from human mem-
Immunol 20:6782 ory B cells. J Immunol 170:261269
32. Wehr C et al (2004) A new CD21low B cell 48. Macallan DC et al (2005) B-cell kinetics in
population in the peripheral blood of patients humans: rapid turnover of peripheral blood
with SLE. Clin Immunol 113:161171 memory cells. Blood 105:36333640
33. Odendahl M et al (2000) Disturbed peripheral 49. van Gent R et al (2009) Refined characterization
B lymphocyte homeostasis in systemic lupus and reference values of the pediatric T- and B-cell
erythematosus. J Immunol 165:59705979 compartments. Clin Immunol 133:95107
34. Wirths S, Lanzavecchia A (2005) ABCB1 50. Kruetzmann S et al (2003) Human immuno-
transporter discriminates human resting naive globulin M memory B cells controlling
B cells from cycling transitional and memory B Streptococcus pneumoniae infections are gener-
cells. Eur J Immunol 35:34333441 ated in the spleen. J Exp Med 197:939945
35. Palanichamy A et al (2009) Novel human tran- 51. Griffin DO, Holodick NE, Rothstein TL (2011)
sitional B cell populations revealed by B cell Human B1 cells in umbilical cord and adult
depletion therapy. J Immunol 182:59825993 peripheral blood express the novel phenotype
36. Pascual V et al (1994) Analysis of somatic CD20+CD27+CD43+CD70RDDDDD.
mutation in five B cell subsets of human tonsil. J Exp Med 208:6780
J Exp Med 180:329339 52. Wei C et al (2007) A new population of cells
37. Wellmann U et al (2005) The evolution of lacking expression of CD27 represents a nota-
human anti-double-stranded DNA autoantibod- ble component of the B cell memory compart-
ies. Proc Natl Acad Sci USA 102:92589263 ment in systemic lupus erythematosus.
J Immunol 178:66246633
38. Tsuiji M et al (2006) A checkpoint for autore-
activity in human IgM+ memory B cell devel- 53. Fecteau JF, Cote G, Neron S (2006) A new
opment. J Exp Med 203:393400 memory CD27-IgG+ B cell population in
peripheral blood expressing VH genes with low
39. Cappione A 3rd et al (2005) Germinal center frequency of somatic mutation. J Immunol
exclusion of autoreactive B cells is defective in 177:37283736
human systemic lupus erythematosus. J Clin
Invest 115:32053216 54. Klein U, Rajewsky K, Kuppers R (1998)
Human immunoglobulin (Ig)M+ IgD+ periph-
40. Vinuesa CG, Sanz I, Cook MC (2009) eral blood B cells expressing the CD27 cell sur-
Dysregulation of germinal centres in autoim- face antigen carry somatically mutated variable
mune disease. Nat Rev Immunol 9:845857 region genes: CD27 as a general marker for
41. Crotty S, Ahmed R (2004) Immunological mem- somatically mutated (memory) B cells. J Exp
ory in humans. Semin Immunol 16:197203 Med 188:16791689
42. Mamani-Matsuda M et al (2008) The human 55. Qian Y et al (2010) Elucidation of seventeen
spleen is a major reservoir for long-lived vaccinia human peripheral blood B-cell subsets and
virus-specific memory B cells. Blood 111: quantification of the tetanus response using a
46534659 density-based method for the automated
6 Multiparameter Flow Cytometry and Bioanalytics 133
polyclonal activation of human memory B cells. 88. Herzenberg LA et al (2006) Interpreting flow
Science 298:21992202 cytometry data: a guide for the perplexed.
82. Jacobi AM et al (2010) Effect of long-term Nat Immunol 7:681685
belimumab treatment on B cells in systemic 89. Eisen MB et al (1998) Cluster analysis and
lupus erythematosus: extension of a phase II, display of genome-wide expression patterns.
double-blind, placebo-controlled, dose-ranging Proc Natl Acad Sci USA 95:1486314868
study. Arthritis Rheum 62:201210 90. Dhaeseleer (2005) How does gene expression
83. Moir S et al (2008) Normalization of B cell clustering work? Nat Biotechnol 23:
counts and subpopulations after antiretroviral 14991501
therapy in chronic HIV disease. J Infect Dis 91. Quackenbush J (2001) Computational analy-
197:572579 sis of microarray data. Nat Rev Genet 2:
84. Jacobi AM et al (2008) Activated memory B 418427
cell subsets correlate with disease activity in sys- 92. Misra J et al (2002) Interactive exploration of
temic lupus erythematosus: delineation by microarray gene expression patterns in a
expression of CD27, IgD, and CD95. Arthritis reduced dimensional space. Genome Res 12:
Rheum 58:17621773 11121120
85. Habib LK, Finn WG (2006) Unsupervised 93. Lugli E et al (2007) Subject classification
immunophenotypic profiling of chronic lym- obtained by cluster analysis and principal com-
phocytic leukemia. Cytometry B Clin Cytom ponent analysis applied to flow cytometric data.
70:124135 Cytometry A 71:334344
86. Diaz-Romero J et al (2010) Hierarchical clus- 94. Lee FE et al (2011) Circulating human anti-
tering of flow cytometry data for the study of body-secreting cells during vaccinations and
conventional central chondrosarcoma. J Cell respiratory viral infections are characterized by
Physiol 225:601611 high specificity and lack of bystander effect.
87. Cappione AJ et al (2004) Lupus IgG VH4.34 J Immunol 186:55145521
antibodies bind to a 220-kDa glycoform of 95. Wei et al (2011) OMIP-003: Phenotypic analy-
CD45/B220 on the surface of human B sis of human memory B cells. Cytometry A
lymphocytes. J Immunol 172:42984307 79:894896
Chapter 7
Abstract
Mouse models of lupus have for many years provided accessible and reliable research systems for the
pathogenesis and therapy of systemic autoimmune disease, spanning a spectrum of inbred strains that
develop spontaneous disease to experimentally induced, sometimes genetically manipulated animals.
Nearly all the models share in common the development of glomerulonephritis and autoantibodies, includ-
ing antinuclear and DNA specificities, the most common endpoints examined in experimental studies, but
exhibit specific differences in the incidence of other end-organ manifestations such as hemolytic anemia,
arthritis, dermatitis, and vasculitis. This chapter contrasts the clinical characteristics of these various mod-
els, providing an outline for their use and analysis.
Key words: Lupus, Mice, inbred, Mice, mutant, Mice, knockout, Mice, transgenic, Autoimmune
diseases, Autoantibodies, Glomerulonephritis, Arthritis, Hemolytic anemia
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_7, Springer Science+Business Media New York 2012
135
136 S.L. Peng
1.1. Lupus Models The vast majority of mouse lupus models develop autoantibodies
in Mice such as antinuclear antibodies (ANA) and anti-DNA antibodies, as
well as renal disease, particularly glomerulonephritis, and this com-
bination of manifestations is often used as the primary outcome
measure in experimental studies. Several models, however, develop
additional lupus-like features such as dermatitis, arthritis, cytope-
nias, vasculitis, and neurological manifestations; the pathogenesis
of such manifestations has been less well studied, perhaps in part
because of a less consistent incidence of these manifestations in the
models (Table 1). Because the specific details of these different
models have been well detailed in several prior reviews (26), the
listings here only outline key similarities and differences by which
to guide experimental model selection.
1.1.1. Inbred and The most well studied of the mouse lupus models include poly-
Spontaneous Models genic, inbred mouse strains which spontaneously develop lupus-
like syndromes, particularly the New Zealand (NZ) group of
strains, in addition to Murphys Recombinant Large (MRL) and
BSXB.
New Zealand Group The NZ group of mouse strains includes a number of different
strains that alone or in combination produce spontaneous lupus-
like disease models. In addition, other otherwise normal strains
which have been hybridized with NZ mice to generate other mouse
lupus models are included here since their clinical disease and
pathogenesis resembles the NZ strains. These include the
following:
NZB NZW F1. NZ Bielschowsky black (NZB/Bl) female NZ
White (NZW) male F1 (BW) mice, perhaps the most com-
monly studied hybrid, are considered by many researchers to
resemble the human disease most closely (2, 79). NZB mice
themselves are characterized by B cell hyperactivity and thymic
loss, and spontaneously develop a fatal autoimmune hemolytic
anemia due to anti-erythrocyte antibodies, sometimes in asso-
ciation with a typically mild glomerulonephritis (10). NZW
mice are clinically healthy in isolation but confer lupus in hybrid
with several other lupus-prone strains; this strain therefore
Table 1
Differentiating characteristics of commonly used mouse lupus models
proliferative arthritislupus
overlap
NZW BXSB Atherosclerosis Coronary infarcts Cardiolipin Resembles antiphos-
137
F1 pholipid syndrome
Adapted from ref. 2
138 S.L. Peng
MRL and CD95 Mutants This group of lupus models includes a handful of strains which are
characterized by diffuse autoimmunity, more extensive than the
NZ group, including, in some cases, lymphoproliferation:
MRL/Mp-+/+. These mice, derived from LG/J, C3H,
C57BL/6, and AKR strains, develop late-life lupus including
glomerulonephritis, ANA, and anti-DNA (69). In contrast to
NZ strains, they develop a broader spectrum of autoantibod-
ies, including anti-Sm and rheumatoid factor, as well as more
diverse organ involvement, including arthritis, sialoadenitis,
and ocular disease; many researchers therefore consider this
model a more severe or widespread lupus than NZ. B cell
hyperactivity is characteristic, but T cell dependence has been
demonstrated for only some of the autoimmune manifesta-
tions, such as nephritis and autoantibody production.
Faslpr (lpr). This mutation in the apoptosis-related CD95 gene
arose spontaneously during inbreeding of the MRL/Mp strain
(79, 15). In the MRL/Mp background, it confers dramatic
lymphadenopathy due to the expansion of CD4+ and
B220+CD4CD8 T cells which are unable to undergo normal
apoptosis, correlating with a significant acceleration of the
underlying lupus autoimmunity and resulting in early death
from glomerulonephritis. Lymphoproliferation is also seen
7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 139
Other Inbred Mouse Strains Several additional inbred or spontaneously arising mice develop
lupus-like syndromes:
BXSB. These mice, derived from SB/Le and C57BL/6 strains,
develop lupus similar to NZ animals, consisting also of glom-
erulonephritis, ANA, and anti-DNA, associated with B cell
hyperactivity and T cell-dependent autoantibody production,
and are the most well characterized of this group of strains
(79). However, in these animals, males develop early, acceler-
ated disease, while females develop late disease, due to the
presence of the Y chromosome-linked autoimmune accelerator
(Yaa), a duplication in and overexpression of the toll-like
receptor Tlr7 gene (25, 26).
NZW SB/Le F1. NZW female SB/Le male F1 hybrid ani-
mals also develop lupus-like disease, virtually identical to BXSB
(27).
NZW BSXB F1. These hybrid mice develop accelerated coro-
nary artery disease in addition to anti-DNA and anti-phospho-
lipid antibodies, sometimes associated with glomerulonephritis,
and may therefore be considered a model for antiphospholipid
antibody syndrome (28).
BDX2. This recombinant inbred strain derived from a
C57BL/6 DBA2/J hybrid cross develops spontaneous ero-
sive arthritis in addition to generalized autoimmune disease
consisting of glomerulonephritis, rheumatoid factor, and anti-
DNA autoantibodies. Such features of both rheumatoid arthri-
tis and lupus have suggested this strain as a model for
rheumatoid arthritislupus overlap (rhupus) (29).
SCG/Kj. The spontaneous crescentic glomerulonephritis-
forming mouse/Kinjoh (SCG/Kj), derived from a BXSB/
Mp MRL/Mp-lpr/lpr F1 hybrid, interestingly develops anti-
neutrophil cytoplasmic antibodies which cross-react with
DNA, in addition to glomerulonephritis (30, 31).
Palmerston North (PN). This mouse strain develops necrotiz-
ing vasculitis of the small and medium arteries, in addition to
proliferative glomerulonephritis in association with autoanti-
bodies (32).
140 S.L. Peng
Table 2
Targeted genetic mutations which produce lupus-like phenotypes in mice
Table 2
(continued)
1.2. Selection The diverse array of inbred and genetically altered mice provides
of Mouse Lupus many models for systemic lupus erythematosus, each with particu-
Models lar clinical manifestations and unique pathogeneses (Table 1).
Investigators should therefore choose models based upon areas or
phenomena of interest within the lupus autoimmune spectrum.
Some key issues for consideration include the following:
End-organ disease manifestations. Whereas almost all the mod-
els develop anti-DNA antibodies and glomerulonephritis and
therefore may be used to model the effect of a therapeutic
intervention of interest on lupus nephritis, only some of the
models (e.g., MRL, BDX2, pristane) develop arthritis and so
may be preferable over the NZ strains when arthritis is of
experimental interest. Similarly, only some of the models
develop significant cutaneous or vascular involvement, or non-
DNA autoantibodies such as ribonucleoprotein or phospho-
lipid specificities.
Non-lupus-like traits. Several mouse lupus models exhibit
characteristics that do not resemble typical human lupus: e.g.,
the male predominance of BXSB, or the extreme lymphade-
nopathy conferred by lpr or gld. Depending on the specific
context, these issues may detract from the scientific impact of a
given study.
Timeframe. Although many of the lupus models require sev-
eral months for maturation of disease, some models, such as
MRL/Mp-lpr/lpr, develop rapid disease, allowing earlier
assessment of interventions (e.g., 23 versus 69 months of
age). At the same time, the overall timing of experimental
setup and execution should be considered: for instance, the
study of knockouts or other genetic manipulations in the
inbred lupus-prone backgrounds requires extensive backcross-
ing, preferably with congenic genotyping to confirm strain
homozygosity at target strain chromosomes, and this typically
requires at least 1518 months or more simply to generate the
desired derivative strain. In contrast, the pristane/hydrocar-
bon model, although requiring several months for disease phe-
notypes to manifest, may require less total experimental set up
and execution time since it does not require a backcrossed
genetic locus.
Existing reagents or knowledge. The most well-studied mouse
lupus systems include the NZ, MRL, and BXSB strains: many
studies have already explored the impact of several therapeutic
interventions (e.g., T cell depletion or other costimulatory
144 S.L. Peng
2. Materials
2.3. Analysis of Serum Alkaline phosphatase substrate (e.g., Sigma 104105 tablets).
2.3.1. Immunoglobulin Antibodies:
Isotype Titers by ELISA Capture (e.g., from Southern Biotechnology Associates, SBA):
Goat F(ab)2 anti-mouse Ig (e.g., SBA 1012-01)for
IgM, IgA, and IgGs.
Rat anti-mouse IgE (e.g., 23G3SBA 1130-01).
Detection:
Goat anti-mouse IgM-Alkaline phosphatase (AP) (e.g., SBA
1020-04).
Goat anti-mouse IgA-AP (e.g., SBA 1040-04).
Goat anti-mouse IgG1-AP (e.g., SBA 1070-04).
Goat anti-mouse IgG2a-AP (e.g., SBA 1080-04).
Goat anti-mouse IgG2b-AP (e.g., SBA 1090-04).
Goat anti-mouse IgG3-AP (e.g., SBA 1100-04).
Rat anti-mouse IgE-AP (e.g., SBA 1130-04).
Standards:
Purified mouse IgM, IgA, IgG1, IgG2a, IgG2b, IgG3, IgE
(e.g., myeloma proteins).
Buffers:
2 % Bovine serum albumin in PBS.
Carbonate buffer (325 mL 0.1 M NaHCO3, 50 mL 0.1 M
Na2CO3).
DEA buffer: mix 49 mg MgCl26H2O and 96 mL dietha-
nolamine in 800 mL H2O.
146 S.L. Peng
2.3.4. Total Rheumatoid Alkaline phosphatase substrate (e.g., Sigma 104105 tablets).
Factor ELISA Antibodies:
Purified murine IgG1, ; IgG2a, ; IgG2b, ; and IgG3,
(e.g., myeloma proteins).
Anti-mouse Ig-AP (e.g., SBA 1050-04).
Buffers:
2 % Bovine serum albumin in phosphate-buffered saline (PBS).
Carbonate buffer (325 mL 0.1 M NaHCO3, 50 mL 0.1 M
Na2CO3).
DEA buffer: mix 49 mg MgCl26H2O and 96 mL dietha-
nolamine in 800 mL H2O, titrate to pH 9.8 by HCl, raise
to 1 L store at 4 C in foil, protected from light.
PBS: PBS-Tween (PBS with 0.05 % Tween-20).
7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 147
3. Methods
3.2. Lupus-Oriented 1. Collect urine for analysis, if desired (see Note 3)based on
Mouse Necropsy established protocols (46).
3.2.1. Tissue
(a) Cover the bottom of an empty mouse cage (i.e., with no
HarvestBased in Part
bedding) with plastic wrap.
on Established Protocols (b) Place mouse on plastic wrap.
(44, 45) (c) After 1015 s, remove the mouse.
(d) Collect urine with a micropipette or Pasteur pipette.
2. Collect blood by retro-orbital approach (see Note 4)based
in part on established protocols (47).
(a) Anesthetize the mouse.
(b) Apply pressure to the external jugular vein caudal to the
mandible with thumb, and gently elevate the upper eyelid
with the index finger of the same hand.
(c) Insert a hematocrit tube into the medial canthus of the eye.
(d) Gently direct the hematocrit tube in a ventrolateral direc-
tion until blood is obtained.
150 S.L. Peng
3.2.2. Tissue Processing 1. Serum isolation from whole blood (see Note 7).
(a) Allow the blood to clot at 4 C for 2 h to overnight.
(b) Microcentrifuge the sample at maximum speed (e.g.,
20,800 g) for 5 min.
(c) Collect the serumthe beige-colored, clear supernatant
with a micropipette.
2. Routine histopathology-buffered formalin (see Note 8).
(a) Fix tissues of interest in 30 volume equivalents of 10 %
buffered formalin for 810 h at room temperature, or
24 h at 4 C.
(b) Wash once with PBS (224 h) and transfer to 70 % etha-
nol. Store at room temperature or 4 C until ready for
processing.
3. Frozen tissue specimens (see Note 9).
(a) Prepare a dry iceethanol bath.
(b) Cut tissue into pea-sized specimen, but still containing the
anatomy of interest: e.g., for kidney, this usually equals
about one-half of a hemisphere.
(c) Cut off the cap from a microcentrifuge tube, to be used as
a mounting board. Place tissue on the cap (or other
mounting apparatus as desired). Embed with OCT
medium, using just enough as necessary to cover the
tissue.
(d) Immediately place the cap-tissue complex into the super-
chilled ethanol. Store at 70 C or below until
sectioning.
(e) Section and stain per protocol for specific antigens (e.g.,
mouse IgG for kidneys).
4. Specimens to be analyzed in tissue culture or by flow cytome-
try can be rinsed in PBS and/or placed directly in complete
cell culture medium.
7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 153
3.3. Analysis of Serum 1. Coat 96-well plates with 50 L/well of 2 g/mL capture
(See Note 10) antibody in carbonate buffer, 4 C overnight (see Note 11).
3.3.1. Immunoglobulin
2. Remove coating solution by tapping.
Isotype Titers by ELISA 3. Block with 200 L/well 2 % Bovine serum albumin in PBS,
37 C, 2 h (see Note 12).
4. Remove blocking solution by tapping.
5. Make serial dilutions of each serum sample, such as 1:100,
1:200, 1:400, etc. up to 1:102,400 in PBS, and add 50 L per
well in duplicate (see Note 13).
6. Make serial dilutions of isotype standard control (e.g., 4,000,
2,000, 1,000, 500, 250, 125 ng/mL) and add 50 L per well
in duplicate. Use a separate standard curve for each plate (see
Note 14).
7. Incubate at room temperature for 12 h.
8. Wash each well 46 times with 200 L PBS-Tween.
9. Dilute detection antibody 1:1,0001:2,000 in PBS (according
to manufacturers instructions) and add 50 L per well.
Incubate at room temperature for 4560 min.
10. Wash each well 46 times with 200 L PBS-Tween.
11. Dissolve phosphatase substrate in DEA buffer to 1 mg/mL
(e.g., one 5 mg tablet in 5 mL), and add 50 L per well. Watch
yellow color development in standard curve.
12. Read OD405 in a spectrophotometer. Interpolate and extrapo-
late Ig concentration in each sample using the standard curve
and dilutions, averaging the duplicate samples. Serial OD read-
ings may be necessary to obtain interpretable results for every
sample. Discard sample readings that are outside the range of
the standard curve. Note that OD and concentration is a semi-
log relationship (see Note 15).
3.3.2. Fluorescent 1. For screening, dilute test serum 1:40 or 1:50 in PBS, and add
Antinuclear Antibody Test 50 L to a slide well of fixed, permeabilized HEp-2 cells.
(FANA) (See Note 16) Incubate for 3045 min, room temperature.
2. Wash slide 46 times with PBS in a Coplin jar, 5 min each. Blot
the excess fluid away after the last wash.
3. Dilute FITC-anti-mouse IgG 1:500 in PBS or as directed by
manufacturer, and add 50 L to each well. Incubate for
3045 min, room temperature, protected from light.
4. Wash slide 46 times with PBS in a Coplin jar, 5 min each. Blot
the excess fluid away after the last wash.
5. Add 35 drops of mounting medium and coverslip to the slide.
Observe slide immediately by fluorescence microscopy.
6. Titers less than 1:40 are generally considered negative. Positive
samples, identified by the presence of any type of apple-green
154 S.L. Peng
3.3.3. Anti-dsDNA 1. Dilute test serum 1:10 in PBS, add 25 L to a slide well of
Assessment: Crithidia fixed, permeabilized Crithidia. Incubate for 3045 min, room
luciliae temperature.
Immunofluorescence (See 2. Wash slide 46 times with PBS in a Coplin jar, 5 min each. Blot
Note 17) the excess fluid away after the last wash.
3. Dilute FITC-anti-mouse IgG 1:500 in PBS or as directed by
manufacturer, and add 25 L to each well. Incubate for
3045 min, room temperature, protected from light.
4. Wash slide 46 times with PBS in a Coplin jar, 5 min each. Blot
the excess fluid away after the last wash.
5. Add 35 drops of mounting media and coverslip to the slide.
Observe slide immediately by fluorescence microscopy.
6. Titers of less than 1:10 are generally considered negative.
Positive samples are identified by the presence of apple-green
fluorescence of both the nucleus (indicating positive antinu-
clear antibodies) and kinetoplast, the dsDNA-containing
organelle near the base of the flagellum.
3.3.4. Total Rheumatoid 1. Coat 96-well plates with 50 L/well of 0.5 g/mL each of
Factor Assessment (ELISA) IgG1, ; IgG2a, ; IgG2b, ; and IgG3, in carbonate buffer,
(See Note 18) 4 C overnight.
2. Remove coating solution by tapping.
3. Block with 200 L/well 2 % bovine serum albumin in PBS,
37 C, 2 h.
4. Remove blocking solution by tapping.
5. Make serial dilutions of each serum sample, such as 1:100,
1:200, 1:400, etc. up to 1:102,400 in PBS, and add 50 L per
well in duplicate.
6. Make serial dilutions of isotype standard control (e.g., 4,000,
2,000, 1,000, 500, 250, 125 ng/mL) and add 50 L per well
in duplicate. Use a separate standard curve for each plate.
7. Incubate at room temperature for 12 h.
8. Wash each well 46 times with 200 L PBS-Tween.
9. Dilute alkaline phosphatase-conjugated anti-mouse Ig anti-
body 1:1,0001:2,000 in PBS (according to manufacturers
instructions) and add 50 L per well. Incubate at room tem-
perature for 4560 min.
10. Wash each well 46 times with 200 L PBS-Tween.
7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 155
3.4.2. Immunofluorescence 1. Place the frozen kidney in OCT in the cryostat chamber for
for Immune Deposits 1020 min to allow the block to equilibrate in temperature.
This is critical for quality of sections.
2. Place the tissue block on the cryostat specimen disk. Face the
block until desired tissue is exposed.
3. Cut 57 m sections of frozen kidney in OCT, placing them
on a poly-L-lysine-coated slide (see Note 20).
4. Fix sections in cold acetone, 20 C, 2 min.
5. Wash slide 3 in PBS, room temperature.
6. Add FITC-anti-mouse IgG, diluted 1:500 in PBS, just enough
to cover the tissue (typically 2550 L). Incubate in a
humidified chamber, room temperature, protected from light
(see Note 21).
7. Wash slide three times in PBS, room temperature.
8. Add 35 drops of mounting media and coverslip to the slide.
Observe slide immediately by fluorescence microscopy.
9. Positive samples are identified by apple-green fluorescence at
the glomeruli (see Note 22).
3.4.3. Surrogate Assays 1. Serum analysis can be performed using commercially available
for Renal Function kits for creatinine and/or urea nitrogen (e.g., Sigma), if
desired.
2. Urine protein excretion can be quantified by various protein
assays (e.g., Pierce), if desired.
4. Notes
References
1. Rahman A, Isenberg DA (2008) Systemic in mice explained by defects in Fas antigen that
lupus erythematosus. New Engl J Med mediates apoptosis. Nature 356:314317
358:929939 16. Kelley VE, Roths JB (1985) Interaction of
2. Hahn BH, Singh RR (2007) Animal models mutant lpr gene with background strain
of systemic lupus erythematosus. In: Wallace influences renal disease. Clin Immunol
DJ, Hahn BH (eds) Dubois lupus erythema- Immunopathol 37:220229
tosus. Lippincott Williams & Wilkins, 17. Eisenberg RA, Craven SY, Fisher CL et al
Philadelphia, PA (1989) The genetics of autoantibody produc-
3. Davidson A, Aranow C (2009) Lupus nephri- tion in MRL/lpr lupus mice. Clin Exp
tis: lessons from murine models. Nat Rev Rheumatol 7:S35S40
Rheumatol 6:1320 18. Peng SL, Craft J (1999) Lessons from knock-
4. Ghoreishi M, Dutz JP (2009) Murine models out and transgenic lupus-prone mice (chapter
of cutaneous involvement in lupus erythema- 10). In: Kammer GM, Tsokos GC (eds) Lupus:
tosus. Autoimmun Rev 8:484487 molecular and cellular pathogenesis. Humana
5. Rottman JB, Willis CR (2010) Mouse models Press Inc., Totowa, NJ, pp 152166
of systemic lupus erythematosus reveal a com- 19. Matsuzawa A, Moriyama T, Kaneko T et al
plex pathogenesis. Vet Pathol 47:664676 (1990) A new allele of the lpr locus, lprcg, that
6. Peng SL (2004) Experimental use of murine complements the gld gene in induction of
lupus models. Methods Mol Med 102:227272 lymphadenopathy in the mouse. J Exp Med
171:519531
7. Izui S, McConahey PJ, Dixon FJ (1978)
20. Kimura M, Mohri H, Shimada K et al (1990)
Increased spontaneous polyclonal activation of
Serological and histological characterization
B lymphocytes in mice with spontaneous auto-
of the new mutant strain of lpr mice, CBA/
immune disease. J Immunol 121:22132219
KlJms-lprcg/lprcg. Clin Exp Immunol
8. Andrews BS, Eisenberg RA, Theofilopoulos 79:123129
AN et al (1978) Spontaneous murine lupus-
21. Adachi M, Suematsu S, Kondo T et al (1995)
like syndromes. Clinical and immunopatho-
Targeted mutation in the Fas gene causes
logical manifestations in several strains. J Exp
hyperplasia in peripheral lymphoid organs and
Med 148:11981215
liver. Nat Genet 11:294300
9. Dixon FJ, Andrews BS, Eisenberg RA et al
22. Roths JB, Murphy ED, Eicher EM (1984) A
(1978) Etiology and pathogenesis of a spon-
new mutation, gld, that produces lymphopro-
taneous lupus-like syndrome in mice. Arthritis
liferation and autoimmunity in C3H/HeJ
Rheum 21:S64S67
mice. J Exp Med 159:120
10. Bielschowsky M, Helyer BJ, Howie JB (1959) 23. Lynch DH, Watson ML, Alderson MR et al
Spontaneous haemolytic anemia in mice of (1994) The mouse Fas-ligand gene is mutated
the NZB/BL strain. Proc Univ Otago Med in gld mice and is part of a TNF family gene
Sch 37:9 cluster. Immunity 1:131136
11. Morel L, Rudofsky UH, Longmate JA et al 24. Takahashi T, Tanaka M, Brannan CI et al
(1994) Polygenic control of susceptibility to (1994) Generalized lymphoproliferative dis-
murine systemic lupus erythematosus. ease in mice, caused by a point mutation in
Immunity 1:219229 the Fas ligand. Cell 76:969976
12. Kaliyaperumal A, Mohan C, Wu W et al 25. Pisitkun P, Deane JA, Difilippantonio MJ et al
(1996) Nucleosomal peptide epitopes for (2006) Autoreactive B cell responses to RNA-
nephritis-inducing T helper cells of murine related antigens due to TLR7 gene duplica-
lupus. J Exp Med 183:24592469 tion. Science 312:16691672
13. Dumont F, Monier JC (1983) Sex-dependent 26. Subramanian S, Tus K, Li QZ et al (2006) A
systemic lupus erythematosus-like syndrome Tlr7 translocation accelerates systemic auto-
in (NZB SJL)F1 mice. Clin Immunol immunity in murine lupus. Proc Natl Acad Sci
Immunopathol 29:306317 USA 103:99709975
14. Vidal S, Gelpi C, Rodriguez-Sanchez JL 27. Izui S, Masuda K, Yoshida H (1984) Acute
(1994) (SWR SJL)F1 mice: a new model SLE in F1 hybrids between SB/Le and NZW
of lupus-like disease. J Exp Med 179: mice; prominently enhanced formation of
14291435 gp70 immune complexes by a Y chromosome-
15. Watanabe-Fukunaga R, Brannan CI, Copeland associated factor from SB/Le mice. J Immunol
NG et al (1992) Lymphoproliferation disorder 132:701704
164 S.L. Peng
autoimmunity in MRL-lpr mice. J Immunol 68. Choi Y, Ramnath VR, Eaton AS et al (1999)
162:63226330 Expression in transgenic mice of dominant
54. Mizutani A, Shaheen VM, Yoshida H et al interfering Fas mutations: a model for human
(2005) Pristane-induced autoimmunity in autoimmune lymphoproliferative syndrome.
germ-free mice. Clin Immunol 114:110118 Clin Immunol 93:3445
55. Hoff J (2000) Methods of blood collection in 69. Murga M, Fernandez-Capetillo O, Field SJ
the mouse. Lab Anim 29:4753 et al (2001) Mutation of E2F2 in mice causes
56. Hem A, Smith AJ, Solberg P (1998) Saphenous enhanced T lymphocyte proliferation, leading
vein puncture for blood sampling of the to the development of autoimmunity.
mouse, rat, hamster, gerbil, guinea pig, ferret Immunity 15:959970
and mink. Lab Anim 32:364368 70. Salvador JM, Hollander MC, Nguyen AT et al
57. Furner RL, Mellett LB (1975) Mouse restrain- (2002) Mice lacking the p53-effector gene
ing chamber for tail-vein injection. Lab Animal Gadd45a develop a lupus-like syndrome.
Sci 25:648 Immunity 16:499508
58. Durschlag M, Wurbel H, Stauffacher M et al 71. Zhang Y, Schlossman SF, Edwards RA et al
(1996) Repeated blood collection in the labo- (2002) Impaired apoptosis, extended dura-
ratory mouse by tail incisionmodification of tion of immune responses, and a lupus-like
an old technique. Phys Behavior autoimmune disease in IEX-1-transgenic
60:15651568 mice. Proc Natl Acad Sci USA 99:878883
59. Hijmans W, Radema H, van Es L et al (1969) 72. Woelfel M, Bixby J, Brehm MA et al (2006)
Cryoglobulins in New Zealand Black mice. Transgenic expression of the viral FLIP
Clin Exp Immunol 4:227239 MC159 causes lpr/gld-like lymphoprolifera-
tion and autoimmunity. J Immunol
60. Reininger L, Berney T, Shibata T et al (1990) 177:38143820
Cryoglobulinemia induced by a murine IgG3
rheumatoid factor: skin vasculitis and glom- 73. Balomenos D, Martin-Caballero J, Garcia MI
erulonephritis arise from distinct pathogenic et al (2000) The cell cycle inhibitor p21 con-
mechanisms. Proc Natl Acad Sci USA trols T-cell proliferation and sex-linked lupus
87:1003810042 development. Nat Med 6:171176
61. Dammacco F, Sansonno D, Piccoli C et al 74. Lawson BR, Kono DH, Theofilopoulos AN
(2001) The cryoglobulins: an overview. Eur J (2002) Deletion of p21 (WAF-1/Cip1) does
Clin Invest 31:628638 not induce systemic autoimmunity in female
BXSB mice. J Immunol 168:59285932
62. Peng SL, Craft J (2008) Antinuclear antibod-
ies (chapter 50). In: Firestein GS, Budd RC, 75. Di Cristofano A, Kotsi P, Peng YF et al (1999)
Harris ED et al (eds) Kellys textbook of rheu- Impaired Fas response and autoimmunity in
matology, 8th edn. Elsevier, Inc., Philadelphia, Pten+/ mice. Science 285:21222125
PA 76. Suzuki A, Yamaguchi MT, Ohteki T et al
63. Shlomchik MJ, Madaio MP, Ni D et al (1994) (2001) T cell-specific loss of Pten leads to
The role of B cells in lpr/lpr-induced autoim- defects in central and peripheral tolerance.
munity. J Exp Med 180:12951306 Immunity 14:523534
64. Peng SL, Cappadona J, McNiff JM et al 77. Prodeus AP, Goerg S, Shen LM et al (1998) A
(1998) Pathogenesis of autoimmunity in T critical role for complement in maintenance of
cell-deficient lupus-prone mice. Clin Exp self-tolerance. Immunity 9:721731
Immunol 111:107116 78. Scott RS, McMahon EJ, Pop SM et al (2001)
65. Strasser A, Whittingham S, Vaux DL et al Phagocytosis and clearance of apoptotic cells
(1991) Enforced BCL2 expression in is mediated by MER. Nature 411:207211
B-lymphoid cells prolongs antibody responses 79. Nash JT, Taylor PR, Botto M et al (2001)
and elicits autoimmune disease. Proc Natl Immune complex processing in C1q-deficient
Acad Sci USA 88:86618665 mice. Clin Exp Immunol 123:196202
66. Bouillet P, Metcalf D, Huang DC et al (1999) 80. Botto M, DellAgnola C, Bygrave AE et al
Proapoptotic Bcl-2 relative Bim required for (1998) Homozygous C1q deficiency causes
certain apoptotic responses, leukocyte homeo- glomerulonephritis associated with multiple
stasis, and to preclude autoimmunity. Science apoptotic bodies. Nat Genet 19:5659
286:17351738 81. Napirei M, Karsunky H, Zevnik B et al (2000)
67. Bouillet P, Purton JF, Godfrey DI et al (2002) Features of systemic lupus erythematosus in
BH3-only Bcl-2 family member Bim is Dnase1-deficient mice. Nat Genet 25:177181
required for apoptosis of autoreactive thymo- 82. Bolland S, Ravetch JV (2000) Spontaneous
cytes. Nature 415:922926 autoimmune disease in FcRIIB-deficient
166 S.L. Peng
mice results from strain-specific epistasis. 96. Waterhouse P, Penninger JM, Timms E et al
Immunity 13:277285 (1995) Lymphoproliferative disorders with
83. Boes M, Schmidt T, Linkemann K et al (2000) early lethality in mice deficient in Ctla-4.
Accelerated development of IgG autoantibod- Science 270:985988
ies and autoimmune disease in the absence of 97. Han B, Moore PA, Wu J et al (2007)
secreted IgM. Proc Natl Acad Sci USA Overexpression of human decoy receptor 3 in
97:11841189 mice results in a systemic lupus erythemato-
84. A-Gonzalez N, Bensinger SJ, Hong C et al sus-like syndrome. Arthritis Rheum
(2009) Apoptotic cells promote their own 56:37483758
clearance and immune tolerance through acti- 98. Le LQ, Kabarowski JH, Weng Z et al (2001)
vation of the nuclear receptor LXR. Immunity Mice lacking the orphan G protein-coupled
31:245258 receptor G2A develop a late-onset autoim-
85. Hanayama R, Tanaka M, Miyasaka K et al mune syndrome. Immunity 14:561571
(2004) Autoimmune disease and impaired 99. Yoshinaga SK, Zhang M, Pistillo J et al (2000)
uptake of apoptotic cells in MFG-E8-deficient Characterization of a new human B7-related
mice. Science 304:11471150 protein: B7RP-1 is the ligand to the co-stim-
86. Xue D, Shi H, Smith JD et al (2003) A lupus- ulatory protein ICOS. Int Immunol 12:
like syndrome develops in mice lacking the Ro 14391447
60-kDa protein, a major lupus autoantigen. 100. Nishimura H, Nose M, Hiai H et al (1999)
Proc Natl Acad Sci USA 100:75037508 Development of lupus-like autoimmune dis-
87. Bickerstaff MC, Botto M, Hutchinson WL eases by disruption of the PD-1 gene encod-
et al (1999) Serum amyloid P component con- ing an ITIM motif-carrying immunoreceptor.
trols chromatin degradation and prevents anti- Immunity 11:141151
nuclear autoimmunity. Nat Med 5:694697 101. Wilkinson R, Lyons AB, Roberts D et al
88. Lu Q, Lemke G (2001) Homeostatic regula- (2002) Platelet endothelial cell adhesion mol-
tion of the immune system by receptor ecule-1 (PECAM-1/CD31) acts as a regula-
tyrosine kinases of the Tyro 3 family. Science tor of B-cell development, B-cell antigen
293:306311 receptor (BCR)-mediated activation, and
89. OKeefe TL, Williams GT, Davies SL et al autoimmune disease. Blood 100:184193
(1996) Hyperresponsive B cells in CD22- 102. Seshasayee D, Valdez P, Yan M et al (2003)
deficient mice. Science 274:798801 Loss of TACI causes fatal lymphoproliferation
90. Stunz LL, Busch LK, Munroe ME et al (2004) and autoimmunity, establishing TACI as an
Expression of the cytoplasmic tail of LMP1 in inhibitory BLyS receptor. Immunity
mice induces hyperactivation of B lympho- 18:279288
cytes and disordered lymphoid architecture. 103. Wen L, Roberts SJ, Viney JL et al (1994)
Immunity 21:255266 Immunoglobulin synthesis and generalized
91. Mehling A, Loser K, Varga G et al (2001) autoimmunity in mice congenitally deficient
Overexpression of CD40 ligand in murine in (+) T cells. Nature 369:654658
epidermis results in chronic skin inflammation 104. Wang JH, Avitahl N, Cariappa A et al (1998)
and systemic autoimmunity. J Exp Med Aiolos regulates B cell activation and matura-
194:615628 tion to effector state. Immunity 9:543553
92. Higuchi T, Aiba Y, Nomura T et al (2002) 105. Shankar M, Nixon JC, Maier S et al (2007)
Cutting edge: ectopic expression of CD40 Anti-nuclear antibody production and auto-
ligand on B cells induces lupus-like autoim- immunity in transgenic mice that overexpress
mune disease. J Immunol 168:912 the transcription factor Bright. J Immunol
93. Majeti R, Xu Z, Parslow TG et al (2000) An 178:29963006
inactivating point mutation in the inhibitory 106. Krawczyk C, Bachmaier K, Sasaki T et al
wedge of CD45 causes lymphoproliferation (2000) Cbl-b is a negative regulator of recep-
and autoimmunity. Cell 103:10591070 tor clustering and raft aggregation in T cells.
94. Li DH, Winslow MM, Cao TM et al (2008) Immunity 13:463473
Modulation of peripheral B cell tolerance by 107. Stohl W, Xu D, Kim KS et al (2005) Humoral
CD72 in a murine model. Arthritis Rheum autoimmunity in mice overexpressing B cell
58:31923204 surface CD19: vital role for MHC class II.
95. Tivol EA, Borriello F, Schweitzer AN et al Clin Immunol 116:257264
(1995) Loss of CTLA-4 leads to massive lym- 108. Hsiao HW, Liu WH, Wang CJ et al (2009)
phoproliferation and fatal multiorgan tissue Deltex1 is a target of the transcription factor
destruction, revealing a critical negative regu- NFAT that promotes T cell anergy. Immunity
latory role of CTLA-4. Immunity 3:541547 31:7283
7 Experimental Use of Mouse Models of Systemic Lupus Erythematosus 167
109. Zhu B, Symonds AL, Martin JE et al (2008) auto-immunity in mice lacking protein kinase
Early growth response gene 2 (Egr-2) con- C. Nature 416:865869
trols the self-tolerance of T cells and prevents 123. Layer K, Lin G, Nencioni A et al (2003)
the development of lupuslike autoimmune Autoimmunity as the consequence of a spon-
disease. J Exp Med 205:22952307 taneous mutation in Rasgrp1. Immunity
110. Shim GJ, Kis LL, Warner M et al (2004) 19:243255
Autoimmune glomerulonephritis with spon- 124. Ishimaru N, Arakaki R, Yoshida S et al (2008)
taneous formation of splenic germinal centers Expression of the retinoblastoma protein
in mice lacking the estrogen receptor gene. RbAp48 in exocrine glands leads to Sjgrens
Proc Natl Acad Sci USA 101:17201724 syndrome-like autoimmune exocrinopathy.
111. Zhang L, Eddy A, Teng YT et al (1995) An J Exp Med 205:29152927
immunological renal disease in transgenic 125. Espinosa A, Dardalhon V, Brauner S et al (2009)
mice that overexpress Fli-1, a member of the Loss of the lupus autoantigen Ro52/Trim21
ets family of transcription factor genes. Mol induces tissue inflammation and systemic auto-
Cell Biol 15:69616970 immunity by disregulating the IL-23-Th17
112. Yu CC, Yen TS, Lowell CA et al (2001) pathway. J Exp Med 206:166171
Lupus-like kidney disease in mice deficient in 126. Yu D, Tan AH-M, Hu X et al (2007) Roquin
the Src family tyrosine kinases Lyn and Fyn. represses autoimmunity by limiting inducible
Curr Biol 11:3438 T-cell co-stimulator messenger RNA. Nature
113. Li H, Dai M, Zhuang Y (2004) A T cell intrin- 450:299303
sic role of Id3 in a mouse model for primary 127. Finetti F, Pellegrini M, Ulivieri C et al (2008)
Sjgrens syndrome. Immunity 21:551560 The proapoptotic and antimitogenic protein
114. Hida S, Ogasawara K, Sato K et al (2000) p66SHC acts as a negative regulator of lym-
CD8(+) T cell-mediated skin disease in mice phocyte activation and autoimmunity. Blood
lacking IRF-2, the transcriptional attenuator 111:50175027
of interferon-/ signaling. Immunity 13: 128. Savino MT, Ortensi B, Ferro M et al (2009) Rai
643655 acts as a negative regulator of autoimmunity by
115. Pflegerl P, Vesely P, Hantusch B et al (2009) inhibiting antigen receptor signaling and lym-
Epidermal loss of JunB leads to a SLE pheno- phocyte activation. J Immunol 182:301308
type due to hyper IL-6 signaling. Proc Natl 129. Shultz LD, Schweitzer PA, Rajan TV et al
Acad Sci USA 106:2042320428 (1993) Mutations at the murine motheaten
116. Sommers CL, Park CS, Lee J et al (2002) A locus are within the hematopoietic cell pro-
LAT mutation that inhibits T cell develop- tein-tyrosine phosphatase (Hcph) gene. Cell
ment yet induces lymphoproliferation. Science 73:14451454
296:20402043 130. Tsui HW, Siminovitch KA, de Souza L et al
117. Hibbs ML, Tarlinton DM, Armes J et al (1993) Motheaten and viable motheaten mice
(1995) Multiple defects in the immune sys- have mutations in the haematopoietic cell
tem of Lyn-deficient mice, culminating in phosphatase gene. Nat Genet 4:124129
autoimmune disease. Cell 83:301311 131. Fujimoto M, Tsutsui H, Xinshou O et al
118. Nishizumi H, Taniuchi I, Yamanashi Y et al (2004) Inadequate induction of suppressor of
(1995) Impaired proliferation of peripheral B cytokine signaling-1 causes systemic autoim-
cells and indication of autoimmune disease in mune diseases. Int Immunol 16:303314
lyn-deficient mice. Immunity 3:549560 132. Sun H, Lu B, Li RQ et al (2001) Defective T cell
119. Cornall RJ, Cyster JG, Hibbs ML et al (1998) activation and autoimmune disorder in Stra13-
Polygenic autoimmune traits: Lyn, CD22, deficient mice. Nat Immunol 2:10401047
and SHP-1 are limiting elements of a bio- 133. Drappa J, Kamen LA, Chan E et al (2003)
chemical pathway regulating BCR signaling Impaired T cell death and lupus-like autoim-
and selection. Immunity 8:497508 munity in T cell-specific adapter protein-
120. Hurov JB, Stappenbeck TS, Zmasek CM et al deficient mice. J Exp Med 198:809821
(2001) Immune system dysfunction and auto- 134. Lech M, Kulkarni OP, Pfeiffer S et al (2008)
immune disease in mice lacking Emk (Par-1) Tir8/Sigirr prevents murine lupus by suppress-
protein kinase. Mol Cell Biol 21:32063219 ing the immunostimulatory effects of lupus
121. Yoh K, Itoh K, Enomoto A et al (2001) Nrf2- autoantigens. J Exp Med 205:18791888
deficient female mice develop lupus-like auto- 135. Mackay F, Woodcock SA, Lawton P et al
immune nephritis. Kidney Int 60:13431353 (1999) Mice transgenic for BAFF develop
122. Miyamoto A, Nakayama K, Imaki H et al lymphocytic disorders along with autoimmune
(2002) Increased proliferation of B cells and manifestations. J Exp Med 190:16971710
168 S.L. Peng
136. Gross JA, Johnston J, Mudri S et al (2000) 146. Carballo E, Lai WS, Blackshear PJ (1998)
TACI and BCMA are receptors for a TNF Feedback inhibition of macrophage tumor
homologue implicated in B-cell autoimmune necrosis factor- production by tristetrapro-
disease. Nature 404:995999 lin. Science 281:10011005
137. Khare SD, Sarosi I, Xia XZ et al (2000) Severe 147. Taylor GA, Carballo E, Lee DM et al (1996)
B cell hyperplasia and autoimmune disease in A pathogenetic role for TNF in the syn-
TALL-1 transgenic mice. Proc Natl Acad Sci drome of cachexia, arthritis, and autoimmu-
USA 97:33703375 nity resulting from tristetraprolin (TTP)
138. Seery JP (2000) IFN- transgenic mice: clues deficiency. Immunity 4:445454
to the pathogenesis of systemic lupus erythe- 148. Dang H, Geiser AG, Letterio JJ et al (1995)
matosus? Arthritis Res 2:437440 SLE-like autoantibodies and Sjgrens syn-
139. Sadlack B, Lohler J, Schorle H et al (1995) drome-like lymphoproliferation in TGF-
Generalized autoimmune disease in knockout mice. J Immunol 155:32053212
interleukin-2-deficient mice is triggered by an 149. Yaswen L, Kulkarni AB, Fredrickson T et al
uncontrolled activation and proliferation of (1996) Autoimmune manifestations in the
CD4+ T cells. Eur J Immunol 25:30533059 transforming growth factor-1 knockout
140. Willerford DM, Chen J, Ferry JA et al (1995) mouse. Blood 87:14391445
Interleukin-2 receptor chain regulates the 150. Gorelik L, Flavell RA (2000) Abrogation of
size and content of the peripheral lymphoid TGF signaling in T cells leads to spontane-
compartment. Immunity 3:521530 ous T cell differentiation and autoimmune
141. Suzuki H, Kundig TM, Furlonger C et al disease. Immunity 12:171181
(1995) Deregulated T cell activation and 151. Cheng J, Turksen K, Yu QC et al (1992)
autoimmunity in mice lacking interleukin-2 Cachexia and graft-vs.-host-disease-type
receptor . Science 268:14721476 skin changes in keratin promoter-driven
142. Erb KJ, Ruger B, von Brevern M et al (1997) TNF transgenic mice. Genes Dev
Constitutive expression of interleukin (IL)-4 6:14441456
in vivo causes autoimmune-type disorders in 152. Green RS, Stone EL, Tenno M et al (2007)
mice. J Exp Med 185:329339 Mammalian N-glycan branching protects
143. Vosters JL, Landek-Salgado MA, Yin H et al against innate immune self-recognition and
(2009) Interleukin-12 induces salivary gland inflammation in autoimmune disease patho-
dysfunction in transgenic mice, providing a genesis. Immunity 27:308320
new model of Sjgrens syndrome. Arthritis 153. Demetriou M, Granovsky M, Quaggin S et al
Rheum 60:36333641 (2001) Negative regulation of T-cell activa-
144. Shen L, Zhang C, Wang T et al (2006) tion and autoimmunity by Mgat5
Development of autoimmunity in IL-14- N-glycosylation. Nature 409:733739
transgenic mice. J Immunol 177:56765686 154. Xiao C, Srinivasan L, Calado DP et al (2008)
145. Wang J, Lo JC, Foster A et al (2001) The Lymphoproliferative disease and autoimmu-
regulation of T cell homeostasis and autoim- nity in mice with increased miR-17-92 expres-
munity by T cell-derived LIGHT. J Clin Invest sion in lymphocytes. Nat Immunol
108:17711780 9:405414
Chapter 8
Abstract
CD4+ T cell DNA hypomethylation may contribute to the development of drug induced and idiopathic
human lupus. Inhibiting DNA methylation in mature CD4+ T cells causes MHC-specific autoreactivity
in vitro. The lupus-inducing drugs hydralazine and procainamide also inhibit T cell DNA methylation and
induce autoreactivity, and T cells from patients with active lupus have hypomethylated DNA and a similarly
autoreactive T cell subset. Further, T cells treated with DNA methylation inhibitors demethylate the same
sequences that demethylate in T cells from patients with active lupus. The pathologic significance of the
autoreactivity induced by inhibiting T cell DNA methylation has been tested by treating murine T cells
in vitro with drugs which modify DNA methylation, then injecting the cells into syngeneic female mice.
Mice receiving CD4+ T cells demethylated by a variety of agents including procainamide and hydralazine
develop a lupus-like disease. Further, transgenic mice with an inducible T cell DNA methylation defect also
develop lupus-like autoimmunity. This chapter describes the protocols for inducing autoreactivity in
murine T cells in vitro and for inducing autoimmunity in vivo using an adoptive transfer approach or trans-
genic animal models.
Key words: Lupus, Drug-induced lupus, DNA methylation, Animal models, Autoimmunity
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_8, Springer Science+Business Media New York 2012
169
170 B. Richardson et al.
1.3. T Cell DNA The pathologic significance of 5-azaC induced autoreactivity has
Hypomethylation been tested in animal models. The approach is to treat stimulated
and Autoimmunity CD4+ T cells with 5-azaC in vitro, culture for at least 12 cell
cycles, and then inject the treated cells into syngeneic recipients.
5-azaC and other DNA methylation inhibitors prevent the methy-
lation of newly synthesized DNA during S phase, referred to as
passive demethylation. Thus, these agents are only effective when
added to dividing cells. Further, 12 rounds of cell division are
often required before changes in gene expression are observed
(18). Adoptive transfer of murine CD4+ T cells, made autoreactive
either by treatment with 5-azaC or by transfection with CD18,
causes a lupus-like disease in syngeneic recipients (15). The disease
induced closely resembles chronic graft-vs-host disease in mice, in
which features of lupus-like autoimmunity are also induced by
CD4+ T cells responding to host class II MHC molecules (19).
The DNA hypomethylation model has been used successfully
with polyclonal CD4+ T cells in DBA/2 mice (20), cloned Th2
cells in AKR mice (21), and cloned Th1 cells in B10.A mice (22).
We have also used a panel of DNA methylation inhibitors, includ-
ing 5-azaC, procainamide, hydralazine, and the ERK pathway
inhibitor U0126 to induce autoimmunity (23, 24). 5-azaC and
procainamide are DNA methyltransferase inhibitors (18, 25), while
hydralazine and the ERK pathway inhibitors prevent the upregula-
tion of Dnmt1 and Dnmt3a during T cell stimulation (24). All the
DNA hypomethylation models develop anti-DNA antibodies but
vary to some degree with respect to the histologic changes induced,
either due to the different repertoire of effector functions displayed
by the treated cells, or due to host-specific genetic influences. The
mechanism common to all models is promiscuous killing of host
macrophages (M). This may contribute to the development of
anti-DNA antibodies by increasing the total amount of potentially
antigenic apoptotic material (26), and/or by removing the cells
responsible for removing apoptotic debris, analogous to knock-
out mice with defective clearance of apoptotic material, which
develop anti-DNA antibodies (27).
Next, we designed experiments to provide further evidence
that the T cell ERK signaling defect and DNA hypomethylation
described in lupus patients plays a pathogenic role rather than
merely being a result of the disease process. We generated trans-
genic mice that express a dominant negative MEK (dnMEK) under
the control of a promoter sequence that contains multiple tetracy-
cline-responsive elements. By breeding these mice onto another
transgenic mouse strain that expresses a reverse tetracycline trans-
activator (rtTA) under the control of a CD2 promoter, we can
induce T cell-specific expression of the dominant negative MEK by
adding doxycycline to the drinking water of the double transgenic
mice (B6.dnMEK/CD2-rtTA). This allowed us to study the effects
172 B. Richardson et al.
1.4. Relevance At least six lines of evidence support the contention that the DNA
to Human Lupus hypomethylation model has relevance to human lupus. First, the
two drugs that most clearly cause a lupus-like disease in people,
procainamide and hydralazine, are T cell DNA methylation inhibi-
tors (30). Cloned murine Th2 cells treated with these drugs induce
a lupus-like disease identical to that caused by 5-azaC (23), sug-
gesting a mechanism by which they might cause lupus in humans.
Second, T cells from patients with active lupus have decreased lev-
els of total genomic dmC, similar to 5-azaC treated cells (31).
Third, T cells from patients with active lupus overexpress LFA-1
on an autoreactive T cell subset (14), and the overexpression is
associated with hypomethylation of the same sequences flanking
the CD11a promoter that demethylate following 5-azaC treatment
(17). Fourth, T cells from patients with active lupus have a selec-
tive defect in ERK pathway signaling, the pathway inhibited by
hydralazine (32), and inhibiting this pathway with hydralazine or
U0126 causes a lupus-like disease in the adoptive transfer model
and in the dnMEK/CD2-rtTA mouse model (2428). Fifth,
LFA-1 overexpressing T cells isolated from patients with active
lupus spontaneously kill autologous monocytes with a specificity
identical to experimentally hypomethylated T cells (14), and by the
same mechanisms (FasL, TRAIL, and TWEAK) as experimentally
hypomethylated T cells (33, 34). Finally, the X-linked methylation
sensitive gene CD40LG is overexpressed in CD4+ T cells from
female but not male lupus patients (35). The overexpression of
CD40LG in female patients with active lupus is associated with
hypomethylation of the CD40LG promoter sequence in both
alleles, allowing for transcriptional activity of the CD40LG
gene from the normally heavily methylated and inactive X chro-
mosome (35). These findings are consistent with a gene-dose effect
on the X-chromosome as demonstrated by the dnMEK/CD2-rtTA
mice experiments and suggest that chromosomal sex is perhaps
more important than hormonal sex in explaining the female pre-
dominance of lupus. Together, these studies strongly suggest that
similar mechanisms contribute to the development of autoimmu-
nity in the DNA hypomethylation models and in drug induced and
idiopathic human lupus.
174 B. Richardson et al.
2. Materials
2.1. Mice Young (68 weeks) female AKR and B10.A mice are obtained from
Jackson Labs, and DBA/2 mice from Charles River. The dnMEK
transgenic mice were generated by subcloning a dominant negative
MEK1 into the pTRE2 construct (Clontech), containing multiple
tetracycline response elements and a minimal CMV promoter.
These mice were then bred with CD2-rtTA mice, obtained from
Dr. Rose Zamoyska, to produce dnMEK/CD2-rtTA double trans-
genic mice. Doxycycline, added to the drinking water at a concen-
tration of 2 mg/ml, was used to induce expression of the dominant
negative MEK in vivo.
2.2. Cell Lines D10.G4.1 (D10) cells are obtained from the American Type
Culture Collection (ATCC). AE7 cells were obtained from Dr.
Ronald Schwartz.
2.4. IL-2 The IL-2 secreting T cell line MLA-144, obtained from the ATCC,
was cultured in RPMI 1640 supplemented with 3 % fetal calf serum
(FCS). Three times a week the cells were sedimented by centrifu-
gation, the conditioned media filtered to remove any remaining
cells, and then stored frozen at 20 C.
2.5.2. D10 Cells Clicks medium supplemented with 10 % FCS, 40 % IL-2 contain-
ing conditioned media, 2 mM glutamine, 100 IU/ml penicillin,
100 mg/ml streptomycin, and 5 105 M 2-mercaptoethanol.
3. Methods
3.1. Cells Spleens are removed from young (6- to 8-week old) female
and 5-Azacytidine DBA/2 (H-2d) mice and dissociated with forceps followed by
Treatment forcing through a sterile disposable plastic screen with a syringe
piston. Splenocytes are then isolated by density gradient centrifu-
3.1.1. Polyclonal CD4+ T
gation through Lympholyte-M (Cedarlane). CD8+ cells are
Cell Lines
depleted with magnetic beads (Miltenyi) according to the manu-
facturers instructions, then 106 CD4+ cells are cultured in 2 ml of
IL-2 containing media (see Subheading 2) and stimulated with
either 106 irradiated (3000R) allogeneic splenocytes (e.g.,
C57BL/6, H-2b) or 5 mg/ml Concanavalin A (Pharmacia) using
flat-bottom 24-well plates. The cultures are maintained at 37 C
in 5 % CO2 and humidified atmosphere, and rocked on a rocker
platform (Bellco) at 56 cycles/min (see Note 2). The lines are
maintained by the addition of fresh IL-2 containing media every
23 days, and restimulating every 710 days, using ~106 cells/
well and equal numbers of irradiated allogeneic splenocytes or
0.51.0 106 irradiated syngeneic splenocytes + 1 mg/ml
Concanavalin A, as appropriate. One day after restimulation the
cells are treated with 5-azaC and 6 days later the cells tested for
autoreactivity and/or used for adoptive transfer. The cells should
also be tested for CD4 and CD8 expression by flow cytometry at
this point to exclude overgrowth by CD8+ cells.
3.1.2. D10 Cells D10.G4.1 (D10) cells (American Type Culture Collection) are
maintained in IL-2 containing media (see Subheading 2) using flat-
bottomed 24-well plates and a rocker platform as for polyclonal cells.
The line is maintained by challenging 0.11.0 106 D10 cells with
5 105 irradiated (3000R) AKR splenocytes and 100 mg/ml conal-
bumin (Sigma) every 710 days. The D10 line contains an autoreac-
tive subset (see Note 3) and must be subcloned by limiting dilution
and a nonautoreactive subclone selected prior to use. The cells are
treated with 5-azaC and used for functional characterization or given
in adoptive transfer at least 6 days after treatment.
3.1.3. AE7 Cells AE7 cells are maintained in IL-2 containing media (see
Subheading 2) and stimulated weekly with irradiated syngeneic
(B10.A) splenocytes and antigen (100 mg/ml pigeon cytochrome
C) on a rocker platform as described for polyclonal CD4+ cells and
D10 cells. To induce autoreactivity the cells are treated with 5-aza-
2-deoxycytidine (see Note 4) and used 6 days later.
176 B. Richardson et al.
3.2. Autoreactivity For D10 cells, 2 104 treated or untreated cells are cultured with
Assays graded numbers (210 104) of irradiated syngeneic (AKR) sple-
nocytes in 200 ml of the same media but without IL-2, with or
3.2.1. Proliferation Assays
without 100 mg/ml conalbumin, using round-bottom 96-well
plates. Proliferation is tested 45 days later by adding 1 mCi triti-
ated thymidine/well and 6 h later determining 3H incorporation
into DNA. Polyclonal CD4+ T cells are similarly tested, using
5 104 T cells and ~105 irradiated syngeneic splenocytes/well
(range 5 1045 105), using allogeneic splenocytes or Conca-
navalin A as appropriate for the positive control. In all cases deter-
minations are performed in triplicate or quadruplicate.
3.2.2. Cytotoxicity Assays For D10 cells, thioglycollate elicited syngeneic (AKR) M are labeled
with 100 mCi 51Cr in 1 ml of RPMI/10 % FCS for 1 h 37 C in a
round-bottom culture tube. The cells are washed, then 5,000 labeled
M cultured with 125,000 D10 cells with or without 100 mg/ml
conalbumin in a total volume of 200 ml of sterile media lacking IL-2,
using round-bottom microtiter plates. 18 h later chromium release
is measured using a scintillation spectrometer (33). AE7 killing
assays are performed similarly, except that an effectortarget ratio of
10:1 is used, and the antigen (positive control) is 100 mg/ml pigeon
cytochrome C. Splenocyte killing assays are similarly performed,
using an effectortarget ratio of 25:1. Percent cytotoxicity is calcu-
lated as [(experimental background release)/(total incorpora-
tion background release)] 100 (20) (see Note 6).
3.3. Adoptive Transfer All adoptive transfer models are performed similarly. The treated
of Autoreactive Cells cells are washed, dead cells removed by centrifugation through
Lympholyte M, then 5 106 viable cells are suspended in 0.2 ml
sterile PBS and injected into the tail vein of young (<12 weeks)
syngeneic female mice, using a 26-gauge needle. A total of six
injections are performed, spaced 2 weeks apart. The rationale for
repeated adoptive transfers derives from the observation that
5-azaC induced autoreactivity is self-limited (12). Four weeks after
the last injection the mice are euthanized and studied for the devel-
opment of serologic and histologic evidence of autoimmunity. The
development of proteinuria and hematuria may be monitored by
holding Chemstrips (Boehringer Mannheim) under the mouse
while picking it up (see Note 7).
3.4. IgG, IgM, and Total serum IgG and IgM concentrations are measured using
Anti-DNA Antibody Immulon 4 plates (Dynatech Laboratories) coated with 2.5 mg
Assays anti-mouse IgG or IgM (Sigma) in 100 ml 0.01 M PBS, pH 7.4 for
18 h at 4 C. The plates are washed 3 with PBS containing 0.05 %
Tween 20, then 200 ml of PBS supplemented with 3 % BSA, 0.1 %
gelatin and 0.05 % Tween 20 are added and incubated 2 h 23 C.
8 Murine Models of Lupus Induced by Hypomethylated T Cells 177
4. Notes
3. D10 Cells: With prolonged culture, D10 cells tend to lose the
restriction for antigen and proliferate to syngeneic APC with-
out added antigen. The mechanism is unknown, but adoptive
transfer of these cells does not induce autoimmunity (Bruce
Richardson, MD, PhD, University of Michigan). The cells also
tend to lose the requirement for IL-2 for sustained growth
over time, causing high backgrounds in proliferation assays.
Consequently, it is necessary to repeatedly subclone this line
and select antigen-specific cells. Alternatively, multiple aliquots
of quality tested subcloned cells may be stored in liquid nitro-
gen, and thawed as needed.
4. AE7 Cells: AE7 cells are more refractory to the induction of
autoreactivity than normal T cells or D10 cells. Treatment with
5-aza-2-deoxycytidine is required, and concentrations up to
8 mM are sometimes needed (22). Also, in our hands, prolif-
eration assays are less reliable than cytotoxicity assays for both
antigen reactivity and autoreactivity.
5. Variations: Activated T cells can be modified with other DNA
methylation inhibitors, or by transfection as needed. Our
group has found D10 cells to be the best suited for these stud-
ies, and have successfully compared procainamide with
N-acetylprocainamide, and hydralazine with phthalazine in this
model (23). Similarly, we have used the ERK pathway inhibitor
U0126 (24) and used D10 cells transfected with CD18 (15).
Other modifications of the cells may be similarly tested.
6. If desired, MHC specificity of the autoreactivity assays may be
tested using monoclonal antibodies to the relevant class II
MHC molecules, or congenic mouse strains.
7. Following injection of 5-azaC treated polyclonal CD4+ T cells
from DBA/2 mice, hematuria is first seen between weeks 1
and 3, and usually lasts 714 days then resolves. Proteinuria,
defined as >30 mg/dl, was more persistent. Immunofluorescent
evidence of renal Ig deposition correlates with active hematu-
ria and resolves at later time points.
8. Sometimes the control sera give a relatively high background
in the anti-DNA ELISAs. The specificity of the ELISAs may be
tested by adding 5 mg/ml of soluble ss-DNA or ds-DNA as
appropriate to replicate wells. Lack of inhibition is indicative of
nonspecificity, while inhibition is indicative of autoantibodies.
References
1. Attwood JT, Yung RL, Richardson BC (2002) 3. Okano M, Bell DW, Haber DA, Li E (1999)
DNA methylation and the regulation of gene DNA methyltransferases Dnmt3a and Dnmt3b
transcription. Cell Mol Life Sci 59:241257 are essential for de novo methylation and mam-
2. Li E, Bestor TH, Jaenisch R (1992) Targeted malian development. Cell 99:247257
mutation of the DNA methyltransferase gene 4. Taylor SM, Jones PA (1979) Multiple new
results in embryonic lethality. Cell 69: phenotypes induced in 10T1/2 and 3T3 cells
915926 treated with 5-azacytidine. Cell 17:771779
8 Murine Models of Lupus Induced by Hypomethylated T Cells 179
5. Golbus J, Palella TD, Richardson BC (1990) 18. Jones PA (1984) Gene activation by 5-azacyti-
Quantitative changes in T cell DNA methyla- dine. In: Razin A, Cedar H, Riggs A (eds)
tion occur during differentiation and ageing. DNA methylation biochemistry and biological
Eur J Immunol 20:18691872 significance. Springer-Verlag, New York,
6. Young HA (1996) Regulation of interferon- pp 165187
gamma gene expression. J Interferon Cytokine 19. van der Veen FM, Rolink AG, Gleichmann E
Res 16:563568 (1982) Autoimmune disease strongly resem-
7. Santangelo S, Cousins DJ, Winkelmann NE, bling systemic lupus erythematosus (SLE) in
Staynov DZ (2002) DNA methylation changes F1 mice undergoing graft-versus-host reaction
at human Th2 cytokine genes coincide with (GVHR). Adv Exp Med Biol 149:669677
DNase I hypersensitive site formation during 20. Quddus J, Johnson KJ, Gavalchin J et al
CD4(+) T cell differentiation. J Immunol (1993) Treating activated CD4+ T cells with
169:18931903 either of two distinct DNA methyltransferase
8. Floess S, Freyer J, Siewert C et al (2007) inhibitors, 5-azacytidine or procainamide, is
Epigenetic control of the foxp3 locus in regula- sufficient to cause a lupus-like disease in syn-
tory T cells. PLoS Biol 5:e38 geneic mice. J Clin Invest 92:3853
9. Young HA, Ghosh P, Ye J et al (1994) 21. Yung RL, Quddus J, Chrisp CE, Johnson KJ,
Differentiation of the T helper phenotypes by Richardson BC (1995) Mechanism of drug-
analysis of the methylation state of the IFN- induced lupus. I. Cloned Th2 cells modified with
gamma gene. J Immunol 153:36033610 DNA methylation inhibitors in vitro cause auto-
10. Lu Q, Wu A, Ray D, Deng C, Attwood J, immunity in vivo. J Immunol 154:30253035
Hanash S, Pipkin M, Lichtenheld M, 22. Yung R, Kaplan M, Ray D et al (2001)
Richardson B (2003) DNA methylation and Autoreactive murine Th1 and Th2 cells kill
chromatin structure regulate T cell perforin syngeneic macrophages and induce autoanti-
gene expression. J Immunol 170:51245132 bodies. Lupus 10:539546
11. Richardson BC, Liebling MR, Hudson JL (1990) 23. Yung R, Chang S, Hemati N, Johnson K,
CD4+ cells treated with DNA methylation inhib- Richardson B (1997) Mechanisms of drug-
itors induce autologous B cell differentiation. induced lupus. IV. Comparison of procain-
Clin Immunol Immunopathol 55:368381 amide and hydralazine with analogs in vitro
12. Richardson B (1986) Effect of an inhibitor of and in vivo. Arthritis Rheum 40:14361443
DNA methylation on T cells. II. 5-Azacytidine 24. Deng C, Lu Q, Zhang Z et al (2003) Hydralazine
induces self-reactivity in antigen-specific T4+ may induce autoimmunity by inhibiting extra-
cells. Hum Immunol 17:456470 cellular signal-regulated kinase pathway signal-
13. Richardson B, Powers D, Hooper F, Yung RL, ing. Arthritis Rheum 48:746756
ORourke K (1994) Lymphocyte function- 25. Scheinbart LS, Johnson MA, Gross LA, Edelstein
associated antigen 1 overexpression and T cell SR, Richardson BC (1991) Procainamide inhibits
autoreactivity. Arthritis Rheum 37:13631372 DNA methyltransferase in a human T cell line.
14. Richardson BC, Strahler JR, Pivirotto TS et al J Rheumatol 18:530534
(1992) Phenotypic and functional similarities 26. Mevorach D, Zhou JL, Song X, Elkon KB
between 5-azacytidine-treated T cells and a T (1998) Systemic exposure to irradiated apop-
cell subset in patients with active systemic lupus totic cells induces autoantibody production.
erythematosus. Arthritis Rheum 35:647662 J Exp Med 188:387392
15. Yung R, Powers D, Johnson K et al (1996) 27. Walport MJ (2000) Lupus, DNase and defec-
Mechanisms of drug-induced lupus. II. T cells tive disposal of cellular debris. Nat Genet
overexpressing lymphocyte function-associated 25:135136
antigen 1 become autoreactive and cause a 28. Sawalha AH, Jeffries M, Webb R et al (2008)
lupuslike disease in syngeneic mice. J Clin Defective T-cell ERK signaling induces inter-
Invest 97:28662871 feron-regulated gene expression and overex-
16. Kaplan MJ, Beretta L, Yung RL, Richardson pression of methylation-sensitive genes similar
BC (2000) LFA-1 overexpression and T cell to lupus patients. Genes Immun 9:368378
autoreactivity: mechanisms. Immunol Invest 29. Strickland FM, Hewagama R, Lu Q, Johnson
29:427442 K, Webb R, Sawalha A, Richardson BC (2010)
17. Lu Q, Kaplan M, Ray D, Zacharek S, Gutsch Both Estrogen and female sex contribute to
D, Richardson B (2002) Demethylation of disease development in an epigenetic model of
ITGAL (CD11a) regulatory sequences in lupus. Lupus 19:91
systemic lupus erythematosus. Arthritis Rheum 30. Cornacchia E, Golbus J, Maybaum J, Strahler
46:12821291 J, Hanash S, Richardson B (1988) Hydralazine
180 B. Richardson et al.
and procainamide inhibit T cell DNA methyla- 33. Kaplan MJ, Lewis EE, Shelden EA et al (2002)
tion and induce autoreactivity. J Immunol The apoptotic ligands TRAIL, TWEAK, and Fas
140:21972200 ligand mediate monocyte death induced by autol-
31. Richardson B, Scheinbart L, Strahler J, Gross ogous lupus T cells. J Immunol 169:60206029
L, Hanash S, Johnson M (1990) Evidence for 34. Kaplan MJ, Ray D, Mo RR, Yung RL,
impaired T cell DNA methylation in systemic Richardson BC (2000) TRAIL (Apo2 ligand)
lupus erythematosus and rheumatoid arthritis. and TWEAK (Apo3 ligand) mediate CD4+ T
Arthritis Rheum 33:16651673 cell killing of antigen-presenting macrophages.
32. Deng C, Kaplan MJ, Yang J et al (2001) J Immunol 164:28972904
Decreased Ras-mitogen-activated protein 35. Lu Q, Wu A, Tesmer L, Ray D, Yousif N,
kinase signaling may cause DNA hypomethyla- Richardson B (2007) Demethylation of CD40LG
tion in T lymphocytes from lupus patients. on the inactive X in T cells from women with
Arthritis Rheum 44:397407 lupus. J Immunol 179:63526358
Chapter 9
Abstract
This chapter describes methods utilized in establishing a mouse model of neuropsychiatric lupus encompassing
both cognitive and emotional dysfunction, and a model of the influence of maternal antibody on the devel-
oping brain. The antibody of interest binds the N-methyl-D-aspartate receptor (NMDAR), a receptor for
glutamate that is a major excitatory neurotransmitter in the brain involved in synaptic plasticity, in memory
and learning, and in emotional responses.
We introduce basic concepts of these models and provide protocols for the following: (1) the induction
of anti-dsDNA, anti-NMDAR antibodies, (2) testing serum antibody titer by ELISA, (3) breaching blood
brain barrier (BBB) integrity with LPS and epinephrine, (4) passive transfer of pathology by injecting
human and mouse brain-reactive antibodies into adult mouse as well as injecting the antibody into gestat-
ing mice and transfer of antibody from dam to fetus, (5) blocking NMDAR-mediated pathogenicity
in vivo, (6) evacuation of blood from the brain by cardiac perfusion to preserve the brain for histology,
(7) evaluating injured/apoptotic neurons in brain histology, (8) preparing membrane-enriched brain
fractions for NMDAR analysis.
Key words: CNS lupus, Neuropsychiatric lupus, Anti-NMDA receptor antibodies, Blood brain barrier,
Pathogenic maternal autoantibody
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_9, Springer Science+Business Media New York 2012
181
182 C. Kowal and B. Diamond
2. Materials
2.1.1. Immunization Animals. 6 8 week old BALB/c female mice (see Note 1).
Equipment. Eppedorf centrifuge, two pairs of glass syringes and
3-way Luer-lock stopcock for emulsifying the antigens, glass
184 C. Kowal and B. Diamond
2.2. Testing Serum ELISA assays. The following protocols adapted from Scripps
Antibody Titer Research Laboratory Manual (La Jolla, CA) for phage combinato-
by ELISA rial libraries are used in our laboratory for the analysis of serum
titers from immunized mice as well as for the analysis of patients
sera for the presence of anti-dsDNA, anti-NMDAR antibodies. We
use these protocols with slight modifications for other antigens, for
quantification of immunoglobulin in the serum human and in
other media and for an inhibition ELISA.
2.2.1. ELISA Assays Equipment. Costar 3690 plates (see Note 4), polyvinyl chloride
plates (see Note 5), ELISA plate reader (see Note 6), ELISA plate
washer (see Note 7), multichannel pipettes and plastic tips, any
kind of plastic reagent reservoir.
Reagents and buffers. Antigens used in immunization (see
Subheading 2.1.1), DWEYS peptide, DWEYSVWLSN peptide,
extracellular domain of the NMDARrecombinant protein (see
Note 8), calf thymus double stranded DNA (dsDNA) (see Note
9), 0.1 M NaHCO3, pH 8.6 (see Note 10), PBS-Tween 20, 0.05 %
(PBS-T), PBS, 1 % bovine serum albumin (BSA) in PBS (see Note
11), alkaline phosphatase (AP)-labeled secondary antibody (see
Note 12), AP substrate, AP substrate buffer: 0.001 M magnesium
chloride (MgCl2) plus 0.05 M sodium carbonate (Na2Co3) (see
Note 13).
2.3. Opening the BBB We based our experiments of the opening of the BBB on the
by LPS and by groundbreaking work done by Bank and colleagues (23, 24).
Epinephrine in NPSLE There is a significant strain and age dependent sensitivity to LPS
Mouse Model toxicity so the conditions described here are for young BALB/c
mice. This is still work in progress so here we present effective pro-
cedures but by no means the final ones.
2.3.1. Opening the BBB Animals. BALB/c female mice immunized with MAP-
DWEYSVWLSN peptide and with MAP-core.
Reagents and buffers. Bacterial LPS (see Note 14), epinephrine
(see Note 15), saline.
Equipment. Water bath sonicator, freezing vials, eppendorf tubes,
1 ml plastic syringes for injections.
9 Aspects of CNS Lupus 185
2.4. Passive Transfer Passive transfer studies are an integral part of any animal model of
of Pathology antibody-mediated disease. We started by injecting of R4A, a mono-
by Intravenous clonal antibody that binds dsDNA and NMDAR, and the isotype
Injection of Human control antibody directly into mouse cortex and hippocampus using
and Mouse Brain- stereotaxic coordinates. Mice exposed to NMDAR-reactive anti-
Reactive Antibodies body but not control mice analyzed 1 and 2 days post injection
into Adult Mouse showed neuronal loss (25). Similar results were obtained by direct
and into the Embryo injection of lupus IgG affinity purified on DWEYSVWLSN peptide
via Maternal column, with normal IgG purified on a protein G column as con-
Circulation at Different trol. We also tested and confirmed NMDAR-mediated pathogenic-
Gestational Stages
ity by direct injection to the brain of undiluted, NMDAR-reactive
CSF from a lupus patient and normal CSF as control as well as anti-
body purified from the frozen brain tissue of a lupus patient with
neuropsychiatric symptoms and normal IgG subjected to the same
procedures (13, 25). We then established a more physiological
model and intravenously injected NMDAR-reactive antibodies
from lupus patient purified on protein G and antibodies from simi-
larly treated normal serum. In the same model, we intravenously
injected the same lupus serum depleted of NMDAR-reactivity on a
DWEYSVWLSN peptide column as control. The animals were
treated subsequently with LPS to compromise the BBB. Mice that
received pathogenic, NMDAR-specific antibody showed neuronal
deficits primarily in the hippocampus and, consequently, deficits in
hippocampus-dependent cognitive tasks (13). For the embryonal
model, we injected timed-pregnant mice with anti-dsDNA, anti-
NMDAR-reactive antibody, R4A, and isotype control on day E12
and analyzed the embryonal brain by histology at E15, just before
birth and postnatally. We observed cortical abnormalities and behav-
ioral deficits in the offspring of mice exposed to the anti-NMDAR
antibody in utero but not in control animals. Similar results were
obtained in fetuses exposed to human monoclonal anti-dsDNA,
anti-NMDAR reactive antibody, G11, using monoclonal B1 anti-
body that did not bind these antigens as a control (19).
saline, 0.3 mg/ml LPS (see above), protein G slurry or Nunc spin
column, Amicon centrifugal units, purified antibodies, sera, CSF,
all ELISA reagents (see Subheading 2.2.1).
2.5. Blocking NMDAR- In parallel to establishing the animal model of CNS lupus, we tested
Mediated the blocking of antibody-mediated damage as a potential future
Pathogenicity In Vivo therapy. We have successfully tested two blocking reagents, the
NMDARs specific blocker memantine (see Note 19) and a D-isoform
of DWEYS peptide (see Note 20) in a MAP-DWEYSVWLSN
immunized mouse (5). We are currently developing new blocking
reagents (26). We describe here a simple dose and injection sched-
ule that resulted in successful inhibition of pathology.
2.5.1. Blocking NMDAR- Animals. Immunized 68-week-old female BALB/c mice (as
Mediated Pathogenicity described in paragraph 3.1 in this chapter) (see Note 21 and
In Vivo Note 65).
Equipment. Insulin syringes (see Note 22), isoflurane anesthesia
machine (see Note 23), small volume, 0.250.5 ml, with a needle
gauge 2729.
Reagents. Ringers solution, memantine hydrochloride, D-DWEYS
peptide.
2.6. Evacuation Cardiac perfusion. In order to perform brain histology the blood
of Blood from the needs to be evacuated from the animals brain. We use transcardiac
Brain by Cardiac perfusion that is effective and relatively easy to learn.
Perfusion and Fixing
the Brain for Histology
2.6.1. Cardiac Perfusion Equipment. Anesthesia machine for isoflurane with oxygen tank
(see Note 23), peristaltic pump (see Note 24), proper tubing (see
Note 25) with the needle adaptor on one side, stainless steel con-
tainer for collecting the fluids during procedure, stainless steel
mesh to cover the container, 27 G 3/8 needle, rongeur for remov-
ing the skull (see Note 26), surgical scissors (see Note 27), spatula,
50 ml conical tubes, eppendorf tubes, glass capillaries, small slotted
spoon (see Note 28).
Reagents and buffers. Pre-perfusion buffer: 0.9 % sodium chloride
(NaCl), 0.5 % sodium nitrate, 1,000 U Heparin (see Note 29);
perfusion buffer: 4 % paraformaldehyde, 0.1 M sodium phosphate
buffer (PB) pH 7.4 (see Note 30); 30 % sucrose (see Note 31),
PBS, isoflurane.
2.8.2. Western Blot For western blot (WB) we use Invitrogens (Carlsbad, CA) system
Analysis of NMDARs for electrophoresis and protein transfer and their gels and buffers.
188 C. Kowal and B. Diamond
3. Methods
the other syringe. Add 100 l extra for lubrication (you may
need more for bigger syringes). Remove the air bubbles using
the plunger and add the antigen in saline through the open
syringe. Secure the open syringe with the plunger and carefully
remove the air from the syringe by opening the stopper. Close
the stopper and start emulsification by pushing the solution
from one syringe to the other until emulsion is homogenous
and you start feeling the resistance (see Note 50). Test the emul-
sion by placing a drop on water in a small container. The prop-
erly prepared emulsion is thick and does not disperse on water.
4. Transfer emulsion for immunization by removing one glass
syringe and replacing it by Leur-lock 1 ml plastic syringe. Push
the proper amount of emulsion. Place 26 G 3/8 needle on the
top. Remove air by delicately tapping the syringe on the hard
surface. Check the air bubble in a syringe against good light
and press the air out through the needle.
5. First immunization. Inject BALB/c female mice intraperitone-
ally (see Note 51), with 100 l of emulsion/mouse. Wait for
2 weeks.
6. First boost. Repeat intraperitoneal injection of the same
amount of antigen in Incomplete Freunds Adjuvant. Wait for
2 weeks.
7. Second boost. Repeat intraperitoneal injection of the same
amount of antigen in Incomplete Freunds Adjuvant.
8. Bleeding. Obtain about 50100 l of blood at desired time
point. Test the serum titer (see Note 52).
3.3. Opening 1. Prepare LPS solution at 0.3 mg/ml in saline (see Note 14).
of the BBB by LPS 2. Inject BALB/c mice intraperitoneally using 3 mg/kg (see
and by Epinephrine Note 56). Observe the animals for septic effects. Use saline for
3.3.1. Opening of the Blood
control animals. Wait for 48 h.
Brain Barrier Using LPS 3. Repeat injection using the same dose (see Note 57). Wait for
the proper time for neuronal loss analysis or for behavioral
experiments. We have observed neuronal loss at day 7 and did
not see any increase in neuronal loss past 30 days after LPS
injection. Behavioral experiments were performed 48 weeks
after opening of the BBB.
4. Perform cardiac perfusion (described below) for histological
analysis of the brain at the conclusion of the experiment.
3.3.2. Opening of the Blood 1. Prepare epinephrine solution in saline at the dose of
Brain Barrier Using 200 nM/100 l per mouse (see Note 58).
Epinephrine 2. Inject BALB/c mice intraperitoneally using 200 nM/100 l
per mouse (see Note 59). Wait for 48 h.
3. Repeat injection using the same dose. Wait for the proper time
for neuronal loss analysis or for behavioral experiments. We
performed the same 7 and 30 day analysis as with LPS and see
similar neuronal loss but in different brain areas. Behavioral
studies were done 48 weeks after opening of the BBB.
4. Perform cardiac perfusion (described below) for histological
analysis of the brain at the conclusion of the experiment.
3.4. Passive Transfer 1. Grow monoclonal antibody in 10 % ultra low FBS in DMEM
medium using Petri dish until you obtain 1 108 hybridoma
3.4.1. Production of
cells.
Monoclonal Antibodies
Using Hollow Fiber 2. Equilibrate the Hollow Fiber Bioreactor with PBS and DMEM
Bioreactor medium according to manufacturers instruction.
9 Aspects of CNS Lupus 191
3.4.2. Purification 1. Prepare the column using Protein G sepharose for gravity
of Antibodies from Serum purification or use commercially available Protein G spin col-
and Culture Supernatant umns following manufacturers instruction. Wash the columns
and from Brain Tissue with Protein G binding buffer or PBS.
2. Filter (0.2 m) serum/supernatant and load the column. Do
not exceed column capacity.
3. Let the antibody flow through the column by gravity or spin
the column if using the Nunc system.
4. Wash the column with neutral buffer (PBS) until no protein is
detectable in the flowthrough.
5. Elute antibody using 0.1 M glycine pH 2.5 with or without
0.15 M sodium chloride (see Note 60) and immediately neu-
tralize each fraction using 2 M TrisCl pH 9.0 (see Note 61).
6. Dialyze or exchange buffer using Amicon filtration system to
experimental buffer or PBS.
7. Check the concentration of purified antibody using any avail-
able method (see Note 62). If the antibody concentration is
high, dilute it in PBS before use (see Note 63).
8. Test antibody binding by ELISA, remove the aggregates by
centrifugation at 1,000 x g for 10 min and filter-sterilize it.
For extraction of antibody from frozen brain tissue we followed
the excellent method of Owens et al. (27) and then purified IgG
using Protein G as above.
3.4.3. Passive Transfer Use sterile solutions for all of the injections.
of Pathogenicity in Mouse
1. For direct injection to the brain parenchyma use 15 g in
Model of CNS Lupus
12 l volume. Inject slowly (0.25 l/min) using stereotaxic
frame and coordinates. Use precision peristaltic pump and glass
capillaries.
2. For intravenous injection of timed-pregnant mice use 200
400 g of purified monoclonal antibody in 100 l (see
Note 64). For adult model we injected 2 mg of antibodies
192 C. Kowal and B. Diamond
3.6. Cardiac Perfusion 1. Prepare the buffers, 50 ml each, pre-perfusion and 4 % para-
formaldehyde perfusion buffer/mouse.
2. Prepare all of the equipment: anesthesia machine, peristaltic
pump and surgical instruments.
3. Put 27 G 3/8 needle at the end of the Tygon tubing of the
peristaltic pump and the other end in 50 ml conical tube con-
taining pre-perfusion buffer. Flush small volume of pre-perfu-
sion buffer to remove the air from the tubing.
4. Preset the oxygen level at 1.51.7 ml/min and the isoflurane
at the higher level (44.5 is that higher level of isoflurane on
the anesthesia machine, no subheadings 44.5) at the start so
that the animal is anesthetized quickly (see Note 65).
5. Anesthetize the mouse (see Note 66).
6. Place the mouse nose cone (see Note 67) on the animal and
turn the switch to allow anesthetic delivery and continuing
anesthesia until the animal expires.
7. Open abdominal cavity at the edge of the rib cage. Make a
wide cut from one side to the other using sharp surgical scis-
sors avoiding any organ injury.
9 Aspects of CNS Lupus 193
3.7. Histology Preparing brain sections. Select the proper thickness for the sec-
tions of the brain and the method of staining.
(a) For thin, 1020 m sections use cryostat or microtome and
Super Frost microscope slides. Equilibrate frozen tissue to cry-
ostat temperature before starting sectioning. You may brush
the slide with water for better adherence, but you will need to
dry these sections longer (see Note 71). Place thin sections on
microscope slides and let it adhere for at least 1 h. You can
freeze the slides in a slide cassette at 80 C until use.
(b) For thicker sections, use microtome or vibratome and free-
floating binding. Use M-1 embedding matrix for microtome
to freeze the brain and to adhere the brain to the platform for
194 C. Kowal and B. Diamond
3.7.1. Staining Brain 1. Equilibrate the frozen slides to room temperature for at least
Sections Using Microscope 20 min if frozen.
Slides 2. Rehydrate the slide in PBS in a histology jar for 5 min.
3. Remove an excess of PBS by tilting the slide on the edge and
using Kimwipes or paper towel.
4. Make a circle around the section using hydrophobic slide
marker (see Note 72).
5. Place the slide in humidified, light-proof, flat chamber and put
the proper volume of blocking buffer (see Note 73) on a sec-
tion; usually 100 l is sufficient for mouse brain section.
6. Block for 30 min to 1 h at room temperature.
7. Remove blocking solution by using low vacuum suction or by
Kimwipe.
8. Apply primary antibody in the blocking buffer or in PBS-T,
same volume as above (see Note 74).
9. Incubate for 12 h at room temperature or overnight at 4 C
in a light-proof chamber (see Note 75).
10. Wash the slides in a battery histology jars filled with PBS.
Typically, wash 35 times for 10 min each time. Remove an
excess of washing buffer after final wash.
11. Apply secondary antibody in the blocking buffer or in PBS-T
(see Note 74).
12. Incubate for 0.51 h at room temperature.
13. Wash 35 times for 10 min each time. Remove an excess of the
washing buffer. For staining without DAPI move on directly
to Subheading 3.7.1 step 15.
14. For staining with DAPI, cover the tissue with 1 g/ml of
DAPI in PBS in 100150 l PBS, wait for 12 min, remove
DAPI solution, and move on to Subheading 3.7.1 step 15.
15. Apply the mounting medium near the tissue, place the glass
coverslip carefully on the top avoiding air bubbles and press
delicately to remove the excess of the mounting medium.
Drain an excess of the mounting medium by tilting the slide
on the edge and using Kimwipe or Whatman paper (see
Note 76). Seal the coverslip using nail polish.
16. Analyze sections under the microscope.
9 Aspects of CNS Lupus 195
3.7.2. Free-Floating 1. Collect the sections in the tissue culture dish containing
Section Staining Used 0.1 M PB buffer, pH 7.4.
for Thicker Sections
2. Prepare the blocking solution (see Note 73).
for Detecting Antibody
Deposition in the Brain 3. Place the proper volume of blocking solution in the wells of
ceramic dish or in the tissue culture dish (see Note 35). Do not
fill up, as the binding is done with slow rotating shaker.
4. Using camel hair brush place the sections in the blocking solu-
tion containing wells.
5. Incubate at room temperature for 1 h with delicate rotating
shaking.
6. Transfer the sections to new wells containing primary antibody
(see Note 74).
7. Incubate at room temperature for few hours or overnight at
4 C on a slow rotating shaker.
8. Wash the sections by transferring them to the well containing
PBS washing buffer and incubate with rotating for 510 min.
9. Repeat three times.
10. Transfer the sections to the wells containing secondary anti-
body (see Note 74).
11. Incubate for 2 h at room temperature or overnight at 4 C.
12. Wash three times for 510 min as in step 8.
13. Mount on a glass or gelatinized slide as in 3.7.1 step 15. For
DAPI staining see step 14 in 3.7.1.
14. Analyze in the microscope.
3.8. Membrane- 1. Snap-freeze freshly harvested brains using liquid nitrogen (see
Enriched Brain Note 78). For the whole brain use metal beaker or 50 ml coni-
Fractions and WB cal tube, for a fraction of a brain you may use slotted spoon or
Analysis of NMDARs eppendorf tube. Use cold reagents and tools through the
preparation.
3.8.1. Membrane-Enriched
Fractions 2. Make a powder of the frozen brain tissue in porcelain mortar
and pestle. Small fraction of a brain can be homogenized
directly.
3. Transfer the brain powder or small piece of the brain to the
glass homogenizing tube using ten times brain volume of
homogenizing buffer. For mouse brain, the volume will be
45 ml.
4. Homogenize tissue using Teflon pestle: 1015 strokes.
5. Transfer homogenate to spinning tube and spin at 1,000 g for
15 min. Save the supernatant.
6. Transfer the supernatant to high speed ultracentrifuge tube
and spin in 60 Ti rotor at 50,000 for 30 min or an equivalent
(see Note 43). Save the pellet.
7. Resuspend the pellet in 2 ml of homogenizing buffer and
repeat the spin (see Note 79). Save the pellet.
8. Resuspend the pellet in the lysis buffer (see Note 80) or freeze
the pellet at 80 C until use.
9. Check the protein concentration using any method that is
compatible with the buffer components or dialyze the product
against PBS (see Note 81).
10. Freeze the aliquots of the preparation at 80 C until use.
3.8.2. Western Blot 1. Prepare 1025 g of brain proteins in 1015 l total volume
Analysis of NMDARs Using in 1 loading buffer.
Membrane Enriched Brain 2. Heat 10 min at 70 C. Place on ice.
Fractions
3. Load the gel using protein standard and run the gel at 180
200 V (see Note 46) until the blue line reaches the bottom of
the gel.
4. Prepare the transfer system and transfer proteins at 30 V for
12 h. You may check the progress by checking the transfer of
the protein marker without disassembling the system.
5. Rinse the membrane with PBS and block it for 1 h at room
temperature in a blocking buffer (see Note 47).
6. Incubate membrane in primary antibody in a blocking buffer;
typically, overnight at 4 C with shaking. We use anti-NR1
antibody at 1:500 dilution, anti-NR2A at 1:200 dilution, and
anti-NR2B antibody at 1:500 dilution.
7. Wash the membrane 34 times 10 min each, in PBS-T with
shaking.
198 C. Kowal and B. Diamond
4. Notes
42. Any centrifuge that provides the proper volume and speed is
fine. The volume depends on the size of the brain; mouse
whole brain is 0.40.5 g, so the volume is 45 ml; the speed
is 1,000 g. For a fraction of a brain you may use
microcentrifuge.
43. You will need an ultracentrifuge. We currently use Beckman
TLA120.2 at 75 K rpm (gmax 245000), for 15 min. For bigger
volumes, use bigger rotors. Use Beckman speed converter to
calculate the speed in your rotor.
44. Prepare higher concentration stock solutions of each compo-
nent and mix them accordingly. This is particularly important
for low solubility products such as sodium vanadate. Do not
add it as a powder; it is difficult to solubilize when the other
components are already present in the buffer.
45. We use Roches Complete Mini (Roche Applied Science,
Indianapolis, IN).
46. There are a variety of gels and buffer systems available. We use
one of the earlier systems, NuPAGE Bis-Tris gel and NuPAGE
MES SDS running buffer. The voltage and running time
depend on the gel-buffer system and so are the bands positions
of the markers. Invitrogen provides the templates for their
markers in different buffers. Watch the temperature of the gel.
Avoid overheating. You may run the gel in an ice-water bath.
47. Blocking time may vary and is quite flexible. We use LICORs
buffer diluted 1:1 with PBS, but 5 % milk or 1 % BSA in PBS
are good alternatives.
48. For MAP-peptides use few microliters of saturated Tris-base,
do not exceed 5 l/ml, as this slows down emulsification.
MAP-core dissolves easily without Tris. Map-AAAAAVWLSN
peptide is hardly soluble in water solution; you may use DMSO
or add the powder to CFA/IFA solution. Solubility in water-
based solution may depend on the purity of the product.
49. Do not use small syringes. They are leaky and you will lose too
much of the emulsion.
50. MAP-core emulsifies very easily; the other antigens require
more time. Remember not to use too much Tris.
51. Originally, we published a different schedule for immuniza-
tion, but we now routinely use the version described here.
52. Initially, we bled the animals 2 weeks after each immunization
to establish the time course of immunization and later reduced
the bleeding to pre-bleed and a final bleed at the conclusion of
the experiment.
53. Dry-coating for dsDNA is important as otherwise dsDNA may
not sufficiently bind to the plastic plate. An insufficient amount
of antigen may give too low a reading.
9 Aspects of CNS Lupus 203
79. For smaller size of tissue you may skip the second spin. The
quality of this preparation is good for most of the applications
after single spin.
80. The volume depends on the size of the brain. We resuspend
the whole brain preparation in 1 ml and dilute or concentrate
if necessary.
81. You may use the Amicon filtration system (Millipore, Billerica,
MA) for exchanging buffer or any dialysis system. Amicon filter
units come with different cut-offs and sizes and are very con-
venient. Protein loss in this system is minimal, as long as you
do not exceed three spins per unit.
82. The Odyssey secondary antibody can be used at two wave-
lengths 700 and 800 nm, red and green, respectively, that
allows simultaneous detection of two proteins. The recom-
mended concentration of the secondary antibody by manufac-
turer is effective. We use infrared labeled rabbit anti-mouse
(700) and goat anti-mouse 800 nm.
83. You may continue washing and checking if the background is
too high.
References
1. Alexander JJ, Quigg RJ (2007) Systemic lupus double-stranded DNA (dsDNA) induces
erythematosus and the brain: what mice are autoantibody production and renal immuno-
telling us. Neurochem Int 50:511 globulin deposition. J Exp Med 188:2938
2. Banks WA (2010) Mouse models of neurologi- 9. Hawkins BT, Davis TP (2005) The blood
cal disorders: a view from the bloodbrain bar- brain barrier/neurovascular unit in health and
rier. Biochim Biophys Acta 1802:881888 disease. Pharmacol Rev 57:173185
3. Denburg SD, Larocque L, Denburg JA (1999) 10. Paolinelli R, Corada M, Orsenigo F, Dejana E
Cognitive dysfunction in systemic lupus ery- (2011) The molecular basis of the blood brain
thematosus. In: Systemic Lupus Erythematosus, barrier differentiation and maintenance. Is it
Fourth Edition, Lahita RG (ed.). Academic still a mystery? Pharmacol Res 63:165171
Press, San Diego, pp 611629 11. Daneman R, Zhou L, Kebede AA, Barres BA
4. Hanly JG (2007) New insights into central (2010) Pericytes are required for bloodbrain
nervous system lupus: a clinical perspective. barrier integrity during embryogenesis. Nature
Curr Rheumatol Rep 9:116124 468:562566
5. Huerta PT, Kowal C, DeGiorgio LA, Volpe 12. Banks WA, Erickson MA (2010) The blood
BT, Diamond B (2006) Immunity and behav- brain barrier and immune function and dys-
ior: antibodies alter emotion. Proc Natl Acad function. Neurobiol Dis 37:2632
Sci USA 103:678683 13. Kowal C, Degiorgio LA, Lee JY, Edgar MA,
6. Kowal C, DeGiorgio LA, Nakaoka T, Huerta PT, Volpe BT, Diamond B (2006)
Hetherington H, Huerta PT, Diamond B, Volpe Human lupus autoantibodies against NMDA
BT (2004) Cognition and immunity; antibody receptors mediate cognitive impairment. Proc
impairs memory. Immunity 21:179188 Natl Acad Sci USA 103:1985419859
7. Gaynor B, Putterman C, Valadon P, Spatz L, 14. Lahita RG (1988) Systemic lupus erythemato-
Scharff MD, Diamond B (1997) Peptide inhi- sus: learning disability in the male offspring of
bition of glomerular deposition of an anti-DNA female patients and relationship to laterality.
antibody. Proc Natl Acad Sci USA 94: Psychoneuroendocrinology 13:385396
19551960 15. McAllister DL, Kaplan BJ, Edworthy SM,
8. Putterman C, Diamond B (1998) Martin L, Crawford SG, Ramsey-Goldman R,
Immunization with a peptide surrogate for Manzi S, Fries JF, Sibley J (1997) The influence
206 C. Kowal and B. Diamond
of systemic lupus erythematosus on fetal 24. Xaio H, Banks WA, Niehoff ML, Morley JE
development: cognitive, behavioral, and health (2001) Effect of LPS on the permeability of the
trends. J Int Neuropsychol Soc 3:370376 bloodbrain barrier to insulin. Brain Res
16. Neri F, Chimini L, Bonomi F, Filippini E, 896:3642
Motta M, Faden D, Lojacono A, Rebaioli CB, 25. DeGiorgio LA, Konstantinov KN, Lee SC,
Frassi M, Danieli E, Tincani A (2004) Hardin JA, Volpe BT, Diamond B (2001) A
Neuropsychological development of children subset of lupus anti-DNA antibodies cross-
born to patients with systemic lupus erythema- reacts with the NR2 glutamate receptor in sys-
tosus. Lupus 13:805811 temic lupus erythematosus. Nat Med 7:
17. Ross G, Sammaritano L, Nass R, Lockshin M 11891193
(2003) Effects of mothers autoimmune dis- 26. Diamond B, Bloom O, Al Abed Y, Kowal C,
ease during pregnancy on learning disabilities Huerta PT, Volpe BT (2011) Moving towards
and hand preference in their children. Arch a cure: blocking pathogenic antibodies in sys-
Pediatr Adolesc Med 157:397402 temic lupus erythematosus. J Intern Med 269:
18. Tincani A, Danieli E, Nuzzo M, Scarsil M, 3644
Motta M, Cimaz R, Lojacono A, Nacinovich 27. Owens GP, Burgoon MP, Devlin ME, Gilden
R, Taddei F, Doria A, Brucato A, Meroni P DH (1997) Extraction and purification of
(2006) Impact of in utero environment on the active IgG from SSPE and MS brain. J Virol
offspring of lupus patients. Lupus 15:801807 Methods 68:119125
19. Lee JY, Huerta PT, Zhang J, Kowal C, Bertini 28. Fernandes AM, Maurer-Morelli CV, Campos
E, Volpe BT, Diamond B (2009) Neurotoxic CB, Mello ML, Castilho RF, Langone F (2004)
autoantibodies mediate congenital cortical Fluoro-Jade, but not Fluoro-Jade B, stains
impairment of offspring in maternal lupus. Nat non-degenerating cells in brain and retina of
Med 15:9196 embryonic and neonatal rats. Brain Res
20. Arinuma Y, Yanagida T, Hirohata S (2008) 1029:2433
Association of cerebrospinal fluid anti-NR2 29. Schmued LC, Albertson C, Slikker W Jr (1997)
glutamate receptor antibodies with diffuse neu- Fluoro-Jade: a novel fluorochrome for the sen-
ropsychiatric systemic lupus erythematosus. sitive and reliable histochemical localization of
Arthritis Rheum 58:11301135 neuronal degeneration. Brain Res 751:3746
21. Fragoso-Loyo H, Cabiedes J, Orozco-Narvaez 30. Ogawa Y, Kanoh S (1984) Enhancement of
A, Davila-Maldonado L, Atisha-Fregoso Y, endotoxicity and reactivity with carbocyanine
Diamond B, Llorente L, Sanchez-Guerrero J dye by sonication of lipopolysaccharide.
(2008) Serum and cerebrospinal fluid autoanti- Microbiol Immunol 28:13131323
bodies in patients with neuropsychiatric lupus 31. Gao B, Wang Y, Tsan MF (2006) The heat sen-
erythematosus. Implications for diagnosis and sitivity of cytokine-inducing effect of lipopoly-
pathogenesis. PLoS One 3:e3347 saccharide. J Leukoc Biol 80:359366
22. Yoshio T, Onda K, Nara H, Minota S (2006) 32. Nagano I, Takao T, Nanamiya W, Takemura T,
Association of IgG anti-NR2 glutamate recep- Nishiyama M, Asaba K, Makino S, De Souza
tor antibodies in cerebrospinal fluid with neu- EB, Hashimoto K (1999) Differential effects
ropsychiatric systemic lupus erythematosus. of one and repeated endotoxin treatment on
Arthritis Rheum 54:675678 pituitaryadrenocortical hormones in the
23. Banks WA (2001) Enhanced leptin transport mouse: role of interleukin-1 and tumor necro-
across the blood-brain barrier by alpha sis factor-alpha. Neuroimmunomodulation 6:
1-adrenergic agents. Brain Res 899:209217 284292
Chapter 10
Abstract
In this chapter we present methods for the isolation and characterization of mononuclear phagocytes from
the kidneys of mice with SLE. Activation of these cells is associated with the onset of clinical disease in mice
and infiltration with these cells is associated with poor prognosis in humans. Using magnetic beads fol-
lowed by flow cytometric sorting, pure populations of cells are obtained that are functional in a variety of
assays. Sufficient numbers of cells are obtained for genomic characterization. An analysis of the function of
these cells should lead to a better understanding of the inflammatory processes that cause renal impairment
in SLE and other renal inflammatory diseases.
Key words: Lupus, Nephritis, Macrophages, Dendritic cells, Cell activation, Murine models
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_10, Springer Science+Business Media New York 2012
207
208 R. Bethunaickan et al.
1.1. Pathogenesis SLE nephritis is most often initiated by the glomerular deposition
of SLE Nephritis of immune complexes directed at a variety of antigens including
DNA, glomerular extracts, alpha actinin and laminin (911) and is
characterized by varying degrees of glomerular and tubulointersti-
tial inflammation. Although in most cases autoantibody deposition
in the kidneys is required to initiate a cascade of inflammatory
events, signals from the innate immune system and cellular immu-
nity are also crucial contributors to renal disease (Fig. 1). These
signals include complement activation (reviewed in ref. (12),
engagement of activating Fc receptors on mononuclear cells (13),
activation of resident renal cells through Toll-like receptors (14)
and recruitment of inflammatory cells to the kidney. Inflammatory
cytokines and other mediators induce endothelial cells to express
adhesion molecules, increasing the probability that they will recruit
inflammatory cells after contact with immune complexes (15). In
MRL/lpr mice T-cell mediated interstitial renal disease and vascu-
litis together with mild glomerular changes occur even in the com-
plete absence of immunoglobulins (16). Similarly, in human SLE
some pauci-immune forms of nephritis have been observed (6).
Microvascular damage and thromboses also occur in the setting of
SLE nephritis and may be more common in patients with anti-
phospholipid antibodies (17).
In both murine and human SLE, B-cells, T-cells, macrophages
and dendritic cells are recruited into the inflamed kidney (1820).
We have shown in NZB/W mice, that treatment of established
nephritis with a combination of Cytoxan and the costimulatory
antagonist CTLA4Ig does not alter renal immune complex deposi-
tion but does induce remission (21, 22). The mechanism for remis-
sion induction includes downregulation of local chemokine
expression in conjunction with a decrease in migration of lympho-
cytes and dendritic cells to the kidney, a decrease in renal endothe-
lial cell activation and a decrease in the activation state of resident
renal macrophages/DCs (21). Thus, the initiation of nephritis by
immune complexes can be delayed if downstream effector mecha-
nisms such as cell activation, migration or cell death are altered.
This concept offers new therapeutic approaches.
10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 209
Fig. 1. Mechanisms for renal damage in SLE: renal damage is initiated by deposition of
immune complexes and complement in the glomeruli and may be amplified by high serum
levels of soluble cytokines and other inflammatory mediators. This is followed by endothe-
lial activation and recruitment of lymphoid cells. Activation of intrinsic renal cells such as
mesangial cells and intrinsic renal macrophages contribute to loss of integrity of the
glomerulus with damage to podocytes and development of proteinuria. Interstital
inflammation results in tubular damage and fibrosis.
1.2. Macrophage Macrophages and dendritic cells are known to be key players in
and Dendritic Cell acute renal inflammation (19, 2326). Macrophages have a high
Subtypes degree of plasticity and have complex responses to inflammatory
stimuli with distinct activation patterns and functions depending
on the stimuli to which they are exposed (2730). Inflammatory
or classically activated (M1) macrophages are induced during cell
mediated immune responses in response to IFN (or IFN) and
TNF and produce large amounts of proinflammatory cytokines
including IL-1, IL-6, IL-12, and IL-23 and inflammatory media-
tors including iNOS and ROS. These inflammatory macrophages
derive from peripheral Gr1+/CCR2+ monocytes that have a very
short half-life and egress from the blood during acute inflammation
or infection (3133). M1 macrophages help recruit neutrophils to
sites of inflammation and induce the differentiation of TH1 and
TH17 cells. Alternatively activated (M2) macrophages that are
induced by IL-4 secrete protective cytokines and promote wound
healing. They derive from Gr1/CD62L/CX3CR1+ monocytes
that have a longer half-life and patrol the endothelium (34). These
cells extravasate very rapidly upon tissue damage and take on an
inflammatory phenotype for a short time but then appear to switch
to the alternative M2 macrophage differentiation program with a
210 R. Bethunaickan et al.
Two other small populations have been described but have not
been well characterized. One is high for CD11c and is also CD11b
positive. The other includes cells that are CD11c+/CD11b (45).
Finally a small number of plasmacytoid dendritic cells can be found
in the kidneys (46).
1.4. Murine Models Given the heterogeneity of disease course, outcome and response to
of SLE Nephritis therapy in SLE patients, even in those patients with comparable
renal histology, and the lack of sequential biopsy material from
humans, it remains essential to study informative animal models of
disease. Animal models of SLE nephritis offer the advantage that
disease pathogenesis can be studied in a timed manner and new
therapies tested at various disease stages (47). Two main categories
of mouse models exist, namely spontaneous models and induced
models. Of these, the spontaneous models have been extensively
studied and well characterized (48, 49). The classical models of
spontaneous lupus include the F1 hybrid between New Zealand
Black (NZB) and New Zealand White (NZW), MRL/lpr mice,
BXSB.Yaa mice, and NZM 2410. Not surprisingly, striking differ-
ences in responses to immunologic interventions have been observed
in the different mouse models (13, 5052) suggesting that multiple
animal models will be needed to study responses to new therapies
and to dissect pathogenetic mechanisms of SLE nephritis.
Despite the clear association of mononuclear phagocyte
infiltration in the kidneys with poor disease outcome in SLE, little
is known about the function of these cells in the kidneys in lupus
nephritis. Our preliminary studies in NZB/W mice show that renal
macrophages and dendritic cells may behave differently in mice
with lupus nephritis than in normal mice with acute renal
inflammation and that there is some variability between models. In
contrast to the findings in models of acute renal inflammation, the
inflammatory macrophage population (32) does not increase in
the nephritic kidneys of the three mouse models that we have stud-
ied in detail (NZB/W, NZW/BXSB.yaa, and NZM2410), nor is
there infiltration with neutrophils. These findings suggest that the
short-term inflammatory model of nephritis induced by immune
complexes (53) is not an adequate model for chronic SLE nephritis.
Chronic models of SLE nephritis include the following:
(a) Diffuse proliferative glomerulonephritis with anti-DNA anti-
bodies: NZB/W F1 is the oldest classical model with a pheno-
type comparable to lupus patients. Female mice develop
lymphadenopathy, splenomegaly, elevated antinuclear autoanti-
bodies including anti-dsDNA and immune complex mediated
glomerulonephritis (54). Nephritic kidneys are characterized
by extensive infiltration of activated renal macrophages
(F4/80hi) in the tubulointerstitial region and accumulation of
T and B cells along with dendritic cells (CD11chi) in perivascular
and periglomerular aggregates (21, 46). Complete remission
212 R. Bethunaickan et al.
1.5. Induction Remission induction studies were carried out in NZB/W mice
of Remission in with new onset proteinuria >300 mg/dl using a single dose of
NZB/W Mice cyclophosphamide with or without a 2-week course of murine
is Associated CTLA4Ig in combination with or without anti-CD154 (CD40L).
with Alterations Sixty to eighty percent of mice treated with the triple combination
in Renal Mononuclear entered remission, and this remission could be reinduced following
Phagocytes relapse. Triple therapy treatment induced remission for a period of
up to 20 weeks but did not alter anti-DNA antibody titers or ongo-
ing renal immune complex deposition. Flow cytometry and
ELISpot studies revealed that there was an initial decrease in the
frequency of anti-DNA producing B cells and activated T cells but
this persisted for only 36 weeks after treatment. Light microscopy
studies of kidneys from the mice which entered into remission
revealed a complete reversal of renal pathology, with complete
absence of glomerular proliferation, interstitial infiltrates and casts;
there was a gradual return of pathologic changes over time. These
data suggested that remission was associated with a decrease in the
renal inflammatory response to immune complex deposition (21).
To further investigate the reasons for the decrease in renal
inflammation, real-time PCR studies were carried out for 61
inflammatory molecules on RNA obtained from perfused kidneys
of treated and untreated mice at progressive disease stages. Only a
limited set of inflammatory markers were upregulated in the kid-
neys of prenephritic NZB/W mice. These markers include the
chemokine CXCL13 and its receptor CXCR5, CCL20, CCR6 and
CCR8. When age matched mice with and without proteinuria were
compared, both macrophage/DC markers (IL-1, TNF-, IL-6,
BAFF, IL-10 along with CCL3, CCL9 and CCR2) and endothe-
lial activation markers (VCAM-1, E-selectin, P-selectin) were
found to be upregulated. Flow cytometry experiments confirmed
both an increase in the number of renal mononuclear phagocytes
in the kidneys and an increase in CD11b expression on these cells.
When mice entered remission, a significant decrease was noted in
the expression of IL-1, IL-10, TNF-, BAFF, Ox40L, CCL5, and
CCL2. Reductions of these markers were associated with a sig-
nificant decrease in the renal macrophage-DC population and a
decrease in CD11b expression (68).
214 R. Bethunaickan et al.
Fig. 2. Flow cytometric analysis of renal mononuclear phagocytes: gating strategy to identify three major subpopulations
of CD11b positive renal cells in a young (16W) and nephritic (3640W) NZB/W mice. After exclusion of clumps and dead
cells, cells are gated on CD11b and then on CD11c and F4/80. The three populations include F4/80hi (right), F4/80lo (lower
left) and CD11chi (upper left). Note the differences in staining for MHCII and VLA4 between the F4/80hi and CD11chi popu-
lations and the appearance of the CD11chi, VLA4hi, MHCIIlo population in nephritic mice.
2. Materials
2.2. Kits QuantiChrom Urea Assay Kit: Bio assay systems (Cat #DUIR-500).
Diff-Quick stain Kit: IMEB Inc. (Cat #K7128).
BRDU FITC flow kit: BD Pharmingen (Cat #559619).
PicoPure RNA isolation kit: Arcturus (Cat #KIT0202/KIT0204).
RNeasy micro Kit: Qiagen (Cat #74004).
Superscript III: Invitrogen (Cat #18080-051).
216 R. Bethunaickan et al.
2.4. Chemical Multistix 10SG: Reagent Strips for Urinalysis (Cat #2161).
Reagents Collagenase Type I, CLS I: Worthington (Cat #4197, specific
activity 230 U/mg).
DMEM, High glucose: GIBCO (Cat #10313).
BRDU: Sigma (Cat #B5002-5G).
2 % Paraformaldehyde: Tousimis (Cat #1108A).
Prosense 680: VISEN (Part #10003).
MMPsense 680: VISEN (Part #10126).
Dako Glycergel Mounting medium (Ref #C0563).
Superscript III, Invitrogen company (Cat #18080-051).
LightCycler480 SYBR Green I master: Roche (Cat #04707516001).
Trizol Reagent: Invitrogen (Cat #15596-018).
Griess Reagent Kit: Invitrogen (Cat #G-7921).
IL4: R&D SYSTEMS INC (Cat #404-ML-010).
FN: R&D SYSTEMS INC (Cat #485-MI-100).
M-CSF: Invitrogen (Cat #PMC2044).
GM-CSF: Invitrogen (Cat #PMC2015).
L-Arginine: SIGMA-ALDRICH (Cat #A8094).
Manganese chloride: SIGMA-ALDRICH (Cat #244589).
Urea: SIGMA-ALDRICH (Cat #U5378).
a-Isonitrosopropiophenone: SIGMA-ALDRICH (Cat #I3502).
3. Methods
3.1. Kidney Damage 1. Collect overnight urine from each individual mouse in a meta-
Assessment bolic cage.
2. Test the protein level in the urine with Multistix 10SG (Reagent
Strips for Urinalysis). Grade protein levels in the urine as 1+,
2+, 3+, or 4+ equivalent to 30, 100, 300 and 2,000 or more
mg/dl respectively. Note: all normal mice have 1+ proteinuria
by dipstick.
3. Test the mice twice a week for proteinuria.
4. Alternately, when metabolic cages are not available, bladder
massage can be used to obtain urine. The mouse is held in the
upright position. Use the thumb of the other hand to gently
milk the abdomen from top to the bottom. The urine bead is
then directly placed on the strip. Make sure the protein marker
square is completely covered with urine (see Note 1).
218 R. Bethunaickan et al.
3.1.1. Albumin Estimation Mouse albumin can also be measured in 12 or 24 h urine collections
in Urine by ELISA (Kit from Bethyl Laboratories Catalog #NC9285735).
In our experience, values obtained by ELISA fall within the range
indicated by the Multistix.
3.1.2. Blood Urea Nitrogen Blood urea nitrogen (BUN) levels in the blood are measured with
the Quantichrom Urea assay Kit (see Note 2).
1. Transfer 5 l of serum (fresh or stored) to a 96-well flat bot-
tom plate.
2. Prepare serial dilutions of the standards ranging from 100 to
0 mg/dl from the kits master stock. Transfer 5 l of the each
of the standards to the 96-well plate. Use 5 l of deionized
water as blank.
3. Mix equal volumes of Reagent A and Reagent B prior to assay.
From this mix dispense 200 l to the samples, standards and
the blank wells on the plate.
4. Gently tap the plate on the sides to mix the solutions and incu-
bate the plate at RT for exactly 30 min.
5. Read the plate at 470550 nm optical density.
6. The urea concentration is calculated by using the formula
shown below.
OD sample OD blank
N [STD]
OD standard OD blank
OD sample, OD blank, and OD standard are the OD values of
the sample, water and the standard respectively, N is the dilu-
tion factor and [STD] = 50 Urea std concentrations (mg/dl).
7. To obtain the BUN values the following conversion is made.
Urea (mg/dl) = BUN (mg/dl) 2.14.
3.2. Kidney Harvest 1. Anesthetize the mouse and perfuse with 60 ml of cold PBS
over 35 min through the left ventricle after snipping the right
atrium and observe for pale white color change in liver and
kidney. If needed, repeat perfusion with another 60 ml of cold
PBS (see Note 3a).
2. Carefully remove and cut the kidneys into 12 mm3 pieces,
excluding any adjoining renal fat.
3. Incubate the slices in DMEM containing 2 mg/ml Collagenase
Type I (Worthington) for 30 min at 37 C (use 10 ml per two
kidneys).
4. Gently disrupt the tissue by pipetting up and down sequen-
tially through 25 ml, 10 ml, 5 ml pipettes to obtain a fine cell
suspension.
5. Filter the cell suspension through a BD cell strainer (40 m)
into a conical tube.
10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 219
3.3. Flow Cytometric 1. Reuspend the kidney cells in Fc block (Table 1) for 15 min,
Analysis and Cell adjusting the total suspension volume to approximately 100 l
Sorting per stain. Use a maximum of 1 ml (ten stains) for one whole
kidney.
3.3.1. Flow Cytometry
2. Distribute the cells accordingly into a 96-well V bottom plate
(100 l per well).
3. Add biotinylated antibodies (1/200 dilution), mix gently and
incubate for 30 min on ice. Keep the plates in the dark through-
out the incubation.
4. Meanwhile, mix the next cocktail of antibodies of your choice
together with the streptavidin fluorochrome in a single tube
(1/200 dilution of each antibody). Include a set of stains using
isotype controls.
5. To remove the unbound or excess stain add 100 l of ice cold
PBS to the wells after 30 min of incubation. Centrifuge the
plate for 5 min at 1,200 rpm (300 g) at 4 C.
6. Discard the liquid by inverting the plate once without disturb-
ing the cell pellet.
7. Add the cocktail of antibodies from step 4 to the pellet (100 l/
well) and gently resuspend using a multichannel pipette.
Incubate the plate for another 30 min on ice.
8. Repeat steps 5 and 6.
9. Resuspend the cells in 200 l of 2 % paraformaldehyde, trans-
fer and store in FACS tubes in the dark until the time of acqui-
sition on the flow cytometer (within 24 h).
220 R. Bethunaickan et al.
Table 1
Antibodies used for flow cytometric characterization of kidney macrophages
and DCs
Table 1
(continued)
3.3.2. Cell Sorting 1. Suspend the cells from both kidneys from one mouse in 2 ml
of FACS buffer with Fc block and incubate on ice for 20 min.
2. Add 7 l of CD11c-biotin antibody to the tube and incubate
for 30 min on ice.
3. Centrifuge the cells at 1,200 rpm (300 g) for 5 min and
decant the supernatant.
4. Resuspend the cell pellet in 2 ml of FACS buffer containing
anti-CD11b APC, F4/80 FITC, and Streptavidin PerCP.
The cocktail should also include a combination of antibodies
to facilitate the exclusion of unwanted cells, such as PE anti-
CD3, CD5, B220, CD138 (optional) and CD49b (57 l
each); incubate for another 30 min.
5. Wash the cells with 2 ml of ice cold PBS and centrifuge at
1,200 rpm (300 g) for 5 min.
6. Resuspend the cell pellet in 500 l of FACS buffer and filter
the suspension using a strainer cap FACS tube. Later, adjust
the volume for the cells based on the number of events acquired
per second on the cell sorter (FACSAria IIu).
7. Just before sorting the cells, add 2 l of DAPI (1 g/ml) to
exclude the dead cells.
8. Pass the cells through the sorter with a flow rate optimized to
5,0007,000 events per second and maintain the efficiency of
the sort at between 85 and 95 %.
9. Use FSC and SSC to set a monocyte/lymphocyte gate and
exclude the unwanted cells in the dump gate and the dead cells
in the DAPI+ gate. Gate on the CD11b+ and/or CD11c+
cells from the live cell gate (see Note 3c).
10. From the CD11b cell gate, sort the CD11chi, F4/80hi and
F4/80lo cells (Fig. 2) into FACS buffer and process them based
on further study requirements (RNA isolation, morphological
and functional characterization, western blot analysis).
222 R. Bethunaickan et al.
3.4. Enrichment 1. Resuspend the pellet obtained from Subheading 3.2, step 11
of Renal Macrophages in PEB containing Fc Block (1:200) for 30 min on ice. Use
and Dendritic Cells 1 ml volume for two kidneys.
Using Magnetic Beads 2. Add required quantity of CD11c coated MACS beads from
Milltenyi Biotec (as per manufacturers recommendation) to
the suspension and incubate for 15 min followed by CD11b
coated MACS beads for 15 min. (For a cell count of ten mil-
lion, add 10 l of each type of bead.)
3. After the incubation period, set aside a few microliters of the
suspension for use as unstained control during sorting.
4. Add 1015 ml PEB to the sample, and centrifuge for 10 min
at 1,500 rpm (570 g).
5. Discard the supernatant by aspiration and resuspend pellet in
5001,000 l of antibody cocktail for 15 min on ice. Filter the
suspension through a 30 m filter and load in Automacs. Set
aside a few microliters for quantifying the percentage of desired
cells in the total suspension.
6. Separate the labeled cells using either POSSEL_S or POSSEL_D
program in Automacs. (POSSEL-S is a sensitive mode in posi-
tive selection, used when we need to have more recovery of the
sample. To achieve high purity, a double positive selection
mode, POSSEL-D is used.)
7. Add DAPI 12 l (1 g/l), to the cell suspension before
sorting to exclude the dead cells.
8. Purify populations from the positively selected fraction using
the FACS-ARIA cell sorter as described in Subheading 3.3.2,
step 10.
9. Cells gated from live cells can be sorted at a flow rate of 2.5
3.0 with a sheath pressure of 34.0. Evaluate purity of the sorted
populations by a post sort analysis.
10. Collect the sorted cells in 1 ml DMEM containing 10 % FBS
for better viability.
11. For calibration of different fluorochromes, use spleen cells as a
reference. This alleviates the need to stain kidney cells for pre-
paring single color controls. Appropriate color controls are
CD4-FITC, CD4-PE, and B220-APC.
12. Spin the post sort cells at 1,500 rpm (570 g) for 10 min and
discard the supernatant.
13. Add 1 ml of fresh medium and re-suspend the cells. Perform a
cell count and plate for cell culture in desired media at required
cell density.
14. Wash the pellet several times in PBS and then use the cells as
starting material for biochemical analyses or for purification of
RNA.
10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 223
3.5. Morphological 1. Wash sorted kidney cells (100,000 cells) in cold PBS once and
Studies dilute in 1 ml of cold PBS. Keep all samples on ice.
3.5.1. Cytospin 2. Use 100 l of the cells for each cytospin funnel. Centrifuge for
35 min at a maximum speed of 600 rpm (see Note 4).
3. Fix and process the slides using the desired stain such as
WrightGiemsa stain or Diff-Quick stain.
3.5.2. Scanning Electron 1. Quick fix the pellet of sorted cells in 1 % osmium tetroxide,
Microscopy SAMPLE PREP 0.1 M sodium cacodylate, 0.2 M sucrose, 5 mM MgCl2 pH 4
Osmium Quick Fix (SEM buffer) for 10 s, followed by two changes of 2.5 % glu-
taraldehyde in 1 SEM buffer. Secondary fix cells with 1 %
osmium tetroxide, in 1 SEM buffer for 30 min.
2. Dehydrate through a graded series of ethanol.
3. Critical-point-dry using liquid carbon dioxide in a Tousimis
Samdri 795 Critical Point Drier (Rockville MD).
4. Sputter-coat with gold-palladium in a Denton Vacuum Desk-2
Sputter Coater (Cherry Hill, NJ).
5. Examine in a JEOL JSM6400 Scanning Electron Microscope
(Peabody, MA), using an accelerating voltage of 10 kV.
3.6. Cellular Turn Over 1. Freshly prepare a solution of 1 mg BrdU/100 l of PBS. Filter-
Studies sterilize using a 0.45-m filter. Inject 1 mg per mouse i.p. as a
loading dose.
2. Prepare a filter-sterilized solution of 0.8 mg BrdU/ml in drink-
ing water, using 0.45-m filters, and provide it ad libitum to
mice in dark glass water bottles or wrapped with aluminum foil
until time of analysis (typically 760 days).
224 R. Bethunaickan et al.
3.7. Functional Studies In vivo assessment of Cathepsins (B, L, and S) and matrix metal-
of Renal loproteinases (MMPs; MMP2, MMP3, MMP9, and MMP13).
Macrophages/
1. Dilute 5 nmol of the probe in 100 l of sterile PBS. Filter-
Dendritic Cells sterilize using a 0.45-m filter. Perform i.p. injection into the
3.7.1. Cathepsin and mouse 24 h before sacrifice.
Metalloproteinase Activity 2. Anesthetize the mice, perfuse, and collect the kidneys. Prepare
single cell suspensions of kidney cells as described in
Subheading 3.2.
3. Stain the cells for flow cytometry with the cocktail of antibod-
ies against CD11b, CD11c and F4/80. Acquire the fixed cells
on the LSRII with the following instructions:
(a) Use the 695/40 filter and 685/LP dichroic filter con-
figuration to detect Prosense-680 and MMPsense-680.
(b) Analyze the cells positive for Prosense-680 and
MMPsense-680 in the Alexafluor 700 channel. Acquire at
least 100,000200,000 cells per sample (see Note 5).
3.7.2. Culturing 1. Flush mice bone marrow in DMEM followed by RBC lysis
of BM-Derived using 5 ml of ice-cold 0.17 M NH4Cl for 5 min over ice.
Macrophages (Mf) 2. Culture cells in M-CSF (10 ng/ml) or GM-CSF (10 ng/ml)
(negative control).
3. Replace media after 6 days. Harvest and reculture cells in fresh
media in triplicates with M-CSF at a cell density of 2.55.0 105
per well.
4. Treat cells with different cytokines as per requirement.
5. Add IFN- at 10 ng/ml for 48 h.
6. Next day wash one set of IFN- treated cells and add LPS at
10 ng/ml for 30 min. This gives classically activated (CA-) M.
7. Add IL-4 at the required concentration and incubate for 24 h.
This gives alternatively activated (AA-) M.
10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 225
3.7.3. Assay for i-NOS 1. Culture sorted cells with M-CSF (10 ng/ml) with IFN-
Activity gamma (10 ng/ml) LPS (100 ng/ml) for 48 h as above.
2. Carefully remove the media from the wells and spin at
5,000 rpm (2300 g) for 5 min to remove any cell/debris.
3. In a 96-well plate add 130 l of deionized water followed by
130 l of cell supernatant.
4. In a set of wells set up standard nitrite solutions with a water
blank (alternatively, use media for diluting nitrite solutions) to
obtain dilutions between 5 and 100 M concentration.
5. Add 20 l of Griess reagent to the wells and incubate at room
temperature in the dark for 30 min.
6. Read the absorbance at 540 nm in a microplate reader. Use
standards for calculating the amount of nitrite.
7. Wash the cells in the wells several times (at least three times)
with PBS. Aspirate out any remaining solution.
8. Add 100150 l of 0.1 % triton-X with protease inhibitors and
incubate at room temperature for 30 min.
9. Collect the cell lysate and proceed to protein quantification
and arginase assay.
3.7.4. Arginase Assay 1. Take 2550 l of the cell lysate and add equal volume of
10 mM MnCl2 in Tris buffer (50 Mm, pH 7.5).
2. Use a reference set of lysates and add 10 mM EDTA in Tris
buffer as above.
3. Incubate the solutions for 10 min at 55 C to activate the argi-
nase enzyme.
4. Add 2512.5 l of 0.5 M L-Arginine (pH 9.7) solutions to
each tube. Prepare a blank with only water and L-arginine.
5. Incubate for 60 min at 37 C.
6. Remove 50 l of the mix and add 200 l of acid mix
(H2SO4:H3PO4: H2O, 1:3:7).
7. Add 25 l of 9 % -isonitrosopropiophenone (ISPF) prepared
in absolute alcohol.
8. Prepare urea solution in the range of 525 g/ml.
9. Take 50 l of urea solution and add the acid mix and ISPF
solution.
226 R. Bethunaickan et al.
10. Incubate the tubes at 95 C for 45 min. Then cool for 10 min.
to RT.
11. Take 50 l of the solution and assay at 540 nm in a microplate
reader.
12. There should be no significant absorbance in samples with
EDTA incubation.
13. Subtract the EDTA values which account for any non-specific
reaction.
14. Use the urea standards to calculate the amount of urea
released.
15. Express activity in terms of moles of urea released per hour or
per minute.
3.8. Gene Expression 1. Snap freeze one perfused kidney from the mouse immediately
Studies on dry ice, add 3 ml of Trizol and homogenize at once (take
care that the tissue is not thawed during this procedure).
3.8.1. RNA Isolation from
the Kidney by TRIZOL 2. Transfer the homogenate to a 5 ml polypropylene round-bot-
Method tom tube. In a fume hood, add 0.6 ml of chloroform (0.2/ml
per ml of Trizol), mix well by shaking for 15 s and incubate at
room temperature for 5 min.
3. Centrifuge the tube containing the homogenate at a high
speed of 12,000 rpm (16,000 g) for 15 min at 4 C.
4. Transfer the aqueous phase to a new RNase free 5 ml polypro-
pylene round-bottom tube. Add 1,500 l of isopropyl alcohol
(500 l for 1 ml of Trizol reagent).
5. Incubate the sample at RT for 10 min.
6. Centrifuge at 12,000 rpm (16,000 g) for 10 min.
7. Remove supernatant and wash the pellet once with 75 % etha-
nol in RNase free water.
8. Vortex and mix the sample and centrifuge at 7,500 rpm
(6,300 g) for 5 min at 4 C.
9. Remove supernatant from the tube without disturbing the
pellet and air-dry the pellet completely.
10. Resuspend the pellet with 1 ml of 70 % ethanol and transfer it
to an 1.5 ml Eppendorf.
11. Centrifuge at 13,000 rpm (19,000 g) for 10 min at 4 C.
12. Air-dry the pellet in the tube and dissolve it with 100 l of
RNase/DNase free water. Incubate at 55 C for 10 min.
13. Analyze the quality of the isolated RNA by Bioanalyser and
store it at 80 C.
14. To isolate RNA from sorted kidney cells, use RNA isolation
kits such as the RNeasy micro kit from Qiagen or PicoPure
RNA isolation kit which yield high quality and quantity of
RNA.
10 Analysis of Renal Mononuclear Phagocytes in Murine Models of SLE 227
3.8.2. cDNA Synthesis 1. Use 5 g of RNA from the whole kidney for cDNA synthesis.
If using sorted cells from the kidney, use 25100 ng as starting
material (see Note 7).
2. Use Superscript III, Invitrogen (Cat #18080-051) for cDNA
synthesis and follow the manufacturers instructions.
3.8.3. Real-Time PCR 1. Identify the optimal concentration of cDNA and perform the
reaction on a 384 well plate run on the LightCycler 480
(Roche).
2. Prepare the reaction mix for a total of 8 l volume per well.
The details of the mix are as follows (see Note 8):
2 Mastermix SYBR Green 4 l per well
Primer (both forward 2 l per well
and reverse) (1 l forward + 1 l reverse)
cDNA 2 l per well
3. Add the above reagents to the wells, seal the plate and centri-
fuge at 1,200 rpm (300 g) for 5 min.
4. Load the plate onto the machine and run using a standard
protocol for SYBR Green. Briefly, the PCR cycle has the fol-
lowing steps:
Denaturation 1 cycle95 C for 5 min
Amplification 45 cycles95 C for 10 s
55 C for 10 s
72 C for 10 s
Melt curve 1 cycle95 C for 20 s
60 C for 30 s
95 C for continuous mode
Cool down 1 cycle40 C for 10 s
3.9. Immunohistologic 1. Cut slides from OCT samples at 80 C and freeze until use.
Studies 2. Thaw slides in a closed chamber at RT until the slides are
thawed (approximately 20 min) (see Note 9).
3. Immerse the slides in cold acetone (pre chilled at 20 C) for
5 min in a Coplin jar. Keep the slides in the dark by covering
the Coplin jar with aluminum foil.
4. Transfer the slides to another Coplin jar and rinse with cold
PBS for 5 min. Leave covered.
5. Repeat step 3 to ensure complete removal of acetone.
6. Prepare Blocking solution (3 % BSA in PBS with Fc
block50 l/10 ml).
7. Apply blocking solution (about 200 l/sample) over the slide
and incubate for 30 min inside the humidified chamber.
228 R. Bethunaickan et al.
3.10. Protein 1. Wash sorted cells 34 times in PBS to remove the BSA (or
Quantification media if cells collected in media) and pellet by centrifuging at
and Analysis 2,500 rpm (600 g) for 5 min.
2. Resuspend the cell pellet in lysis buffer on ice for an hour.
3. After lysis, centrifuge at 14,000 rpm (16,000 g) for 20 min.
4. Discard the pellet and carefully remove the supernatant for fur-
ther analysis.
5. Quantify protein in lysates using Bradford dye binding (1975)
assay using immunoglobulin or bovine serum albumin as the
standard reference.
6. Perform SDS-PAGE using a 12 % resolving and 5 % stacking
gel using the protocol of Laemilli (1976).
7. Carry out immunoblot of proteins after transfer to nitrocellu-
lose membranes using desired antibodies after blocking the
membrane with 5 % skimmed milk.
4. Notes
References
29. Mosser DM, Edwards JP (2008) Exploring the and in experimental glomerulonephritis. J Am
full spectrum of macrophage activation. Nat Soc Nephrol 15:613621
Rev Immunol 8:958969 43. Soos TJ, Sims TN, Barisoni L, Lin K, Littman
30. Hume DA (2008) Differentiation and hetero- DR, Dustin ML, Nelson PJ (2006) CX3CR1+
geneity in the mononuclear phagocyte system. interstitial dendritic cells form a contiguous
Mucosal Immunol 1:432441 network throughout the entire kidney. Kidney
31. Tacke F, Randolph GJ (2006) Migratory fate Int 70:591596
and differentiation of blood monocyte subsets. 44. Segerer S, Heller F, Lindenmeyer MT, Schmid H,
Immunobiology 211:609618 Cohen CD, Draganovici D, Mandelbaum J,
32. Li L, Huang L, Sung SS, Vergis AL, Rosin DL, Nelson PJ, Grone HJ, Grone EF, Figel AM,
Rose CE Jr, Lobo PI, Okusa MD (2008) The Nossner E, Schlondorff D (2008) Compartment
chemokine receptors CCR2 and CX3CR1 specific expression of dendritic cell markers in
mediate monocyte/macrophage trafficking in human glomerulonephritis. Kidney Int 74:
kidney ischemia-reperfusion injury. Kidney Int 3746
74:15261537 45. Kurts C (2006) Dendritic cells: not just another
33. Swaminathan S, Griffin MD (2008) First cell type in the kidney, but a complex immune
responders: understanding monocyte-lineage sentinel network. Kidney Int 70:412414
traffic in the acutely injured kidney. Kidney Int 46. Bethunaickan R, Berthier CC, Ramanujam M,
74:15091511 Sahu R, Zhang W, Sun Y, Bottinger EP,
34. Auffray C, Fogg D, Garfa M, Elain G, Join- Ivashkiv L, Kretzler M, Davidson A (2011)
Lambert O, Kayal S, Sarnacki S, Cumano A, A unique hybrid renal mononuclear phago-
Lauvau G, Geissmann F (2007) Monitoring of cyte activation phenotype in murine systemic
blood vessels and tissues by a population of lupus erythematosus nephritis. J Immunol:
monocytes with patrolling behavior. Science 186:49945003
317:666670 47. Ramanujam M, Davidson A (2008) Targeting
35. Geissmann F, Auffray C, Palframan R, Wirrig of the immune system in systemic lupus erythe-
C, Ciocca A, Campisi L, Narni-Mancinelli E, matosus. Expert Rev Mol Med 10:e2
Lauvau G (2008) Blood monocytes: distinct 48. Singh RR, Saxena V, Zang S, Li L, Finkelman
subsets, how they relate to dendritic cells, and FD, Witte DP, Jacob CO (2003) Differential
their possible roles in the regulation of T-cell contribution of IL-4 and STAT6 vs STAT4 to
responses. Immunol Cell Biol 86:398408 the development of lupus nephritis. J Immunol
36. Skold M, Behar SM (2008) Tuberculosis trig- 170:48184825
gers a tissue-dependent program of differentia- 49. Santiago ML, Fossati L, Jacquet C, Muller W,
tion and acquisition of effector functions by Izui S, Reininger L (1997) Interleukin-4 pro-
circulating monocytes. J Immunol 181: tects against a genetically linked lupus-like auto-
63496360 immune syndrome. J Exp Med 185:6570
37. Martinez FO, Sica A, Mantovani A, Locati M 50. Matsumoto K, Watanabe N, Akikusa B,
(2008) Macrophage activation and polariza- Kurasawa K, Matsumura R, Saito Y, Iwamoto
tion. Front Biosci 13:453461 I, Saito T (2003) Fc receptor-independent
38. Wu L, Liu YJ (2007) Development of den- development of autoimmune glomerulone-
dritic-cell lineages. Immunity 26:741750 phritis in lupus-prone MRL/lpr mice. Arthritis
39. Liu K, Waskow C, Liu X, Yao K, Hoh J, Rheum 48:486494
Nussenzweig M (2007) Origin of dendritic 51. Ehlers M, Fukuyama H, McGaha TL, Aderem
cells in peripheral lymphoid organs of mice. A, Ravetch JV (2006) TLR9/MyD88 signal-
Nat Immunol 8:578583 ing is required for class switching to pathogenic
40. Auffray C, Emre Y, Geissmann F (2008) IgG2a and 2b autoantibodies in SLE. J Exp
Homeostasis of dendritic cell pool in lymphoid Med 203:553561
organs. Nat Immunol 9:584586 52. Wu X, Peng SL (2006) Toll-like receptor 9 sig-
41. Kamath AT, Henri S, Battye F, Tough DF, naling protects against murine lupus. Arthritis
Shortman K (2002) Developmental kinetics Rheum 54:336342
and lifespan of dendritic cells in mouse lym- 53. Fu Y, Du Y, Mohan C (2007) Experimental
phoid organs. Blood 100:17341741 anti-GBM disease as a tool for studying sponta-
42. Kruger T, Benke D, Eitner F, Lang A, Wirtz neous lupus nephritis. Clin Immunol 124:
M, Hamilton-Williams EE, Engel D, Giese B, 109118
Muller-Newen G, Floege J, Kurts C (2004) 54. Perry D, Sang A, Yin Y, Zheng YY, Morel L
Identification and functional characterization (2011) Murine models of systemic lupus erythe-
of dendritic cells in the healthy murine kidney matosus. J Biomed Biotechnol 2011:271694
232 R. Bethunaickan et al.
Abstract
T-helper cell 17 (Th17) cells play an important role in the pathogenesis of many autoimmune disorders
including Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE). In this chapter we
describe a murine model where deregulated production of IL-17 and IL-21 can lead to either lupus-like
disease or RA-like symptoms depending on the genetic background. We delineate the key techniques that
can be used to dissect the mechanisms responsible for the pathogenesis of these diseases at both a cellular
and molecular level including in vitro Th17 cell differentiation, chromatin immunoprecipitation assays,
and retroviral transduction experiments. We also describe the methodologies that can be utilized to moni-
tor the classic clinical findings of RA and SLE in murine models. Given the broad involvement of deregu-
lated production of IL-17 and IL-21 in autoimmunity, many of these techniques could also be valuable for
the investigation of these pathways in murine models of other autoimmune diseases.
Key words: Arthritis, Systemic lupus erythematosus, Autoimmunity, Animal models, Interleukin-17,
Interleukin-21
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_11, Springer Science+Business Media New York 2012
233
234 P.S. Biswas et al.
2. Materials
2.7. Clinical 1. Positive control serum: Serum from diseased MRL/lpr mice.
Assessment of Lupus: 2. Negative control serum: Serum from healthy wild-type mice.
ANA Staining
3. MBL-BION ANA (HEp-2) Antigen substrate slides (MBL
Bion).
4. FITC conjugated anti-mouse IgG (Jackson Immunoresearch).
5. Phosphate Buffer Saline (Mediatech Inc.).
6. Vectashield Mounting medium for Fluorescence (Vector
Lab).
3. Methods
3.1. T-Helper Cell 17 1. Sacrifice 810-week-old mice and collect spleen and LNs in
Differentiation of ice-cold media.
Mouse Nave CD4+ T 2. Prepare single cell suspension of spleen and LNs by mashing
Cells (See Notes 14) between two frosted microscopic slides followed by passing the
3.1.1. Isolation and cell suspension through 70-m nylon mesh to remove any cell
Purification of Total CD4+ clumps and debris.
T Cells from Mouse Spleen 3. Centrifuge cell suspension for 10 min at 300 g at 4 C.
and Peripheral Lymph 4. Discard the supernatant and lyse the RBCs using RBC lysis
Nodes buffer. Add ice-cold PBS (ten times the volume of RBC lysis
buffer) and centrifuge for 10 min at 300 g at 4 C.
5. Wash the cell pellets twice with ice-cold PBS.
6. Determine viable cell count by Trypan blue exclusion.
7. Proceed to negative selection of total CD4+ T cells using CD4+
T cell Isolation kit following the manufacturers protocol.
238 P.S. Biswas et al.
3.1.2. FACS Sorting 1. Wash MACS-purified total CD4+ T cells with ice-cold MACS
of Nave CD4+ T Cells Buffer.
from Total CD4+ T Cells
2. Determine viable cell count by Trypan blue exclusion.
3. Resuspend the cell pellet in complete Clicks medium at a cell
concentration of ten million cells/ml.
4. Stain the cells with fluorochrome-conjugated antibodies (anti-
mouse CD4 PeCy7, anti-mouse CD25 PE, anti-mouse CD44
FITC, and anti-mouse CD62L APC) for 30 min on ice.
5. Wash the cells twice with ice-cold MACS buffer and finally
resuspend in complete Clicks media at a cell concentration of
1020 million cells/ml.
6. Proceed to cell sorting.
3.1.3. TH17 Differentiation 1. Coat 24-well tissue culture plates with anti-CD3 antibody at a
of Nave CD4+ T Cells concentration of 1 g/ml in PBS at 37 C for 2 h.
2. In the meantime wash the FACS-sorted nave (CD4+CD25-
CD44intCD62Lhi) CD4+ T cells with ice-cold PBS.
3. Resuspend the cell pellet in complete Clicks media.
4. For Th0, Th1, Th2, and Th17 differentiating conditions, use
the following antibody and cytokine cocktails in complete
Clicks medium:
Th0: Anti-CD28 (2 g/ml).
Th1: Anti-CD28 (2 g/ml), Anti-IL-4 (10 g/ml), recombi-
nant mouse IL-12 (10 ng/ml).
Th2: Anti-CD28 (2 g/ml), Anti-IFN- (10 g/ml), recom-
binant mouse IL-4 (10 ng/ml).
Th17: Anti-CD28 (2 g/ml), Anti-IFN- (10 g/ml),
Anti-IL-4 (10 g/ml), recombinant mouse IL-6 (20 ng/
ml), and recombinant human TGF- (5 ng/ml).
5. Wash the anti-CD3-coated plates with PBS to remove any
unbound antibody.
6. Plate the cells with respective differentiating medium at a con-
centration of 11.5 million cells/ml and culture for 35 days.
3.1.4. Detection of IL-17 Following stimulation of nave CD4+ T cells in Th17 skewing con-
by ELISA and Intracellular ditions, production of IL-17 can be quantified by two methods:
FACS Analyses
1. Supernatant ELISA: After 35 days of stimulation in Th17 dif-
ferentiating conditions, harvest the supernatants and subject
them to an IL-17 and IL-21 ELISA using a commercially avail-
able kit from eBioscience and R&D Systems, respectively, as
per the manufacturers protocol.
11 A Murine Autoimmune Model of Rheumatoid Arthritis 239
3.2. Chromatin 1. Stimulate mouse CD4+ T cells (10 ~ 20 106 cells) in the con-
Immunoprecipitation dition of interest.
Assays (See Note 5) 2. Cross-link chromatin by incubating the cells with 1 % formal-
dehyde in PBS for 10 min at 37 C. Stop the cross-linking by
adding glycine to a final concentration of 0.1 M. Rinse the cells
twice with ice-cold PBS.
3. Resuspend the cell pellets in 0.4 ml of cell lysis buffer (5 mM
Pipes [pH 8.0], 85 mM KCl, and 0.5 % NP40) containing pro-
tease inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 g/ml
aprotinin, 10 g/ml leupeptin, and 1 g/ml pepstatin A) and
incubate on ice for 10 min. Centrifuge the samples for 5 min
at 2,700g at 4 C to pellet the nuclei.
4. Resuspend the nuclear pellets in 0.4 ml of nuclear lysis buffer
(50 mM TrisHCl [pH 8.0], 1 % SDS, and 10 mM EDTA)
supplemented with the above-stated protease inhibitors. Keep
the samples on ice for 10 min.
5. Sonicate the nuclear lysates for four to five 30 s pulses at the
maximum setting of 5, with 30 s of cooling on ice between
the pulses, to shear DNA into small fragments. Centrifuge the
nuclear lysate for 10 min at 18,000g at 4 C.
6. Collect the supernatant fraction (containing cross-linked chro-
matin) and dilute in Chromatin dilution buffer (1 % Triton
X-100, 0.01 % SDS, 2 mM EDTA, 150 mM NaCl, 20 mM
240 P.S. Biswas et al.
3.3.2. Preparation of DNA 1. Thaw the DNA and spin at 20,000g for 2 min.
for Transfection
2. Set up two sterile polypropylene tubes for each DNA to be
precipitated and label the tubes Tube # 1 and Tube # 2.
3. To Tube # 1 add 0.5 ml 1 HBSS and 10 l of phosphate
solution.
4. To Tube # 2, add 0.43 ml of ultrapure water minus the volume
of DNA. Total DNA should equal 20 g.
5. Gently mix the DNAs in the water.
6. Add 60 l of calcium solution and mix gently.
3.3.4. Transfection of 293T 1. After 20 min, mix the precipitate well by repeated pipetting
Cells and vortexing.
2. Add 1 ml of DNA suspension to 100 mm plate containing
293T cells. The suspension must be added slowly and drop-
wise while gently swirling the media in the plate.
3. Put the plates back in the incubator for 24 h.
4. Twenty-four hours post transfection remove the media con-
taining precipitate and replace it with 6 ml pre-warmed com-
plete media.
3.3.5. Isolation and 1. Forty-eight hours post transfection purify total CD4+ T cells
Activation of CD4+ T Cells from 8- to 10-week-old mouse spleen and LNs using CD4+ T
cells isolation kit as per manufacturers protocol.
2. Activate the CD4+ T cells in the presence of plate-bound anti-
CD3 (1 g/ml) and soluble anti-CD28 (2 g/ml) at a cell
concentration of 1.5 million cells/ml in a 24-well plate.
3.3.6. Retroviral Infection 1. Seventy-two hours post transfection, check for reporter gene
of CD4+ T Cells expression under fluorescent microscope (YFP+, GFP+, or
RFP+). Collect the virus-containing media from 293T cells and
add Polybrene (10 g/ml) to it before passing it though a
0.22 m syringe filter.
2. Add 6 ml of pre-warmed complete media and put the plates
containing 293T cells back in the incubator for 2 h.
3. Two hours later very carefully and slowly replace complete
media on CD4+ T cells with virus-containing media from 293T
cells.
242 P.S. Biswas et al.
3.3.7. Resting 1. Twenty four hours following the last infection (i.e., 120 h post
of Retrovirally Infected transfection of 293T cells), collect the activated CD4+ T cells
CD4+ T Cells and wash three times with ice-cold PBS.
2. Rest the CD4+ T cells in complete Clicks media containing
recombinant murine IL-2 (20 ng/ml) in 12-well plates for
3 days.
3.3.8. Restimulation 1. Following resting in IL-2, collect the cells and wash several
of Retrovirally Infected times with ice-cold PBS.
CD4+ T Cells 2. Proceed to sorting the retrovirally infected CD4+ T cells on the
basis of reporter gene expression (YFP+, GFP+, or RFP+ cells).
3. Restimulate sorted CD4+ T cells in complete Clicks media in
the presence of plate-bound anti-CD3 (1 g/ml) and soluble
anti-CD28 (2 g/ml) for 48 h.
4. Collect the supernatants for cytokine ELISAs.
5. Alternatively, 96 h post transfection cells can be restimulated
with PMA (50 ng/ml) and ionomycin (1 M) for 46 h in the
presence of Golgi Stop (1 M) and subject to intracellular
staining for the production of cytokines.
3.4. Flow Cytometric 1. Sacrifice mice and collect spleen and peripheral LNs in ice-cold
Analyses of T and B media.
Cell Compartments 2. Prepare single cell suspension of spleen and LNs by mashing
in Spleen and Lymph between two frosted microscopic slides followed by passing the
Nodes (See Notes cell suspension through 70-m nylon mesh to remove any cell
1113) clumps and debris.
3. Centrifuge cell suspension for 10 min at 300 g at 4 C.
4. Discard the supernatant and lyse the RBCs using RBC lysis
buffer. Add ice-cold PBS (ten times the volume of RBC
lysis buffer) and centrifuge for 10 min at 300 g at 4 C.
5. Wash the cell pellets twice with ice-cold PBS.
6. Determine viable cell count by Trypan blue exclusion.
7. For surface staining of T and B cells take one million cells/well
in a 96-well plate.
8. Spin the plate at 300 g for 3 min and discard the super-
natant.
11 A Murine Autoimmune Model of Rheumatoid Arthritis 243
Fig. 1. Representative macroscopic images of the arthritis that develops in Def6-deficient DO11.10 mice (representative
clinical scores of 4 are shown).
3.8. Serologic 1. Using separate pipette tips, apply one drop (2030 l) of each
Assessment of Lupus: mouse serum dilution and positive and negative controls to
ANA Staining (See individual wells of the slide. Make sure not to touch the anti-
Notes 1721) gen surface with the pipette tip and prevent mixing of samples
between wells.
2. Incubate in a moist chamber at room temperature (2025 C)
for 30 min.
3. Remove slides from moist chamber and rinse gently with PBS
using a squeeze wash bottle.
4. Place the slides in coplin jars and wash the slides three times
with PBS.
5. Remove slides from the wash one at a time, shake off excess
PBS, dry around outside edges if necessary, and return each
slide to the moist chamber.
6. Apply 1020 l of FITC conjugated anti-mouse IgG (Jackson
Immunoresearch Lab) (1:1,000 dilution in PBS) with DAPI
counterstain (1:5,000 dilution) (Invitrogen) to each well of
each slide.
7. Incubate in a moist chamber at room temperature (2025 C)
for 30 min in the dark.
8. Remove slides from moist chamber and rinse gently with PBS
using a squeeze wash bottle.
246 P.S. Biswas et al.
9. Place the slides in coplin jars and wash the slides five times with
PBS.
10. Shake off the excess PBS and immediately mount the slides with
Vectashield Mounting medium for fluorescence (Vector Labs).
11. Examine stained slides as soon as possible using a properly
equipped fluorescence microscope.
12. The slides are graded on the basis of fluorescence intensity
based on the following protocol:
Fluorescent intensity may be semi-quantitated by following
the guidelines established by the Centers for Disease Control,
Atlanta, Georgia:
4+ = Maximal fluorescence; brilliant yellow-green.
3+ = Less brilliant yellow-green fluorescence.
2+ = Definite but dull yellow-green fluorescence.
1+ = Very dim subdued fluorescence.
The degree of fluorescent intensity is not clinically relevant and
has only limited value as an indicator of titer.
4. Assessing
Histologic Severity
of Immune-
Complex-Mediated The various histopathologic alterations in different compartments
Glomerulonephritis of the kidney, including the severity of inflammation, can be graded
in a semiquantitative manner on microscopic examination of
11 A Murine Autoimmune Model of Rheumatoid Arthritis 247
Fig. 2. Representative images of the renal histopathologic findings observed in Def6-deficient mice (on a 129/B6 back-
ground). Left panel : Representative H&E-stained sections from the kidneys of WT and Def6-deficient mice; photomicro-
graphs were taken at a magnification of 400. Deposition of immunoglobulin complexes in the glomeruli was detected by
immunofluorescence with anti-IgG (middle panel ) and anti-C3 (right panel ) staining. Magnification of 400 is shown.
5. Notes
15. After each reagent addition gently tap the plate to mix the well
contents prior to beginning incubation.
16. Dilute serum samples in working sample diluent according to
expected autoantibody levels.
17. Nonspecific binding may occur if the reagent is allowed to dry
on the slide.
18. Do not touch the pipette tip in the wells.
19. Take care not to mix the samples between wells.
20. Do not focus the PBS stream directly onto the wells. To prevent
cross contamination tilt slide first toward wells 16 and, run-
ning PBS stream along the midline of the slide, allow the PBS
to run off the top edge of the slide. Then tilt the slide toward
wells 712, and repeat this procedure, allowing the PBS to run
off the bottom edge of the slide.
21. Examine stained slides as soon as possible using a properly
equipped fluorescence microscope. It is recommended that
slides be examined on the same day they are stained.
22. To prevent nonspecific staining never let the slides to dry after
the blocking step.
23. Optimal dilution of fluorochrome-conjugated antibody should
be worked out to prevent background staining.
Acknowledgments
This work was supported by the Alliance for Lupus Research, the
Mary Kirkland Center for Lupus Research, and the NIH (HL62215
and AI076474 to A.P.).
References
1. McInnes IB, Schett G (2007) Cytokines in the 6. Stamp LK, James MJ, Cleland LG (2004)
pathogenesis of rheumatoid arthritis. Nat Rev Interleukin-17: the missing link between T-cell
Immunol 7:429442 accumulation and effector cell actions in rheu-
2. Toh ML, Miossec P (2007) The role of T cells matoid arthritis? Immunol Cell Biol 82:19
in rheumatoid arthritis: new subsets and new 7. Korn T, Bettelli E, Gao W, Awasthi A, Jager A,
targets. Curr Opin Rheumatol 19:284288 Strom TB, Oukka M, Kuchroo VK (2007)
3. Sarkar S, Cooney LA, Fox DA (2010) The role IL-21 initiates an alternative pathway to induce
of T helper type 17 cells in inflammatory arthri- proinflammatory T(H)17 cells. Nature 448:
tis. Clin Exp Immunol 159:225237 484487
4. Tesmer LA, Lundy SK, Sarkar S, Fox DA 8. Nurieva R, Yang XO, Martinez G, Zhang Y,
(2008) Th17 cells in human disease. Immunol Panopoulos AD, Ma L, Schluns K, Tian Q,
Rev 223:87113 Watowich SS, Jetten AM, Dong C (2007)
5. Koenders MI, Joosten LA, van den Berg WB Essential autocrine regulation by IL-21 in the
(2006) Potential new targets in arthritis therapy: generation of inflammatory T cells. Nature
interleukin (IL)-17 and its relation to tumour 448:480483
necrosis factor and IL-1 in experimental arthri- 9. Zhou L, Ivanov II, Spolski R, Min R, Shenderov
tis. Ann Rheum Dis 65(Suppl 3):iii29iii33 K, Egawa T, Levy DE, Leonard WJ, Littman
250 P.S. Biswas et al.
DR (2007) IL-6 programs T(H)-17 cell of proinflammatory IL-17+ T helper cells. Cell
differentiation by promoting sequential 126:11211133
engagement of the IL-21 and IL-23 pathways. 22. Laurence A, Tato CM, Davidson TS, Kanno Y,
Nat Immunol 8:967974 Chen Z, Yao Z, Blank RB, Meylan F, Siegel R,
10. Leonard WJ, Spolski R (2005) Interleukin-21: Hennighausen L, Shevach EM, OShea J
a modulator of lymphoid proliferation, apopto- (2007) Interleukin-2 signaling via STAT5 con-
sis and differentiation. Nat Rev Immunol 5: strains T helper 17 cell generation. Immunity
688698 26:371381
11. Mehta DS, Wurster AL, Grusby MJ (2004) 23. Yang XO, Panopoulos AD, Nurieva R, Chang
Biology of IL-21 and the IL-21 receptor. SH, Wang D, Watowich SS, Dong C (2007)
Immunol Rev 202:8495 STAT3 regulates cytokine-mediated generation
12. Crispin JC, Kyttaris VC, Terhorst C, Tsokos of inflammatory helper T cells. J Biol Chem
GC (2010) T cells as therapeutic targets in 282:93589363
SLE. Nat Rev Rheumatol 6:317325 24. Yang XO, Pappu BP, Nurieva R, Akimzhanov
13. Afzali B, Lombardi G, Lechler RI, Lord GM A, Kang HS, Chung Y, Ma L, Shah B,
(2007) The role of T helper 17 (Th17) and Panopoulos AD, Schluns KS, Watowich SS,
regulatory T cells (Treg) in human organ trans- Tian Q, Jetten AM, Dong C (2008) T helper
plantation and autoimmune disease. Clin Exp 17 lineage differentiation is programmed by
Immunol 148:3246 orphan nuclear receptors ROR alpha and ROR
14. Herber D, Brown TP, Liang S, Young DA, gamma. Immunity 28:2939
Collins M, Dunussi-Joannopoulos K (2007) 25. Brustle A, Heink S, Huber M, Rosenplanter C,
IL-21 has a pathogenic role in a lupus-prone Stadelmann C, Yu P, Arpaia E, Mak TW,
mouse model and its blockade with IL-21R.Fc Kamradt T, Lohoff M (2007) The develop-
reduces disease progression. J Immunol 178: ment of inflammatory T(H)-17 cells requires
38223830 interferon-regulatory factor 4. Nat Immunol
15. Sawalha AH, Kaufman KM, Kelly JA, Adler 8:958966
AJ, Aberle T, Kilpatrick J, Wakeland EK, Li 26. Chen Q, Yang W, Gupta S, Biswas P, Smith P,
QZ, Wandstrat AE, Karp DR, James JA, Merrill Bhagat G, Pernis AB (2008) IRF-4 Binding
JT, Lipsky P, Harley JB (2008) Genetic asso- Protein inhibits interleukin-17 and interleu-
ciation of IL-21 polymorphisms with systemic kin-21 production by controlling the activity of
lupus erythematosus. Ann Rheum Dis 67: IRF-4 transcription factor. Immunity 299:
458461 899911
16. Young DA, Hegen M, Ma HL, Whitters MJ, 27. Huber M, Brustle A, Reinhard K, Guralnik A,
Albert LM, Lowe L, Senices M, Wu PW, Sibley Walter G, Mahiny A, von Low E, Lohoff M
B, Leathurby Y, Brown TP, Nickerson-Nutter (2008) IRF4 is essential for IL-21-mediated
C, Keith JC Jr, Collins M (2007) Blockade of induction, amplification, and stabilization of
the interleukin-21/interleukin-21 receptor the Th17 phenotype. Proc Natl Acad Sci U S A
pathway ameliorates disease in animal models 105:2084620851
of rheumatoid arthritis. Arthritis Rheum 56: 28. Matsuyama T, Grossman A, Mittrucker H,
11521163 Siderovski D, Kiefer F, Kawakami T, Richardson
17. Bettelli E, Oukka M, Kuchroo VK (2007) C, Taniguchi T, Yoshinaga S, Mak T (1995)
T(H)-17 cells in the circle of immunity and Molecular cloning of LSIRF, a lymphoid-
autoimmunity. Nat Immunol 8:345350 specific member of the interferon regulatory
18. Ivanov II, Zhou L, Littman DR (2007) factor family that binds the interferon-stimu-
Transcriptional regulation of Th17 cell differ- lated response element (ISRE). Nucl Acid Res
entiation. Semin Immunol 19:409417 23: 21272136
19. Dong C (2008) TH17 cells in development: an 29. Rengarajan J, Mowen KA, Mcbride KD, Smith
updated view of their molecular identity and ED, Singh H, Glimcher LH (2002) Interferon
genetic programming. Nat Rev Immunol 8: regulatory factor 4 (IRF4) interacts with
337348 NFATc2 to modulate interleukin 4 gene expres-
sion. J Exp Med 195:10031012
20. Takatori H, Kanno Y, Chen Z, OShea JJ
(2008) New complexities in helper T cell fate 30. Gupta S, Lee A, Hu C, Fanzo J, Goldberg I,
determination and the implications for autoim- Cattoretti G, Pernis AB (2003) Molecular
mune diseases. Mod Rheumatol 18: 533541 cloning of IBP, a SWAP-70 homologous GEF,
which is highly expressed in the immune sys-
21. Ivanov II, McKenzie BS, Zhou L, Tadokoro tem. Hum Immunol 64:389401
CE, Lepelley A, Lafaille JJ, Cua DJ, Littman
DR (2006) The orphan nuclear receptor 31. Hotfilder M, Baxendale S, Cross MA, Sablitzky
RORgammat directs the differentiation program F (1999) Def-2, -3, -6, -8, novel mouse genes
11 A Murine Autoimmune Model of Rheumatoid Arthritis 251
differentially expressed in the hematopoietic 34. Murphy KM, Heimberger AB, Loh DY (1990)
system. Br J Haematol 106:335344 Induction by antigen of intrathymic apoptosis
32. Tanaka Y, Bi K, Kitamura R, Hong S, Altman of CD4 + CD8 + TCRlo thymocytes in vivo.
Y, Matsumoto A, Tabata H, Lebedeva S, Science 250:17201723
Bushway PJ, Altman A (2003) SWAP-70-like 35. Biswas PS, Gupta S, Chang E, Song L, Stirzaker
adapter of T cells, an adapter protein that regu- RA, Liao JK, Bhagat G, Pernis AB (2010)
lates early TCR-initiated signaling in Th2 lin- Phosphorylation of IRF4 by ROCK2 regulates
eage cells. Immunity 18:403414 IL-17 and IL-21 production and the develop-
33. Fanzo JC, Yang W, Jang SY, Gupta S, Chen Q, ment of autoimmunity in mice. J Clin Invest
Siddiq A, Greenberg S, Pernis AB (2006) Loss 120:32803295
of IRF-4-binding protein leads to the sponta- 36. Brand DD, Kang AH, Rosloniec EF (2004)
neous development of systemic autoimmunity. The mouse model of collagen-induced arthri-
J Clin Invest 116:703714 tis. Methods Mol Med 102:295312
Chapter 12
Abstract
The transfer of homozygous C57Bl/6 (B6) or DBA/2 (DBA) parental strain T cells into normal B6D2F1
mice in the parent-into-F1 (p F1) model results in a graft-vs.-host disease (GVHD) that takes one of the
following two forms: (a) acute GVHD seen with B6 F1 mice and mediated by donor CD8 cytotoxic T
cells that eliminate host lymphocytes and (b) a chronic lupus-like GVHD seen with DBA F1 mice and
mediated by donor CD4 T cell cognate help to autoreactive B cells resulting in autoantibody production
and renal disease similar to human lupus. Importantly, these two phenotypes can be distinguished by flow
cytometry as early as 2 weeks after donor cell transfer. The p F1 model can be used to screen for agents
that alter lupus development. Additionally, the model is useful for preclinical screening of biologic agents
with immunomodulatory potential. Agents that selectively inhibit CD8 T cell function will convert acute
GVHD to chronic GVHD in B6 F1 mice. Conversely, agents that promote CD8 CTL function will
convert chronic GVHD to acute GVHD in DBA F1 mice. Agents that completely suppress T cell function
will block both phenotypes. The model is also useful for examining the effects of T cell mutations by transferring
mutant T cells into wild-type hosts and assessing the effects on disease phenotype. Differences observed
from wild-type T cells F1 can be directly ascribed to alterations in mutant T cell function. Because of the
early 2-week phenotype development, the p F1 model is well suited to screening of potential immuno-
modulatory therapeutic compounds and the assessment of T cell mutations on in vivo function.
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_12, Springer Science+Business Media New York 2012
253
254 K. Soloviova et al.
is intrinsic to the donor T cells and not to a defect in the non-T cell
donor population. Lastly, donor T cell function can be further dis-
sected by purifying donor CD4 and CD8 T cell subsets separately
from both wild-type and mutant strains and recombining them in
mix-and-match fashion so that all four possible combinations are
transferred. The two control groups are the following: (a) WT
CD4 + WT CD8 F1 and (b) mutant CD4 + mutant CD8 F1,
both of which are used to confirm that neither the WT nor the
mutant GVHD phenotype seen using unfractionated donor sple-
nocytes is altered as a result of the purification and recombination
procedure. The two experimental groups are the following: (c) WT
CD4 + mutant CD8 F1 which tests the ability of mutant CD8
T cells to mature into effector CTL when provided with a normal
source of CD4 T cell help and (d) mutant CD4 + WT CD8 F1
which tests the ability of mutant CD4 T cells to provide help for
normal CD8 CTL maturation. This mix-and-match approach
requires testing of several doses of donor cells to ensure that the
dose is off plateau. Typically, we use a donor CD4:CD8 ratio in the
range of 1.5:1 to 2:1. If we have observed that unfractionated
mutant cells inhibit CD8 CTL function and mitigate acute GVHD
phenotype, then we use a dose of donor cells just above the thresh-
old for acute GVHD induction, i.e., 68 106 CD4 T cells and
45 106 CD8 T cells (9), to demonstrate that at a dose in which
control WT F1 mice exhibit a robust acute GVHD phenotype,
host splenocyte elimination is defective for mutant F1 mice.
Alternatively, if we have observed potentiation of acute GVHD by
mutant T cells, then the donor cell dosage is reduced to levels just
below the threshold of acute GVHD induction, i.e., 4 106 CD4
and 2 CD8 T cells (10), and potentiation is demonstrated if a
typical 2-week acute GVHD phenotype ensues for mutant F1
mice but not for WT F1 mice. For both the inhibition or poten-
tiation analyses, further titration of donor cell numbers up or
down, respectively, should be carried out to determine the donor
cell dose range for the effect.
1.1.3. Investigating Mutant The previous section outlines the investigation of possible altera-
T Cell Help to B Cells tions in CD4 T cell help for CD8 CTL maturation. CD4 T cell
function can be further dissected by testing the ability of mutant
CD4 T cells to provide help to B cells and induce chronic GVHD.
In this approach, donor CD8 T cells are depleted prior to transfer
and chronic GVHD phenotype assessed at both 2 weeks and long
term (i.e., lupus-like ICGN). Significant elevation (~20-fold over
control) of autoantibodies such as anti-ssDNA can be seen at
4 weeks following the transfer of as few as 8 106 B6 donor CD4
T cells. More substantial elevations (~75-fold) are seen at 12 106
CD4 T cells (9); however, renal disease is histologically mild at
this dose and typically 1520 106 B6 CD4 T cells are required
for significantly elevated glomerular scores (11). By contrast,
unfractionated DBA splenocytes containing as few as 1214 106
12 The Parent-into-F1 Murine Model in the Study of Lupus-Like 257
1.1.4. Mutations The BDF1 is less well suited for the study of mutations in B cells or
in Host Cells APC as this requires breeding an F1 with the required mutation on
both the B6 and DBA parental strains. Although many mutations
are available on the B6 background, very few are available on the
DBA strain. A useful substitute is the (Balb/c B6)F1 (CB6F1)
because of the larger number of mutations present on the Balb/c
background vs. the DBA background. B6 CB6F1 and
Balb/c CB6F1 mice exhibit acute and chronic GVHD pheno-
types, respectively; however, the lupus-like renal disease in
Balb/c CB6F1 mice can require 6 months to develop following
a single transfer of donor cells (12) and is not as well characterized
as that seen in DBA BDF1 chronic GVHD mice.
1.2. Major Procedures General. All procedures should be approved by the Institutional
Animal Care and Use Committee beforehand. Both donor and
1.2.1. GVHD Induction
host mice should be young adults preferably close to 810 weeks
of age and of the same sex to avoid donor or host recognition of
H-y antigens, although in acute GVHD transfers this is not an
absolute requirement because donor anti-host MHC alloantigen
recognition will supercede any host anti-donor H-y recognition.
Donor cell transfer: Any source of T cells is acceptable (e.g.,
lymph node, thymus); however, we routinely use splenocytes exclu-
sively for donor transfers because of the ease of preparation.
Additionally, unfractionated splenocytes contain stem cells that can
mitigate mortality by permitting long-term donor repopulation
and stable chimerism (13). Our group exclusively uses a single
injection of unfractionated splenocytes or T cells purified from
splenocytes and transferred intravenously. The use of multiple
transfers and/or intraperitoneal injection is also effective; however,
the single transfer permits a kinetic evaluation of a single popula-
tion of donor T cells and standardization of the phases of donor
T cell activation and contraction kinetics (1). Additionally, using the
single dose allows correlation of both the short-term and long-
term phenotypes to the single population of donor cells.
1.2.2. GVHD Assessment Because the spleen is the major filter for the blood and because our
transfers are i.v., we primarily analyze F1 spleens after transfer.
Liver and lymph node also exhibit donor T cell infiltration within
the first 2 weeks (14); however, the spleen is well suited for early
analysis of the effects of biologics or mutant donor T cells and liver
and lymph nodes offer no experimental advantages. Acute murine
GVHD can also exhibit attacks on a variety of other organs similar
to human acute GVHD, although this generally requires >2 weeks
for organ damage (14).
Day 14: Flow cytometric analysis of splenic lymphocyte populations.
Acute and chronic GVHD phenotypes can be determined by
258 K. Soloviova et al.
2. Materials
2.1.3. Positive 1. Isolation Buffer: PBS (without Ca+ and Mg+) with 10 % BSA
T Cell Isolation and 2 mM EDTA, pH 7.4.
2. Culture Media: RPMI-1640 with 10 % FCS.
3. Dynabeads: Mouse CD8 (Lyt2), Mouse CD4 (Lyt2),
Invitrogen (Carlsbad, CA).
4. Dynal MPC-6, Dynal MPC-50, Invitrogen (Carlsbad, CA).
Surface Staining Antibodies 1. The following mAbs are used: CD4, CD8, B220, H-2Kb/H-
2Kd, I-Ab/I-Ad, Cd11b, Cd11c, CD44, CD62L, CXCR5,
ICOS, FAS, PD1, CCR7, KLRG, CD80, CD86, CD107,
FASL, CD107a. Typically, we use 45-color flow cytometry
using the following fluorochromes: Alexa Fluor;
Allophycocyanin-Cy-7-; Biotin, Phycoerythrin, Texas Red,
Peridinin Chlorophyll Protein Complex, Pacific Blue-
conjugated and Pacific orange. All are available from BD
Biosciences (San Jose, CA), BioLegend (San Diego, CA),
eBioscience (San Diego, CA), or Invitrogen (Carlsbad, CA).
CFSE Staining 1. Cell Trace CFSE Cell Proliferation Kit, Molecular Probes, Inc.
(Eugene, OR).
2.2.3. ELISA 1. High binding capacity polystyrene flat bottom 96-well micro-
titer plates (Thermo scientific NUNC Maxi-Sorp, Thermo
Fisher Scientific, Rochester, NY).
2. DNA, single stranded from calf thymus, lyophilized powder
(Sigma-Aldrich, Atlanta, GA).
3. Double stranded DNA plates (QuantaLITE, INOVA
Diagnostics, Inc. San Diego, CA).
4. Goat anti-mouse IgGAlkaline Phosphatase antibody (Sigma-
Aldrich, Atlanta, GA).
5. Alkaline Phosphatase substrate kit (Bio-Rad, Hercules, CA).
6. Tween-20, BSA (Sigma-Aldrich, Atlanta, GA).
7. 1 PBS (Quality Biological Inc., Gaithersurg, MD).
8. DNA-stock solution: 1 mg single-stranded DNA, 1 ml DEPC-
treated water with 1 mM EDTA.
9. DNA working solution: 20 l of DNA stock solution in 1 ml
of 1 PBS.
12 The Parent-into-F1 Murine Model in the Study of Lupus-Like 261
3. Methods
3.2. Splenocyte 1. Place spleen in Petri dish with RPMI without FCS.
Isolation (for Donor Cell 2. Homogenize spleen by gently pressing capsule with either
Transfer or Analysis) proximal end of sterile 3 cc syringe or glass mesh homogenizer.
3. Filter splenocyte mixture through cell strainer into a 50 ml
polypropylene conical tube.
4. Centrifuge for 10 min, resuspend cells in 2 ml RPMI, and
count using hemocytometer. Adjust to desired concentration.
3.3. GVHD Induction 1. For transfers using unfractionated splenocytes use cells as pre-
pared in Subheading 3.2 and determine the relative percentage
3.3.1. Splenocyte
of CD4 and CD8 T cells by flow cytometry (see below).
Preparation
2. Adjust donor inoculum to desired numbers of CD4 and CD8
T cells.
3.3.2. Donor T Cell The protocol is based on 108 WBC/ml and follows the manufac-
Purification: Negative turers protocol.
Isolation
1. Wash Dynabeads before use and resuspend in vial.
2. Transfer Dynabeads to a 15 ml conical tube, and add 5 ml
Buffer 1.
3. Place tube in a magnet for 1 min and then discard supernatant
using transferring pipette. Do not remove tube from magnet
during this step.
4. Remove tube from magnet and resuspend Dynabeads in 10 ml
of Buffer 1.
5. Prepare cells from mouse spleen as described above and after
centrifuging resuspend cells in Buffer 1.
6. To the splenocytes, add 200 l of heat-inactivated FCS and
200 l of desired mAb mix.
7. Mix well and incubate for 20 min at between 2 and 8 C with
Dynal bi-directional rotator.
8. Add 30 ml Buffer 1 to each tube and centrifuge at 3,000 rpm
(1876.9 g) between 4 and 8 C for 8 min.
9. Resuspend cells in 15 ml of Buffer 1 and add prewashed beads
at 2 ml beads per 100 106 leucocytes. Incubate for 20 min at
room temperature (RT) with bidirectional rotation.
10. Resuspend cells in 5 ml Buffer 1, place tube into magnet for
2 min, and then transfer supernatant into the clean 50 ml tube.
11. Wash beads 2 with Buffer 1.
12. Wash cells 3 with RPMI-1640 and then resuspend in desired
volume of RPMI-1640.
13. Determine the purity of depletion by flow cytometry prior to
tail vein injection.
12 The Parent-into-F1 Murine Model in the Study of Lupus-Like 263
3.3.5. IV Injection Assessment of donor CD4 and CD8 T cells by flow cytometry
should be performed prior to transfer. Live dead gating can be
used but typically the live cells are >90 % and often >95 %. The
donor inocula should be adjusted so that experimental and
controls receive comparable numbers of CD4 and CD8 T cells.
1. Prior to injection, place the cage of mice to be injected under
a heat lamp to increase circulation to the surface blood vessels
and make the tail veins more visible.
2. Prepare syringe for injection and be sure all bubbles are
removed.
3. Place mouse in the restrainer, and hold the tail gently tugging
back to prevent movement during the injection.
4. Line up the needle (bevel side up) exactly in the line with vein.
5. Insert the needle into the vein keeping the needle parallel to
the tail so as not to penetrate out the other side of the vein.
264 K. Soloviova et al.
3.4.2. Splenocyte Flow All mAb should be titrated prior to use for optimal staining.
Cytometry: General Protocol All of these staining protocols can be performed in either tubes
for Surface Staining or plates. The following protocol is for tube staining. For plate
staining, make these substitutions:
1. Use 5 105 cells/well.
2. Decrease concentration of Ab in two times.
3. Use plate-shaker instead of vortex.
4. Do not overload wells during the washing step.
3.4.4. Splenocyte Flow The protocol below can also be used to analyze cellular prolifera-
Cytometry: Intracellular tion by substituting anti-KI67 ab of anti-Foxp3 ab. Both anti-KI67
Staining Protocol for Foxp3 and anti-Foxp3 ab can be stained simultaneously using this proto-
and Ki67 col assuming that appropriate fluorochromes are chosen.
1. Stain cell-surface antigen following the Surface Staining
Protocol but do not fix in 1 % PFA, rather proceed after final
washing step to add Fixation/Permeabilization as described
below.
2. Dilute Fixation/Permeabilization Concentrate (one part) into
the Fixation/Permeabilization Diluent (three parts) to make a
Fixation/Permeabilization Working Solution. Do not store
buffer more than 1 day.
3. After last wash of surface staining, resuspend cell pellet, add
1 ml of Fixation/Permeabilization Working Solution, and
vortex.
4. Incubate for 118 h (overnight) at +4 C in the dark. For
convenience, we usually incubate overnight; however shorter
periods will suffice.
5. Wash twice with 2 ml of 1 Permeabilization Buffer. Optional:
If cell yields are a problem at this stage, it may be that fixation
and permeabilization has rendered them more buoyant.
Centrifugation at speeds of 2,000 rpm (834.2 g) on standard
tabletop centrifuge can improve the cell yield. We generally do
not find this necessary.
6. Add anti-Foxp3 ab or isotype control in 100 l total of 1
Permeabilization Buffer; incubate for 20 min, +4 C.
7. Wash 2 with 2 ml of 1 Permeabilization Buffer.
8. Resuspend in 500 l of Facs-buffer and analyze in flow
cytometer.
9. Gates and voltages may need to be modi fi ed due to the
fi xation and permeabilization procedure which can alter the
FSC/SSC distribution of the cell population compared to
that of live cells.
3.4.5. Splenocyte Flow 1. Stain cell-surface antigen following the Surface Staining
Cytometry: Annexin V Protocol.
Staining Protocol 2. Wash cells 2 with cold PBS.
3. Resuspend in 1 Binding Buffer at concentration 106 cells/ml.
4. Transfer 100 l of the solution to 5 ml culture tube.
266 K. Soloviova et al.
3.4.6. Renal Histology 1. Initially, place harvested kidney into polystyrene round-bottom
(Formalin and Liquid tube with PBS (one per tube).
Nitrogen Prep) 2. Once all kidneys are harvested, one at a time place them in
Petri dish and using forceps, remove the capsule and then cut
in axial plane.
3. Place half of kidney into a 2 ml tube with 2 ml of 10 %
formalin.
4. Place the other half of kidney into the center of foil piece
(pre-labeled), and apply Histoprep on the top of kidney. Fold
the foil to create a secure envelope.
5. Place envelope into liquid nitrogen (in paper box)/or dry ice
for 10 min, and then store in 80 C.
3.4.7. ELISA The following is for anti-ssDNA. For anti-dsDNA, the DNA prep-
aration can be treated with S1 endonuclease or commercial dsDNA
plates can be purchased and the assay performed according to the
manufacturers instructions.
1. Coat 96-well plates with 100 l of DNA working solution and
incubate at 4 C overnight.
2. Remove coating solution by tapping the plate face down against
a paper towel. Wash the plate three times with washing buffer,
400 l/well. After each washing, tap the plates against a paper
towel to ensure no residue on the bottom of the wells.
3. Add 100 l of blocking buffer into each well and incubate for
1 h at 37 C, 5 % CO2.
4. Remove the blocking buffer by tapping and wash three times
with washing buffer, 400 l/well.
5. Dilute each serum sample 1:40 in washing buffer in upper
wells of the plate, and make three serial dilutions (1:80, 1:160,
1:320). Do duplicate dilutions for each sample.
6. Dilute standard control serum 1:100 in washing buffer in upper
wells of the plate, and make serial dilutions (1:200, 1:400, 1:800,
1:1,600, 1:3,200, 1:6,400). Do duplicate dilutions of standard
control. Leave the last wells in the standard control columns
blank. Make a separate standard control for each plate.
7. Incubate for 1 h at 37 C, 5 % CO2.
8. Wash three times with washing buffer, 400 l/well.
9. Dilute detection antibody 1:4,000 in blocking buffer and add
100 l/well.
12 The Parent-into-F1 Murine Model in the Study of Lupus-Like 267
4. Notes
4.1. Tail Vein Injections 1. Make sure that you have a comfortable position during the
injection. Our injections are performed in a hood but this is
not absolutely required.
This procedure is difficult to master and requires frequent
practice.
2. Do not expose animals too long to the heat lamp. When their
activity level increases, they are ready to inject.
3. Visualization of the tail veins can be increased by curling the
end of the tail. Swabbing the tail with 70 % isopropyl alcohol
immediately prior to injection may also improve visualization
of the tail vein.
4. Keep in mind that the veins are located laterally. A dark line
can often be seen running down the back of the tail. This is
not a vein.
5. Be sure to mix splenocyte preparation before drawing up in
syringe as settling of cells occurs.
References
1. Puliaev R, Puliaeva I, Welniak LA et al (2008) 8. Via CS, Shustov A, Rus V et al (2001) In vivo
CTL-promoting effects of CD40 stimulation neutralization of TNF-alpha promotes humoral
outweigh B cell-stimulatory effects resulting in autoimmunity by preventing the induction of
B cell elimination and disease improvement in a CTL. J Immunol 167:68216826
murine model of lupus. J Immunol 9. Puliaeva I, Puliaev R, Shustov A et al (2008)
181:4761 Fas expression on antigen-specific T cells has
2. Via CS (2010) Advances in lupus stemming costimulatory, helper, and down-regulatory
from the parent-into-F1 model. Trends functions in vivo for cytotoxic T cell responses
Immunol 31:236245 but not for T cell-dependent B cell responses.
3. Foster AD, Haas M, Puliaeva I et al (2010) J Immunol 181:59125929
Donor CD8 T cell activation is critical for 10. Puliaeva I, Soloviova K, Puliaiev M et al (2011)
greater renal disease severity in female chronic Enhancement of suboptimal CD8 cytotoxic T
graft-vs.-host mice and is associated with cell effector function in vivo using antigen-
increased splenic ICOS(hi) host CD4 T cells specific CD80 defective T cells. J Immunol
and IL-21 expression. Clin Immunol 186:291304
136:6173 11. Rus V, Nguyen V, Puliaev R et al (2007) T cell
4. Via CS, Sharrow SO, Shearer GM (1987) Role TRAIL promotes murine lupus by sustaining
of cytotoxic T lymphocytes in the prevention of effector CD4 Th cell numbers and by inhibit-
lupus-like disease occurring in a murine model ing CD8 CTL activity. J Immunol
of graft-vs-host disease. J Immunol 178:39623972
139:18401849 12. Tschetter JR, Mozes E, Shearer GM (2000)
5. Puliaeva I, Puliaev R, Via CS (2008) Therapeutic Progression from acute to chronic disease in a
potential of CD8+ cytotoxic T lymphocytes in murine parent-into-F1 model of graft-versus-
SLE. Autoimmun Rev 8:219223 host disease. J Immunol 165:59875994
6. Via CS, Rus V, Nguyen P et al (1996) 13. Hakim FT, Sharrow SO, Payne S et al (1991)
Differential effect of CTLA4Ig on murine Repopulation of host lymphohematopoietic
graft-versus-host disease (GVHD) develop- systems by donor cells during graft-versus-host
ment: CTLA4Ig prevents both acute and reaction in unirradiated adult F1 mice injected
chronic GVHD development but reverses only with parental lymphocytes. J Immunol
chronic GVHD. J Immunol 157:42584267 146:21082115
7. Via CS, Rus V, Gately MK et al (1994) IL-12 14. Niculescu F, Niculescu T, Nguyen P et al
stimulates the development of acute graft-ver- (2005) Both apoptosis and complement mem-
sus-host disease in mice that normally would brane attack complex deposition are major fea-
develop chronic, autoimmune graft-versus-host tures of murine acute graft-vs.-host disease.
disease. J Immunol 153:40404047 Exp Mol Pathol 79:136145
Chapter 13
Abstract
Genetic and environmental factors contribute in the pathogenesis of systemic lupus erythematosus (SLE).
Lupus nephritis, the most common and severe manifestation of SLE, involves inflammation in the kidney
leading to loss of renal function. However, it is not clear what controls the progression of lupus nephritis;
this is an important research question, considering its implications in clinical treatment of lupus nephritis.
Finding genes that underlie the development and progression of lupus nephritis will shed light on this
question. NZM2328 is a spontaneous mouse model for SLE. Most NZM2328 female mice develop
autoantibodies (e.g., antinuclear antibody and anti-dsDNA antibody), glomerulonephritis (GN), and
severe proteinuria between 5 and 12 months of age. In contrast, C57L/J mice fail to exhibit similar signs
of autoimmune disease. We used classical genetics to map and identify SLE genes in offspring generated
by backcrossing C57L/J to NZM2328. Quantitative trait loci (QTL) controlling acute (Agnz1 and
Agnz2) and chronic (Cgnz1) GN features were uncovered by the analysis. To verify the Cgnz1 and Agnz1
on distal mouse chromosome 1, we produced the NZM23238.C57Lc1 (Lc1) congenic strain, which
replaced NZM2328 Cgnz1 and Agnz1 alleles with those derived from C57L/J. The development of acute
GN and chronic GN was markedly reduced in Lc1 mice, confirming the linkage findings. Further mapping
by the generation of intrachromosomal recombinants of NZM2328.Lc1 support the thesis that acute GN
and chronic GN are under separate genetic control.
Key words: Congenic strain, End-stage renal disease, Genetic mapping, Genotyping,
Glomerulonephritis, Linkage analysis, Lupus nephritis, Marker-assisted selection protocol,
Microsatellite marker, NZM2328, Quantitative trait loci, QTL mapping, Speed congenic, Systemic
lupus erythematosus
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_13, Springer Science+Business Media New York 2012
271
272 Y. Ge et al.
1.1. Genetic During the last six decades, extensive data have been accumulated,
Contributions suggesting that both genetic and environmental factors play
in Human SLE significant role in the pathogenesis of SLE. Twin studies have pro-
vided definitive evidence that genetics as well as environment play
important roles in human SLE: the concordance rate of SLE in
monozygotic twins is 2458 %, whereas in dizygotic twins or sib-
lings, the rate was 25 % (3). SLE also displays familial aggrega-
tion, as the risk of disease among siblings of SLE patients is about
29 times higher than that among the general population (4).
Recent genome-wide association studies on lupus susceptibility
genes have identified more than 30 candidate genes that have rela-
tive minor impacts on SLE (58). Evidence has also been obtained
showing ethnic variation in lupus clinical manifestations and the
effects of social-economic factors on this disease (9). These data
suggest the complexity of lupus genetics. The heterogeneity of the
human population is still a major obstacle in genetic studies in
man. It is also evident that to delineate the pathogenetic factors in
a complex disease with protean clinical presentations present a for-
midable challenge. Studies on mouse models on lupus in general
and lupus nephritis in particular have helped significantly in our
understanding of this disorder. They will remain useful comple-
menting human studies (10, 11). In this chapter, the genetic
approaches in mapping genes contributing to lupus nephritis in
mouse are reviewed briefly. Our studies on NZM2328 are dis-
cussed in detail to illustrate the methods for mapping genes that
contribute to the development of lupus nephritis in mouse.
1.2. Mouse Models New Zealand mice were the first mouse model described for human
of Spontaneous lupus (reviewed in (12, 13)). (NZB NZW)F1 females develop
Lupus Nephritis glomerulonephritis (GN) that resembles human proliferative lupus
nephritis with immune complex deposition. New Zealand mice
have been studied extensively. Subsequently, two other models,
MLR/lpr and BXSB have been described. Although these three
mouse models have important similarities, they differ significantly
in their natural history, autoantibody profiles, organ involvement,
and sex predilection (11, 14). It was recognized early that the
background genes influence phenotypic expression. Thus, MRL/lpr
has severe autoimmune phenotypes with autoantibody production,
13 Genetic Approach to Study Lupus Glomerulonephritis 273
vacuities, skin disease, and severe GN while B6.lpr has very few
autoimmune traits. During the past two decades, recombinant
inbred strains have been established from a (NZB NZW)F2 or
(NZB NZW)F1 NZW mating (15). These strains have approxi-
mately 75 % of NZW with 25 % NZB genes. They have ANA and
varying degree of GN (16). These strains are very useful for map-
ping genes contributing to lupus GN.
The aforementioned spontaneous lupus-prone models have
been used in mapping genes contributing to various autoimmune
phenotypes (reviewed in (10, 11, 1719)). Typically, genetic analy-
sis was carried out to identify genetic segments with quantitative
trait loci (QTL) that contribute specifically to particular autoim-
mune phenotypes as distinguished by a quantitative trait by the
study of either a cohort of (lupus-prone strain non-lupus prone
strain)F1 backcrossed to the non-lupus strain or a cohort of inter-
cross (F2) progenies. The identified QTL needs to be substantiated
by the generation of congenic strains in which the genomic region
corresponding to the QTL of interest is introgressed to the non-
autoimmune strain. This approach was taken by Wakeland, Morel
and colleagues in their studies of Sle1, Sle2, and Sle3. In this case,
Sle 1, Sle2, and Sle3 on chromosome 1, 4, and 7, respectively, were
identified to be linked to GN by the analysis of a cohort of
(NZM2410 C57BL/6)F1 C57BL/6, in which NZM2410 is a
lupus-prone strain while C57BL/6 (B6) is the non-lupus prone
strain. Although autoantibody production was one of the traits of
interest, no such QTL was identified. None of the congenics,
B6.Sle1, B6.Sle2, and B6.Sle3 have GN; however, the tricongenic
B6. Sle1Sle2 Sle3 has severe GN. Although the functions of these
three loci in B6-based lupus cogenics have been studied exten-
sively, the nature of the genes conferring the GN phenotypes
remains elusive (reviewed in (11, 19)).
As an alternative approach, we have chosen NZM2328 as the
lupus-prone strain and C57L as the non-lupus strain. As detailed
below, we have identified a single locus Cgnz1 controlling chronic
GN on chromosome 1, a single locus Adnz1 on chromosome 4
linked to ANA and anti-dsDNA production, and three loci, Agnz1
on chromosome 1, the H-2 complex and a locus, Agnz2, distal to
the H-2 complex on chromosome 17 to be linked to acute GN.
The phenotypes of NZM2328-base congenic strains, NZM2328.
Lc1 and MZN2328.Lc4 confirm the genetic data (20, 21).
These two contrasting approaches underline the importance of
the choice of strains and the defining of traits in the mapping of
genetic traits for lupus nephritis.
1.4. Systemic Lupus To identify genes that contribute to the development of lupus
Nephritis Loci in nephritis, we conducted a genome-wide linkage analysis using a
NZM2328 backcross cohort derived by crossing SLE-prone NZM2328 and
non-lupus C57L mice. The backcross offspring were PCR geno-
typed with a genome-wide panel of simple sequence length poly-
morphism (SSLP) markers; they were also characterized in terms
of lupus traits. SLE trait analysis involved following individual off-
spring until 12 months of age or when severe proteinuria devel-
oped, whichever occurred first. As stated above, QTL for acute and
chronic GN (SLE traits) were genetically mapped; acute GN QTLs
Agnz1 and Agnz2 were positioned on chromosomes 1 and 17,
respectively, and a chronic GN QTL Cgnz1 was mapped on distal
chromosome 1. In addition, a single locus, Adnz1 was located on
chromosome 4 (20). Because Cgnz1and Adnz1 were monogenic
for association with ANA/anti-dsDNA and anti-nucleosome anti-
body production and chronic GN, respectively, congenic lines,
NZM2328.Lc1 and NZM2328.Lc4 were generated by introgress-
ing the genetic regions containing these two loci into NZM2328.
These congenic lines have been informative and are discussed sepa-
rately (21).
13 Genetic Approach to Study Lupus Glomerulonephritis 275
1.6. Cgnz1 and Agnz1 The findings of Cgnz1 and Agnz1 agree with previous genetic
Verification and studies on murine SLE, as the Cgnz1 and Agnz1 interval overlap
SLE-Gene Precision with Sle1 and Sle1 was found to be linked to GN in a genome-wide
Mapping on linkage analysis of a (NZM2410 C57BL/6)F1 NZM2410
Chromosome 1 backcross cohort (20).
To verify the biological function of the Cgnz1 and Agnz1 loci,
we generated NZM2328.C57Lc1 (Lc1) congenic mice by intro-
gressing a 24-cM chromosome 1 fragment of C57L/J, which spans
Cgnz1 and Agnz1 loci, onto the NZM2328 background. The Lc1
mice had significantly reduced incidences of both acute and chronic
GN, autoantibody production, severe proteinuria, and early mor-
tality (21); these observations were consistent with our linkage
findings (20). The successful generation of this important congenic
model to study lupus GN in animals with disparate chromosome 1
intervals was largely due to the congenic approach we employed
(i.e., using NZM2328 as the recipient background instead of a
non-lupus strain). On the other hand, GN cannot be directly
examined in congenic strains produced by the opposite approach
as discussed and detailed in Subheading 3.2.
We have generated multiple intrachromosomal recombinant
strains of NZM2328.Lc1 by monitoring cohorts of (NZM2328
NZM2328.Lc1)F2 for recombinations. Preliminary data indicate
that replacing the regions of Agnz1 had no effect on the develop-
ment of both acute and chronic GN and the replacement of the
regions controlling chronic GN inhibited the development of
chronic GN despite the manifestation of acute GN with immune
complex deposition (24). Furthermore, the region controlling
chronic GN has been mapped to a 1.34 Mb segment of chromo-
some 1 (22). This paves the way for the identification of the gene
controlling chronic GN by transgenesis or by knock-in approach.
This aspect of mapping the genes for lupus nephritis is not dis-
cussed further in this chapter.
276 Y. Ge et al.
2. Materials
3. Methods
3.1. Classical Genetic 1. Breed lupus-prone NZM2328 with non-lupus C57L/J mice
Analysis and SLE Trait to generate (NZM2328 C57L/J)F1 progeny (see Note 1 for
Mapping in NZM2328 discussion of mating direction). F1 males and females should
Backcross Mice be kept for further backcrossing and phenotype analysis. All F1
pedigree records (e.g., ID of breeding cage and ID of both
3.1.1. Generation of
parents) must be maintained for future reference.
NZM2328 Backcross Mice
and SLE Trait Development 2. Breed NZM2328 with (NZM2328 C57L/J)F1 mice to
generate [(NZM2328 C57L/J)F1 NZM2328] backcross
offspring (and see Note 1). Alternatively, use a brother sister
(NZM2328 C57L/J)F1 intercross mating scheme to gener-
ate F2 offspring for genetic analysis (see Note 2).
3. Monitor SLE traits (i.e., severe proteinuria and kidney histol-
ogy) in NZM2328 backcross females over the period of
12 months after birth (see Note 3).
3.1.2. Extraction of Mouse 1. Clip 23 mm tail tissue from control NZM2328 and C57L
Tail Genomic DNA mice and backcross offspring and store in a microcentrifuge
tube (1.5 ml) labeled with the mouse ID at 20 C.
2. Add 300 ml Cell Lysis Solution (Gentra) and 1.5 ml Puregene
Proteinase K (final conc. 0.1 mg/ml) to mouse tail tissue (fresh
or frozen) and mix gently by inverting 25 times.
3. Incubate at 55 C overnight or until the tissue has completely
dissolved. Invert tube periodically during the incubation.
When tail tissue is not completely dissolved after overnight
digestion, additional Proteinase K may be added, followed by
12 h further incubation at 55 C.
4. To quickly cool the tail lysate, incubate on ice for 1 min. Add
100 ml Protein Precipitation Solution (Gentra), and mix well
by vortexing vigorously for 20 s at high speed.
5. Centrifuge sample at ~16,000 g for 3 min at room tempera-
ture. The protein precipitate should form a tight pellet.
However, if the pellet is loose, incubate on ice for 5 min and
repeat the centrifugation.
6. In a clean microcentrifuge tube, add 300 ml isopropanol.
Carefully decant the supernatant from step 5 into the tube
with isopropanol. Note that the protein pellet should not be
dislodged during pouring.
7. Mix sample gently by inverting 50 times, centrifuge sample at
~16,000 g for 1 min at room temperature. The DNA precipi-
tate may form a visible small white pellet. Carefully discard the
supernatant, and drain the tube by inverting on a clean piece of
absorbent paper. Be careful not to lose the DNA pellet during
the process, as it might be loose and easily dislodged.
8. Add 300 ml of 70 % ethanol and mix gently by inverting several
times to wash the DNA pellet.
278 Y. Ge et al.
3.1.3. Simple Sequence Detection of large and small effect QTLs in a genome-wide link-
Length Polymorphism and age analysis requires genetic markers spaced at ~20-cM distances,
Single Nucleotide and no more than 10-cM from chromosome ends, for all chromo-
Polymorphism Marker somes in the genome (25). Microsatellite repeat sequences in the
Selection for Genome- mouse genome work well for the purpose of generating SSLP
Wide Linkage Analysis markers to readily distinguish allele variants in common laboratory
strains of mice, including NZB and NZW (20, 26). SNP markers
to distinguish NZB and NZW mouse alleles have been published
(20, 26).
1. Published SSLP (also referred to as PCR marker) and SNP
markers for 89 strains of mice may be obtained through the
Mouse Genome Informatics (MGI), which can be accessed at
http://www.informatics.jax.org/. Detailed marker informa-
tion includes mouse chromosome position, product (allele)
size and amplification primers (PCR markers), and nucleotide
differences for SNP markers.
2. When strain-specific allele information is not available in public
databases for potentially informative and useful SSLP and SNP
markers based on their chromosome location, these must be
tested against the control progenitor strains of interest using
the methods described in Subheadings 3.1.4 and 3.1.5.
3. In practice, one should assemble and test the entire panel of
select genetic markers, which can readily distinguish SSLP and
SNP allele differences in select progenitor strains and their
hybrid offspring. We have routinely used SSLP markers to reli-
ably and accurately genotype alleles distinguished by 2+ bp dif-
ferences in backcross and intercross progenies.
4. All hybrid offspring must be genotyped using the approved
panel of genetic markers.
3.1.4. PCR Genotyping Mouse tail DNA genotyping by PCR is carried out with select
Mouse Tail DNA with SSLP SSLP markers as discussed in Subheading 3.1.3.
Markers
1. Prepare PCR master mix sans tail DNA based on number of
mouse tail DNA samples to be genotyped per SSLP marker.
13 Genetic Approach to Study Lupus Glomerulonephritis 279
2. Aliquot PCR master mix into individual PCR tubes, add tail
DNA, and carry out PCR on a thermal cycler according to the
conditions given in step 3.
Volume per Final
Component reaction (ml) concentration
3.1.5. Size Analysis Two methods to separate and visualize SSLP PCR-amplified mark-
of PCR-Amplified SSLP ers are detailed here.
Markers
MetaPhor Agarose Gel MetaPhor agarose (Cambrex Bio Science Rockland, Inc., Rockland,
Electrophoresis-Based Maine) has an intermediate melting temperature of 75 C and
SSLP Size Analysis offers outstanding resolving capacity for separation of PCR-
amplified SSLP markers which differ by 5+ bp. To insure accurate
genotypes, we routinely run SSLP PCR with progenitor strain (i.e.,
NZM2328 and C57L/J) controls on the same MetaPhor gel with
all offspring PCR products under analysis (27). To achieve opti-
mum resolution, users must adhere strictly to manufacturers
instructions when preparing MetaPhor agarose gels.
1. Add sufficient quantity of MetaPhor agarose slowly to chilled
1 TBE buffer in a beaker with continuous swirling to make a
2 % solution.
2. After soaking agarose in cold 1 TBE buffer for ~15 min,
weigh the beaker and solution, cover the beaker with plastic
wrap with a small hole, and heat the beaker in a microwave
oven until all the particles are dissolved.
3. Add sufficient heated distilled water (75 C) to obtain the
beaker and mix thoroughly. After cooling the solution to
13 Genetic Approach to Study Lupus Glomerulonephritis 281
3.1.6. Genetic Mapping of To map chromosome locations for SLE QTL, NZM2328 back-
SLE Quantitative Trait Loci cross genotypes and lupus trait data were analyzed using
MapMangerQT (20). Other very useful QTL mapping programs
include QTL Cartographer, R/qtl, and MapQTL (28, 29).
Single-QTL genome scans using regression analysis typically
detect chromosome map locations ranging from 10 to 30 cM based
on likelihood ratio test statistic (LRS) or logarithm of the odds
(LOD) scores. For a QTL, the mapping resolution depends on the
number of recombination events in the mapping cohort. When
assessing whether a QTL is statistically significant, Lander and
Kruglyacks guidelines are standard practice: Highly-significant
refers to p < 0.001, significant p < 0.05, and suggestive p < 0.63,
after correcting for multiple testing in genome-wide scans (30).
For a backcross genome-wide linkage analysis, the LOD score
threshold for suggestive linkage is 1.9, and significant linkage 3.3
(30). When reporting QTL mapping results, the LOD scores, the
peak positions, and the estimated confidence intervals should be
provided. Once identified, one may further refine significant QTL
locations by typing the initial backcross or intercross cohort with
additional SSLP markers targeting these regions of interest, and
then rerunning the QTL mapping program. Additionally, preci-
sion mapping for candidate loci may be carried out as described in
Subheading 3.3.
3.2.1. Selection of Genetic A critical first step toward precision SLE gene mapping and later
Background for Congenic functional studies is to select a desirable genetic background for the
Strains production of congenic strains. This is because background genetic
factors may interact with or contribute to donor QTL allele effects,
which may further influence whether a given QTL effect is manifest.
Recently, we produced NZM2328 chromosome 1-congenic
mice with non-lupus alleles derived from C57L/J mice (21).
An important feature in the NZM2328.C57Lc1 (also referred to
as Lc1) mice is that only the select chromosome 1 interval is
responsible for changes in disease progression normally observed
in NZM2328 females. Thus, we can directly monitor the impact of
Agnz1 and Cgnz1 on SLE traits in these mice. Inhibition of both
acute and chronic GN was observed in Lc1 females, thus confirming
that NZM2328 mice harbor one or more SLE QTL on chromo-
some 1 which contribute to disease (20).
In contrast to our approach, several related studies have used
B6 mice, which do not develop lupus features, as recipients to gen-
erate congenic strains with donor alleles derived from lupus-prone
mouse strains (e.g., NZM2410, NZW, and BXSB) (31, 32). An
inherent weakness of the latter approach is that lupus traits have
not manifested in the B6 background. For example, although Sle1
and Nba2 were initially mapped for GN, neither B6.Sle1 nor
B6.Nba2 congenic strains developed GN. Instead, B6.Sle1 mice
developed increased levels of IgG ANA, and B6.Nba2 congenic
mice developed IgG anti-DNA and anti-chromatin autoantibodies
(33, 34). Due to the lack of GN in B6.Sle1, the subsequent fine
mapping of Sle1 had to use autoantibody production as the trait to
approximate GN (35). This approach has made Morel and col-
leagues to postulate the presence of Sle1d that confers the original
phenotype of GN with unknown location within the Sle1 region
(35). Another weakness of the approach is that it potentially intro-
duced a whole new set of gene interactions between non-lupus
background and donor lupus alleles, complicating an already com-
plex research question. In this regard, the non-lupus prone B6 has
been shown to harbor a gene contributing to autoantibody pro-
duction (36). Thus, we conclude that the introgression of non-
lupus alleles into SLE-prone NZM2328 is highly useful and
informative for the purpose of QTL verification and further study
of acute and chronic GN and ESRD.
3.2.2. Speed Congenic The traditional protocol for generating congenic strains is to back-
Protocol cross a donor locus to a recipient genetic background for 10+ gen-
erations, accompanied by screening and selection for progeny
carrying the donor locus at each generation. The founders are gen-
erated by intercrossing the mice of the last backcross and then
selecting for homozygosity at the target region. Though straight-
forward, completion of this protocol takes 34 years (37). The
marker-assisted selection protocol (MASP)-based breeding strategy,
13 Genetic Approach to Study Lupus Glomerulonephritis 283
3.3. Precision Mapping A major goal in precision mapping is to identify a minimal critical
for SLE Genes interval spanning the gene(s) responsible for a QTL effect, which
on Chromosome 1 is flanked or bounded by recombination crossovers. We have used
this approach before to establish critical genomic regions needed
in viral resistance, which subsequently allowed us to identify the
relevant genes (3843). More recently, we used this approach to
pinpoint distinct locations for Agnz1 and Cgnz1 SLE loci within
the larger (24-cM) chromosome 1 interval based on crossovers in
a panel of Lc1 recombinant congenic lines (20, 22). Importantly,
these results demonstrate that precision mapping is an important
step in positional cloning and gene identification, even in narrow
gene regions, based on findings obtained for three different loca-
tions in the mouse genome. As an example, the following method
describes the generation of Lc1 recombinant congenic sublines.
1. Breed NZM2328.C57Lc1 mice, which are heterozygous for
the Lc1 interval, in a brother x sister mating scheme. Most
offspring produced in this breeding scheme will exhibit
heterozygosity or homozygosity over the entire 24-cM Lc1
congenic interval. However, a fraction of the offspring will
have a crossover on one or both chromosomes.
2. Genotype all offspring with select SSLP markers according to
protocol in Subheading 3.1.4 to screen for recombination
crossovers within the Lc1 congenic interval. Subheading 3.1.3
provides detailed information about how to obtain informative
SSLP markers to distinguish alleles in the relevant strains.
(a) When useful markers are unavailable, novel SSLP markers
can be generated as we described previously (26, 42, 44).
(b) In brief, download relevant genomic sequence covering
the region of interest from the National Center for
Biotechnology Information and manually inspect it for
microsatellite repeat sequences.
(c) Design sequence-specific primers to PCR amplify select
microsatellite sequences.
(d) Test SSLP marker primers to verify specific amplification
of the select microsatellite, the capacity to distinguish the
13 Genetic Approach to Study Lupus Glomerulonephritis 285
3.4.1. Acute GN Acute GN was scored blindly using a scale of 04, based on the
severity of glomerular hypercellularity, mesangial matrix expansion,
focal necrosis, and epithelial cell crescents (Fig. 1 B1, C1, D1, and
E1). Grade 0 indicates the absence of signs of acute GN. Grade 1
indicates mild focal lesions with proliferative changes that are pri-
marily in the mesangium. Grades 2 and 3 represent diffuse involve-
ment of moderate severity in the glomeruli, whereas grade 4
indicates widespread changes of more severity (20).
3.4.2. Chronic GN Chronic GN was scored blindly using a scale of 04, based on the
severity of glomerulosclerosis (focal to global), tubular atrophy,
dilated tubules with hyaline casts, and interstitial fibrosis (Fig. 1
B2, C2, D2, and E2). Grade 0 indicates no signs of chronic GN.
Grade 1 represents mild focal glomerulosclerosis and interstitial
inflammation. Grades 2 and 3 represent moderate glomerulo-
sclerosis, tubule atrophy, and interstitial fibrosis. Grade 4 indi-
cates severe glomerulosclerosis, tubule atrophy, and interstitial
fibrosis (20).
Fig. 1. Stages of acute and chronic GN: Normal Kidney (A) from a 12-week-old NZM2328 female mouse. Kidneys from
24- to 28-week-old NZM2328 female mice with acute GN: (B1) stage 1 acute GN kidney showing mesangial expansion
and some hypercellularity with normal tubular cells. Note that the glomerulus is bigger than that in normal kidneys,
13 Genetic Approach to Study Lupus Glomerulonephritis 287
4. Notes
Fig. 1. (continued) (C1) stage 2 acute GN showing more cellular infiltration in the glomeruli, (D1) stage 3 acute GN showing
loci of interstitial infiltration with hypercellular glomerulus, and (E1) stage 4 acute GN showing segmental scleroses in the
glomerulus without collapsing of the Bowmans capsule. The tubules are essentially normal. Kidneys from NZM2328
female mice older than 40 weeks with severe proteinuria and chronic GN: (B2) stage 1 chronic GN showing affected glom-
erulus with tubule dilatation and noncellular cast in the tubules, (C2) stage 2 chronic GN showing area of collapsed
Bowmans capsule and a red cell cast in one of the tubules, (D2) stage 3 chronic GN showing generalized tubular dilatation
and more extensive collapse of the Bowmans capsules, and (E2) stage 4 chronic GN showing sclerotic and necrotic glom-
erulus, with tubular dilatation and interstitial fibrosis. All photos were taken at 400 magnification.
288 Y. Ge et al.
Acknowledgments
References
1. Cameron JS (1999) Lupus nephritis. J Am Soc 8. Kaiser R, Criswell LA (2010) Genetics research
Nephrol 10(2):413424 in systemic lupus erythematosus for clinicians:
2. Houssiau FA, Vasconcelos C, DCruz D, methodology, progress, and controversies.
Sebastiani GD, de Ramon GE et al (2004) Curr Opin Rheumatol 22(2):119125
Early response to immunosuppressive therapy 9. Borchers AT, Naguwa SM, Shoenfeld Y,
predicts good renal outcome in lupus nephritis: Gershwin ME (2010) The geoepidemiology of
lessons from long-term followup of patients in systemic lupus erythematosus. Autoimmun Rev
the euro-lupus nephritis trial. Arthritis Rheum 9(5):A277A287
50(12):39343940 10. Jorgensen TN, Gubbels MR, Kotzin BL (2004)
3. Deapen D, Escalante A, Weinrib L, Horwitz D, New insights into disease pathogenesis from
Bachman B et al (1992) A revised estimate of mouse lupus genetics. Curr Opin Immunol
twin concordance in systemic lupus erythema- 16(6):787793
tosus. Arthritis Rheum 35(3):311318 11. Morel L (2010) Genetics of SLE: evidence
4. Alarcon-Segovia D, Alarcon-Riquelme ME, from mouse models. Nat Rev Rheumatol 6(6):
Cardiel MH, Caeiro F, Massardo L et al (2005) 348357
Familial aggregation of systemic lupus erythe- 12. Theofilopoulos AN, Dixon FJ (1985) Murine
matosus, rheumatoid arthritis, and other auto- models of systemic lupus erythematosus. Adv
immune diseases in 1,177 lupus patients from Immunol 37:269390
the GLADEL cohort. Arthritis Rheum 52(4): 13. Borchers A, Ansari AA, Hsu T, Kono DH,
11381147 Gershwin ME (2000) The pathogenesis of
5. Deng Y, Tsao BP (2010) Genetic susceptibility autoimmunity in New Zealand mice. Semin
to systemic lupus erythematosus in the genomic Arthritis Rheum 29(6):385399
era. Nat Rev Rheumatol 6(12):683692 14. Andrews BS, Eisenberg RA, Theofilopoulos
6. Gualtierotti R, Biggioggero M, Penatti AE, AN, Izui S, Wilson CB et al (1978) Spontaneous
Meroni PL (2010) Updating on the pathogen- murine lupus-like syndromes. Clinical and
esis of systemic lupus erythematosus. immunopathological manifestations in several
Autoimmun Rev 10(1):37 strains. J Exp Med 148(5):11981215
7. Delgado-Vega A, Sanchez E, Lofgren S, 15. Rudofsky UH, Evans BD, Balaban SL, Mottironi
Castillejo-Lopez C, Alarcon-Riquelme ME VD, Gabrielsen AE (1993) Differences in
(2010) Recent findings on genetics of systemic expression of lupus nephritis in New Zealand
autoimmune diseases. Curr Opin Immunol mixed H-2z homozygous inbred strains of mice
22(6):698705 derived from New Zealand black and New
13 Genetic Approach to Study Lupus Glomerulonephritis 289
Zealand white mice. Origins and initial charac- 30. Lander E, Kruglyak L (1995) Genetic dissec-
terization. Lab Invest 68(4):419426 tion of complex traits: guidelines for interpret-
16. Rudofsky UH, Lawrence DA (1999) New zea- ing and reporting linkage results. Nat Genet
land mixed mice: a genetic systemic lupus ery- 11(3):241247
thematosus model for assessing environmental 31. Morel L, Rudofsky UH, Longmate JA,
effects. Environ Health Perspect 107(Suppl 5): Schiffenbauer J, Wakeland EK (1994) Polygenic
713721 control of susceptibility to murine systemic
17. Kono DH, Theofilopoulos AN (2006) Genetics lupus erythematosus. Immunity 1(3):219229
of SLE in mice. Springer Semin Immunopathol 32. Drake CG, Rozzo SJ, Hirschfeld HF,
28(2):8396 Smarnworawong NP, Palmer E et al (1995)
18. Theofilopoulos AN, Kono DH (2001) Genetics Analysis of the new zealand black contribution
of systemic autoimmunity and glomerulone- to lupus-like renal disease. Multiple genes that
phritis in mouse models of lupus. Nephrol Dial operate in a threshold manner. J Immunol
Transplant 16(Suppl 6):6567 154(5):24412447
19. Fairhurst AM, Wandstrat AE, Wakeland EK 33. Mohan C, Alas E, Morel L, Yang P, Wakeland
(2006) Systemic lupus erythematosus: multiple EK (1998) Genetic dissection of SLE patho-
immunological phenotypes in a complex genesis. Sle1 on murine chromosome 1 leads to
genetic disease. Adv Immunol 92:169 a selective loss of tolerance to H2A/H2B/
20. Waters ST, Fu SM, Gaskin F, Deshmukh US, DNA subnucleosomes. J Clin Invest 101(6):1
Sung SS et al (2001) NZM2328: a new mouse 3621372
model of systemic lupus erythematosus with 34. Rozzo SJ, Allard JD, Choubey D, Vyse TJ, Izui
unique genetic susceptibility loci. Clin Immunol S et al (2001) Evidence for an interferon-
100(3):372383 inducible gene, Ifi202, in the susceptibility to
21. Waters ST, McDuffie M, Bagavant H, systemic lupus. Immunity 15(3):435443
Deshmukh US, Gaskin F et al (2004) Breaking 35. Morel L, Blenman KR, Croker BP, Wakeland
tolerance to double stranded DNA, nucleosome, EK (2001) The major murine systemic lupus
and other nuclear antigens is not required for erythematosus susceptibility locus, Sle1, is a
the pathogenesis of lupus glomerulonephritis. J cluster of functionally related genes. Proc Natl
Exp Med 199(2):255264 Acad Sci U S A 98(4):17871792
22. Ge Y, Fu SM. MS in preparation. In press. 36. Heidari Y, Fossati-Jimack L, Carlucci F, Walport
23. Bagavant H, Fu SM (2005) New insights from MJ, Cook HT et al (2009) A lupus-susceptibil-
murine lupus: disassociation of autoimmunity ity C57BL/6 locus on chromosome 3 (Sle18)
and end organ damage and the role of T cells. contributes to autoantibody production in 129
Curr Opin Rheumatol 17(5):523528 mice. Genes Immun 10(1):4755
24. Ge Y, Jiang C, Gaskin F, Sung SJ, Bagavant H 37. Wakeland E, Morel L, Achey K, Yui M,
et al (2009) Pathogenesis of proliferative lupus Longmate J (1997) Speed congenics: a classic
nephritis: different genetic control for acute technique in the fast lane (relatively speaking).
and chronic glomerulonephritis and new insight Immunol Today 18(10):472477
into the mechanism of immune complex medi- 38. Scalzo AA, Brown MG, Chu DT, Heusel JW,
ated nephritis. Arthritis Rheum 60:2019 Yokoyama WM et al (1999) Development of
25. Darvasi A (1998) Experimental strategies for intra-natural killer complex (NKC) recombi-
the genetic dissection of complex traits in ani- nant and congenic mouse strains for mapping
mal models. Nat Genet 18(1):1924 and functional analysis of NK cell regulatory
loci. Immunogenetics 49(3):238241
26. Rodriguez MR, Lundgren A, Sabastian P, Li
Q, Churchill G et al (2009) A Cmv2 QTL on 39. Scalzo AA, Wheat R, Dubbelde C, Stone L,
chromosome X affects MCMV resistance in Clark P et al (2003) Molecular genetic charac-
New Zealand male mice. Mamm Genome terization of the distal NKC recombination
20(7):414423 hotspot and putative murine CMV resistance
control locus. Immunogenetics 55(6):370378
27. Asif M, Rahman M, Mirza JI, Zafar Y (2008)
High resolution metaphor agarose gel elecctro- 40. Brown MG, Zhang J, Du Y, Stoll J, Yokoyama
phoresis for genotyping with microsatellite WM et al (1999) Localization on a physical
markers. Pak J Agric Sci 45(1):7579 map of the NKC-linked Cmv1 locus between
Ly49b and the prp gene cluster on mouse chro-
28. Manly KF, Olson JM (1999) Overview of QTL mosome 6. J Immunol 163(4):19911999
mapping software and introduction to map
manager QT. Mamm Genome 10(4):327334 41. Brown MG, Dokun AO, Heusel JW, Smith
HR, Beckman DL et al (2001) Vital involve-
29. Broman KW, Wu H, Sen S, Churchill GA ment of a natural killer cell activation receptor
(2003) R/qtl: QTL mapping in experimental in resistance to viral infection. Science 292(5518):
crosses. Bioinformatics 19(7):889890 934937
290 Y. Ge et al.
42. Xie X, Stadnisky MD, Brown MG (2009) cell resistance to murine cytomegalovirus.
MHC class I dk locus and Ly49G2+ NK cells Proc Natl Acad Sci U S A 107(19):
confer H-2k resistance to murine cytomegalo- 87548759
virus. J Immunol 182(11):71637171 44. Rodriguez M, Sabastian P, Clark P, Brown MG
43. Xie X, Stadnisky MD, Coats ER, Ahmed (2004) Cmv1-independent antiviral role of NK
Rahim MM, Lundgren A et al (2010) MHC cells revealed in murine cytomegalovirus-
class I D(k) expression in hematopoietic and infected New Zealand white mice. J Immunol
nonhematopoietic cells confers natural killer 173(10):63126318
Chapter 14
Abstract
Primary biliary cirrhosis (PBC) is a female-predominant autoimmune disease of the liver characterized by
immune-mediated destruction of the intrahepatic bile ducts and the presence of antimitochondrial anti-
bodies (AMAs). There have been limited advances in understanding the molecular pathogenesis of the
disease because of the difficulty in accessing human tissues and the absence of appropriate animal models.
Recently, several unique murine models that manifest the serological, biochemical, and histological fea-
tures similar to human PBC have been described. In this article, we discuss the current data on three
spontaneous and two induced murine models of PBC. The spontaneous models are: (a) NOD.c3c4,
(b) dominant negative TGF- receptor II (dnTGFRII), and (c) IL-2R/ mouse line models. The two
induced models are: (a) xenobiotic and (b) Novosphingobium aromaticivorans immunized mice. These
animal models provide various important platforms to further investigate the etiology and mechanisms of
pathogenesis in PBC. Laboratory methodologies and the protocols that are used in evaluating these animal
models are described. Finally, we stress the importance of realizing the strengths and limitations of the
animal models are essential in data analysis and their application in therapeutic studies.
Key words: Antimitochondrial antibodies, Biliary epithelial cells, Cytokine analysis, E2 subunit of
pyruvate dehydrogenase, ELISA, Flow cytometry, Immunohistochemical staining, Liver lesions,
Primary biliary cirrhosis, Western Blot
Abbreviations
PBC Primary biliary cirrhosis
AMA Antimitochondrial antibody
PDC-E2 E2 subunits of pyruvate dehydrogenase
BCOADC-E2 2-Oxo acid dehydrogenase
OGDC-E2 2-Oxo-glutarate dehydrogenase
NOD Non-obese diabetic
dnTGFRII Dominant negative TGF- receptor II
* Supported in part by National Institutes of Health grants DK074768 DK090019, DK067003, and
DK39588.
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_14, Springer Science+Business Media New York 2012
291
292 P.S.C. Leung et al.
1. Introduction
2. Mouse Models
2.1. NOD.ABD Mouse Non-obese diabetic (NOD) mice exhibit susceptibility to sponta-
Lines neous development of autoimmune type I diabetes (T1D) (13).
Genetic loci associated with susceptibility to T1D have been
defined through the development of congenic mouse strains, which
Table 1
Comparison between human PBC and murine models of PBC
2-OA-
BSA + alpha
14
Granuloma +-++ + + +
Eosinophilia + +
(continued)
293
Table 1
294
(continued)
2-OA-
BSA + alpha
PBC IL-2Ra/ 2-OA-BSA glyceramide Novosphingobium aromati-
patients NOD.ABD mice dnTGF-bRII mice mice immunization immunization civorans immunization
P.S.C. Leung et al.
Remarks Also develop Spontaneously Also develop Proof of hypothesis The only The remarkable sequence
common bile develop inflammatory that molecular murine homology between
duct dilation inflammatory bowel mimics of model of lipoylated proteins
and biliary bowel disease disease, lipoylated PBC that of xenobiotic metaboliz-
epithelial cell unless main- 2050 % die PDC-E2 in the exhibits ing N. aromaticivorans
proliferation tained by a from environment can fibrosis in combinationwith
Helicobacter free hemolytic lead to loss of the natural liver tropism
diet and anemia at tolerance to of NKT cells and the
antibiotics in 820 weeks PDC-E2 and accumulation of
drinking water of age autoimmune N. aromaticivorans
cholangitis in the liver likely explains
the liver specificity
of destructive responses
and AMA.
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 295
2.2. Dominant Negative Shortly after the discovery of the NOD.c3c4 mouse model, Oertelt
TGF-b Receptor II Mice et al. described another mouse model with PBC-like liver pathology
and AMAs, viz. the dnTGFRII mice (21). The dnTGFII mice
have an over-expression of a dominant-negative form of TGF-
receptor type II under the control of the CD4 promoter (22).
TGF- receptor II is critical for signal transduction of TGF-,
which regulates the activation of lymphocytes (23, 24). Deficiency
in TGF- results in various pleiotrophic immunological abnormali-
ties including colitis and early death (2527). dnTGFRII mice are
maintained by mating male dnTGFRII mice with normal B6
females (since female dnTGFRII mice experience reproductive
failure), to produce heterozygote dnTGFRII mice and normal
B6 offspring. To maintain the colony, genotyping is performed at
4 weeks of age by purifying DNA collected from ear punches.
dnTGFRII mice are fed a sterile rodent helicobacter diet and sterile
drinking water including sulfratrim, and are maintained individu-
ally in ventilated cages under specific pathogen-free conditions.
dnTGFRII mice exhibit major serologic and histological
characteristics of human PBC suggesting that the dnTGFRII
pathway is important in the pathogenesis of PBC (21, 28). They are
100 % AMA positive with autoantibodies directed against PDC-E2,
BCOADC-E2, and OGDC-E2, the major mitochondrial autoanti-
gens in human PBC. The liver and serum cytokine levels reflect a
Th1 profile. The liver histology of dnTGFRII mice manifests
lymphoid cell infiltration in the portal tracts of 100 % of the mice
including CD4, CD8, and CD19 positive cells as seen in human
PBC. This is accompanied by bile duct injury in 2550 % of mice up
to 22 weeks of age, which is also seen in human PBC (28).
Similar to patients with PBC, there is an elevated level of CD8/
CD4 T cells in the livers of dnTGFRII mice (21). To understand
the role of CD4+ and CD8+ T cells in liver pathology in these
mice, we performed adoptive transfer studies by transferring dnT-
GFRII mice-derived splenic CD4+ and/or CD8+ T cells derived
into Rag1/ recipients. Rag1/ recipients of dnTGFRII mice
unfractionated splenocytes developed features of liver disease
similar to human PBC, suggesting that splenic T and B cell loss of
tolerance causes autoimmune cholangitis. Furthermore, the transfer
of dnTGFRII-derived CD8+ T cells into Rag1/ recipients
resulted in liver-specific autoimmunity, whereas CD4+ T cell transfer
led to colitis, indicating that CD8+ T cells are the primary contribu-
tors for bile duct destruction in this model (28).
Although the presence of high titers of AMAs is present in
95 % of patients with PBC, there is no direct correlation between
AMAs and pathogenesis (29, 30). We have investigated the role of
AMAs in disease pathology in the dnTGFRII mice model of PBC.
Briefly, dnTGFRII mice were crossed with B cell-deficient mice
(Ig/), and were evaluated for the development of liver
inflammation, as well as the severity of accompanying colitis.
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 297
2.3. IL-2Ra / Mice IL-2 is critical for the development and peripheral expansion of
CD4+ CD25+ Tregs, which promote self-tolerance by suppressing
T cell responses in vivo (38, 39). Previously, it was reported that a
child with a genetic deficiency of IL-2R developed clinical
manifestations similar to PBC (40). Interestingly, C57BL/6 J
IL-2R/ mice had serological and pathological characteristics
resembling those of chronic nonsuppurative destructive cholangitis,
298 P.S.C. Leung et al.
3. 2-Octynoic
Acid-BSA-
Immunized Mice
Our laboratory has hypothesized that xenobiotic modification of
the native lipoyl moiety of the major mitochondrial autoantigen
PDC-E2, may lead to the loss of self-tolerance in PBC (43). This
thesis is based on the findings of readily detectable levels of immu-
noreactivity of PBC sera against extensive panels of protein microarrays,
which mimics the inner lipoyl domain of PDC-E2 and subsequent
quantitative structureactivity relationships (QSARs) (44). Previous
studies showed that rabbits immunized with one such xenobiotic,
6-bromohexanoate conjugated to bovine serum albumin (BSA),
produced AMAs to PDC-E2, BCOADC-E2, and OGDC-E2, but
without any PBC-like liver pathology (45). In 2008, Wakabayashi
et al. reported that murine immunization with 2-octynoic acid
(2-OA) coupled to BSA induced AMAs and cholangitis. When
using 2-OA-BSA as an immunogen in B6 mice and NOD.1101
(NOD.B6 Idd10 Idd18r2) mice, it led to high titer AMAs, portal
inflammation, and autoimmune cholangitis similar to human PBC
(46). Using this mouse model, we investigated the role of B cells
in PBC by depleting B cells using two different monoclonal anti-
bodies, CD20 and CD79. The results of the experiment revealed
that B cell depletion led to exacerbated cholangitis, with higher
T cell infiltrates and inflammatory cytokines, indicating a protec-
tive role of B cells in PBC (32).
Taking advantage of our experience in this xenobiotic induced
model of PBC, we have investigated the role of innate immunity
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 299
4. Materials
4.6. Frozen Tissue OCT embedding compound (Sakura Finetek, Tokyo, Japan).
Preparation 2-Methylbutane (Sigma Aldrich, St. Louis, MO).
4.11. Serum Cytokine Mouse Inflammatory (Containing IL-6, IL-10, MCP-1, IFN-,
Analysis TNF, and IL-12p70) and Th1/Th2/Th17 (Containing IL-2,
IL-4, IL-6, IFN-, TNF, IL-17A, IL-10) Cytometric Bead
Array kit (BD Biosciences, San Jose, CA).
Mouse IL-12p40, IL-23p19 ELISA kit (R&D system, Minneapolis,
MN).
Mouse IL-27 (p28//EBI3, IL27) ELISA Kit (eBioscience, San
Diego, CA).
Mouse IL-18 ELISA Kit (MBL International, Woburn, MA).
4.12. Liver Cytokine TNE buffer (1 % NP 40, 10 mM TrisHCl pH 7.5, 150 mM NaCl,
and Chemokine 1 mM EDTA) containing protease inhibitor and phosphatase
Analysis inhibitor cocktail (Roche, Indianapolis, IN).
BCA protein assay kit (Pierce Biotechnology, Rockford, IL).
Mouse Inflammatory (Containing IL-6, IL-10, MCP-1, IFN-,
TNF, and IL-12p70) and Th1/Th2/Th17 (Containing IL-2,
IL-4, IL-6, IFN-, TNF, IL-17A, IL-10) Cytometric Bead
Array kit (BD Biosciences, San Jose, CA).
5. Methods
Tg+
1.44 Tg-
OD of PDC-E2 by ELISA
OD of OGDC by ELISA
OD of BCKD by ELISA
0.99 0.94
1.04
0.69 0.74
0.64
Fig. 1. Serological Ig Reactivity to PDC-E2, OGDC-E2, or BCOADC-E2. Serum samples from dnTGFRII mice and control B6
littermates were analyzed against recombinant proteins of PDC-E2, OGDC-E2, or BCOADC-E2 by ELISA. Serum samples
were diluted 1/500. Sera from 4- to 10-week-old mice (dnTGFRII n = 11, B6 n = 10), 12- to 16-week-old mice (dnTGFRII
n = 10, B6 n = 9), and 22- to 39-week-old mice (dnTGFRII n = 19, B6 n = 10) were tested. Threshold values are set by
mean + 2 SD of values for B6 controls. p < 0.001. Tg+ dnTGFRII transgenic mice, Tg littermate controls.
3. ELISA
Note: A typical comparison of antibody titer to PDC-E2 in AMA
positive mice versus control mice is shown in Fig. 1.
(a) Prepare coating antigens from stock antigen (recombinant
PDC-E2) such that the final concentration of coating anti-
gens is 10 g/ml in carbonate coating buffer (0.5 M
carbonate bicarbonate buffer, pH 9.6).
(b) To a 96-well plate, add 100 l of coating antigen to each
well and store at 4 C.
(c) Remove coating solution. Wash plate three times with
300 l of 0.05 % PBST (0.15 M PBS, pH 7.2 with 0.05 %
Tween 20) per well. Then wash two times with 300 l
of PBS.
(d) Remove liquid by flicking inverted plate over sink and pat
dry on paper towels.
(e) Block entire plate with 150 l of 3 % skim milk in PBS and
incubate for 1 h at room temperature.
(f) Decant blocking solution from the wells and pat dry on
paper towels.
(g) Add 100 l of serum samples (diluted 1/250 with 3 % milk
in PBS) to plate and incubate for 1 h at room temperature.
Make sure to include sera from nave mice as negative con-
trols and blank wells without sera for background.
(h) Decant serum samples from plate. Wash plate three times
with 300 l of 0.05 % PBST per well. Then wash two times
with 300 l of PBS, and pat dry on paper towels
(i) Add 100 l of horseradish peroxidase-conjugated anti-
mouse IgG (at a predetermined dilution in 3 % milk with
PBS) per well and incubate for 1 h at room temperature.
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 307
(j) Remove solution from plate. Wash plate three times with
300 l of 0.05 % PBST per well. Then wash two times
with 300 l of PBS, and pat dry on paper towels.
(k) Add 100 ml of ELISA developing substrate. (For BD
OptEIA, mix substrate A and B in 1:1 ratio.) Incubate at
room temperature for 10 min.
(l) Stop the reaction by adding 50 l of 2N H2SO4 to plate.
(m) Read the optical density of the plate at 450 nm using a
plate reader.
5.3. Paraffin Tissue 1. Excise the liver from the peritoneal cavity immediately after
Preparation animal is sacrificed.
2. Using sterile scissors, dissect the liver into approximately
46 mm cubes or other desired portion and immediately fix
the tissues by transferring the liver cubes to 10 % neutral
buffered formalin or 4 % PFA solution.
3. Fix the liver with fixative for 2 days at room temperature.
4. Wash the tissue with 0.1 M PBS several times.
5. Place the sliced tissue in a histology cassette.
6. Dehydrate the tissue through a series of ascending ethanol
percentage, via 70100 % ethanol, xylene, and paraffin
infiltration by automatic tissue processor (Sakura).
1 5 min 70 % ethanol.
1 5 min 100 % ethanol.
5 60 min 100 % ethanol.
3 60 min xylene.
1 420 min paraffin infiltration.
7. Embedded in paraffin using embedding molds and make
paraffin-embedded tissue blocks with histology cassettes.
8. Cut the tissue block on a microtome in 34 m thickness.
5.4. Frozen Tissue 1. Label the plastic tissue mold with proper tissue identification
Preparation and fill partly with OCT embedding compound.
2. Note: If freezing unfixed tissue, remove tissue from animal,
dab on towel to remove any fluid, and immediately place tissue
mold in with OCT and continue with protocol.
3. Note: Choose the best orientation for the tissue to be frozen
in, and let it settle to the bottom. The bottom of the mold is
where the cutting will begin.
4. Add more OCT on top of tissue to cover it completely and fill
the mold.
5. Fill a plastic or metal container half way with 2-methylbutane.
6. Add several small pieces of dry ice and wait a few moments for
the temperature to drop (40 C).
308 P.S.C. Leung et al.
7. Grasp the edge of the mold with forceps and dip into
2-methylbutane.
8. Note: Dip only the most bottom part of the plastic mold. Do
not submerge the mold yet. The OCT will begin to turn white
as it freezes.
9. When all of the OCT is frozen, drop the mold into the cold
2-methylbutane to freeze thoroughly (~5 min).
10. Remove the mold from the freezing liquid, wrap the mold in
foil, and immediately store at 80 C.
5.5. H&E Staining 1. Place paraffin specimens in a slide holder and place the slide
holder into a staining jar.
2. Deparaffinize the specimen via xylene, 10080 % ethanol and
rehydrate with distilled water.
3 3 min xylene (blot excess xylene before going into ethanol).
3 3 min 100 % ethanol.
1 3 min 95 % ethanol.
1 3 min 80 % ethanol.
1 5 min deionized H2O.
3. Blot excess water from slide holder.
4. Apply Meyers hematoxylin solution for 5 min.
5. Rinse with running water, warm water, followed by distilled
water.
6. Blot excess water from slide holder.
7. Apply Eosin solution for 5 min.
8. Dehydrate via ethanol and xylene.
3 5 min 95 % ethanol.
3 5 min 100 % ethanol (blot excess ethanol before going into
xylene).
3 15 min xylene.
9. Place coverslip over the slides by placing a drop of Permount
(xylene based) on the slide using a glass rod, taking care to
leave no bubbles.
10. Angle the coverslip and let it fall gently onto the slide.
11. Allow the Permount to spread beneath the coverslip, covering
all the tissue.
12. Dry overnight in the hood.
5.6. Immunohisto- Note: A histological staining of liver portal tracts with lymphocytic
chemical Staining infiltrations, specifically CD4+ and CD8+, is illustrated in Fig. 2.
for CD4 and CD8 Cells
1. Place paraffin specimens in a slide holder.
2. Place slide holder with paraffin specimens in a 60 C oven
beforehand.
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 309
Fig. 2. Histological features of the 2428 week liver of dnTGFRII mice. (ac) Different degrees of lymphocytic infiltration
(red arrows) surrounding the small bile ducts were detected within the portal tracts (H&E staining); (df) individual cells
(red arrows) of cell types composing the portal tract infiltrates were stained with Abs against cell markers: (d) biliary cells
identified by cytokeratin 7; (e) CD4+ lymphocytes; (f) CD8+ lymphocytes.
5.7. Cell Preparation 1. Asphyxiate a mouse by CO2 overdose, and then dip the entire
for Flow Cytometry mouse in a beaker containing 70 % ethanol.
Analysis 2. In a sterile hood, carefully cut the skin of the mouse with sterile
instruments. Make an incision through the peritoneal layer and
remove the whole spleen (and other lymphoid organs if need).
3. Place the entire spleen in a 60 mm petri dish containing 5 ml
of 0.2 % BSA-PBS.
4. Transfer the spleen into a 100-m nylon cell strainer. Use the
plunger end of the syringe to gently mash and press the spleen
through the cell strainer into the petri dish.
5. Pipet up and down several times to separate the cells to the
single cell suspension using a 1 ml transfer pipette.
6. Filter the suspended cells through a nylon mesh mounted on a
15 ml conical tube in order to remove the debris and other
connective tissues.
7. Pellet the cells at 1,500 rpm (or 514 g) for 5 min in a table
top centrifuge. Discard the supernatant and then resuspend
the cells in 8 ml HBSS (0.2 % BSA).
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 311
a Liver b Spleen
Tg+ Tg- Tg+ Tg-
CD3
CD3
c FSC d FSC
CD4
CD4
CD8 CD8
Fig. 3. Flow cytometry analysis of lymphoid cells from liver and spleen of dnTGFRII transgenic mice (Tg+) and littermate
controls (Tg). Lymphoid cells isolated from liver and spleen of dnTGFRII mice were stained with fluorochrome-conjugated
mAbs and analyzed by flow cytometry. (a, b) Frequency of CD3+ lymphocytes in total lymphocyte population. (c, d)
The percentage of CD4+ and CD8+ cells in total CD3+ population.
5.8. Flow Cytometry Note: A representative analysis of the liver and spleen CD3+, CD4+,
of Liver and Spleen and CD8+ cells are shown in Fig. 3.
Cell Populations
1. Aliquot 106 cells from liver or spleen samples into a 1.5 ml
Eppendorf tube.
2. Centrifuge at 6,000 rpm (or 3,300 g) for 2 min.
3. Aspirate supernatant.
4. Add 25 l of staining buffer (PBS containing 0.2 % BSA and
0.1 % sodium azide) that includes 0.5 l of the blocking Ab
(anti-CD16/32 Ab, Biolegend) to each tube.
5. Vortex the tube to resuspend cells.
6. Incubate at 4 for 10 min.
7. Add 25 l of staining buffer that includes Ab to each tube and
vortex the tube.
312 P.S.C. Leung et al.
5.9. Serum Cytokine 1. Warm the mouse with heating lamp for about 10 min.
Analysis 2. Prepare cotton soaked with alcohol, a razor, and tubes in the
mean time.
3. Place a mouse in a restraining tube. Apply alcohol cotton to
the ventral side of the tail to clean.
4. After visualizing the ventral artery, use a razor to make a shallow
incision on the ventral side of the tail from the tip of the tail
(about 23 cm from the tip of the tail).
5. Collect the blood in an Eppendorf tube as drops appear. About ten
drops will be sufficient for serum AMA and cytokine detection.
6. Apply pressure to stop the bleeding.
7. Keep the collected blood for 34 h at room temperature.
8. Centrifuge at 10,000 rpm (or 9,200 g) for 5 min.
9. Collect serum in a new Eppendorf tube.
10. Measure cytokine by ELISA kit following the manufacturers
directions.
5.10. Liver Cytokine and 1. Sacrifice mice by CO2 overdose in a CO2 chamber.
Chemokine Analysis 2. Cut open the abdominal cavity and remove the liver and place
in a 60 mm culture dish.
3. Make TNE buffer and keep the buffer on ice prior use.
4. Cut 100200 mg liver tissue off and put the liver tissue into a
14 ml round bottom tube, keep the sample on ice.
5. Add 500 l of TNE buffer into the tube.
6. Homogenize tissue on ice using a homogenizer.
7. Clean, rinse, and dry the probe after homogenizing each sample.
8. Centrifuged sample at 12,000 rpm (or 13,300 g) for 20 min
at 4 C.
9. Collect the supernatant in a new tube for protein concentra-
tion assay (store protein at 80 C if not use).
10. Measure protein concentration using BCA protein assay kit
following the manufacturers directions.
11. Use 50100 mg of total protein to determine the cytokine
expression levels by an inflammatory cytokine and Th1/Th2/
Th17 CBA kit, or an ELISA kit according to the instructions
provided by the manufacturers.
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 313
References
1. Gershwin ME, Ansari AA, Mackay IR, Colombo S, Casella G, Morini L, Caporaso N,
Nakanuma Y, Nishio A, Rowley MJ, Coppel Colli A, Spinzi G, Montanari R, Gregersen PK,
RL (2000) Primary biliary cirrhosis: an orches- Heathcote EJ, Hirschfield GM, Siminovitch
trated immune response against epithelial cells. KA, Amos CI, Gershwin ME, Seldin MF
Immunol Rev 174:210225 (2010) Genome-wide meta-analyses identify
2. Leung PS, Chuang DT, Wynn RM, Cha S, three loci associated with primary biliary cir-
Danner DJ, Ansari A, Coppel RL, Gershwin rhosis. Nat Genet 42:658660
ME (1995) Autoantibodies to BCOADC-E2 11. Lleo A, Bowlus CL, Yang GX, Invernizzi P,
in patients with primary biliary cirrhosis recog- Podda M, Van de Water J, Ansari AA, Coppel
nize a conformational epitope. Hepatology RL, Worman HJ, Gores GJ, Gershwin ME
22:505513 (2010) Biliary apotopes and anti-mitochondrial
3. Moteki S, Leung PS, Dickson ER, Van Thiel antibodies activate innate immune responses in
DH, Galperin C, Buch T, Alarcon-Segovia D, primary biliary cirrhosis. Hepatology
Kershenobich D, Kawano K, Coppel RL et al 52:987998
(1996) Epitope mapping and reactivity of autoan- 12. Oertelt S, Rieger R, Selmi C, Invernizzi P,
tibodies to the E2 component of 2-oxoglutarate Ansari AA, Coppel RL, Podda M, Leung PS,
dehydrogenase complex in primary biliary cir- Gershwin ME (2007) A sensitive bead assay for
rhosis using recombinant 2-oxoglutarate dehy- antimitochondrial antibodies: chipping away at
drogenase complex. Hepatology 23:436444 AMA-negative primary biliary cirrhosis.
4. Van de Water J, Ansari A, Prindiville T, Coppel Hepatology 45:659665
RL, Ricalton N, Kotzin BL, Liu S, Roche TE, 13. Kikutani H, Makino S (1992) The murine
Krams SM, Munoz S, Gershwin ME (1995) autoimmune diabetes model: NOD and related
Heterogeneity of autoreactive T cell clones strains. Adv Immunol 51:285322
specific for the E2 component of the pyruvate 14. Chen YG, Scheuplein F, Osborne MA, Tsaih
dehydrogenase complex in primary biliary cir- SW, Chapman HD, Serreze DV (2008)
rhosis. J Exp Med 181:723733 Idd9/11 genetic locus regulates diabetogenic
5. Ishibashi H, Shimoda S, Gershwin ME (2005) activity of CD4 T-cells in nonobese diabetic
The immune response to mitochondrial autoan- (NOD) mice. Diabetes 57:32733280
tigens. Semin Liver Dis 25:337346 15. Fox CJ, Paterson AD, Mortin-Toth SM,
6. Chuang YH, Ridgway WM, Ueno Y, Gershwin Danska JS (2000) Two genetic loci regulate T
ME (2008) Animal models of primary biliary cell-dependent islet inflammation and drive
cirrhosis. Clin Liver Dis 12:333347, ix autoimmune diabetes pathogenesis. Am J Hum
7. Folci M, Meda F, Gershwin ME, Selmi C Genet 67:6781
(2011) Cutting-edge issues in primary biliary 16. Fraser HI, Dendrou CA, Healy B, Rainbow
cirrhosis. Clin Rev Allergy Immunol DOI DB, Howlett S, Smink LJ, Gregory S, Steward
10.1007/s12016-011-82533 CA, Todd JA, Peterson LB, Wicker LS (2010)
8. Gershwin ME, Mackay IR (2008) The causes Nonobese diabetic congenic strain analysis of
of primary biliary cirrhosis: convenient and autoimmune diabetes reveals genetic complex-
inconvenient truths. Hepatology 47:737745 ity of the Idd18 locus and identifies Vav3 as a
9. Leung PS, Coppel RL, Gershwin ME (2005) candidate gene. J Immunol 184:50755084
Etiology of primary biliary cirrhosis: the search 17. Aoki CA, Borchers AT, Ridgway WM, Keen
for the culprit. Semin Liver Dis 25:327336 CL, Ansari AA, Gershwin ME (2005) NOD
10. Liu X, Invernizzi P, Lu Y, Kosoy R, Bianchi I, mice and autoimmunity. Autoimmun Rev
Podda M, Xu C, Xie G, Macciardi F, Selmi C, 4:373379
Lupoli S, Shigeta R, Ransom M, Lleo A, Lee 18. Koarada S, Wu Y, Fertig N, Sass DA, Nalesnik
AT, Mason AL, Myers RP, Peltekian KM, M, Todd JA, Lyons PA, Fenyk-Melody J,
Ghent CN, Bernuzzi F, Zuin M, Rosina F, Rainbow DB, Wicker LS, Peterson LB, Ridgway
Borghesio E, Floreani A, Lazzari R, Niro G, WM (2004) Genetic control of autoimmunity:
Andriulli A, Muratori L, Muratori P, Almasio protection from diabetes, but spontaneous
PL, Andreone P, Margotti M, Brunetto M, autoimmune biliary disease in a nonobese dia-
Coco B, Alvaro D, Bragazzi MC, Marra F, betic congenic strain. J Immunol 173:
Pisano A, Rigamonti C, Colombo M, Marzioni 23152323
M, Benedetti A, Fabris L, Strazzabosco M, 19. Irie J, Wu Y, Wicker LS, Rainbow D, Nalesnik
Portincasa P, Palmieri VO, Tiribelli C, Croce L, MA, Hirsch R, Peterson LB, Leung PS, Cheng
Bruno S, Rossi S, Vinci M, Prisco C, Mattalia C, Mackay IR, Gershwin ME, Ridgway WM
A, Toniutto P, Picciotto A, Galli A, Ferrari C, (2006) NOD.c3c4 congenic mice develop
314 P.S.C. Leung et al.
autoimmune biliary disease that serologically 30. Kim WR, Poterucha JJ, Jorgensen RA, Batts
and pathogenetically models human primary KP, Homburger HA, Dickson ER, Krom RA,
biliary cirrhosis. J Exp Med 203:12091219 Wiesner RH, Lindor KD (1997) Does antimi-
20. Yang GX, Wu Y, Tsukamoto H, Leung PS, tochondrial antibody status affect response to
Lian ZX, Rainbow DB, Hunter KM, Morris treatment in patients with primary biliary cir-
GA, Lyons PA, Peterson LB, Wicker LS, rhosis? Outcomes of ursodeoxycholic acid ther-
Gershwin ME, Ridgway WM (2011) CD8 T apy and liver transplantation. Hepatology
cells mediate direct biliary ductule damage in 26:2226
nonobese diabetic autoimmune biliary disease. 31. Moritoki Y, Zhang W, Tsuneyama K, Yoshida
J Immunol 186:12591267 K, Wakabayashi K, Yang GX, Bowlus C,
21. Oertelt S, Lian ZX, Cheng CM, Chuang YH, Ridgway WM, Ueno Y, Ansari AA, Coppel RL,
Padgett KA, He XS, Ridgway WM, Ansari AA, Mackay IR, Flavell RA, Gershwin ME, Lian ZX
Coppel RL, Li MO, Flavell RA, Kronenberg (2009) B cells suppress the inflammatory
M, Mackay IR, Gershwin ME (2006) Anti- response in a mouse model of primary biliary
mitochondrial antibodies and primary biliary cirrhosis. Gastroenterology 136:10371047
cirrhosis in TGF-beta receptor II dominant- 32. Dhirapong A, Lleo A, Yang GX, Tsuneyama K,
negative mice. J Immunol 177:16551660 Dunn R, Kehry M, Packard TA, Cambier JC,
22. Gorelik L, Flavell RA (2000) Abrogation of Liu FT, Lindor K, Coppel RL, Ansari AA,
TGFbeta signaling in T cells leads to spontane- Gershwin ME (2011) B cell depletion therapy
ous T cell differentiation and autoimmune dis- exacerbates murine primary biliary cirrhosis.
ease. Immunity 12:171181 Hepatology 53:527535
23. Taylor AW (2009) Review of the activation of 33. Chuang YH, Lian ZX, Tsuneyama K, Chiang
TGF-beta in immunity. J Leukoc Biol BL, Ansari AA, Coppel RL, Gershwin ME
85:2933 (2006) Increased killing activity and decreased
24. Yoshimura A, Wakabayashi Y, Mori T (2010) cytokine production in NK cells in patients
Cellular and molecular basis for the regulation with primary biliary cirrhosis. J Autoimmun
of inflammation by TGF-beta. J Biochem 26:232240
147:781792 34. Chuang YH, Lian ZX, Yang GX, Shu SA,
25. Ebert EC, Panja A, Das KM, Praveen R, Geng Moritoki Y, Ridgway WM, Ansari AA,
X, Rezac C, Bajpai M (2009) Patients with Kronenberg M, Flavell RA, Gao B, Gershwin
inflammatory bowel disease may have a trans- ME (2008) Natural killer T cells exacerbate
forming growth factor-beta-, interleukin (IL)- liver injury in a transforming growth factor
2- or IL-10-deficient state induced by intrinsic beta receptor II dominant-negative mouse
neutralizing antibodies. Clin Exp Immunol model of primary biliary cirrhosis. Hepatology
155:6571 47:571580
26. Kel JM, Girard-Madoux MJ, Reizis B, Clausen 35. Yoshida K, Yang GX, Zhang W, Tsuda M,
BE (2010) TGF-beta is required to maintain Tsuneyama K, Moritoki Y, Ansari AA, Okazaki
the pool of immature Langerhans cells in the K, Lian ZX, Coppel RL, Mackay IR, Gershwin
epidermis. J Immunol 185:32483255 ME (2009) Deletion of interleukin-12p40 sup-
presses autoimmune cholangitis in dominant
27. Perruche S, Zhang P, Maruyama T, Bluestone negative transforming growth factor beta
JA, Saas P, Chen W (2009) Lethal effect of receptor type II mice. Hepatology 50:
CD3-specific antibody in mice deficient in 14941500
TGF-beta1 by uncontrolled flu-like syndrome.
J Immunol 183:953961 36. Nakamura A, Yamazaki K, Suzuki K, Sato S
(1997) Increased portal tract infiltration of
28. Yang GX, Lian ZX, Chuang YH, Moritoki Y, mast cells and eosinophils in primary biliary cir-
Lan RY, Wakabayashi K, Ansari AA, Flavell RA, rhosis. Am J Gastroenterol 92:22452249
Ridgway WM, Coppel RL, Tsuneyama K,
Mackay IR, Gershwin ME (2008) Adoptive 37. Lan RY, Cheng C, Lian ZX, Tsuneyama K,
transfer of CD8(+) T cells from transforming Yang GX, Moritoki Y, Chuang YH, Nakamura
growth factor beta receptor type II (dominant T, Saito S, Shimoda S, Tanaka A, Bowlus CL,
negative form) induces autoimmune cholangi- Takano Y, Ansari AA, Coppel RL, Gershwin
tis in mice. Hepatology 47:19741982 ME (2006) Liver-targeted and peripheral blood
alterations of regulatory T cells in primary bil-
29. Invernizzi P, Crosignani A, Battezzati PM, iary cirrhosis. Hepatology 43:729737
Covini G, De Valle G, Larghi A, Zuin M, Podda
M (1997) Comparison of the clinical features 38. Buckner JH (2010) Mechanisms of impaired
and clinical course of antimitochondrial anti- regulation by CD4(+)CD25(+)FOXP3(+) reg-
body-positive and -negative primary biliary cir- ulatory T cells in human autoimmune diseases.
rhosis. Hepatology 25:10901095 Nat Rev Immunol 10:849859
14 Animal Models of Primary Biliary Cirrhosis: Materials and Methods 315
58. Kinjo Y, Wu D, Kim G, Xing GW, Poles MA, Peterson LB, Wicker LS (2000) The NOD
Ho DD, Tsuji M, Kawahara K, Wong CH, Idd9 genetic interval influences the pathoge-
Kronenberg M (2005) Recognition of bacterial nicity of insulitis and contains molecular vari-
glycosphingolipids by natural killer T cells. ants of Cd30, Tnfr2, and Cd137. Immunity
Nature 434:520525 13:107115
59. Mattner J, Debord KL, Ismail N, Goff RD, 62. Wicker LS, Leiter EH, Todd JA, Renjilian RJ,
Cantu C 3rd, Zhou D, Saint-Mezard P, Wang Peterson E, Fischer PA, Podolin PL, Zijlstra
V, Gao Y, Yin N, Hoebe K, Schneewind O, M, Jaenisch R, Peterson LB (1994) Beta
Walker D, Beutler B, Teyton L, Savage PB, 2-microglobulin-deficient NOD mice do not
Bendelac A (2005) Exogenous and endogenous develop insulitis or diabetes. Diabetes
glycolipid antigens activate NKT cells during 43:500504
microbial infections. Nature 434:525529 63. Koarada S, Wu Y, Yim YS, Wakeland EW,
60. Mattner J, Savage PB, Leung P, Oertelt SS, Ridgway WM (2004) Nonobese diabetic CD4
Wang V, Trivedi O, Scanlon ST, Pendem K, lymphocytosis maps outside the MHC locus
Teyton L, Hart J, Ridgway WM, Wicker LS, on chromosome 17. Immunogenetics
Gershwin ME, Bendelac A (2008) Liver auto- 56:333337
immunity triggered by microbial activation of 64. Podolin PL, Denny P, Armitage N, Lord CJ,
natural killer T cells. Cell Host Microbe Hill NJ, Levy ER, Peterson LB, Todd JA,
3:304315 Wicker LS, Lyons PA (1998) Localization of
61. Lyons PA, Hancock WW, Denny P, Lord CJ, two insulin-dependent diabetes (Idd) genes to
Hill NJ, Armitage N, Siegmund T, Todd JA, the Idd10 region on mouse chromosome 3.
Phillips MS, Hess JF, Chen SL, Fischer PA, Mamm Genome 9:283286
Chapter 15
Abstract
Current models for systemic sclerosis have many limitations for mimicking human disease. We have recently
described a novel model based on chronic stimulation of skin by poly(I:C), a form of double-stranded
RNA known to stimulate innate immune responses. This model shows inflammation and extracellular
matrix deposition, and upregulated expression of genes associated with fibrosis and vascular injury. We
detail here the methodology for this model, utilizing osmotic pumps to deliver innate immune stimuli to
subcutaneous tissue. Subcutaneous osmotic pumps provide a flexible system for studying innate immunity
in the skin as well as the affects of other ligands on skin inflammation and fibrosis.
Key words: Skin, Innate immunity, Osmotic pump, Toll-like receptor, Systemic sclerosis, Scleroderma
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_15, Springer Science+Business Media New York 2012
317
318 M. Dimarzio et al.
1.2. Modeling Innate Skin in patients with systemic sclerosis (SSc) is characterized by
Immune-Mediated collagen deposition and varying degrees of inflammation. Poly(I:C)
Inflammation and is a synthetic double-stranded RNA that acts as a ligand for toll-like
Fibrosis receptor 3 (TLR3), but is also a cytosolic sensor, melanoma differ-
entiation-associated protein-5 (MDA5) (2). Poly(I:C) is a particularly
potent stimulus of innate immune responses by dermal fibroblasts,
upregulating both pro-inflammatory and pro-fibrotic gene expres-
sion (3, 4). We have recently described utilization of osmotic pump
delivery of poly(I:C) as a model for understanding the effect of
chronic innate immune activation in the skin. We have shown using
this model that poly(I:C) delivered subcutaneously over 7 days
induces expression of both interferon- and transforming growth
factor--regulated genes (3, 4). This pattern of gene expression is
similar to what we have seen in the skin of patients with SSc,
suggesting that this model reproduces some of the relevant patho-
logical features of this disease. In addition, if 28-day osmotic pumps
are used matrix deposition, fat loss, and myofibroblast accumula-
tion is seen, thus reproducing all the typical features of SSc skin.
Although we will be focusing on the methodology used for the
poly(I:C) model of SSc, we have used the same basic approach
with other ligands, in particular TGF, but also in ongoing work
using other protein and nucleic acid ligands (ref. 4 and R. Lafyatis
unpublished).
1.3. Summary The simplicity of this system is its major advantage. However, careful
of Methodology attention to sterility and consideration of outcomes to be assessed
(histology, immunofluorescence, and gene expression) are key
features to success. The goal of this chapter is to describe how
specific placement of poly(I:C) in osmotic pumps can be employed
15 Modeling Innate Immunity in Murine Skin 319
2. Materials
3. Methods
3.1. Preparation Prior These procedures are carried out in a tissue culture hood.
to Surgery: Selection
1. Pumps and modulators are removed from supplied packaging
and Loading Osmotic
and dropped into sterile petri dish(es). Typically five pumps
Pumps, and can be placed into one 100 mm petri dish. Pumps should be
Preparation of divided so that pumps in each petri dish will be filled with the
Anesthesia/Analgesia same ligand (see Note 2).
2. Poly(I:C) 0.5 mg/ml or other ligand at the appropriate con-
centration (to be determined empirically) to be placed into the
pump is prepared by dissolving in sterile PBS in sterile
Eppendorf or equivalent.
3. The person to load pumps dons sterile gloves.
4. A second person opens a sterile 1 ml syringe, handling only the
package and delivers the syringe sterilely to the pump loader.
5. While the sterile syringe is held steady by the pump loader, the
filling tube included with the pumps is opened and pushed
onto the sterile syringe by holding and then removing the needle
cap by the second person.
6. The pump loader draws up the sterile solution of PBS, poly(I:C)
or other ligand sufficient to fill one or several pumps (~250 l/
pump).
7. The needle-like filling tube is carefully inserted into the top
of the pump and the pump filled (see Note 3).
8. The regulator is carefully inserted into the pump top.
9. Steps 68 are repeated, filling all pumps with the same ligand
solution.
10. If pumps containing another ligand are to be loaded, then
steps 49 are repeated with other ligands and/or control PBS
(see Note 4), using a new filing tube for each ligand.
11. During the procedure above, the second person assists the
gloved pump loader by opening and closing petri dishes and
vials of ligands.
12. Petri dishes with loaded pumps are all covered, gloves are
removed.
13. 0.5 ml Xylazine (20 mg/ml stock), 1.0 ml ketamine (100 mg/
ml stock), and 4 ml buprenorphine (0.3 mg/ml stock) are
drawn by syringe/needle sterilely from vials and mixed into
14.5 ml PBS.
14. The diluted anesthetic/analgesic solution is sterilely drawn
into 3 ml syringes (~2.5 ml/syringe).
15 Modeling Innate Immunity in Murine Skin 321
3.2. Induction These procedures are carried out in a laminar flow hood in the
of Anesthesia animal facility. The hood surface is cleaned with 70 % ethanol.
and Pump Insertion
1. Mice are weighed (see Note 5).
2. 0.02 ml/g mouse of the diluted anesthetic/analgesic solution
(0.5 ml for typical 25 g, 68 week mouse), is injected intrap-
eritoneally, low on the abdomen directed caudally, after brief
spray sterilization of the mouse abdomen with 70 % ethanol.
3. Mice typically fall to sleep in 23 min.
4. While mice are falling asleep, the sterilized instruments are
placed into the 50 ml conical holding ~40 ml 70 % ethanol.
5. After the mice are sleeping, an area approximately 1 cm 2 cm
across the mid scapula is shaved (see Note 6). Typically pumps
can be inserted in up to five mice in parallel (a batch). After
shaving, the hair needs to be completely removed from the
hood so that it does not contaminate the operating field. Mice
should be placed on a towel to help maintain body heat during
the remainder of the procedure.
6. The shaved area on each mouse is generously sterilized with
betadine solution either on a swab or gauze. In young mice the
ears might also be sterilized.
7. Using forceps and scissors, a 78 mm incision is made over the
upper back tangentially to spine and laterally to the midline
(see Fig. 1). The incision needs to pierce the subcutaneous
tissue, but not enter the underlying muscle (see Note 7).
Fig. 1. Pump insertion. A region over the midscapula is shaved and an incision made with scissors (panel a). Hemostats or
needle holders are used for blunt dissection, forming a pocket for the pump (panel b). The pump is inserted through the
incision into the subcutaneouspocket (panel c) and stapled closed with 23 staples (panel d). Panel (e) shows pump in
place with wound completely closed.
322 M. Dimarzio et al.
Fig. 2. Histologic evaluation of skin from poly(I:C) treated mice. Formalin fixed, H&E stained skin 7 days after placement of
7-day osmotic pumps containing PBS (panel a) or poly(I:C) (panel b, bold arrows shows inflammation of fat, narrow arrow
shows early fibrosis), or 28 days after placement of 28-day pump: low power (panel c) or high power (panel d, arrows
indicate area of fibrosis).
3.4. Assessment 1. The critical step for RNA preparation from skin is vigorous and
of Gene Expression immediate homogenization of the tissue. Tissue cut from the
in Skin area of the pump outlet is placed on aluminum foil and minced
thoroughly with scissors.
2. The minced skin is submerged in 2.0 ml Trizol in a 15 ml coni-
cal tube and homogenized vigorously, typically 1520 s until
no visible pieces of skin are visible. At this point the homoge-
nized tissue solution may be stored at 80 C.
3. Thaw the homogenized Trizol/tissue homgenate at 37 C for
>5 min to permit complete dissociation of nucleoprotein com-
plexes and then centrifuge at >2,000 rpm (1,800 g) in clinical
centrifuge to pellet debris.
4. Aliquot and refreeze 1 ml of the clarified tissue/Trizol solution for
back-up if needed. Transfer the remaining 1.0 ml tissue/Trizol
solution to a 1.5 ml Eppendorf, and continue RNA purification
according to the protocol included with Trizol reagent.
5. Resuspend RNA in 50 l RNase-free H2O and store at
80 C.
6. Prepare cDNA and analyze for gene expression by real-time
q-PCR using standard methods.
324 M. Dimarzio et al.
4. Notes
References
1. http://www.alzet.com/products/guide_to_ 4. Farina GA, York MR, Di Marzio M, Collins
use/pump_selection.html CA, Meller S, Homey B, Rifkin IR, Marshak-
2. Kawai T, Akira S (2008) Toll-like receptor and Rothstein A, Radstake TR, Lafyatis R (2010)
RIG-I-like receptor signaling. Ann NY Acad Poly(I:C) drives type I IFN- and TGFbeta-
Sci 1143:120 mediated inflammation and dermal fibrosis
3. Farina G, York M, Collins C, Lafyatis R (2010) simulating altered gene expression in sys-
dsRNA activation of endothelin-1 and markers temic sclerosis. J Invest Dermatol 130:
of vascular activation in endothelial cells and 25832593
fibroblasts. Ann Rheum Dis 70:544550
Chapter 16
Abstract
Scleroderma is an autoimmune disease characterized by the progressive and dysregulated accumulation of
collagen in the skin and internal organs. Pulmonary complications including interstitial lung disease have
emerged as the greatest cause of mortality in this disease. Because treatments are limited, new areas of
investigation are sorely needed. An emerging area of interest in this field is a potential role for fibrocytes as
biomarkers or mediators of disease. Fibrocytes are monocyte-derived mesenchymal progenitor cells that
exhibit features of extracellular matrix production and wound contraction in addition to immunologic
functions such as cytokine and chemokine production, antigen presentation, leukocyte trafficking, and
modulation of angiogenesis. Fibrocytes could participate in the pathogenesis of scleroderma lung disease
through any or all of these functions and may be useful biomarkers of disease activity. This chapter presents
protocols that have been developed for the study of fibrocytes obtained from human circulation and tis-
sues. Protocols for the quantification of fibrocytes in murine models also are described, along with discus-
sion of common technical challenges. It is hoped that this information will allow further investigation of
the role that fibrocytes might play in Scleroderma-related lung disease and perhaps lead to new areas of
study in this difficult-to-treat and deadly disease.
Key words: Biomarker, Collagen, Extracellular matrix, Fibrocyte, Flow cytometry, Scleroderma,
Pulmonary fibrosis
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_16, Springer Science+Business Media New York 2012
327
328 T.M. Russell et al.
1.1. Fibrocytes: fibrocytes might play important roles in tissue repair and remodel-
Disease Associations ing. These cells are identified by their co-expression of leukocyte
markers such as CD45, extracellular matrix proteins such as
Collagen-1, and stem cell markers such as CD34 (8). An increas-
ing amount of data highlight the association between peripheral
blood fibrocyte abnormalities in diverse forms of autoimmune dis-
ease such as rheumatoid arthritis (9) autoimmune thyroiditis (10),
amyopathic antisynthetase syndrome (11), and scleroderma (12,
13). Fibrocytes also are present in many forms of chronic
inflammatory disorders that are not classically associated with auto-
immunity including idiopathic pulmonary fibrosis (IPF) (1416),
asthma (1719), nephrogenic systemic fibrosis (20), cardiovascular
disease (21), pulmonary hypertension (22), and even normal aging
(12). Furthermore, animal modeling implicates fibrocytes in the
development of organ fibrosis for which fibroblasts, and their acti-
vated counterpart, the myofibroblast, are believed to be centrally
involved including the kidney (23, 24), liver (25), heart (2628),
vasculature (29), and lungs (15, 16, 30, 31). In many of these
studies fibrocytes appeared to function as a biomarker of disease
activity, which has led to speculation that similar parallels may be
drawn in patients with SSc-ILD. For all of these reasons, the study
of fibrocytes has become particularly important and timely.
1.3. Fibrocytes: Insight into fibrocyte function may be gleaned from an under-
Differentiation standing of the factors promoting their differentiation and recruit-
and Homing ment. Fibrocytes differentiate from a precursor population present
within the CD14+ monocyte fraction of peripheral blood (37).
Further enrichment for CD11b(+) CD115(+) Gr1(+) expressing
monocytes increases the monocyte-to-fibrocyte transition in cul-
tured murine cells; these effects are promoted by direct contact
with activated CD4+ lymphocytes via an mTOR-PI3 kinase-depen-
dent pathway (38). Other studies have determined that the fibrocyte
precursor expresses the Fc receptor (39) and that inhibition of this
receptor with the short pentraxin protein serum Amyloid P
significantly reduces fibrocyte outgrowth in human (39, 40), rat
(41), and murine samples (30) via an ITIM-dependent mechanism
(42). Fibrocyte differentiation from CD14+ precursors is also
reduced by the TH1 cytokines IFN, TNF, and IL-12 and is aug-
mented by the TH2 cytokines IL-4 and IL-13 (43), TGF-1, and
engagement of the 1 integrin subunit (13, 22). These latter effects
require Erk phosphorylation (22). However, careful lineage
tracing studies will be required to confirm the monocyte origin of
fibrocytes under different fibrogenic stimuli.
Murine fibrocytes express the chemokine receptors CCR2,
CCR7, and CXCR4 which mediate the recruitment of fibrocytes to
injured tissue (31, 44, 45). Human fibrocytes also express the
chemokine receptors CCR3 (eotaxin receptor) and CCR5 (MCP-1
receptor) as well as CD29 and Semaphorin 7a (46). While there are
only limited data regarding the mediators that affect fibrocyte
recruitment in humans, our own work demonstrates an association
between concentrations of soluble factors such as TNF, IL-10,
330 T.M. Russell et al.
Table 1
Fibrocyte marker expression
1.4. Fibrocytes: It has been postulated that the ultimate phenotype of fibrocytes is
Functions the contractile myofibroblast (31, 37, 46, 48), a hypothesis that is
based on the finding that cultured fibrocytes respond to TGF-1
by expressing alpha-Smooth Muscle Actin (-SMA) and contract-
ing collagen gels in vitro (31, 37, 46, 48). However, the ability of
fibrocytes to differentiate into myofibroblasts in vivo is less clear.
Studies using bone marrow transplantation show only a minimal
contribution of fibrocytes to -SMA production in some models
(25, 49, 50), implying that this differentiation pathway may not
necessarily be a dominant feature of fibrocytes in the tissue remod-
eling response.
While a specific contribution to disease pathogenesis has yet to
be established, fibrocytes exhibit many functions that would be
expected to play a role in different autoimmune diseases (Fig. 1).
Human fibrocytes respond to Interleukin-1 beta (IL-1) by
increasing secretion of Interleukin-6 (IL-6), Interleukin-8 (IL-8),
Chemokine (C-C motif) ligand 2 (CCL2, also called monocyte
chemotactic protein-1 or MCP-1), and Chemokine (C-C motif)
ligand 3 (CCL3, also called Macrophage inflammatory protein-
1) and by increasing expression of Intercellular adhesion mole-
cule-1 (ICAM-1) which would be expected to recruit and activate
leukocytes (51). Porcine fibrocytes respond to Toll-like receptor
(TLR) agonists and viral infection to increase expression of MHC
I and II, and the costimulatory proteins CD80 and CD86 which
allow efficient antigen presentation and subsequent activation of
cytotoxic CD8+ cells (52). Human fibrocytes demonstrate these
properties as well (34). In addition to these proinflammatory func-
tions, fibrocytes also respond to IL-1 by increasing Interleukin-10
(IL-10) production (51) which would be expected to reduce
inflammation. Fibrocytes could influence repair and remodeling
through their ability to express -SMA and enact wound contrac-
tion in ex vivo wound chambers (8). Fibrocytes also exhibit a dis-
tinctive pattern of ECM production characterized by high levels of
Collagen V, hyaluronan, versican, and perlecan (36); this profile
would be expected to promote recruitment of inflammatory cells
332 T.M. Russell et al.
Fig. 1. Potential contributions of fibrocytes to autoimmune pathogenesis. In the setting of autoantigen exposures, acute
injury, IL-1, serum factors, and innate immune stimuli with TLRs and viral infection fibrocytes adopt a proinflammatory
phenotype characterized by secretion of IFN, IL-6, IL-8, CCL3, and CCL4. Leukocyte trafficking is enhanced via ICAM-1.
Production of ECM components is reduced by exposure to SAP via Fc-mediated effects. Antigen presentation to T cells is
performed by CD80, CD86, MHCI, and MHCII. Local tissue destruction may be increased by expression of MMPs. As the
local milieu begins to favor repair and remodeling (or perhaps concurrent with ongoing injury in the right biological context)
fibrocytes evolve into a more reparative cell, perhaps in response to exogenous or autocrine secretion of IL-10. Here
TGF-1 stimulates fibrocyte development via noncanonical pathways mediated by Semaphorin 7a and CD29, though other
TGF-1 signaling pathways may also be involved. Sema 7a could activate monocytes and dendritic cells while dampening
T cell responses. ECM production is further stimulated by TH2 cytokines such as IL-4 and IL-13 as well as by exposure to
apoptotic cells. Transformation of tissue-resident fibroblasts into activated myofibroblasts is promoted by TGF-1 pro-
duced by fibrocytes, though fibrocytes may also adopt -SMA expression in response to TGF-1 and ET-1. PDGF, IL-10,
VEGF, HGF, and b-HGF support neoangiogenesis, and recruitment to sites of injury is promoted via expression of chemokine
receptors such as CXCR4.
2. Materials
3. Methods
3.1. Human Peripheral 1. Collect peripheral blood in sterile sodium heparin tubes (10 ml
Blood Mononuclear draw).
Cell Collection and 2. Perform the following procedures in hood with sterile
Staining technique.
3.1.1. Separation 3. Dilute blood with PBS 1:2 blood to PBS.
of Peripheral Blood 4. Layer diluted blood over histopaque: 1:3 histopaque to blood/
Mononuclear Cells PBS (typically 10 ml of histopaque to 30 ml blood/PBS).
from Plasma
5. Centrifuge at 10,625 g for 22 min at 12 C with acceleration,
deceleration curves at (1,1) (most gradual).
6. Remove peripheral blood mononuclear cells (PBMCs) in the
buffy coat layer (layer between plasma and histopaque) with
pipettor.
7. Wash with PBS twice, and centrifuge at 10,625 g for 5 min at
4 C with acceleration, deceleration curves at (9,9).
8. Count cells with hemocytometer and trypan blue and resus-
pend at one million cells/ml.
9. If needed, separate cells for RNA and DNA analysis prior to
FACS Staining (one million cells/Eppendorf, individual
Eppendorfs for RNA and DNA).
(a) RNA and DNA analysis (non-sterile).
(b) Centrifuge cells at 10,625 g for 1 min.
(c) Pipette off supernatant.
(d) Store DNA pellet at 80 C.
(e) Resuspend RNA pellet in 350 l of TRIzol buffer and
store at 80 C.
3.1.2. Staining of Cells 1. Place approximately 1 106 cells/tube. Prepare the following
for CD45 and Pro- tubes:
Collagen-1 Expression (a) No Stain.
(b) CD45-FITC + PerCP isotype antibody.
(c) CD45 PerCP (2 l) + Isointracellular + 2Ab.
(d) CD45 PerCP (2 l), Rat Anti-human Procol-I FITC.
336 T.M. Russell et al.
3.1.3. Human Lung Cell Lung biopsy is rarely performed for the diagnosis of lung disease in
Isolation and Staining patients with scleroderma. However, the following protocol is suit-
able for use in explanted scleroderma lungs, lung samples obtained
from nonfibrotic controls and from lung biopsies performed for
other types of fibrotic lung disease (autoimmune and idiopathic).
1. Lung sample should be placed in DMEM + 10 % FBS and trans-
ported to the laboratory from the pathology department.
2. Upon arrival in laboratory remove from medium, place in
100 mm Petri Dish, and cut into 1 cubes.
16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 337
Fig. 2. Flow cytometric identification of fibrocytes in human peripheral blood. (a) Live cells are selected based on FSC vs.
SSC. (b, c) FITC-detected intracellular isotype control (X axis) vs. anti-CD45-PerCP (Y axis). The negative gate for PerCP is
set (b). PerCP-stained CD45+ve cells are selected and the negative gate for intracellular FITC staining is chosen (c).
(d) This gate is then applied to the sample stained with FITC-detected Pro-Collagen-I (X axis) vs. anti-CD45-PerCP. The
dual-positive cells in the right upper quadrant represent fibrocytes. The proportion of dual-positive cells in the right upper
quadrant in the negative control (c) is subtracted from the proportion of dual-positive cells in the sample stained with Pro-
Collagen-I (d) to determine the overall percentage of double-positive cells. (eh) Importance of choosing correct controls.
(e) Extracellular FITC isotype control (X axis) vs. anti-CD45-PerCP (Y axis) on an unpermeabilized sample from the subject
shown in panel (d). As can be seen, there is only minimal shift of the CD45+ve cells to the right. When this gate is applied
to the sample shown in (d), nearly 100 % of the cells fall into the right upper quadrant (f). This problem demonstrates the
importance of permeabilizing all of the control samples. Another commonly encountered problem is the comparison of
samples from different individuals run at the same time. Panel (g) demonstrates a sample from a normal control that was
stained with FITC-detected intracellular isotype control (X axis) vs. anti-CD45-PerCP (Y axis). There is a much less pro-
nounced rightward shift of the CD45+ve cells. When this gate is applied to the scleroderma subject, again a very large
number of cells fall into the double-positive gate. This commonly encountered problem can be avoided by preparing a
CD45/intracellular isotype control for each subject.
3.2. Mouse Sacrifice Murine modeling allows mechanistic studies of the pathways regu-
and Isolation of Blood lating fibrocyte biology. For this reason, assessment of circulating
and Lung Cells and/or tissue fibrocytes has emerged as an important tool in mouse
models of scleroderma lung disease caused by either TGF-1 over-
expression (30) or Bleomycin administration (31). Presented here
is a detailed approach to harvest and preparation of the murine
blood and lung for flow cytometric analysis of circulating and intra-
pulmonary fibrocytes. Users should modify the detailed outline
below for their own particular tissue.
3.2.1. Sacrifice of Mouse 1. Anesthetize mouse so that it is unresponsive to paw pinch and
is breathing deeply. Do not perform cervical dislocation.
2. Place mouse in supine position with limbs extended and immo-
bilized on a foil covered Styrofoam board that has been cov-
ered with paper towels. Spray mouse with 70% ethanol.
3. With the mouse head at the top of the board, use scissors and
sharp forceps to create a 1 cm incision above trachea.
4. Use blunt forceps to separate tissue covering trachea. Be care-
ful not to transect the blood vessels that run vertically along
the trachea.
5. Use sharp forceps to cleanly and carefully lift exposed trachea.
With opposite hand use the second set of forceps to pass suture
material underneath.
6. Loosely tie suture in knot around trachea.
7. Insert 23 gauge angiocatheter directly into exposed trachea,
advance 1 cm, remove metal stylet, and tie suture tightly to
stabilize catheter.
(a) Note: Advancing the catheter too far will puncture the tra-
chea. For this reason, it may be useful to make a small
mark on the angiocatheter about 1 cm from the bottom.
When this mark is reached, the catheter should be advanced
no further.
Fig. 3. Flow cytometric analysis of fibrocytes in fibrotic human lungs. (a) Live cells are gated on based on FSC (X axis) vs.
SSC (Y axis). (b) Unstained cells demonstrate mild to moderate autofluorescence in the FL1 (X axis) and FL2 (Y axis) chan-
nels. (c) FITC-positive control (X axis) vs. PE-negative control (Y axis). This gate is used to set the negative gate for PE.
(d, e) FITC-detected intracellular isotype control (X axis) vs. anti-CD45-PE (Y axis). CD45+ve cells are gated on (d) and the
negative gate for FITC is set (e). (f) FITC-detected intracellular Pro-Collagen-I staining (X axis) vs. anti-CD45-PE (Y axis).
The dual-positive cells in the right upper quadrant meet flow cytometric criteria for fibrocytes. These panels demonstrate
acceptable staining for fibrocytes. Panels (gi) demonstrate unacceptable staining. (g) Unstained cells from a poorly pre-
pared specimen demonstrate high autofluorescence in the FL-1 (X axis) and FL-2 (Y axis) channels. (h) FITC-detected
intracellular isotype control (X axis) vs. anti-CD45-PE (Y axis) on this sample. The diagonal line evident in the left upper
quadrant indicates that the baseline autofluorescence of the sample has made compensation difficult. (i) FITC-detected
intracellular Pro-Collagen-I staining (X axis) vs. anti-CD45-PE (Y axis). The dual-positive population, which represents more
than 50 % of the CD45+ve cells, demonstrates a similar diagonal morphology and represents false positive staining.
2. Rotate mouse 180 so that feet are now at the top of the board
and make vertical incision from below xyphoid/rib cage up to
and including the incision in the neck, cut through ribs and
expose heart (still beating) and lungs, and transect inferior
vena cava. Exsanguination will occur.
3. Insert a 25 gauge needle attached to a 10 ml syringe containing
50 l of EDTA into the left ventricle. Remove as much blood
340 T.M. Russell et al.
3.2.3. Removal 1. Tie off lungs, use scissors to remove, and place on ice for
and Enzymatic Digestion 2 min.
of Lungs 2. Following incubation on ice, transfer lungs to a 14 ml Falcon
tube eppendorf tube containing 3 ml prewarmed collagenase
and incubate at 37 C 25 min.
3. Remove lungs from collagenase and mince into small pieces
with scissors.
4. Place lung pieces in 100 mm Petri dish containing 7 ml DMEM
supplemented with 10 % NGS and 100 U DNAseI/ml (lung
dissociation buffer) and gently use surgical instruments to
homogenize tissue.
5. Add an additional 510 ml of lung dissociation buffer to Petri
dish in increments and then pour over filter (gently pushing
tissue through between increments).
(a) Note: In the fibrotic lung there will be some areas that
do not digest as well as others; these are remodeled areas
containing increased extracellular matrix proteins.
6. Centrifuge at 10,625 g for 5 min at 4 C with acceleration,
deceleration curves at (9,9).
7. Resuspend in x ml (based on the estimate of cell count) of
FACS Buffer and use a hemocytometer or Coulter Counter to
perform cell count.
8. Resuspend cells so that concentration is 1 106 cells/ml.
16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 341
3.2.5. Staining of Murine 1. Divide cells up to 1 106 cells/test tube using 22 m Nytex
Blood and Dissociated mesh to help de-aggregate the cells. Samples are as follows:
Lung Cells for Fibrocyte (a) Unstained.
Identification
(b) CD45 FITC (0.5 l) and PerCP isotype control.
(c) CD45 PerCP + isointracellular + 2Ab.
(d) Stain 1 [CD45 PercP (0.5 l)], Collagen-1 FITC.
2. Centrifuge cells at 10,625 g for 5 min at 4 C with accelera-
tion, deceleration curves at (9,9).
3. Resuspend cells in 100 l of 10 % NGS FACS Buffer and 5 ml/
sample of FCR blocker.
4. Add appropriate antibodies (amount noted above) for extra-
cellular staining.
5. Incubate at 4 C for 30 min.
6. Wash with FACS Buffer twice, and centrifuge at 10,625 g for
5 min at 4 C with acceleration, deceleration curves at (9,9).
7. Add 100200 l of cytofix/cytoperm to the remaining samples
and incubate at 4 C for 15 min.
8. Wash with P/W, and centrifuge at 10,625 g for 5 min at 4 C
with acceleration, deceleration curves at (9,9).
9. Fix all samples that were only stained with extracellular anti-
bodies in 100200 l of 4 % PFA and store at 4 C.
10. Resuspend intracellular samples in 100 l of P/W and add 1 l
of Anti-collagen antibody.
11. Incubate in fridge for 30 min.
12. Wash all with P/W and centrifuge at 10,625 g for 5 min at
4 C with acceleration, deceleration curves at (9,9).
13. Add 100 l of Fluorescein dilution (1:1,000 Fluorescein to
P/W) to all intracellular samples [Secondary antibody step
(2Ab)].
14. Incubate at 4 C for 30 min.
342 T.M. Russell et al.
15. Wash with P/W, then wash with FACS Buffer, and centrifuge
at 10,625 g for 5 min at 4 C with acceleration, deceleration
curves at (9,9).
16. Fix all intracellular samples with 100200 l of 4 % PFA and
store at 4 C.
3.3. Flow Cytometry 1. Open Cell Quest, connect to the cytometer, and make sure
that the setup box is checked.
3.3.1. Flow Cytometry of
Stained Samples 2. Voltages for the FACS Calibur are set using the non-stain
control.
(a) Adjust the Forward Scatter (FSC) (controls lateral move-
ment of events on graph if FSC is X-axis) and Side Scatter
(SSC) (controls vertical tilt if SSC is Y-axis) to record as
many of the cells of interest as possible.
(b) Using histograms of each of the channels (FL1, FL3) or a
dot plot, adjust voltages so that the majority of events
occur between 0 and 101.
3. Compensations for the FACS Calibur are set using the single-
color controls.
(a) Using the FITC control, adjust the compensation so that
the majority of events occur in the FITC channel (it will
bleed into PE).
(b) Using the PerCP control, adjust the compensations so
that the majority of events occur in the PerCP channel
(it will bleed into PE and APC).
4. Uncheck the setup box, set the location to record all samples,
and acquire all samples (including controls).
3.4. Analysis of Flow 1. Open the flow cytometry analysis software (in this case, Flowjo)
Cytometry Data and import FACS files.
3.4.1. FACS Analysis for 2. First a gate is set for all of the live cells based on the FSC and
Fibrocytes SSC and applied to all samples (Fig. 2a). It is important to
include only live cells as dead/dying cells can increase
autofluorescence. In addition, it is important to be consistent
in the populations being analyzed throughout samples.
3. Quadrant gating is established through comparing the single-
color positive controls to the isotype controls (ideally single-color
positive controls should be about 90 % positive, and isotype con-
trols should be greater than 97 % negative) (Fig. 2b, c).
4. The negative gate for fibrocyte analysis is determined by stain-
ing a CD45-stained sample with intracellular isotype control
and secondary antibody (Fig. 2c).
5. Once the negative gate for Col-I staining of CD45+ cells is
established, this gating strategy should be applied to the
sample(s) of interest (Fig. 2d).
16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 343
4. Notes
Acknowledgements
References
1. Steen VD, Medsger TA (2007) Changes in 8. Bucala R, Spiegel LA, Chesney J, Hogan M,
causes of death in systemic sclerosis, 1972 Cerami A (1994) Circulating fibrocytes define
2002. Ann Rheum Dis 66:940944 a new leukocyte subpopulation that mediates
2. Goh NS, Desai SR, Veeraraghavan S, Hansell tissue repair. Mol Med 1:7181
DM, Copley SJ, Maher TM, Corte TJ, Sander 9. Galligan CL, Siminovitch KA, Keystone EC,
CR, Ratoff J, Devaraj A et al (2008) Interstitial Bykerk V, Perez OD, Fish EN (2010) Fibrocyte
lung disease in systemic sclerosis: a simple stag- activation in rheumatoid arthritis. Rheumatology
ing system. Am J Respir Crit Care Med 177: (Oxford) 49:640651
12481254 10. Douglas RS, Afifiyan NF, Hwang CJ, Chong
3. Tashkin DP, Elashoff R, Clements PJ, Goldin J, K, Haider U, Richards P, Gianoukakis AG,
Roth MD, Furst DE, Arriola E, Silver R, Strange Smith TJ (2010) Increased generation of
C, Bolster M et al (2006) Cyclophosphamide fibrocytes in thyroid-associated ophthalmopa-
versus placebo in scleroderma lung disease. N thy. J Clin Endocrinol Metab 95:430438
Engl J Med 354:26552666 11. Peng XP et al (2011) Local apoptosis promotes
4. Swigris JJ, Olson AL, Fischer A, Lynch DA, collagen production by monocyte derived cells.
Cosgrove GP, Frankel SK, Meehan RT, Brown Fibrogen Tissue Repair 4:12
KK (2006) Mycophenolate mofetil is safe, well 12. Mathai SK, Gulati M, Peng X, Russell TR,
tolerated, and preserves lung function in Shaw AC, Rubinowitz AN, Murray LA, Siner
patients with connective tissue disease-related JM, Antin-Ozerkis DE, Montgomery RR et al
interstitial lung disease. Chest 130:3036 Circulating monocytes from systemic sclerosis
5. Daoussis D, Liossis SN, Tsamandas AC, patients with interstitial lung disease show an
Kalogeropoulou C, Kazantzi A, Sirinian C, enhanced profibrotic phenotype. Lab Invest
Karampetsou M, Yiannopoulos G, Andonopoulos 6:812823
AP (2010) Experience with rituximab in sclero- 13. Gan Y, Reilkoff RA, Peng X, Russell TR, Chen
derma: results from a 1-year, proof-of-principle QC, Mathai SK, Gulati M, Homer RJ, Elias JA,
study. Rheumatology (Oxford) 49:271280 Bucala RJ et al (2011) Role of Semaphorin 7a
6. DOvidio F, Singer LG, Hadjiliadis D, Pierre in TGF 1 induced lung fibrosis, fibrocyte dif-
A, Waddell TK, de Perrot M, Hutcheon M, ferentiation, and scleroderma-related intersti-
Miller L, Darling G, Keshavjee S (2005) tial lung disease 63:24842494
Prevalence of gastroesophageal reflux in 14. Moeller A, Gilpin SE, Ask K, Cox G, Cook D,
end-stage lung disease candidates for lung Gauldie J, Margetts PJ, Farkas L, Dobranowski
transplant. Ann Thorac Surg 80:12541260 J, Boylan C et al (2009) Circulating fibrocytes
7. DOvidio F, Mura M, Tsang M, Waddell TK, are an indicator for poor prognosis in idiopathic
Hutcheon MA, Singer LG, Hadjiliadis D, pulmonary fibrosis. Am J Respir Crit Care Med
Chaparro C, Gutierrez C, Pierre A et al (2005) 7:588594
Bile acid aspiration and the development of 15. Mehrad B, Burdick MD, Zisman DA, Keane
bronchiolitis obliterans after lung transplanta- MP, Belperio JA, Strieter RM (2007)
tion. J Thorac Cardiovasc Surg 129:11441152 Circulating peripheral blood fibrocytes in
16 Flow Cytometric Identification of Fibrocytes in Scleroderma Lung Disease 345
human fibrotic interstitial lung disease. Biochem marrow-derived fibroblast precursors mediate
Biophys Res Commun 353:104108 ischemic cardiomyopathy in mice. Proc Natl
16. Mehrad B, Burdick MD, Strieter RM (2009) Acad Sci USA 103:1828418289
Fibrocyte CXCR4 regulation as a therapeutic 28. Haudek SB, Trial J, Xia Y, Gupta D, Pilling D,
target in pulmonary fibrosis. Int J Biochem Entman ML (2008) Fc receptor engagement
Cell Biol 41:17081718 mediates differentiation of cardiac fibroblast
17. Schmidt M, Sun G, Stacey MA, Mori L, Mattoli precursor cells. Proc Natl Acad Sci USA 105:
S (2003) Identification of circulating fibrocytes 1017910184
as precursors of bronchial myofibroblasts in 29. Buday A, Orsy P, Godo M, Mozes M, Kokeny
asthma. J Immunol 171:380389 G, Lacza Z, Koller A, Ungvari Z, Gross ML,
18. Wang CH, Huang CD, Lin HC, Lee KY, Lin Benyo Z et al (2010) Elevated systemic TGF-
SM, Liu CY, Huang KH, Ko YS, Chung KF, beta impairs aortic vasomotor function through
Kuo HP (2008) Increased circulating fibrocytes activation of NADPH oxidase-driven superox-
in asthma with chronic airflow obstruction. Am ide production and leads to hypertension, myo-
J Respir Crit Care Med 178: 583591 cardial remodeling, and increased plaque
19. Nihlberg K, Larsen K, Hultgardh-Nilsson A, formation in apoE(/) mice. Am J Physiol
Malmstrom A, Bjermer L, Westergren- Heart Circ Physiol 299:H386H395
Thorsson G (2006) Tissue fibrocytes in patients 30. Murray LA, Chen Q, Kramer MS, Hesson DP,
with mild asthma: a possible link to thickness of Argentieri RL, Peng X, Gulati M, Homer RJ,
reticular basement membrane? Respir Res 7:50 Russell T, van Rooijen N et al TGF-beta driven
20. Vakil V, Sung JJ, Piecychna M, Crawford JR, lung fibrosis is macrophage dependent and
Kuo P, Abu-Alfa AK, Cowper SE, Bucala R, blocked by serum amyloid P. Int J Biochem
Gomer RH (2009) Gadolinium-containing Cell Biol 43:154162
magnetic resonance image contrast agent pro- 31. Phillips RJ, Burdick MD, Hong K, Lutz MA,
motes fibrocyte differentiation. J Magn Reson Murray LA, Xue YY, Belperio JA, Keane MP,
Imaging 30:12841288 Strieter RM (2004) Circulating fibrocytes
21. Falk E (2006) Pathogenesis of atherosclerosis. traffic to the lungs in response to CXCL12 and
J Am Coll Cardiol 47:C7C12 mediate fibrosis. J Clin Invest 114:438446
22. Nikam VS, Wecker G, Schermuly R, Rapp U, 32. Yang L, Scott PG, Giuffre J, Shankowsky HA,
Szelepusa K, Seeger W, Voswinckel R (2011) Ghahary A, Tredget EE (2002) Peripheral
Treprostinil inhibits adhesion and differentia- blood fibrocytes from burn patients:
tion of fibrocytes via cAMP and Rap dependent identification and quantification of fibrocytes in
ERK inactivation. Am J Respir Cell Mol Biol adherent cells cultured from peripheral blood
45:692703 mononuclear cells. Lab Invest 82:11831192
23. Niedermeier M, Reich B, Rodriguez Gomez 33. Pilling D, Fan T, Huang D, Kaul B, Gomer RH
M, Denzel A, Schmidbauer K, Gobel N, Talke (2009) Identification of markers that distin-
Y, Schweda F, Mack M (2009) CD4+ T cells guish monocyte-derived fibrocytes from mono-
control the differentiation of Gr1+ monocytes cytes, macrophages, and fibroblasts. PLoS One
into fibrocytes. Proc Natl Acad Sci USA 106: 4:e7475
1789217897 34. Chesney J, Bacher M, Bender A, Bucala R
24. Katebi M, Fernandez P, Chan ES, Cronstein (1997) The peripheral blood fibrocyte is a
BN (2008) Adenosine A2A receptor blockade potent antigen-presenting cell capable of prim-
or deletion diminishes fibrocyte accumulation ing naive T cells in situ. Proc Natl Acad Sci
in the skin in a murine model of scleroderma, USA 94:63076312
bleomycin-induced fibrosis. Inflammation 31: 35. Bellini A, Mattoli S (2007) The role of the
299303 fibrocyte, a bone marrow-derived mesenchymal
25. Kisseleva T, Uchinami H, Feirt N, Quintana- progenitor, in reactive and reparative fibroses.
Bustamante O, Segovia JC, Schwabe RF, Lab Invest 87:858870
Brenner DA (2006) Bone marrow-derived 36. Bianchetti L, Barczyk M, Cardoso J, Schmidt
fibrocytes participate in pathogenesis of liver M, Bellini A, Mattoli S (2011) Extracellular
fibrosis. J Hepatol 45:429438 matrix remodeling properties of human
26. Haudek SB, Cheng J, Du J, Wang Y, Hermosillo- fibrocytes. J Cell Mol Med 3:483495
Rodrigues J, Trial J, Taffet GE, ML E (2010) 37. Abe R, Donnelly SC, Peng T, Bucala R, Metz
Monocytic fibroblast precursors mediate fibrosis CN (2001) Peripheral blood fibrocytes: differ-
in angiotensin-II-induced cardiac hypertrophy. entiation pathway and migration to wound
J Mol Cell Cardiol 49:499507 sites. J Immunol 166:75567562
27. Haudek SB, Xia Y, Huebener P, Lee JM, 38. Niedermeier M, Reich B, Gomez MR, Denzel
Carlson S, Crawford JR, Pilling D, Gomer RH, A, Schmidbauer K, Gobel N, Talke Y, Schweda
Trial J, Frangogiannis NG et al (2006) Bone F, Mack M (2009) CD4+ T cells control the
346 T.M. Russell et al.
Abstract
Autoimmune Type 1 A Diabetes (T1D) is characterized by dependence on exogenous insulin consequential
to the autoimmune attack and destruction of insulin-producing islet beta cells. Pancreatic islet cell
inflammation, or insulitis, precedes beta cell death and T1D onset. In the insulitic lesion, innate immune
cells produce chemokines and cytokines that recruit and activate adaptive immune cells (Eizirik D et al.,
Nat Rev Endocrinol 5:219226, 2009). Locally produced cytokines not only increase immune surveillance
of beta cells (Hanafusa T and Imagawa A, Ann NY Acad Sci 1150:297299, 2008), but also cause beta cell
dysfunction and decreased insulin secretion due to the generation of reactive oxygen species (ROS) and
reactive nitrogen species (RNS) by the beta cells. This, coupled to the high levels of ROS and RNS
secreted by activated macrophages and the low antioxidant capacities of beta cells (Huurman VA, PLoS
One 3:e2435, 2008; Schatz D, Pediatr Diabetes 5:7279, 2004; Verge CF, Diabetes 44:11761179,
1995), implicates free radicals as important effectors in T1D pathogenesis (Eizirik D et al., Nat Rev
Endocrinol 5:219226, 2009; Hanafusa T and Imagawa A, Ann NY Acad Sci 1150:297299, 2008;
Eisenbarth GS and Jeffrey J, Arq Bras Endocrinol Metabol 52:146155, 2008; Pietropaolo M et al.,
Pediatr Diabetes 6:184192, 2005).
Key words:, Reactive oxygen species, Reactive nitrogen species, Pancreatic beta cells, Diabetes,
Mitochondria, Apoptosis, Necrosis
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_17, Springer Science+Business Media New York 2012
347
348 Y.L. Lightfoot et al.
Fig. 1. Oxidative stress-induced beta cell dysfunction and death. (1) Alloxan (A) is a cytotoxic ROS generating glucose
analogue that preferentially accumulates in beta cells via the GLUT2 glucose transporter. Alloxan prevents glucose-
stimulated insulin secretion by inhibiting glucokinase activity, the enzyme responsible for the rate-limiting step of glucose
catabolism, as well as enzymes associated with mitochondrial ATP production. ROS are generated in a cyclic reaction
between alloxan and its reduced product, dialuric acid (AH2). Autoxidation of dialuric acid generates superoxide radicals
( O 2 ), hydrogen peroxide (H2O2), and, in the presence of a metal catalyst through the Fenton reaction, hydroxyl radicals
(OH). (2) Glutathione (GSH) is consumed within the cell for redox cycling, thereby producing oxidized glutathione (GSSG).
However, because beta cells display low glutathione reductase activity, beta cells are unable to maintain redox balance and
undergo necrotic cell death in response to ROS challenge. (3) Beta cells also exhibit low levels of the H2O2-inactivating
enzymes catalase and glutathione peroxidase. This contributes to the high susceptibility of beta cells to ROS. (4) Exogenous
Nitric Oxide (NO) production promotes beta cell dysfunction by preventing increases in ATP/ADP ratios through the inhibi-
tion of aconitase, a tricarboxylic acid (TCA) cycle enzyme, and electron transport chain complex 4. NO causes necrotic beta
cell death. (5) The combination of IL1 and IFN leads to the induction of NFB-responsive stress genes such as inducible
Nitric Oxide Synthase (iNOS) by the beta cell, further driving dysfunction and necrosis by endogenous NO production.
(6) When added together, IL1, IFN, and TNF cause changes in the mitochondrial membrane potential (m) and
mitochondrial outer membrane permeabilization (MOMP), which allows the release of pro-apoptotic proteins such as
cytochrome c (Cyt c) and subsequent activation of the caspase cascade via the apoptosome (Cyt c, Apaf-1, and Caspase
9). Ultimately, effector caspases (Caspase 3) activate Caspase-Activated Deoxyribonuclease (CAD), a DNase enzyme. DNA
cleavage promotes apoptotic cell death. (7) Mitochondrial ROS production induced by the interaction of TNF with TNFR1
or FasL with Fas contributes to Caspase 8 activation, potentiating the caspase cascade.
2. Materials
3. Methods
3.1. Islet Cell Isolation, 1. Perform common bile duct cannulation with a 27-gauge nee-
Culture, and Oxidative dle connected with tygon R-3603 laboratory tubing to a
Damage Induction 10 mL syringe containing the collagenase solution.
3.1.1. Isolation and Culture 2. Inflate pancreas with collagenase solution (2 mL per mouse); pre-
of Mouse Pancreatic Islets vent leakage into the intestine by using a straight bulldog clamp
to seal the entrance of the common bile duct into duodenum.
3. Remove pancreata and place no more than two in a 50 mL
conical tube.
4. Incubate in a 37 C water bath for 19 min.
5. Add 30 mL of HBSS and shake vigorously; mix by pipeting
with a 10 mL pipette several times.
6. Wash five times with HBSS by centrifugation at 200 g for
5 min.
7. Resuspend in 15 mL Histopaque 1119 and place in a clean
50 mL conical tube; use a 10 mL syringe with a 20-gauge
blunt-end needle for the gradient layering steps.
8. Gently layer on 10 mL of Histopaque 1100.
9. Gently layer on 10 mL of Histopaque 1080.
10. Gently layer on 10 mL of Histopaque 1060.
11. Centrifuge at room temperature for 18 min at 1,000 g.
12. Take caution as not to disturb the layers; islets will be between
the two middle layers.
13. Remove and wash these two layers with HBSS in a 50 mL con-
ical tube.
14. Hand pick for purity, place in tissue culture dishes containing
VC-LG-DMEM, and maintain at 37 C in a humidified atmo-
sphere with 5 % CO2.
354 Y.L. Lightfoot et al.
3.2.3. Treatment with 1. Preculture islets for 6 days in VC-LG-DMEM; change into
Alloxan and Inhibitors fresh medium every 48 h.
of Oxidative Stress 2. Prepare a 1 M stock alloxan solution in PBS pH 2.0, and dilute
to target concentrations with culture medium. Prepare 0, 1, 2,
and 3 mM alloxan solutions.
3. Prepare 5 mM L-NMMA solution in VC-LG-DMEM.
4. Prepare 5 mM NAC solution in VC-LG-DMEM.
5. Prepare 34 M FBC-007 solution in VC-LG-DMEM.
6. Place ten islets per well; seed triplicate wells per treatment and
control group.
7. Incubate islets in alloxan solutions at 37 C, 5 % CO2 for 5 min.
17 Oxidative Stress and Beta Cell Dysfunction 355
3.3.4. Insulin Content 1. Incubate stimulated islets from above in acidethanol for 18 h
of Islet Cells at 4 C.
2. Extract protein with TCA.
3. Neutralize with 3 N NaOH (10 L 3 N NaOH to 200 L
acidethanol) before testing for insulin and DNA
concentration.
3.3.5. Insulin ELISA Quantification of the stimulated insulin secreted as well as the
insulin content of the harvested islets is performed using the
ALPCO Insulin (Mouse) Ultrasensitive EIA Kit according to
the manufacturers instructions, and read with a SpectraMax M5
plate reader. Insulin standard curves are established in each experi-
ment; low and high controls should also be included. Samples
often need to be diluted in the range of 10 to 50; it is recom-
mended that the required dilution factors be established before
running all the samples.
356 Y.L. Lightfoot et al.
3.3.6. DNA Quantification The Quant-iT PicoGreen dsDNA reagent system is used to quan-
Assay tify the total DNA in acidethanol extracted samples, according to
the manufacturers instructions. Fluorescence is then read with a
SpectraMax M5 plate reader.
1. Prepare a low-range standard curve of dsDNA from 25 pg/
mL to 25 ng/mL using a sample of known concentration.
2. Add 100 L of standard curve or neutralized test sample to
their corresponding wells in a 96-well, clear bottom, black
plate.
3. Add 100 L of working solution of Quant-iT PicoGreen
reagent to each well.
4. Incubate for 5 min at room temperature in the dark.
5. Read fluorescence and calculate concentrations based on the
prepared standard curve.
6. Normalize insulin concentration measurements from above
per nanogram of DNA.
3.4. Measurement The ApoGlow Assay Kit employs the luciferase reaction, which uti-
of Intracellular ATP lizes ATP to catalyze the oxidation of luciferin and generate light.
and Relative ADP: Thus light intensity is directly proportional to the ATP concentra-
ATP Ratio tion. Conversion of ADP to ATP, and subsequent detection with
luciferase, allows the measurement of ADP levels in the test sam-
ples. The reactions are performed according to the manufacturers
instructions and luminescence read with a 1-s integrated reading
using a SpectraMax M5 plate reader.
1. Place 50 islets per well; seed triplicate wells per treatment and
control groups.
17 Oxidative Stress and Beta Cell Dysfunction 357
3.5. Quantification GSH availability within the islets is detected and quantified with
of Glutathione the GSH-Glo Glutathione Assay kit following the manufacturers
in the Islet Cells instructions. The luminescence-based assay uses the GSH-
dependent conversion of a luciferin derivate to luciferin and the
latter is detected as described before.
1. Place 30 islets per well; seed triplicate wells per treatment and
control groups.
2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-induc-
ing stimuli for the specified time period.
3. Prepare kit reagents, including standard curve, as indicated
and allow reagents and samples to equilibrate to room
temperature.
4. Harvest islets by centrifugation and collect the supernatant for
other studies.
5. Resuspend in 50 L of PBS and add to their corresponding
wells.
6. Add 50 L of GSH-Glo reagent 2 to each well and mix by
shaking at 150 rpm on an orbital microplate shaker for 30 s.
7. Incubate at room temperature for 30 min.
8. Pipette 100 L of luciferin detection reagent to each well and
mix by shaking at 150 rpm on an orbital microplate shaker for
30 s.
9. Incubate at room temperature for 15 min.
10. Read luminescence. The standard curve should demonstrate a
linear relationship between luminescence and GSH
concentration.
11. Convert relative luminescence units (RLUs) to M of GSH.
358 Y.L. Lightfoot et al.
3.6. Measurement Nitric oxide production within the islets is indirectly quantified by
of Nitric Oxide measuring nitrite ( N O 2), a stable end product of NO, via the
and Reactive Oxygen Greiss Reagent System. Absorbance is read with a SpectraMax M5
Species Production plate reader.
3.6.1. Nitrate Assay 1. The supernatants collected in Subheading 3.4, step 4, will be
used for analysis.
2. Add 100 L of sample media and standards (0, 1, 2, 4, 8, 16,
32, 64, 128 M NaNO2) to plate in duplicate.
3. Prepare a 1:1 solution of Greiss Reagent 1:Greiss Reagent 2.
4. Add 100 L of this solution to each well.
5. Read absorbance at 550 nm and calculate concentration.
3.6.2. MitoSox Red 1. Place 50 islets per well in a 96-well, clear bottom, black plate;
and CM-H2DCFDA-Based seed triplicate wells per treatment and control groups.
Detection of ROS 2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-induc-
ing stimuli for the specified time period.
3. As positive control, include islet cells treated with 1.5 % H2O2
10 min before performing assay.
4. Harvest islets and wash with phenol red-free LG-DMEM.
5. Add MitoSox Red at a final concentration of 5 M or
CM-H2DCFDA at a final concentration of 10 M.
6. Allow dye loading for 30 min in the dark at 37 C, 5 % CO2.
7. Read MitoSox Red fluorescence with an excitation wavelength
of 520 nm and emission wavelength of 590 nm.
8. Read CM-H2DCFDA fluorescence with an excitation wave-
length of 488 nm and emission wavelength of 560 nm.
3.7. Viability 1. Place 100 islets per well; seed triplicate wells per treatment and
and Caspase Enzyme control groups.
Activity Assays 2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-induc-
3.7.1. MTT Assay ing stimuli for the specified time period in a total volume of
200 L.
3. Remove 20 L and add 20 L of MTT solution for a final
concentration of 0.5 mg/mL.
4. Incubate at 37 C, 5 % CO2 for 15 h to allow MTT to be
metabolized to insoluble formazan crystals.
5. Remove media and blot on paper towels.
6. Dissolve crystals with acidisopropanol by shaking at 150 rpm
on an orbital microplate shaker for 5 min.
7. Read absorbance at 560 nm and subtract background at
670 nm. Intensity of purple color is directly proportional to
live cell number.
17 Oxidative Stress and Beta Cell Dysfunction 359
3.7.2. Annexin V and PI 1. Place 50 islets per well; seed triplicate wells per treatment and
Staining of Islet Cells control groups.
2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-induc-
ing stimuli for the specified time period.
3. Harvest and wash in PBS.
4. Incubate for 10 min at 37 C in Ca2+- and Mg2-free PBS con-
taining 0.2 mg/mL EDTA to disperse into single cells.
5. Inject islet cells through 16- to 22-gauge needles to further
disperse islets into a single-cell suspension (16-, 18-, 20-,
22-gauge needles).
6. Incubate for 15 min with PI (10 g/mL) and APC-annexin V
(5 L) in the dark.
7. Visualize and estimate cell death using a Zeiss Axioskop
Microscope with fluorescence capabilities.
3.7.3. Caspase-8 and -3 Protein-based fluorometric assay kits are used in parallel to mea-
Fluorometric Assays sure caspase-8 and -3 activity inductions. Active caspases cleave
caspase-specific peptides conjugated to a fluorochrome, thereby
releasing the molecules that, when excited at 400 nm, fluoresce at
505 nm. Fluorescence is read with a SpectraMax M5 plate reader.
1. Place 1,000 islets per well; seed triplicate wells per treatment
and control groups.
2. Incubate islets at 37 C, 5 % CO2 with oxidative stress-inducing
stimuli for the specified time period.
3. Harvest islet cells and discard supernatant.
4. Add 60 L of cold lysis buffer.
5. Incubate on ice for 10 min.
6. Centrifuge at 10,000 g for 1 min and collect supernatant.
7. Determine protein concentration.
3.7.4. BCA Protein Assay 1. Prepare working reagent by mixing 50 parts of BCA Reagent
A with 1 part BCA Reagent B.
2. Prepare diluted albumin (BSA) standards with a concentration
range of 02,000 g/mL.
3. Remove 10 L of test sample from above and dilute to run in
triplicate wells.
4. Pipette 10 L of each standard or test sample into a well;
include two replicates for each.
5. Add 200 L of working reagent to each well and mix by shak-
ing at 150 rpm on an orbital microplate shaker for 30 s.
6. Incubate at 37 C for 30 min.
7. Cool plate to room temperature and read absorbance at 562 nm.
8. Calculate protein concentration and dilute in the range of
24 mg/mL using lysis buffer.
360 Y.L. Lightfoot et al.
References
1. Eizirik DL, Colli ML, Ortis F (2009) The role cell autoimmunity in identical twins of patients
of inflammation in insulitis and beta-cell loss in with type I diabetes. Diabetes 44:
type 1 diabetes. Nat Rev Endocrinol 5: 11761179
219226 10. Lenzen S, Brand FH, Panten U (1988)
2. Hanafusa T, Imagawa A (2008) Insulitis in Structural requirements of alloxan and ninhy-
human type 1 diabetes. Ann N Y Acad Sci drin for glucokinase inhibition and of glucose
1150:297299 for protection against inhibition. Br J Pharmacol
3. Eisenbarth GS, Jeffrey J (2008) The natural 95:851859
history of type 1A diabetes. Arq Bras Endocrinol 11. Lenzen S, Tiedge M, Panten U (1987)
Metabol 52:146155 Glucokinase in pancreatic B-cells and its inhibi-
4. Pietropaolo M, Yu S, Libman IM, Pietropaolo tion by alloxan. Acta Endocrinol (Copenh)
SL, Riley K, LaPorte RE et al (2005) 115:2129
Cytoplasmic islet cell antibodies remain valu- 12. Lenzen S, Freytag S, Panten U (1988)
able in defining risk of progression to type 1 Inhibition of glucokinase by alloxan through
diabetes in subjects with other islet autoanti- interaction with SH groups in the sugar-bind-
bodies. Pediatr Diabetes 6:184192 ing site of the enzyme. Mol Pharmacol 34:
5. Verge CF, Gianani R, Kawasaki E, Yu L, 395400
Pietropaolo M, Jackson RA et al (1996) 13. Lenzen S, Munday R (1991) Thiol-group reac-
Prediction of type I diabetes in first-degree tivity, hydrophilicity and stability of alloxan, its
relatives using a combination of insulin, GAD, reduction products and its N-methyl deriva-
and ICA512bdc/IA-2 autoantibodies. Diabetes tives and a comparison with ninhydrin. Biochem
45:926933 Pharmacol 42:13851391
6. Wang J, Miao D, Babu S, Yu J, Barker J, 14. Lenzen S, Mirzaie-Petri M (1992) Inhibition
Klingensmith G et al (2007) Prevalence of of aconitase by alloxan and the differential
autoantibody-negative diabetes is not rare at all modes of protection of glucose,
ages and increases with older age and obesity. J 3-O-methylglucose, and mannoheptulose.
Clin Endocrinol Metab 92:8892 Naunyn Schmiedebergs Arch Pharmacol 346:
7. Huurman VA, Hilbrands R, Pinkse GG, Gillard 532536
P, Duinkerken G, van de Linde P et al (2008) 15. Grankvist K, Marklund S, Sehlin J, Tljedal I-B
Cellular islet autoimmunity associates with (1979) Superoxide dismutase, catalase and
clinical outcome of islet cell transplantation. scavengers of hydroxyl radicals protect against
PLoS One 3:e2435 toxic action of alloxan on pancreatic islet cells
8. Schatz D, Cuthbertson D, Atkinson M, Salzler in vitro. Biochem J 182:1725
MC, Winter W, Muir A et al (2004) Preservation 16. Grankvist K, Marklund SL, Taljedal IB (1981)
of C-peptide secretion in subjects at high risk CuZn-superoxide dismutase, Mn-superoxide
of developing type 1 diabetes mellitusa new dismutase, catalase and glutathione peroxidase
surrogate measure of non-progression? Pediatr in pancreatic islets and other tissues in the
Diabetes 5:7279 mouse. Biochem J 199:393398
9. Verge CF, Gianani R, Yu L, Pietropaolo M, 17. Grankvist K, Marklund S, Taljedal IB (1981)
Smith T, Jackson RA et al (1995) Late progres- Superoxide dismutase is a prophylactic against
sion to diabetes and evidence for chronic beta- alloxan diabetes. Nature 294:158160
17 Oxidative Stress and Beta Cell Dysfunction 361
18. Asplund K, Grankvist K, Marklund S, Taljedal rophages kill pancreatic syngeneic islet cells via
IB (1984) Partial protection against streptozo- arginine-dependent nitric oxide generation.
tocin-induced hyperglycaemia by superoxide Biochem Biophys Res Commun 175:752758
dismutase linked to polyethylene glycol. Acta 31. Schwizer RW, Leiter EH, Evans R (1984)
Endocrinol (Copenh) 107:390394 Macrophage mediated cytotoxicity against cul-
19. Lenzen S, Drinkgern J, Tiedge M (1996) Low tured pancreatic islet cells. Transplantation
antioxidant enzyme gene expression in pancre- 37:539544
atic islets compared with various other mouse 32. Sandler S, Eizirik DL, Sternesjo J, Welsh N
tissues. Free Radical Bio Med 20:463466 (1994) Role of cytokines in regulation of pan-
20. Tiedge M, Lortz S, Drinkgeer J, Lenzen S creatic B-cell function. Biochem Soc Trans
(1997) Relation between antioxidant enzyme 22:2630
gene-expression and antioxidative defense sta- 33. Corbett JA, McDaniel ML (1994) Reversibility
tus of insulin-producing cells. Diabetes 46: of interleukin-1 beta-induced islet destruction
17331742 and dysfunction by the inhibition of nitric oxide
21. Tiedge M, Lortz S, Munday R, Lenzen S synthase. Biochem J 299(Pt 3):719724
(1998) Complementary action of antioxidant 34. Scarim AL, Heitmeier MR, Corbett JA (1997)
enzymes in the protection of bioengineered Irreversible inhibition of metabolic function
insulin producing rinm5f cells against the tox- and islet destruction after a 36-hour exposure
icity of reactive oxygen species. Diabetes 47: to interleukin-1beta. Endocrinology 138:
15781585 53015307
22. Mathews CE, Leiter EH (1999) Constitutive 35. Augstein P, Heinke P, Salzsieder E, Grimm R,
differences in anti-oxidant defense status dis- Giebel J, Salzsieder C et al (2008) Dominance
tinguish Alloxan Resistant (ALR/Lt) and of cytokine- over FasL-induced impairment of
Alloxan Susceptible (ALS/Lt) mice. Free the mitochondrial transmembrane potential
Radical Bio Med 27:449455 (Deltapsim) in the pancreatic beta-cell line
23. Mathews CE, Leiter EH (1999) Resistance of NIT-1. Diab Vasc Dis Res 5:198204
ALR/Lt Islets to free radical mediated diabeto- 36. Steer SA, Scarim AL, Chambers KT, Corbett
genic stress is inherited as a dominant trait. JA (2005) Interleukin-1 Stimulates beta-Cell
Diabetes 48:21892196 Necrosis and Release of the Immunological
24. Mathews CE, Suarez-Pinzon WL, Baust JJ, Adjuvant HMGB1. PLoS Med 3:e17
Strynadka K, Leiter EH, Rabinovitch A (2005) 37. Hughes KJ, Chambers KT, Meares GP, Corbett
Mechanisms Underlying Resistance of Pancreatic JA (2009) Nitric oxides mediates a shift from
Islets from ALR/Lt Mice to Cytokine-Induced early necrosis to late apoptosis in cytokine-
Destruction. J Immunol 175:12481256 treated {beta}-cells that is associated with irre-
25. Chen H, Li X, Epstein PN (2005) MnSOD and versible DNA damage. Am J Physiol Endocrinol
catalase transgenes demonstrate that protection Metab 297:E1187E1196
of islets from oxidative stress does not alter 38. Rabinovitch A, Suarez WL, Thomas PD,
cytokine toxicity. Diabetes 54:14371446 Strynadka K, Simpson I (1992) Cytotoxic
26. Li X, Chen H, Epstein PN (2006) effects of cytokines on rat islets: evidence for
Metallothionein and catalase sensitize to diabe- involvement of free radicals and lipid peroxida-
tes in nonobese diabetic mice: reactive oxygen tion. Diabetologia 35:409413
species may have a protective role in pancreatic 39. Rabinovitch A, Sumoski W, Rajotte RV,
beta-cells. Diabetes 55:15921604 Warnock GL (1990) Cytotoxic effects of cytok-
27. Mathews CE, Graser RT, Savinov AY, Serreze ines on human pancreatic islet cells in mono-
DV, Leiter EH (2001) Unusual resistance of layer culture. J Clin Endocrinol Metab
ALR/Lt beta cells to autoimmune destruction: 71:152156
Role for beta cell expressed resistance determi- 40. Nerup J, Mandrup-Poulsen T, Helqvist S,
nants. Proc Natl Acad Sci 98:235240 Andersen HU, Pociot F, Reimers JI et al (1994)
28. Foulis AK, McGill M, Farquharson MA (1991) On the pathogenesis of IDDM. Diabetologia
Insulitis in type 1 (insulin-dependent) diabetes 37(Suppl 2):S82S89
mellitus in manmacrophages, lymphocytes, 41. Corbett JA, McDaniel ML (1995) Intraislet
and interferon-gamma containing cells. J release of interleukin 1 inhibits beta cell func-
Pathol 165:97103 tion by inducing beta cell expression of induc-
29. Foulis AK (1996) The pathology of the endo- ible nitric oxide synthase. J Exp Med 181:
crine pancreas in type 1 (insulin-dependent) 559568
diabetes mellitus. APMIS 104:161167 42. Rabinovitch A, Suarez-Pinzon WL, Sorensen
30. Kroncke KD, Kolb-Bachofen V, Berschick B, O, Bleackley RC (1996) Inducible nitric oxide
Burkart V, Kolb H (1991) Activated mac- synthase (iNOS) in pancreatic islets of nonobese
362 Y.L. Lightfoot et al.
diabetic mice: identification of iNOS- expressing 47. Oliveira HR, Verlengia R, Carvalho CR, Britto
cells and relationships to cytokines expressed in LR, Curi R, Carpinelli AR (2003) Pancreatic
the islets. Endocrinology 137:20932099 beta-cells express phagocyte-like NAD(P)H
43. Rabinovitch A, Suarez-Pinzon W, El-Sheikh A, oxidase. Diabetes 52:14571463
Sorensen O, Power RF (1996) Cytokine gene 48. Morgan D, Oliveira-Emilio HR, Keane D,
expression in pancreatic islet-infiltrating leuko- Hirata AE, Santos da Rocha M, Bordin S et al
cytes of BB rats: expression of Th1 cytokines (2007) Glucose, palmitate and pro-
correlates with beta-cell destructive insulitis inflammatory cytokines modulate production
and IDDM. Diabetes 45:749754 and activity of a phagocyte-like NADPH oxi-
44. Mandrup-Poulsen T, Corbett JA, McDaniel dase in rat pancreatic islets and a clonal beta cell
ML, Nerup J (1993) What are the types and line. Diabetologia 50:359369
cellular sources of free radicals in the pathogen- 49. Newsholme P, Morgan D, Rebelato E, Oliveira-
esis of type 1 (insulin-dependent) diabetes mel- Emilio HC, Procopio J, Curi R et al (2009)
litus? Diabetologia 36:470471 Insights into the critical role of NADPH
45. Corbett JA, Kwon G, Turk J, McDaniel ML oxidase(s) in the normal and dysregulated pan-
(1993) IL-1 beta induces the coexpression of creatic beta cell. Diabetologia 52:24892498
both nitric oxide synthase and cyclooxygenase 50. Suarez-Pinzon WL, Strynadka K, Rabinovitch
by islets of Langerhans: activation of cyclooxy- A (1996) Destruction of rat pancreatic islet
genase by nitric oxide. Biochemistry 32: beta-cells by cytokines involves the production
1376713770 of cytotoxic aldehydes. Endocrinology 137:
46. Takamura T, Kato I, Kimura N, Nakazawa T, 52905296
Yonekura H, Takasawa S et al (1998) Transgenic 51. Shen HM, Lin Y, Choksi S, Tran J, Jin T,
mice overexpressing type 2 nitric-oxide syn- Chang L et al (2004) Essential roles of recep-
thase in pancreatic beta cells develop insulin- tor-interacting protein and TRAF2 in oxidative
dependent diabetes without insulitis. J Biol stress-induced cell death. Mol Cell Biol
Chem 273:24932496 24:59145922
Chapter 18
Abstract
Experimental autoimmune encephalomyelitis (EAE) is an inflammatory demyelinating disease of the
central nervous system that is induced in laboratory animals by the generation of an immune response
against myelin epitopes. It has been used as a prototype of Th1- and/or Th17-driven, organ-specific auto-
immunity and as a model for the human disease, multiple sclerosis. In this chapter we describe two classic
protocols for EAE induction (active immunization and adoptive transfer of Th1- or Th17-polarized cells)
in Subheadings 3.1 and 3.2, respectively. Subheading 3.3 describes methods for rating clinical disease in
symptomatic animals. Subheading 3.4 includes instructions for the isolation of mononuclear cells from the
inflamed spinal cords of mice with EAE. Subheading 3.5 describes a method for performing the enzyme-
linked immunospot assay.
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_18, Springer Science+Business Media New York 2012
363
364 P. Rao and B.M. Segal
Table 1
Sequences of encephalitogenic peptides used to induce
EAE in susceptible mouse strains
Peptidea Sequence
PLP4364 EKLIETYFSKNYQDYEYLINVI
PLP103116 YKTTICGKGLSATV
b
PLP 139151
HSLGKWLGHPDKF
PLP185206 SIAFPSKTSASIGSLCADARMY
PLP180199 WTTCQSIAFPSKTSASIGSL
PLP215232 PGKVCGSNLLSICKTAEF
MBPAc111 Ac-ASQKRPSQRHG
MBP89101 VHFFKNIVTPRTP
MBP84104 VHFFKNIVTPRTPPPSQGKGR
MBP3547 TGILDSIGRFFSG
MOG92106 DEGGYTCFFRDHSYQ
MOG3555 MEVGWYRSPFSRVVHLYRNGK
a
References are provided in Table 2
b
In order to increase solubility, the synthetic peptide differs from the native sequence by
the substitution of Serine (S) for Cysteine (C) at position 140
Table 2
Peptides used to induce EAE in susceptible mouse strains
2. Materials
2.2. Lymph Node Cell 1. Tissue culture media (TCM) consisting of the following
Culture for Passive ingredients:
Transfer RPMI 1640 with L-glutamine (GIBCO BRL): 1,000 ml.
Fetal Bovine Serum (GIBCO BRL). Heat denature at 58 C
for 45 min and filter sterilize prior to use. Add 100 ml.
2-Mercaptoethanol (Sigma, St. Louis, MO). Prepare stock
at 5 105 M. Add 1 ml.
Sodium pyruvate (100 mM, GIBCO BRL). Add 10 ml.
Non-essential amino acid solution (NEEAS) (10 mM,
GIBCO BRL). Add 10 ml.
Penicillin/streptomycin solution (10,000 U of penicillin
and 10 mg of streptomycin/ml, GIBCO BRL). Add 10 ml.
HEPES (1 M, GIBCO BRL). Add 12.5 ml.
All of the above ingredients should be stored at 4 C except
for Penicillin/Streptomycin and fetal calf serum, both of which
should be stored at 20 C.
2. Sterile Hanks Balanced Salt solution (HBSS).
3. Sterile disposable cell strainers (70 m, Nylon; BD Falcon,
Millville, NJ).
4. Sterile ACK lysing buffer (Invitrogen).
5. 50 ml conical polypropylene centrifuge tubes.
6. Trypan Blue.
7. Hemocytometer.
8. 24-Well plates treated for tissue culture.
9. Synthetic myelin peptides as described above.
2.3. Spinal Cord 1. Peristaltic mini pump with variable flow, medium Flow Rate
Harvest and Isolation (4.085.0 ml/min) (Cat # 54856-075, VWR Scientific, West
of CNS Mononuclear Chester, PA).
Cells 2. Collagenase (Worthington Biochemical corp., Lakewood, NJ).
Prepare stock solution of 8 mg/ml in HBSS or RPMI. Store
aliquots of 5 ml at 80 C.
3. DNAse I (Sigma Chemical Corp, St. Louis, MO). Prepare stock
solution of 20 mg/ml in PBS. Store aliquots of 1 ml at 20 C.
4. Percoll (Amersham, Piscataway, NJ). Store at 4 C.
5. Sterile 15 and 50 ml polypropylene tubes.
2. Medium: TCM.
ELISPOT plates (Model MAIP N4550; Millipore, Billerica, MA).
3. Coating antibodies and biotinylated secondary antibodies for
each cytokine (eBioscience, San Diego, CA).
4. Streptavidin Alkaline phosphatase (Southern Biotech,
Birmingham, AL).
5. Vector Blue substrate solution (Vector Laboratories,
Burlingame, CA).
6. 100 mM TrisHCl, pH 8.2.
7. CTL ImmunoSpot Analyzer (Cellular Technology, Cleveland,
OH).
3. Methods
There are two basic approaches for induction of EAE: active immu-
nization and passive transfer. In active immunization, the entire
disease process, from autoreactive T cell priming to CNS infiltration
and demyelination, takes place in the same animal. Depending on
the mouse strain and myelin epitope, it is sometimes necessary to
inject the recipient with pertussis toxin to attain a high rate of inci-
dence and to synchronize the course between experimental sub-
jects. The mechanism of action of pertussis toxin is unknown, but
it is widely believed that it acts by increasing the permeability of the
bloodbrain barrier, thereby expediting migration of effector cells
into the brain and spinal cord (19).
Adoptive transfer allows the separation of the induction and
effector phases, which might be advantageous depending on the
experimental question(s) posed. Myelin peptide-primed lymph
node cells are reactivated with antigen in vitro for 96 h prior to
disease transfer. The in vitro stimulation step is critical for success-
ful disease transfer. Presumably it allows for the selective expansion
of myelin-reactive T cells and/or their terminal differentiation into
encephalitogenic effector cells.
3.1. Induction of EAE 1. Estimate the amount of peptide and CFA you will need:
by Active Irrespective of the specific model, we find that immunization
Immunization of mice with 100 g of myelin peptide in 100 l of an emul-
sion with CFA is sufficient for the reproducible induction of
EAE at high incidence. Therefore the total amount of peptide
you will need in microgram equals 100 n, where n is the
number of mice to be immunized. Dilute the appropriate
amount of peptide (from stock solution) with sterile PBS to a
final concentration of 1 g/l. To prepare the emulsion, CFA
18 Experimental Autoimmune Encephalomyelitis 369
Table 3
Requirementa of pertussis toxin in active immunization
models of EAE
3.2. Induction of EAE 1. Immunize donor mice as described in Subheading 3.1 but do
by Passive Transfer not inject pertussis toxin.
(Fig. 1) 2. Sacrifice mice between days 10 and 16 post immunization.
3. Harvest draining lymph nodes: Four axillary (the proper and
accessory axillary on each side) and two inguinal nodes under
aseptic conditions and place in HBSS.
4. Prepare a single-cell suspension by pressing the lymph node
cells through a cell strainer or mesh screen with a plunger from
a sterile 3 or 5 ml syringe. We use disposable nylon cell strain-
ers (70 M) that fit over a 50 ml conical tube. (Alternatively
use a 100-mesh screen from Fisher. Clean and flame sterilize the
screen prior to each use.) During the preparation of the suspen-
sion, periodically douse the strainer with 12 ml aliquots of
sterile HBSS to wash adherent cells through into the 50 ml
conical tube. Remove debris and connective tissue as they
accumulate on the screen with sterile forceps.
5. Once the single-cell suspension is finished, centrifuge the 50 ml
tube at 300 g at 4 C for 7 min.
6. Typically lymph node preparations do not contain significant
number of erythrocytes. In case there is a significant number
oft RBCs, resuspend the pellets in 7.5 ml of ACK lysing buffer
18 Experimental Autoimmune Encephalomyelitis 371
3. Next, while holding the tail between thumb and index finger,
flip the animal on its back and time how long it takes her to
assume an upright position. A healthy mouse will turn herself
over immediately. A delay suggests hind limb weakness.
4. Place the mouse on a wire cage top or metal grid and observe
as she crosses from one side to the other. Pay particular atten-
tion to whether the hind limbs slip between the bars.
5. Observe for complete paralysis in the limbs. This observation
is critical to distinguish between a score of 3 and 4. Again,
please refer to the video files and observe partial hind limb
activity in the mouse with a score of 3.
Notice complete hind limb paralysis in one or both limbs
in a mouse with a score of 4.
6. Score according to the following 5 point scale:
0: Healthy mouse. No signs of neurological dysfunction.
1: Limp tail only. The tail remains flaccid when the mouse is
picked up.
2: Hind limb paresis, but without frank leg dragging. The
mouse fails the backflip test and has a waddling gait. When
placed on a wire cage top, the mouse frequently slips, with
one or more limbs falling in between the bars.
3: Partial hind limb weakness with one of both hind limbs
dragging, but some movement preserved.
4: Complete hind limb paralysis. (Depicted in Fig. 2.)
5: Moribund. The mouse is paralyzed in both hind limbs and
possibly one forelimb. Inevitably, there is weight loss.
Breathing appears labored.
Occasionally, mice develop EAE displaying signs of an atypical
form of the disease characterized by ataxia. This happens when
lesions develop in the brain stem and cerebellum. Such mice typi-
cally cannot right themselves, are tilted to one side with either
complete or partial paralysis of all limbs, and typically move around
in circles. It is seen sometimes in the active immunization model
but more typically when wild-type (WT) Th17- or Th17-polarized
IFN--deficient cells are used for adoptive transfer to WT recipi-
ents. Please refer to the video displaying symptoms of atypical EAE.
The scoring scale for atypical form of EAE is as follows (21):
0: Healthy.
1: Mouse partly tilted, feet fall into cage fence.
2: Tilted and tumbles.
3: Mouse heavily tilted and moves in circles.
4: Inability to walk, mouse is only rolling.
5. Moribund.
374 P. Rao and B.M. Segal
Fig. 2. Photograph of a mouse with EAE. The arrow points to a female SJL mouse 15 days
after injection with 50 106 PLP139151 reactive cells (according to the protocol described
in Subheading 3.2). At this point, the mouse had reached a clinical score of 4 (see
Subheading 3.3). Neurological signs initially presented as a flaccid tail on day 10 after cell
transfer and then evolved into hind limb paralysis. A nave, healthy littermate is shown for
the sake of comparison.
HBSS
Myelin debris
30 % Percoll
MNCs
70 % Percoll
RBCs/dead cells
Fig. 3. Isolation of mononuclear cells from spinal cords of mice with EAE using a Percoll
gradient (described in Subheading 3.4).
3.5. ELISPOT Assay 1. Day 1 (under sterile conditions): Dilute coating antibody to
3 g/ml in PBS, add 100 l/well, and leave the plate either at
RT for 2 h or at 4 C overnight.
2. Day 2 (under sterile conditions):
(a) Wash plate 3 with PBS (200 l/well), aspirate.
(b) Block with 200 l of PBS + 1 % BSA, 1 h RT.
(c) Wash plate 3 with PBS.
(d) Prepare 100 g/ml myelin antigen/mitogen in TCM
medium and add 100 l/well. The final concentration of
the peptide will be 50 g/ml.
(e) Resuspend MNC in TCM at 2 106 cells/100 l. Make
serial dilutions as desired.
(f) Add 100 l cell suspension/well to give a total volume
of 200 l. Add the cells with a steady hand in the center
of the well. This ensures a uniform distribution of
ELISPOTs.
(g) Do not mix the cells and peptide. Try to hold the plate as
steady as possible before placing it in the incubator. Incubate
at least for 24 h at 37 C. The incubation times will vary
according to the cytokine being analyzed.
3. Day 3 (can be carried out under non-sterile conditions from
here on):
(a) Wash plate 3 with PBS (200 l/well).
(b) Wash plate 3 with PBS-Tween (200 l/well).
4. Dilute biotinylated secondary antibody to 3 g/ml in PBS-
TBSA. Add 100 l/well and leave the plate either at RT for
2 h or at 4 C overnight.
5. Day 4:
(a) Wash plate 4 with PBS-Tween (200 l/well).
(b) Add StreptavidinAP (1:1,000) 100 l/well diluted in
PBS-TBSA. Leave the plate at RT for 2 h.
(c) Wash plate 3 with PBS (200 l/well).
(d) Add 100 l/well developer (two drops of each solution
per 5 ml 100 mM TrisHCl pH 8.2). Place the plate in a
dark spot and monitor spot formation. Do not let a
significant background develop as counting spots will
become a problem.
(e) Rinse plate with water, and let it air-dry completely before
reading in a counter.
6. Please refer notes 1922 for further suggestions.
18 Experimental Autoimmune Encephalomyelitis 377
4. Notes
References
1. Raine CS, Barnett LB, Brown A, Behar T, 3. Wujek JR, Bjartmar C, Richer E, Ransohoff
McFarlin DE (1980) Neuropathology of exper- RM, Yu M, Tuohy VK, Trapp BD (2002) Axon
imental allergic encephalomyelitis in inbred loss in the spinal cord determines permanent
strains of mice. Lab Invest 43(2):150157 neurological disability in an animal model of
2. Trapp BD, Peterson J, Ransohoff RM, Rudick multiple sclerosis. J Neuropathol Exp Neurol
R, Mork S, Bo L (1998) Axonal transection in 61(1):2332
the lesions of multiple sclerosis [Comment]. N 4. Baron JL, Madri JA, Ruddle NH, Hashim G,
Engl J Med 338(5):278285 Janeway CA Jr (1993) Surface expression of
18 Experimental Autoimmune Encephalomyelitis 379
27. Zamvil SS, Mitchell DJ, Powell MB, Sakai K, 31. Montgomery NI, Rauch HC (1982)
Rothbard JB, Steinman L (1988) Multiple dis- Experimental allergic encephalomyelitis (EAE)
crete encephalitogenic epitopes of the autoan- in mice: primary control of EAE susceptibility
tigen myelin basic protein include a determinant is outside the H-2 complex. J Immunol
for I-E class II-restricted T cells. J Exp Med 128(1):421425
168(3):11811186 32. Tuohy VK, Sobel RA, Lees MB (1988) Myelin
28. Whitham RH, Bourdette DN, Hashim GA, proteolipid protein-induced experimental aller-
Herndon RM, Ilg RC, Vandenbark AA, Offner gic encephalomyelitis. Variations of disease
H (1991) Lymphocytes from SJL/J mice immu- expression in different strains of mice. J Immunol
nized with spinal cord respond selectively to a 140(6):18681873
peptide of proteolipid protein and transfer relaps- 33. Lyons JA, Ramsbottom MJ, Trotter JL,
ing demyelinating experimental autoimmune Cross AH (2002) Identification of the
encephalomyelitis. J Immunol 146(1):101107 encephalitogenic epitopes of CNS proteo-
29. Zamvil SS, Mitchell DJ, Moore AC, Kitamura lipid protein in BALB/c mice. J Autoimmun
K, Steinman L, Rothbard JB (1986) T-cell 19(4):195201
epitope of the autoantigen myelin basic protein 34. Sobel RA, Tuohy VK, Lees MB (1991) Parental
that induces encephalomyelitis. Nature MHC molecule haplotype expression in
324(6094):258260 (SJL/J SWR) F1 mice with acute experimen-
30. Endoh M, Rapoport SI, Tabira T (1990) Studies tal allergic encephalomyelitis induced with two
of experimental allergic encephalomyelitis in old different synthetic peptides of myelin proteo-
mice. J Neuroimmunol 29(13):2131 lipid protein. J Immunol 146(2):543549
Chapter 19
Abstract
Experimental autoimmune encephalomyelitis (EAE) and Theilers Murine Encephalitis Virus-Induced
Demyelinating Disease (TMEV-IDD) are two clinically relevant murine models of multiple sclerosis (MS).
Like MS, both are characterized by mononuclear cell infiltration into the CNS and demyelination. EAE is
induced by either the administration of myelin protein or peptide in adjuvant or by the adoptive transfer
of encephalitogenic T cell blasts into nave recipients. The relative merits of each of these protocols are
compared. Depending on the type of question being asked, different mouse strains and peptides are used.
Different disease courses are observed with different strains and different peptides in active EAE. These
variations are also addressed. Additionally, issues relevant to clinical grading of EAE in mice are discussed.
In addition to EAE induction, useful references for other disease indicators such as DTH, in vitro prolif-
eration, and immunohistochemistry are provided. TMEV-IDD is a useful model for understanding the
possible viral etiology of MS. This section provides detailed information on the preparation of viral stocks
and subsequent intracerebral infection of mice. Additionally, virus plaque assay and clinical disease assess-
ment are discussed. Recently, recombinant TMEV strains have been created for the study of molecular
mimicry which incorporate various 30 amino acid myelin epitopes within the leader region of TMEV.
Key words: Multiple sclerosis, Experimental autoimmune encephalomyelitis, EAE, Emulsion, Active
induction, Adoptive transfer, T cell blasts, Encephalitogenic, Neurodegeneration, Theilers murine
encephalomyelitis virus-induced demyelinating disease, PLP, MOG, Myelin, MBP, VP2, VP3,
Relapsingremitting, Epitope spreading
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_19, Springer Science+Business Media New York 2012
381
382 D.P. McCarthy et al.
(e) isolate antigen-specific T cells from the CNS (27, 28). Passive
EAE induction is a valuable tool for delineating the relative contri-
butions of T helper subsets in disease, and it is considered to be a
more direct way of characterizing T cell effector function in the
CNS. In the SJL/J mouse, both active induction and adoptive
transfer of disease typically take a relapsingremitting form, while
the C57BL/6 mouse displays chronicprogressive disease following
active or passive EAE induction.
TMEV-IDD has been defined as a mouse model for human
multiple sclerosis (29, 30). TMEV is a natural mouse pathogen
that belongs to the cardiovirus group of the Picornaviridae family
(31, 32), and is composed of a single, positive-strand RNA genome
surrounded by a capsid containing viral proteins, VP1, VP2, and
VP3. TMEV is divided into two subgroups based on the patho-
genesis of the viruses. The first subgroup, which includes GDVII,
is highly virulent and induces fatal encephalitis in infected mice.
The second group, which is defined as the Theilers original subgroup,
includes Daniels (DA) and BeAn 8386 strains that have low viru-
lence and do not induce severe encephalitis, but do establish per-
sistent infections of the CNS associated with immune-mediated
demyelination (33).
TMEV-IDD is an immune-mediated demyelinating disease
dependent on persistent virus infection of the macrophages, micro-
glia, and astrocytes within the CNS (34, 35). TMEV-IDD is asso-
ciated with a mononuclear cell infiltrate consisting predominantly
of CD4+ T cells, macrophages, and B cells. The chronic phase of
TMEV-IDD is mediated by a PLP139151-specific CD4+ Th1 type T
cell response that can be initially detected at approximately
4555 days post-infection (36). As the disease progresses, epitope
spreading leads to autoimmune responses to additional myelin
antigens (37). The inflammation and demyelination observed in
TMEV-IDD are similar to the pathological descriptions in MS
patients (38, 39). Importantly, epidemiological studies suggest a
viral etiology for MS providing additional importance for TMEV-
IDD as a relevant model for multiple sclerosis (40, 41). Infection
of SJL/J mice with the BeAn strain has been directly associated
with the development of a chronicprogressive demyelinating
disease arising approximately 3035 days post-infection characterized
by spastic hind limb paralysis and primary demyelination (42).
In this article, we address the basic methods for inducing both
active and adoptive transfer of EAE and the induction of TMEV-
IDD. While different mouse strains have various susceptibilities to
both models of disease, we focus primarily on the induction in the
most commonly used strains of mice, the SJL/J and C57BL/6.
Additionally, the induction of TMEV-IDD is described using the
BeAn strain of TMEV in the SJL/J mouse. Mouse strain susceptibili-
ties and variation in disease are discussed in the Notes section 5.
384 D.P. McCarthy et al.
2. Materials
8. Centrifuge tubes.
9. Polyethylene glycol (PEG) (Sigma).
10. Tris base (Sigma).
11. Sodium chloride, NaCl (Sigma).
12. Sodium dodecyl sulfate, SDS (Sigma).
13. Sucrose (Sigma).
14. 21-, 23-, and 27-gauge needles.
15. Cesium sulfate, Cs2SO4 (Sigma).
16. PBS.
17. 60 mm tissue culture dishes (Nunc).
18. Noble agar (Sigma).
19. Penicillin/streptomycin (Life technologies).
20. Crystal violet (Sigma).
21. ClaI restriction enzyme (Promega).
22. DH5 max efficient E. coli (Invitrogen).
23. Sp6/T7 in vitro transcription kit (Roche).
24. Lipofectin reagent (Gibco BRL).
25. SJL/J mice (Harlan Laboratories).
3. Methods
3.1. Induction EAE can be induced in susceptible strains of mice by using proteo-
of Active lipid protein (PLP), myelin basic protein (MBP), myelin oligoden-
and Passive EAE drocyte glycoprotein (MOG), or peptides corresponding to the
encephalitogenic portions of these proteins. Peptides (>98 % purity
3.1.1. Induction
based on mass spectrophotometry) or spinal cord homogenate to
of Active EAE Disease
be used in priming are first dissolved in PBS and irradiated at
6,000 rads for sterilization purposes. For peptides that are insolu-
ble in PBS (pH 7.0), the pH can be raised until the peptide dis-
solves. The pH can then be lowered to physiologic levels; however,
pH has little influence on disease induction with peptide in CFA.
Peptide should be diluted in PBS to a concentration of 1 mg/ml if
inducing disease with PLP139151 in the SJL/J mouse. MOG3555-
induced disease in the C57BL/6 mouse requires a concentration
of 4 mg/ml in PBS as do all other peptides used to induce C57BL/6
or SJL/J disease. An equal volume of peptide/PBS is added to
complete Freunds adjuvant (containing 4 mg/mldesiccated M.
tuberculosis, H37 RA in Incomplete Freunds Adjuvant). This is
then thoroughly mixed to form a thick peptide/CFA emulsion.
For small volumes, the emulsion can be prepared directly between
two 1 ml tuberculin syringes using a 3-way stopcock with a Luer
386 D.P. McCarthy et al.
Table 1
Mouse strain and encephalitogenic peptides in active EAE
3.1.3. Clinical Grading Following priming, mice should be monitored every other day for
of Active and Passive EAE the development of disease. The appearance of EAE disease induced
by active immunization varies considerably based on mouse strain
and peptide used. For most strains and peptides, disease appears
between the second and fourth week following priming. The disease
is characterized by an ascending hind limb paralysis that begins in
the tail and spreads to involve the hind limbs and forelimbs. The
disease is graded on a 05 scale, though depending on strain and
peptide, mice do not always reach the higher disease grades before
disease resolution or disease plateau. Grade 0: there is no observ-
able difference from nave animals. Grade 1: assigned to mice that
have lost tail tonicity or show hind limb weakness (but not both).
Loss of tail tonicity is judged in mice that when held aloft by the
base of the tail show sagging of the tail and the tail cannot be lifted.
Additionally, the tip of the tail fails to curl. Hind limb weakness is
defined by the objective criterion that when placed on the wire
screen of the cage, the animals legs fall through as it tries to walk.
A waddling gait can also be observed as the animal walks on a flat
surface. The rear limbs are splayed and the rear posture lowered.
Grade 2: assigned to mice that present both a limp tail and show
hind limb weakness as defined above. Grade 3: assigned to mice
that show partial hind limb paralysis defined as the ability of a
mouse to move one or both hind limbs to some extent but not
maintain posture or walk. Grade 4: assigned to mice that cannot
move their hind limbs. The animal moves only by dragging itself
with its front limbs. A spastic paralysis and atrophy of the hind
limbs and lower body are often observed at this point. Mice at this
stage are given food on the cage floor (that can be moistened),
bottles with long sipper tubes, and daily injections of subcutaneous
saline to prevent death by dehydration. Grade 5: assigned to the
most severe end-stage assessment of EAE. These mice show a com-
plete inability to move due to paralysis in all limbs. In addition, any
animals that consistently show high grades and die (death by EAE)
should be given a grade of five. Mice that reach this stage and are
moribund with EAE should be sacrificed for humane reasons.
Histopathologically, the disease can be characterized by CD4+
T cell and F4/80+ (macrophage) inflammatory cell infiltrates that
can be found in both diffuse and focal patterns. In most EAE
models, the pattern of infiltrate tends to concentrate in the tho-
racic section of the spinal cord with less involvement in other
regions of the cord or the brain. However, recent studies have
revealed distinct differences in the histopathological features and
infiltration profile induced by Th1 and Th17 cells (23, 46).
3.1.4. Clinical Disease The first clinical episode is referred to as acute-phase disease which
Course is preceded by pronounced weight loss. Mice will experience this
acute episode for variable times depending on whether the disease
is relapsing and remitting (R/R), monophasic, or chronic/progressive
19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 391
Fig. 1. Histopathologic evaluation of 1 m thick Epon embedded spinal cord sections. Panel (a)spinal cord section from
a normal mouse. Note the presence of profuse and evenly distributed ringed structures reflecting myelinated axons, with
no infiltrating immune cells. Panel (b)spinal cord section from an SJL/J mouse with severe EAE. Note the few and
unevenly distributed myelinated axonal ringed structures with large bare areas, along with large numbers of infiltrating
immune cells throughout the section, appearing as dense and dark spots. Magnification: 220.
392 D.P. McCarthy et al.
3.2. Induction Virus is produced in BHK-21 cells (ATCC). BHK-21 cells are
of TMEV-IDD grown in DMEM (Sigma) supplemented with 10 % FCS, 0.295 %
tryptose phosphate broth (Sigma), 1.0 % gentamycin (Gibco BRL),
3.2.1. TMEV Infecting
and 1 % antimycoticantibiotic (Gibco BRL). Cells are maintained
Stock
in culture at 37 C and 5 % CO2 and grown to confluence. BHK-
21 cells are removed from the flask by rinsing with versene (1:5,000)
(Gibco BRL) or 1 TrypsinEDTA Solution (Sigma), resuspended
in complete medium (above), and seeded (1:10 split) in a new
flask. These BHK-21 cells are grown to confluence (23 days), and
washed with DMEM without serum or supplements.
Medium is removed from the cells, and TMEV, BeAn 8386
strain, is added at an MOI of 5 in a minimal volume of serum-free
medium ensuring that the cell monolayer is covered with medium.
The infected cells are incubated overnight at 33 C at 5 % CO2 until
BHK-21 cells detach from the flask surface indicating lysis. The
medium is transferred to a conical for centrifugation to pellet the cell
debris. The cell lysate is removed and stored on ice, leaving a small
volume on top of the pelleted cells. The pelleted cells are then soni-
cated using brief pulses to completely lyse the cells, and the resulting
cell debris is again pelleted by centrifugation. The lysate is added to
the stored lysate collected from the first spin and this is aliquoted
into small volumes and stored at 70 C. The titer of the infecting
virus stock is determined by plaque assay (described below).
3.2.2. Purification of TMEV Virus is produced in large stocks as described in Subheading 3.2.1
until the point of removing the supernatant from the infected cells.
The supernatant is removed from the infected cells and the pH is
adjusted with HCl to a pH below 7.0 and then frozen in bottles at
20 C. The bottles are thawed in 37 C shaking water bath with-
out allowing the supernatant to become too warm. To each 500 ml
of lysate, 14.5 g NaCl and 30 g PEG are added and the lysate is
stirred overnight at 4 C. The precipitated lysate is centrifuged at
7,000g in a Sorvall HB-4 swinging bucket rotor for 45 min at 4 C.
19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 393
3.2.3. TMEV Plaque Assay BHK-21 cells are cultured in 35 mm culture dishes (Thermo
Fischer: Nunc, Rochester, NY) to 90 % confluence as described
above. The BHK-21 cells are washed twice with serum-free
DMEM. Dilutions of the virus stock or tissue homogenate are
made in serum-free DMEM and 0.5 ml of each dilution is added
to the BHK-21 cells in duplicate. The cells are incubated at room
394 D.P. McCarthy et al.
3.2.4. Construction The cDNA for BeAn genome has been inserted into pGEM
of TMEV Containing plasmid for molecular manipulations. A restriction enzyme site,
Molecular Mimics of ClaI, was inserted into the leader sequence of the BeAn genome
Myelin Peptides along with a 23 amino acid deletion. Molecular mimic sequences
for myelin epitopes as previously described (48) are inserted into
the ClaI restriction site. PCR mutagenesis was conducted to insert
ClaI sites flanking the sequence to be inserted into the virus
genome. The mimic sequences are 30 amino acids in length to
restore the deletion in the leader protein. The mimic sequence is
ligated into the ClaI site in the BeAn cDNA, and DH5 E. coli are
transformed with the ligated product to produce a BeAn cDNA
containing the mimic sequence in the correct orientation. Next,
in vitro transcription of the BeAn cDNA containing the mimic
sequence is driven by an upstream T7 promoter using an Sp6/T7
in vitro transcription kit (Roche). This produces a single positive-
stranded RNA. The resulting RNA is transfected into BHK-21
cells in a 60 mm culture dish with DMEM supplemented with 2 %
FBS using lipofectin reagent (Gibco BRL) as described by the
manufacturers protocol. The transfected cells are incubated over-
night at 33 C. Following the incubation, the medium is removed
from the cells, replaced with DMEM containing 2 % serum, and
incubated for 23 additional days at 33 C until the cells began to
lyse, indicating virus production. Virus is isolated from the cells
following the procedure described above (Subheading 3.2.1). The
virus is amplified beginning with very small volumes until the virus
titer reaches 104 PFU/ml, and then larger volumes can be used to
produce the recombinant virus for infecting stocks. The viral titer
of the recombinant viruses is determined by plaque assay as
described above (Subheading 3.2.3).
19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 395
3.2.5. Induction TMEV is a naturally endemic infection in mice spread through the
of TMEV-Induced fecal oral route, which results in a 5060 % incidence of TMEV-
Demyelinating Disease IDD in disease-susceptible animals. Experimentally, disease-suscep-
tible SJL/J mice can be infected intracranially in order to increase
the incidence of TMEV-IDD development to approximately 90 % of
all infected animals. Six- to seven-week-old female SJL/J mice are
anesthetized with aerosolized isoflurane (Abbott Laboratories) and
inoculated with either 5 106 PFU of wild-type TMEV (BeAn 8386
strain) infecting stock (produced as described in Subheading 3.2.1)
or recombinant mimic-expressing viruses (produced as described in
Subheading 3.2.4), in 30 l in the right cerebral hemisphere by free-
hand injection with a 24 gauge needle. The cap of the needle remains
on the needle during injection but is cut down so that approximately
23 mm of the needle is exposed for the injection. This same needle
guard is used in all injections so that the virus is injected at the same
depth in each mouse injected. Mice are marked to allow for indi-
vidual evaluation of clinical and histological disease.
3.2.6. Clinical Assessment The clinical disease presentation seen in susceptible mouse strains,
of TMEV-Induced such as SJL/J, depends upon the strain of TMEV used for infec-
Demyelinating Disease tion. Following inoculation with the brain-derived DA strain of
virus, mice first develop a flaccid paralysis. Mice recover in approxi-
mately 2 weeks indicating that this phase of disease is self-limiting.
However, 23 weeks after infection, mice then develop a spastic
paresis of the hind limbs, which, in SJL/J mice, has a chronic dis-
ease course resulting in severe spastic paralysis (42). In contrast,
infection of SJL/J mice with the tissue culture-adapted BeAn 8386
strain does not produce clinical evidence of a first-phase disease.
Infected mice begin to show signs of clinical disease between 30
and 40 days post-TMEV infection and develop a chronic, progres-
sive paralysis with no recovery or remitting episodes, similar to
primary progressive MS. Unlike EAE, clinical signs develop slowly,
with no drastic changes in gait from day to day. Mice are moni-
tored for disease progression 23 times per week continuing for
100 days post-infection. Each mouse is assigned a numerical score
between 0 and 5, based on the severity of its impairment: 0, asymp-
tomatic; 1, mild gait abnormalities; 2, severe gait abnormalities; 3,
loss of ability to right itself associated with mild spastic paralysis; 4,
spastic paralysis in both hind limbs combined with urinary inconti-
nence and dehydration; 5, moribund. Infection with recombinant
infection with recombinant TMEV containing different molecular
mimic sequences leads to different disease profiles depending upon
the epitope expressed. Mice infected with the leader deletion
recombinant virus, ClaI-BeAn, do not develop signs of demyeli-
nating autoimmune disease (48). In contrast, mice infected with
the recombinant PLP139-BeAn virus exhibit an earlier onset and
more severe clinical disease, with onset between days 7 and 10
post-infection (48).
396 D.P. McCarthy et al.
4. Notes
References
1. Brown A, McFarlin DE, Raine CS (1982) 11. Traugott U, McFarlin DE, Raine CS (1986)
Chronologic neuropathology of relapsing Immunopathology of the lesion in chronic
experimental allergic encephalomyelitis in the relapsing experimental autoimmune encepha-
mouse. Lab Invest 46:171185 lomyelitis in the mouse. Cell Immunol
2. Karcher D, Lassmann H, Lowenthal A, Kitz K, 99:395410
Wisniewski HM (1982) Antibodies-restricted 12. Boyle EA, McGeer PL (1990) Cellular immune
heterogeneity in serum and cerebrospinal fluid response in multiple sclerosis plaques. Am J
of chronic relapsing experimental allergic Pathol 137:575584
encephalomyelitis. J Neuroimmunol 2:93106 13. McCallum K, Esiri MM, Tourtellotte WW,
3. Lassmann H (1983) Chronic relapsing experi- Booss J (1987) T cell subsets in multiple scle-
mental allergic encephalomyelitis: its value as rosis. Gradients at plaque borders and differ-
an experimental model for multiple sclerosis. J ences in nonplaque regions. Brain
Neurol 229:207220 110:12971308
4. Tan LJ, Kennedy MK, Miller SD (1992) 14. Renno T, Krakowski M, Piccirillo C, Lin JY,
Regulation of the effector stages of experimen- Owens T (1995) TNF-alpha expression by resi-
tal autoimmune encephalomyelitis via dent microglia and infiltrating leukocytes in the
neuroantigen-specific tolerance induction. II. central nervous system of mice with experimen-
Fine specificity of effector T cell inhibition. J tal allergic encephalomyelitis. Regulation by
Immunol 148:27482755 Th1 cytokines. J Immunol 154:944953
5. Eng LF, Ghirnikar RS, Lee YL (1996) 15. Swanborg RH (1995) Experimental autoim-
Inflammation in EAE: role of chemokine/ mune encephalomyelitis in rodents as a model
cytokine expression by resident and infiltrating for human demyelinating disease [see com-
cells. Neurochem Res 21:511525 ments) [Review) [99 refs). Clin Immunol
6. Miller SD, Karpus WJ (1994) The immunop- Immnopathol 77:413
athogenesis and regulation of T-cell mediated 16. Epstein LG, Prineas JW, Raine CS (1983)
demyelinating diseases. Immunol Today Attachment of myelin to coated pits on mac-
15:356361 rophages in experimental allergic encephalo-
7. Williams KC, Ulvestad E, Hickey WF (1994) myelitis. J Neurol Sci 61:341348
Immunology of multiple sclerosis [Review) 17. Smith ME (1993) Phagocytosis of myelin by
[300 refs). Clin Neurosci 2:229245 microglia in vitro. J Neurosci Res 35:480487
8. Lublin FD (1985) Relapsing experimental 18. Sommer MA, Forno LS, Smith ME (1992)
allergic encephalomyelitis. An autoimmune EAE cerebrospinal fluid augments in vitro
model of multiple sclerosis. Springer Semin phagocytosis and metabolism of CNS myelin
Immunopathol 8:197208 by macrophages. J Neurosci Res 32:384394
9. Arnason BG (1983) Relevance of experimental 19. Colover J (1988) Immunological and cytologi-
allergic encephalomyelitis to multiple sclerosis. cal studies of autoimmune demyelination and
Neurol Clin 1:765782 multiple sclerosis. Brain Behav Immun
10. Traugott U, Stone SH, Raine CS (1979) 2:341345
Chronic relapsing experimental allergic enceph- 20. Yednock TA, Cannon C, Fritz LC, Sanchez-
alomyelitis. Correlation of circulating lympho- Madrid F, Steinman L, Karin N (1992)
cyte fluctuations with disease activity in Prevention of experimental autoimmune
suppressed and unsuppressed animals. J Neurol encephalomyelitis by antibodies against 41
Sci 41:1729 integrin. Nature 356:6366
19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 399
21. Kawakami N, Lassmann S, Li Z, Odoardi F, model identify the location of possible immu-
Ritter T, Ziemssen T, Klinkert WE, Ellwart JW, nogenic sites. Ann NY Acad Sci 540:652653
Bradl M, Krivacic K, Lassmann H, Ransohoff 32. Pevear DC, Calenoff M, Rozhon E, Lipton HL
RM, Volk HD, Wekerle H, Linington C, Flugel (1987) Analysis of the complete nucleotide
A (2004) The activation status of neuroantigen- sequence of the picornavirus Theilers murine
specific T cells in the target organ determines encephalomyelitis virus indicates that it is closely
the clinical outcome of autoimmune encepha- related to cardioviruses. J Virol 61:15071516
lomyelitis. J Exp Med 199:185197 33. Lipton HL (1980) Persistent Theilers murine
22. Lees JR, Golumbek PT, Sim J, Dorsey D, encephalomyelitis virus infection in mice depends
Russell JH (2008) Regional CNS responses to on plaque size. J Gen Virol 46:169177
IFN-gamma determine lesion localization pat- 34. Clatch RJ, Miller SD, Metzner R, Dal Canto
terns during EAE pathogenesis. J Exp Med MC, Lipton HL (1990) Monocytes/mac-
205:26332642 rophages isolated from the mouse central ner-
23. Kroenke MA, Carlson TJ, Andjelkovic AV, Segal vous system contain infectious Theilers murine
BM (2008) IL-12- and IL-23-modulated T cells encephalomyelitis virus (TMEV). Virology
induce distinct types of EAE based on histology, 176:244254
CNS chemokine profile, and response to cytokine 35. Peterson JD, Karpus WJ, Clatch RJ, Miller SD
inhibition. J Exp Med 205:15351541 (1993) Split tolerance of Th1 and Th2 cells in
24. Cross AH, Raine CS (1990) Serial adoptive tolerance to Theilers murine encephalomyeli-
transfer of murine experimental allergic enceph- tis virus. Eur J Immunol 23:4655
alomyelitis: successful transfer is dependent on 36. Katz-Levy Y, Neville KL, Padilla J, Rahbe SM,
active disease in the donor. J Neuroimmunol Begolka WS, Girvin AM, Olson JK, Vanderlugt
28:2737 CL, Miller SD (2000) Temporal development
25. Raine CS, Mokhtarian F, McFarlin DE (1984) of autoreactive Th1 responses and endogenous
Adoptively transferred chronic relapsing exper- antigen presentation of self myelin epitopes by
imental autoimmune encephalomyelitis in the CNS-resident APCs in Theilers virus-infected
mouse. Neuropathologic analysis. Lab Invest mice. J Immunol 165:53045314
51:534546 37. Miller SD, Vanderlugt CL, Begolka WS, Pao
26. Zamvil S, Nelson P, Trotter J, Mitchell D, W, Yauch RL, Neville KL, Katz-Levy Y,
Knobler R, Fritz R, Steinman L (1985) T-cell Carrizosa A, Kim BS (1997) Persistent infec-
clones specific for myelin basic protein induce tion with Theilers virus leads to CNS autoim-
chronic relapsing paralysis and demyelination. munity via epitope spreading. Nat Med
Nature 317:355358 3:11331136
27. Kim C, Tse HY (1993) Adoptive transfer of 38. Prineas J (1975) Pathology of the early lesion
murine experimental autoimmune encephalo- in multiple sclerosis. Hum Pathol 6:531554
myelitis in SJL.Thy-1 congenic mouse strains. 39. Dal Canto MC, Lipton HL (1975) Primary
J Neuroimmunol 46:129136 demyelination in Theilers virus infection. An
28. Howard LM, Miller SD (2001) Autoimmune ultrastructural study. Lab Invest 33:626637
intervention by CD154 blockade prevents T 40. Nathanson N, Miller A (1978) Epidemiology
cell retention and effector function in the tar- of multiple sclerosis: critique of evidence for a
get organ. J Immunol 166:15471553 viral etiology. Am J Epidemiol 107:451461
29. Dal Canto M, Lipton HL, Miller SD, 41. Kurtzke JF, Raine CS, McFarlin HF,
Melvold RW, Capen CC, Jones TC, Migaki G Tourtellotte WW (1997) The epidemiology of
(1986) Theilers murine encephalomyelitis multiple sclerosis. In: Multiple sclerosis: clinical
virus (TMEV) infection in mice as a model for and pathogenetic basis. Chapman and Hall,
MS. In: Capen CC, Jones TC, Migaki G (eds.). London, pp 91139
In Registry of comparative pathology, hand- 42. Lipton HL (1975) Theilers virus infection in
book of animal models of human disease, VIIth mice: an unusual biphasic disease process leading
fascicle. AFIP, Washington, D.C. to demyelination. Infect Immun 11:11471155
30. Miller SD (1995) Pathogenesis of Theilers 43. Walker MR, Mannie MD (2002) Acquisition of
murine encephalomyelitis virus-induced demy- functional MHC class II/peptide complexes by
elinating disease a model of multiple sclerosis. T cells during thymic development and CNS-
ACLAD Newslett 16:46 directed pathogenesis. Cell Immunol
31. Pevear DC, Borkowski J, Luo M, Lipton H 218:1325
(1988) Sequence comparison of a highly viru- 44. Segal BM, Shevach EM (1996) IL-12 unmasks
lent and a less virulent strain of Theilers virus. latent autoimmune disease in resistant mice.
Amino acid differences on a three- dimensional J Exp Med 184:771775
400 D.P. McCarthy et al.
45. Axtell RC, de Jong BA, Boniface K, van der 55. McRae BL, Vanderlugt CL, Dal Canto MC,
Voort LF, Bhat R, De Sarno P, Naves R, Han Miller SD (1995) Functional evidence for
M, Zhong F, Castellanos JG, Mair R, Christakos epitope spreading in the relapsing pathology of
A, Kolkowitz I, Katz L, Killestein J, Polman experimental autoimmune encephalomyelitis. J
CH, de Waal Malefyt R, Steinman L, Raman C Exp Med 182:7585
(2010) T helper type 1 and 17 cells determine 56. Begolka WS, Vanderlugt CL, Rahbe SM, Miller
efficacy of interferon-beta in multiple sclerosis SD (1998) Differential expression of
and experimental encephalomyelitis. Nat Med inflammatory cytokines parallels progression of
16:406412 central nervous system pathology in two clini-
46. Stromnes IM, Cerretti LM, Liggitt D, Harris cally distinct models of multiple sclerosis. J
RA, Goverman JM (2008) Differential regula- Immunol 161:44374446
tion of central nervous system autoimmunity 57. Sakai K, Zamvil SS, Mitchell DJ, Lim M,
by T(H)1 and T(H)17 cells. Nat Med Rothbard JB, Steinman L (1988)
14:337342 Characterization of a major encephalitogenic T
47. Dal Canto MC, Melvold RW, Kim BS, Miller cell epitope in SJL/J mice with synthetic oligo-
SD (1995) Two models of multiple sclerosis: peptides of myelin basic protein. J
experimental allergic encephalomyelitis (EAE) Neuroimmunol 19:2132
and Theilers murine encephalomyelitis virus 58. Tuohy VK, Thomas DM (1993) A third
(TMEV) infection a pathological and immu- encephalitogenic determinant of myelin prote-
nological comparison. Microsc Res Tech olipid protein (PLP) for SJL/J mice. J Immunol
32:215229 150:194A
48. Olson JK, Croxford JL, Calenoff M, Dal Canto 59. Greer JM, Kuchroo VK, Sobel RA, Lees MB
MC, Miller SD (2001) A virus-induced molec- (1992) Identification and characterization of a
ular mimicry model of multiple sclerosis. J Clin second encephalitogenic determinant of myelin
Invest 108:311318 proteolipid protein (residues 178191) for SJL
49. Clatch RJ, Lipton HL, Miller SD (1986) mice. J Immunol 149:783788
Characterization of Theilers murine encepha- 60. Greer JM, Sobel RA, Sette A, Southwood S,
lomyelitis virus (TMEV)-specific delayed-type Lees MB, Kuchroo VK (1996) Immunogenic
hypersensitivity responses in TMEV-induced and encephalitogenic epitope clusters of myelin
demyelinating disease: correlation with clinical proteolipid protein. J Immunol 156:371379
signs. J Immunol 136:920927 61. Amor S, Groome N, Linington C, Morris MM,
50. Gerety SJ, Clatch RJ, Lipton HL, Goswami Dornmair K, Gardinier MV, Matthieu JM,
RG, Rundell MK, Miller SD (1991) Class Baker D (1994) Identification of epitopes of
II-restricted T cell responses in Theilers murine myelin oligodendrocyte glycoprotein for the
encephalomyelitis virus-induced demyelinating induction of experimental allergic encephalo-
disease. IV. Identification of an immunodomi- myelitis in SJL and Biozzi AB/H mice. J
nant T cell determinant on the N-terminal end Immunol 153:43494356
of the VP2 capsid protein in susceptible SJL/J 62. Zamvil SS, Mitchell DJ, Moore AC, Kitamura
mice. J Immunol 146:24012408 K, Steinman L, Rothbard JB (1986) T-cell
51. Jin YH, Kang B, Kim BS (2009) Theilers virus epitope of the autoantigen myelin basic protein
infection induces a predominant pathogenic that induces encephalomyelitis. Nature
CD4+ T cell response to RNA polymerase in 324:258260
susceptible SJL/J mice. J Virol 63. Whitham RH, Jones RE, Hashim GA, Hoy
83:1098110992 CM, Wang RY, Vandenbark AA, Offner H
52. Miller SD, Olson JK, Croxford JL (2001) (1991) Location of a new encephalitogenic
Multiple pathways to induction of virus- epitope (residues 43 to 64) in proteolipid pro-
induced autoimmune demyelination: lessons tein that induces relapsing experimental auto-
from Theilers virus infection. J Autoimmun immune encephalomyelitis in PL/J and (SJL
16:219227 PL)F1 mice. J Immunol 147:38033808
53. Duong TT, Finkelman FD, Singh B, Strejan 64. Mendel I, Kerlero DR, Ben-Nun A (1995) A
GH (1994) Effect of anti-interferon-gamma myelin oligodendrocyte glycoprotein peptide
monoclonal antibody treatment on the devel- induces typical chronic experimental autoim-
opment of experimental allergic encephalomy- mune encephalomyelitis in H-2b mice: fine
elitis in resistant mouse strains. J Neuroimmunol specificity and T cell receptor V beta expression
53:101107 of encephalitogenic T cells. Eur J Immunol
54. Zamvil SS, Steinman L (1990) The T lympho- 25:19511959
cyte in experimental allergic encephalomyelitis. 65. Tompkins SM, Padilla J, Dal Canto MC, Ting
Annu Rev Immunol 8:579621 JP, Van Kaer L, Miller SD (2002) De novo cen-
19 Mouse Models of Multiple Sclerosis: Experimental Autoimmune 401
tral nervous system processing of myelin anti- cryptic encephalitogenic T cell determinant for
gen is required for the initiation of experimental SJL/J mice. J Neuroimmunol 56:161170
autoimmune encephalomyelitis. J Immunol 74. Skundric DS, Kim C, Tse HY, Raine CS (1993)
168:41734183 Homing of T cells to the central nervous system
66. Tuohy VK, Lu ZJ, Sobel RA, Laursen RA, Lees throughout the course of relapsing experimen-
MB (1988) A synthetic peptide from myelin tal autoimmune encephalomyelitis in Thy-1
proteolipid protein induces experimental aller- congenic mice. J Neuroimmunol 46:113121
gic encephalomyelitis. J Immunol 75. Fritz RB, Zhao ML (1994) Encephalitogenicity
141:11261130 of myelin basic protein exon-2 peptide in mice.
67. Endoh M, Kunishita T, Nihei J, Nishizawa M, J Neuroimmunol 51:16
Tabira T (1990) Susceptibility to proteolipid 76. Segal BM, Raine CS, McFarlin DE, Voskuhl
apoprotein and its encephalitogenic determi- RR, McFarland HF (1994) Experimental aller-
nants in mice. Int Arch Allergy Appl Immunol gic encephalomyelitis induced by the peptide
92:433438 encoded by exon 2 of the MBP gene, a peptide
68. Muller DM, Pender MP, Greer JM (2000) A implicated in remyelination. J Neuroimmunol
neuropathological analysis of experimental 51:719
autoimmune encephalomyelitis with predomi- 77. Pettinelli CB, McFarlin DE (1981) Adoptive
nant brain stem and cerebellar involvement and transfer of experimental allergic encephalomy-
differences between active and passive induc- elitis in SJL/J mice after in vitro activation of
tion. Acta Neuropathol (Berl) 100:174182 lymph node cells by myelin basic protein:
69. Maron R, Hancock WW, Slavin A, Hattori M, requirement for Lyt 1+ 2 T lymphocytes. J
Kuchroo V, Weiner HL (1999) Genetic suscep- Immunol 127:14201423
tibility or resistance to autoimmune encephalo- 78. Pettinelli CB, Fritz RB, Chou CHJ, McFarlin
myelitis in MHC congenic mice is associated DE (1982) Encephalitogenic activity of guinea
with differential production of pro- and anti- pig myelin basic protein in the SJL mouse. J
inflammatory cytokines. Int Immunol Immunol 129:12091211
11:15731580 79. Shaw MK, Kim C, Hao HW, Chen F, Tse HY
70. Slavin A, Ewing C, Liu J, Ichikawa M, Slavin J, (1996) Induction of myelin basic protein-
Bernard CC (1998) Induction of a multiple specific experimental autoimmune encephalo-
sclerosis-like disease in mice with an immu- myelitis in C57BL/6 mice: mapping of T cell
nodominant epitope of myelin oligodendrocyte epitopes and T cell receptor V beta gene seg-
glycoprotein. Autoimmunity 28:109120 ment usage. J Neurosci Res 45:690699
71. McRae BL, Kennedy MK, Tan LJ, Dal Canto 80. Clark RB, Grunnet M, Lingenheld EG (1997)
MC, Miller SD (1992) Induction of active and Adoptively transferred EAE in mice bearing the
adoptive chronic-relapsing experimental auto- lpr mutation. Clin Immunol Immnopathol
immune encephalomyelitis (EAE) using an 85:315319
encephalitogenic epitope of proteolipid pro- 81. Mendel I, Shevach EM (2002) Differentiated
tein. J Neuroimmunol 38:229240 Th1 autoreactive effector cells can induce
72. Miller SD, Tan LJ, Kennedy MK, Dal Canto experimental autoimmune encephalomyelitis in
MC (1991) Specific immunoregulation of the the absence of IL-12 and CD40/CD40L inter-
induction and effector stages of relapsing EAE actions. J Neuroimmunol 122:6573
via neuroantigen-specific tolerance induction. 82. Segal BM, Dwyer BK, Shevach EM (1998) An
Ann NY Acad Sci 636:7994 Interleukin (IL)-10/IL-12 immunoregulatory
73. Tuohy VK, Thomas DM (1995) Sequence circuit controls susceptibility to autoimmune
104-117 of myelin proteolipid protein is a disease. J Exp Med 187:537546
Chapter 20
Abstract
Multiple sclerosis is an inflammatory demyelinating and neurodegenerative disorder of the central nervous
system (CNS). The primary cause of the disease remains unknown, but an altered immune regulation with
features of autoimmunity has generally been considered to play a critical role in the pathogenesis.
Historically, lesion development has been attributed to activation of CD4 and CD8 T lymphocytes, B
lymphocytes, and monocytes in the peripheral circulation and the migration of these cells through the
bloodbrain barrier to exert direct or indirect cytotoxic effects on myelin, oligodendrocytes and neuronal
processes in the CNS. This broadly accepted concept was significantly influenced by the experimental
autoimmune encephalitis (EAE) model, in which either immunization with myelin antigens or injection of
a myelin antigen-specific T cell line into a recipient results in inflammatory demyelination in the CNS.
More recent studies reveal that the loss of oligodendrocytes and neurons begins in the earliest stages of the
disease and may not always be associated with blood-derived inflammatory cells. The pathology affects
both the white and the gray matters and the clinical disability best correlates with the overall neurodegen-
erative process. These newer observations prompted several revisions of the classical concept of MS and
facilitated a shift from using EAE to using other model systems. This chapter summarizes the classical and
more contemporary concepts of MS, and provides methodologies for employing the cuprizone model for
further explorations of the pathogenesis and treatment of the disease.
1. Inflammation,
Demyelination,
and Neuro-
degeneration in Multiple sclerosis is an inflammatory process associated with demy-
Multiple Sclerosis elination and neurodegeneration in the central nervous system
(CNS). The etiology remains unknown, but the development of
1.1. Immune-Mediated the disease has been linked to an altered immune response qualified
Mechanisms of MS by many measures for autoimmunity (1).
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_20, Springer Science+Business Media New York 2012
403
404 P. Acs and B. Kalman
Box 1
The EAE Model
1.2. The Histo- Since most histopathology studies have to rely on sampling at a
pathology of MS single time point, the temporal sequence of events and the exact
role of blood-derived mononuclear cells (MNCs) in lesion development
20 Pathogenesis of Multiple Sclerosis 407
1.2.1. Heterogeneity of MS Using biopsied and postmortem MS tissues with early lesions
Lesions and Loss of Lucchinetti et al. (24) proposed four intra-individually consistent
Oligodendrocytes: The subtypes of acute demyelinating lesions. Type I and II lesions express
Concept of Histological signs of autoimmunity with perivenular inflammation and demyeli-
Heterogeneity of MS nation. Type I lesions are only characterized by MNC infiltration.
Evolved in Two Directions Type II lesions also have MNC infiltration, but with signs of immu-
noglobulin and complement deposition. In contrast to type I and II
Interindividual
lesions, type III and IV lesions have sparse inflammatory cell
Heterogeneity and
infiltration and instead of autoimmunity, they express signs of oligo-
Intra-individual
dendrocytopathy. The loss of oligodendrocytes is best characterized
Homogeneity of MS
in type III lesions. Type IV lesions with oligodendrocyte loss are the
Lesions
least frequent among all MS lesions (Box 2a).
Sequence of Lesion A more uniform mechanism was proposed for early plaque forma-
Development and Temporal tion with heterogeneity over time by Barnett and Prineas (25)
Heterogeneity (Box 2b). Studying new symptomatic lesions in patients who died
shortly after the onset of a relapse, these authors observed exten-
sive oligodendrocyte apoptosis with nuclear condensation but
without caspase 3 activation. The loss of oligodendrocytes in dis-
crete, still myelinated tissue regions was associated with microglial
activation in the absence of MNC infiltration. Myelin edema and
tissue vacuolization appeared to follow the loss of oligodendro-
cytes, and to trigger the immigration of activated lymphocytes.
A subsequent analysis by the same group confirmed that oligo-
dendrocyte apoptosis is the earliest event in prephagocytic lesions
with still intact myelin and without T and B cell infiltration in tis-
sue bordering rapidly expanding MS lesions. Phagocytic lesions
include macrophages associated with myelin fragmentation, where
the role of innate immune response is to scavenge injured myelin.
408 P. Acs and B. Kalman
Box 2
Loss of Oligodendrocytes
(continued)
20 Pathogenesis of Multiple Sclerosis 409
Box 2
(continued)
1.2.2. Neuroaxonal Oligodendrocytes are not the only cell types undergoing apoptosis
Degeneration in MS in MS brains. Trapp et al. (20) demonstrated axonal transection in
association with inflammation in the white matter by using immu-
nohistochemistry and confocal microscopy. In contrast, apoptotic
neurons and transection of axonal and dendritic processes were
described in areas of demyelination but in the absence of notable
MNC infiltration in cortical MS lesions (29). Only activated
microglia surrounded injured dendrites, neuritis, and neuronal
pericaria, suggesting that the neuroaxonal loss may be related to or
followed by microglial activation (22). Demyelinating lesions and
neuronal loss were observed not only in cortical but also in deep
gray matter regions (30). Observations suggest that the loss of
410 P. Acs and B. Kalman
1.2.3. Possible Causes Thus far, no primary endogenous or exogenous causes of oligoden-
of Cell Injury in MS Brains drocyte and neuronal apoptosis have been identified. Some of the
factors contributing to cell and tissue injury developing secondary
to inflammation are reviewed in Box 2c. Direct cytotoxic mecha-
nisms mediated by cytokines and their receptors have been compre-
hensively explored in experimental systems but direct evidence is
sparse in MS (32, 33). Our team proposed that inflammation ignites
a mitochondrion-driven mechanism that contributes to tissue
degeneration in MS (3437). Activated monocytes and microglia
in MS express inducible nitric oxide synthase (iNOS) and produce
increased amounts of NO, which damages proteins by generating
nitration adducts (e.g., nitrotyrosine). NO can also react with O2
(a component of reactive oxygen speciesROS) resulting in a toxic
intermediate called peroxinitrite (38, 39). ROS are produced in
increased amounts by activated inflammatory cells in MS and give a
ring-like appearance on MRI representing macrophages around
acute plaques (40, 41). NO and ROS cause oxidative damage to
macromolecules in the site of inflammation (34). While evidence
suggests that scavengers of NO and physiologic antioxidants can
provide significant therapeutic effects in EAE (42), upregulation of
such molecules was not detected in MS lesions. In contrast, a
significant accumulation of oxidative damage to DNA and a
decreased activity of Complex I were found in chronic active plaques
(34). Despite the accumulated oxidative damage to mitochondrial
(mt)DNA, this decreased Complex I activity was not related to an
accelerated accumulation of somatic mtDNA deletions in chronic
active plaques compared to other brain regions or to age-matched
controls (43). Altered mRNA and protein expressions of mito-
chondrial molecules and impaired activity of other mitochondrial
enzymes were also observed in MS cortex and plaques (4446).
Using histochemistry, Mahad et al. (47) found impaired function
of Complex IV (cytochrome c oxidaseCOX) in Balos type of
concentric sclerosis and in pattern III but not in pattern II MS
lesions (24). This defect in enzyme activity was accompanied by a
decreased expression of the catalytic component (COX-I) of
Complex IV in oligodendrocytes, hypertrophied astrocytes, and
axons (46, 47). These observations suggest that Complex IV func-
tional defect may contribute to the injury of mature oligodendro-
cytes in subtypes of demyelinating MS lesions. In addition to
inflammation, demyelination may directly contribute to a mito-
chondrial mechanism facilitating axonal loss. This mechanism
results from the increased expression and redistribution of the
voltage-gaited Na-channels from the node of Ranvier to the entire
20 Pathogenesis of Multiple Sclerosis 411
2. Cuprizone-
Induced
Oligodendrocyte
Apoptosis and In the past few decades, the cuprizone-induced demyelination
Demyelination model attracted prominent interest, since contrary to other models
of MS, this one provides a highly reproducible system of primary
2.1. The Utility of the oligodendrocyte (OL) apoptosis and secondary demyelination.
Cuprizone Model in The administration of the copper chelating agent cuprizone
(bis-cyclohexanone oxaldihydrazone) to mice induces spatially and
Studying MS
temporally well-defined histopathological alterations in the CNS.
The earliest event is the appearance of megamitochondria (52),
followed by oligodendrocyte apoptosis. The peak of the apoptotic
events is between the third and tenth days (53) of the cuprizone
challenge, but apoptotic oligodendrocytes can be detected during
the entire administration, and even during the recovery period
12 weeks posttreatment (54). The exact mechanism of oligoden-
drocyte apoptosis is not fully understood, and is often debated.
However, it is generally accepted that cuprizone induces metabolic
disturbances in oligodendrocytes which leads to caspase-independent,
apoptosis inducing factor (AIF)-mediated cell death, involving a
mitochondrial mechanism (36, 55).
The massive OL apoptosis is followed by extensive demyelina-
tion. The loss of myelin is preceded and accompanied by a down-
regulation of myelin-related proteins with varying kinetics. For
example, a down-regulation of MAG expression can be seen in a
few days after the initiation of cuprizone administration, while a
complete demyelination of the corpus callosum is usually observed
after 6 weeks of treatment. While demyelination was thought to
affect only particular white matter tracts (i.e., corpus callosum,
superior cerebellar peduncle) (56), recent studies reveal that other
regions including the hippocampus, putamen, cerebellum, and
even distinct gray matter areas in the cortex also undergo demyeli-
nation (57).
412 P. Acs and B. Kalman
3. Materials
3.1. Methods As the cuprizone model is suitable for asking a wide variety of
scientific questions, here we only aim to describe the basic method-
ology applicable to different experimental systems. Using these
methods, investigators will be able to investigate general patho-
logical features of oligodendrocyte apoptosis, demyelination, and
remyelination, to test and evaluate the effectiveness of cuprizone
treatment, and to learn a general scenario that occurs in most of
the cuprizone experiments. Before starting a cuprizone experi-
ment, we recommend performing pilot experiments and determin-
ing the kinetics of pathological events in order to have a baseline
for subsequent studies.
3.1.1. Mice The most commonly used strain is the C57BL/6J, and the most
suitable region for the evaluation of demyelination is the corpus
callosum. Therefore, the description below is focused on the alter-
ations found in the corpus callosum of 8-week-old, C57BL/6J,
male mice. It has to be noticed that the cuprizone treatment
induces highly reliable and reproducible demyelination in the CNS
(Fig. 1), although strain, gender, and age differences influence the
distribution and extent of demyelination in mice (Fig. 2; for details
see Subheading 2.3.5) (59, 60).
3.1.3. In Vivo Monitoring Cuprizone experiments commonly last for several weeks, during
of the Cuprizone Effect which the pathological effects of the toxin are monitored without
sacrificing animals.
Measuring the Weight The optimal weight range to start the cuprizone administration is
of the Mice between 20 and 24 g in case of male mice. A loss of 34 g in the
first 2 weeks of treatment is characteristic of cuprizone challenge.
However, the weight loss is not an essential element of a successful
cuprizone experiment. After the omission of cuprizone, a rapid
weight gain is expected.
Note: If the weight of mice is to be precisely recorded, it is useful
to change the standard pellet chow to milled chow a couple of days
before the beginning of the experiment, as the switch to the milled
chow itself may induce a minor weight loss (12 g).
20 Pathogenesis of Multiple Sclerosis 417
3.1.4. Tissue Harvesting The choice of tissue harvesting techniques may depend on the aim
of the experiment. Generally, transcardial perfusion is recom-
mended for histochemistry, but this method of fixation is not suit-
able for protein or RNA studies. The detailed description of tissue
harvesting processes for protein and RNA assays is shown in the
corresponding Subheadings 2.3.8 and 2.3.9.
Transcardial perfusion
1. Mice are pretreated with 0.1 ml low-molecular-weight heparin
30 min prior to the perfusion.
2. Mice are anesthetized by intraperitoneal injection of diazepam
(5 mg/kg) and ketamine (80 mg/kg).
3. Mice are pinned to the procedure table, the chest is opened,
and the left ventricle is punctured with a green butterfly nee-
dle, while the right atrium is opened.
4. The fixative is introduced into the main circulation via the
green butterfly needle placed into the left ventricle.
5. 4 % Paraformaldehyde solution in PBS is used for fixation.
6. The perfusion is followed by a careful dissection of the brain
and an overnight postfixation in the same fixative.
7. For frozen sections, the brains are placed first into a 10 %, and
then into a 15 % sucrose solution for 1-1 h and subsequently
soaked in a 20 % sucrose solution overnight.
8. For paraffin sections, the brains are routinely dehydrated and
embedded into paraffin.
Note: If low-molecular-weight heparin is unavailable, the perfu-
sion may be started with PBS or physiological saline to wash the
blood out of the vessels. The exact duration of this initial PBS/
saline wash may vary, but as a general rule, the perfusion with the
fixative can be started when the fluid leaking out of the dissected
right atrium becomes water clear.
Fig. 6. MBP immunoblot from the dissected corpus callosum after 4 weeks of cuprizone
administration. MBP expression is almost absent in the sample from the cuprizone-treated
mouse. Even protein loadings were confirmed by an anti-Akt antibody and immunoblotting.
3.1.9. Gene Expression The selective regional vulnerability is one of the major characteris-
Analyses of Myelin Basic tic features of cuprizone model. The physiological and pathologi-
Protein by Real-Time PCR cal mechanisms causing this phenomenon are still unknown. The
combination of micropunch technique with qRT PCR allows us to
investigate anatomically closely localized but functionally different
brain regions.
1. Mice are rapidly decapitated and brains are quickly removed
and placed on dry ice.
2. Samples are obtained by micropunching technique (66). While
this method may require higher numbers of animals (since the
amounts of the obtained samples are small), it provides the best
quality of RNA and allows the most precise localization of the
acquired punched samples. Samples obtained by this technique
will exclusively contain tissue from a highly selected brain region.
3. Two-hundred micrometer thick serial sections are made in a
cryostat (Leica CM3050 S).
4. The slides are immediately placed on dry ice for transfer, and
kept at 80 C until needed.
5. The micropunching is performed with a punching needle
(200 mm in diameter) on dry ice, under stereomicroscopic
control. Samples can be obtained from the corpus callosum or
other selected parts of the brain, and kept at 80 C until
further experiments.
6. Total RNA is isolated using the NucleoSpin kit (Macherey-
Nagel, Germany) according to the manufacturers protocol.
7. The concentration of RNA is extrapolated from the OD260
spectrophotometric measurement. The quality of RNA may be
assessed based on the OD260/280 ratio and the integrity of
20 Pathogenesis of Multiple Sclerosis 427
4. Conclusion
References
1. Bennett JL, Stve O (2009) Update on model for testing of immunosuppressive agents
inflammation, neurodegeneration, and immuno- for the treatment of autoimmune central ner-
regulation in multiple sclerosis: therapeutic impli- vous system disease. J Pharmacol Exp Ther
cations. Clin Neuropharmacol 32(3):121132 242(2):614620
2. Linthicum DS, Munoz JJ, Blaskett A (1982) 4. Steinman L (2010) Mixed results with modula-
Acute experimental autoimmune encephalomy- tion of TH-17 cells in human autoimmune dis-
elitis in mice. I. Adjuvant action of Bordetella eases. Nat Immunol 11(1):4144
pertussis is due to vasoactive amine sensitiza- 5. Crome SQ, Wang AY, Levings MK (2010)
tion and increased vascular permeability of the Translational mini-review series on TH17 cells:
central nervous system. Cell Immunol function and regulation of human T helper 17
73(2):299310 cells in health and disease. Clin Exp Immunol
3. Westarp ME, Wekerle H, Ben-Nun A et al 159(2):109119
(1987) T lymphocyte line-mediated experimen- 6. OConnor RA, Taams LS, Anderton SM (2010)
tal allergic encephalomyelitis a pharmacologic Translational mini-review series on Th17 cells:
20 Pathogenesis of Multiple Sclerosis 429
CD4 T helper cells: functional plasticity and 18. Hauser SL, Waubant E, Arnold DL et al (2008)
differential sensitivity to regulatory T cell- HERMES Trial Group. B-cell depletion with
mediated regulation. Clin Exp Immunol rituximab in relapsing-remitting multiple scle-
159(2):137147 rosis. N Engl J Med 358(7):676688
7. Correale J, Villa A (2010) Role of regulatory 19. Bar-Or A, Calabresi PA, Arnold D et al (2008)
CD8+CD25+FoxP3+ T cells in multiple scle- Rituximab in relapsing-remitting multiple scle-
rosis. Ann Neurol 67(5):625638 rosis: a 72-week, open-label, phase I trial. Ann
8. Cepok S, Rosche B, Grummel V et al (2005) Neurol 63(3):395400
Short-lived plasma blasts are the main B cell 20. Trapp BD, Peterson J, Ransohoff RM et al
effector subset during the course of multiple (1998) Axonal transection in the lesions of
sclerosis. Brain 128:16671676 multiple sclerosis. N Engl J Med 338:278285
9. Qin Y, Duquette P, Zhang Y et al (1998) Clonal 21. Trapp BD, Bo L, Mork S, Chang A (1999)
expansion and somatic hypermutation of Vh Pathogenesis of tissue injury in MS lesions. J
genes of B cells from the cerebrospinal fluid in Neuroimmunol 98:4956
multiple sclerosis. J Clin Invest 102:10451050 22. Peterson JW, Bo L, Mork S, Chang A et al
10. Owens GP, Burgoon MP, Anthony J et al (2001) (2002) VCAM-1-positive microglia target oligo-
The immunoglobulin G heavy chain repertoire dendrocytes at the border of multiple sclerosis
in multiple sclerosis plaques is distinct from the lesions. J Neuropathol Exp Neurol 61:539546
heavy chain repertoire in peripheral blood lym- 23. Kutzelnigg A, Lucchinetti CF, Stadelmann C
phocytes. Clin Immunol 98:258263 (2005) Cortical demyelination and diffuse
11. Monson NL, Brezinschek HP, Brezinschek RI white matter injury in multiple sclerosis. Brain
et al (2005) Receptor revision and atypical 128(Pt 11):27052712
mutational characteristics in clonally expanded 24. Lucchinetti C, Brck W, Parisi J et al (2000)
B cells from the cerebrospinal fluid of recently Heterogeneity of multiple sclerosis lesions:
diagnosed multiple sclerosis patients. J implications for the pathogenesis of demyelina-
Neuroimmunol 158(12):170181 tion. Ann Neurol 47(6):707717
12. Lambracht-Washington D, OConnor KC, 25. Barnett MH, Prineas JW (2004) Relapsing and
Cameron EM et al (2007) Antigen specificity remitting multiple sclerosis: pathology of the
of clonally expanded and receptor edited cere- newly forming lesion. Ann Neurol 55:
brospinal fluid B cells from patients with relaps- 458468
ing remitting MS. J Neuroimmunol 26. Henderson AP, Barnett MH, Parratt JD,
186(12):164176 Prineas JW et al (2009) Multiple sclerosis: dis-
13. Serafini B, Rosicarelli B, Magliozzi R et al tribution of inflammatory cells in newly form-
(2004) Detection of ectopic B-cell follicles with ing lesions. Ann Neurol 66:739753
germinal centers in the meninges of patients 27. Barnett MH, Sutton I (2006) The pathology of
with secondary progressive multiple sclerosis. multiple sclerosis: a paradigm shift. Curr Opin
Brain Pathol 14(2):164174 Neurol 19:242247
14. Willis SN, Stadelmann C, Rodig SJ et al (2009) 28. Breij EC, Brink BP, Veerhuis R et al (2008)
EpsteinBarr virus infection is not a character- Homogeneity of active demyelinating lesions in
istic feature of multiple sclerosis brain. Brain established multiple sclerosis. Ann Neurol
132(Pt 12):33183328 63(1):1625
15. Lucchinetti C, Brck W, Noseworthy J (2001) 29. Peterson JW, Bo L, Mork S et al (2001)
Multiple sclerosis: recent developments in neu- Transected neurites, apoptotic neurons, and
ropathology, pathogenesis, magnetic resonance reduced inflammation in cortical multiple scle-
imaging studies and treatment. Curr Opin rosis lesions. Ann Neurol 50:389400
Neurol 14(3):259269
30. Vercellino M, Plano F, Votta B et al (2005)
16. Stoeckle C, Tolosa E (2009) Antigen processing Grey matter pathology in multiple sclerosis.
and presentation in multiple sclerosis. Results J Neuropathol Exp Neurol 64:11011107
Probl Cell Differ 2010;51:149172
31. Wegener C, Esiri MM, Chance SA et al (2006)
17. Goodin DS, Cohen BA, OConnor P et al Neocortical neuronal, synaptic, and glial loss in
(2008) Therapeutics and Technology Assessment multiple sclerosis. Neurology 67:960967
Subcommittee of the American Academy of
Neurology. Assessment: the use of natalizumab 32. Zipp F (2000) Apoptosis in multiple sclerosis.
(Tysabri) for the treatment of multiple sclerosis Cell Tissue Res 301(1):163171
(an evidence-based review): report of the 33. Hestvik AL, Skorstad G, Vartdal F et al (2009)
Therapeutics and Technology Assessment Idiotype-specific CD4+ T cell induced apoptosis
Subcommittee of the American Academy of of human oligodendrocytes. J Autoimmun
Neurology. Neurology 71(10):766773 32(2):125132
430 P. Acs and B. Kalman
in endogenous cell repair of the CNS. Future reveal strain and gender pattern differences in
Neurol 2:689697 demyelination. Brain Pathol 19:467479
63. Morell P, Barrett CV, Mason JL et al (1998) 65. Hiremath MM, Saito Y, Knapp GW et al (1998)
Gene expression in brain during cuprizone- Microglial/macrophage accumulation during
induced demyelination and remyelination. Mol cuprizone-induced demyelination in C57BL/6
Cell Neurosci 12:220227 mice. J Neuroimmunol 92:3849
64. Taylor LC, Gilmore W, Matsushima GK (2009) 66. Palkovits M (1983) Punch sampling biopsy
SJL mice exposed to cuprizone intoxication technique. Methods Enzymol 103:368376
Chapter 21
Abstract
Inflammatory diseases of the mucosal surfaces are rising worldwide and particularly in the Western world
that is witnessing unprecedented increases in the number and incidence of both asthma and inflammatory
bowel disease. The laboratory mouse allows the application of the full panoply of modern genetic, immu-
nological and biochemical tools to increase our understanding of how inflammation arises and how it
might be controlled at mucosal surfaces. Here we provide a detailed description of how to systematically
assess inflammatory disease in the lung and intestines of the laboratory mouse. We provide histopathology
examples from SHIP mutant mice that are the only known genetic mutant to suffer from pulmonary con-
solidation, asthma, and Crohns disease. The intent of this chapter is to facilitate increased surveillance of
mucosal inflammation in studies where the laboratory mouse is utilized so that we can better understand
the cell types, genes, and microorganisms that contribute to mucosal inflammatory disease and thereby
develop more effective therapies and preventive strategies.
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_21, Springer Science+Business Media New York 2012
433
434 R.W. Engelman and W.G. Kerr
Fig. 1. (a) Insufflated small intestine rolled to fit as a Swiss roll into a cassette for
histological processing. (b) Full-length small intestine thin section preparation for microscopic
assessment from an SHIP/ mouse with regional thickening of the ileum. (c) Insufflated
lungs ready for cassette placement and histological processing.
Fig. 2. Eosinophilic crystalline pneumonia of SHIP/ mice with (a) large crystals in bronchiolar airways, focal mixed
leukocyte infiltration, bronchiolar subepithelial fibrosis, hypertrophy, and mucous metaplasia of bronchiolar epithelium,
numerous alveolar macrophages, and multinucleate cells, which result in patchy (b) to lobar (c) pulmonary consolidation.
Fig. 3. Histopathological appearance of the small intestine of SHIP/ mice with CD-like disease assessed as inflammatory
(a) grade 1 with mild polymorphonuclear (PMN) leukocyte infiltrations of the lamina propria and enteric lymph nodule
(arrow); (b) grade 2 with moderate PMN leukocyte infiltrations; (c) grade 3 with marked PMN infiltrations extending below
the muscularis mucosa; (d) grade 4 with marked infiltrations extending into the tunica muscularis; (e) grade 5 with marked
transmural leukocyte infiltrations; (f) grade 6 with transmural leukocyte infiltrations extending into the mesentery.
Acknowledgements
This work was supported in part by grants from the NIH (RO1
HL72523, R01 HL085580, R01 HL107127) and the Paige
Arnold Butterfly Run. WGK is the Murphy Family Professor of
Childrens Oncology Research and an Empire Scholar of the State
University of NY. The staffs of the Weiskotten (SUNY) and Moffitt
(USF) vivariums are thanked for their very capable assistance with
tissue harvesting and processing.
References
6. Barrett JC, Hansoul S, Nicolae DL et al (2008) of toll-like receptor 3 (TLR3) and TLR4 in
Genome-wide association defines more than 30 inflammatory bowel disease. Infect Immun
distinct susceptibility loci for Crohns disease. 68:70107017
Nat Genet 40:955962 21. Pizarro TT, Michie MH, Bentz M et al (1999)
7. Sollid LM, Johansen FE (2008) Animal models IL-18, a novel immunoregulatory cytokine, is
of inflammatory bowel disease at the dawn of up-regulated in Crohns disease: expression
the new genetics era. PLoS Med 5:e198 and localization in intestinal mucosal cells.
8. Bouma G, Strober W (2003) The immunologi- J Immunol 162:68296835
cal and genetic basis of inflammatory bowel 22. Olson TS, Reuter BK, Scott KG et al (2006)
disease. Nat Rev Immunol 3:521533 The primary defect in experimental ileitis origi-
9. Shanahan F (2002) Crohns disease. Lancet nates from a nonhematopoietic source. J Exp
359:62 Med 203:541552
10. Monteleone G, Biancone L, Marasco R et al 23. Heazlewood CK, Cook MC, Eri R, Price GR,
(1997) Interleukin 12 is expressed and actively Tauro SB et al (2008) Aberrant mucin assem-
released by Crohns disease intestinal lamina bly in mice causes endoplasmic reticulum stress
propria mononuclear cells. Gastroenterology and spontaneous inflammation resembling
112:11691178 ulcerative colitis. PLoS Med 5(3):e54
11. Fuss IJ, Neurath M, Boirivant M et al (1996) 24. Van der Sluis M, De Koning BA, De Bruijn
Disparate CD4+ lamina propria (LP) lym- AC, Velcich A, Meijerink JP et al (2006) Muc2-
phokine secretion profiles in inflammatory deficient mice spontaneously develop colitis,
bowel disease. Crohns disease LP cells mani- indicating that MUC2 is critical for colonic
fest increased secretion of IFN-gamma, whereas protection. Gastroenterology 131:117129
ulcerative colitis LP cells manifest increased 25. Strober W, Fuss IJ, Blumberg RS (2002) The
secretion of IL-5. J Immunol 157:12611270 immunology of mucosal models of
12. Duerr RH, Taylor KD, Brant SR et al (2006) A inflammation. Annu Rev Immunol 20:
genome-wide association study identifies 495549
IL23R as an inflammatory bowel disease gene. 26. Matsumoto S, Okabe Y, Setoyama H et al
Science 314:14611463 (1998) Inflammatory bowel disease-like enteri-
13. Yen D, Cheung J, Scheerens H et al (2006) tis and caecitis in a senescence accelerated
IL-23 is essential for T cell-mediated colitis and mouse P1/Yit strain. Gut 43:7178
promotes in fl ammation via IL-17 and IL-6. 27. Kontoyiannis D, Pasparakis M, Pizarro TT et al
J Clin Invest 116:13101316 (1999) Impaired on/off regulation of TNF
14. Singh B, Read S, Asseman C et al (2001) biosynthesis in mice lacking TNF AU-rich ele-
Control of intestinal inflammation by regula- ments: implications for joint and gut-associated
tory T cells. Immunol Rev 182:190200 immunopathologies. Immunity 10:387398
15. Kazemi-Shirazi L, Gasche CH, Natter S et al 28. Kerr WG, Park MY, Maubert M, Engelman
(2002) IgA autoreactivity: a feature common RW (2010) SHIP deficiency causes Crohns
to inflammatory bowel and connective tissue disease-like ileitis. Gut 10:11361148
diseases. Clin Exp Immunol 128:102109 29. Kosiewicz MM, Nast CC, Krishnan A et al
16. Dieckgraefe BK, Korzenik JR (2002) Treatment (2001) Th1-type responses mediate spontane-
of active Crohns disease with recombinant ous ileitis in a novel murine model of Crohns
human granulocyte-macrophage colony-stimu- disease. J Clin Invest 107:695702
lating factor. Lancet 360:14781480 30. Marini M, Bamias G, Rivera-Nieves J et al
17. Lopez-Cubero SO, Sullivan KM, McDonald (2003) TNF-alpha neutralization ameliorates
GB (1998) Course of Crohns disease after the severity of murine Crohns-like ileitis by
allogeneic marrow transplantation. abrogation of intestinal epithelial cell apopto-
Gastroenterology 114:433440 sis. Proc Natl Acad Sci U S A 100:83668371
18. Hisamatsu T, Suzuki M, Reinecker HC et al 31. Ho J, Kurtz CC, Naganuma M et al (2008) A
(2003) CARD15/NOD2 functions as an anti- CD8+/CD103high T cell subset regulates
bacterial factor in human intestinal epithelial TNF-mediated chronic murine ileitis. J Immunol
cells. Gastroenterology 124:9931000 180:25732580
19. Cadwell K, Liu JY, Brown SL et al (2008) A 32. Liu L, Damen JE, Ware M et al (1997) A new
key role for autophagy and the autophagy gene player in cytokine-induced signalling. Leukemia
Atg16l1 in mouse and human intestinal Paneth 11:181184
cells. Nature 456:259263 33. Kerr WG (2008) A role for SHIP in stem cell
20. Cario E, Podolsky DK (2000) Differential biology and transplantation. Curr Stem Cell
alteration in intestinal epithelial cell expression Res Ther 3:99106
21 Assessing Inflammatory Disease at Mucosal Surfaces in Murine Genetic Models 441
34. Wang JW, Howson JM, Ghansah T et al (2002) and Src homology 2-containing inositol
Influence of SHIP on the NK repertoire and 5-phosphatase -are required for regulation
allogeneic bone marrow transplantation. of CD4+CD25+ T cell development. J Immunol
Science 295:20942097 176:39583965
35. Ghansah T, Paraiso KH, Highfill S et al (2004) 41. Kerr WG (2011) Inhibitor and activator: dual
Expansion of myeloid suppressor cells in SHIP- functions for SHIP in immunity and cancer.
deficient mice represses allogeneic T cell Ann N Y Acad Sci 1217:117
responses. J Immunol 173:73247330 42. Imielinski M, Baldassano RN, Griffiths A et al
36. Paraiso KH, Ghansah T, Costello A et al (2007) (2009) Common variants at five new loci asso-
Induced SHIP deficiency expands myeloid reg- ciated with early-onset inflammatory bowel dis-
ulatory cells and abrogates graft-versus-host ease. Nat Genet 41:13351340
disease. J Immunol 178:28932900 43. Helgason CD, Damen JE, Rosten P et al
37. Rauh MJ, Ho V, Pereira C et al (2005) SHIP (1998) Targeted disruption of SHIP leads to
represses the generation of alternatively acti- hemopoietic perturbations, lung pathology,
vated macrophages. Immunity 23:361374 and a shortened life span. Genes Dev 12:
38. Collazo MM, Wood D, Paraiso KH et al (2009) 16101620
SHIP limits immunoregulatory capacity in the 44. Guo L, Johnson RS, Schuh JC (2000)
T-cell compartment. Blood 113:29342944 Biochemical characterization of endogenously
39. Locke NR, Patterson SJ, Hamilton MJ et al formed eosinophilic crystals in the lungs of
(2009) SHIP regulates the reciprocal develop- mice. J Biol Chem 275:80328037
ment of T regulatory and Th17 cells. J Immunol 45. Hoenerhoff MJ, Starost MF, Ward JM (2006)
183:975983 Eosinophilic crystalline pneumonia as a major
40. Kashiwada M, Cattoretti G, McKeag L et al cause of death in 129S4/SvJae mice. Vet Pathol
(2006) Downstream of tyrosine kinases-1 43:682688
Chapter 22
Abstract
The model of experimental autoimmune uveitis (EAU) in mice and in rats is described. EAU targets
immunologically privileged retinal antigens and serves as a model of autoimmune uveitis in humans as well
as a model for autoimmunity in a more general sense. EAU is a well-characterized, robust, and reproduc-
ible model that is easily followed and quantitated. It is inducible with synthetic peptides derived from reti-
nal autoantigens in commonly available strains of rats and mice. The ability to induce EAU in various
gene-manipulated, including HLA-transgenic, mouse strains makes the EAU model suitable for the study
of basic mechanisms as well as in clinically relevant interventions.
Key words: Uveitis, Uveoretinitis, EAU, Autoimmunity, T cells, Tolerance, Th1, Th2, IRBP, S-Ag
1. Introduction
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_22, Springer Science+Business Media New York 2012
443
444 R.K. Agarwal et al.
2. Materials
3. Methods
3.1. EAU in Rats The model of EAU, originally established in the guinea pig using
homologous uveal tissue (3537), was adapted to the rat in 1973
by Wacker and Kalsow (38) using whole retinal extracts, and was
subsequently refined by de Kozak et al. using the retinal S-Ag (39).
IRBP was shown to be uveitogenic in rats (40).
Susceptibility of EAU varies among different rat strains. The
strain most commonly used for EAU studies is the Lewis rat. In the
Lewis rat, the disease is of monophasic course, which develops
448 R.K. Agarwal et al.
Table 1
Susceptibility to EAU of some inbred rat strainsa
Source Nickname (if any) Positionb a.a. sequencec Minimal dosed Reference
Fig. 1. Clinical appearance of EAU in the Lewis rat by anterior chamber examination. (a) Normal eye; translucent appear-
ance; pupil and iris blood vessels are clearly visible and the vessels are not congested. (b) Uveitic eye; the eye appears
larger due to swelling and proptosis; red reflex is absent and pupil is obscured.
22 Rodent Models of Experimental Autoimmune Uveitis 451
Table 3
Clinical grading of EAU in the rat
Gradea Criteria
Table 4
Scoring EAU histopathologically in the rat
Area of retinal
Grade section affected Criteria
3.1.1. Expected Course The course of disease in the Lewis rat is typically acute and of short
of Disease duration. The time of onset will vary depending on severity of the
developing disease, the antigen and the antigen dose. S-Ag and its
peptides tend to give more severe disease but a later onset than
22 Rodent Models of Experimental Autoimmune Uveitis 453
Quantitation Clinical. Onset of disease in the albino Lewis rat can be recognized
by inspecting the eyes with the aid of a good flashlight (Fig. 1).
The normal eye appears translucent and reflects the light (red
reflex). The first sign of uveitis is engorgement of blood vessels in
the iris and an irregular pupil that cannot contract in response to
light (caused by the iris adhering to the lens). Leukocyte infiltration
and deposition of fibrin is first seen as dulling of the red reflex,
progressing to complete opacification of the anterior chamber. The
eye swells and can protrude from its socket (proptosis). In very
severe cases hemorrhages in the anterior chamber and even perfo-
ration of the cornea can occur. In the latter case the animal should
be euthanized. We grade clinical EAU on a scale of 0 (no disease)
to 4 (severe disease), using the criteria listed in Table 3.
Histopathology. EAU is defined primarily as a posterior segment
disease, because the target antigens reside in the retina. Lewis rats
can develop very severe anterior chamber inflammation, which can
lead to corneal perforation. Therefore, although clinical follow-up
by anterior chamber inflammation is important and yields valuable
information, the final readout should be recorded by histopathol-
ogy. We grade EAU by histopathology based on number and extent
of lesions on an arbitrary scale of 0 (none) to 4 (maximum sever-
ity), in half-point increments, as shown in Table 4 (42). Typical
EAU vs. normal histology is shown in Fig. 2. Although the Figure
shows EAU histopathology in the mouse, it is representative of
what is seen in rat.
3.2. EAU in Mice Mice are highly resistant to EAU induction with S-Ag, but some
strains can develop severe EAU after immunization with IRBP
(33). As in rats, age and sex do not appear to have a major influence
on susceptibility to disease. Table 5 lists some common EAU sus-
ceptible mouse strains and IRBP epitopes that have been found to
induce disease in each. The epitopes of the IRBP molecule
454 R.K. Agarwal et al.
Fig. 2. Clinical appearance of EAU in the B10.RIII mouse by fundoscopic exam. Eyes were photographed with a fundus
camera during the acute phase of disease (day 1421). The range of disease severity scores parallel the pathological
scores in Fig. 3. (Figure adapted from reference 69).
22 Rodent Models of Experimental Autoimmune Uveitis 455
Table 5
Susceptibility of different mouse strains to IRBP-EAUa
Expected
Strain H-2 disease score Epitope, position, and reference
pathogenic for the H-2b, H-2r, and H-2k haplotypes have been
identified (4648). In the B10.A mouse (Kk, Ak, Ek, Dd) MHC
control of susceptibility in mice has been tentatively mapped to the
I-A region with modifying influences from the I-E (13). In addi-
tion to a susceptible H-2 haplotype that can bind and present the
uveitogenic epitopes, the strain must also have a permissive
background to express disease. Studies with MHC-congenic mice
showed that a nonpermissive background can completely prevent
expression of disease in mice having a susceptible H-2. Known
susceptible H-2 haplotypes include H2r, H-2k, H-2a (shares class II
subregion with H-2k), H-2b, H-2q, and H-2d. The last two, initially
thought to be resistant, can in fact be shown to be EAU-susceptible
in situations where IFN-g is neutralized or knocked out (ref. 23
and Grajewski and Caspi, unpublished).
The most susceptible mouse strain currently known is B10.
RIII (H-2r) (Table 5). Unlike other mouse strains, this strain does
not require PT to develop disease by active immunization either
with IRBP or with its major pathogenic epitope, residues 161180
of human IRBP. Human epitopes 171190 and 541560 require
PT, as does the murine sequence of peptide 161180. This is likely
due to thymic elimination of high-affinity T cells reacting with the
endogenous version of peptide 161180 (46). In less susceptible
mouse strains, such as B10.A or C57BL/6, a higher dose of IRBP
(50150 mg) or of the peptide appropriate for the haplotype in
CFA is injected s.c., concurrently with PT (0.30.4 mg) given i.p.
456 R.K. Agarwal et al.
Table 6
Clinical scoring of EAU in the mouse
Grade Criteria
0 No change
0.5 (trace) Few (12) very small, peripheral focal lesion;
minimal vasculitis/vitritis
1 Mild vasculitis; <5 small focal lesions; 1 linear lesion
2 Multiple (>5) chorioretinal lesions and/or
infiltrations; severe vasculitis (large size, thick wall,
infiltrations); few linear lesions (<5)
3 Pattern of linear lesions; large confluent lesions;
subretinal neovascularization; retinal hemorrhages;
papilledema
4 Large retinal detachment; retinal atrophy
Table 7
Grading EAU histopathologically in the mouse
Grade Criteria
0 No change
0.5 (trace) Mild inflammatory cell infiltration. No tissue damage
1 Infiltration; retinal folds and focal retinal detachments; few
small granulomas in choroid and retina, perivasculitis
2 Moderate infiltration; retinal folds, detachments and focal
photoreceptor cell damage; small to medium sized
granulomas, perivasculitis and vasculitis
3 Medium to heavy infiltration; extensive retinal folding with
detachments, moderate photoreceptor cell damage;
medium sized granulomatous lesions; subretinal
neovascularization
4 Heavy infiltration; diffuse retinal detachment with serous
exudate and subretinal bleeding; extensive photoreceptor
cell damage; large granulomatous lesions; subretinal
neovascularization
3.2.1. Expected Course The severity and clinical course of EAU induced by active immuni-
of Disease zation (CFA-EAU) with peptide 161180 in B10.RIII mice is
typically monophasic, resembling the Lewis rat, and lasts for
23 weeks. High intensity immunization results in an acute form
of disease with onset as early as day 8 or 9 and widespread photo-
receptor damage, whereas lower intensity of immunization will
result in milder disease with progressively later onset (24). In other
22 Rodent Models of Experimental Autoimmune Uveitis 461
Fig. 3. Histopathology of EAU in the B10.RIII mouse. Eyes were collected from B10.RIII mice 21 days after uveitogenic
immunization with IRBP, representing a range of disease scores. (Figure adapted from references 49, 69). EAU in the rat
shows essentially the same type of histopathology.
3.3. Support Protocols Fundoscopic examination can be used to detect and evaluate EAU
in pigmented animals, provided that the anterior segment of the
3.3.1. Fundoscopic
eye remains clear. In this procedure, retina of the eye is examined
Examination
through the dilated pupil under a microscope. Fundoscopy is a
good tool for determining the onset and clinical grading of disease
in pigmented animals but not in albinos.
Materials
Ophthalmic dilating solutions: 1 % Tropicamide and
Phenylephrine.
Sterile physiological solution (either normal saline or PBS or
artificial tears).
Binocular microscope with coaxial illumination.
Microscope coverslip
Anesthetize the animals and dilate the pupil with one to two
drops of dilating solution. It takes several minutes for the
22 Rodent Models of Experimental Autoimmune Uveitis 463
Fig. 4. Protocol for induction of autoimmune uveitis in B10.RIII mice with antigen-pulsed DCs (54).
3.3.2. Handling of Eyes Eyes should be collected within 15 min of euthanasia; otherwise,
for Histopathology autolysis may preclude correct evaluation of the results. Enucleation
in rats should be performed by carefully dissecting the globe from
the periocular tissues and the optic nerve without applying pres-
sure on the globe, to avoid maceration of delicate ocular tissues
that become even more fragile when inflamed. In mice for enucle-
ation, the eye should be made to protrude by applying pressure on
the skull, and plucked free of the tissue with a curved forceps.
Freshly enucleated eyes are fixed in 4 % phosphate-buffered glu-
taraldehyde for 1 h and then transferred into 10 % phosphate-buffered
formalin at least overnight or until processing. The brief fixation in
4 % glutaraldehyde prevents artifactual detachment of the retina
from the choroid. However, leaving the eyes in glutaraldehyde for
too long will cause excessive hardening of the lens, which will make
sectioning difficult. The grading is conveniently done on methacry-
late or paraffin-embedded tissue sections cut up to 8 mm in thick-
ness, stained with hematoxylin and eosin. To arrive at the final
grading, several sections cut through the pupillary-optic nerve axis
should be examined for each eye. It should be remembered that in
this type of visual scoring, there is always an element of subjectivity.
Therefore, it is important that the results be read in a masked fash-
ion, preferably always by the same person. Whenever possible, both
eyes should be evaluated for the disease, as disease may be unilat-
eral. If it becomes experimentally necessary to take only one eye,
always collect the same eye to average out this random variation.
464 R.K. Agarwal et al.
3.3.3. Complete Freunds 100 mg heat killed M. tuberculosis (strain H37Ra; Difco) is crushed
Adjuvant into a fine powder using a porcelain mortar and pestle. Mix with
70 ml of CFA (1.0 mg/ml of M. tuberculosis). Store at 4 C until
used. The suspension must be thoroughly mixed before each use as
the mycobacterial particles settle quickly to the bottom.
4. Notes
Although the EAU model is very robust, problems can arise with
the technique at a number of levels. If no disease is obtained, the
following questions should be considered (some may seem trivial
or obvious, but we have encountered all!):
Has the antigen been uveitogenic in previous experiments? If
this is a new synthesis, a synthesis error might have changed
the pathogenic epitope.
Has a sufficient dose of antigen been used?
Has the adjuvant been prepared correctly: enough mycobacte-
ria, mixed before sampling (mycobacteria may have settled),
well-prepared (thick) emulsion?
Has the correct strain and substrain of mouse or rat been used?
Substrains of the same strain, and even the same strain from a
different vendor, may vary in their susceptibility.
22 Rodent Models of Experimental Autoimmune Uveitis 465
References
tinct clinical signature and is driven by unique GJ, Gery I (1988) Synthetic peptides derived
effector mechanisms: initial encounter with from IRBP induce EAU and EAP in Lewis rats.
autoantigen defines disease phenotype. J Curr Eye Res 7:727735
Immunol 178:55785587 64. Kotake S, de Smet MD, Wiggert B, Redmond
55. Knapp JE, Liu D (2004) Hydrodynamic deliv- TM, Chader GJ, Gery I (1991) Analysis of the
ery of DNA. Methods Mol Biol 245:245250 pivotal residues of the immunodominant and
56. Chan CC, Caspi RR, Ni M, Leake WC, Wiggert highly uveitogenic determinant of interphoto-
B, Chader GJ, Nussenblatt RB (1990) receptor retinoid-binding protein. J Immunol
Pathology of experimental autoimmune uveo- 146:29953001
retinitis in mice. J Autoimmun 3:247255 65. de Smet MD, Bitar G, Roberge FG, Gery I,
57. Bjorck P (2001) Isolation and characterization Nussenblatt RB (1993) Human S-Antigen:
of plasmacytoid dendritic cells from Flt3 ligand Presence of multiple immunogenic and
and granulocyte-macrophage colony-stimulat- immunopathogenic sites in the Lewis rat.
ing factor-treated mice. Blood 98:35203526 J Autoimmun 6:587599
58. Vremec D, Pooley J, Hochrein H, Wu L, 66. Merryman CF, Donoso LA, Zhang XM,
Shortman K (2000) CD4 and CD8 expression Heber-Katz E, Gregerson DS (1991)
by dendritic cell subtypes in mouse thymus and Characterization of a new, potent, immunop-
spleen. J Immunol 164:29782986 athogenic epitope in S-antigen that elicits T
59. Donoso LA, Yamaki K, Merryman CF, cells expressing V beta 8 and V alpha 2-like
Shinohara T, Yue S, Sery TW (1988) Human genes. J Immunol 146:7580
S-antigen: characterization of uveitopathogenic 67. Gregerson DS, Merryman CF, Obritsch WF,
sites. Curr Eye Res 7:10771085 Donoso LA (1990) Identification of a potent
60. Singh VK, Nussenblatt RB, Donoso LA, new pathogenic site in human retinal S-antigen
Yamaki K, Chan CC, Shinohara T (1988) which induces experimental autoimmune uveo-
Identification of a uveitopathogenic and lym- retinitis in LEW rats. Cell Immunol 128:
phocyte proliferation site in bovine S-antigen. 209219
Cell Immunol 115:413419 68. Donoso LA, Merryman CF, Sery T, Sanders R,
61. Donoso LA, Merryman CF, Sery TW, Shinohara Vrabec T, Fong SL (1989) Human interstitial
T, Dietzschold B, Smith A, Kalsow CM (1987) retinoid binding protein. A potent uveitopatho-
S-antigen: characterization of a pathogenic genic agent for the induction of experimental
epitope which mediates experimental autoim- autoimmune uveitis. J Immunol 143:7983
mune uveitis and pinealitis in Lewis rats. Curr 69. Horai R, Caspi RR (eds) (2010) Retinal
Eye Res 6:11511159 inflammation: uveitis/uveoretinitis. Animal
62. Kotake S, Redmond TM, Wiggert B, Vistica B, models for retinal diseases, Neuromethods, vol
Sanui H, Chader GJ, Gery I (1991) Unusual 46. Humana, New York, NY
immunologic properties of the uveitogenic 70. Mattapallil MJ, Wawrousek EF, Chan CC,
interphotoreceptor retinoid-binding protein- Zhao H, Roychoudhury J, Ferguson TA,
derived peptide R23. Invest Ophthalmol Vis Caspi RR (2012) The rd8 mutation of the
Sci 32:20582064 Crb1 gene is present in vendor lines of
63. Sanui H, Redmond TM, Hu LH, Kuwabara T, C57BL/6N mice and embryonic stem cells,
Margalit H, Cornette JL, Wiggert B, Chader and confounds ocular induced mutant pheno-
types. Invest Ophthalmol Vis Sci. (in press)
Chapter 23
Abstract
A master control of both the innate and adaptive immune system is the bodys ability to distinguish
between self and foreign entities. This is accomplished by the elimination of autoreactive leukocytes
through a series of checkpoints both in the thymus (central deletion) and in the circulating periphery
(peripheral tolerance), thus establishing tolerance to self-antigens. When one or more of these controls is
disrupted, there is the potential for the development of autoimmune disease. Current available therapies
for these diseases often rely on global immune suppression or expensive treatments that are not affordable
to all. Herein, we describe a novel therapeutic technique in which tolerance to self can be re-established
via B-cell delivered antigen-specific tolerogenic gene constructs. Furthermore, this technique shows prom-
ise in the gene therapeutic treatment of monogenic disorders and the acceptance of tissue transplants.
Key words: Autoimmunity, Immune tolerance, B cells, Gene therapy, Immunoglobulin fusion protein
1. Introduction
1.1. Self Tolerance and It has been well established that the immune system is designed to
Autoimmune Disease recognize and eliminate foreign invaders from the host and at the
same time largely maintain the homeostasis of the body. One of the
many challenges the immune system faces is recognition of self
from non-self. It has evolved the ability to discriminate selfness
from foreignness; thus, self-tolerance is considered the basic tenet
of immune function. This tolerance to self is maintained in at least
two levels. The first mechanism, known as central tolerance,
involves autoreactive T cells being deleted in the thymus before
reaching the periphery. Similar events for clonal deletion or recep-
tor editing of immature autoreactive B cells occur in the bone mar-
row. Central deletion is not foolproof, however, and some
autoreactive cells escape deletion in the thymus/bone marrow, and
must be controlled in the periphery; otherwise, autoimmunity is
likely to occur. Some of the safety mechanisms designed to
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4_23, Springer Science+Business Media New York 2012
471
472 R.J. Rossi et al.
1.3. B-Cell Delivered Based on the ability of B cells to function as tolerogenic APCs as
Gene Therapy for well as the excellent tolerogenicity of IgG as protein carrier, our lab
Tolerance Induction developed a novel B-cell gene therapy platform for antigen-specific
tolerance induction (9, 10). Thus, the target antigen (peptide or
23 Tolerance Induction via B-Cell Delivered Gene Therapy 473
Table 1
Common autoimmune diseases and their corresponding antigenic targets
for gene therapy
2. Materials
26. 96-Well flat bottom cell culture plates (Corning Inc., Corning
NY).
27. Complete Freunds adjuvant (Sigma-Aldrich, St. Louis, MO).
28. Anti-Thy 1 supernatant (clone 30H12, Rat IgG2b).
29. Anti-CD4 supernatant (clone GK1.5, Rat IgG2b).
30. Anti-CD8 supernatant (clone 3.155, Rat IgM).
31. Low-Tox M rabbit complement (Cedarlane Labs, Burlington,
NC).
32. Lympholyte M (Cedarlane Labs, Burlington, NC).
33. Lipopolysaccharide (LPS) from Escherichia coli 055:B5 (Sigma-
Aldrich, St. Louis, MO).
34. Red blood cell lysis buffer (buffered ammonium chloride):
4.15 g NH4Cl, 0.5 g KHCO3 dissolved in 500 mL H2O with
pH adjusted to 7.2 by gassing with 10 % CO2.
35. Media supplement for complete RPMI and DMEM: Stock 1:
0.1 M b-mercaptoethanol; combine 6.5 mL PBS with 50 ml
b-mercaptoethanol. Stock 2: 100 mL of 100 mM sodium
pyruvate, 100 mL of 100 solution MEM nonessential amino
acids, 100 mL of 1 M HEPES buffer, 100 mL of
200 mM L-glutamine, 100 mL of 100 Penicillin-Streptomycin
(Cellgro by Mediatech, Manassas, VA). Add 5 mL of stock 1 to
stock 2 and stir to combine. Sterile filter, aliquot, and freeze at
20 C.
36. Complete RPMI-1640 medium: 500 mL RPMI with 25 mL
media supplement from above and 5 % FCS (Atlanta Biologicals,
Lawrenceville, GA).
37. b-Mercaptoethanol (Sigma-Aldrich, St. Louis, MO).
38. Phosphate-buffered saline (PBS): 8.5 g NaCl, 0.57 g Na2HPO4,
0.14 g KH2PO4 dissolved in 1 L of H2O with pH adjusted to
7.4.
39. Anti-B220 (clone RA36B2, Rat IgG2c k).
40. Biomag goat a-rat IgG (Polysciences Inc., Warrington, PA).
41. Magnetic separator.
42. Centrifuge.
43. Rotating mixer.
44. Falcon cell strainer (Fisher Scientific, Pittsburgh, PA).
45. Conical tubes (Fisher Scientific, Pittsburgh, PA).
46. [3H] thymidine (Perkin Elmer, Waltham, MA).
47. Glass fiber filtermats.
48. Sample bag for filtermats.
49. Heat sealer.
50. Scintillation fluid.
476 R.J. Rossi et al.
3. Methods
3.1. Insertion of Gene 3.1.1. Using the NotI and Xho1 restriction sites, insert the PCR
of Interest into product of gene of interest into BSSK cassette which
BSSK-Ig contains a murine IgG1 heavy chain cDNA.
1. PCR amplify your GOI such that restriction sites NotI
and XhoI are at the 5 and 3 ends, respectively. In design-
ing the 5 primer ensure that translation of the fusion
protein will occur in frame by adding one additional
nucleotide between the NotI site and the start of your
GOI (see vector sequence below).
3.2. Isolation 3.2.1. Digest the BSSK-Ig-gene of interest construct with Sal I.
and Sub-cloning Run digestion product on a 1.22 % agarose gel to isolate
of Ig-Gene of Interest the GOI away from pBluescript. It is necessary to deter-
into MBAE or MSCV mine the length of the fusion product, so that you will know
where the target fragment will be separated in the gel. The
pBluescript is approximately 2,800 bp. If the GOI is less
than 800 bp or more than 1,500 bp, it is likely that a 1.2 %
gel will work. Otherwise, it may be necessary to digest the
pBluescript with an additional enzyme not found in the
GOI. This will allow for better separation of pBluescript
from the gene insert. Purify the Ig-gene of interest with
a suitable gel purification kit (QIAquick from Qiagen).
3.2.2. Linearize the MBAE vector by digesting with Sal I. In order
to prevent self-ligation of the vector, it is necessary to
treat with calf intestinal alkaline phosphatase (CIAP) for
478 R.J. Rossi et al.
3.3. Stable Day 1: Add 1 106 GP + E-86 fibroblast cells in complete DMEM
Transfection with 10 % FCS to two 100 mm tissue culture plates for every
of GP + E-86 construct being transfected.
Fibroblasts
Day 2: Introduce DNA into plated GP + E-86 cells by calcium
phosphate precipitation. (It is also possible to follow another
transfection method such as using FuGENE kit).
1. Add 1050 mg DNA to a polystyrene tube.
2. Add enough sterile water to bring the volume to 450 ml.
3. Add 50 ml of 2.5 M CaCl2 and gently vortex the mix.
4. To a clean polystyrene tube, add 500 ml of 2 HEPES buffered
saline.
5. While gently vortexing, add the DNA in a dropwise fashion to
the 2 HEPES buffered saline.
6. Let stand at room temperature (RT) for 45 min to 1 h to allow
precipitate to form.
7. Add DNA-HEPES mixture to plated GP + E-86 cells, again in
a dropwise fashion.
8. Incubate overnight at 37 C + 7 % CO2.
Day 3: Remove complete DMEM and add media containing G418
selection with a starting concentration of 0.5 mg/mL and increase
the concentration of G418 to 1 mg/mL on day 4 or day 5.
3.4. Isolation 3.4.1. Approximately 1014 days post retroviral DNA transfection,
of GP + E-86 Viral colonies should be visible on the tissue culture plates. While
Producing Colonies there are several different ways to pick colonies from the plate,
our lab utilizes sterile cloning disks from Bel-Art Products.
23 Tolerance Induction via B-Cell Delivered Gene Therapy 479
3.5. Viral Titers Day 1: Harvest supernatant and freeze in 2 mL aliquots until
needed. Thaw each vial one time only. As an alternative, you
3.5.1. Harvest Viral
can change media on sub-confluent packaging cells and use
Supernatant from
fresh media on day 2. Be sure there is no selection component
Packaging Cell Line
such as G418 in the media.
3.5.2. Titer of Viral Day 1: Split confluent NIH 3T3 cells and plate 5 105 cells in
Supernatant 60 mm dishes in complete DMEM. You will need to set up
three plates for every viral cell line checked.
Day 2:
1. Label plates with construct name and dilutions. Discard media
and add 1 mL fresh complete DMEM.
2. Perform serial dilutions of viral supernatant by adding 100 ml
to plate 1. This will be a 1:10 dilution or 0.1 mL virus. Gently
mix supernatant into plated media until it has been well
distributed.
3. Remove 100 ml media from plate 1 and add it to plate 2. Mix
following the same procedure as above. This is a 1:100
480 R.J. Rossi et al.
3.6. B-Cell Isolation 1. Aseptically harvest spleens from mice and make into single cell
and Activation suspension by preferred method.
2. Wash one time with RPMI and centrifuge at 250 g for 5 min.
3. Pour off supernatant and re-suspend cell pellet.
4. Lyse red blood cells by adding 5 mL of buffered ammonium
chloride per spleen. Mix well and allow to incubate at room
temperature for approximately 5 min.
5. Following 5 min incubation, add 1015 mL RPMI media and
centrifuge for 5 min at 250 g.
6. Prepare anti-T cell cocktail using a 1:1:1 ratio of anti-Thy 1
(clone 30H12), anti-CD4 (clone GK1.5), and anti-CD8 (clone
3.155). You will need 1 mL of cocktail for each spleen.
7. Harvest pelleted spleen cells with gentle pipetting and filter
through a cell strainer. Centrifuge for 5 min at 250 g.
23 Tolerance Induction via B-Cell Delivered Gene Therapy 481
3.8. Proliferation 1. Immunize animals in one hind footpad and the base of tail
Assay with peptide or antigen emulsified in CFA.
2. After 1014 days, euthanize mice and remove popliteal and
inguinal lymph nodes on the side of the body that the immu-
nization was given. Working sterilely in a biological safety cabi-
net, mash lymph nodes into single cell suspension, and suspend
in complete RPMI with 2.5 % FCS or X-VIVO serum-free
medium.
3. Wash cells two times. Centrifuge at 250 g for 5 min.
4. Re-suspend cells at a concentration of 5 106 cells/mL.
5. Prepare proliferation plates by adding 100 ml medium per well
in 96-well flat bottom cell culture plate. Each well contains no
or different concentrations of stimulation antigen. Typically an
antigen titration is set up in the range of 100 ng to 30 mg,
6. Add 100 ml cells (5 106 cells/mL) per well, so that final cul-
ture volume is 200 mL and final cell number is 0. 5 106.
Incubate plates at 37 C + 7 % CO2.
7. After 48 h, add 0.5 mCi/well [3H] thymidine to each well and
incubate an additional 1418 h.
8. Harvest cells from plate onto glass fiber filters using a cell
harvester.
9. Add scintillation fluid and read filter using scintillation counter.
23 Tolerance Induction via B-Cell Delivered Gene Therapy 483
Harvest spleen
Purify B cells from spleen Collect blood serum for
Activate B cells with LPS, cytokine analysis/antibody
-IgM, or CD40L for 24 hours response using ELISA
14 Days Harvest draining lymph nodes
for T cell proliferation assay, FACS,
and/or ELISPOT
Inject mice sub cutaneaously Clinical scoring of paralysis in
Co-culture activated B cells with antigen/CFA in footpad Experimental Autoimmune
with virus producing packaging Encephalomyelitis (EAE)
cells
Packaging cells produce tolerogenic 7-10 Days Measure blood glucose levels in
construct consisting of IgG heavy chain type 1 diabetes (T1D)
backbone containing in frame target antigen
Fig. 1. Schematic representation of protocol for B-cell delivered gene therapy. Splenic B cells from donor mice are purified
and activated with either LPS, a-IgM, or CD40 ligand (CD40L) for 24 h. Irradiated viral packaging cells transfected with
tolerogenic construct are then co-cultured with activated B cells which allows transduction of tolerogenic construct into B
cells. Transduced B cells can then be injected intravenously or intraperitoneally into recipient mice who are subsequently
challenged with immunogenic form of a disease-specific antigen emulsified in Complete Freunds adjuvant. Blood collec-
tion for serum analysis, EAE clinical disease scoring, and blood glucose levels (T1D) can be monitored. Approximately
2 weeks post footpad injection, draining lymph nodes can be removed for proliferation assay, ELISA, and FACS.
3.9. Blood Collection Circulating cytokines and/or antibodies can be detected in blood
and ELISA serum by enzyme linked immunosorbent assay (ELISA).
3.9.1. Serum Collection 1. Collect blood into microcentrifuge tubes via retro-orbital eye
bleed utilizing glass capillary tubes that DO NOT contain hep-
arin or other anticoagulants.
2. Place tubes containing blood on ice for 30 min to allow blood
to clot. Centrifuge for 1015 min at 1,000 g to separate
serum from other blood components.
3. After centrifugation, pipette clear top layer (serum) into a clean
microcentrifuge tube and assay immediately or freeze at 20 C.
3.10. ELISPOT 1. Use coating buffer to dilute capture antibody and add 100 ml
to each well of an ELISPOT plate.
2. Incubate at 4 C overnight.
3. Aspirate coating antibody and wash wells 12 with blocking
solution (see Note 1).
4. Block any nonspecific binding by adding 200 ml of blocking
solution/well. Incubate at room temperature for 2 h.
5. Aspirate blocking solution and wash plate.
6. Dilute capture antibody in complete medium and add 100 ml/
well. Add 100 ml lymph node single cell suspension(s) to plate.
Incubate plate at 37 C + 5 % CO2 1824 h.
7. After incubation, remove cells and wash plate with 280 ml
deionized water 2, soaking wells for 5 min between washes.
Wash wells three times with 280 ml wash buffer I.
8. Add 100 ml diluted detection (e.g., biotinylated) antibody to
each well and incubate for 2 h at room temperature.
9. After incubation with detection antibody, aspirate wells and
wash plate with 280 ml/well wash buffer I, allowing wells to
soak for 2 min between washes.
10. Dilute streptavidin-HRP enzyme conjugate in dilution buffer and
add 100 ml/well. Incubate plate for 1 h at room temperature.
11. Aspirate plate and wash 4 with 280 ml/well wash buffer I,
allowing plate to soak for 2 min between washes.
23 Tolerance Induction via B-Cell Delivered Gene Therapy 485
3.12. Fluorescence- 1. Euthanize animals and remove draining lymph node as in pro-
Activated Cell Sorting liferation assay.
Analysis 2. Place a cell strainer atop a 50 mL conical tube. Wet cell strainer
with 12 mL of RPMI media and place lymph nodes inside.
Crush lymph nodes using a syringe plunger and wash cells
through cell strainer using RPMI, collecting eluate in bottom
of 50 mL conical tube (see Note 3).
3. Centrifuge single cell suspension for 5 min at 250 g.
4. Pour off supernatant and vortex pellet.
5. Re-suspend cells in 1020 mL of FACS buffer and centrifuge
one more time as above.
486 R.J. Rossi et al.
4. Notes
References
1. Caspi RR (2008) Immunotherapy of autoim- 7. Eynon EE, Parker DC (1992) Small B cells as
munity and cancer: the penalty for success. Nat antigen-presenting cells in the induction of tol-
Rev Immunol 8:970976 erance to soluble protein antigens. J Exp Med
2. Scott DW (1994) The Nature of Immunologic 175:131138
Tolerance. R.G. Landes, Austin, TX 8. Fuchs EJ, Matzinger P (1992) B cells turn off
3. Billingham RE, Brent L, Medawar PB (1953) virgin but not memory T cells. Science 258:
Actively acquired tolerance of foreign cells. 11561159
Nature 172:603606 9. Zambidis ET, Barth RK, Scott DW (1997)
4. Borel Y, Lewis RM, Stollar BD (1973) Both resting and activated B lymphocytes
Prevention of murine lupus nephritis by carrier- expressing engineered peptide-Ig molecules
dependent induction of immunologic tolerance serve as highly efficient tolerogenic vehicles in
to denatured DNA. Science 182:7678 immunocompetent adult recipients. J Immunol
5. Borel Y (1980) Haptens bound to self IgG 158:21742182
induce immunologic tolerance, while when cou- 10. Kang Y, Melo M, Deng E, Tisch R, El-Amine
pled to syngeneic spleen cells they induce immune M, Scott DW (1999) Induction of hypore-
suppression. Immunol Rev 50:71104 sponsiveness to intact foreign protein via
6. Lassila O, Vainio O, Matzinger P (1988) Can B retroviral-mediated gene expression: the
cells turn on virgin T cells? Nature 334:253255 IgG scaffold is important for induction and
23 Tolerance Induction via B-Cell Delivered Gene Therapy 487
Andras Perl (ed.), Autoimmunity: Methods and Protocols, Methods in Molecular Biology, vol. 900,
DOI 10.1007/978-1-60761-720-4, Springer Science+Business Media New York 2012
489
AUTOIMMUNITY: METHODS AND PROTOCOLS
490 Index
Autoantibody methods
anti-ssDNA ............................................................... 256 blood collection and ELISA ........................ 483484
BXSB......................................................................... 139 BSSK ................................................................... 477
high-titer, SLE .................................................. 110111 description ................................................... 476477
hydrocarbon oil .......................................................... 140 EAE .................................................................... 485
lupus-specific ............................................................. 254 ELISPOT.................................................... 484485
mediated pathogenicity.............................................. 181 FACS analysis .............................................. 485486
MRL and CD95 mutants .......................................... 138 GP + E-86 fibroblasts, stable
production ......................................................... 271273 transfection and isolation ....................... 478479
profiles ....................................................................... 272 isolation and activation ................................ 480481
SLE ............................................................................. 91 MBAE/MSCV ............................................ 477478
Autoantigen (IgG) proliferation assay ................................................ 482
clinical diagnosis ............................................................ 5 transduction ......................................... 481482, 483
exposures ........................................................... 331, 332 viral titers ..................................................... 479480
Autoimmune diseases self tolerance and autoimmune
antigenic targets ......................................................... 473 disease .......................................................... 471472
fibrocytes contributions ..................................... 331, 332 B cell receptor (BCR)
organ-specific................................................................. 4 antigen engagement ................................................... 114
system ............................................................................ 3 CD10 marker ............................................................ 111
Autoimmunity IgD/IgM ................................................................... 115
B cell .......................................................................... 406 immunoglobulins (Ig) ................................................ 110
EAU .......................................................................... 445 transitional stage ..................................................... 111
endocytic recycling role (see Endocytic B cells
recycling, SLE) APC .................................................................. 255, 257
mitochondrial dysfunction assessment CD4 T cell................................................................. 254
(see Mitochondrial dysfunction assessment, deficient mice............................................................. 296
SLE patients) depletion .................................................................... 298
mouse lupus models (see Mouse lupus models) epitopes...................................................................... 292
pathogenesis and spectrum flow cytometric analyses ............................ 236, 242243
antigens.................................................................... 5 GVHD assessment .................................................... 258
causes, diseases ..................................................... 45 hyperactivity
description ........................................................... 1, 3 BXSB................................................................... 139
diseases, organ-specific autoimmune ....................... 4 MRL mutants ...................................................... 138
exogenous and endogenous factors ...................... 2, 3 NZB mice .................................................... 136, 138
gene expression profiling.......................................... 7 immunity ........................................................... 293294
organ systems ........................................................... 3 investigating mutant .................................................. 256
theory....................................................................... 5 loss, tolerance ............................................................. 296
TNF....................................................................56 role, antigen presentation............................................. 13
SLE (see Systemic lupus erythematosus (SLE)) SLE
and T cell DNA hypomethylation ..................... 171172 antibody-secreting cells (ASC) .................... 117118
T cell signaling abnormalities (see T cell BCR (see B cell receptor (BCR))
signaling abnormalities, human SLE) CD27+ memory ........................................... 115116
type I and II lesions ................................................... 407 characterization, memory B cells ................. 111113
double-negative (DN) ................................. 116117
B GC............................................................... 114115
Bacillus Calmette-Guerin (BCG) ................................... 140 high-titer autoantibody ................................ 110111
BBB. See Blood brain barrier (BBB) immunoglobulins (Ig) .......................................... 110
B-cell delivered gene therapy, tolerance induction signature ...................................................... 118119
adoptive transfer ........................................................ 473 transitional and mature-nave ...................... 111, 114
antigen-specific .......................................................... 472 BCG. See Bacillus Calmette-Guerin (BCG)
autoimmune diseases and antigenic BCR. See B cell receptor (BCR)
targets .................................................................. 473 Beta cell dysfunction. See Oxidative stress
functions ............................................................ 472473 and beta cell dysfunction
materials ............................................................ 474476 Beta cells.................................................................. 348350
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
491
Blood brain barrier (BBB) WB analysis ................................................. 197198
BALB/c mice............................................................. 184 NMDAR ........................................................... 186, 192
epinephrine ................................................................ 190 passive transfer
LPS ........................................................................... 190 antibody purification............................................ 191
NPSLE mouse model ................................................ 184 description ........................................................... 185
B lymphocyte.................15, 61, 109, 121, 127, 405, 406, 428 materials ...................................................... 185186
Bovine serum albumin (BSA) monoclonal antibodies production............... 190191
alpha glyceramide immunization ....................... 293294 pathogenicity, mouse model ......................... 191192
2-OA-BSA, immunized mice (see 2-Octynoic physical barrier........................................................... 182
acid-BSA-immunized mice) staining brain sections ............................................... 194
BSA. See Bovine serum albumin (BSA) WB analysis, NMDARs .................................... 187188
Cerebrospinal fluid (CSF)
C GM-CSF................................................................... 332
Calcium response................................................... 26, 3436 MS patients
Calf intestinal alkaline phosphatase (CIAP) ........... 477478 CD8 lymphocytes ................................................ 405
Caspases .................................................62, 8182, 350, 359 CD19-plasma cells .............................................. 405
CD. See Crohns disease (CD) CFA. See Complete Freunds adjuvant (CFA)
CD3 .................................................................... 50, 93, 95 Chemokine
Cell activation C-C motif.................................................................. 331
and lysis ................................................................. 36, 40 CXCR4, 331, 332
mitochondrial checkpoints, T cell.......................... 6263 and liver cytokine............................................... 303, 312
pathogenesis, SLE nephritis ...................................... 208 receptors ............................................................ 329, 330
Cellular immunology, SLE T cell signaling molecules ChIP assay. See Chromatin immunoprecipitation
confocal microscopy............................................... 5556 (ChIP) assay
flow cytometry Chromatin immunoprecipitation (ChIP) assay
cell surface staining .......................................... 5152 cross-linking and preparation, cell extract.................... 48
intracellular staining ........................................ 5253 description ................................................................... 47
fluorescence microscopy......................................... 5355 phosphorylated transcription factors ........................... 49
Central nervous system (CNS) lupus procedure ............................................................... 4849
antibodies .................................................................. 183 salmon sperm DNA-blocked
BALB/c ..................................................................... 183 sepharose protein A/G........................................... 48
BBB (see Blood brain barrier (BBB)) CIAP. See Calf intestinal alkaline
cardiac perfusion phosphatase (CIAP)
description ........................................................... 186 ClaI restriction enzyme ................................................... 394
materials .............................................................. 186 CNS. See Central nervous system (CNS)
methods ....................................................... 192193 CNS lupus. See Central nervous system (CNS) lupus
description ......................................................... 181182 Collagen
detecting antibody deposition .................................... 195 CD45 and procollagen-1 expression.................. 335336
detecting preapoptotic neurons .................................. 196 collagen-1 .................................................................328
ELISA assays collagen V .................................................................. 330
description ........................................................... 184 extracellular matrix components ................................ 328
methods ....................................................... 189190 I/III/IV, vimentin and tenascin.................................. 330
testing serum antibody titer ................................. 184 procollagen-I staining ........................................ 338, 339
epinephrine injection ................................................. 182 Complement
FluoroJade, staining ................................................... 196 components ................................................................. 12
free-floating section staining ..................................... 195 Low-Tox M rabbit ............................................. 475, 481
immunization proteins and toll-like receptors .................................... 13
description ........................................................... 183 serum ......................................................................... 188
induction...................................................... 183184 targeted genetic mutations ......................................... 141
methods ....................................................... 188189 Complete Freunds adjuvant (CFA)
injured/apoptotic neurons and antigen emulsified ...................................................... 482
immunoglobulin deposition ......................... 186187 EAE
LPS ........................................................................... 182 emulsion .............................................................. 369
membrane-enriched brain fractions estimation ............................................................ 368
preparation........................................................... 187 Mycobacterium tuberculosis .....................................366
AUTOIMMUNITY: METHODS AND PROTOCOLS
492 Index
Insulin Lupus
organ-specific autoimmune diseases .............................. 4 ANA/anti-dsDNA, anti-nucleosome antibody
secretion and content production and GN ............................................. 275
arginine-stimulated .............................................. 355 ANA staining ............................................................ 237
DNA quantification assay .................................... 356 Cgnz1 and Agnz1...................................................... 275
ELISA ......................................................... 355356 C57L/J, F1 progeny ................................................... 287
glucose-stimulated ............................................... 355 CNS (see Central nervous system (CNS) lupus)
islet cells............................................................... 355 cytokines ........................................................................ 6
KCl-stimulated .................................................... 355 directional dominance................................................ 287
syringe VWR ............................................................. 259 exogenous and endogenous factors ................................ 2
Insulin-dependent diabetes mellitus (IDDM).............4, 5, 7, flow cytometry and bioanalytics
292, 295 (see Multiparameter flow cytometry
Interferon and bioanalytics, SLE)
cellular signaling/regulation ....................................... 142 genetic contributions, human SLE ............................ 272
DNA methylation...................................................... 170 genome-wide screening ............................................. 288
IFN- ..................................................................404405 immunofluorescence staining..................................... 237
signaling pathway ........................................................ 15 materials .................................................................... 276
Interferon-gamma (IFN) ............................................... 333 methods
Interferon regulatory factor (IRF) generation, Non-SLE NZM2328 (see Non-SLE
association, IRF5 ......................................................... 15 NZM2328 generation)
cellular signaling/regulation ....................................... 142 genetic analysis and SLE trait mapping, NZM2328
Interphotoreceptor retinoid-binding protein (IRBP) (see Genetic analysis)
bovine and human ............................................. 448, 449 murine (see Murine)
and EAU ................................................................... 455 precision mapping, chromosome 1............... 284285
epitopes.............................................................. 453454 mouse models (see Mouse lupus models)
148 kDa protein......................................................... 444 mouse models, nephritis .................................... 272273
Lewis rat ............................................................ 448, 449 murine lupus models (see T cell DNA hypomethylation,
peptide ....................................................... 456, 457458 murine lupus models)
residues ...................................................................... 456 nephritis loci, NZM2328 .......................................... 274
Interstitial lung disease (ILD) ......................... 327328, 331 NZM2328 females .................................................... 287
Intestinal epithelial cell (IEC) ......................... 433435, 436 NZM2328, SLE ................................................ 273274
IRBP. See Interphotoreceptor retinoid-binding serologic assessment........................................... 245246
protein (IRBP) serum levels, anti-dsDNA antibody................... 237, 245
IRF. See Interferon regulatory factor (IRF) SLE ....................................................271272, 472, 473
Luxol Fast Blue (LFB) staining
L cuprizone-induced demyelination ............................. 415
Large Lupus Association Study 2 (LLAS2) evaluation, demyelination .................................. 418420
approaches ................................................................... 18 gender differences, mouse .......................................... 416
collaborators and role............................................. 1618 Lymphokine .......................................................64, 350, 451
description ................................................................... 16
M
genotyping ................................................................... 18
papers published .......................................................... 19 Macrophages (M)
project .................................................................... 1819 antibodies use, flow cytometry ........................... 220221
Lewis rat, EAU induction enrichment, magnetic beads use ........................ 222223
active immunization .......................................... 448451 and fibroblasts ............................................................ 329
adoptive transfer ................................................ 451452 functional studies, and renal DCs (see Dendritic
clinical appearance ..................................................... 450 cells (DCs))
pathogenic ................................................................. 449 granulocytemacrophage ........................................... 332
LFB staining. See Luxol Fast Blue (LFB) staining inflammatory ............................................................. 209
Linkage disequilibrium ................................................ 15, 20 inflammatory protein 1 .............................................331
Lipopolysaccharide (LPS) ....................................... 182, 190 regulatory................................................................... 210
LLAS2. See Large Lupus Association Study 2 (LLAS2) renal ................................................................... 210211
LPS. See Lipopolysaccharide (LPS) RNS........................................................................... 348
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
497
T cells ........................................................................ 258 PBMC separation ............................................ 6970
xanthine oxidase system ............................................. 348 materials
Major histocompatibility complex (MHC) caspase substrate peptides ...................................... 68
DNA methylation...................................................... 170 cytokines ................................................................ 66
non MHC control ..................................... 444445, 448 flow cytometer ....................................................... 67
Mammalian target of rapamycin (mTOR) luminometer .......................................................... 67
endocytic pathway, SLE T cells ................................... 92 monoclonal antibody ............................................. 69
MHP ........................................................................... 63 pharmacological targeting
Matrix metalloprotease (MMP) ..............................224, 229, GSH/GSSG ratios ................................................ 66
233, 332, 405 NO production ...................................................... 66
Mature-Nave B cell ................................................ 111, 114 oxidative stress, transcription factors................ 64, 66
MBP. See Myelin basic protein (MBP) propidium iodide (PI)............................................ 8485
Memory B cell ROI production, measurement .............................. 7374
CD27+ ............................................................... 115116 T cell activation and apoptosis (see T cells)
gating strategy ........................................................... 124 Mitochondrial hyperpolarization (MHP)
9G4, staining ..................................................... 123124 elevation, ym............................................................... 63
MHC. See Major histocompatibility complex (MHC) mTOR, activation........................................................ 63
MHP. See Mitochondrial hyperpolarization (MHP) pharmacological targeting...................................... 64, 66
Mitochondria ROI production ........................................................... 62
ABCB1 transporter ................................................... 114 Mitochondrial transmembrane potential
AMA ......................................................................... 292 elevation................................................................. 6263
ATP production......................................................... 349 flow cytometric analysis ......................................... 7273
autoantigens............................................................... 292 hyperpolarization ......................................................... 63
autoantigens, immunoreactivity determination MHP ........................................................................... 63
ELISA ......................................................... 306307 mTOR activity ............................................................ 92
recombinant PDC-E2 ................................. 304305 ROI production ........................................................... 62
western blot ......................................................... 305 signaling abnormalities, T cell death............................ 64
dysfunction ................................................................ 348 Mitochondrion ...................................................62, 410, 422
MHP ........................................................................... 92 MMP. See Matrix metalloprotease (MMP)
PDC-E2 .................................................................... 298 MOG. See Myelin oligodendrocyte glycoprotein (MOG)
ROS production ........................................................ 350 Mouse blood and lung cells isolation
Mitochondrial dysfunction assessment, SLE patients analysis, flow cytometry data (see Flow cytometry)
AFC calibration curve ................................................. 85 bronchoalveolar lavage and perfusion ................ 338340
ATP assay flow cytometry
and ADP, assessment ............................................. 79 stained samples .................................................... 342
cell collection ......................................................... 75 preparation of blood, flow cytometry ......................... 341
precautions............................................................. 77 removal and enzymatic digestion, lungs..................... 340
protocol............................................................ 7778 sacrifice ...................................................................... 338
reagents .................................................................. 77 staining, murine ................................................. 341342
caspase enzyme assays............................................ 8082 Mouse lupus models
description ............................................................. 6162 blood collection, alternatives .............................. 158159
ETC activity (see Electron transport chain (ETC)) description ......................................................... 135136
flow cytometric analysis, ym ................................. 7273 diagnosis ............................................................ 156157
fluorochromes .............................................................. 84 ELISA systems .................................................. 160161
HPLC assay, glutathione levels.............................. 7980 experimentally induced models
intracellular pH measurements .............................. 7475 BCG injection ..................................................... 140
Lowry assay ................................................................. 76 chronic GVHD ................................................... 140
luciferase reaction ........................................................ 75 genetically targeted animals ......................... 140143
lymphocyte hydrocarbon oil .................................................... 140
CD3/CD28 co-stimulation, PBL .......................... 70 16/6 idiotype........................................................ 140
description ............................................................. 70 IgG2a reagents .......................................................... 161
Fas-mediated cell death, PBL................................ 71 implementation
H2O2 treatment ..................................................... 71 chronic GVHD ........................................... 148149
monitoring, cell death ...................................... 7172 hydrocarbon oil ............................................ 147148
monocytes and PBL separation ............................. 70 materials .............................................................. 144
AUTOIMMUNITY: METHODS AND PROTOCOLS
498 Index
O P
2-Octynoic acid-BSA-immunized mice Parent-into-F1 murine model
MHC restriction ....................................................... 299 analysis, biological agents .......................................... 255
mitochondrial autoantigen......................................... 298 B6 F1 mice ........................................................... 254
N. aromaticivorans ...............................................299300 DBA F1 mice ....................................................... 254
NKT .................................................................. 298299 description ......................................................... 253254
PDC-E2 .................................................................... 300 GVHD (see Graft-vs.-host disease (GVHD))
xenobiotic .......................................................... 298299 intracellular staining, cytokine production ................. 269
OL. See Oligodendrocyte (OL) investigating mutant T cell ................................ 256257
Oligodendrocyte (OL) mutations, host cells................................................... 257
apoptosis splenocyte isolation.................................................... 262
cuprizone induced........................................ 411412 tail vein injections ...................................................... 269
electron microscopic image .................................. 422 testing, T cell mutations .................................... 255256
neuropathological features ................................... 420 tissue harvesting ........................................................ 261
pathological features ............................................ 415 Pathogenesis and spectrum
direct cytotoxic effects ............................................... 404 description ................................................................. 1, 3
early loss .................................................................... 408 exogenous and endogenous factors ............................ 2, 3
mechanisms, loss................................................ 408409 PBC. See Primary biliary cirrhosis (PBC)
and myelin damage .................................................... 409 PBLs. See Peripheral blood lymphocytes (PBLs)
type III and IV lesions ............................................... 408 PBMC. See Peripheral blood mononuclear cells (PBMC)
Osmotic pump..................................................... 6, 317325 PBS. See Phosphate-buffered saline (PBS)
Oxidative stress PCA. See Principal component analysis (PCA)
and beta cell dysfunction (see Oxidative stress PCR. See Polymerase chain reaction (PCR)
and beta cell dysfunction) Peripheral blood lymphocytes (PBLs)
inflammation-induced ................................. 408409 human lymphocytes preparation.................................. 99
pharmacological targeting, mitochondrial ................... 64 recycling assay .................................................... 100101
Oxidative stress and beta cell dysfunction Peripheral blood mononuclear cells (PBMC)............ 99, 100
CTLs and MHC ....................................................... 350 Phosphate-buffered saline (PBS) .................................... 249
cytokines .................................................................... 350 PLP. See Proteo lipid protein (PLP)
cytosolic Fe2+ .............................................................. 349 Polymerase chain reaction (PCR)
death .................................................................. 348, 349 buffer ......................................................................... 276
FPIR.......................................................................... 348 genotyping mouse tail DNA, SSLP .................. 278279
AUTOIMMUNITY: METHODS AND PROTOCOLS
Index
501
marker ....................................................................... 278 Q
size analysis, amplified SSLP
capillary polymer-based ............................... 279280 QTL. See Quantitative trait loci (QTL)
metaphor agarose gel electrophoresis ........... 280281 Quantitative trait loci (QTL)
Primary biliary cirrhosis (PBC) acute and chronic GN ............................................... 274
AMAs........................................................................ 292 allele effects ............................................................... 282
eppendorf................................................................... 320 autoimmune phenotype ............................................. 273
materials genetic mapping, SLE ............................................... 281
ELISA ................................................................. 301 mapping software ...................................................... 276
flow cytometry analysis ................................ 302303 validity ....................................................................... 281
flow cytometry, liver and spleen ........................... 303 X-linked .................................................................... 287
frozen tissue preparation ...................................... 301
R
H&E staining ...................................................... 302
immunohisto chemical staining, CD4 Reactive oxygen intermediates (ROI) ....................6264, 66,
and CD8, 302 6971, 7374, 84, 92, 94
liver cytokine and chemokine analysis ................. 303 Real-time PCR........................................................ 426427
paraffin tissue preparation.................................... 301 Receptor recycling ..................................................... 94, 104
recombinant PDC-E2, PAGE ............................ 301 Relapsing-remitting................................................. 389, 407
recombinant proteins preparation ................ 300301 Renal mononuclear phagocytes, SLE
serum cytokine analysis........................................ 303 arginase and iNOS assays .......................................... 229
western blot ......................................................... 301 cDNA synthesis ......................................................... 229
methods cell
flow cytometry analysis ................................ 310311 purification .......................................................... 229
flow cytometry, liver and spleen ................... 311312 sorting .................................................................. 221
frozen tissue preparation .............................. 307308 cellular turn over studies .................................... 223224
H&E staining ...................................................... 308 cytospin ..................................................................... 229
immunohistochemical staining, CD4 damage assessment
and CD8 ................................................ 308310 albumin estimation, urine .................................... 218
immunoreactivity, mitochondrial autoantigens blood urea nitrogen (BUN) ................................. 218
(see Mitochondria) description ........................................................... 217
liver cytokine and chemokine analysis ................. 312 DCs (see Dendritic cells (DCs))
paraffin tissue preparation.................................... 307 flow cytometry ................................................... 219221
recombinant proteins preparation ................ 303304 functional studies ............................................... 224226
serum cytokine analysis........................................ 312 gene expression studies
mice treated ............................................................... 324 cDNA synthesis ................................................... 227
mouse models real-time PCR ..................................................... 227
dominant negative TGF- receptor II mice RNA isolation, Trizol method ............................. 226
(see TGF- receptor) harvest ............................................................... 218219
IL-2R-/-Mice ............................................ 297298 immunohistologic studies .................................. 227228
NOD.ABD.................................................. 292, 295 macrophages (see Macrophages (M))
Novosphingobium aromaticivorans ................................292 magnetic beads .................................................. 222223
2-OA-BSA-immunized mice (see 2-Octynoic materials
acid-BSA-immunized mice) buffers .................................................................. 216
organ-specific autoimmune diseases .............................. 4 chemical reagents ................................................. 216
osmotic pumps........................................................... 323 equipment ............................................................ 215
sterile solution ........................................................... 320 kits.. ............................................................. 215216
Principal component analysis (PCA) plasticware ................................................... 216217
dimension-reduction technique ......................... 129130 morphological studies ................................................ 223
generic workflow................................................ 125126 murine models, nephritis (see Murine models,
Proteo lipid protein (PLP) SLE nephritis)
active EAE ................................................................ 386 nephritis............................................................. 207208
EAE pathogenesis, nephritis
active immunization models ................................ 370 autoantibody deposition....................................... 208
induce, susceptible mouse strains ......................... 365 mechanisms, renal damage........................... 208, 209
EAE adoptive transfer models................................... 389 remission induction ............................................. 208
myelin epitope ........................................................... 396 protein quantification and analysis ............................ 228
AUTOIMMUNITY: METHODS AND PROTOCOLS
502 Index