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1. Targeting genetically modified macrophages to the glomerulus..................................................................

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Targeting genetically modified macrophages to the glomerulus

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Abstrak: Macrophages are key players in the development of the majority of renal diseases and are therefore
ideal cellular vectors for site specifically targeting gene therapy to inflamed glomeruli. Macrophages can be
genetically modified using viral vectors ex vivo then re-introduced into the body where they can home to the
diseased site. This review summarises current experience in efficiently targeting modified macrophages to the
inflamed glomerulus focussing on the factors controlling macrophage localisation, macrophage gene transfer
methods, in vivo gene delivery and results of recent investigations using modified macrophage gene therapy for
glomerular disease.
Teks lengkap: Headnote
Key Words
Macrophages * Inflammation * Glomerulonephritis . Adenovirus . Gene
Abstract
Macrophages are key players in the development of the majority of renal diseases and are therefore ideal
cellular vectors for site specifically targeting gene therapy to inflamed glomeruli. Macrophages can be
genetically modified using viral vectors ex vivo then re-introduced into the body where they can home to the
diseased site. This review summarises current experience in efficiently targeting modified macrophages to the
inflamed glomerulus focussing on the factors controlling macrophage localisation, macrophage gene transfer
methods, in vivo gene delivery and results of recent investigations using modified macrophage gene therapy for
glomerular disease.
Introduction
Macrophage infiltration of renal tissue is a feature of many forms of nephrological disease ranging from classical
inflammatory diseases such as rapidly progressive glomerulonephritis to more indolent 'non-inflammatory'
processes such as diabetic nephropathy. The classical view has been that these cells are primarily injurious,
and that inhibiting their function or preventing entry into renal tissue is the main goal of therapy. Macrophages
are, however, heterogeneous and perform distinct functions; increasingly their role in the down-regulation and
resolution of inflammation has been identified [1]. This is most clearly seen in skin wounds where inhibition of
macrophage entry prolongs inflammation and delays healing [2]. This raises the potential to utilise macrophages
as therapeutic cells to down-regulate renal inflammation and restore normal function. Critical to achieving this is
to establish what determines their function, what factors control macrophage localisation to inflamed renal
tissue, and how their properties can be modified to ameliorate inflammation.
Functions of Macrophages in Renal Inflammation
Macrophages arc critical adaptors in the immune response, able to both drive the immune response by antigen
presentation to T cells and respond to cytokines produced by the specific immunity; they are both conductor and
player in the inflammatory orchestra. Macrophage infiltration into renal tissue is an early feature of
glomerulonephritis and contributes to the development of necrosis, crescent formation and subsequent scarring.
Macrophage function in the early stages of inflammation is geared towards promoting inflammation as within the
inflamed environment macrophages are exposed to a wide range of stimuli including pro-inflammatory cytokines
such as IL-1[beta], TNF-[alpha] and IFN-[gamma], lipid mediators, reactive oxygen and nitrogen products,
chemokines and necrotic and apoptotic cells. The importance of a number of these factors in experimental
models of glomerular inflammation has been established with inhibition of cytokines such as IL-1[beta], TNF[alpha] and macrophage migration inhibitory factor (MIF) reducing disease severity. This pro-inflammatory
activation of macrophages renders them unresponsive to anti-inflammatory cytokines such as transforming
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growth factor-[beta] (TGF-[beta]) or IL-4 [3], and re-orientating this response is a critical approach to resolve
inflammation. The crosstalk between infiltrating macrophages and their environment is important in
inflammation. Suto et al. [4] have demonstrated that injection of activated macrophages into glomeruli resulted
in the up-regulation of matrix metalloproteinase 9 (MMP-9) in normal glomeruli but expression was downregulated when activated macrophages were injected into regenerating glomeruli from rats with Thy-1 nephritis
where TGF-[beta] production was high. Thus, local expression of TGF-[beta] from resident mesangial cells may
suppress activated macrophages and macrophage factors may influence the regulation of genes in resident
glomerular cells [5].
Factors Controlling Macrophage Localisation to Inflamed Renal Tissue
The first stage of monocyte/macrophage involvement in glomerular inflammation is adhesion to activated
endothelium and transmigration in response to a chemotactic gradient. There is increasing evidence that
different vascular beds and different forms of inflammation utilise distinct molecules to enable macrophage
localisation and these need to be understood for glomerular inflammation to allow efficient homing of genetically
modified inflammatory cells. Initial work demonstrated that blockade of classical integrin molecules, intracellular
adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule (VCAM) by antibodies decreased
inflammation in nephrotoxic nephritis (NTN) [6], however, there was no consistent evidence of a reduction in
glomerular macrophage numbers and the reduction in injury may have reflected alteration of trafficking of
inflammatory cells in other organs such as spleen or regional lymph nodes. Recent studies have focused on
direct visualisation of leukocyte adhesion. De Vrcise et al. [7] showed that inhibition of selectins did not affect
glomerular leukocyte adhesion, while inhibition of [beta]^sub 2^ integrin CD11b (counter-receptor to ICAM-1)
prevented glomerular leukocyte accumulation.
Recently, chemokines have also been show to have roles in adhesion. Fractalkine is a CX3C chemokine that
exists as a soluble chemotactic agent but it also has a transmembrane domain and is expressed on activated
endothelium. Inhibition of fractalkine receptor (CX3CR1) in NTN reduced glomerular macrophage infiltration and
attenuated histological and biochemical measures of injury [8]. The chemokine GRO-[alpha] can also be
immobilised to cell surface proteoglycans to support adhesion of monocytes to mesangial cells in vitro. In
addition, blockade of GRO-[alpha] or fractalkine receptors on macrophages injected directly into the renal artery
of rats with NTN suppressed their localisation to inflamed glomeruli [9]. Thus, adhesion to glomerular
endothelium in experimental models appears to involve chemoadhesin interaction followed by integrin-mediated
adhesion.
Once adherent to endothelium, macrophages transmigrate across the glomerular basement membrane in
response to chemokines. A wide range of approaches have been used to study which chemokines are preeminent in the control of renal inflammation [reviewed in 10] and as with adhesion, it appears to depend on both
the nature of injury and at what point during the immune response they are assessed. Two chemokines for
which the clearest role is defined are monocyte chemotactic protein-1 (MCP-1 - binds to CCR2) and regulated
on activation normal T cell expressed and secreted, RANTES (binds to CCR1, 3 and 5). Early studies showed
that inhibition of MCP-1 by anti-bodies reduced macrophage glomerular infiltration and proteinuria in NTN and
expression of MCP-1 and CCR2 are early features of immune complex glomerulonephritis, NTN and murine
lupus nephritis. We have also demonstrated that direct inhibition of CCR2 receptor on macrophages reduced
their glomerular localisation in rats with NTN [9]. Similarly, RANTES is found in many models of glomerular
inflammation and inhibition by the antagonists met-RANTES or aminoxypentane-RANTES reduces macrophage
infiltration in NTN and anti-Thy-1 nephritis.
Expression of chemokines has also been identified in human renal biopsies and a recent study by Segerer et al.
[11] showed that in crescentic glomerulonephritis, CCR2 was mainly expressed on glomerular macrophages
while CCR5 was found mainly on interstitial T cells. This suggests that specific chemokine expression directs
macrophages and T cells to separate compartments with distinct functional consequences. Thus, at present,
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MCP-1 and its counter-receptor CCR2 are prominent in the early stages of glomerular inflammation and provide
a target to direct macrophages to inflamed glomeruli. Further work is required to determine the orchestration of
chemokines during glomerulonephritis, particularly those expressed during the resolution of inflammation.
Macrophage Manipulation
Macrophages are ideally suited to alter inflammatory disease due to their preferential localisation to inflamed
tissue, the properties they can develop and their interaction with other cells of the immune response. Thus far,
work has focused on altering macrophage function ex vivo before re-administration into animals with renal
inflammation. A number approaches exist to transduce macrophages to express specific transgenes including
viral vectors such as adenoviruses, retroviruses and lentiviruses as well as non-viral methods including cationic
DNA carrier molecules, electroporation and receptor-mediated DNA uptake [reviewed in 12]. Non-viral
approaches give a low efficiency of transfection compared to viral methods, thus the latter have been favoured
for in vivo manipulation. Adenoviral vectors are at present the most popular method of genetically modifying
macrophages; large genes (up to ~ 10 kb) can be transferred in standard vectors and a high level of expression
of the transgene achieved. Adenoviruses are relatively inefficient at transducing monocytes due to the low
expression of adenoviral receptor integrins [alpha]^sub v^[beta]^sub 3^ and [alpha]^sub v^[beta]^sub 5^, but
infection rates can be increased to greater than 90% if primary cultures of monocytes are incubated with
macrophage colony-stimulating factor which up-regulates integrin expression. Adenoviral transduced primary
cultures of bone-derived macrophages (BMDM) or macrophage cell lines such as RAW 267.4 and NR8383 cells
express high levels of transgene within 24 h of transduction and this remains stable for 7-10 days [13] allowing
efficient short-term expression in vivo.
Retroviruses transduce rapidly dividing cells by stably incorporating DNA into the host cell genome, however,
the transduction efficiency is low due to the low rate of proliferation. To overcome the problem, CD34^sup +^
bone marrow stem cells, precursors of macrophages, have been used that are capable of cell division and
retroviral infection. This allows reconstitution with genetically modified bone marrow cells that permits longlasting expression of the relevant transgene. Recombinant lentiviruses have a natural tropism for macrophages
and can stably transduce these cells. Despite their high level of gene transfer and expression, concerns
regarding safety issues such as generation of replicon-competent virus during production of vectors have so far
limited their use to in vitro work.
In vivo Localisation of Genetically Modified Macrophages
Site specifically targeting genetically modified macrophages to the glomerulus and limiting expression in nontarget tissues is a fundamental requirement for their use as vehicles for gene therapy. Macrophages have been
injected intravenously or directly into the renal artery where they have a concentrated exposure to the
inflammatory site. The main concern with intravenous injection is that only a small proportion of the modified
macrophages localise to inflamed glomeruli due to their hold up in the lung and trafficking to the spleen. Direct
injection into the renal artery by contrast, allows highly efficient first-pass localisation and we have shown that
adenoviral transduced macrophages localise preferentially in the presence of glomerular inflammation in rats
and when macrophages are activated with lipopolysaccharide [13].
Yokoo et al. [14] have shown that CD11b^sup +^CD18^sup +^ (ligand for ICAM-1) bone marrow-derived cells
transduced with recombinant adenovirus then re-injected systemically into mice localise to glomeruli when the
animals were treated with lipopolysaccharide, which up-regulates ICAM-1 expression, and on induction of
glomerulonephritis [15]. Thus the state of activation of macrophages and the endothelium, as well as the
method of delivery, affects the efficiency of glomerular macrophage localisation. The transgene expressed by
macrophages can also affect the properties of cells as we found that transduction with adenoviruses expressing
IL-4 markedly enhanced the ability of macrophages to localise to inflamed glomeruli compared to expression of
inert transgene [16]. The most likely mechanism for this is up-regulation of CXCR1 and 2 expression by
macrophages, which adhere to GRO-[alpha], a recognised chemoadhesin on glomerular endothelium. Thus,
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glomerular-specific genetically modified macrophage localisation can be achieved effectively.

Effect of Genetically Modified Macrophages on Renal Inflammation

The transduction of macrophages to alter the development of inflammatory disease has thus far focused on
expression of cytokines with principally anti-inflammatory and/or regulatory effects. Systemic injection of
macrophages transduced with recombinant adenovirus to express IL-1 receptor antagonist (IL-1ra) reduced the
severity of glomerular inflammation in mice with NTN and reduced interstitial macrophage infiltration in a model
of unilateral urcteric obstruction [15, 17]. We have studied the effects of macrophages transduced to express
anti-inflammatory cytokincs IL-4, IL-10 and TGF-[beta]. Initial work transduced the rat alveolar macrophage cell
line NR8383 with adenovirus to express IL-4. The cells localised to glomeruli of rats with NTN, produced the
cytokine in vivo and reduced the level of albuminuria, histological markers of glomerular inflammation and
macrophage infiltration [16] demonstrating that macrophage cell lines can be modified efficiently and have
therapeutic effects in vivo. By contrast, injection of NR8383 cells transduced to express active TGF-[beta] had
limited impact on the development of renal injury in rats with NTN [unpubl. data]. The properties of macrophage
cell lines arc different from native macrophages and may express transgenes in a different manner and
subsequent work focused on utilising primary cultures of BMDM from inbred strains of rats. Macrophages
transduced to express IL-10 localised preferentially to glomeruli of rats with NTN after renal artery injection and
produced a profound reduction in the severity of glomerular inflammation reducing both macrophage infiltration
and their state of activation as assessed by both MHC class II and ED3 expression [18].
Interestingly, in all these experiments, delivery of macrophages transduced with an inert gene ([beta]galactosidase) to inflamed glomeruli resulted in a reduction in injury as assessed by albuminuria but only up to
48 h after disease induction. The most likely mechanisms for this are either transduction of macrophages
causes them to release antiinflammatory molecules (e.g. IL-10) or that infusion of the transduced macrophages
prevents infiltration of pathogenic macrophages by competition for adhesion sites. This further highlights how
genetically modified macrophages have a complex interaction with glomerular inflammation.
The injection of IL-4 and IL-10 expressing macrophages resulted in highly effective localisation to glomeruli of a
single kidney with transduced cells seen infrequently in the contralateral kidney. Despite this, we have found
that injection into a single kidney markedly attenuated the development of inflammation in the contralateral
kidney. This implies that the expression of cytokine expressing macrophages at one inflammatory site can
decrease injury both at that site and at distant sites. These changes could not be mimicked by systemic

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administration of IL-10 or IL-4 expressing macrophages and there was no evidence of increased systemic levels
of the relevant cytokines. In light of the specific requirement of glomerular localisation in producing this
response, we hypothesise the effect is due to high local concentrations of cytokines altering the properties of
macrophages, dendritic cells and T cells trafficking through the glomerulus that consequently modify the
development of the immune response or that the transduced macrophages themselves migrate to regional
lymph tissue and regulate the systemic development of the immune response (fig. 1). As many autoimmune
diseases arc multifocal, these results raise the potential that disease modified by macrophages at one site may
be able to ameliorate inflammation at distant sites, a potentially powerful tool in therapy.
Regulation of Macrophage Gene Expression
The work so far has demonstrated the feasibility of macrophages to down-regulate inflammation and has largely
used adenoviral transduction to achieve this. However, a critical issue in macrophage-mediated gene therapy is
how to control gene expression, in particular, how macrophage activation can lead to regulated transgene
expression. Yokoo et al. [19] used double transduced macrophages that only express target gene at the site of
inflammation. To achieve this, macrophages were transduced with an adenovirus containing the IL-1[beta]
promoter which controls expression of Cre recombinase that, in turn, excised DNA from a second target
adenovirus carrying a reporter gene under the control of strong viral promoter, thus leading to reporter gene
expression. Following injection, the vehicle cells were detected in various organs including the kidney but the
reporter gene was expressed only in the kidney following the induction of nephritis. The double transduction
requirement for this method, however, reduce its general applicability and an alternative approach using
tetracycline sensitive promoters in a single vector could control transgene expression in vivo.
An additional problem with use of adenovirus is that expression of the transgene is short-lived. Thus to continue
to suppress ongoing inflammation would potentially require repeat injections of modified macrophages.
However, retroviral transformation of bone marrow stem cells leads to stable incorporation of the gene of
interest into the chromosomal DNA and subsequent daughter cells. This approach has been used to generate
CD34^sup +^ stem cells from mice expressing IL-1ra and following re-infusion in irradiated mice around 10-20%
of bone marrow and spleen cells were derived from donor cells [20]. When NTN was induced in these mice
there was a reduced severity of disease, although it is unclear to what extent this effect was a consequence of
IL-1ra expression by glomerular macrophages as opposed to effects elsewhere in the immune system.
However, combining long-term expression with regulation of transgene activation would provide the optimum
system to control genetically modified macrophages in inflammation.
Future
The experimental work to date has shown the potential for macrophages to ameliorate immune-mediated
glomerular disease (fig. 1). They provide a powerful tool to study the effects of both macrophages and cytokines
on the evolution of inflammatory disease. Therapies using genetically modified macrophages are in the
preliminary stages and many problems need to be addressed before moving to the clinical setting. In spite of
this, cell-based therapy to treat immune-mediated diseases is a clear objective. With the increasing use of stem
cells and other bone marrow-derived cells in the treatment of autoimmune diseases, it could be feasible to
augment the anti-inflammatory and reparative properties of macrophages using genetic manipulation. The
administration of such genetically enhanced macrophages could provide a powerful complimentary therapy for
glomerulonephritis and other forms of inflammation.
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Sidebar
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Fax + 41 61 306 12 34
E-Mail karger@karger.ch
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www.karger.com
(C) 2003 S. Karger AG, Basel
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Accessible online at:
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References
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adenovirus. Gene Ther 2000; 7:263-270.
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Inflamed site-specific gene delivery using bone marrow-derived CD11b^sup +^ CD18^sup +^ vehicle cells in
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15 Yokoo T, Ohashi T, Utsunomiya Y, Kojima H, Imasawa T, Kogure T, Hisada Y, Okabe M, Eto Y, Kawamura
T, Hosoya T: Prophylaxis of antibody-induced acute glomerulonephritis with genetically modified bone marrowderived vehicle colls. Hum Gene Ther 1999;10: 2673-2678.
16 Kluth DC, Ainslie CV, Pearce WP, Clarke D, Anegon I, Rees AJ: Macrophages transfected with adenovirus
to express IL-4 reduce inflammation in experimental glomerulonephritis. J Immunol 2001;166:4728-4736.
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17 Yamagishi H, Yokoo T, Imasawa T, Mitarai T, Kawamura T, Utsunomiya Y: Genetically modified bone

marrow-derived vehicle cells site specifically deliver an anti-inflammatory cytokine to inflamed interstitium of
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AuthorAffiliation
H.M. Wilson D.C. Kluth
Department of Medicine and Therapeutics, Institute of Medical Sciences, Foresterhill, Aberdeen, Scotland
AuthorAffiliation
H.M. Wilson
Department of Medicine and Therapeutics
Institute of Medical Sciences
Foresterhill, Aberdeen AB25 2ZD (UK)
Tel. +44 1224 55835, Fax +44 1224 555766, E-Mail d.c.kluth@abdn.ac.uk
MeSH: Adenoviridae -- genetics, Animals, Cell Movement -- physiology, Gene Transfer Techniques, Genetic
Engineering -- methods, Glomerulonephritis -- pathology, Glomerulonephritis -- therapy, Humans,
Immunotherapy, Adoptive -- methods, Macrophages -- virology, Cell Movement -- genetics (utama), Kidney
Glomerulus -- pathology (utama), Macrophage Activation -- genetics (utama), Macrophages -- physiology
(utama), Macrophages -- transplantation (utama)
Judul: Targeting genetically modified macrophages to the glomerulus
Pengarang: Wilson, H M; Kluth, D C
Judul publikasi: Nephron
Volume: 94
Edisi: 4
Halaman: e113-8
Tahun publikasi: 2003
Tanggal publikasi: Aug 2003
Tahun: 2003
Bagian: Minireview
Penerbit: S. Karger AG
Tempat publikasi: Basel
Negara publikasi: Switzerland
Subjek publikasi: Medical Sciences

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ISSN: 00282766
CODEN: NPRNAY
Jenis sumber: Scholarly Journals
Bahasa publikasi: English
Jenis dokumen: Journal Article
Nomor aksesi: 12972709
ID dokumen ProQuest: 274309492
URL Dokumen: http://search.proquest.com/docview/274309492?accountid=166961
Hak cipta: Copyright S. Karger AG Aug 2003
Terakhir diperbarui: 2014-04-18
Basis data: ProQuest Medical Library

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Daftar Pustaka
Citation style: APA 6th - American Psychological Association, 6th Edition
Wilson, H. M., & Kluth, D. C. (2003). Targeting genetically modified macrophages to the glomerulus. Nephron,
94(4), e113-8. Retrieved from http://search.proquest.com/docview/274309492?accountid=166961

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