Leukocyte Tetraspanin CD53 Restrains α3

Integrin Mobilization and Facilitates

This information is current as
of June 14, 2020.
Cytoskeletal Remodeling and Transmigration
in Mice
Louisa Yeung, Jeremy M. L. Anderson, Janet L. Wee, Maria
C. Demaria, Michaela Finsterbusch, Yuxin S. Liu, Pam Hall,
Brodie C. Smith, Wendy Dankers, Kirstin D. Elgass, Ian P.
Wicks, Hang Fai Kwok, Mark D. Wright and Michael J.
Hickey

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J Immunol published online 12 June 2020
http://www.jimmunol.org/content/early/2020/06/11/jimmun
ol.1901054

Supplementary http://www.jimmunol.org/content/suppl/2020/06/11/jimmunol.190105
Material 4.DCSupplemental

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Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Published June 12, 2020, doi:10.4049/jimmunol.1901054
The Journal of Immunology

Leukocyte Tetraspanin CD53 Restrains a3 Integrin

Mobilization and Facilitates Cytoskeletal Remodeling and
Transmigration in Mice

Louisa Yeung,*,† Jeremy M. L. Anderson,* Janet L. Wee,*,† Maria C. Demaria,†

Michaela Finsterbusch,* Yuxin S. Liu,* Pam Hall,* Brodie C. Smith,* Wendy Dankers,*
Kirstin D. Elgass,‡ Ian P. Wicks,x,{,‖ Hang Fai Kwok,# Mark D. Wright,†,1 and
Michael J. Hickey*,1
The importance of tetraspanin proteins in regulating migration has been demonstrated in many diverse cellular systems. However,
the function of the leukocyte-restricted tetraspanin CD53 remains obscure. We therefore hypothesized that CD53 plays a role in

Downloaded from http://www.jimmunol.org/ at BMC-libraries on June 14, 2020

regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflam-
matory stimuli. Deletion of CD53 significantly reduced neutrophil recruitment to the acutely inflamed peritoneal cavity. Intravital
microscopy revealed that in response to several inflammatory and chemotactic stimuli, absence of CD53 had only minor effects on
leukocyte rolling and adhesion in postcapillary venules. In contrast, Cd532/2 mice showed a defect in leukocyte transmigration
induced by TNF, CXCL1 and CCL2, and a reduced capacity for leukocyte retention on the endothelial surface under shear flow.
Comparison of adhesion molecule expression in wild-type and Cd532/2 neutrophils revealed no alteration in expression of b2
integrins, whereas L-selectin was almost completely absent from Cd532/2 neutrophils. In addition, Cd532/2 neutrophils showed
defects in activation-induced cytoskeletal remodeling and translocation to the cell periphery, responses necessary for efficient
transendothelial migration, as well as increased a3 integrin expression. These alterations were associated with effects on inflam-
mation, so that in Cd532/2 mice, the onset of neutrophil-dependent serum-induced arthritis was delayed. Together, these findings
demonstrate a role for tetraspanin CD53 in promotion of neutrophil transendothelial migration and inflammation, associated with
CD53-mediated regulation of L-selectin expression, attachment to the endothelial surface, integrin expression and trafficking, and
cytoskeletal function. The Journal of Immunology, 2020, 205: 000–000.

R
ecruitment of circulating leukocytes is of fundamental
importance in protective and reparative responses fol-
lowing tissue infection or injury (1, 2). Leukocyte re-
cruitment in most settings requires leukocytes to undergo a
sequence of interactions with the endothelium lining the vascu-
lature at the affected site, progressing through the steps of rolling,
adhesion, intravascular crawling, and transmigration (1). Several
families of adhesion molecules expressed by leukocytes and en-
dothelial cells are required for these interactions, mediating in-
teractions by binding to ligands expressed on the corresponding
cell type. The importance of these interactions is demonstrated by
the severe immune deficiencies that afflict patients with mutations
in adhesion molecules and functionally linked molecules (3, 4). In
addition to classical adhesion molecules, more recent studies have
demonstrated that the tetraspanin family of cell surface molecules
also contributes to leukocyte recruitment (5–11). The mechanisms
underlying the actions of tetraspanins are distinct from those
mediated by adhesion molecules in that tetraspanins have no
known ligands and do not mediate cell–cell interactions via ligand
binding. In contrast, tetraspanins organize partner molecules in the
cell membrane into functionally linked microdomains through
which they mediate functional effects on adhesion molecules and

*Centre for Inflammatory Diseases, Monash University Department of Medicine,
Monash Medical Centre, Clayton, Victoria 3168, Australia; †Department of Immu-
nology, Monash University, Alfred Research Alliance, Melbourne, Victoria 3004,
Australia; ‡Monash Micro Imaging, Monash University, Clayton, Victoria 3800,
Australia; xWalter and Eliza Hall Institute of Medical Research, Parkville, Victoria
3052, Australia; {Department of Medical Biology, The University of Melbourne,
Parkville, Victoria 3050, Australia; ‖Department of Rheumatology, The Royal Mel-
bourne Hospital, Parkville, Victoria 3050, Australia; and #Institute of Translational
Medicine, Faculty of Health Sciences, University of Macau, Taipa, Macau Special
Administrative Region, China
1
M.D.W. and M.J.H. contributed equally to this study.
ORCIDs: 0000-0003-1233-1168 (L.Y.); 0000-0001-6015-5651 (M.C.D.); 0000-0003-
2606-5585 (Y.S.L.); 0000-0002-4180-0472 (W.D.); 0000-0002-6349-4517 (H.F.K.);
0000-0002-2177-5214 (M.D.W.); 0000-0003-2354-357X (M.J.H.).
Received for publication August 29, 2019. Accepted for publication May 15, 2020.
This work was supported by funding from National Health and Medical Research
Council (NHMRC) Australia Project Grant 1033198 (to M.D.W.), Program Grant
1113577 (to I.P.W.), Senior Research Fellowship 1042775 (to M.J.H.), NHMRC
Medical Research Future Fund Practitioner Fellowship 1154325 (to I.P.W.), The Reid
Charitable Trusts (to I.P.W.), the Rebecca L. Cooper Medical Research Foundation
and ANZ Trustees (to M.J.H.), Austrian Science Fund Erwin Schroedinger Fellow-
ship J 3752-B28, (to M.F.), and funding from the Science and Technology De-
velopment Fund, Macau Special Administrative Region (0055/2019/A1; to H.F.K.).
This study was made possible through Victorian State Government Operational In-
frastructure Support and the Australian Government NHMRC Independent Research
Institute Infrastructure Support scheme.
L.Y., J.M.L.A., J.L.W., M.C.D., M.F., Y.S.L., P.H., B.C.S., and W.D. performed
research and analyzed data. K.D.E., I.P.W., and H.F.K. contributed vital new reagents
and analytical tools. L.Y., M.D.W., and M.J.H. designed the research and wrote the
paper.
Address correspondence and reprint requests to Prof. Michael J. Hickey, Centre for
Inflammatory Diseases, Monash University Department of Medicine, Block E, Monash
Medical Centre, 246 Clayton Road, Clayton, VIC 3168, Australia. E-mail address:
michael.hickey@monash.edu
The online version of this article contains supplemental material.
Abbreviations used in this article: PFA, paraformaldehyde; RT, room temperature;
WT, wild-type.
Copyright Ó 2020 by The American Association of Immunologists, Inc. 0022-1767/20/$37.50

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901054
2 CD53 PROMOTES TRANSMIGRATION
the cytoskeleton (12, 13). We have recently demonstrated that the
leukocyte-expressed tetraspanin CD37 promotes leukocyte re-
cruitment via regulation of b2 integrin–mediated adhesion (10).
However, whether CD53, the other member of the tetraspanin
family expressed exclusively on immune cells, acts in a similar
manner is unknown.
CD53 was originally identified as a cell surface glycoprotein
expressed on all immune cells (14, 15). Predictive biochemical
analyses determined that this protein spanned the cell membrane
four times and was therefore a member of the tetraspanin family
(16). The function of CD53 has remained unclear, although two
studies provide evidence that CD53 has important functions in
the human immune system. Examination of neutrophils from
members of a family affected by recurrent microbial infections
revealed that the only detectable phenotypic abnormality was
absence of CD53 (17). A more recent genome-wide association
study of patients with increased susceptibility to mycobacterial
infection identified an association with mutations in the CD53
locus (18). Together, these studies infer that mutations in CD53
result in immune deficiency. A range of mechanisms were pos-
tulated to explain this effect, including modulation of cytokine
expression, MHC class I and MHC class II expression and cel-
lular adhesion, and defective immune cell signaling (19–23).
However, these hypotheses are yet to be supported by functional
in vivo data.
The phenotype associated with CD53 deficiency in humans has
similarities to that of leukocyte adhesion deficiency patients,
raising the possibility of a role for CD53 in immune cell adhesion
and trafficking. Further evidence comes from studies of cross-
linking CD53 on immune cells, which increases cell–cell ad-
hesion, potentially via effects on the b2 integrin LFA-1 (20, 24,
25). However, in vivo substantiation of these findings is lacking.
Two recent studies have investigated the effect of genetic de-
letion of CD53 on the adaptive immune system, demonstrating
roles for CD53 in early B cell development (26) and lymphocyte
recirculation via stabilization of L-selectin on the lymphocyte
cell surface (27). However, the role of CD53 in myeloid cell
function remains unclear. In this study we investigated a role for
CD53 as a regulator of neutrophil and monocyte trafficking. The
results indicate that CD53 contributes to myeloid cell recruitment
by selectively promoting their transmigration.
Materials and Methods
Mice
The generation of Cd53 2/2 mice has been described previously (28).
In brief, Cd532/2 mice were generated using a Cre/lox approach,
introducing LoxP sites in the mouse Cd53 gene downstream of exon 1
and upstream of exon 6. Thereby, exons 2 to 5 were deleted upon
crossing a Cd53fl/fl mouse with a ubiquitous Cre deleter mouse (29).
Deletion of the Cd53 gene was verified by PCR analysis and via flow
cytometric analysis of CD53 protein abundance on blood leukocytes.
Cd532/2 mice develop normally and are phenotypically normal, apart
from reductions in L-selectin expression on some immune cell populations
(27, 28). Cd532/2 mice were fully backcrossed to the C57BL/6J background
prior to their use in this study. C57BL/6J wild-type (WT) mice were obtained
from Monash Animal Research Platform, Monash University. Cd532/2
mice were bred in-house. Itgam2/2 mice were kindly provided by Prof.
K. Peter (Baker Heart & Diabetes Institute, Melbourne, Australia). Mice
were maintained in specific pathogen–free conditions and used for experi-
ments at 8–16 wk of age. Experiments were performed in male mice. All
animal experiments were approved by the Monash Medical Centre Animal
Ethics Committee or the Alfred Medical Research and Education Precinct
Animal Ethics Committee.
Abs
The following Abs and reagents were used (from BD Biosciences, North Ryde,
NSW, Australia, unless otherwise stated): anti-Ly-6G-PE (clone 1A8);
anti-Ly-6G-V450 (1A8; ProSci, Poway, CA); anti-CD62L-PE or FITC
(MEL-14); anti-LFA-1-AF647 (M17/4; BioLegend, San Diego, CA);
anti-Mac-1-FITC (M1/70); anti-SMA-Cy3 (1A4; Merck, Bayswater,
VIC, Australia); anti-MRP14-AF488 (2B10; Abcam, Melbourne, VIC,
Australia); anti-CD31 (MEC13.3); anti-CD49c (42/CD49c); anti-CD49f-
AF488 (GoH3; BioLegend); anti-Gr-1-DyLight 650 or DyLight 568
(RB6-8C5, conjugated in-house); anti-ADAM17-AF568 [A9(B8) (30),
conjugated in-house]; goat anti-rat IgM-AF488 (Invitrogen); anti–b-
actin (clone AC-15; Sigma-Aldrich, St. Louis, MO); goat anti-mouse IgG
HRP (Cayman Chemical, Ann Arbor, MI); phalloidin–AF488 (Thermo Fisher
Scientific, Waltham, MA).
Peritonitis
Peritonitis was induced using a modification of a previously published
technique (10). Mice were inoculated i.p. with 500 ml of 3% thioglycollate
(Sigma-Aldrich). Four hours later, mice were euthanized, and cells were
harvested by peritoneal lavage. Counts of total immune cells and neu-
trophils were performed, identifying neutrophils on the basis of Ly-6G
expression as determined by flow cytometry (LSRFortessa cell analyzer;
BD Biosciences).
Intravital microscopy
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Leukocyte–endothelial cell interactions were assessed via intravital mi-
croscopy of the cremaster muscle, as previously described (10, 31). In
brief, mice were anesthetized via i.p. injection of a xylazine hydrochloride
(10 mg/kg) and ketamine hydrochloride (150 mg/kg) solution prior to
surgery. A catheter was inserted into the right jugular vein, allowing
for immediate delivery of additional anesthetic. The left testis was
exposed over a microscope pedestal. A coverslip was placed over the
muscle and held in place with vacuum grease. Under basal conditions,
constant superfusion buffer (13 bicarbonate saline [pH 7.2–7.4], 37˚C) was
supplied to exposed tissue. At least two postcapillary venules (25–40 mm)
were examined in each experiment.
Vessels were visualized using intravital microscopy, using a ZEISS
Axioplan 2 microscope with a 253 long working distance lens and 103
eyepiece. Recordings of vessels were made using a Sony SSC-DC50AP
video recorder, and images were projected onto a Sony Trinitron monitor.
Recordings were stored on a Panasonic DMR-EH57 DVD Recorder,
allowing for playback of recordings for further analysis. Leukocyte re-
cruitment parameters assessed for each vessel consisted of rolling flux,
rolling velocity, adhesion, and emigration. A rolling cell was defined as a
cell that appeared to be attached to the endothelium, moving at a rate
slower than the surrounding blood. Rolling velocity (micrometers per
second) was measured as the time taken for the cell to travel 100 mm.
Rolling flux (cells per minute) was defined as the number of cells rolling
past a certain point each minute. An adherent cell was defined as a cell
that remained stationary for 30 or more seconds within a 100-mm stretch
of vessel (expressed as cells/100 mm), whereas transmigration was de-
fined as the number of cells located outside the vessel within the field of
view (expressed as cells per field of view).
Assessment of leukocyte recruitment in response to cytokine/
chemokine stimulation
Leukocyte–endothelial cell interactions were assessed in three different
inflammatory models. To examine the response to an inflammatory stim-
ulus incorporating endothelial activation, local TNF was used. TNF (50 ng
in 170 ml of saline), or a saline control, was administered via s.c. intra-
scrotal injection 4 h prior to imaging (31). Leukocyte–endothelial cell
interactions were assessed 4 h later as stated above. To investigate the
response to a neutrophil-selective chemotactic stimulus, the acute response
to CXCL1 was assessed (10). In these experiments, a baseline recording was
taken before replacement of the superfusion buffer with solution containing
mouse CXCL1 (7.5 or 3.75 nM). Recordings were taken at 30 and 60 min
post-CXCL1 application, with recruitment parameters analyzed as per above.
To examine the response to a monocyte-selective stimulus, CCL2 was used
(32). Mouse CCL2 (JE, 333 ng in 175 ml of saline, 138 nM) was administered
via intrascrotal injection 4 h prior to imaging. Preparations were assessed as
for the TNF experiments above.
In some experiments using CXCL1, Mac-1 activation on adherent
neutrophils was assessed as previously described (33). In brief, on the day
of the experiment, 2 3 109 polystyrene microspheres (1 mm, yellow-green
fluorescent, FluoSpheres; Molecular Probes) were incubated in BSA
(1 mg/ml in PBS, 300 ml) for 2 h, prior to sonication. A total of 1 3 109
beads (150 ml) of the mixture were administered i.v. into mice undergoing
intravital microscopy and CXCL1 exposure, along with anti-Gr1-PE (2 mg) to
aid identification of neutrophils. After 10 min, cremaster preparations
The Journal of Immunology
were visualized using spinning disk confocal microscopy, recording
brightfield images along with sequential green and red fluorescent signals.
Data were assessed as the number of attached microspheres per adherent
leukocyte within a 100-mm length of venule.
Intravascular crawling and cell fate analysis
To examine the behavior of adherent neutrophils in the vasculature, time-
lapse intravital microscopy was used to examine cremasteric postcapillary
venules during CXCL1 superfusion (34). Images were captured using a
Ultraview VoX spinning disk microscope system (PerkinElmer) running
Volocity (version 6.3.0). Brightfield images were captured every 10 s for
60 min to visualize intravascular migration of leukocytes following arrest
on the endothelium. Analysis was performed using the Manual Tracking
plug-in in ImageJ (version 1.44), assessing the distance the cell traveled
(in micrometers) following its attachment to the endothelium until de-
tachment or transmigration. From the distance traveled, cell velocity
(micrometers per minute) was calculated based on the amount of time
from attachment until detachment or transmigration. One hundred cells
were assessed in each experiment from n = 6 mice per group.
Confocal microscopy analysis of CXCL1-stimulated
cremaster muscle
Immunohistochemistry of whole-mounted cremaster muscles was used to
assess neutrophil localization in response to CXCL1 stimulation using a
previously described technique (35). In brief, following 60-min CXCL1
superfusion, cremaster muscles were fixed in 4% paraformaldehyde (PFA)
for 30 min at room temperature (RT). Subsequently, the muscles were
blocked and permeabilized in 10% FCS, 10% goat serum, 5% mouse se-
rum, and 1% Triton X-100 in PBS for 2 h at RT. Tissues were then stained
(overnight, 4˚C) with anti-SMA-Cy3 (to identify pericytes), anti-MRP14-
AF488 (to identify neutrophils), and anti-CD31-AF647 (to identify endo-
thelial cells). Stained tissues were mounted on microscope slides and
imaged using a Nikon C1 inverted confocal microscope equipped with a
603 water immersion lens. The location of neutrophils within the vascu-
lature was assessed according to a previously published technique (36).
Leukocytes were defined as follows: 1) intravascular (within the anti-CD31
stain), 2) within the vascular wall (with the anti-SMA staining), and 3)
extravascular (beyond the anti-SMA staining). For purposes of analyses,
leukocytes in categories 1 and 2 were combined and deemed as intravas-
cular. Intravascular and extravascular neutrophil infiltration were deter-
mined by volumetric analysis using Imaris (Bitplane, Zurich, Switzerland)
image analysis software. Because in some instances it was not possible
to resolve individual neutrophils in confocal images, the total volume of
recruited neutrophils was quantified using volumetric analysis in Imaris
(version 7.6.4). In brief, following background subtraction, individual
surfaces were generated for vascular (SMA) and neutrophil (MRP14)
channels. The SMA surface was then used as a layer mask to define
intravascular and extravascular compartments, and the volume statistics for
the neutrophil surfaces in each compartment were generated. Data were
analyzed for two to three vessels per mouse, from n = 8 mice per group.
Flow cytometric analysis of neutrophil adhesion
molecule expression
Expression of L-selectin, LFA-1, and Mac-1 on WT and Cd532/2 neu-
trophils was assessed via flow cytometry. In brief, leukocytes were isolated
from blood or bone marrow of WT and Cd532/2 mice and stained with
anti-Ly-6G-V450 (to identify neutrophils) and anti-CD62L-PE, or anti-Ly-
6G-PE, anti-CD11b-FITC, and anti-LFA-1-AF647, for assessment of
Mac-1 and LFA-1. For assessment of surface and intracellular L-selectin,
nonpermeabilized neutrophils were first stained for surface L-selectin us-
ing PE-conjugated anti–L-selectin at a concentration (8 mg/ml) determined
in pilot experiments to minimize any further staining achieved by subsequent
exposure to anti-L-selectin–FITC (data not shown). Cells were subsequently
washed, fixed, permeabilized (BD Cytofix/Cytoperm, BD Biosciences), and
stained for intracellular L-selectin using FITC-conjugated anti–L-selectin
(5 mg/ml). Surface and intracellular L-selectin levels were determined via
flow cytometric analysis, identifying neutrophils as Ly-6G+ cells. Samples
were analyzed using either FACSCanto II or LSRFortessa cell analyzer (BD
Biosciences) flow cytometers. Data were analyzed using FlowJo (version
VX; FlowJo, Ashland, OR).
ADAM17 activity
ADAM17 activity was determined on cell membrane fractions from WT and
Cd532/2 neutrophils using the SensoLyte 520 TACE (a-Secretase) Ac-
tivity Assay Kit (AnaSpec, Fremont, CA), according to the manufacturer’s
instructions. In brief, cell membrane fractions were prepared by five
3
rounds of freezing–thawing and pelleted by centrifugation at 20,000 3 g.
Cell membrane fractions from 1 3 105 neutrophils were assayed in du-
plicate over 60 min at RT. Controls run with the ADAM17 inhibitor TAPI-
0 showed no activity, confirming the specificity of the assay.
Mobilization of a3 and a6 integrins
Mobilization of integrin a3 and a6 in neutrophils was performed as pre-
viously described on 2% BSA (control)- or PECAM-1 (2 mg/ml)–, ICAM-
1 (1 mg/ml)–, and CXCL1 (4 ng/ml)-coated slides (36). Neutrophils were
isolated from WT and Cd532/2 bone marrow via MACS-based negative
selection (Miltenyi Biotec, Macquarie Park, NSW, Australia) and allowed
to adhere to coated slides for 30 min at 37˚C. Cells were then fixed with
2% PFA, blocked in 2% BSA, and when examining intracellular integrin
distribution, permeabilized with 0.1% Triton X-100/2% BSA. Cells were
stained with anti-CD49c (anti-a3, 42/CD49c, 5 mg/ml), anti-CD49f-AF488
(anti-a6, 8 mg/ml), anti-Gr-1-DL650, and Hoechst 33342. Anti-CD49c was
detected using goat anti-mouse AF568. Cells were imaged by confocal
microscopy as stated above, and images were analyzed using ImageJ. For
assessment of total surface expression for each of the integrins, mean
fluorescence intensity for each channel was measured. In brief, maximum
intensity projections of the z-stacks were generated for each channel/
integrin. Gr-1+ cells were outlined as regions of interest, and mean in-
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tensities of a3 and a6 integrin staining were then assessed within the cell
margins. Approximately 40 cells per mouse from eight mice per condition
were assessed. Analysis of integrin cluster number and size was performed
in ImageJ using a custom-written algorithm. The maximal projection in-
tensity for each stain was obtained, after which the algorithm counted
individual integrin a3 and a6 clusters and measured their corresponding
areas. Ring formation analysis was conducted using Imaris. Cells were
considered positive for ring formation when a3 and/or a6 staining was
visually localized to the periphery of the cell for at least ∼25% of the cell.
Approximately 50 neutrophils were examined per treatment group.
Western blotting analysis
Western blotting for a3 integrin was performed using protein samples from
bone marrow neutrophils from WT and Cd532/2 mice isolated via MACS-
based negative selection as described above. Neutrophils were lysed in
RIPA buffer (150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate,
0.1% SDS, 50 mM Tris–HCL [pH 8] in dH2O) and protease inhibitor and
centrifuged at 13,000 rpm (10 min at RT). Lysates were mixed with
NuPage LDS Sample Buffer (Thermo Fisher Scientific) and heated at 70˚C
for 10 min. Protein samples were loaded into 3–8% Tris–Glycine gels with
Tris–Acetate Running Buffer and the HiMark Prestained Protein Standard
(all from Thermo Fisher Scientific), after which samples were transferred
to PVDF membranes (Merck). Membranes were blocked in 5% milk
powder and then cut to probe for a3 integrin and b-actin separately.
Membranes were then incubated with mouse anti-mouse CD49c (clone
42/CD49c; BD Biosciences) or anti-mouse b-actin (clone AC-15; Sigma-
Aldrich), followed by incubation with goat anti-mouse IgG HRP (Cayman
Chemical). b-actin served as a loading control. The blots were developed
using Amersham ECL Western Blotting Detection Reagent (GE Healthcare
Life Sciences, Marlborough, MA). Blots were imaged using a GE ImageQuant
LAS 4000 and ImageQuant software (GE Healthcare Life Sciences).
Subsequent quantitative analysis was performed using ImageJ. Intensity
plots for a3 and b-actin were generated, and the ratio of the area of the a3
to b-actin plots was calculated. This was performed three times for each
sample to obtain an average ratio. Each sample average was then nor-
malized to the average of the WT ratios for each set of experiments.
F-actin redistribution and colocalization of CD53 and
ADAM17 in mouse neutrophils
For F-actin assessment, isolated WT and Cd532/2 neutrophils underwent
adhesion to the substrates described above. Cells were then stained with
phalloidin-AF488, anti-Gr-1-AF568, and Hoechst 33342 and imaged by
confocal microscopy. Circularity based on F-actin staining was measured
using the ImageJ Circularity plug-in. Approximately 60 neutrophils were
examined per treatment group. Colocalization of CD53 and ADAM17 in
neutrophils was performed on 2% BSA substrate. Neutrophils were iso-
lated from WT mouse bone marrow via MACS-based negative selection
and allowed to adhere to coated slides (37˚C for 20 min). Cells were then
activated with PMA (50 ng/ml) for 2.5, 5, or 15 min prior to fixation with
2% PFA. Cells were then blocked in 2% BSA and permeabilized with
0.5% saponin/PBS. Cells were stained with OX-79 (anti-CD53), A9(B8)–
AF568 (anti-ADAM17), anti-Gr-1-DL650, and Hoechst 33342. OX-79 was
detected using goat anti-rat AF488. Cells were imaged by confocal mi-
croscopy as stated above. Analysis of CD53 and ADAM17 colocalization
4 CD53 PROMOTES TRANSMIGRATION

was performed in ImageJ using the JACoP plug-in, and Mander correlation
coefficients were obtained for each cell (37). Approximately 40 neutrophils
were examined per mouse for each time point.
K/BxN serum-induced arthritis
Mice underwent the K/BxN serum-transfer model of arthritis using a
modification of a previously published technique (38, 39). In brief, mice
were administered K/BxN serum (75 ml, i.p.) on days 0 and 2. Baseline
measurements of body weight, ankle thickness, and general activity were
taken prior to the first dose and assessed daily thereafter. Mice were scored on
a five-point scale in which 0 = normal paw, 1 = one toe swollen, 2 = more
than one toe swollen, 3 = entire paw swollen, and 4 = ankylosed paw. Scores
of all four paws were added to obtain a composite clinical score (maximum
clinical score = 16). Mice were euthanized, and joints were taken for histo-
logical assessment at either day 2 or 7. Joint pathology was assessed in H&E-
stained sections of ankle joints from mice undergoing the K/BxN model using
a four-point scoring method, assessing synovitis and joint space exudate in
which 0 = no area affected, 1 = one area affected, 2 = multiple small areas
affected, and 3 = large areas affected. Cartilage damage was assessed using
Safranin O/Fast Green staining and scored on a four-point scale in which 0 =
fully stained cartilage, 1 = slight loss of cartilage staining, 2 = severe loss of
cartilage staining, and 3 = completely destained cartilage (38, 40). For all
between the strains in these parameters (Fig. 1A, 1B). Similarly,
leukocyte and neutrophil abundance in the bone marrow did not
differ between the strains (Supplemental Fig. 1). These findings
are supported by similar observations reported in a recent inde-
pendent analysis of Cd532/2 mice (26).
We subsequently examined the role of CD53 in leukocyte traf-
ficking in thioglycollate peritonitis. Four hours after thioglycollate
injection, WT mice showed a robust recruitment of leukocytes, of
which 60–70% were neutrophils (Fig. 1C). In Cd532/2 mice,
the numbers of both total leukocytes and neutrophils were sig-
nificantly lower than those in WT mice (Fig. 1C), demonstrating
a role for CD53 in leukocyte recruitment.
To investigate the role of CD53 within the microvasculature, we
next used intravital microscopy of the cremaster muscle to compare
leukocyte–endothelial cell interactions in WT and Cd532/2 mice.
In the absence of exogenous stimuli, leukocyte rolling flux and
adhesion did not differ between WT and Cd532/2 mice, although
a minor reduction in leukocyte rolling velocity was observed in
Cd532/2 mice after 60 min of observation (Supplemental Fig. 2).

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assessments, the operator was blinded to the mouse genotype.
Statistical analysis
GraphPad Prism 7 (GraphPad Software, San Diego, CA) was used to conduct
data analysis. Significance was determined using a Mann–Whitney U test,
a Holm–Sidak multiple test, an unpaired two-tailed t test, repeated mea-
sures one-way ANOVA with multiple comparisons, or two-way ANOVA
with multiple comparisons. Statistical significance was set at p , 0.05.
Results
Absence of CD53 impairs leukocyte recruitment via an effect
on transmigration
We first compared the number of circulating leukocytes and neu-
trophils in WT and Cd532/2 mice, demonstrating no differences
These findings indicate that the absence of CD53 does not have a
marked effect on basal leukocyte–endothelial cell interactions. We
next examined the effect of CD53-deficiency on leukocyte trafficking
induced by local administration of TNF. In both WT and Cd532/2
mice, TNF markedly reduced leukocyte rolling flux and rolling
velocity and significantly increased adhesion and transmigration
(Fig. 1D–G). The changes in rolling and adhesion were similar in WT
and Cd532/2 mice (Fig. 1D–F). However, TNF-induced leukocyte
transmigration was significantly lower in Cd532/2 mice (Fig. 1G),
indicating a selective role for CD53 in promotion of transmigration.
To examine this effect in more detail, we next assessed responses
induced by chemokines CXCL1 and CCL2, which promote
transmigration of neutrophils and monocytes, respectively (32, 41).

FIGURE 1. CD53 modulates leukocyte recruitment via ef-

fects on transmigration. (A and B) Total circulating leukocytes
(A) and neutrophils (B) in WT and Cd532/2 mice. (C) Thio-
glycollate-induced peritoneal leukocyte recruitment in WT and
Cd532/2 mice 4 h after thioglycollate injection, with data
shown for total leukocytes and neutrophils as enumerated by
flow cytometry. For (A)–(C), data points represent individual
mice. Mean 6 SEM are also shown. (D–G) Leukocyte re-
cruitment parameters in cremasteric postcapillary venules after
local (intrascrotal) administration of TNF assessed in WT and
Cd532/2 mice (or saline as control in WT mice only). Data are
shown for leukocyte rolling flux (D), rolling velocity (E), ad-
hesion (F), and transmigration (G). Data are shown as mean 6
SEM, from n = 4–6 mice per group. *p , 0.05, **p , 0.01 by
Mann–Whitney test.
The Journal of Immunology 5

In WT mice exposed to CXCL1, rolling flux decreased over the

observation period, with no change in rolling velocity (Fig. 2A,
2B). In contrast, CXCL1 induced increases in adhesion and
transmigration by 30 min, with transmigration progressively
increasing for the remainder of the experiment (Fig. 2C, 2D). In
Cd532/2 mice, rolling flux but not velocity was significantly
reduced relative to WT mice at all time points examined
(Fig. 2A, 2B). The reduction in rolling in Cd532/2 mice did not
translate to an alteration in the number of adherent leukocytes
(Fig. 2C). However, Cd532/2 mice showed a significant reduction
in the number of transmigrated leukocytes (Fig. 2D). A similar
trend was seen in response to CCL2 in that there were no differ-
ences in rolling flux, velocity, or adhesion, whereas transmigration
was significantly decreased in Cd532/2 mice (Fig. 2E–H). Together,
these findings indicate that CD53 plays a previously unrecognized
role in myeloid leukocyte transmigration.

CD53 promotes both retention on the endothelium and

migration across the endothelial barrier

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The selective effect of CD53 deficiency on transmigration indicates
that the major effect of this tetraspanin on leukocyte behavior
occurs subsequent to the adhesion step. After chemokine-induced
arrest, leukocytes undergo intravascular migration, or crawling, in
search of an optimal location to cross the endothelial barrier (34).
To investigate whether CD53 impacted on this process, we used
time-lapse imaging to assess neutrophil intravascular crawling
induced by CXCL1. Leukocyte migration velocity and distance
did not differ significantly between WT and Cd532/2 mice at the
normal concentration of CXCL1 (Supplemental Fig. 3A, 3B).
However, at a lower concentration at which leukocyte migration
was less likely to be impeded by adherent cells, Cd532/2 leuko-
cytes crawled at a faster velocity than WT cells (Supplemental
Fig. 3C), indicating that CD53 acts to slow intravascular crawling.
Moreover, the proportion of crawling cells that failed to proceed
to transmigration, instead detaching from the endothelial sur-
face, was significantly elevated in Cd532/2 mice (Supplemental
Fig. 3E). Together, these findings indicate that in response to
CXCL1, CD53 acts to stabilize the attachment of leukocytes to
the endothelial surface.
We also examined the impact of CD53 on the capacity of
neutrophils to navigate across the specific layers of the vascular
wall in response to CXCL1, using whole mount immunohisto-
chemistry and confocal microscopy to identify the location of
neutrophils relative to the endothelial and pericyte layers (42, 43).
In WT mice, neutrophils were present throughout the vascular
wall, with many having migrated well clear of the pericyte layer
(Fig. 3A). In contrast, in Cd532/2 mice, neutrophils were pre-
dominantly present within the vascular lumen or retained between
endothelial cells, consistent with a reduced capacity of these cells
to penetrate the pericyte layer (Fig. 3A). These observations were
supported by quantitation of neutrophil volume, either inside
blood vessels or retained in the endothelial layer (defined as in-
travascular), which revealed a significant elevation of intravascular
neutrophils in Cd532/2 mice (Fig. 3B).
Marked decrease in surface L-selectin expression of
Cd532/2 neutrophils
Given the alterations in leukocyte–endothelial cell interactions, we
next assessed whether neutrophil adhesion molecules that partic-
ipate in transmigration were affected by the absence of CD53. As
tetraspanins are recognized for their ability to regulate integrin
function (5, 10, 44–47), we first focused on the b2 integrins. LFA-
1 and Mac-1 were expressed at equivalent levels on circulating
neutrophils from WT and Cd532/2 mice (Fig. 4A–D). As Mac-1
FIGURE 2. CD53 selectively promotes leukocyte transmigration in re-
sponse to chemokine stimulation. Leukocyte–endothelial cell interactions
in cremasteric postcapillary venules of WT and Cd532/2 mice were ana-
lyzed during a 60-min acute exposure to CXCL1 (7.5 nM) commencing
immediately after the 0 min time point (A–D) or 4 h after local CCL2
administration (138 nM, intrascrotal) (E–H). Data are shown for leukocyte
rolling flux (A and E), rolling velocity (B and F), adhesion (C and G), and
transmigration (D and H). Data are shown as mean 6 SEM, for n = 6–9
mice per group. *p , 0.05, **p , 0.01, ***p , 0.001 by Holm–Sidak
multiple t test or unpaired two-tailed t test.
facilitates CXCL1-induced intravascular crawling and transmi-
gration (34), we also examined the functional state of Mac-1 on
adherent neutrophils in CXCL1-exposed venules, assessing their
capacity to capture albumin-coated microbeads from the circula-
tion (Fig. 4E) (33). This assay serves as a readout of the capacity
of Mac-1 to bind one of its recognized ligands, albumin, stemming
from conformational change of the integrin to its high-affinity
form as a result of inside-out signaling (48, 49). Experiments in
Itgam2/2 mice demonstrated that microbead capture was Mac-1–
dependent (Fig. 4F). Microbead capture was similar in WT and
Cd532/2 neutrophils (Fig. 4F), providing evidence that the func-
tional state of Mac-1 on adherent neutrophils did not differ be-
tween these strains.
Another immune cell–expressed molecule with a less well-
recognized role in transmigration is L-selectin (50, 51). There-
fore, we compared L-selectin expression on WT and Cd532/2
neutrophils, finding that L-selectin was almost completely absent
from the surface of circulating neutrophils from Cd532/2 mice
(Fig. 5A, 5B). Bone marrow neutrophils from Cd532/2 mice
6 CD53 PROMOTES TRANSMIGRATION
FIGURE 3. CD53 promotes neutrophil transendothelial migration in
response to CXCL1. (A) Representative confocal microscopy images
(original magnification 31000) of whole mounts of CXCL1-stimulated
cremaster muscles from WT and Cd532/2 mice after 60-min CXCL1
superfusion showing the locations of neutrophils relative to the structural
elements of a postcapillary venule. Shown in separate panels are staining
for PECAM-1 (endothelial cells, blue), a-smooth muscle actin (a-SMA)
(pericyte layer, red) and MRP14 (neutrophils, green), with the merged
images shown below (A). (B) Quantitation of location of neutrophils as
either intravascular (defined as restricted to the endothelial and pericyte
layers) or extravascular (located outside the pericyte layer). In this
showed the same phenotype (Fig. 5C, 5D), indicating that the
reduction in L-selectin developed prior to neutrophils entering the
bloodstream. This phenotype did not stem from increased reten-
tion of L-selectin within Cd532/2 neutrophils, as flow cytometric
assessment revealed that intracellular L-selectin was not elevated
but in fact significantly lower in Cd532/2 neutrophils relative to
WT neutrophils (Supplemental Fig. 4).
L-selectin is typically shed from the leukocyte surface upon
activation, a process mediated by the metalloprotease ADAM17
(52). Furthermore, in monocytic cells, the tetraspanin CD9 has
been shown to associate with ADAM17 and regulate its function
(53). We therefore reasoned that CD53 might promote retention of
surface L-selectin by associating with ADAM17 to regulate its
activity. To address this possibility, we assessed colocalization of
CD53 and ADAM17 in WT neutrophils using confocal micros-
copy. Both CD53 and ADAM17 were detectable on the surface of
neutrophils (Fig. 5E), with ∼40% of the ADAM17 being colo-
calized with CD53 (Fig. 5F). Exposure to PMA, a stimulus that
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induces L-selectin shedding, resulted in a significant reduction in
the proportion of ADAM17 colocalizing with CD53 (Fig. 5E, 5F),
providing evidence that association of CD53 with ADAM17 in
neutrophils is reduced upon activation. To assess whether absence
of CD53 impacted on ADAM17 enzymatic activity, we used a
fluorogenic substrate assay, assessing ADAM17 activity in cell
membrane fractions prepared from WT and Cd532/2 neutrophils.
These experiments revealed no difference in ADAM17 activity
between WT and Cd532/2 neutrophils (Fig. 5G). Together, these
findings indicate that, whereas there is evidence of an association
between CD53 and ADAM17 in the cell membrane, total
ADAM17 activity is unaffected by the absence of CD53.
Cd532/2 neutrophils display premature a3 integrin
redistribution, increased a3 integrin expression, and impeded
F-actin reorganization
We next investigated the effect of CD53 deficiency on neutrophil-
expressed integrins with roles in migration through the venular
vascular wall, focusing on a3 and a6 integrins (36, 54, 55). In
unstimulated neutrophils, and following neutrophil adhesion to an
activating substrate, total surface expression of a3 integrins, as
assessed using nonpermeabilized cells, did not differ between the
strains (Fig. 6A, 6B). In addition, there were no differences in the
number of surface-expressed a3 integrin clusters or the cluster
area between WT and Cd532/2 neutrophils at baseline or fol-
lowing activation (Fig. 6C, 6D). However, previous studies have
shown that upon adhesion to substrates that mimic early molecular
events in transmigration (PECAM-1/ICAM-1/CXCL-1), these
integrins move from intracellular stores to the cell periphery, with
this redistribution thought to be critical for transmigration (36).
Therefore, we used this approach to examine the redistribution of
the a3 integrin in WT and Cd532/2 neutrophils, making this
assessment in permeabilized cells. In WT neutrophils, the a3
integrin underwent redistribution from an intracellular location to
the cell periphery upon activation, as denoted by a significant in-
crease in integrin ring formation (Fig. 6E, 6F). In contrast, Cd532/2
neutrophils showed an elevated level of a3 ring formation in resting
cells, which did not increase further upon stimulation (Fig. 6E, 6F).
analysis, because of aggregation of neutrophils leading to difficulties re-
solving individual cells, these parameters were assessed by quantitation of
the volume of MRP14 staining in the various vascular compartments. Data
are shown as mean 6 SEM for intravascular and extravascular neutrophils
from n = 8 mice per group. **p , 0.01 by Mann–Whitney U test.
The Journal of Immunology
FIGURE 4. Absence of CD53 does not affect neutrophil b2 integrin
expression or Mac-1 function. (A–D) Flow cytometric analysis of surface
LFA-1 (A and B) and Mac-1 (C and D) expression on WT and Cd532/2
neutrophils. Data are shown as histograms from representative experi-
ments, with isotype control staining shown as a dashed line (A and C) and
group data of results from individual experiments as measured by the
relative ratio to the average WT mean fluorescence intensity (B and D).
Data are shown as mean 6 SEM of (n = 13) mice per group. (E and F)
Analysis of Mac-1–dependent binding of albumin-coated beads to adher-
ent neutrophils in CXCL1-exposed postcapillary venules of WT, Cd532/2,
and Itgam2/2 mice. (E) Spinning disk confocal microscopy image of a
postcapillary venule following CXCL1 exposure, with neutrophils labeled
by i.v. anti–Gr-1 (red). Albumin-coated microbeads (green, arrows) are
visible attached to two neutrophils (original magnification 3400). (F)
Quantitation of bead interactions with neutrophils (number per cell) in WT,
Cd532/2, and Itgam2/2 mice. Data are shown as mean 6 SEM of (n = 6–9)
mice per group. *p , 0.05, ***p , 0.001 by Mann–Whitney U test or one-
way ANOVA with multiple comparisons.
Given this alteration in a3 integrin distribution in Cd532/2 neutrophils,
we also compared a3 integrin expression via Western blot, observing a
significant elevation of the integrin in Cd532/2 neutrophils (Fig. 6G,
6H). Taken together, these data indicate that in resting neutrophils,
CD53 acts to inhibit expression of the a3 integrin and its trans-
location to the cell periphery.
Examination of the a6 integrin revealed similar findings in
regard to surface expression and integrin cluster formation in
that these did not differ between WT and Cd532/2 neutrophils
(Fig. 7A–D). The a6 integrin showed a trend toward premature
ring formation in Cd532/2 neutrophils as seen with the a3
integrin, although this did not reach significance (Fig. 7E, 7F).
Another key requisite for transmigration is the cytoskeletal
remodeling required for leukocytes to migrate through the vascular
endothelium (56). Therefore, we next investigated the contribution
of CD53 to activation-induced cytoskeletal remodeling, using
confocal microscopy to examine F-actin distribution (10). Upon
adhesion to ICAM-1/PECAM-1/CXCL1, WT neutrophils showed
increased polarization (Fig. 7G) as demonstrated by a significant
decrease in the circularity ratio (Fig. 7H). In CD53-deficient
neutrophils, activation failed to induce a significant change in
circularity (Fig. 7G, 7H). Taken together with the a3 integrin data,
these findings indicate that CD53 acts to maintain neutrophils in
an unactivated state prior to exposure to stimuli associated with
transmigration.
7
Delayed onset of acute K/BxN serum-induced arthritis in
Cd532/2 mice
Finally we addressed the question of whether absence of CD53
impacted neutrophil-dependent inflammation, making use of the
K/BxN serum-induced model of arthritis, in which neutrophil
infiltration is central to joint inflammation (38). Cd532/2 mice
displayed a significant delay in the development of arthritis, as
demonstrated by reduced total joint swelling on days 2 and 4
(Fig. 8A). However, by day 7, joint swelling in Cd532/2 mice was
comparable to that in WT mice. Assessment of joint pathology at
day 7 revealed a significant reduction in joint damage in Cd532/2
compared with WT mice, but the extent of synovitis and exudate
observed did not differ between the strains (Fig. 8B, 8C).
Discussion
The most notable feature of the phenotype of CD53-deficient
myeloid cells observed in this study was their reduced capacity
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to undergo transendothelial migration. As the tetraspanin family
has been shown to modulate the function of numerous molecular
pathways, it is likely that the mechanism underlying this CD53-
dependent effect is multifactorial. However, given that integrins
are one of the most common targets of tetraspanin-mediated
regulation, we initially focused on leukocyte integrins involved
in neutrophil recruitment and transmigration. For transmigration,
timely redistribution of integrins, including LFA-1, a3, and a6,
during the process is crucial (36, 57). Particularly for the a3
integrin, translocation from its primary location in intracellular
secretory vesicles to the cell surface supports neutrophil extrava-
sation via promotion of cell elongation (55, 58). In the current
study, in CD53-deficient neutrophils, the a3 integrin had already
undergone translocation to the periphery of the cell prior to ini-
tiation of recruitment, demonstrating that CD53 constitutively acts
as a “brake” on a3 integrin redistribution (or recycling). Given the
importance of the appropriate timing of a3 integrin translocation,
its presence adjacent to the cell membrane of Cd532/2 neutrophils
prior to initiation of transmigration is likely to have contributed
to the reduced capacity of these cells to exit the vasculature. In
contrast, we saw no difference in expression of LFA-1 or Mac-1
or the functional state of Mac-1 in CD53-deficient neutrophils,
demonstrating a level of specificity in the actions of CD53 and
distinguishing it from CD37, a tetraspanin which modulates b2
integrin function (10).
We also observed that absence of CD53 led to a significant
increase in total cellular expression of a3 integrin in Cd532/2
neutrophils. The mechanism whereby loss of CD53 impacts on
total expression of this integrin is not immediately clear. In some
previous studies, tetraspanin deletion has resulted in the opposite
effect, reducing expression of partner proteins. For example, ab-
sence of tetraspanin CD81 in B cells led to decreased expression
of CD19 because of a role for the tetraspanin in supporting CD19
through processing in the endoplasmic reticulum (59). In the case
of CD53, our findings clearly demonstrate that normal a3 integrin
trafficking is disrupted in the absence of the tetraspanin. Based on
that observation, it is tempting to speculate that this alteration
impacted the conventional intracellular processing and degrada-
tion of this integrin, and that this led to its aberrant accumulation
within neutrophils. Further experiments will be required to cast
additional light on this issue.
Leukocyte shape change via cytoskeletal remodeling is central to
transmigration. Tetraspanins, including CD81, CD82, and CD37,
are known to modulate cytoskeletal dynamics via association with
molecules such as Rho GTPases and ERM proteins (9, 10, 60–62).
In this study we observed reduced cytoskeletal function in neutrophils
8 CD53 PROMOTES TRANSMIGRATION

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FIGURE 5. Absence of CD53 results in loss of surface L-selectin expression on Cd532/2 circulating and bone marrow neutrophils. (A–D) Flow
cytometric analysis of surface L-selectin expression on WT and Cd532/2 neutrophils from blood (A and B) and bone marrow (C and D). Data are
shown as histograms from representative experiments, with isotype control data shown as dotted lines (A and C) and group data of results from
individual experiments as measured by the relative ratio to the average WT mean fluorescence intensity (B and D). Data are shown as mean 6 SEM of
(n = 6) mice per group. **p , 0.01, ***p , 0.001 by Mann–Whitney U test. (E) Representative confocal microscopy images of WT neutrophils
stained for CD53 (green) and ADAM17 (red) before and after stimulation with PMA assessed at time points shown above the images. The lower
panels show the merged images [original magnification in (A) and (E) 31150]. (F) Quantitation of colocalization of CD53 and ADAM17 in WT
neutrophils, as determined using Pearson coefficient. Data are shown as mean 6 SEM of ∼50 neutrophils per mouse from (Figure legend continues)
The Journal of Immunology 9

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FIGURE 6. Absence of CD53 results in alteration in mobilization and expression of a3 integrin in neutrophils. (A–D) Surface expression of a3 integrin
on WT and Cd532/2 neutrophils as assessed by immunofluorescence and confocal microscopy of nonpermeabilized cells. (A) Representative confocal
microscopy images of WT and Cd532/2 neutrophils on either 2% BSA (control substrate) or PECAM-1/ICAM-1/CXCL1 substrate, stained for a3 integrin.
(B) Quantitation of total surface expression as measured by mean fluorescence intensity of a3 integrin. (C and D) Surface clustering of a3 integrin on WT
and Cd532/2 neutrophils, showing quantitation of number (C) and area (D) of a3 integrin clusters. (E and F) a3 integrin ring formation in WT and Cd532/2
neutrophils. Representative confocal microscopy images (E) and quantitation (F) of a3 integrin ring formation in WT and Cd532/2 neutrophils on either 2%
BSA control substrate or PECAM-1/ICAM-1/CXCL1 substrate stained under Triton X-100–permeabilized conditions [original magnification in (A) and (E)
31150]. Data in (A)–(F) were derived from n = 7–9 mice per group and ∼40 neutrophils per mouse and are shown as mean 6 SEM. **p , 0.01 by Holm–
Sidak multiple t test. (G and H) Total a3 integrin expression in WT and Cd532/2 neutrophils as determined using Western blot. (G) Example blot showing
a3 integrin expression in three WT samples and three Cd532/2 samples. b-actin bands from the same gel are shown as loading controls. (H) Quantitation of
Western blot results, shown as ratios of the intensities of a3 integrin and b-actin bands for each sample. Data are shown as mean 6 SEM for n = 9 mice per
group. *p , 0.05.

lacking CD53, as evidenced by a reduction in the capacity to
undergo shape change in response to activation, indicating that
this member of the tetraspanin family also modulates leukocyte
function via this pathway.
In addition to its well-recognized contribution to leukocyte
rolling, L-selectin also promotes leukocyte transmigration (50).
L-selectin ligation has been shown to promote intracellular sig-
naling via pathways, including PKC, MAPK and Syk, leading to
alterations in cytoskeletal function and b2 integrins and increased
transendothelial migration and chemotaxis (63–65). More re-
cently, L-selectin on monocytes was shown to promote polariza-
tion and cell membrane protrusion required for transmigration
across endothelial cell monolayers (51, 66). Given the reduction in
expression of L-selectin on neutrophils in Cd532/2 mice, it is
reasonable to surmise that signals derived from L-selectin ligation
are impaired in these cells. The reduction in these signals is
therefore likely to have contributed to some degree to the observed
deficit in transmigration.
Nevertheless, effects on the canonical role of L-selectin in
mediating leukocyte rolling could also have had an impact on
leukocyte recruitment in Cd532/2 mice. Indeed, we observed
significant reductions in leukocyte rolling in Cd532/2 mice both
under basal conditions, after prolonged observation, and during
stimulation with CXCL1. However, although rolling was reduced
during these responses, substantial residual rolling remained. These
findings are consistent with our previous observations of leuko-
cyte rolling in L-selectin–deficient mice, in which the absence of
L-selectin reduced but failed to eliminate rolling in cremasteric
postcapillary venules. In this context, P-selectin–mediated inter-
actions accounted for the bulk of the residual rolling (67). Fur-
thermore, in both L-selectin2/2 mice, and as seen in this study in
Cd532/2 mice, leukocyte adhesion was not reduced, indicating
that this partial decrease in leukocyte rolling was insufficient to
significantly impact the number of leukocytes able to undergo arrest
in the inflamed microvasculature. Taken together, these findings
indicate that any potential contribution of the loss of L-selectin on
the impaired transmigration of Cd532/2 leukocytes is likely to have
occurred downstream of leukocyte arrest.
The mechanism underlying the early loss of L-selectin from
neutrophils remains to be determined. We addressed the possibility
that CD53 mediates this effect by controlling the function of
ADAM17, the major metalloprotease responsible for cleavage of
the L-selectin ectodomain from the surface of activated leukocytes
(68, 69). ADAM17-mediated cleavage of TNF is subject to reg-
ulation by another member of tetraspanin family, CD9, in both
monocytic cell lines and endothelial cells (53). In this setting, cell
activation reduces the ADAM17/CD9 association at the same time
as it increases ADAM17 activity, suggesting a mechanism of
regulation of ADAM17 function related to the colocalization of
these molecules in the cell membrane (53). In this study, we

n = 4 mice. *p , 0.05 by repeated measures one-way ANOVA with multiple comparisons. (G) ADAM17 activity in cell membrane fractions prepared from
WT and Cd532/2 neutrophils, as determined using a fluorogenic substrate assay. Data are shown as mean 6 SEM of preparations from n = 9 mice per group
for both genotypes.
10
FIGURE 7. CD53 regulates cytoskeletal function but not surface ex-
pression and mobilization of a6 integrin in neutrophils. (A–D) Surface
expression of a6 integrin on WT and Cd532/2 neutrophils as assessed by
immunofluorescence and confocal microscopy of nonpermeabilized cells.
(A) Representative confocal microscopy images of WT and Cd532/2
neutrophils on either 2% BSA (control substrate) or PECAM-1/ICAM-1/
CXCL1 substrate, stained for a6 integrin. (B) Quantitation of total surface
expression as measured by mean fluorescence intensity of a6 integrin.
(C and D) Surface clustering of a6 integrin on WT and Cd532/2 neutro-
phils, showing quantitation of number (C) and area (D) of a6 integrin
clusters. (E and F) a6 integrin ring formation in WT and Cd532/2 neu-
trophils. Representative confocal microscopy images (E) and quantitation
(F) of a6 integrin ring formation in WT and Cd532/2 neutrophils on either
2% BSA control substrate or PECAM-1/ICAM-1/CXCL1 substrate,
stained under Triton X-100–permeabilized conditions. Data in (A)–(F)
were derived from n = 7–9 mice per group, and ∼40 neutrophils per mouse
and are shown as mean 6 SEM. (G and H) Polarization of WT and Cd532/2
neutrophils as determined by assessment of F-actin distribution. (G) Repre-
sentative confocal images of WT and Cd532/2 neutrophils on 2% BSA
CD53 PROMOTES TRANSMIGRATION
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FIGURE 8. CD53 ablation attenuates cartilage injury in K/BxN arthritis.
WT and Cd532/2 mice underwent the K/BxN model of serum-transfer
arthritis and were assessed over the following 7 d. (A) Combined joint
pathology scores for WT and Cd532/2 mice over a 7-d period following
initiation of model. (B) Representative H&E and Safranin O/Fast Green–
stained histological sections depicting day 7 WT and Cd532/2 ankle joints.
Arrow indicates area of cartilage degradation (original magnification 3100).
(C) Blinded histological scoring of day 7 WT and Cd532/2 ankle joints. Data
are shown as mean 6 SEM of (n = 12–13) mice per group. *p , 0.05 be-
tween WT and Cd532/2 by two-way ANOVA with multiple comparisons (A)
or unpaired t test (B).
showed via confocal microscopy that in resting neutrophils a
consistent proportion of CD53 colocalized with ADAM17 in the
cell membrane. In response to neutrophil activation, this degree
of colocalization diminished, coincident with an increase in the
L-selectin–shedding activity of ADAM17. However, despite this
evidence of molecular association, comparison of ADAM17
enzyme activity in WT and Cd532/2 neutrophils by measuring
ADAM17-dependent cleavage of a non–cell-associated ligand
revealed no difference between the strains. It should be noted
that, although this assay does assess total ADAM17 activity, it
does not reveal the capacity of ADAM17 to access endogenous
control substrate or PECAM-1/ICAM-1/CXCL1 substrate stained for F-
actin [original magnification in (A), (E), and (G) 31150]. (H) Quantitation
of circularity of WT and Cd532/2 neutrophils under resting and stimulated
conditions. Data are shown as mean 6 SEM of ∼60 neutrophils per mouse
from n = 7 mice per group. ***p , 0.001, as determined using two-way
ANOVA with multiple comparisons.
The Journal of Immunology
substrates within the cell membrane. This is important, as recent
work on the regulation of ADAM10 by the Tspan C8 subfamily
of tetraspanins suggests that tetraspanins may regulate metal-
loprotease function by interacting with the ADAM and regu-
lating its trafficking and substrate accessibility and specificity
(70, 71). In regard to CD53, such a mechanism would be con-
sistent with our own observations of an activation-induced de-
crease in the interaction of ADAM17 and CD53. Moreover, this
raises the possibility that CD53 may negatively regulate access
of ADAM17 to its substrate L-selectin. It is also notable that
L-selectin can also be cleaved by other proteases, and these may
be alternative targets of the actions of CD53 (72, 73). As such,
more work is required to dissect the molecular mechanism by
which CD53 regulates L-selectin shedding.
Finally, we assessed whether this action of CD53 on myeloid
cell transmigration resulted in an impact on a clinically relevant
model of inflammation, the K/BxN serum-transfer model of
arthritis. The absence of CD53 led to a significant delay in
development of joint pathology. Moreover, at the termination of
the model, Cd532/2 mice displayed reduced cartilage damage.
The finding that cartilage damage was reduced at a time point
when there was no clear difference in neutrophil infiltration
raises the intriguing possibility that CD53 may regulate neutrophil-
derived proteases responsible for cartilage damage. These and
other functions of CD53 will be the subject of future investiga-
tions. However, together, these studies reveal a previously unap-
preciated role for the tetraspanin CD53 in promotion of myeloid
leukocyte recruitment, mediated primarily via a selective effect on
transmigration.
Acknowledgments
We wish to acknowledge the kind assistance of Dr. Tim Gottschalk (Depart-
ment of Immunology and Pathology, Monash University) for assistance with
flow cytometry, Prof. Karlheinz Peter (Baker Heart and Diabetes Institute)
for provision of Itgam2/2 mice, Monash Micro Imaging (Monash Univer-
sity) for the provision of instrumentation, training, and technical support,
and Dr. Olga Barriero (Harvard Medical School) for careful reading of the
manuscript.
Disclosures
The authors have no financial conflicts of interest.
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