Leukocyte Tetraspanin CD53 Restrains Α3 Integrin Mobilization

Leukocyte Tetraspanin CD53 Restrains α3 Integrin Mobilization and Facilitates Cytoskeletal Remodeling and Transmigration in Mice This information is current as of September 25, 2021. 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 Downloaded from J Immunol published online 12 June 2020 http://www.jimmunol.org/content/early/2020/06/11/jimmun ol.1901054 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2020 by The American Association of Immunologists, Inc. All rights reserved. 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 regulating leukocyte recruitment and tested this hypothesis by examining responses of CD53-deficient mice to a range of inflammatory 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. 2/2 Comparison of adhesion molecule expression in wild-type and Cd53 neutrophils revealed no alteration in expression of b2 http://www.jimmunol.org/ 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 inflammation, 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.

ecruitment of circulating leukocytes is of fundamental the severe immune deﬁciencies that afﬂict patients with mutations by guest on September 25, 2021 importance in protective and reparative responses fol- in adhesion molecules and functionally linked molecules (3, 4). In R lowing tissue infection or injury (1, 2). Leukocyte re- addition to classical adhesion molecules, more recent studies have cruitment in most settings requires leukocytes to undergo a demonstrated that the tetraspanin family of cell surface molecules sequence of interactions with the endothelium lining the vascu- also contributes to leukocyte recruitment (5–11). The mechanisms lature at the affected site, progressing through the steps of rolling, underlying the actions of tetraspanins are distinct from those adhesion, intravascular crawling, and transmigration (1). Several mediated by adhesion molecules in that tetraspanins have no families of adhesion molecules expressed by leukocytes and en- known ligands and do not mediate cell–cell interactions via ligand dothelial cells are required for these interactions, mediating in- binding. In contrast, tetraspanins organize partner molecules in the teractions by binding to ligands expressed on the corresponding cell membrane into functionally linked microdomains through cell type. The importance of these interactions is demonstrated by which they mediate functional effects on adhesion molecules and

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

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1901054 2 CD53 PROMOTES TRANSMIGRATION the cytoskeleton (12, 13). We have recently demonstrated that the anti-Ly-6G-V450 (1A8; ProSci, Poway, CA); anti-CD62L-PE or FITC leukocyte-expressed tetraspanin CD37 promotes leukocyte re- (MEL-14); anti-LFA-1-AF647 (M17/4; BioLegend, San Diego, CA); cruitment via regulation of b integrin–mediated adhesion (10). anti-Mac-1-FITC (M1/70); anti-SMA-Cy3 (1A4; Merck, Bayswater, 2 VIC, Australia); anti-MRP14-AF488 (2B10; Abcam, Melbourne, VIC, However, whether CD53, the other member of the tetraspanin Australia); anti-CD31 (MEC13.3); anti-CD49c (42/CD49c); anti-CD49f- family expressed exclusively on immune cells, acts in a similar AF488 (GoH3; BioLegend); anti-Gr-1-DyLight 650 or DyLight 568 manner is unknown. (RB6-8C5, conjugated in-house); anti-ADAM17-AF568 [A9(B8) (30), CD53 was originally identified as a cell surface glycoprotein conjugated in-house]; goat anti-rat IgM-AF488 (Invitrogen); anti–b- actin (clone AC-15; Sigma-Aldrich, St. Louis, MO); goat anti-mouse IgG expressed on all immune cells (14, 15). Predictive biochemical HRP (Cayman Chemical, Ann Arbor, MI); phalloidin–AF488 (Thermo Fisher analyses determined that this protein spanned the cell membrane Scientific, Waltham, MA). four times and was therefore a member of the tetraspanin family (16). The function of CD53 has remained unclear, although two Peritonitis studies provide evidence that CD53 has important functions in Peritonitis was induced using a modification of a previously published the human immune system. Examination of neutrophils from technique (10). Mice were inoculated i.p. with 500 ml of 3% thioglycollate members of a family affected by recurrent microbial infections (Sigma-Aldrich). Four hours later, mice were euthanized, and cells were harvested by peritoneal lavage. Counts of total immune cells and neu- revealed that the only detectable phenotypic abnormality was trophils were performed, identifying neutrophils on the basis of Ly-6G absence of CD53 (17). A more recent genome-wide association expression as determined by flow cytometry (LSRFortessa cell analyzer; study of patients with increased susceptibility to mycobacterial BD Biosciences). infection identified an association with mutations in the CD53 Intravital microscopy locus (18). Together, these studies infer that mutations in CD53 Downloaded from result in immune deficiency. A range of mechanisms were pos- Leukocyte–endothelial cell interactions were assessed via intravital mi- tulated to explain this effect, including modulation of cytokine croscopy of the cremaster muscle, as previously described (10, 31). In expression, MHC class I and MHC class II expression and cel- brief, mice were anesthetized via i.p. injection of a xylazine hydrochloride (10 mg/kg) and ketamine hydrochloride (150 mg/kg) solution prior to lular adhesion, and defective immune cell signaling (19–23). surgery. A catheter was inserted into the right jugular vein, allowing However, these hypotheses are yet to be supported by functional for immediate delivery of additional anesthetic. The left testis was

in vivo data. exposed over a microscope pedestal. A coverslip was placed over the http://www.jimmunol.org/ The phenotype associated with CD53 deficiency in humans has 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 similarities to that of leukocyte adhesion deficiency patients, supplied to exposed tissue. At least two postcapillary venules (25–40 mm) raising the possibility of a role for CD53 in immune cell adhesion were examined in each experiment. and trafficking. Further evidence comes from studies of cross- Vessels were visualized using intravital microscopy, using a ZEISS linking CD53 on immune cells, which increases cell–cell ad- Axioplan 2 microscope with a 253 long working distance lens and 103 hesion, potentially via effects on the b integrin LFA-1 (20, 24, eyepiece. Recordings of vessels were made using a Sony SSC-DC50AP 2 video recorder, and images were projected onto a Sony Trinitron monitor. 25). However, in vivo substantiation of these findings is lacking. Recordings were stored on a Panasonic DMR-EH57 DVD Recorder, Two recent studies have investigated the effect of genetic de- allowing for playback of recordings for further analysis. Leukocyte re- letion of CD53 on the adaptive immune system, demonstrating cruitment parameters assessed for each vessel consisted of rolling flux, by guest on September 25, 2021 roles for CD53 in early B cell development (26) and lymphocyte 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 recirculation via stabilization of L-selectin on the lymphocyte slower than the surrounding blood. Rolling velocity (micrometers per cell surface (27). However, the role of CD53 in myeloid cell second) was measured as the time taken for the cell to travel 100 mm. function remains unclear. In this study we investigated a role for Rolling flux (cells per minute) was defined as the number of cells rolling CD53 as a regulator of neutrophil and monocyte trafficking. The past a certain point each minute. An adherent cell was defined as a cell m results indicate that CD53 contributes to myeloid cell recruitment that remained stationary for 30 or more seconds within a 100- m stretch of vessel (expressed as cells/100 mm), whereas transmigration was de- by selectively promoting their transmigration. fined as the number of cells located outside the vessel within the field of view (expressed as cells per field of view). Materials and Methods Assessment of leukocyte recruitment in response to cytokine/ Mice chemokine stimulation The generation of Cd532/2 mice has been described previously (28). 2 2 Leukocyte–endothelial cell interactions were assessed in three different In brief, Cd53 / mice were generated using a Cre/lox approach, inflammatory models. To examine the response to an inflammatory stim- introducing LoxP sites in the mouse Cd53 gene downstream of exon 1 ulus incorporating endothelial activation, local TNF was used. TNF (50 ng and upstream of exon 6. Thereby, exons 2 to 5 were deleted upon in 170 ml of saline), or a saline control, was administered via s.c. intra- crossing a Cd53fl/fl mouse with a ubiquitous Cre deleter mouse (29). scrotal injection 4 h prior to imaging (31). Leukocyte–endothelial cell Deletion of the Cd53 gene was verified by PCR analysis and via flow interactions were assessed 4 h later as stated above. To investigate the cytometric analysis of CD53 protein abundance on blood leukocytes. 2 2 response to a neutrophil-selective chemotactic stimulus, the acute response Cd53 / mice develop normally and are phenotypically normal, apart to CXCL1 was assessed (10). In these experiments, a baseline recording was from reductions in L-selectin expression on some immune cell populations 2 2 taken before replacement of the superfusion buffer with solution containing Cd53 / (27, 28). mice were fully backcrossed to the C57BL/6J background mouse CXCL1 (7.5 or 3.75 nM). Recordings were taken at 30 and 60 min prior to their use in this study. C57BL/6J wild-type (WT) mice were obtained 2 2 post-CXCL1 application, with recruitment parameters analyzed as per above. from Monash Animal Research Platform, Monash University. Cd53 / 2 2 To examine the response to a monocyte-selective stimulus, CCL2 was used mice were bred in-house. Itgam / mice were kindly provided by Prof. (32). Mouse CCL2 (JE, 333 ng in 175 ml of saline, 138 nM) was administered K. Peter (Baker Heart & Diabetes Institute, Melbourne, Australia). Mice via intrascrotal injection 4 h prior to imaging. Preparations were assessed as were maintained in specific pathogen–free conditions and used for experi- for the TNF experiments above. ments at 8–16 wk of age. Experiments were performed in male mice. All In some experiments using CXCL1, Mac-1 activation on adherent animal experiments were approved by the Monash Medical Centre Animal neutrophils was assessed as previously described (33). In brief, on the day Ethics Committee or the Alfred Medical Research and Education Precinct 9 of the experiment, 2 3 10 polystyrene microspheres (1 mm, yellow-green Animal Ethics Committee. fluorescent, FluoSpheres; Molecular Probes) were incubated in BSA 9 Abs (1 mg/ml in PBS, 300 ml) for 2 h, prior to sonication. A total of 1 3 10 beads (150 ml) of the mixture were administered i.v. into mice undergoing The following Abs and reagents were used (from BD Biosciences, North Ryde, intravital microscopy and CXCL1 exposure, along with anti-Gr1-PE (2 mg) to NSW, Australia, unless otherwise stated): anti-Ly-6G-PE (clone 1A8); aid identification of neutrophils. After 10 min, cremaster preparations The Journal of Immunology 3 were visualized using spinning disk confocal microscopy, recording rounds of freezing–thawing and pelleted by centrifugation at 20,000 3 g. brightfield images along with sequential green and red fluorescent signals. Cell membrane fractions from 1 3 105 neutrophils were assayed in du- Data were assessed as the number of attached microspheres per adherent plicate over 60 min at RT. Controls run with the ADAM17 inhibitor TAPI- leukocyte within a 100-mm length of venule. 0 showed no activity, confirming the specificity of the assay.

Intravascular crawling and cell fate analysis Mobilization of a3 and a6 integrins

To examine the behavior of adherent neutrophils in the vasculature, time- Mobilization of integrin a3 and a6 in neutrophils was performed as pre- lapse intravital microscopy was used to examine cremasteric postcapillary viously described on 2% BSA (control)- or PECAM-1 (2 mg/ml)–, ICAM- venules during CXCL1 superfusion (34). Images were captured using a 1(1mg/ml)–, and CXCL1 (4 ng/ml)-coated slides (36). Neutrophils were Ultraview VoX spinning disk microscope system (PerkinElmer) running isolated from WT and Cd532/2 bone marrow via MACS-based negative Volocity (version 6.3.0). Brightfield images were captured every 10 s for selection (Miltenyi Biotec, Macquarie Park, NSW, Australia) and allowed 60 min to visualize intravascular migration of leukocytes following arrest to adhere to coated slides for 30 min at 37˚C. Cells were then fixed with on the endothelium. Analysis was performed using the Manual Tracking 2% PFA, blocked in 2% BSA, and when examining intracellular integrin plug-in in ImageJ (version 1.44), assessing the distance the cell traveled distribution, permeabilized with 0.1% Triton X-100/2% BSA. Cells were (in micrometers) following its attachment to the endothelium until destained with anti-CD49c (anti-a3, 42/CD49c, 5 mg/ml), anti-CD49f-AF488 tachment or transmigration. From the distance traveled, cell velocity (anti-a6,8mg/ml), anti-Gr-1-DL650, and Hoechst 33342. Anti-CD49c was (micrometers per minute) was calculated based on the amount of time detected using goat anti-mouse AF568. Cells were imaged by confocal from attachment until detachment or transmigration. One hundred cells microscopy as stated above, and images were analyzed using ImageJ. For were assessed in each experiment from n =6micepergroup. assessment of total surface expression for each of the integrins, mean fluorescence intensity for each channel was measured. In brief, maximum Confocal microscopy analysis of CXCL1-stimulated intensity projections of the z-stacks were generated for each channel/ cremaster muscle +

integrin. Gr-1 cells were outlined as regions of interest, and mean in- Downloaded from Immunohistochemistry of whole-mounted cremaster muscles was used to tensities of a3 and a6 integrin staining were then assessed within the cell assess neutrophil localization in response to CXCL1 stimulation using a margins. Approximately 40 cells per mouse from eight mice per condition previously described technique (35). In brief, following 60-min CXCL1 were assessed. Analysis of integrin cluster number and size was performed superfusion, cremaster muscles were fixed in 4% paraformaldehyde (PFA) in ImageJ using a custom-written algorithm. The maximal projection in- for 30 min at room temperature (RT). Subsequently, the muscles were tensity for each stain was obtained, after which the algorithm counted blocked and permeabilized in 10% FCS, 10% goat serum, 5% mouse se- individual integrin a3 and a6 clusters and measured their corresponding rum, and 1% Triton X-100 in PBS for 2 h at RT. Tissues were then stained areas. Ring formation analysis was conducted using Imaris. Cells were considered positive for ring formation when a3 and/or a6 staining was http://www.jimmunol.org/ (overnight, 4˚C) with anti-SMA-Cy3 (to identify pericytes), anti-MRP14- ∼ AF488 (to identify neutrophils), and anti-CD31-AF647 (to identify endo- visually localized to the periphery of the cell for at least 25% of the cell. thelial cells). Stained tissues were mounted on microscope slides and Approximately 50 neutrophils were examined per treatment group. imaged using a Nikon C1 inverted confocal microscope equipped with a Western blotting analysis 603 water immersion lens. The location of neutrophils within the vasculature was assessed according to a previously published technique (36). Western blotting for a3 integrin was performed using protein samples from Leukocytes were defined as follows: 1) intravascular (within the anti-CD31 bone marrow neutrophils from WT and Cd532/2 mice isolated via MACS- stain), 2) within the vascular wall (with the anti-SMA staining), and 3) based negative selection as described above. Neutrophils were lysed in extravascular (beyond the anti-SMA staining). For purposes of analyses, RIPA buffer (150 mM NaCl, 1% IGEPAL, 0.5% sodium deoxycholate, leukocytes in categories 1 and 2 were combined and deemed as intravas- 0.1% SDS, 50 mM Tris–HCL [pH 8] in dH2O) and protease inhibitor and cular. Intravascular and extravascular neutrophil infiltration were deter- centrifuged at 13,000 rpm (10 min at RT). Lysates were mixed with by guest on September 25, 2021 mined by volumetric analysis using Imaris (Bitplane, Zurich, Switzerland) NuPage LDS Sample Buffer (Thermo Fisher Scientific) and heated at 70˚C image analysis software. Because in some instances it was not possible for 10 min. Protein samples were loaded into 3–8% Tris–Glycine gels with to resolve individual neutrophils in confocal images, the total volume of Tris–Acetate Running Buffer and the HiMark Prestained Protein Standard recruited neutrophils was quantified using volumetric analysis in Imaris (all from Thermo Fisher Scientific), after which samples were transferred (version 7.6.4). In brief, following background subtraction, individual to PVDF membranes (Merck). Membranes were blocked in 5% milk surfaces were generated for vascular (SMA) and neutrophil (MRP14) powder and then cut to probe for a3 integrin and b-actin separately. channels. The SMA surface was then used as a layer mask to define Membranes were then incubated with mouse anti-mouse CD49c (clone intravascular and extravascular compartments, and the volume statistics for 42/CD49c; BD Biosciences) or anti-mouse b-actin (clone AC-15; Sigma- the neutrophil surfaces in each compartment were generated. Data were Aldrich), followed by incubation with goat anti-mouse IgG HRP (Cayman analyzed for two to three vessels per mouse, from n = 8 mice per group. Chemical). b-actin served as a loading control. The blots were developed Flow cytometric analysis of neutrophil adhesion using Amersham ECL Western Blotting Detection Reagent (GE Healthcare Life Sciences, Marlborough, MA). Blots were imaged using a GE ImageQuant molecule expression LAS 4000 and ImageQuant software (GE Healthcare Life Sciences). Expression of L-selectin, LFA-1, and Mac-1 on WT and Cd532/2 neu- Subsequent quantitative analysis was performed using ImageJ. Intensity trophils was assessed via flow cytometry. In brief, leukocytes were isolated plots for a3 and b-actin were generated, and the ratio of the area of the a3 from blood or bone marrow of WT and Cd532/2 mice and stained with to b-actin plots was calculated. This was performed three times for each anti-Ly-6G-V450 (to identify neutrophils) and anti-CD62L-PE, or anti-Ly- sample to obtain an average ratio. Each sample average was then nor- 6G-PE, anti-CD11b-FITC, and anti-LFA-1-AF647, for assessment of malized to the average of the WT ratios for each set of experiments. Mac-1 and LFA-1. For assessment of surface and intracellular L-selectin, F-actin redistribution and colocalization of CD53 and nonpermeabilized neutrophils were first stained for surface L-selectin using PE-conjugated anti–L-selectin at a concentration (8 mg/ml) determined ADAM17 in mouse neutrophils in pilot experiments to minimize any further staining achieved by subsequent For F-actin assessment, isolated WT and Cd532/2 neutrophils underwent exposure to anti-L-selectin–FITC (data not shown). Cells were subsequently adhesion to the substrates described above. Cells were then stained with washed, fixed, permeabilized (BD Cytofix/Cytoperm, BD Biosciences), and phalloidin-AF488, anti-Gr-1-AF568, and Hoechst 33342 and imaged by stained for intracellular L-selectin using FITC-conjugated anti–L-selectin confocal microscopy. Circularity based on F-actin staining was measured (5 mg/ml). Surface and intracellular L-selectin levels were determined via + using the ImageJ Circularity plug-in. Approximately 60 neutrophils were flow cytometric analysis, identifying neutrophils as Ly-6G cells. Samples examined per treatment group. Colocalization of CD53 and ADAM17 in were analyzed using either FACSCanto II or LSRFortessa cell analyzer (BD neutrophils was performed on 2% BSA substrate. Neutrophils were iso- Biosciences) flow cytometers. Data were analyzed using FlowJo (version lated from WT mouse bone marrow via MACS-based negative selection VX; FlowJo, Ashland, OR). and allowed to adhere to coated slides (37˚C for 20 min). Cells were then ADAM17 activity 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 ADAM17 activity was determined on cell membrane fractions from WTand 0.5% saponin/PBS. Cells were stained with OX-79 (anti-CD53), A9(B8)– Cd532/2 neutrophils using the SensoLyte 520 TACE (a-Secretase) Ac- AF568 (anti-ADAM17), anti-Gr-1-DL650, and Hoechst 33342. OX-79 was tivity Assay Kit (AnaSpec, Fremont, CA), according to the manufacturer’s detected using goat anti-rat AF488. Cells were imaged by confocal mi- instructions. In brief, cell membrane fractions were prepared by five 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 between the strains in these parameters (Fig. 1A, 1B). Similarly, coefficients were obtained for each cell (37). Approximately 40 neutrophils leukocyte and neutrophil abundance in the bone marrow did not were examined per mouse for each time point. differ between the strains (Supplemental Fig. 1). These findings K/BxN serum-induced arthritis are supported by similar observations reported in a recent independent analysis of Cd532/2 mice (26). Mice underwent the K/BxN serum-transfer model of arthritis using a modification of a previously published technique (38, 39). In brief, mice We subsequently examined the role of CD53 in leukocyte traf- were administered K/BxN serum (75 ml, i.p.) on days 0 and 2. Baseline ficking in thioglycollate peritonitis. Four hours after thioglycollate measurements of body weight, ankle thickness, and general activity were injection, WT mice showed a robust recruitment of leukocytes, of taken prior to the first dose and assessed daily thereafter. Mice were scored on which 60–70% were neutrophils (Fig. 1C). In Cd532/2 mice, 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 the numbers of both total leukocytes and neutrophils were sig- of all four paws were added to obtain a composite clinical score (maximum nificantly lower than those in WT mice (Fig. 1C), demonstrating clinical score = 16). Mice were euthanized, and joints were taken for histo- a role for CD53 in leukocyte recruitment. logical assessment at either day 2 or 7. Joint pathology was assessed in H&E- To investigate the role of CD53 within the microvasculature, we stained sections of ankle joints from mice undergoing the K/BxN model using next used intravital microscopy of the cremaster muscle to compare a four-point scoring method, assessing synovitis and joint space exudate in 2/2 which 0 = no area affected, 1 = one area affected, 2 = multiple small areas leukocyte–endothelial cell interactions in WT and Cd53 mice. affected, and 3 = large areas affected. Cartilage damage was assessed using In the absence of exogenous stimuli, leukocyte rolling flux and Safranin O/Fast Green staining and scored on a four-point scale in which 0 = adhesion did not differ between WT and Cd532/2 mice, although fully stained cartilage, 1 = slight loss of cartilage staining, 2 = severe loss of a minor reduction in leukocyte rolling velocity was observed in cartilage staining, and 3 = completely destained cartilage (38, 40). For all 2 2

/ Downloaded from assessments, the operator was blinded to the mouse genotype. Cd53 mice after 60 min of observation (Supplemental Fig. 2). These findings indicate that the absence of CD53 does not have a Statistical analysis marked effect on basal leukocyte–endothelial cell interactions. We GraphPad Prism 7 (GraphPad Software, San Diego, CA) was used to conduct next examined the effect of CD53-deficiency on leukocyte trafficking 2 2 data analysis. Significance was determined using a Mann–Whitney U test, induced by local administration of TNF. In both WT and Cd53 / a Holm–Sidak multiple test, an unpaired two-tailed t test, repeated mea- mice, TNF markedly reduced leukocyte rolling flux and rolling sures one-way ANOVA with multiple comparisons, or two-way ANOVA velocity and significantly increased adhesion and transmigration http://www.jimmunol.org/ with multiple comparisons. Statistical significance was set at p , 0.05. (Fig. 1D–G). The changes in rolling and adhesion were similar in WT and Cd532/2 mice (Fig. 1D–F). However, TNF-induced leukocyte Results transmigration was significantly lower in Cd532/2 mice (Fig. 1G), Absence of CD53 impairs leukocyte recruitment via an effect indicating a selective role for CD53 in promotion of transmigration. on transmigration To examine this effect in more detail, we next assessed responses We first compared the number of circulating leukocytes and neu- induced by chemokines CXCL1 and CCL2, which promote trophils in WT and Cd532/2 mice, demonstrating no differences transmigration of neutrophils and monocytes, respectively (32, 41). by guest on September 25, 2021

FIGURE 1. CD53 modulates leukocyte recruitment via effects 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 recruitment 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), adhesion (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 differences 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 Downloaded from 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 http://www.jimmunol.org/ 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 leukocytes 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 by guest on September 25, 2021 to transmigration, instead detaching from the endothelial sur- FIGURE 2. CD53 selectively promotes leukocyte transmigration in re- 2/2 sponse to chemokine stimulation. Leukocyte–endothelial cell interactions face, was significantly elevated in Cd53 mice (Supplemental 2 2 in cremasteric postcapillary venules of WT and Cd53 / mice were ana- Fig. 3E). Together, these findings indicate that in response to lyzed during a 60-min acute exposure to CXCL1 (7.5 nM) commencing CXCL1, CD53 acts to stabilize the attachment of leukocytes to immediately after the 0 min time point (A–D) or 4 h after local CCL2 the endothelial surface. administration (138 nM, intrascrotal) (E–H). Data are shown for leukocyte We also examined the impact of CD53 on the capacity of rolling flux (A and E), rolling velocity (B and F), adhesion (C and G), and neutrophils to navigate across the specific layers of the vascular transmigration (D and H). Data are shown as mean 6 SEM, for n = 6–9 wall in response to CXCL1, using whole mount immunohisto- mice per group. *p , 0.05, **p , 0.01, ***p , 0.001 by Holm–Sidak chemistry and confocal microscopy to identify the location of multiple t test or unpaired two-tailed t test. 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 facilitates CXCL1-induced intravascular crawling and transmi- (Fig. 3A). In contrast, in Cd532/2 mice, neutrophils were pre- gration (34), we also examined the functional state of Mac-1 on dominantly present within the vascular lumen or retained between adherent neutrophils in CXCL1-exposed venules, assessing their endothelial cells, consistent with a reduced capacity of these cells capacity to capture albumin-coated microbeads from the circula- to penetrate the pericyte layer (Fig. 3A). These observations were tion (Fig. 4E) (33). This assay serves as a readout of the capacity supported by quantitation of neutrophil volume, either inside of Mac-1 to bind one of its recognized ligands, albumin, stemming blood vessels or retained in the endothelial layer (defined as in- from conformational change of the integrin to its high-affinity travascular), which revealed a significant elevation of intravascular form as a result of inside-out signaling (48, 49). Experiments in neutrophils in Cd532/2 mice (Fig. 3B). Itgam2/2 mice demonstrated that microbead capture was Mac-1– dependent (Fig. 4F). Microbead capture was similar in WT and Marked decrease in surface L-selectin expression of 2/2 2/2 Cd53 neutrophils (Fig. 4F), providing evidence that the func- Cd53 neutrophils tional state of Mac-1 on adherent neutrophils did not differ be- Given the alterations in leukocyte–endothelial cell interactions, we tween these strains. next assessed whether neutrophil adhesion molecules that partic- Another immune cell–expressed molecule with a less well- ipate in transmigration were affected by the absence of CD53. As recognized role in transmigration is L-selectin (50, 51). There- tetraspanins are recognized for their ability to regulate integrin fore, we compared L-selectin expression on WT and Cd532/2 function (5, 10, 44–47), we first focused on the b2 integrins. LFA- neutrophils, finding that L-selectin was almost completely absent 1 and Mac-1 were expressed at equivalent levels on circulating from the surface of circulating neutrophils from Cd532/2 mice neutrophils from WT and Cd532/2 mice (Fig. 4A–D). As Mac-1 (Fig. 5A, 5B). Bone marrow neutrophils from Cd532/2 mice 6 CD53 PROMOTES TRANSMIGRATION

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 retention 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 microscopy. Both CD53 and ADAM17 were detectable on the surface of neutrophils (Fig. 5E), with ∼40% of the ADAM17 being colocalized with CD53 (Fig. 5F). Exposure to PMA, a stimulus that

induces L-selectin shedding, resulted in a significant reduction in Downloaded from 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 2/2 membrane fractions prepared from WT and Cd53 neutrophils. http://www.jimmunol.org/ 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. 2/2 Cd53 neutrophils display premature a3 integrin redistribution, increased a3 integrin expression, and impeded

F-actin reorganization by guest on September 25, 2021 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 following 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 2/2 the a3 integrin in WT and Cd53 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 increase in integrin ring formation (Fig. 6E, 6F). In contrast, Cd532/2 FIGURE 3. CD53 promotes neutrophil transendothelial migration in neutrophils showed an elevated level of a ring formation in resting response to CXCL1. (A) Representative confocal microscopy images 3 (original magnification 31000) of whole mounts of CXCL1-stimulated cells, which did not increase further upon stimulation (Fig. 6E, 6F). 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) analysis, because of aggregation of neutrophils leading to difficulties re- (pericyte layer, red) and MRP14 (neutrophils, green), with the merged solving individual cells, these parameters were assessed by quantitation of images shown below (A). (B) Quantitation of location of neutrophils as the volume of MRP14 staining in the various vascular compartments. Data either intravascular (defined as restricted to the endothelial and pericyte are shown as mean 6 SEM for intravascular and extravascular neutrophils layers) or extravascular (located outside the pericyte layer). In this from n = 8 mice per group. **p , 0.01 by Mann–Whitney U test. The Journal of Immunology 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

to undergo transendothelial migration. As the tetraspanin family Downloaded from has been shown to modulate the function of numerous molecular pathways, it is likely that the mechanism underlying this CD53- FIGURE 4. Absence of CD53 does not affect neutrophil b2 integrin dependent effect is multifactorial. However, given that integrins A D expression or Mac-1 function. ( – ) Flow cytometric analysis of surface are one of the most common targets of tetraspanin-mediated A B C D 2/2 LFA-1 ( and ) and Mac-1 ( and ) expression on WT and Cd53 regulation, we initially focused on leukocyte integrins involved neutrophils. Data are shown as histograms from representative experi-

in neutrophil recruitment and transmigration. For transmigration, http://www.jimmunol.org/ 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 timely redistribution of integrins, including LFA-1, a3, and a6, relative ratio to the average WT mean ﬂuorescence intensity (B and D). during the process is crucial (36, 57). Particularly for the a3 Data are shown as mean 6 SEM of (n = 13) mice per group. (E and F) integrin, translocation from its primary location in intracellular Analysis of Mac-1–dependent binding of albumin-coated beads to adher- secretory vesicles to the cell surface supports neutrophil extrava- ent neutrophils in CXCL1-exposed postcapillary venules of WT, Cd532/2, sation via promotion of cell elongation (55, 58). In the current 2/2 E and Itgam mice. ( ) Spinning disk confocal microscopy image of a study, in CD53-deﬁcient neutrophils, the a3 integrin had already postcapillary venule following CXCL1 exposure, with neutrophils labeled undergone translocation to the periphery of the cell prior to ini- by i.v. anti–Gr-1 (red). Albumin-coated microbeads (green, arrows) are tiation of recruitment, demonstrating that CD53 constitutively acts

3 F by guest on September 25, 2021 visible attached to two neutrophils (original magnification 400). ( ) as a “brake” on a integrin redistribution (or recycling). Given the Quantitation of bead interactions with neutrophils (number per cell) in WT, 3 2/2 2/2 6 importance of the appropriate timing of a3 integrin translocation, Cd53 ,andItgam mice. Data are shown as mean SEM of (n = 6–9) 2/2 mice per group. *p , 0.05, ***p , 0.001 by Mann–Whitney U test or one- its presence adjacent to the cell membrane of Cd53 neutrophils way ANOVA with multiple comparisons. 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 2/2 Given this alteration in a3 integrin distribution in Cd53 neutrophils, or the functional state of Mac-1 in CD53-deficient neutrophils, we also compared a3 integrin expression via Western blot, observing a demonstrating a level of specificity in the actions of CD53 and 2/2 significant elevation of the integrin in Cd53 neutrophils (Fig. 6G, distinguishing it from CD37, a tetraspanin which modulates b2 6H). Taken together, these data indicate that in resting neutrophils, integrin function (10). CD53 acts to inhibit expression of the a3 integrin and its trans- We also observed that absence of CD53 led to a significant 2/2 location to the cell periphery. increase in total cellular expression of a3 integrin in Cd53 Examination of the a6 integrin revealed similar findings in neutrophils. The mechanism whereby loss of CD53 impacts on regard to surface expression and integrin cluster formation in total expression of this integrin is not immediately clear. In some that these did not differ between WT and Cd532/2 neutrophils previous studies, tetraspanin deletion has resulted in the opposite (Fig. 7A–D). The a6 integrin showed a trend toward premature effect, reducing expression of partner proteins. For example, ab- 2/2 ring formation in Cd53 neutrophils as seen with the a3 sence of tetraspanin CD81 in B cells led to decreased expression integrin, although this did not reach significance (Fig. 7E, 7F). of CD19 because of a role for the tetraspanin in supporting CD19 Another key requisite for transmigration is the cytoskeletal through processing in the endoplasmic reticulum (59). In the case remodeling required for leukocytes to migrate through the vascular of CD53, our findings clearly demonstrate that normal a3 integrin endothelium (56). Therefore, we next investigated the contribution trafficking is disrupted in the absence of the tetraspanin. Based on of CD53 to activation-induced cytoskeletal remodeling, using that observation, it is tempting to speculate that this alteration confocal microscopy to examine F-actin distribution (10). Upon impacted the conventional intracellular processing and degrada- adhesion to ICAM-1/PECAM-1/CXCL1, WT neutrophils showed tion of this integrin, and that this led to its aberrant accumulation increased polarization (Fig. 7G) as demonstrated by a significant within neutrophils. Further experiments will be required to cast decrease in the circularity ratio (Fig. 7H). In CD53-deficient additional light on this issue. neutrophils, activation failed to induce a significant change in Leukocyte shape change via cytoskeletal remodeling is central to circularity (Fig. 7G, 7H). Taken together with the a3 integrin data, transmigration. Tetraspanins, including CD81, CD82, and CD37, these findings indicate that CD53 acts to maintain neutrophils in are known to modulate cytoskeletal dynamics via association with an unactivated state prior to exposure to stimuli associated with molecules such as Rho GTPases and ERM proteins (9, 10, 60–62). transmigration. In this study we observed reduced cytoskeletal function in neutrophils 8 CD53 PROMOTES TRANSMIGRATION Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

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 Downloaded from

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 2/2 microscopy images of WT and Cd53 neutrophils on either 2% BSA (control substrate) or PECAM-1/ICAM-1/CXCL1 substrate, stained for a3 integrin. B C D ( ) Quantitation of total surface expression as measured by mean fluorescence intensity of a3 integrin. ( and ) Surface clustering of a3 integrin on WT http://www.jimmunol.org/ 2/2 2/2 and Cd53 neutrophils, showing quantitation of number (C) and area (D)ofa3 integrin clusters. (E and F) a3 integrin ring formation in WT and Cd53 2/2 neutrophils. Representative confocal microscopy images (E) and quantitation (F)ofa3 integrin ring formation in WT and Cd53 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– 2/2 Sidak multiple t test. (G and H) Total a3 integrin expression in WT and Cd53 neutrophils as determined using Western blot. (G) Example blot showing 2/2 a3 integrin expression in three WT samples and three Cd53 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. by guest on September 25, 2021 lacking CD53, as evidenced by a reduction in the capacity to findings are consistent with our previous observations of leuko- undergo shape change in response to activation, indicating that cyte rolling in L-selectin–deficient mice, in which the absence of this member of the tetraspanin family also modulates leukocyte L-selectin reduced but failed to eliminate rolling in cremasteric function via this pathway. postcapillary venules. In this context, P-selectin–mediated inter- In addition to its well-recognized contribution to leukocyte actions accounted for the bulk of the residual rolling (67). Fur- rolling, L-selectin also promotes leukocyte transmigration (50). thermore, in both L-selectin2/2 mice, and as seen in this study in L-selectin ligation has been shown to promote intracellular sig- Cd532/2 mice, leukocyte adhesion was not reduced, indicating naling via pathways, including PKC, MAPK and Syk, leading to that this partial decrease in leukocyte rolling was insufficient to alterations in cytoskeletal function and b2 integrins and increased significantly impact the number of leukocytes able to undergo arrest transendothelial migration and chemotaxis (63–65). More re- in the inflamed microvasculature. Taken together, these findings cently, L-selectin on monocytes was shown to promote polariza- indicate that any potential contribution of the loss of L-selectin on tion and cell membrane protrusion required for transmigration the impaired transmigration of Cd532/2 leukocytes is likely to have across endothelial cell monolayers (51, 66). Given the reduction in occurred downstream of leukocyte arrest. expression of L-selectin on neutrophils in Cd532/2 mice, it is The mechanism underlying the early loss of L-selectin from reasonable to surmise that signals derived from L-selectin ligation neutrophils remains to be determined. We addressed the possibility are impaired in these cells. The reduction in these signals is that CD53 mediates this effect by controlling the function of therefore likely to have contributed to some degree to the observed ADAM17, the major metalloprotease responsible for cleavage of deficit in transmigration. the L-selectin ectodomain from the surface of activated leukocytes Nevertheless, effects on the canonical role of L-selectin in (68, 69). ADAM17-mediated cleavage of TNF is subject to reg- mediating leukocyte rolling could also have had an impact on ulation by another member of tetraspanin family, CD9, in both leukocyte recruitment in Cd532/2 mice. Indeed, we observed monocytic cell lines and endothelial cells (53). In this setting, cell significant reductions in leukocyte rolling in Cd532/2 mice both activation reduces the ADAM17/CD9 association at the same time under basal conditions, after prolonged observation, and during as it increases ADAM17 activity, suggesting a mechanism of stimulation with CXCL1. However, although rolling was reduced regulation of ADAM17 function related to the colocalization of during these responses, substantial residual rolling remained. These these molecules in the cell membrane (53). In this study, we

n = 4 mice. *p , 0.05 by repeated measures one-way ANOVAwith 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 CD53 PROMOTES TRANSMIGRATION Downloaded from http://www.jimmunol.org/

FIGURE 8. CD53 ablation attenuates cartilage injury in K/BxN arthritis. by guest on September 25, 2021 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 between 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 FIGURE 7. CD53 regulates cytoskeletal function but not surface ex- cell membrane. In response to neutrophil activation, this degree pression and mobilization of a6 integrin in neutrophils. (A–D) Surface of colocalization diminished, coincident with an increase in the 2/2 expression of a6 integrin on WT and Cd53 neutrophils as assessed by L-selectin–shedding activity of ADAM17. However, despite this immunofluorescence and confocal microscopy of nonpermeabilized cells. evidence of molecular association, comparison of ADAM17 2 2 (A) Representative confocal microscopy images of WT and Cd53 / enzyme activity in WT and Cd532/2 neutrophils by measuring neutrophils on either 2% BSA (control substrate) or PECAM-1/ICAM-1/ ADAM17-dependent cleavage of a non–cell-associated ligand B CXCL1 substrate, stained for a6 integrin. ( ) Quantitation of total surface revealed no difference between the strains. It should be noted expression as measured by mean fluorescence intensity of a6 integrin. that, although this assay does assess total ADAM17 activity, it (C and D) Surface clustering of a integrin on WT and Cd532/2 neutro- 6 does not reveal the capacity of ADAM17 to access endogenous phils, showing quantitation of number (C) and area (D)ofa6 integrin 2/2 clusters. (E and F) a6 integrin ring formation in WT and Cd53 neutrophils. Representative confocal microscopy images (E) and quantitation 2/2 (F)ofa6 integrin ring formation in WT and Cd53 neutrophils on either 2% BSA control substrate or PECAM-1/ICAM-1/CXCL1 substrate, control substrate or PECAM-1/ICAM-1/CXCL1 substrate stained for F- stained under Triton X-100–permeabilized conditions. Data in (A)–(F) actin [original magnification in (A), (E), and (G) 31150]. (H) Quantitation were derived from n = 7–9 mice per group, and ∼40 neutrophils per mouse of circularity of WT and Cd532/2 neutrophils under resting and stimulated and are shown as mean 6 SEM. (G and H)PolarizationofWTandCd532/2 conditions. Data are shown as mean 6 SEM of ∼60 neutrophils per mouse neutrophils as determined by assessment of F-actin distribution. (G) Repre- from n = 7 mice per group. ***p , 0.001, as determined using two-way 2 2 sentative confocal images of WT and Cd53 / neutrophils on 2% BSA ANOVA with multiple comparisons. The Journal of Immunology 11 substrates within the cell membrane. This is important, as recent 10. Wee, J. L., K. E. Schulze, E. L. Jones, L. Yeung, Q. Cheng, C. F. Pereira, A. Costin, G. Ramm, A. B. van Spriel, M. J. Hickey, and M. D. Wright. 2015. work on the regulation of ADAM10 by the Tspan C8 subfamily Tetraspanin CD37 regulates b2 integrin-mediated adhesion and migration in of tetraspanins suggests that tetraspanins may regulate metal- neutrophils. J. Immunol. 195: 5770–5779. loprotease function by interacting with the ADAM and regu- 11. Yeung, L., M. J. Hickey, and M. D. Wright. 2018. The many and varied roles of tetraspanins in immune cell recruitment and migration. Front. Immunol. 9: 1644. lating its trafficking and substrate accessibility and specificity 12. Maecker, H. T., S. C. Todd, and S. Levy. 1997. The tetraspanin superfamily: (70, 71). In regard to CD53, such a mechanism would be con- molecular facilitators. FASEB J. 11: 428–442. sistent with our own observations of an activation-induced de- 13. Kovalenko, O. V., D. G. Metcalf, W. F. DeGrado, and M. E. Hemler. 2005. Structural organization and interactions of transmembrane domains in tetraspa- crease in the interaction of ADAM17 and CD53. Moreover, this nin proteins. BMC Struct. Biol. 5: 11. raises the possibility that CD53 may negatively regulate access 14. Paterson, D. J., J. R. Green, W. A. Jefferies, M. Puklavec, and A. F. Williams. 1987. The MRC OX-44 antigen marks a functionally relevant subset among rat of ADAM17 to its substrate L-selectin. It is also notable that thymocytes. J. Exp. Med. 165: 1–13. L-selectin can also be cleaved by other proteases, and these may 15. Angelisova´,P., C. Vlcek, I. Stefanova´, M. Lipoldova´, and V. Horejsı´. 1990. The be alternative targets of the actions of CD53 (72, 73). As such, human leucocyte surface antigen CD53 is a protein structurally similar to the CD37 and MRC OX-44 antigens. Immunogenetics 32: 281–285. more work is required to dissect the molecular mechanism by 16. Tomlinson, M. G., A. F. Williams, and M. D. Wright. 1993. Epitope mapping of which CD53 regulates L-selectin shedding. anti-rat CD53 monoclonal antibodies. Implications for the membrane orientation Finally, we assessed whether this action of CD53 on myeloid of the Transmembrane 4 Superfamily. Eur. J. Immunol. 23: 136–140. 17. Mollinedo, F., G. Fontań, I. Barasoain, and P. A. Lazo. 1997. Recurrent in- cell transmigration resulted in an impact on a clinically relevant fectious diseases in human CD53 deficiency. Clin. Diagn. Lab. Immunol. 4: model of inflammation, the K/BxN serum-transfer model of 229–231. 18. Omae, Y., L. Toyo-Oka, H. Yanai, S. Nedsuwan, S. Wattanapokayakit, arthritis. The absence of CD53 led to a significant delay in N. Satproedprai, N. Smittipat, P. Palittapongarnpim, P. Sawanpanyalert, development of joint pathology. Moreover, at the termination of W. Inunchot, et al. 2017. Pathogen lineage-based genome-wide association study Downloaded from the model, Cd532/2 mice displayed reduced cartilage damage. identified CD53 as susceptible locus in tuberculosis. J. Hum. Genet. 62: 1015– 1022. The finding that cartilage damage was reduced at a time point 19. Olweus, J., F. Lund-Johansen, and V. Horejsi. 1993. CD53, a protein with four when there was no clear difference in neutrophil infiltration membrane-spanning domains, mediates signal transduction in human monocytes raises the intriguing possibility that CD53 may regulate neutrophil- and B cells. J. Immunol. 151: 707–716. 20. Cao, L., T. Yoshino, N. Kawasaki, I. Sakuma, K. Takahashi, and T. Akagi. derived proteases responsible for cartilage damage. These and 1997. Anti-CD53 monoclonal antibody induced LFA-1/ICAM-1-dependent other functions of CD53 will be the subject of future investiga- and -independent lymphocyte homotypic cell aggregation. Immunobiology http://www.jimmunol.org/ 197: 70–81. tions. However, together, these studies reveal a previously unap- 21. Escola, J. M., M. J. Kleijmeer, W. Stoorvogel, J. M. Griffith, O. Yoshie, and preciated role for the tetraspanin CD53 in promotion of myeloid H. J. Geuze. 1998. Selective enrichment of tetraspan proteins on the internal leukocyte recruitment, mediated primarily via a selective effect on vesicles of multivesicular endosomes and on exosomes secreted by human B- lymphocytes. J. Biol. Chem. 273: 20121–20127. transmigration. 22. Bos, S. D., N. Lakenberg, R. van der Breggen, J. J. Houwing-Duistermaat, M. Kloppenburg, A. J. de Craen, M. Beekman, I. Meulenbelt, and P. E. Slagboom. 2010. A genome-wide linkage scan reveals CD53 as an im- Acknowledgments portant regulator of innate TNF-alpha levels. Eur. J. Hum. Genet. 18: 953–959. We wish to acknowledge the kind assistance of Dr. Tim Gottschalk (Depart- 23. Lee, H., S. Bae, J. Jang, B. W. Choi, C. S. Park, J. S. Park, S. H. Lee, and ment of Immunology and Pathology, Monash University) for assistance with Y. Yoon. 2013. CD53, a suppressor of inflammatory cytokine production, is

associated with population asthma risk via the functional promoter polymor- by guest on September 25, 2021 flow cytometry, Prof. Karlheinz Peter (Baker Heart and Diabetes Institute) phism -1560 C.T. Biochim. Biophys. Acta 1830: 3011–3018. 2/2 for provision of Itgam mice, Monash Micro Imaging (Monash Univer- 24. Lazo, P. A., L. Cuevas, A. Gutierrez del Arroyo, and E. Orué. 1997. Ligation of sity) for the provision of instrumentation, training, and technical support, CD53/OX44, a tetraspan antigen, induces homotypic adhesion mediated by and Dr. Olga Barriero (Harvard Medical School) for careful reading of the specific cell-cell interactions. Cell. Immunol. 178: 132–140. 25. Todros-Dawda, I., L. Kveberg, J. T. Vaage, and M. Inngjerdingen. 2014. The manuscript. tetraspanin CD53 modulates responses from activating NK cell receptors, promoting LFA-1 activation and dampening NK cell effector functions. PLoS One 9: e97844. Disclosures 26. Greenberg, Z. J., D. A. Monlish, R. L. Bartnett, Y. 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Supplemental Figures

Yeung et al.

Leukocyte tetraspanin CD53 restrains 3 integrin mobilization and facilitates cytoskeletal remodelling and transmigration

Supplemental Figures 1-4

Supplemental Figure 1: Bone marrow leukocytes and neutrophils do not differ between

wild-type and Cd53-/- mice. Total cell counts (A) and neutrophil (B) counts from bone

marrow of a single leg (femur & tibia) were determined for wild-type and Cd53-/- mice,

based on hemocytometer counts in combination with flow cytometry to determine the

% of Ly6G+ cells (neutrophils). Data are shown for individual mice and as mean ± sem

derived from n=9 mice/group.

Supplemental Figure 2. CD53 does not modulate basal leukocyte interactions. Leukocyte recruitment parameters were analyzed under basal conditions in the absence of inflammation in postcapillary venules of the cremaster muscle of wild-type and Cd53-/- mice. The parameters quantified were rolling flux (A), rolling velocity (B), adhesion (C), and transmigration (D). Data are shown as mean ± SEM of n=6 mice per group. *, p<0.05 as determined Holm-Sidak multiple t test.

Supplemental Figure 3. CD53 regulates leukocyte crawling velocity and retention on the endothelial surface. Wild-type and Cd53-/- leukocyte intravascular crawling parameters were analyzed using time-lapse imaging over a 60 min period of CXCL1 superfusion at either standard (7.49 nM) (A, B) or low (3.75 nM) concentration (C, D). Data are shown for crawling velocity (A, C) and crawling distance (B, D). E: Assessment of rate of detachment of crawling neutrophils during CXCL1 (7.49 nM) superfusion. Data are shown as mean ± SEM, from n=6 mice/group. In A-D, data are shown for individual cells, while in E, data are shown per mouse. **, p<0.01; ****, p<0.0001, as determined using unpaired t-test.

Supplemental Figure 4: Both surface and intracellular L-selectin are reduced in neutrophils from Cd53-/- mice. A-D: Surface-expressed and intracellular L- selectin were assessed in neutrophils from wild-type and Cd53-/- mice using flow cytometry, as described in the Materials and Methods. A, B: Surface L- selectin, determined using a saturating concentration of PE-conjugated anti-L- selectin prior to permeabilization, shown for wild-type and Cd53-/- mice. C, D: Intracellular L-selectin in the same cells as in A & B, determined using subsequent staining with FITC-conjugated anti-L-selectin following permeabilization, shown for both wild-type and Cd53-/- mice. A & C show representative histograms for wild-type and Cd53-/- neutrophils, in addition to isotype control staining. B & D show group and individual mean fluorescence intensity (MFI) data from n=8-9 mice/group. Mean ± sem are also shown. ** denotes p < 0.01 & **** denotes p < 0.0001 for comparisons shown.