Vivo Recruitment at Sites of Inflammation in Phosphorylation

Endothelial CD47 Promotes Vascular Endothelial-Cadherin Tyrosine Phosphorylation and Participates in T Cell Recruitment at Sites of Inflammation In Vivo This information is current as of September 29, 2021. Veronica Azcutia, Michael Stefanidakis, Naotake Tsuboi, Tanya Mayadas, Kevin J. Croce, Daiju Fukuda, Masanori Aikawa, Gail Newton and Francis W. Luscinskas J Immunol published online 18 July 2012 http://www.jimmunol.org/content/early/2012/07/18/jimmun Downloaded from ol.1103606

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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 © 2012 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published July 18, 2012, doi:10.4049/jimmunol.1103606 The Journal of Immunology

Endothelial CD47 Promotes Vascular Endothelial-Cadherin Tyrosine Phosphorylation and Participates in T Cell Recruitment at Sites of Inﬂammation In Vivo

Veronica Azcutia,*,†,1 Michael Stefanidakis,*,†,1,2 Naotake Tsuboi,*,†,3 Tanya Mayadas,*,† Kevin J. Croce,†,‡ Daiju Fukuda,†,‡ Masanori Aikawa,†,‡ Gail Newton,* and Francis W. Luscinskas*,†

At sites of inflammation, endothelial adhesion molecules bind leukocytes and transmit signals required for transendothelial migration (TEM). We previously reported that adhesive interactions between endothelial cell CD47 and leukocyte signal regulatory protein g (SIRPg) regulate human T cell TEM. The role of endothelial CD47 in T cell TEM in vivo, however, has not been explored. In this study, CD472/2 mice showed reduced recruitment of blood T cells as well as neutrophils and monocytes in Downloaded from a dermal air pouch model of TNF-a–induced inflammation. Reconstitution of CD472/2 mice with wild-type bone marrow cells did not restore leukocyte recruitment to the air pouch, indicating a role for endothelial CD47. The defect in leukocyte TEM in the CD472/2 endothelium was corroborated by intravital microscopy of inflamed cremaster muscle microcirculation in bone marrow chimera mice. In an in vitro human system, CD47 on both HUVEC and T cells was required for TEM. Although previous studies

showed CD47-dependent signaling required Gai-coupled pathways, this was not the case for endothelial CD47 because pertussis http://www.jimmunol.org/ toxin, which inactivates Gai, had no inhibitory effect, whereas Gai was required by the T cell for TEM. We next investigated the endothelial CD47-dependent signaling events that accompany leukocyte TEM. Ab-induced cross-linking of CD47 revealed robust actin cytoskeleton reorganization and Src- and Pyk-2–kinase dependent tyrosine phosphorylation of the vascular endothelial- cadherin cytoplasmic tail. This signaling was pertussis toxin insensitive, suggesting that endothelial CD47 signaling is independent

of Gai. These ﬁndings suggest that engagement of endothelial CD47 by its ligands triggers outside-in signals in endothelium that facilitate leukocyte TEM. The Journal of Immunology, 2012, 189: 000–000.

eukocyte recruitment from the peripheral blood to sites because displacement of AJ proteins, like vascular endothelial-

of inflammation involves the well-established multistep cadherin (VE-cad), is induced transiently by leukocytes (2–4). by guest on September 29, 2021 L adhesion cascade (1). In most models of inflammation, Recent studies have provided insight into the underlying mecha- adherens junctions (AJs) play an important role in regulating leu- nisms of TEM at cell–cell junction (paracellular TEM). The en- kocyte transendothelial cell migration (TEM) at cell–cell junctions gagement of adhesion molecules, such as VCAM-1 and ICAM-1 by their T cell counterreceptors a4b1 and aLb2 integrins, and CD44 *Department of Pathology, Center for Excellence in Vascular Biology, Boston, MA interacting with hyaluronan during TEM, triggers outside-in sig- 02115; †Harvard Medical School, Boston, MA 02115; and ‡Cardiovascular Division, naling in endothelial cells that results in alterations in proteins lo- Department of Medicine, Brigham and Women’s Hospital, Boston, MA 02115 calized at cell junctions (reviewed in Refs. 5–7). For example, 1 V.A. and M.S. contributed equally to this work. cross-linking of ICAM-1 induces endothelial actin–cytoskeleton 2Current address: Department of Ophthalmology, Novartis Institutes for BioMedical remodeling and phosphorylation of cortactin and VE-cad by Src Research, Cambridge, MA. and Pyk2 protein tyrosine kinases in the endothelium (8, 9). Sub- 3Current address: Department of Nephrology, Internal Medicine, Nagoya University Graduate School of Medicine, Nagoya, Japan. sequent studies have implicated tyrosine phosphorylation of the VE-cad cytoplasmic tail as a key event leading to dissociation of the Received for publication December 13, 2011. Accepted for publication June 19, 2012. AJs through an incompletely understood mechanism (reviewed in This work was supported by Award P01HL-03628 (to F.W.L. and T.M.) from the Ref. 6). These latter events, VE-cad phosphorylation, VE-protein National Heart, Lung, and Blood Institute. V.A. and M.S. were supported by Amer- tyrosine phosphatase dissociation from VE-cad, and the formation ican Heart Association postdoctoral awards. D.F. was supported by fellowship grants of a VE-cad complex gap, are considered necessary events in leu- from the Japan Society for the Promotion of Science and the Uehara Memorial Foundation. kocyte paracellular TEM (8, 10–13). The content is solely the responsibility of the authors and does not necessarily CD47 (integrin-associated protein) is a 50-kDa transmembrane represent the official views of the National Heart, Lung, and Blood Institute or the glycoprotein expressed by most cell types (14, 15). In endothelial National Institutes of Health. cells, CD47 is present on the apical surface and is enriched at Address correspondence and reprint requests to Dr. Francis W. Luscinskas, Brigham endothelial cell–cell junctions (16, 17). CD47 has been shown to and Women’s Hospital, 77 Avenue Louis Pasteur, NRB-752P, Boston, MA 02115. E-mail address: fl[email protected] interact in cis with avb3, aIIbb3, and a2b1 integrins and in trans The online version of this article contains supplemental material. with members of the signal regulatory protein (SIRP) family and Abbreviations used in this article: AJ, adherens junction; CHS, contact hypersensi- with thrombospondins (reviewed in Ref. 14). SIRPs are a family tivity; DIC, differential interference contrast; IVM, intravital microscopy; PTX, per- of regulatory membrane proteins expressed mainly by leukocytes tussis toxin; siRNA, small interfering RNA; SIRP, signal regulatory protein; TEM, and neurons. SIRPa and SIRPg are ligands for CD47 (18). SIRPa transendothelial migration; VE-cad, vascular endothelial-cadherin; WT, wild-type. is abundantly expressed on myeloid cells and smooth muscle cells Copyright Ó 2012 by The American Association of Immunologists, Inc. 0022-1767/12/$16.00 and at low levels by cultured murine and human endothelium (16,

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1103606 2 ENDOTHELIAL CD47 MEDIATES T CELL RECRUITMENT IN VIVO

17). SIRPg expression is restricted to T cells, NK cells, and some or PBS-containing murine rTNF-a (500 ng) was injected in each air pouch, B cells (19, 20). Unlike SIRPa, SIRPg does not appear to signal to and 4 or 24 h later cell infiltrates were harvested by lavage with repeated the cytoplasm because its short cytoplasmic tail has no consensus PBS washes. The recovered volume was measured, and the number of recovered cells was determined by hemocytometer. The frequency of signaling motifs (19). + 2 2 CD3 T cells, neutrophils, and monocytes/macrophages was determined by Previous studies in CD47 / mice demonstrated that CD47 staining with primary labeled mAb specific for these cell types and ac- plays a role in neutrophil emigration in a bacteria-induced murine quired on a FACSCalibur flow cytometer (BD Biosciences). FACS data peritonitis model (21), a LPS-induced acute lung injury and bac- were analyzed using FlowJo software (Tree Star, Ashland, OR). terial pneumonia model (22), a TNBS-induced colitis model Bone marrow transplantation protocol (23), hapten-stimulated inflammation (24), and in vitro models of Donor bone marrow (BM) cells were harvested aseptically from femurs and neutrophil TEM of endothelium (25) and epithelium (26), and 2/2 tibias of 8- to 10-wk-old WT mice. Recipient CD47 and WT male mice monocyte TEM of endothelium (27). Recently, we reported that (8 wk old) received a lethal dose of whole body irradiation with a cesium human endothelial CD47 interacting with T cell-expressed SIRPg source (1200 rad in 2 doses, 4 h apart), as described previously (32). Donor is required for T cell TEM under flow conditions in vitro (17). The BM cells (2 3 106 cells in 0.3 ml, 3% FBS in PBS) were transfused by tail 2/2 downstream signals mediated by CD47 in the endothelium during vein injected into irradiated WT or CD47 recipient mice. Recipient mice received normal chow and water containing antibiotics (Sulfatrim) 1 T cell TEM and its potential role in leukocyte recruitment in vivo, wk prior to and 6 wk after transplantation. Air pouch leukocyte recruitment however, have not been explored. studies were performed 8–9 wk post-BM transplantation. To confirm BM Based on reports that endothelial cell adhesion molecules in- engraftment, leukocytes from chimeric and WT mice were stained with volved in leukocyte TEM trigger intracellular signals and elicit FITC rat anti-mouse CD47. Expression levels were acquired on a FACS- Calibur flow cytometer and analyzed using FlowJo software. downstream effects, we hypothesized that engagement of CD47 Downloaded from triggers alterations in the endothelial cell cytoskeleton and VE-cad Intravital microscopy of leukocyte recruitment during phosphorylation, both of which are necessary for TEM. We report microvascular inflammation in vivo that CD472/2 mice have a profound defect in neutrophil, CD3+ Intravital microscopy (IVM) of leukocyte transmigration of postcapillary T cell, and monocyte recruitment in a dermal air pouch model of venules of the mouse cremaster muscle was performed at 2 h following TNF-a–induced inflammation that is dependent on CD47 on pa- intrascrotal injection of mouse TNF-a (500 ng in saline/mouse), as initially renchymal cells, presumably the endothelium, and provide evi- described (33). Mice were anesthetized, and surgical exteriorization of the http://www.jimmunol.org/ dence that endothelial CD47 generates intracellular signals that cremaster muscle was performed as we have recently described (34). are necessary for leukocyte TEM. Microvessel data and physiology indices were obtained using a specialized Olympus FV 1000 intravital microscope (Olympus, Center Valley, PA) fitted with an Olympus 403 water immersion objective (Olympus) (34). Materials and Methods Leukocyte transmigration events were recorded from 8 to 12 vessels per Mice mouse using an Olympus DP71 CCD video camera and Olympus Fluo- View 1000 imaging software (Olympus). Videos were analyzed offline 2/2 CD47 knockout (CD47 ) mice (C57BL/6 strain) have been previously with the National Institutes of Health software package ImageJ (National described (21) and were obtained from E. Brown (Genentech, San Fran- Institutes of Health, Bethesda, MD). The number of transmigrated leu- cisco, CA). Wild-type (WT) C57BL/6 mice were purchased from Charles kocytes per vessel was determined by counting the average number of River Laboratories (Wilmington, MA), and a breeding colony was estab- perivascular cells in a 50 3 100-mm area adjacent to the vessel wall, as by guest on September 29, 2021 lished in our facility for use as WT control animals. Mice were maintained previously described (34, 35). in a specific pathogen-free barrier unit at our institution. Animal care and experimentation were in accordance with the National Institutes of Health Measurement of leukocytes in murine peripheral blood Guidelines for the Care and Use of Laboratory Animals and were approved by the Institutional Animal Care and Use Committee. Peripheral blood from mice was collected in EDTA-coated vials (Sarsdedt) at 4 and 24 h after injections of PBS or PBS–TNF-a. A complete blood Reagents and Abs count with cellular differential was assessed by automated multispecies hematology instrument (Hemavet 950FS; Drew Scientific, Oxford, CT). Recombinant human TNF-a and CXCL12 were from PeproTech (Rocky Hill, NJ). Recombinant murine TNF-a was purchased from BioLegend Isolation of human endothelial cells and T cells and TEM (San Diego, CA). The Src kinase inhibitor PP2 and the p38 MAPK in- under flow conditions hibitor SB203580 were from Calbiochem (San Diego, CA). Pertussis toxin (PTX) was purchased from Sigma-Aldrich (St. Louis, MO). The following Human CD3+ T cells (.95% purity) were isolated by negative selection mAbs were used as purified IgG: mAb B6H12 is a function-blocking mAb from anticoagulated whole blood obtained from healthy volunteers, as to human CD47, and mAb TS1/22 is a function-blocking mAb against previously described (17). Blood was obtained from volunteer donors aLb2 integrin (American Type Culture Collection, Manassas, VA); mAb according to Brigham and Women’s Hospital Institutional Review Board- 2D3 is a nonblocking anti-CD47 mAb (28) obtained from E. Brown; un- approved protocols for protection of human subjects, and all volunteer labeled and FITC-labeled rat anti-mouse CD47 function-blocking mAb subjects gave informed consent, in accordance with the Declaration of (miap301) was from BioLegend; anti–VE-cad mAb Hec-1 (29) was from Helsinki. Pooled HUVEC were isolated and cultured, as described (17). W. Muller (Northwestern University, Chicago, IL); anti-HLA class I (class Confluent HUVEC monolayers were used in all biochemical and TEM 1 MHC) mAb W6/32, anti–ICAM-1 mAB Hu5/3, and function-blocking studies. Confluent HUVEC monolayers on fibronectin-coated glass cov- anti–JAM-A mAb 1H2A9 were as previously described (30). The blocking erslips were stimulated with TNF-a (4 h, 25 ng/ml), followed by treatment mAb to a4 HP2/1 was from Immunotech (Glendale, CA). The anti- with CXCL12 (50 ng/ml) for 15 min, a step that promotes T cell TEM, phosphotyrosine–specific mAb 4G10 was from Millipore (Billerica, MA). prior to insertion into the flow chamber thermostated to 37˚C. T cells (1 3 Phospho-specific polyclonal Abs to VE-cad (pY658 and pY731) and Pyk2 106 in 0.1 ml) were drawn across HUVEC monolayers at 0.8 dynes/cm2, (pY402) were from BioSource International (Camarillo, CA). Phospho- and the accumulation/mm2 and TEM were measured, as described (17). specific and polyclonal anti-Src and anti-p38 MAPK Abs were from The percentage of TEM was calculated as follows: total transmigrated Cell Signaling Technology (Danvers, MA). FITC-conjugated markers for T cells/[total adhered + transmigrated T cells] 3 100. CD3 T cells (145-2cl), monocytes and macrophages (allophycocyanin- conjugated anti-F4/80), neutrophils (allophycocyanin-conjugated GR-1 RNA interference Ab), and isotype control mAbs were from BD Biosciences (San Jose, Predesigned small interfering RNA (siRNA) against CD47 was obtained 9 CA). Alexa-488– or FITC-conjugated F(ab )2 goat anti-mouse IgG and from Qiagen (Valencia, CA). The silencing siRNA target sequences (cat- phalloidin-Alexa 488 were from Invitrogen (Carlsbad, CA). alog SI04894582 and SI03117401) were 59-CCCTTACGTGATTGT- In vivo transmigration assay (s.c. air pouch model) TAGTTA-39 and 59-TGAAACGATCATCGAGCTAAA-39. Both reduced surface-expressed CD47 by 70–80%. A predesigned siRNA oligonucle- Air pouches were created in the dorsal portion of the back of 8- to 10-wk-old otide sequence targeting CD47 but with no inhibitory effect on CD47 ex- C57BL/6 WT and CD472/2 male mice, as previously described (31). PBS pression (target sequence, 59-CACGATAAGTTTACTCCCCCA-39; catalog The Journal of Immunology 3

2 2 SI00028168) was used as a negative control. HUVEC were transfected 22% in CD47 / compared with WT, as shown in Table I (21, 39). with siRNAs (100 nmol/l) using Oligofectamine in Optimem sera-free We conclude that CD47 plays an important role in the recruitment culture medium (Invitrogen). After 92 h, HUVEC were stimulated with of blood T cells, neutrophils, and monocytes in this murine model TNF-a (25 ng/ml, 4 h) and used in studies. of inflammation. Immunofluorescence microscopy of CD47 expression and We next addressed the role of BM and host-expressed CD47 phalloidin staining in leukocyte recruitment in the air pouch model by com- 2/2 HUVEC monolayers transfected with different siRNAs were fixed and paring CD47 and WT mice reconstituted with WT BM (WT stained with B6H12 anti-CD47 mAb, and primary mAb was detected by BM→CD472/2;WTBM→WT mice). The reciprocal reconstitu- staining with Alexa-488–conjugated goat anti-mouse IgG, as previously tion of CD472/2 BM→WT is not feasible because transfused described (36). Confluent resting or 4-h TNF-a–activated HUVEC on 2/2 fibronectin-coated coverslips were treated with anti-CD47 mAb (B6H12) CD47 BM cells are cleared rapidly from the circulation by (30 mg/ml, 30 min) and washed, and then goat anti-mouse secondary mAb splenic macrophages in WT animals, and these mice do not sur- 2 2 (10 mg/ml) was used to cross-link CD47 mAb for 0, 5, 10, 20, and 30 min. vive (40). Reconstituted WT BM→CD47 / mice had comparable As a control, HUVEC were incubated with secondary mAb only. HUVEC levels of CD47 expression on leukocytes as WT BM→WT ani- were fixed and permeabilized, and endogenous F-actin was detected by mals. Analysis of TNF-a–induced leukocyte accumulation in these staining with Alexa-568–phalloidin, according to the manufacturer’s pro- 2/2 tocol (Invitrogen). Actin stress fiber formation was quantified in .100 animals showed that reconstitution of CD47 mice with WT BM 2/2 cells in multiple fields at the 30-min time point. Cells with five or more (WT BM→CD47 ) does not rescue the defect in recruitment of stress fibers were considered positive. Results are representative of four or neutrophils, T cells, or monocytes in response to TNF-a at both more separate experiments. 4 and 24 h (Fig. 2A–D). Because WT BM→CD472/2 chimeric

Flow cytometry and analysis animals have CD47+/+ hematopoietic cells, but lack CD47 in pa- Downloaded from renchymal cells, these data indicate host cell CD47, presumably the Conﬂuent resting or 4-h TNF-a–stimulated HUVEC monolayers were trypsinized and stained for expression of CD47 and ICAM-1 using B6H12 vascular endothelium, is necessary for leukocyte recruitment in vivo. and Hu5/3 mAbs, respectively, followed by FITC-tagged secondary IVM studies in CD472/2 BM chimera mice support a role for F(ab9)2 mAb, as previously described (17). Expression levels were acquired on a FACSCalibur ﬂow cytometer and analyzed using FlowJo software. endothelial CD47 in leukocyte TEM

CD47 Ab cross-linking studies and Western blotting To directly visualize leukocyte TEM in vivo, we performed IVM http://www.jimmunol.org/ experiments in 2-h TNF-a–activated cremaster muscle microcir- Twenty-four–hour TNF-a–stimulated HUVEC in 35-mm diameter wells → 2/2 (6-well culture plate; Corning Life Sciences, Lowell, MA) were serum culation of WT BM CD47 chimera mice. Hemodynamic starved for 2 h in OPTI-MEM I (Life Technologies/Invitrogen) and then parameters and estimated wall shear rates were similar among the incubated for 10 min at 37˚C with Dynabeads (Invitrogen Dynal AS, Oslo, animals after TNF-a treatment (data not shown). As shown in Fig. Norway) prebound to mAb to CD47 (B6H12), ICAM-1 (Hu5/3), class 1 2E, the number of emigrated leukocytes in WT BM→CD472/2 MHC, or murine IgG Ab (10 mg/ml), using a ratio of 10 beads per cell. The HUVEC were washed twice in ice-cold PBS to remove nonadherent beads chimera animals was significantly less as compared with the WT and immediately lysed in 250 ml boiling sample buffer under reducing BM→WT chimera animals. Representative images of a segment 2 2 conditions. Cell lysates were diluted to allow immunoprecipitation with of venules in WT and CD47 / animals depicting transmigrated anti-phosphotyrosine mAb 4G10, as described previously (8, 11). Recov- leukocytes are shown (Fig. 2F, 2G). The results in the cremaster by guest on September 29, 2021 ered proteins were separated by SDS-PAGE, transferred to nitrocellulose model indicate that endothelial cell CD47 contributes directly to membranes, and Western blotted for phospho-Src (Tyr416), phospho-Pyk2 (Tyr402), phosphor-VE-cad (Tyr658), and phosphor-VE-cad (Tyr731) and leukocyte TEM in vivo. detected by an ECL (Pierce Biotechnology, Rockford, IL) following incubation with appropriate HRP-conjugated secondary Abs. Band intensity Both human endothelial and leukocyte CD47 are required for was quantified by NIH Image J software. T cell TEM Statistical analysis Human CD3+ T cells (95 6 11% purity) arrest uniformly on 4- or 18-h TNF-a– or IL-1b–activated HUVEC and transmigrate at cell Data are expressed as mean 6 SEM and were compared using Student t test for experiments with two groups or one-way ANOVAwith Tukey multiple- junctions in the presence of exogenously added apical chemokine comparison posttest for experiments with three or more groups by Prism CXCL12 under shear flow conditions (17, 41). Pretreatment of software v5.00 for Windows (GraphPad, San Diego, CA). Data were TNF-activated HUVEC with blocking mAb to CD47 had no sig- , considered statistically significant at p 0.05. nificant effect on T cell accumulation (Fig. 3A), but significantly reduced T cell TEM (Fig. 3B) as compared with control mAb to Results MHC class I. Function blocking mAb to ICAM-1 in HUVEC (Fig. CD472/2 mice exhibit defective leukocyte recruitment to 3B), or to aLb2 integrin, its ligand on T cells, also significantly TNF-a in a dermal air pouch model of inflammation reduced T cell TEM, whereas anti-a4 integrin-blocking mAb did CD47 has been shown previously to play a role in T cell recruitment not (Supplemental Fig. 1), which is consistent with a previous in vitro under physiologically relevant shear flow conditions (17, report (41). Next, the effect of CD47 blockade in T cells was 37). In this study, the involvement of CD47 in regulating TNF-a– examined in parallel. Treatment of T cells with anti-CD47 mAb induced leukocyte recruitment was investigated using a skin air significantly reduced T cell transmigration without altering T cell pouch model used by other investigators to examine the contri- adhesion. mAb blocking of CD47 in both HUVEC and T cell was bution of adhesion molecules and chemokines to leukocyte no more effective than blocking either cell type alone. These data recruitment (31, 38). In WT mice, TNF-a triggered increased demonstrate that both endothelial and T cell CD47 play an im- leukocyte accumulation in air pouches at 4 and 24 h (Fig. 1A). portant role in T cell TEM in vitro. Flow cytometric analysis showed that recruited leukocytes con- sisted of neutrophils, T cells, and monocytes/macrophages (Fig. siRNA knockdown of CD47 in HUVEC reduces T cell TEM 1B–D). In contrast to WT mice, recruitment of each leukocyte under flow 2 2 type in CD47 / mice was dramatically reduced at both 4 and 24 To corroborate CD47 mAb blocking of TEM in Fig. 3, siRNA h. The peripheral blood leukocyte counts at 0, 4, and 24 h were silencing of CD47 in HUVEC was performed. Silencing of CD47 similar in both lines of mice (Fig. 1E). Consistent with a previous by two different siRNA caused a 70–80% reduction (n =4)in study, the percentage of lymphocytes in blood was reduced by surface CD47 staining in resting (data not shown) and 4-h TNF-a– 4 ENDOTHELIAL CD47 MEDIATES T CELL RECRUITMENT IN VIVO Downloaded from

FIGURE 1. Impaired leukocyte recruitment in CD472/2 mice in s.c. air pouch model. (A) Leukocyte recruitment into air pouches was determined in the lavage fluid from WT (4 mice per condition) or CD472/2 (4 mice per condition) after administration of TNF-a (500 ng/ml in 200 ml PBS) or PBS. (B–D) The http://www.jimmunol.org/ frequency of neutrophils, monocytes/macrophages, and CD3+ T cells in lavage was determined using leukocyte-type specific primary labeled mAb and flow cytometric analysis, as described in Materials and Methods. Total CD3+, neutrophils, and monocytes/macrophages per pouch are shown, and were calculated by multiplying the total cell counts by the percentage of each cell population as deduced by flow cytometry. (E) Blood leukocyte counts were determined by automated multispecies hematology instrument. There was no significant difference in WBC between WT and CD472/2 animals. Data are means 6 SEM for four mice per group and are representative from three independent experiments (Student t test). *p , 0.05, **p , 0.01 for indicated comparisons.

activated HUVEC (Fig. 4A, 4B) as compared with control (non- examine whether CD47 signals through the Gai pathway in en- silencing) siRNA targeting of CD47. As observed with CD47 dothelium during TEM, we preincubated HUVEC with different

mAb blocking, CD47 siRNA significantly reduced T cell TEM concentrations of PTX for 5 h to inactivate Gai subunits and by guest on September 29, 2021 and also did not alter T cell accumulation (Fig. 4C, 4D). However, assessed T cell adhesion and TEM (Fig. 5A, 5B). PTX pretreat- HUVEC transfected with control targeting CD47 siRNA, but with ments of HUVEC had no inhibitory effect on T cell adhesion or no inhibitory effects, supported T cell adhesion and robust TEM TEM, whereas PTX treatment of T cells significantly blocked (Fig. 4C, 4D). Live cell differential interference contrast (DIC) transmigration (Fig. 5C), consistent with earlier reports (41, 43, imaging of TEM showed that adherent T cells polarize and 44). Videomicroscopy of the TEM assay revealed that adherent transmigrate in control siRNA-treated TNF-activated HUVEC, T cells flatten, polarize, and transmigrate in PTX-treated HUVEC whereas treatment of HUVEC with CD47-specific siRNA inhibi- (Fig. 5D, left panel; arrowheads). In contrast, pretreatment of ted T cell TEM (Fig. 4E, arrowheads identify nontransmigrated T cells with PTX abrogated T cell TEM, and most adherent T cells T-cells; arrows, transmigrated T cells). To control for off-target did not flatten or migrate on the HUVEC monolayer (Fig. 5D, effects of siRNA, we observed that silencing CD47 siRNA and right panel; arrowheads). Because PTX treatment of rodent en- nonsilencing control CD47 siRNA-treated HUVEC showed no dothelium has variable inhibitory effects, ranging from no effect in difference in the expression of ICAM-1 (Fig. 4F), or in surface T cell TEM of rat HEV (43) to 80% inhibition of T cell TEM of expression of VE-cad, VCAM-1, or E-selectin in 4-h TNF-a– murine brain endothelial cells in vitro (45) or neutrophil and eo- 2/2 activated HUVEC (data not shown). sinophil TEM of endothelial monolayers isolated from Gai mice (46), we tested the effects of PTX in endothelium derived PTX treatment of endothelial cells does not inhibit T cell TEM from human dermal microvessels and saphenous vein. Consistent under shear flow conditions with the results in HUVEC, T cell adhesion or TEM across either CD47 has been reported to signal through a heterotrimeric Gi- human dermal microvessels or human saphenous vein was not coupled receptor complex in a variety of cell types (14, 15, 42). To inhibited by PTX pretreatment (Supplemental Fig. 2A, 2B).

Table I. Blood neutrophil and lymphocyte differential and total numbers in WT and CD472/2 mice

WBC %NE %LY (3103/ml) NE LY n WT 28.11 6 2.42 64.17 6 2.94 5.02 6 0.07 1.47 6 0.30 3.16 6 0.40 10 CD472/2 41.19 6 3.57** 49.83 6 3.10** 4.95 6 0.56 2.06 6 0.27 2.46 6 0.32 9 A complete blood count with cellular differential was determined by automated multispecies hematology instrument (Hemavet 950FS; Drew Scientiﬁc, Oxford, CT). CD472/2 % NE and % LY are signiﬁcantly different from WT, **p , 0.01 (Student t test). LY, Lymphocyte; NE, neutrophil. The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 2. WT BM→CD472/2 mice have reduced leukocyte recruitment as compared with WT BM→WT mice. (A–D) Dermal air pouches were established in age-matched WT BM→CD472/2 or WT BM→WT chimeric mice (n = 5 mice/group). The total leukocyte number and the total number neutrophils, monocytes/macrophages, and CD3+ T cells per pouch were determined, as described in Fig. 1 legend. TNF-a–induced leukocyte recruitment was not rescued in WT BM→CD472/2 chimeric mice. Data are means 6 SEM for four mice per group and are representative from two independent experiments. *p , 0.05, **p , 0.01 for indicated comparisons (Student t test). (E) Leukocyte transmigration in BM chimera animals. Data are means 6 SEM for four mice per group, and a total 49 WT and 42 CD472/2 venules were examined. From the literature, neutrophils normally represent a minority of circulating leukocytes, but are the predominant rolling and transmigrated leukocytes in this model (61). (F and G) Representative still images of transmigrated leukocytes in each animal type after 2 h of TNF-a treatment. Transmigrated cells in the 50 3 100-mm area adjacent to the vessel wall regions are identiﬁed by arrowheads.

Treatment of HUVEC with PTX had no effect on the TNF-a– CD47 mAb for 10 min, followed by a secondary mAb for dif- induced increase in ICAM-1, E-selectin, or VCAM-1 (data not ferent periods of time (Fig. 6A). Cross-linking with anti-CD47 shown), and the treated and control monolayers maintained the mAb triggers robust stress fiber formation, as assessed by phal- same normal cobblestone morphology throughout the study. In loidin staining, as early as 5 min with most cells responding by 30 addition, PTX treatment of HUVEC had no effect on neutrophil min (Fig. 6A, right panel). In the absence of CD47 cross-linking, TEM under flow conditions (data not shown). These results sug- or cross-linking of endothelial class I MHC mAb (control), gest a CD47-dependent mechanism that is independent of Gai HUVEC showed few stress fibers (data not shown). protein signaling in human endothelium during TEM. ICAM-1 cross-linking by anti–ICAM-1 mAb or mAb-coated polystyrene beads are well-characterized approaches that mimic Cross-linking of endothelial cell CD47 triggers robust ICAM-1 binding by leukocytes. We next assessed the effects of cytoskeletal remodeling and tyrosine phosphorylation of VE- incubating 24-h TNF-a–activated HUVEC with 3 mm Dynal cad beads coated with either anti-CD47 or anti–ICAM-1 mAbs and Rapid remodeling of the endothelial actin cytoskeleton and in- found a 2- to 3-fold increase in phosphorylation of tyrosine 658 creased phosphorylation of tyrosine residues 658 and 731 in the and 731 in the VE-cad cytoplasmic tail (Fig. 6B, 6C) as compared cytoplasmic domain of VE-cad are necessary events in T cell and with medium or anti-class I MHC mAb-coated beads in both neutrophil TEM (8, 10, 11). Our laboratory has previously re- cases. Class I mAb-coated beads bind equally as well as CD47 ported (9) that mAb cross-linking of ICAM-1 leads to a rapid mAb-coated beads to HUVEC monolayers, but did not trigger increase in actin cytoskeletal remodeling in HUVEC. To deter- increased phosphorylation of VE-cad tyrosine 658 or 731. Previ- mine whether engaging endothelial CD47 triggers remodeling of ous studies have shown that engagement of ICAM-1 activates Src the actin cytoskeleton, we preincubated HUVEC with an anti- and Pyk2 family kinases, which lead to tyrosine phosphorylation 6 ENDOTHELIAL CD47 MEDIATES T CELL RECRUITMENT IN VIVO

FIGURE 3. Both endothelial and T cell-expressed CD47 are required for TEM. (A and B) Four-hour or 18-h TNF-a (10 or 25 ng/ml)–activated HUVEC monolayers were preincubated with function-blocking mAb to CD47 (B6H12), ICAM-1 (Hu5/3), or a control binding mAb to class I MHC (W6/32) (each mAb used at 30 mg/ml, 30-min preincubation). Where noted, human T cells were also preincubated with function- blocking mAb to CD47 (Tc). The total number of adherent T cells (T-cells/ mm2) and the percentage of adherent T cells that transmigrated after 10 min were determined by live cell imaging, as described earlier (17). Data Downloaded from are mean 6 SEM, n = 3 independent experiments. *p , 0.003 versus medium or class I for CD47 (EC), p , 0.002 versus medium or class I for CD47 (Tc), p , 0.01 versus medium or class I for CD47 (EC + Tc), and p , 0.02 versus medium or class I for ICAM-1 (one-way ANOVA, followed by the Tukey test). Medium versus class I was not statistically different (p = 0.34). http://www.jimmunol.org/ of several endothelial cell proteins, including VE-cad (8, 10, 36). FIGURE 4. CD47 siRNA-treated HUVEC show reduced T cell trans- To determine whether engaging CD47 also induced Src and Pyk2 migration. (A) Four-hour TNF-a–stimulated HUVEC previously trans- kinase activation, we challenged CD47 and ICAM-1 or as control fected with CD47-targeting siRNA, control (nonsilencing CD47-targeted) class I MHC with appropriate mAb-coated beads. Western blot siRNA, or sham-treated (transfection reagent without siRNA) were analysis of cell lysates with phospho-specific Abs directed against assessed for CD47 expression by flow cytometry, as described in Materials and Methods (data are representative of n = 5; control = nonbinding mouse activation-induced phosphotyrosine pY416 of Src or pY402 of IgG). (B) Fluorescent micrographs of CD47 surface staining in HUVEC Pyk2 was performed. Both anti-CD47 and anti–ICAM-1 mAb- transfected with control or CD47-silencing siRNA. Scale bar, 20 mm. Total coated beads triggered a 2-fold increase in phosphorylation of fluorescence of CD47 signal was reduced by 75%, as assessed by ImageJ by guest on September 29, 2021 both Src and Pyk2 (Fig. 7A). In contrast, class I mAb beads did software. (C and D) TNF-a–stimulated HUVEC were transfected with not alter phosphorylation of either Src or Pyk2. CD47-targeted siRNA, a control, nonsilencing CD47-targeted siRNA or We next used a pharmacological approach to corroborate in- left untreated, or HUVEC were pretreated with function-blocking mAb to volvement of Src and Pyk2 family kinases or PTX to block Gai- CD47 (B6H12), CD47 nonblocking (2D3) or control class I MHC mAb, coupled signaling. Because ICAM-1 cross-linking by mAbs is and adhesion and TEM, assessed as described in Materials and Methods. 6 known to induce MAPK activation (reviewed in Ref. 47), we also Data are the mean SEM of duplicate coverslips for each condition, three , used a p38 MAPK inhibitor to test its effects on ICAM-1– and independent experiments. *p 0.05 for indicated comparisons of CD47 siRNA versus control siRNA and blocking versus nonblocking CD47 mAb CD47-induced VE-cad phosphorylation. Interestingly, ICAM- (Student t test). (E) The morphology of T cells on CD47-targeted siRNA- 1– and CD47-induced VE-cad phosphorylation was not prevented treated (arrowheads) and control, nonsilencing CD47-targeted siRNA by pretreatment of HUVEC with either PTX or the p38 MAPK (arrows) HUVEC is shown as DIC images of live cells at the conclusion of inhibitor, SB203580 (Fig. 7B). However, pretreatment of cells the TEM assay (320 objective). Arrowheads identify T cells that have with Src kinase inhibitor, PP2, significantly inhibited both ICAM- defects in polarization, and arrows identify T cells that completed TEM. 1– and CD47-induced VE-cad phosphorylation at tyrosines 658 (F) HUVEC transfected with CD47-targeting or CD47-targeted, non- and 731 (Fig. 7B). silencing (control) siRNA stimulated with TNF-a for 4 h show no difference in ICAM-1 expression (representative histogram from n = 3). Discussion A number of endothelial-expressed adhesion molecules, including localized to the apical surface and was enriched at cell–cell ICAM-1, VCAM-1, ESAM, CD99, and PECAM-1, are involved junctions, and that adhesive interactions between endothelial cell in adhesive and/or intracellular signaling events required for leu- CD47 and human T cell-expressed SIRPg played an important kocyte transmigration (5–7). In addition, endothelial presented role in T cell TEM under flow conditions in vitro (17). In this chemokines (e.g., IL-8, MCP-1) and chemoattractants (e.g., Leu- study, we examined the contribution of endothelial CD47 in leu- kotrieneB4) promote leukocyte arrest and polarized migration on kocyte recruitment in an experimental animal model of inflam- and across the endothelium and into tissues (48, 49). CD47 is mation and sought to gain insight into signals elicited by CD47 involved in a broad range of cell adhesion processes, including engagement in endothelium that might contribute to TEM events. neutrophil transepithelial and transendothelial migration (25, 26), 2/2 monocyte transendothelial migration (27), T cell transendothelial CD47 animals have defects in leukocyte recruitment migration (17), human platelet adhesion to vascular endothelium A murine dermal air pouch was employed as a model of TNF-a– (50), dendritic cell migration (51, 52), as well as regulation of induced skin inflammation to study the role of CD47 in leukocyte innate and adoptive immune responses (reviewed in Refs. 14, 53). extravasation (31, 38). The data show that injection of TNF-a into In human endothelial cells, we recently reported that CD47 was the dorsal site of WT mice induced a time-dependent accumula- The Journal of Immunology 7

FIGURE 5. PTX pretreatment of HUVEC does not block T cell transmigration. (A) HUVEC were pretreated with PTX (200 ng/ml) for 1 h before addition of TNF-a for 4 h (total incubation = 5 h). T cells were also subjected to PTX treatment for 2 h at the same concentration before assay. T cell Downloaded from accumulation and TEM were not affected when HUVEC or T cells were pretreated with PTX. n = 2 independent experiments with duplicate coverslips. (B) Pretreatment of HUVEC for 5 h with three different concentrations of PTX had no effect on T cell TEM. n = 2 independent experiments with duplicate coverslips. (C) HUVEC were pretreated with PTX (200 ng/ml) for 1 h before addition of TNF-a for 4 h (total incubation = 5 h). The percentage of T cell TEM was assessed, as described in Materials and Methods. Data are means 6 SEM, n = 5 experiments; *p , 0.001 versus medium (Student t test). (D) The majority of T cells polarize, flatten, and transmigrate on PTX-treated endothelium (left, arrowhead) as compared with PTX-treated T cells that are rounded and refractile and did not transmigrate (right, arrowhead), as demonstrated in DIC images of live cells captured at the end of the TEM assay (320 objective). http://www.jimmunol.org/ tion of neutrophils, T cells, and monocytes in the air pouch exu- Two other studies have evaluated the contribution of CD47 dates, whereas recruitment of leukocytes in CD472/2 mice was to T cell recruitment in a dermal contact hypersensitivity (CHS) strikingly reduced and nearly absent. Analysis of BM chimera model of inflammation. The level of inflammation in CD472/2 mice indicates that endothelial cell CD47 plays a significant role animals as compared with WT mice was similar (no reduction) in in leukocyte recruitment. In addition, the analysis of inflamed one CHS study (55) and significantly elevated and also protracted cremaster muscle microcirculation in WT and CD472/2 BM in a second study (24). The CHS, however, is complex, and chimera animals by IVM revealed an important role of endothelial neutrophils and NK cells as well as T cells are present (56). by guest on September 29, 2021 cell CD47 in TEM. These in vivo observations are consistent with Although this might seem at odds with our air pouch data, one our in vitro human T cell TEM results that show function-blocking explanation is that the mechanism(s) of leukocyte recruitment mAb to CD47 on T cells or human endothelial monolayers sig- depends on the nature of the inflammatory stimulus (i.e., induction nificantly reduced T cell TEM (Figs. 3, 4, Supplemental Figs. 2A, of different chemokines or cytokines) and/or that recruited T cells 2B). Silencing of CD47 by siRNA in HUVEC strongly reduced may be different in the CHS site versus the dermal air pouch, and T cell TEM, but not adhesion under flow conditions in vitro, and one subset is more dependent on CD47 than the other. thus corroborates the function-blocking mAb-blocking results. We Human CD47 binds to thrombospondins, SIRPa and SIRPg. The conclude that endothelial CD47 plays a key role in the transmi- interaction of human endothelial CD47 with SIPRg expressed on gration of multiple leukocyte types to sites of TNF-a–induced skin T cells is important for T cell TEM in vitro assays using HUVEC. inflammation and in vitro models of TEM. CD47 on T cells appears In rat, endothelial CD47 has been shown to interact with SIRPa- to also contribute to TEM in vitro, but the in vivo relevance of this expressing monocytes, and this pathway was critical for monocyte could not be addressed because the reciprocal BM reconstitution adhesion and TEM. Currently, a rat or mouse SIRPg ortholog has of WT mice with BM from CD472/2 animals is not feasible due not yet been described or predicted from searches of genomic or to clearance of these cells by splenic macrophages (40). EST sequence databases. However, there is no evidence that ro- Our data demonstrate an important role for CD47 in TEM by dent T cells lack receptors for CD47. To the contrary, functional both the T cell and endothelium. In T cells, CD47 provides several assays, as those described in this study, indicate the presence of stimulatory signals (reviewed in Ref. 14). In particular, CD47 was a CD47 ligand on T cells whose identity remains to be determined. reported to participate in TCR-mediated Jurkat T cell spreading, and in Jurkat T cell adhesion to an endothelial cell line (EA. Endothelial CD47 signaling in leukocyte TEM is Gai hy926) or VCAM-1 under shear flow or static assay conditions independent through regulation of VLA-4 integrins (37, 54). The precise role CD47 has been shown to participate in the regulation of cell–cell that T cell CD47 plays in adhesion and TEM in vivo or in vitro, adhesion and cell migration through reorganization of the actin however, is as yet unexplored and, hence, will require future in- cytoskeleton in epithelial cells, a process mediated by activation depth studies. Interestingly, the combined mAb blocking of T cell of the MAPK pathway (57). Several studies reported that cis and HUVEC CD47 was not additive, and this was unexpected. interactions between CD47 and integrins recruit a PTX-sensitive The reason for this result is not immediately apparent, but may be Gai protein-thrombospondin complex (reviewed in Ref. 14). Of due to the complexity of CD47 interactions with its endothelial relevance to our study, treatment of rodent endothelium with PTX and leukocyte ligands thrombospondins and SIRPs and with its resulted in reduced T cell transmigration; the level of inhibition well-characterized regulation of multiple integrins. Future studies was variable and ranged from no effect in a rat HEV system (43) will be required to address these questions. to an 80% reduction in murine brain endothelial cell migration 8 ENDOTHELIAL CD47 MEDIATES T CELL RECRUITMENT IN VIVO Downloaded from http://www.jimmunol.org/

FIGURE 6. CD47 cross-linking triggers cytoskeletal remodeling and tyrosine phosphorylation of VE-cad HUVEC. (A) CD47 expressed in confluent resting or 4-h TNF-activated HUVEC was cross-linked, washed, fixed, and permeabilized, and endogenous F-actin was detected, as described in Materials and Methods. Actin fiber formation was quantified in .100 cells in multiple fields from three separate experiments at the 30-min time point. Cells that had five or more stress fibers were considered positive, as described previously (9, 36). (B and C) Confluent 24-h TNF-a– FIGURE 7. CD47 cross-linking activates Src and Pyk2 protein tyrosine by guest on September 29, 2021 treated HUVEC were incubated with beads coated with anti-CD47 kinases and tyrosine phosphorylation of VE-cad. (A) Confluent 24-h TNF- (B6H12), anti–ICAM-1 (Hu5/3), or class I MHC (W6/32) mAbs and a–treated HUVEC were serum starved for 2 h and then cross-linked with blotted with phospho-VE-cad–specific Abs to detect tyrosine phosphory- bead-bound mAbs to mouse IgG, class I MHC, ICAM-1, or CD47, and lation induced by cross-linking. A representative blot of each phospho-VE- activation of Src and Pyk2 was measured, as described in Materials and cad Ab is shown. The graphs represent normalized values obtained by Methods. A representative blot for Src and Pyk2 is shown. The graphs densitometry analysis corresponding to phosphotyrosine 658 or 731 VE- (right) represent normalized values obtained by densitometry analysis cad divided by the total VE-cad input. Data are mean 6 SEM, n = 3 in- corresponding to phospho-Src (upper panel) and phospho-Pyk2 (lower dependent experiments; phospho-Y658 graph, *p , 0.0004 versus medium panel) divided by the total Src and b-actin input, respectively. Data are and anti-class I; phospho-Y732, *p , 0.0001 versus medium and anti-class mean 6 SEM from n = 3 independent experiments. Phospho-SrcY416, I (one-way ANOVA, followed by Tukey test). *p , 0.0001 versus IgG and anti-class I MHC; phospho-Pyk-2 Y402, *p , 0.0001 versus IgG and anti-class I (one-way ANOVA, followed by Tukey test). (B)Asin(A), mAb to class I MHC (control mAb), ICAM-1, or CD47 2/2 were conjugated to Dynal beads and used to cross-link these molecules. system (45). In Gai animals, leukocyte recruitment in an allergen-induced lung model was significantly impaired and Prior to cross-linking, HUVEC were preincubated with PTX (200 mg/ml) was dependent on endothelial cell G -dependent signaling (46). for 5 h, PP2 (10 mM) for 30 min, or SB203580 (10 mM) for 1 h. The ai graphs (bottom) represent normalized values obtained by densitometry In these models and in models of leukocyte chemotaxis (41), PTX top . analysis corresponding to phosphorylated VE-cad at tyrosine 658 ( ) and pretreatment essentially blocked 80% of leukocyte migration, tyrosine 731 (lower) divided by the total VE-cad input, respectively. Data indicating a critical role of T cell Gai proteins. However, the role are mean 6 SEM, n = 3 separate experiments. *p , 0.001 versus no of Gai proteins in TEM of human endothelial cells has not been treatments with anti–ICAM-1, *p , 0.001 versus no treatments with anti- reported. In this study, we observed that PTX pretreatment of CD47 mAb (Student t test). endothelium isolated from three different human vascular beds had no effect on T cell adhesion or TEM, indicating that Gai proteins in human endothelial cells are not involved in TEM. gagement of ICAM-1 also activates Pyk2, which collaborates with Src protein kinases to phosphorylate tyrosine 658 and 731 in the CD47 engagement induces actin stress fibers and activates Src cytoplasmic tail of VE-cad (8). Tyrosine phosphorylation of VE- and Pyk2 to phosphorylate VE-cad cad is widely thought to facilitate dissociation of junctional pro- Previous studies have shown that ICAM-1 cross-linking triggers teins during paracellular migration of leukocytes. Previous studies robust actin stress fiber formation in HUVEC by activating Rho A by our laboratory showed that inhibition of Src leads to reduced (58) and induces tyrosine phosphorylation of cortactin (9, 36, 59, leukocyte TEM (36). Our results show that, like ICAM-1, CD47 60) and VE-cad (8, 10, 11), and inhibition of these events sig- ligation triggers actin cytoskeletal remodeling and Src- and Pyk2- nificantly reduces leukocyte TEM (reviewed in Refs. 6, 7). En- mediated VE-cad tyrosine phosphorylation in HUVEC. CD47- The Journal of Immunology 9 induced VE-cad phosphorylation was significantly inhibited by 16. Johansen, M. L., and E. J. Brown. 2007. Dual regulation of SIRPalpha phosphorylation by integrins and CD47. J. Biol. Chem. 282: 24219–24230. a Src inhibitor, but was not affected by PTX or by a p38 MAPK 17. Stefanidakis, M., G. Newton, W. Y. Lee, C. A. Parkos, and F. W. Luscinskas. inhibitor. Taken together, Src can act as the main regulator of 2008. Endothelial CD47 interaction with SIRPgamma is required for human T- CD47- and ICAM-1–induced phosphorylation events required for cell transendothelial migration under shear flow conditions in vitro. Blood 112: 1280–1289. TEM in HUVEC. 18. Barclay, A. N., and M. H. Brown. 2006. The SIRP family of receptors and In conclusion, our in vivo and in vitro studies show a crucial role immune regulation. Nat. Rev. Immunol. 6: 457–464. for endothelial CD47 in T cell transmigration and in recruitment 19. Brooke, G., J. D. Holbrook, M. H. Brown, and A. N. Barclay. 2004. Human lymphocytes interact directly with CD47 through a novel member of the of T cells, neutrophils, and monocytes to extravascular sites of signal regulatory protein (SIRP) family. J. Immunol. 173: 2562–2570. inflammation. We also identify signaling pathways mediated by 20. Piccio, L., W. Vermi, K. S. Boles, A. Fuchs, C. A. Strader, F. Facchetti, M. Cella, and M. Colonna. 2005. Adhesion of human T cells to antigen-presenting cells CD47 that contribute to leukocyte TEM. Uncontrolled recruit- through SIRPbeta2-CD47 interaction costimulates T-cell proliferation. Blood ment of leukocytes and increased endothelial cell permeability 105: 2421–2427. are observed in various pathologies, such as psoriasis, autoimmune 21. Lindberg, F. P., D. C. Bullard, T. E. Caver, H. D. Gresham, A. L. Beaudet, and E. J. Brown. 1996. Decreased resistance to bacterial infection and granulocyte diseases, multiple sclerosis, and rheumatoid arthritis. We propose defects in IAP-deficient mice. Science 274: 795–798. that CD47 and/or the CD47 pathway downstream could serve as 22. Su, X., M. Johansen, M. R. Looney, E. J. Brown, and M. A. Matthay. 2008. potential future targets for the treatment of such inflammatory CD47 deficiency protects mice from lipopolysaccharide-induced acute lung injury and Escherichia coli pneumonia. J. Immunol. 180: 6947–6953. conditions. 23. Fortin, G., M. Raymond, V. Q. Van, M. Rubio, P. Gautier, M. Sarfati, and D. Franchimont. 2009. A role for CD47 in the development of experimental colitis mediated by SIRPalpha+CD1032 dendritic cells. J. Exp. 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