A New Role for -Endothelial Molecule-1 (CD31): Inhibition of TCR-Mediated Signal Transduction

This information is current as Debra K. Newton-Nash and Peter J. Newman of September 26, 2021. J Immunol 1999; 163:682-688; ; http://www.jimmunol.org/content/163/2/682 Downloaded from References This article cites 57 articles, 35 of which you can access for free at: http://www.jimmunol.org/content/163/2/682.full#ref-list-1

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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 © 1999 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. A New Role for Platelet-Endothelial -1 (CD31): Inhibition of TCR-Mediated Signal Transduction1

Debra K. Newton-Nash2 and Peter J. Newman

Platelet-endothelial cell adhesion molecule-1 (PECAM-1) is a 130-kDa transmembrane glycoprotein expressed by endothelial cells, , , , and certain subsets. The PECAM-1 extracellular domain has six Ig-homology domains that share sequence similarity with cellular adhesion molecules. The PECAM-1 cytoplasmic domain contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) that, when appropriately engaged, becomes phosphorylated on tyrosine residues, creating docking sites for nontransmembrane, Src homology 2 domain-bearing tyrosine phosphatase (SHP)-1 and SHP-2. The purpose of the present study was to determine whether PECAM-1 inhibits protein tyrosine kinase (PTK)-dependent signal transduction mediated by the immunoreceptor tyrosine-based activation motif-containing TCR. Jurkat cells, which coexpress ⑀ PECAM-1 and the TCR/CD3 complex, were INDO-1AM-labeled and then incubated with anti-CD3 mAbs, anti-PECAM-1 mAbs, Downloaded from or both, and goat anti-mouse IgG was used to cross-link surface-bound mAbs. Calcium mobilization induced by CD3 cross-linking was found to be attenuated by coligation of PECAM-1 in a dose-dependent manner. PECAM-1-mediated inhibition of TCR signaling was attributable, at least in part, to inhibition of release of calcium from intracellular stores. These data provide evidence that PECAM-1 can dampen signals transduced by ITAM-containing receptors and support inclusion of PECAM-1 within the family of ITIM-containing inhibitors of PTK-dependent signal transduction. The Journal of Immunology, 1999, 163: 682–688. http://www.jimmunol.org/ activation is a process that is controlled, in ciated with the CTLA-4 cytoplasmic domain dephosphorylates part, by regulation of the phosphorylation state of tyrosine tyrosine residues in the ITAMs of the TCR ␨-chain (5). Similarly, T residues in the cytoplasmic domains of TCR-associated KIRs contain an ITIM within their cytoplasmic domain, which, signal transduction units. In the initial activation phase, tyrosine upon tyrosine phosphorylation, supports the binding of the non- residues in immunoreceptor tyrosine-based activation motifs transmembrane PTP, SHP-1 (6, 7). The substrates for KIR-asso- 3 (ITAM) of the signal transduction units of the TCR complex be- ciated SHP-1 have not yet been determined. come phosphorylated by members of the src family of protein Platelet-endothelial cell adhesion molecule-1 (PECAM-1), or tyrosine kinases (PTKs), supporting recruitment of ␨-associated CD31, is a 130-kDa member of the Ig superfamily that is protein of 70 kDa (ZAP-70), which itself becomes activated by expressed on the surface of endothelial cells and circulating plate- by guest on September 26, 2021 tyrosine phosphorylation and enables transmission of downstream lets, monocytes, neutrophils, and certain T cell subsets. PECAM-1 signals for T cell activation (1). The level of protein tyrosine phos- was originally assigned to the family of Ig-like cellular adhesion phorylation is balanced by the activity of protein tyrosine phos- molecules (CAMs) based on sequence similarity between its six phatases (PTPs), which negatively regulate the initial phase of T extracellular Ig domains and those of other Ig domain-containing cell activation. The mechanism by which the initial phase of T cell CAMs (8). An adhesive function for PECAM-1 is supported by the activation is modulated involves active transmission of negative observations that amino-terminal domains 1 and 2 mediate ho- signals through membrane-associated molecules, such as CTLA-4 mophilic interactions (9, 10) that facilitate adhesion between and killer cell inhibitory receptors (KIRs) (2, 3). CTLA-4 contains PECAM-1-expressing cells (11). Recent evaluation of the structure an immunoreceptor tyrosine-based inhibitory motif (ITIM)-like se- and biochemical properties of its cytoplasmic domain, however, quence that supports the binding of Src homology 2 domain-bear- ing PTP (SHP)-2 (4). Upon docking and activation, SHP-2 asso- suggest that PECAM-1 may also belong to the family of Ig-like, ITIM-containing inhibitory receptors (12). The PECAM-1 cyto- plasmic domain contains numerous potential sites for phosphory- Blood Research Institute, Blood Center of Southeastern Wisconsin, Milwaukee, WI lation of serine, threonine, and tyrosine residues (8). Of the five 53233 tyrosine residues in the human PECAM-1 cytoplasmic domain, Received for publication March 4, 1999. Accepted for publication April 29, 1999. two are surrounded by sequences that conform to an ITIM, for The costs of publication of this article were defrayed in part by the payment of page which the consensus sequence is (I/V/L)xpYxx(L/V) (6). The charges. This article must therefore be hereby marked advertisement in accordance PECAM-1 cytoplasmic domain contains two ITIMs, one around with 18 U.S.C. Section 1734 solely to indicate this fact. tyrosine 663 and the other around tyrosine 686 (reviewed in Ref. 1 This work was supported by National Institutes of Health Grant P01 HL44612 and the Blood Center Research Foundation. 12). The long distance between ITIMs in the PECAM-1 cytoplas- 2 Address correspondence and reprint requests to Dr. Debra K. Newton-Nash, Blood mic domain (17 residues) contrasts with the relatively short dis- Research Institute, P.O. Box 2178, Milwaukee, WI 53201-2178. E-mail address: tance (6–8 residues) that separates ITAM sequences, and is con- [email protected] sistent with the ability of the PECAM-1 cytoplasmic domain to 3 Abbreviations used in this paper: ITAM, immunoreceptor tyrosine-based activation support interactions with the relatively widely spaced tandem Src motif; ITIM, immunoreceptor tyrosine-based inhibitory motif; AP, alkaline phospha- tase; BCR, for Ag; CB, calcium buffer; GAM, goat anti-mouse IgG; homology 2 (SH2) domains of PTPs, as opposed to the relatively KIR, killer inhibitory receptor; PECAM-1, platelet-endothelial cell adhesion mole- closely spaced tandem SH2 domains of PTKs (reviewed in Ref. cule-1; PTK, protein tyrosine kinase; PTP, protein tyrosine phosphatase; SHP, Src homology 2 domain-bearing PTP; SH2, Src homology 2; PerCP, peridinin chlorophyl 12). The tyrosine residues at positions 663 and 686 of the PE- protein. CAM-1 cytoplasmic domain become phosphorylated in response

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 The Journal of Immunology 683

to numerous stimuli, including platelet aggregation (13), cross- CA), murine monoclonal anti-SHP-2 (Santa Cruz Biotechnology, Santa linking of PECAM-1-specific mAbs (14, 15), mechanical stimu- Cruz, CA), rabbit polyclonal anti-human PECAM-1 (SEW32-34; prepared lation of endothelial cells (16), engagement of the Fc⑀RI on ba- in our laboratory), and HRP-conjugated GAM (Jackson ImmunoResearch, West Grove, PA) or alkaline phosphatase (AP)-conjugated goat anti-rabbit sophils (17), aggregation of the Ag receptor on T cells (18), and IgG (Zymed Laboratories) Abs were used for Western blot analysis. treatment with the PTP inhibitor pervanadate (19). When phos- phorylated, these tyrosine residues support the binding of SHP-2 Induction and assessment of PECAM-1 tyrosine phosphorylation (13, 18, 20–23) and possibly SHP-1 (18, 22, 23). Thus, both struc- and SHP-2 binding tural and biochemical properties of the PECAM-1 cytoplasmic do- Jurkat cells in log phase growth were pelleted by centrifugation at 300 ϫ main suggest that it may belong to the Ig-like, ITIM-containing g for 10 min at room temperature and resuspended at a final concentration 7 family of inhibitory receptors. of 10 cells/ml in cold RPMI (complete)/10% FBS. Cell suspensions (1 ml) were incubated with no Ab, with the human CD3⑀-specific Ab, UCTH1 (1 The pattern of PECAM-1 expression on T also ␮g/ml), with a human PECAM-1-specific Ab, or with both anti-CD3⑀ and suggests a potential inhibitory function for this molecule. Specif- anti-PECAM-1 for 30 min on ice. To remove unbound Ab, cell suspensions ically, the majority of CD4ϩ (24–27) and half of CD8ϩ T lym- were washed twice with 5 ml of cold 0.145 M sodium chloride containing phocytes (24, 25) lose PECAM-1 expression as they make the 10 mM HEPES, 2.8 mM KCl, 2 mM MgCl2,10mMD-glucose, and 0.1% BSA (calcium buffer (CB)), and 10 mM CaCl2. Cells were resuspended in transition from naive to memory cells. This pattern of expression the same buffer at a final concentration of 2 ϫ 107 cells/ml. is consistent with a role for PECAM-1 in dampening the effector To induce PECAM-1 phosphorylation and SHP-2 binding, cell suspen- function of naive T cells, which are less potent effectors than are sions were first prewarmed at 37°C for 10 min. GAM was added to achieve memory cells (28). Furthermore, PECAM-1-negative CD4ϩ T a final concentration of 20–100 ␮g/ml, which was empirically determined in preliminary experiments to yield maximal stimulation. Following incu- cells provide more effective helper function, in that they respond Downloaded from bation for an additional 3 min at 37°C, the reaction was stopped by the better to recall Ags, secrete more IL-4, and provide better help for ϫ ϩ addition of an equal volume of cold 2 lysis buffer (0.145 M NaCl, 4% B cell Ig production than do PECAM-1-positive CD4 T cells Triton X-100, 0.2 mM MgCl2, 30 mM HEPES, 20 mM EGTA, 4 mM (27). Based on these observations, and given the structural and sodium orthovanadate, 40 ␮g/ml leupeptin, 2 mM PMSF (pH 7.4)), and biochemical properties of the PECAM-1 cytoplasmic domain, we lysates were prepared with gentle rocking for 120 min at 4°C. Lysates were also prepared from unstimulated cells, which were allowed to settle to the hypothesized that PECAM-1 might function as an inhibitory re- bottom of a 15-ml conical tube at 37°C and were not subjected to warming,

ceptor that moderates PTK-mediated signal transduction in T cells. cooling, or centrifugation. Following cell lysis, the Triton-insoluble frac- http://www.jimmunol.org/ To test this hypothesis, we assessed the impact of PECAM-1 co- tion was removed by centrifugation at 15,000 ϫ g for 15 min at 4°C. ligation on TCR-mediated calcium mobilization in T cells. We PECAM-1.1-coated beads were prepared by incubating PECAM-1.1 Ab ␮ found that the PECAM-1 cytoplasmic domain becomes tyrosine (25 g/ml) with protein A-Sepharose beads for2hat4°Cwith gentle rocking. Ab-coated beads were washed three times with lysis buffer to phosphorylated in response to cross-linking either the TCR or remove unbound Ab. Cell lysates were incubated with 100 ␮lofa50% PECAM-1 itself, with subsequent recruitment of SHP-2. When slurry of PECAM-1.1-coated beads with gentle rocking overnight at 4°C. PECAM-1/SHP-2 was brought into close proximity with the TCR, Immunoprecipitates were washed three times with immunoprecipitation it resulted in attenuation of TCR-mediated release of calcium from buffer (50 mM Tris (pH 7.4), containing 150 mM NaCl, 2 mM Na3VO4, 1% Triton X-100, and 1 mM PMSF). Pelleted beads were boiled in 60 ␮l intracellular stores. These data support inclusion of PECAM-1 of 2ϫ SDS-sample reducing buffer. Material eluted from the beads was within the family of ITIM-containing inhibitory receptors. separated on a 7.5% SDS-polyacrylamide gel and transferred to an Immo- by guest on September 26, 2021 bilon-P membrane, which was subjected to Western blot analysis for the Materials and Methods presence of PECAM-1, phosphotyrosine residues, and SHP-2. Cell lines and cell culture Calcium mobilization assays The acute T cell cell line, Jurkat (29), and the mature human T All procedures for measurement of intracellular calcium concentrations by cell lymphoblast line HUT 78 (30, 31), were obtained from the American fluorescence spectrofluorometry were performed in the dark. Jurkat T lym- Type Culture Collection (ATCC, Manassas, VA). Cells were maintained in phocytes (107 cells/ml) were incubated for 30 min at 37°C in RPMI (com- ϫ 5 log phase growth at a density of 1–4 10 cells/ml in a humidified, 37°C, plete) containing 10% FBS and 10 ␮M INDO-1AM (Molecular Probes, 6 5% CO2/95% air atmosphere. Jurkat cells were cultured in RPMI (com- Eugene, OR). Cells were then adjusted to a final concentration of 8 ϫ 10 plete) medium, which is RPMI 1640 medium (BioWhittaker, Walkersville, cells/ml with the addition of cold RPMI (complete)/10%FBS, and cell sus- MD) supplemented with 2 mM L-glutamine (Life Technologies, Gaithers- pensions were placed on ice. Cell suspensions (0.5 ml) containing 4 ϫ 106 burg, MD), 1 mM sodium pyruvate (Life Technologies), 10 IU/ml heparin cells/ml were incubated at 4°C for 30 min in the presence of Abs at the (Pharmacia/Upjohn, Kalamazoo, MI), 25 mM HEPES (Life Technologies), indicated final concentrations. To remove unbound Abs, Ab-bound cells ␮ 50 g/ml gentamicin (Elkins-Sinn, Cherry Hill, NC), 100 U/ml penicillin were washed three times at 4°C with 3 ml of CB (0.145 m NaCl, 10 mM (Life Technologies), and 100 ␮g/ml streptomycin (Life Technologies), HEPES, 2.8 mM KCl, ZmM MgCl2,10mMD-glucose, 0.1% BSA (pH containing 10% FBS (Life Technologies). HUT 78 cells were cultured in 7.2)). Cells were resuspended at a final concentration of 2 ϫ 106 cells/ml IMDM (Life Technologies) supplemented with 4 mM L-glutamine, 1.5 g/L in cold CB containing either 10 mM CaCl2 or 0.1 mM EGTA and main- sodium bicarbonate (Life Technologies) and 20% FBS. tained on ice until calcium flux was induced and measured. Immediately before induction of calcium mobilization, cell suspensions were placed in a 37°C water bath for 2 min. Prewarmed cell suspensions Peridinin chlorophyl protein (PerCP)-conjugated mouse anti-human CD3 were transferred to a cuvette equipped with a magnetic stir bar and trans- (IgG1 isotype), FITC-conjugated goat anti-mouse IgG (GAM), PerCP-con- ferred to the sample chamber, maintained at 37°C, of an SLM 8100 spec- jugated IgG1, and normal mouse IgG1 (Becton Dickinson, San Jose, CA) trofluorometer (SLM-AMINCO, Urbana, IL). To cross-link surface-bound were purchased from Becton Dickinson Immunocytometry Systems. The Abs, GAM was added to achieve a final concentration of 20 ␮g/ml, which mouse anti-human PECAM-1 Abs, PECAM-1.1 (IgG2a), -1.2 (IgG1), and was determined in preliminary experiments to induce maximal stimulation. -1.3 (IgG1), were prepared in our laboratory, and have been previously Fluorescence of stirring cell suspensions was excited at 350 nm and emis- described (9). The mouse anti-human PECAM-1 Ab, 4G6 (IgG2b), was sion of fluorescence at wavelengths of 405 and 490 nm was recorded every generously provided by Dr. Steven Albelda (University of Pennsylvania 3 s over a period ranging from 3 to 10 min in length. Results are presented Medical Center, Philadelphia, PA) (32). as plots of the ratio of fluorescence detected at 405, relative to 490, nm as The human CD3⑀-specific Ab, UCHT1 (mouse IgG1, ␬), containing no a function of time. azide and low endotoxin, was purchased from PharMingen (San Diego, Ј CA). Azide-free GAM F(ab )2 fragments (Accurate Chemical and Scien- Results Ј tific, Westbury, NY) or azide-containing GAM F(ab )2 fragments (Sigma, St. Louis, MO), from which azide was removed by dialysis, were used for Previous studies have shown that cross-linking the TCR leads to cross-linking of cell-surface-associated molecules. HRP-conjugated mouse tyrosine phosphorylation of the cytoplasmic domain of PECAM-1, anti-phosphotyrosine (clone PY20; Zymed Laboratories, San Francisco, creating a docking site for the protein tyrosine phosphatase, SHP-2 684 PECAM-1 INHIBITS TCR-MEDIATED CALCIUM MOBILIZATION Downloaded from FIGURE 1. Jurkat T lymphocytes coexpress CD3 and PECAM-1. Cell surface staining of the acute lymphocytic leukemia T cell line, Jurkat, was evaluated by flow cytometry. Negative controls included cells stained with normal mouse IgG1 (NMIgG1) followed by FITC-conjugated GAM (GAM- FITC) and then with PerCP-conjugated normal mouse IgG1 (IgG1-PerCP) (A). To evaluate cell surface expression of PECAM-1 and CD3, cells were stained with the murine Ab specific for human PECAM-1, PECAM-1.3 (anti-PECAM-1), followed with GAM-FITC and then with PerCP-conjugated mouse anti-human CD3 (CD3-PerCP) (B). http://www.jimmunol.org/

(18). We sought to more thoroughly examine the conditions re- brought into proximity with the TCR upon coligation of TCR and quired for, and the downstream consequences of, PECAM-1 ty- PECAM-1. rosine phosphorylation and PTP binding in T cells. Since T cell Previous studies have shown that coligation of ITIM-bearing expression of PECAM-1 is restricted to certain T cell subsets and inhibitory receptors with ITAM-bearing stimulatory receptors re- maturation states (24), several T cell lines were evaluated for co- sults in attenuation or blunting of cellular activation responses, expression of TCR and PECAM-1. Consistent with previously re- including the rate and/or extent of calcium mobilization (34–39), ported observations (25, 33), the acute T cell leukemia line, Jurkat, IP3 generation (38), and cell-mediated cytotoxic responses (38, by guest on September 26, 2021 expressed both TCR and PECAM-1 (Fig. 1). The HUT78 cell line 39). To determine whether PECAM-1/SHP-2 might be able to expressed TCR, but was PECAM-1-negative by both flow cyto- modulate TCR-mediated signal transduction, we examined the ef- metric and immunoblot analyses (data not shown). Therefore, fect of PECAM-1 coligation with the TCR in INDO-1AM-loaded Jurkat T lymphocytes were chosen for further studies. To optimize the conditions required for PECAM-1 tyrosine phosphorylation and PTP binding in T cells, murine mAbs specific for CD3⑀, PECAM-1, or CD3⑀ and PECAM-1 were bound to the surface of Jurkat T cells and then cross-linked by addition of Ј F(ab )2 fragments of a polyclonal goat Ab specific for murine IgG heavy and chains (GAM). Cells to which no primary Abs were bound served as negative controls. As shown in Fig. 2, a slight increase in PECAM-1 tyrosine phosphorylation was ob- served in cells stimulated by CD3 cross-linking, relative to the no Ab control. However, cross-linking of PECAM-1 itself induced a higher level of PECAM-1 tyrosine phosphorylation, which was further enhanced upon coligation of PECAM-1 with CD3. Ty- rosine phosphorylation of PECAM-1 resulted in the association of SHP-2, and the amount of phosphatase coprecipitated correlated with the degree of PECAM-1 tyrosine phosphorylation (Fig. 2). We also observed a small amount of the related PTP, SHP-1, co- precipitating with PECAM-1, but could not rule out that its visu- alization was due to Ab cross-reactivity with SHP-2 (data not FIGURE 2. Effect of receptor cross-linking on PECAM-1 tyrosine shown). Therefore, we conclude that the major PTP associated phosphorylation and PTP binding in T cells. Jurkat T lymphocytes (107) with PECAM-1 in Jurkat cells is SHP-2. These results extend pre- were treated with no Ab (lane 1), 1 ␮g/ml of murine anti-human CD3⑀ ␮ vious observations (18) by showing that coligation of PECAM-1 (lane 2), 50 g/ml of murine Ab specific for human PECAM-1 (PECAM- 1.3; lane 3), or both anti-CD3⑀ and PECAM-1.3 (lane 4) at 4°C. After and the TCR results in higher levels of PECAM-1 tyrosine phos- prewarming for 10 min at 37°C, receptor cross-linking was induced by phorylation than is observed upon cross-linking of the TCR alone. ␮ Ј addition of 20 (data not shown) or 100 g/ml of GAM F(ab )2. After a Furthermore, the tyrosine-phosphorylated cytoplasmic tail of further incubation for 3 min, cells were lysed in Triton X-100, and PECAM-1 appears to preferentially bind SHP-2. On the basis of PECAM-1 immunoprecipitates were prepared and analyzed by anti- these results, we conclude that SHP-2, which associates with the phosphotyrosine (PY20; top panel), anti-SHP-2 (second panel), and anti- tyrosine-phosphorylated cytoplasmic domain of PECAM-1, can be PECAM-1 (bottom panel) immunoblots. The Journal of Immunology 685

FIGURE 4. PECAM-1 coligation attenuates CD3-induced calcium mo- FIGURE 3. PECAM-1 coligation attenuates the early rise in intracellu- bilization in a dose-dependent manner. Jurkat T lymphocytes loaded with lar calcium but not the late sustained high calcium plateau induced by INDO-1AM were preincubated for 30 min at 4°C with murine anti-human ligation of CD3. Jurkat T lymphocytes loaded with INDO-1AM were pre- CD3⑀ alone (solid line) or together with the murine anti-human PECAM-1 Downloaded from incubated for 30 min at 4°C in the presence of murine anti-human CD3⑀ (PECAM-1.3; hatched lines) at 0.8, 3.1, 12.5, or 50 ␮g/ml, as indicated. (dark solid line), anti-CD3⑀ and a murine Ab specific for human PE- Cells were washed free of unbound Ab and then prewarmed for 2 min at CAM-1, PECAM-1.3 (hatched line), or PECAM-1.3 (gray solid line) 37°C. Changes in intracellular calcium concentrations were analyzed in the Ј alone. Cells were washed free of unbound Ab and then prewarmed for 2 presence of external calcium following addition of GAM F(ab )2. min at 37°C. Changes in intracellular calcium concentrations were ana- lyzed over 10 min in the presence of external calcium following addition Ј of concentrations of GAM F(ab ) determined, in preliminary experiments http://www.jimmunol.org/ 2 ⑀ (data not shown), to induce maximal cross-linking and calcium anti-CD3 -mediated calcium mobilization (Fig. 3), an effect that mobilization. was proportional to the amount of PECAM-1/SHP-2 brought into the TCR complex (Fig. 4). The ability of PECAM-1-specific Abs to inhibit TCR-mediated calcium flux also appeared to be influ- Jurkat cells. A dose of anti-CD3⑀ (50 ng/ml) that induced mea- enced by the Ig homology domain-specificity of the anti- surable but suboptimal increases in intracellular calcium concen- PECAM-1 mAb. Thus, two different Ig domain 6-specific mAbs trations only when cross-linked with GAM, and an optimal con- (PECAM-1.2 and 4G6), and one specific for domain 1 (PECAM- centration of GAM (20–100 ␮g/ml), were identified in preliminary 1.3), effected inhibition of TCR signaling, whereas PECAM-1.1,

dose response studies (data not shown). A suboptimal concentra- which is specific for PECAM-1 Ig domain 5, failed to attenuate the by guest on September 26, 2021 tion of anti-CD3⑀ was used to enable detection of either an en- early rise in intracellular calcium induced by CD3 cross-linking hancing or an inhibitory effect of PECAM-1 on TCR-induced cal- (Table I), most likely due to the orientation with which PECAM- cium mobilization. Furthermore, we chose conditions that required 1.1 interacts with cell surface PECAM-1 (see Discussion). GAM cross-linking so that we could control whether or not The early rise in intracellular calcium reflects both release of PECAM-1 was colocalized with the TCR from the time of initia- calcium from intracellular stores and influx of calcium across the tion of T cell activation. As shown in Fig. 3, cross-linking surface- plasma membrane, whereas the later, sustained elevation of intra- bound anti-PECAM-1 mAb, PECAM-1.3, failed to induce calcium cellular calcium levels is attributable to influx of calcium across mobilization, even though PECAM-1 became tyrosine phosphor- the plasma membrane (40–42). PECAM-1 coligation appeared to ylated and bound SHP-2 under these conditions (Fig. 2). Evalua- inhibit the initial rise in intracellular calcium, whereas the later, tion of three other PECAM-1-specific mAbs revealed that none by sustained elevation of intracellular calcium induced by TCR cross- themselves induced calcium mobilization in Jurkat T lymphocytes linking was largely unaffected by PECAM-1 coligation (Fig. 3). (Table I). In contrast, cross-linking of CD3⑀ induced strong and From these results, we predicted that PECAM-1/SHP-2 acts by rapid biphasic calcium mobilization characterized by a transient, inhibiting calcium release from intracellular stores, but not influx initial rise in intracellular calcium, followed by a lower but more of calcium from extracellular sources. To test this prediction, we sustained elevation of intracellular calcium levels (Fig. 3). Impor- assessed the inhibitory effect of PECAM-1/TCR coligation in the tantly, coligation of PECAM-1/SHP-2 with the TCR attenuated absence vs the presence of extracellular calcium. As shown in Fig. 5, the presence of extracellular EGTA did not affect the inhibitory activity of PECAM-1 on TCR-mediated calcium mobilization. Table I. Ability of PECAM-1-specific Abs to induce calcium Rather, the effect of PECAM-1 inhibition was to attenuate TCR- mobilization or inhibit TCR-mediated calcium mobilization in Jurkat T lymphocytes induced release of calcium from intracellular stores.

Inhibition of Discussion Anti-PECAM-1 Ig Domain Induction of TCR-Mediated mAb Specificity Ca2ϩ Flux Ca2ϩ Flux The purpose of the present study was to evaluate the downstream consequences of PECAM-1 tyrosine phosphorylation and binding PECAM-1.1 5 No Noa of PTPs, to determine whether PECAM-1, like other ITIM-con- PECAM-1.2 6 No Yes taining members of the Ig-superfamily, can function as an inhibitor PECAM-1.3 1 No Yes 4G6 6 No Yes of PTK-dependent signal transduction. To this end, we evaluated the ability of PECAM-1 to inhibit calcium mobilization induced by a Levels of PECAM-1.1 binding to cell surface PECAM-1 and of GAM binding to surface-bound PECAM-1.1, as assessed by flow cytometry, did not differ from TCR cross-linking in Jurkat T lymphocytes. We show in the those observed with PECAM-1.2 and PECAM-1.3 Abs (data not shown). present study that, in response to PECAM-1 or TCR coligation, 686 PECAM-1 INHIBITS TCR-MEDIATED CALCIUM MOBILIZATION

FIGURE 5. PECAM-1 coligation inhibits CD3-induced calcium release from intracellular stores. Jurkat T lymphocytes loaded with INDO-1AM were Downloaded from preincubated for 30 min at 4°C with murine anti-human CD3⑀ alone (solid lines) or with anti-CD3 and the murine Ab specific for human PECAM-1, PECAM-1.2 (dotted lines). Cells were washed free of unbound Ab and then prewarmed for 2 min at 37°C. Changes in intracellular calcium concentrations were analyzed in the presence of extracellular calcium (A) or in the absence of extracellular calcium and in the presence of extracellular EGTA following Ј addition of GAM F(ab )2. Note: Different y-axis scale values are used in A and B because the magnitude of the early rise in intracellular calcium is higher in the presence of extracellular calcium than it is in the absence of extracellular calcium and the presence of extracellular EGTA. http://www.jimmunol.org/

PECAM-1 becomes tyrosine phosphorylated and binds SHP-2 phosphorylation is supported by the observation that low levels of (Fig. 2). Furthermore, we demonstrate that calcium mobilization PECAM-1 tyrosine phosphorylation in Syk-deficient cells could be induced by TCR cross-linking is attenuated by coligation of the reconstituted by the stable transfection of Syk (17), a close homo- TCR with PECAM-1, which specifically affects the early rise, but logue of ZAP-70 in non-T cells. However, Cao et al.(23) have not the late, sustained elevation of intracellular calcium (Fig. 3). recently shown that overexpression of Syk in COS-1 cells is in- PECAM-1-mediated attenuation of TCR-induced calcium mobili- capable of stimulating PECAM-1 tyrosine phosphorylation. Fi- zation is dose-dependent (Fig. 4), affects release of calcium from nally, somewhat surprisingly, members of the Csk family of PTKs intracellular stores (Fig. 5), and is influenced by the domain-spec- are also able to phosphorylate murine PECAM-1 (23). Further by guest on September 26, 2021 ificity of the cross-linking Ab (Table I). Together, these findings studies are required to determine which of the many PTKs capable demonstrate that PTPs associated with the PECAM-1 cytoplasmic of phosphorylating PECAM-1 on tyrosine residues actually do so domain can, upon receptor coligation, be brought into proximity in activated T cells. with the TCR and interfere with PTK-dependent signal transduc- Tyrosine phosphorylation of PECAM-1 results in the creation of tion events in T cells. The importance of the PECAM-1 ITIM for sites with the potential for binding the tandem SH2 domain-con- this inhibitory function, the components of the TCR signal trans- taining PTPs, SHP-1 and SHP-2. Our data show that, in T cells, duction pathway that are affected, and the extent to which PE- SHP-2 preferentially associates with the tyrosine-phosphorylated CAM-1 inhibition of TCR signal transduction normally occurs cytoplasmic domain of PECAM-1. These results are consistent during the course of Ag stimulation are currently under active with those of numerous studies demonstrating coprecipitation of investigation in our laboratory. Nevertheless, these data demon- SHP-2 with tyrosine-phosphorylated PECAM-1 (13, 18, 20, 22, strate that PECAM-1 is able to attenuate cellular activation stim- 23). Although we occasionally observed, as have others (18, 23), ulated by an ITAM-containing stimulatory receptor, and provide coprecipitation of small amounts of SHP-1 with PECAM-1, SHP-2 the first evidence that PECAM-1 contains a functional ITIM. appears to bind preferentially, most likely due to the presence of The identity of the PTK responsible for phosphorylating ty- specific amino acid residues surrounding the phosphorylated ty- rosine residues in the PECAM-1 cytoplasmic domain in T cells is rosines in the PECAM-1 ITIM, with which the SH2 domains of not known. We observed a low level of PECAM-1 tyrosine phos- SHP-1 and SHP-2 must interact. The tandem SH2 domains of phorylation upon cross-linking of the TCR, and higher levels upon SHP-1 and SHP-2 are structurally similar, enabling both to recog- cross-linking of PECAM-1 either alone or when coligated with nize the consensus ITIM sequence (44). The PECAM-1 ITIM se-

TCR. The PTKs that may be responsible for PECAM-1 tyrosine quence of VxpY663xxVx17TxpY686xxV (12) is like that of another phosphorylation under these conditions include members of the member of the Ig-like, ITIM-containing family of inhibitory re- Src family of PTKs (p59fyn and p56lck) and ZAP-70, all of which ceptors, Ly49, in that it possesses a valine instead of a leucine can be recruited to the TCR signaling complex and activated fol- residue at the ϩ3 position relative to the tyrosine residues in its lowing TCR ligation (1). Ample evidence supports a role for mem- ITIM (45). The ability of the Ly49 ITIM to activate SHP-1 is bers of the Src family of PTKs in PECAM-1 tyrosine phosphor- reduced relative to that of another inhibitory receptor, p58 KIR, ylation. Specifically, Fyn coprecipitates with PECAM-1 (21) and and this difference has been attributed to the substitution of valine c-Src both coprecipitates with and can phosphorylate the cytoplas- for leucine at ITIM position ϩ3 (45). In contrast, the binding of mic domain of bovine (16, 21, 43) or murine PECAM-1 (23). In SHP-2 to phosphorylated ITIMs appears not to be affected by the addition, overexpression of Src family members, p56lck, p56lyn, substitution of valine for leucine at position ϩ3, as peptides con- and p53lyn in COS-1 cells resulted in tyrosine phosphorylation of taining either amino acid at this position were selected equally well murine PECAM-1 (23). A role for ZAP-70 in PECAM-1 tyrosine from a degenerate peptide library by the SH2 domains of SHP-2 The Journal of Immunology 687

(44). Thus, the preferential binding of SHP-2, relative to SHP-1, to localization of NF-AT (54), induction of IL-2 transcription (55), the PECAM-1 ITIM may similarly be attributable to the existence and irreversible commitment of T cells to full activation (56). of valine, as opposed to leucine, residues at the ϩ3 positions of the Thus, the inability of PECAM-1 coligation to reduce the magni- PECAM-1 ITIM. tude of the late, sustained calcium signal may influence its ability We found that PECAM-1 oligomerization resulted in PECAM-1 to affect the magnitude of a T cell effector response. However, tyrosine phosphorylation and SHP-2 binding, but not calcium mo- stimuli that delay the onset of a rise in intracellular calcium fol- bilization in Jurkat T cells. Our observations are consistent with lowing initial T cell activation, as does PECAM-1, also delay the the previous observation of PECAM-1 tyrosine phosphorylation in onset of a T cell proliferative response (57). Thus, we predict that response to binding of the PECAM-1-specific Ab, PECAM-1.2 PECAM-1 coligation with the TCR may delay the onset of T cell (15). They are, however, inconsistent with the previous observa- effector responses, but not affect their ultimate magnitude. That the tion that the PECAM-1-specific mAb, 4G6, induces calcium mo- overall level of inhibition caused by PECAM-1/TCR coligation is bilization in human endothelial cells and PECAM-1-transfected incomplete is consistent with the observation that PECAM-1-de- REN cells (46). This difference could be due to different signal ficient mice do not display any overt defects in T cell maturation transduction properties for PECAM-1 in T cells relative to endo- or homing (58). It is possible that an effect of PECAM-1 on T cell thelial cells, or to a difference in the assays used for assessment of responses other than maturation and migration may have been calcium mobilization. We conclude that PECAM-1 tyrosine phos- missed in preliminary characterizations of the PECAM-1-deficient phorylation and SHP-2 binding, at least in T cells, are not coupled mice. However, it is also important to consider that T cell activa- to signal transduction pathways that elevate intracellular calcium. tion is controlled by other inhibitory receptors, including KIRs and

Thus, though a positive signaling function for SHP-2 has been CTLA-4. If the function of PECAM-1 in T cells is subtle, so as to Downloaded from proposed based upon its ability to bind Grb2/SOS and activate the delay the onset of, or raise the threshold required for activation, it Ras/MAPK signal transduction pathway (47–51), our results sug- is possible that the effect of PECAM-1 deficiency may be observ- gest that positive signaling effected by SHP-2 is either not active able only in the absence of these other inhibitory receptors. Studies in T cells or, if active, is not coupled to signaling cascades that of T cell function in PECAM-1-deficient mice that are also het- induce calcium mobilization. In either case, our results support the erozygous for CTLA-4 deficiency are currently ongoing in our

hypothesis that induction of protein tyrosine phosphorylation and laboratory to explore this possibility. http://www.jimmunol.org/ activation of signal transduction pathways that result in calcium Upon interaction with appropriate APC, T cells receive signals mobilization can be independently regulated (52). through both activating and inhibitory receptors. The relative Whereas PECAM-1 oligomerization, tyrosine phosphorylation strength of the signal delivered by each of these two classes of and SHP-2 binding were not sufficient to induce calcium mobili- receptors determines whether the degree of activation of the signal zation in T lymphocytes, they did interfere with calcium mobili- transduction pathway reaches a critical threshold required for com- zation induced by TCR cross-linking. Only one of four PECAM- mitment to activation. In general, the function of inhibitory recep- 1-specific Abs tested, PECAM-1.1, failed to produce this effect. It tors is to recruit phosphatases that counteract the activity of ki- is possible that the orientation of PECAM-1.1, when bound to nases recruited to activating receptors, thus making it more PECAM-1, imposes geometrical constraints on the receptor that difficult for a given T cell to reach the critical activation threshold. by guest on September 26, 2021 interfere with the ability of the GAM cross-linker to either dimer- We show here that PECAM-1, a molecule that is expressed on the ize PECAM-1 or to bring it productively into proximity with Ab- surfaces of both endothelial cells and certain T cell subsets, and bound CD3⑀. All of the other PECAM-1-specific Abs tested, how- that mediates homophilic interactions, is capable of interfering ever, supported PECAM-1-mediated inhibition of TCR-induced with TCR-mediated signal transduction. On the basis of this un- calcium mobilization. derstanding, we propose that one context in which PECAM-1 ex- The characteristics of PECAM-1-mediated inhibition of TCR- erts its inhibitory function may involve T cell transmigration induced calcium mobilization differed somewhat from the effects across endothelial cell barriers, during which process PECAM-1 of other Ig-ITIM inhibitory receptors. Specifically, PECAM-1/ may serve to prevent T cells from becoming aberrantly activated. TCR coligation inhibited release of calcium from intracellular Studies of in vitro T cell effector activity, as well as in vivo studies stores only incompletely and failed to block or reduce the late, involving comparisons of the immune reactivity of wild-type and sustained elevation in intracellular calcium concentration that is PECAMϪ/Ϫ mice, are currently ongoing in our laboratory to test normally observed after TCR ligation. This pattern of inhibition the validity of this newly demonstrated function for PECAM-1. differs from that of Fc␥RIIb, which inhibits B cell receptor (BCR)- induced influx of calcium from extracellular sources, a property Acknowledgments ␥ that is consistent with the ability of Fc RIIb, upon tyrosine phos- We thank Cecilia Hillard for helpful discussions and gratefully acknowl- phorylation, to recruit the 5Ј-inositol phosphatase, SHIP (reviewed edge the excellent technical assistance of Kathleen Kennedy, Matthew in Ref. 53). The inhibitory effect of PECAM-1 differed less dra- Armstrong, and Shradha Oza. This manuscript is dedicated to the memory matically from those of inhibitory receptors that, like PECAM-1, of Ed Moser, coordinator of the High School Student Research Mentorship recruit protein tyrosine phosphatases to the membrane upon ty- Program of The Blood Center of Southeastern Wisconsin. rosine phosphorylation. 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