Denise E. Jackson

The unfolding tale of PECAM-1.

Platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31) is a member of the immunoglobulin (Ig) superfamily that has distinctive features of an immunoreceptor based upon its genomic structure and the presence of intrinsic immunoreceptor tyrosine inhibitory motifs (ITIMs) in its ligand binding polypeptide. This has lead to its subclassification into the Ig‐ITIM superfamily. Its amino‐terminal Ig‐like domain of PECAM‐1 is necessary for its homophilic binding, which plays an important role in cell–cell interactions. Its intracellular ITIMs serve as scaffolds for recruitment of signalling molecules including protein‐tyrosine phosphatases to mediate its inhibitory co‐receptor activity. Increasing evidence has implicated PECAM‐1 in a plethora of biological phenomena, including modulation of integrin‐mediated cell adhesion, transendothelial migration, angiogenesis, apoptosis, cell migration, negative regulation of immune cell signalling, autoimmunity, macrophage phagocytosis, IgE‐mediated anaphylaxis and thrombosis. In this review, we discuss some of the new developments attributed to this molecule and its unique roles in biology.

Src Homology 2 Domain-Containing Protein-Tyrosine Phosphatases, SHP-1 and SHP-2, Are Required for Platelet Endothelial Cell Adhesion Molecule-1/CD31-Mediated Inhibitory Signaling

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is a newly assigned member of the Ig immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. To test whether PECAM-1 is capable of delivering inhibitory signals in B cells and the functional requirement of protein-tyrosine phosphatases (PTPs) for this inhibitory signaling, we generated chimeric FcγRIIB1-PECAM-1 receptors containing the extracellular and transmembrane portions of murine FcγRIIB1 and the cytoplasmic domain of human PECAM-1. These chimeric receptors were stably expressed in chicken DT40 B cells either as wild-type or mutant cells deficient in SHP-1−/−, SHP-2−/−, SHIP−/−, or SHP-1/2−/− and then assessed for their ability to inhibit B cell Ag receptor (BCR) signaling. Coligation of wild-type FcγRIIB1-PECAM-1 with BCR resulted in inhibition of intracellular calcium release, suggesting that the cytoplasmic domain of PECAM-1 is capable of delivering an inhibitory signal that blocks BCR-mediated activation. This PECAM-1-mediated inhibitory signaling correlated with tyrosine phosphorylation of the FcγRIIB1-PECAM-1 chimera, recruitment of SHP-1 and SHP-2 PTPs by the phosphorylated chimera, and attenuation of calcium mobilization responses. Mutational analysis of the two tyrosine residues, 663 and 686, constituting the immunoreceptor tyrosine-based inhibitory motifs in PECAM-1 revealed that both tyrosine residues play a crucial role in the inhibitory signal. Functional analysis of various PTP-deficient DT40 B cell lines stably expressing wild-type chimeric FcγRIIB1-PECAM-1 receptor indicated that cytoplasmic Src homology 2-domain-containing phosphatases, SHP-1 and SHP-2, were both necessary and sufficient to deliver inhibitory negative regulation upon coligation of BCR complex with inhibitory receptor.

CEACAM1 negatively regulates platelet-collagen interactions and thrombus growth in vitro and in vivo.

Carcinoembryonic antigen cell adhesion molecule-1 (CEACAM1) is a surface glycoprotein expressed on various blood cells, epithelial cells, and vascular cells. CEACAM1 possesses adhesive and signaling properties mediated by its intrinsic immunoreceptor tyrosine-based inhibitory motifs that recruit SHP-1 protein-tyrosine phosphatase. In this study, we demonstrate that CEACAM1 is expressed on the surface and in intracellular pools of platelets. In addition, CEACAM1 serves to negatively regulate signaling of platelets by collagen through the glycoprotein VI (GPVI)/Fc receptor (FcR)-gamma-chain. ceacam1(-/-) platelets displayed enhanced type I collagen and GPVI-selective ligand, collagen-related peptide (CRP), CRP-mediated platelet aggregation, enhanced platelet adhesion on type I collagen, and elevated CRP-mediated alpha and dense granule secretion. Platelets derived from ceacam1(-/-) mice form larger thrombi when perfused over a collagen matrix under arterial flow compared with wild-type mice. Furthermore, using intravital microscopy to ferric chloride-injured mesenteric arterioles, we show that thrombi formed in vivo in ceacam1(-/-) mice were larger and were more stable than those in wild-type mice. GPVI depletion using monoclonal antibody JAQ1 treatment of ceacam1(-/-) mice showed a reversal in the more stable thrombus growth phenotype. ceacam1(-/-) mice were more susceptible to type I collagen-induced pulmonary thromboembolism than wild-type mice. Thus, CEACAM1 acts as a negative regulator of platelet-collagen interactions and of thrombus growth involving the collagen GPVI receptor in vitro and in vivo.

Absence of Platelet Endothelial Cell Adhesion Molecule-1 (CD31) Leads to Increased Severity of Local and Systemic IgE-Mediated Anaphylaxis and Modulation of Mast Cell Activation

Platelet endothelial cell adhesion molecule-1 (PECAM-1) is a newly assigned member of the Ig-immunoreceptor tyrosine-based inhibitory motif superfamily, and its functional role is suggested to be an inhibitory receptor that modulates immunoreceptor tyrosine-based activation motif-dependent signaling cascades. In this study, we hypothesized that PECAM-1 plays an essential in vivo role as a counterregulator of immediate hypersensitivity reactions. We found that PECAM-1 was highly expressed on the surface of immature bone marrow mast cells and at a lower density on mature peritoneal mast cells. Examination of skin biopsies from PECAM-1+/+ and PECAM-1−/− mice revealed that absence of PECAM-1 did not affect mast cell development or the capacity of mast cells to populate tissues. To examine whether the absence of PECAM-1 would influence immediate hypersensitivity reactions, PECAM-1+/+ and PECAM-1−/− mice were presensitized with anti-DNP mouse IgE and then challenged 20 h later with DNP-BSA or PBS. PECAM-1−/− mice exhibited elevated serum histamine concentrations after Ag stimulation compared with PECAM-1+/+ mice, indicating an increased severity of systemic IgE-mediated anaphylaxis. PECAM-1−/− mice have increased sensitivity to local cutaneous IgE-dependent anaphylaxis compared with PECAM-1+/+ mice, as assessed by greater tissue swelling of their ears and mast cell degranulation in situ. PECAM-1−/− bone marrow mast cells showed enhanced dense granule serotonin release after FcεRI cross-linking in vitro. These results suggest that PECAM-1 acts as a counterregulator in allergic disease susceptibility and severity and negatively modulates mast cell activation.

A platelet tetraspanin superfamily member, CD151, is required for regulation of thrombus growth and stability in vivo

Summary.u2002 Background:u2002This study was designed to determine the role of CD151 in platelet thrombus formation in vivo and define the contribution of platelet vs. endothelial CD151 in regulating platelet thrombus formation in vivo. Methods and Results: Using intravital microscopy and ferric chloride (FeCl3) injury of mesenteric arterioles, we found that thrombi formed in CD151+/− and CD151−/− mice were smaller and less stable, than those formed in CD151+/+ mice, with a tendency for embolization. Similarly, in Folt’s FeCl3−induced carotid injury model, both CD151+/− and CD151−/− mice showed more prolonged times to 95% vessel occlusion than CD151+/+ mice. In addition, laser‐induced injury of cremaster muscle arterioles showed that thrombi formed in CD151+/− and CD151−/− mice were smaller and less stable than those formed in CD151+/+ mice. Following platelet depletion/reconstitution with ex vivo‐labeled donor platelets, platelet‐depleted CD151+/+ mice that received reconstitution with CD151−/− platelets had smaller thrombi that were unstable and embolized. In contrast, platelet‐depleted CD151−/− mice that received reconstitution with CD151+/+ platelets had normal thrombi that were stable. Conclusions: These data provide evidence that platelet CD151 is required for regulating thrombus formation in vivo.

The inhibitory co-receptor, PECAM-1 provides a protective effect in suppression of collagen-induced arthritis.

Studies of PECAM-1−/− mice have identified that PECAM-1 functions as an inhibitory co-receptor to modulate immunological responsiveness. In this study, we describe the in vivo consequences of PECAM-1 deficiency in mouse models of collagen-induced arthritis (CIA) and K/BxN passive transfer model that resembles many of the features of human rheumatoid arthritis. Immunization of PECAM-1−/− C57BL/6 (H-2b) mice with chicken collagen type II induced CIA with an incidence of 82% by day 49, while 33%; of wild-type and 100% of DBA/1 mice developed arthritis in a similar time frame. The mean onset of disease for PECAM-1−/− C57BL/6 mice was day 32 compared to day 51 for wild-type C57BL/6 mice and day 18 for DBA/1 mice (H-2q susceptible). In terms of disease severity, the mean maximal arthritic index for PECAM-1−/− C57BL/6 mice was comparable to DBA/1 mice (8.91 ± 0.91 vs 11.67 ± 0.82). This mean maximal index in PECAM-1−/− C57BL/6 mice was significantly higher than wild-type C57BL/6 mice (5.00 ± 0.73). IgG1 and IgG2b antibody responses against CII were elevated in arthritic PECAM-1−/− C57BL/6 mice compared to wild-type C57BL/6 mice. Histological examination of arthritic paws of PECAM-1−/− C57BL/6 mice revealed inflammatory infiltrates of lymphocytic/monocytic cells and cartilage/bone destruction similar to CIA-induced DBA/1 arthritic paws. In the K/BxN model, the arthritis was not augmented in PECAM-1−/− mice compared to wild-type mice. In contrast, in active CIA, PECAM-1−/− mice developed severe disease comparable to susceptible DBA/1 mice and profoundly more severe than C57BL/6 mice, where only one third developed a mild/moderate disease. Together these observations suggest that PECAM-1 plays a crucial role in the suppression of development of autoimmune arthritis.

Proteolytic cleavage of platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31) is regulated by a calmodulin-binding motif

Homophilic engagement of platelet endothelial cell adhesion molecule‐1 (PECAM‐1/CD31) induces ‘outside‐in’ signal transduction that results in phosphorylation events and recruitment and activation of signalling molecules. The formation of signalling scaffolds with PECAM‐1 are important signalling events that modulate platelet secretion, aggregation and platelet thrombus formation. In this study, we describe a novel interaction between PECAM‐1 and cytosolic calmodulin (CaM) in platelets. Reciprocal co‐immunoprecipitation studies revealed that cytosolic CaM is constitutively associated with PECAM‐1 in resting, thrombin activated and aggregated human platelets. Our studies demonstrate that CaM directly interacts with a PECAM‐1 peptide (594–604) C595A containing the sequences 594KAFYLRKAKAK604. This CaM:PECAM‐1 interaction has a threefold higher affinity than CaM:GPVI interaction. It is potentiated by the addition of calcium ions, and dissociated by the CaM inhibitor, trifluoperazine. Treatment of platelets with CaM inhibitors triggers cleavage of PECAM‐1 in a time‐ and dose‐dependent manner. Furthermore, this membrane proximal portion of PECAM‐1 is conserved across mammalian species and the helical representation of basic/hydrophobic residues reveals a charge distribution analogous to other CaM‐binding motifs in other proteins. Taken together, these results suggest that this highly charged cluster of amino acids in the PECAM‐1 cytoplasmic domain directly interacts with CaM and this novel interaction appears to regulate cleavage of PECAM‐1.

Regulation of B cell activation by PECAM-1: Implications for the development of autoimmune disorders

Regulation of B-cell development and activation is imperative to the myriad of activities that perpetuate humoral immunity. This T-cell dependent immune mechanism often relies upon the maintenance of T-cell tolerance, such that the maturity of the antigen-presentating cell, its function and molecular mimicry are contributing factors. Recent findings have implicated the involvement of the B-cell and their corresponding surface co-receptors in regulating autoimmune disease. One candidate receptor, PECAM-1, has demonstrated the ability to downregulate both B and T-cell signalling pathways. The deletion of PECAM-1 in mice has led to a hyper-responsive B-cell phenotype with abnormal B-cell development. Additionally, in vivo functional studies have found that absence of PECAM-1 results in an increased susceptibility to autoimmune disorders of encephalomyelitis and Type I hypersensitivity reactions. Taken together, these findings indicate that PECAM-1 may have an important role in maintaining B-cell tolerance and regulatory function in preventing the onset of autoimmune disease. Elucidating the mechanisms of PECAM-1 function in autoimmune disorders could facilitate development of novel therapeutics.