Differential association of cytoplasmic signalling molecules SHP-1, SHP-2, SHIP and phospholipase C-gamma1 with PECAM-1/CD31.

Recent studies have shown that, in addition to its role as an adhesion receptor, platelet endothelial cell adhesion molecule 1/CD31 becomes phosphorylated on tyrosine residues Y663 and Y686 and associates with protein tyrosine phosphatases SHP-1 and SHP-2. In this study, we screened for additional proteins which associate with phosphorylated platelet endothelial cell adhesion molecule 1, using surface plasmon resonance. We found that, besides SHP-1 and SHP-2, platelet endothelial cell adhesion molecule 1 binds the cytoplasmic signalling proteins SHIP and PLC-gamma1 via their Src homology 2 domains. Using two phosphopeptides, NSDVQpY663TEVQV and DTETVpY686SEVRK, we demonstrate differential binding of SHP-1, SHP-2, SHIP and PLC-gamma1. All four cytoplasmic signalling proteins directly associate with cellular platelet endothelial cell adhesion molecule 1, immunoprecipitated from pervanadate-stimulated THP-1 cells. These results suggest that overlapping immunoreceptor tyrosine-based inhibition motif/immunoreceptor tyrosine-based activation motif-like motifs within platelet endothelial cell adhesion molecule 1 mediate differential interactions between the Src homology 2 containing signalling proteins SHP-1, SHP-2, SHIP and PLC-gamma1.

Homophilic PECAM-1(CD31) interactions prevent endothelial cell apoptosis but do not support cell spreading or migration.

PECAM-1 (CD31) is a highly abundant cell surface glycoprotein expressed on haemopoietic and endothelial cells. As well as mediating homophilic (PECAM-1/PECAM-1) adhesion, PECAM-1 can also bind the integrin alphavbeta3. Both PECAM-1 and alphavbeta3 have been shown to have roles in regulating angiogenesis, endothelial tube formation and in the case of alphavbeta3, endothelial cell apoptosis. In this study we show that despite being expressed at equivalent levels, endothelial alphavbeta3 is not a ligand for PECAM-1. Rather, PECAM-1 supports homophilic binding on HUVEC with similar characteristics to those we have previously reported for leukocytes and becomes tyrosine phosphorylated after homophilic PECAM-1 and integrin/fibronectin engagement. Immunoprecipitation studies show that in addition to SHP-2, tyrosine phosphorylated PECAM-1 can interact with at least four other phosphoproteins in pervanadate stimulated HUVEC. While PECAM-1/PECAM-1 interactions support robust endothelial cell adhesion, they do not support cell spreading or migration. In addition PECAM-1 homophilic adhesion rescues HUVEC from serum deprivation-induced apoptosis. Taken together our results indicate that PECAM-1 homophilic interactions play an important role in interendothelial cell adhesion, survival and signalling.

C1q-mediated chemotaxis by human neutrophils: involvement of gClqR and G-protein signalling mechanisms.

C1q, the first component of the classical pathway of the complement system, interacts with various cell types and triggers a variety of cell-specific cellular responses, such as oxidative burst, chemotaxis, phagocytosis, etc. Different biological responses are attributed to the interaction of C1q with more than one putative cell-surface C1q receptor/C1q-binding protein. Previously, it has been shown that C1q-mediated oxidative burst by neutrophils is not linked to G-protein-coupled fMet-Leu-Phe-mediated response. In the present study, we have investigated neutrophil migration brought about by C1q and tried to identify the signal-transduction pathways involved in the chemotactic response. We found that C1q stimulated neutrophil migration in a dose-dependent manner, primarily by enhancing chemotaxis (directed movement) rather than chemokinesis (random movement). This C1q-induced chemotaxis could be abolished by an inhibitor of G-proteins (pertussis toxin) and PtdIns(3,4,5)P3 kinase (wortmannin and LY294002). The collagen tail of C1q appeared to mediate chemotaxis. gC1qR, a C1q-binding protein, has recently been reported to participate in C1q-mediated chemotaxis of murine mast cells and human eosinophils. We observed that gC1qR enhanced binding of free C1q to adherent neutrophils and promoted C1q-mediated chemotaxis of neutrophils by nearly seven-fold. Our results suggests C1q-mediated chemotaxis involves gC1qR as well as G-protein-coupled signal-transduction mechanisms operating downstream to neutrophil chemotaxis.

Mapping the homotypic binding sites in CD31 and the role of CD31 adhesion in the formation of interendothelial cell contacts.

CD31 is a member of the immunoglobulin superfamily consisting of six Ig-related domains. It is constitutively expressed by platelets, monocytes, and some lymphocytes, but at tenfold higher levels on vascular endothelial cells. CD31 has both homotypic and heterotypic adhesive properties. We have mapped the homotypic binding sites using a deletion series of CD31-Fc chimeras and a panel of anti-CD31 monoclonal antibodies. An extensive surface of CD31 is involved in homotypic binding with domains 2 and 3 and domains 5 and 6 playing key roles. A model consistent with the experimental data is that CD31 on one cell binds to CD31 on an apposing cell in an antiparallel interdigitating mode requiring full alignment of the six domains of each molecule. In addition to establishing intercellular homotypic contacts. CD31 binding leads to augmented adhesion via beta 1 integrins. The positive cooperation between CD31 and beta 1 integrins can occur in heterologous primate cells (COS cells). The interaction is specific to both CD31 and beta 1 integrins. Neither intercellular adhesion molecule-1 (ICAM-1)/leukocyte function-associated antigen-1 (LCAM-1) nor neural cell adhesion molecule (NCAM)/NCAM adhesion leads to recruitment of beta 1 integrin adhesion pathways. Establishment of CD31 contacts have effects on the growth and morphology of endothelial cells. CD31(D1-D6)Fc inhibits the growth of endothelial cells in culture. In addition, papain fragments of anti-CD31 antibodies (Fab fragments) disrupt interendothelial contact formation and monolayer integrity when intercellular contacts are being formed. The same reagents are without effect once these contacts have been established, suggesting that CD31-CD31 interactions are critically important only in the initial phases of intercellular adhesion.

Endothelial cell-associated platelet-activating factor primes neutrophils for enhanced superoxide production and arachidonic acid release during adhesion to but not transmigration across IL-1 beta-treated endothelial monolayers.

The role of platelet-activating factor (PAF) in stimulating neutrophils during interaction with HUVEC has been investigated using the specific PAF receptor antagonists WEB 2086 and YM 264. PAF antagonists at concentrations up to 40 microM had little effect on the adhesion of neutrophils to control or IL-1 beta-stimulated HUVEC monolayers. However, polarization of neutrophils on adhesion to IL-1-treated HUVEC was markedly diminished in the presence of PAF antagonists. In addition, the PAF antagonists WEB 2086 and YM 264 strongly inhibited the migration of neutrophils across IL-1-treated endothelial monolayers. Priming of neutrophil respiratory burst and arachidonic acid release caused by contact with IL-1-treated endothelial cells was also inhibited by PAF antagonists. However, priming as a consequence of transmigration was not inhibited by either WEB 2086 or YM 264. These results suggest that HUVEC-associated PAF plays a central role in the polarization and subsequent transmigration of neutrophils during interaction with HUVEC monolayers. Moreover, the results further suggest that PAF is responsible for priming neutrophils during contact with HUVECs. However, after transmigration, factors other than PAF are responsible for the priming of neutrophil function.

Studies of lymphocyte transendothelial migration: analysis of migrated cell phenotypes with regard to CD31 (PECAM-1), CD45RA and CD45RO.

CD31 is a 130,000 MW cell-surface glycoprotein expressed on endothelial cells, polymorphonuclear leucocytes, monocytes and about 50% of peripheral blood lymphocytes, and it has been proposed that it plays a role in transendothelial migration. If it is involved in endothelial transmigration of lymphocytes then the proportion of CD31+ cells should be increased in the lymphocyte population which has crossed an endothelial monolayer. This was tested using two endothelial types, namely human umbilical vein endothelial cells (HUVEC) and rat high endothelial venule (RHEV) cells. As a control, lymphocyte CD45RA and CD45RO expression was also determined since there is a correlation between lymphocytes bearing these isoforms and different migratory patterns. Double labelling techniques showed a close correlation between CD31 and CD45RA expression. With HUVEC monolayers, the transmigrated lymphocyte population was depleted of CD31+ cells. This depletion was even more marked if the HUVEC monolayers had been stimulated with interleukin-1 beta (IL-1 beta). The migrated lymphocytes were enriched for CD31-CD45RO+ cells but depleted of CD31+CD45RA+ cells. In addition, lymphocyte populations depleted of CD31+ cells by immunopanning were also able to migrate across HUVEC monolayers. Taken together these data suggest that lymphocyte CD31 expression is not necessary for transmigration across HUVEC monolayers and, if anything, is negatively correlated with transmigration. With the second endothelial cell type, RHEV cells, there was no consistent change in the proportion of CD31+ lymphocyte in the transmigrated population, suggesting neither a positive nor a negative correlation between CD31+ expression and lymphocyte transmigration across RHEV cells. However, with both endothelial cell types, the migrated lymphocyte populations were enriched for the marker CD45RO. In conclusion, lymphocyte surface expression of CD31 is not necessary for transmigration across the endothelial cell types used in this study, but with both cell types an enrichment of CD45RO+ lymphocytes is seen in the migrated population.

Differential regulation of early phase and late phase responses in human neutrophils by cAMP.

The elevation of intracellular levels of cyclic AMP by forskolin stimulation of adenylate cyclase regulates early and late phase neutrophil responses differentially. Early phase neutrophil responses as measured by shape change in response to chemotactic factors, transmigration across a polycarbonate membrane and priming were unaffected by forskolin-induced elevation of intracellular cAMP. Late phase neutrophil responses such as release of superoxide anions, activation of phospholipase A2 and platelet activating factor (PAF) synthesis were inhibited by increasing intracellular cAMP through the addition of 10 microM forskolin for 10 min prior to stimulation. N-Formyl-methionyl-leucyl-phenylalanine-stimulated arachidonic acid release fell from 9.3% (untreated cells) to 4.6% in forskolin-treated cells. PAF generation was also inhibited from 430 pg/10(6) cells in untreated cells to background levels in forskolin-treated cells (110 pg/10(6) cells). Also, the reduction of cytochrome c by superoxide anions fell from 4.2 nmol/10(6) cells in the absence of forskolin to 2.0 nmol/10(6) cells following forskolin treatment. These results indicate that in neutrophils the elevation of cAMP acts differentially on cellular responses, not affecting early activation events, but markedly inhibiting late events such as the release of inflammatory mediators.

Tumour necrosis factor induction of ELAM-1 and ICAM-1 on human umbilical vein endothelial cells--analysis of tumour necrosis factor-receptor interactions.

Induction of the adhesion molecules ELAM-1 and ICAM-1 on endothelial cells is a key pro-inflammatory effect of tumour necrosis factor (TNF). Earlier work in non-human systems has suggested that unlike other cell types, endothelial cells interact with the N-terminus of the TNF molecule, thereby implying novel TNF receptors on endothelial cells. This is also supported by 125I-TNF cross-linking studies on bovine endothelial cells. The present study aimed to see whether TNF induction of ELAM-1 and ICAM-1 on human umbilical vein endothelial cells (HUVECs) involved novel TNF-receptor interactions. Three approaches were employed. First, antibodies directed at different sites on the TNF molecule were tested for inhibition of TNF-induction of ELAM-1 and ICAM-1 on HUVECs. Inhibition was seen only with antibodies reacting with epitopes outside the N-terminal region. Second, an N-terminal TNF peptide (residues 1-26) failed to induce ELAM-1 and ICAM-1 on HUVECs or antagonise TNF induction of these molecules. Third, HUVEC/125I-TNF cross-linking revealed a major complex characteristic of the known 55 kDa TNF receptor: this was confirmed with receptor-specific monoclonal antibodies. It is concluded that (a) the same part of the TNF molecule interacts with TNF-receptors on HUVECs and other cell types and (b) TNF induction of ELAM-1 and ICAM-1 on HUVECs is mediated via the well-characterized 55 kDa TNF receptor.