PECAM-1–dependent neutrophil transmigration is independent of monolayer PECAM-1 signaling or localization

Abstract

Platelet endothelial cell adhesion molecule-1 (PECAM-1/CD31), a tyrosine phosphoprotein highly expressed on endothelial cells and leukocytes, is an important component in the regulation of neutrophil transendothelial migration. Engagement of endothelial PECAM-1 activates tyrosine phosphorylation events and evokes prolonged calcium transients, while homophilic engagement of neutrophil PECAM-1 activates leukocyte ␤-integrins. Although PECAM-1 modulates polymorphoneutrophil transmigration via homophilic PECAM-1-PECAM-1 interaction, the mechanisms underlying endothelial PECAM-1 function are unknown. Proposed mechanisms include (1) formation of a haptotactic gradient that "guides" neutrophils to the cell-cell border, (2) service as a "passive ligand" for neutrophil PECAM-1, ultimately mediating activation of neutrophil ␤ integrins, (3) regulation of endothelial calcium influx, and (4) mediation of SH2 protein association, and/or (5) catenin and non-SH2 protein interaction. Utilizing PECAM-1-null "model" endothelial cells (REN cells), we developed a neutrophil transmigration system to study PECAM-1 mutations that specifically disrupt PECAM-1-dependent signaling and/or PECAM-1 cell localization. We report that interleukin-1␤ (IL-1␤) elicits PECAM-1-dependent transmigration that requires homophilic PECAM-PECAM-1 engagement, but not heterophilic neutrophil PECAM-1 interactions, and is intercellular adhesion molecule-1 dependent. Conversely, whereas IL-8 and leukotriene-B 4 -mediated transmigration is PECAM-1-independent, PECAM-1 and IL-8-dependent transmigration represent separable and additive components of cytokine-induced transmigration. Surprisingly, neither monolayer PECAM-1-regulated calcium signaling, cell border localization, nor the PE-CAM-1 cytoplasmic domain was required for monolayer PECAM-1 regulation of neutrophil transmigration. We conclude that monolayer (endothelial cell) PECAM-1 functions as a passive homophilic ligand for neutrophil PECAM-1, which after engagement leads to neutrophil signal transduction, integrin activation, and ultimately transmigration in a stimulus-specific manner. IntroductionAlthough the mechanisms regulating initial cell contact and adhesion have been well described, 1 the mechanisms underlying endothelial regulation of polymorphoneutrophil (PMN) transmigration are not as well understood. It has been proposed that neutrophil transmigration through endothelial cell (EC) monolayers involves 2 distinct pathways. The first, called type I transendothelial migration (TEM), requires direct neutrophil activation with chemotactic agents such as N-formyl-methionyl-leucinyl-phenylalanine (FMLP), leukotriene-B 4 (LTB 4 ), or interleukin-8 (IL-8). The second, type II TEM ("endothelium dependent"), involves endothelial prestimulation by inflammatory mediators, such as IL-1␤, that induce endothelial expression of surface proteins and secretion of factors that ultimately activate neutrophils and promote transmigration. 2 The mechanisms regulating neutrophil transmigration are thus stimulus-and ...

Ultrastructural Localization of Platelet Endothelial Cell Adhesion Molecule (PECAM-1, CD31) in Vascular Endothelium

“…A recent report (O'Brien et al 2003) describes five mechanisms by which CD31 could modulate the neutrophil migration across endothelial cell monolayers that is induced by cytokines (e.g., IL-1␤). The authors also note that transendothelial migration induced by neutrophil chemoattractants (LTB 4 ) or chemokines (IL-8) is CD31-independent.…”

“…Therefore, attachment of leukocytes to endothelium is not dependent on CD31, whereas transmigration of leukocytes is, at least for certain stimuli. Blocking antibodies could also act by interfering with cell signaling mechanisms (Newton-Nash and Newman 1999; Henshall et al 2001;Ilan et al 2001;Newman et al 2001;O'Brien et al 2003).…”

Ninjurin1 Deficiency Attenuates Susceptibility of Experimental Autoimmune Encephalomyelitis in Mice

“…Antibodies-For a custom-made anti-moue Ninjurin1 antibody, rabbits were immunized with keyhole limpet hemocyanin-conjugated synthetic peptide-bearing mouse Ninjurin1 residues 26 -37 (Ab 26 -37 ) and 1-15 (Ab [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] ) following standard procedures (Peptron, Inc. and Abfrontier, Inc.); these antiNinjurin1 antibodies were purified in each case with antigenspecific affinity chromatography. The isotype rabbit IgG purified with a Protein A column (Upstate Biotechnology) from the serum of normal rabbits was used for the in vivo blocking experiment in the EAE model.…”

“…Each siRNA was transfected in Raw264.7 cells by Nucleofector (Amaxa) at a concentration of Ͻ200 pmol per sample. After 24 h, the Ninjurin1 knockdown efficiency was determined by Western blotting of anti-GFP and the custommade anti-Ninjurin1 antibody (Ab [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] ).…”

Folate Deficiency Enhances Delayed Neuronal Death in the Hippocampus After Transient Cerebral Ischemia

“…Therefore, initial sPECAM-1 might be of predictive value for the short-term outcome of stroke ( Zaremba and Losy, 2002b). O'Brien et al(2003) demonstrated that PECAM-1 mediates neutrophil migration through IL-1 beta stimulated endothelial cells. It has shown that hyperhomocysteinemia at moderate level activates human monocyte and induces cytokine expression including tumor necrosis alpha, IL-1 beta, IL-6, IL-8, and IL-12 (Su et al, 2005).…”