SummaryDuring the inflammatory response, the adhesion molecule PECAM plays a crucial role in transendothehal migration, the passage of leukocytes across endothelium. We report here an additional role for PECAM in the subsequent migration of monocytes through the subendothelial extraceHular matrix. PECAM has six immunoglobulin (Ig) superfamily domains. Monoclonal antibodies whose epitopes map to domains 1 and/or 2 selectively block monocyte migration through the endothelial junction, whereas those that map to domain 6 block only the migration through the extracellular matrix, trapping the monocyte between the endothelium and its basal lamina. Therefore, transendothelial migration (diapedesis) and passage through extracellnlar matrix (interstitial migration) are distinct and separable phases of monocyte emigration. Furthermore, distinct and separate Ig domains of PECAM are involved in mediating these two steps.

The inflammatory response involves sequential adhesive interactions between cell adhesion molecules of leukocytes and the endothelium. Unlike the several adhesive steps that precede it, transendothelial migration (diapedesis), the step in which leukocytes migrate between apposed endothelial cells, appears to involve primarily one adhesion molecule, platelet–endothelial cell adhesion molecule (PECAM, CD31). Therefore, we have focused on PECAM as a target for antiinflammatory therapy. We demonstrate that soluble chimeras made of the entire extracellular portion of PECAM, or of only the first immunoglobulin domain of PECAM, fused to the Fc portion of IgG, block diapedesis in vitro and in vivo. Furthermore, the truncated form of the PECAM-IgG chimera does not bind stably to its cellular ligand. This raises the possibility of selective anti-PECAM therapies that would not have the untoward opsonic or cell-activating properties of antibodies directed against PECAM.

We have reported that overexpression of Neu leads to heregulin-stimulated neurite outgrowth and the tyrosine-phosphorylation of Neu and other cellular proteins in PC12 cells. Considering that Neu/ErbB2 alone is not able to functionally couple to heregulin, we looked for the possible involvement of ErbB3 in these neurite outgrowth and tyrosine phosphorylation responses. We found that heregulin stimulates the tyrosine phosphorylation of endogenous ErbB3 protein in PC12 cells and that this phosphorylation, like that of Neu, is greatly enhanced in cells that overexpress Neu. Furthermore, overexpression of ErbB3 in PC12 cells led to heregulin-stimulated neurite extension. In addition to becoming tyrosine-phosphorylated, Neu/ErbB2 and ErbB3 associate with each other, and each associates with the 85-kDa regulatory subunit (p85) of phosphatidylinositol 3-kinase in a heregulin-dependent manner. Thus, Neu/ErbB2 and ErbB3 appear to cooperate to mediate the heregulin signal in PC12 cells. Like heregulin, epidermal growth factor (EGF) also stimulate the tyrosine phosphorylation of both Neu and ErbB3. However, there are clear differences between the EGF- and heregulin-stimulated phosphorylations of ErbB3. In the heregulin response, two tyrosine-phosphorylated forms of ErbB3 are detected. Of these, only the more quickly migrating form (on SDS-polyacrylamide gel electrophoresis) is found to be associated with Neu, whereas the other, more slowly migrating form is uniquely capable of forming stable complexes with p85. In the EGF response, at least two tyrosine-phosphorylated forms of ErbB3 are detected, but these phosphoproteins have distinctly lower apparent molecular weights compared with the heregulin-stimulated ErbB3 phosphoproteins and do not complex with p85. Thus the formation of a stable ErbB3-p85 complex in PC12 cells is a unique outcome of heregulin signaling that correlates with the differences in cell morphology induced by the activated EGF receptor and the Neu tyrosine kinase.