INTEGRIN ENGAGEMENT INDUCES MONOCYTE PROCOAGULANT ACTIVITY AND TUMOR-NECROSIS-FACTOR PRODUCTION VIA INDUCTION OF TYROSINE PHOSPHORYLATION

The specific interactions of monocytes with the endothelial cell surfa ce and underlying extracellular matrix proteins are mediated via surfa ce adhesion molecules, particularly those of the integrin family. Rece nt studies have suggested that these interactions may be important in modulating gene expression and thus cell function. We tested the hypot hesis that engagement of surface integrins on monocytes, simulating in tegrin/ligand interactions, might modulate monocyte procoagulant activ ity (PCA) and tumor necrosis factor (TNF) production. Mononuclear cell s from pooled rat blood were incubated with a mouse anti-rat VLA-4 (be ta 1 integrin) antibody (20 mu g/ml), or a mouse anti-rat Mac-1 (beta 2 integrin) antibody (20 mu g/ml), washed, and then cell surface integ rins were crosslinked with a goat anti-mouse IgG antibody (20 mu g/ml) . After incubation at 37 degrees C for 4 hr, supernatants were aspirat ed and tested for TNF by ELISA and cell pellets were freeze-thawed for measurement of PCA in a one-stage clotting assay. Crosslinking of the beta 1 integrin VLA-4 or the beta 2 integrin Mac-1 on monocytes signi ficantly increased cellular procoagulant activity and TNF production ( PCA mU/10(6) cells: control, 30 +/- 3; anti-VLA-4, 131 +/- 33; anti-Ma c-1, 152 +/- 29; TNF pg/ml: control, 60 +/- 4; anti-VLA-4, 548 +/- 38; anti-Mac-1, 701 +/- 134). Since tyrosine phosphorylation participates in macrophage signaling, we studied whether integrin ligation might s timulate this pathway. By Western blot analysis, crosslinking of the i ntegrin VLA-4 was shown to induce the accumulation of tyrosine phospho rylated proteins, an effect which was inhibited by the tyrosine kinase inhibitor genistein. In parallel studies, genistein inhibited cellula r PCA. Considered together, these studies suggest that monocyte activa tion following integrin engagement is induced by stimulation of tyrosi ne kinase activity. Thus, in addition to mediating cell adhesion, surf ace integrins may play a role in modulating cell function at sites of inflammation. (C) 1996 Academic Press, Inc.