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 surface and underlying extracellular matrix proteins are mediated via surface adhesion molecules, particularly those of the integrin family. Recent studies have suggested that these interactions may be important in modulating gene expression and thus cell function. We tested the hypothesis that engagement of surface integrins on monocytes, simulating integrin/ligand interactions, might modulate monocyte procoagulant activity (PCA) and tumor necrosis factor (TNF) production. Mononuclear cells from pooled rat blood were incubated with a mouse anti-rat VLA-4 (beta1 integrin) antibody (20 microg/ml), or a mouse anti-rat Mac-1 (beta2 integrin) antibody (20 microg/ml), washed, and then cell surface integrins were crosslinked with a goat anti-mouse IgG antibody (20 microg/ml). After incubation at 37 degrees C for 4 hr, supernatants were aspirated 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 beta1 integrin VLA-4 or the beta2 integrin Mac-1 on monocytes significantly increased cellular procoagulant activity and TNF production (PCA mU/10(6) cells: control, 30 +/- 3; anti-VLA-4, 131 +/- 33; anti-Mac-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 stimulate this pathway. By Western blot analysis, crosslinking of the integrin VLA-4 was shown to induce the accumulation of tyrosine phosphorylated proteins, an effect which was inhibited by the tyrosine kinase inhibitor genistein. In parallel studies, genistein inhibited cellular PCA. Considered together, these studies suggest that monocyte activation following integrin engagement is induced by stimulation of tyrosine kinase activity. Thus, in addition to mediating cell adhesion, surface integrins may play a role in modulating cell function at sites of inflammation.
Dackiw
A P
AP
Department of Surgery, Toronto Hospital, University of Toronto, Canada.
Nathens
A B
AB
Marshall
J C
JC
Rotstein
O D
OD
D000818:Animals; D001779:Blood Coagulation Factors / Q000096:biosynthesis*; D002478:Cells, Cultured / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004790:Enzyme Induction / Q000187:drug effects / Q000502:physiology; D016023:Integrins / Q000187:drug effects / Q000502:physiology*; D009000:Monocytes / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis*; D051381:Rats; D017207:Rats, Sprague-Dawley; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis*
D001779:Blood Coagulation Factors; D003432:Cross-Linking Reagents; D016023:Integrins; D014409:Tumor Necrosis Factor-alpha; C045072:macrophage procoagulant activity; D011505:Protein-Tyrosine Kinases