The Binding of Type I Collagen to Lymphocyte Function-associated Antigen (LFA) 1 Integrin Triggers the Respiratory Burst of Human Polymorphonuclear Neutrophils

Abstract

Monoclonal antibodies to the α(L)β2 integrin inhibit the binding of type I collagen to PMN (polymorphonuclear neutrophil leukocytes) as well as the subsequent stimulation of superoxide production and enzyme secretion elicited by this collagen. Pepsinized collagen still binds PMN but no longer stimulates them. The I domain of the a chain of the integrin is involved in the binding. Two sequences of the α1(I) polypeptide chain of collagen participate in the process. Experiments of competitive inhibition by synthetic peptides showed that the sequence RGD (915-917) is used for binding to the cells and DGGRYY (1034-1039) serves to stimulate PMN. Experiments of radioactive labeling of the cells and affinity chromatography on Sepharose- collagen confirmed the presence in PMN extracts of two proteins, 95 and 185 kDa, respectively, corresponding to the molecular weights of the β2 and α(L) chains of the integrin and recognized by their specific monoclonal antibodies. The transduction pathways depending on the α(L)β2 integrin do not involve a G protein (ruled out by the use of cholera and pertussis toxins), whereas the cytoskeleton was found to participate in the process, as evidenced by inhibition by cytochalasin B. After collagen stimulation, cytoplasmic inositol trisphosphate and calcium ion increased sharply for less than 2 min. The use of the inhibitors staurosporine and calphostin C demonstrated that protein kinase C was involved. Evaluation of the activity of this enzyme showed that, upon stimulation of PMN with collagen I, it was translocated to plasma membrane. Acrylamide gel electrophoresis of the protein bands corresponding to the integrin α(L)β2 followed by immunoblotting using monoclonal antibodies to phosphotyrosine, permitted us to demonstrate that, prior to stimulation by type I collagen, there was no phosphorylation, whereas after stimulation, both α(L) β2 chains were stained by anti-phosphotyrosine antibodies. The adhesion of PMN to pepsinized type I collagen triggered tyrosine phosphorylation of the β2 chain of the integrin, without stimulating O2/. production by these cells, whereas their stimulation by complete type I collagen induced the tyrosine phosphorylation of both α(L) and β2 subunits. The tyrosine phosphorylation of both integrin subunits during transduction of stimuli is a heretofore undescribed phenomenon that may correspond to a new system of transmembrane communication.