THE BINDING OF TYPE-I COLLAGEN TO LYMPHOCYTE FUNCTION-ASSOCIATED ANTIGEN (LFA)-1 INTEGRIN TRIGGERS THE RESPIRATORY BURST OF HUMAN POLYMORPHONUCLEAR NEUTROPHILS - ROLE OF CALCIUM SIGNALING AND TYROSINE PHOSPHORYLATION OF LFA-1

Monoclonal antibodies to the alpha(L) beta(2) integrin inhibit the bin ding of type I collagen to PMN (polymorphonuclear neutrophil leukocyte s) 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 o f the integrin is involved in the binding, Two sequences of the alpha( 1)(I) polypeptide chain of collagen participate in the process, Experi ments of competitive inhibition by synthetic peptides showed that the sequence RGD (985-917) is used for binding to the cells and DGGRYY (10 34-1039) serves to stimulate PMN, Experiments of radioactive labeling of the cells and affinity chromatography on Sepharose-collagen confirm ed the presence in PMN extracts of two proteins, 95 and 185 kDa, respe ctively, corresponding to the molecular weights of the beta(2) and alp ha(L) chains of the integrin and recognized by their specific monoclon al antibodies. The transduction pathways depending on the alpha(L) bet a(2) integrin do not involve a G protein (ruled out by the use of chol era and pertussis toxins), whereas the cytoskeleton was found to parti cipate in the process, as evidenced by inhibition by cytochalasin B. A fter collagen stimulation, cytoplasmic inositol trisphosphate and calc ium ion increased sharply for less than 2 min, The use of the inhibito rs staurosporine and calphostin C demonstrated that protein kinase C w as involved, Evaluation of the activity of this enzyme showed that, up on stimulation of PMN with collagen I, it was translocated to plasma m embrane. Acrylamide gel electrophoresis of the protein bands correspon ding to the integrin alpha(L) beta(2), followed by immunoblotting usin g monoclonal antibodies to phosphotyrosine, permitted us to demonstrat e that, prior to stimulation by type I collagen, there was no phos pho rylation, whereas after stimulation, both cY,and beta(2) chains were s tained by anti-phosphotyrosine antibodies, The adhesion of PMN to peps inized type I collagen triggered tyrosine phosphorylation of the beta( 2) chain of the integrin, without stimulating O-2radical anion product ion by these cells, whereas their stimulation by complete type I colla gen induced the tyrosine phosphorylation of both alpha(L) and beta(2) subunits, The tyrosine phosphorylation of both integrin subunits durin g transduction of stimuli is a heretofore undescribed phenomenon that may correspond to a new system of transmembrane communication.