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_Lβ₂ 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 α 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_Lβ₂ 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_Lβ₂, 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_Land 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 O⨪₂ 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.