This information is current as (CD11c-CD18, gp150-95) Integrin 2 β x α I Collagen Through

The transduction pathways depending on the ␣ L ␤ 2 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 ␣ L ␤ 2 , 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 ␣ L and ␤ 2 chains were stained by anti-phosphotyrosine antibodies. The adhesion of PMN to pepsinized type I collagen triggered tyrosine phosphorylation of the ␤ 2 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 ␣ L and ␤ 2 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.Type I collagen, a major component of the extra cellular matrix, promotes the adhesion of a variety of cells in solid tissues, influencing many processes such as proliferation, differentiation, migration, and cell shape changes (1). Several receptor molecules have been demonstrated as promoting the adhesion to type I collagen. Among these receptors, some belong to the family of integrins, for instance ␣ 1 ␤ 1 , ␣ 2 ␤ 1 , and ␣ 3 ␤ 1 (2). On the other hand, some types of mobile cells also interact with collagens, for instance polymorphonuclear neutrophils (PMN), 1 a variety of leukocytes circulating in blood, capable of crossing the vascular wall in order to invade the inflamed tissues and to participate in defenses against bacteria or foreign molecules through their property of phagocytosis. In several previous papers, we demonstrated that PMN and type I collagen do interact and began to describe their interactions (3-5).Purified type I collagen is able to bind to PMN in vitro. This binding is followed by the stimulation of some main functions of PMN, such as emission of pseudopods, secretion of lytic enzymes, and liberation of superoxide. We demonstrated that the stimulation of PMN by collagen occurs through two sequences of the ␣ 1 (I) chain, both located in the C-terminal region of the molecules, an RGD sequence corresponding to residues 915-917, and a DGGRYY sequence corresponding to residues 1034 -1039, located at the C-terminal extremity of the chain. The type I collagen molecule, either fibrillar or denatured, is active, whereas pepsinized collagen, lacking the C...

We previously demonstrated that the alpha 1(I) polypeptide chain of collagen can bind and activate polymorphonuclear neutrophils (PMN). In the present experiments, performed in culture grade 96-well plastic plates coated with collagen, fibronectin, or other proteins, adhesion was assessed by staining the adhering cells after 30 min with crystal violet and measuring absorbance at 560 nm, and activation of PMNs was assessed by measuring the amount of O2-formed. Adhesion occurred at 17 and 37 degrees C but activation at 37 degrees C only. Monoclonal antibody anti-CD 18 inhibited adhesion, showing that the receptor of collagen I on PMNs is a beta 2 integrin. On the other hand, adhesion of PMNs to fibronectin was inhibited by monoclonal antibodies to CD18 and to CD11b.