Complement Receptor 3 ( CR 3 , Mac-l , Integrin OtMfl 2 , CDllb / CD 18 ) Is Required for Tyrosine Phosphorylation of Paxillin in Adherent and Nonadherent Neutrophils

Expression of the leukocyte (f12) integrins is required for many functions of activated neutrophils (PMN), even when there is no recognized ligand for any/52 integrin. To investigate the hypothesis that/52 integrins may be involved in a signal transduction pathway related to cytoskeletal reorganization, we examined whether/52 integrins have a role in tyrosine phosphorylation of the cytoskeletal protein paxillin. Treatment of PMN in suspension with phorbol esters, f-Met-Leu-Phe, and TNF-ct resulted in paxillin tyrosine phosphorylation. However, treatment of/5zdeficient (LAD) PMN failed to induce paxillin tyrosine phosphorylation. Normal PMN phosphorylated paxillin in response to adhesion to immune complexes, while the LAD PMN did not. Adhesion of phorbol ester activated-LAD PMN to the extracellular matrix proteins fibronectin, laminin, and vitronectin failed to induce paxillin tyrosine phosphorylation. Treatment of activated normal PMN with mAb directed against the/52 integrin a chains demonstrated that CR3 (o~Mfl2) was required for paxillin phosphorylation. Transfection of the cell line K562 with CR3 confirmed that CR3 ligation resulted in paxillin tyrosine phosphorylation. As a control, K562 transfected with CR2 (CD21) which bound equally avidly to the same complement C3-derived ligand (C3bi) as the CR3 transfectants, showed no enhanced tyrosine phosphorylation of paxillin upon receptor ligation. While both CR2 and CR3 transfectants showed efficient adhesion to a C3bi-coated surface, only the CR3 transfectants spread during adhesion and phosphorylated paxillin. Together these data demonstrate that CR3 is required for paxillin phosphorylation during activation of both adherent and nonadherent PMN. Even PMN activated in suspension or by adhesion to immune complexes, when no CR3 ligand is apparent, still require CR3 for a signal transduction pathway leading to paxillin tyrosine phosphorylation. This pathway is likely to be important for PMN function in inflammation and host defense. C IRCULATING PMN are quiescent cells which became activated in response to a variety of soluble stimuli including cytokines, formyl-methionine-leucinephenylalanine (fMLP), t and phorbol esters. After activation polymorphonuclear neutrophils (PMN) migrate to inflammatory sites, spread on extracellular matrix, activate the NADPH oxidase and arachidonate metabolism, and markedly increase the efficiency of ingestion of opsonized Please address all correspondence to Dr. Irene Graham, Infectious Diseases Division, Rm. 1102, St. Louis Children's Hospital, 1 Children's Place, St. Louis, MO 63110. Telephone: (314) 454-2890; FAX: (314) 454-2836. The current address of V. Michael Holers is Division of Rheumatology, University of Colorado, Denver, CO. 1. Abbreviations used in this paper: AI, attachment index; CR3, complement receptor 3; fMLP, formyl-methionine-leucine-phenylalanine; LAD, leukocyte adhesion deficiency; PDBu, phorbol dibutyrate; PMN, polymorphonuclear neutrophils; TNF-a, tumor necrosis factor-a. particles. Many of these functions of activated PMN are dependent on the presence of complement receptor 3 (CR3), a member of the t2 (CD18) integrin family, even when there is no known CR3 ligand present (20). However, the mechanism of involvement of CR3 in these diverse functions is incompletely understood. A common component of many CR3-dependent functions is the requirement for reorganization of the cytoskeleton. Thus, a testable hypothesis is that one essential role for CR3 in PMN activation is in a signal transduction pathway for cytoskeletal reorganization. A potential target for such a signal transduction event is the microfilament-associated protein paxiUin (15, 26). Paxillin is a vinculin-binding protein which becomes tyrosine phosphorylated in fibroblasts after integrin ligation and which is present in focal contacts (7, 27). In addition, tyrosine phosphorylation of paxiUin increases during IgG-mediated phagocytosis by macrophages and after bombesin activation of 3T3 cells (18, 34). Paxillin also binds to the SH3 domain © The Rockefeller University Press, 0021-9525/94/1 i/1139/9 $2.00 The Journal of Cell Biology, Volume 127, Number 4, November 1994 1139-1147 1139 on July 9, 2017 jcb.rress. org D ow nladed fom of the src family of tyrosine kinases, suggesting it may play a role in localizing signal transduction molecules to sites of organized cytoskeleton (30). We therefore hypothesized that paxillin phosphorylation may be associated with 82 integrin-dependent signal transduction in PMN. In this work, we have investigated whether tyrosine phOsphorylation of paxillin increases during PMN activation, and we have tested the hypothesis that 82 integrins are required for this event. Our results demonstrate that tyrosine phosphorylation of paxillin is induced by a variety of soluble and adhesive stimuli in PMN. In all cases, this phosphorylation is absent in PMN genetically deficient in B2 integrins. Monoclonal antibodies against the various/~2-associated ct chains demonstrate that CR3, the most abundant/32 integrin on PMN, is the essential integrin required for paxillin tyrosine phosphorylation. K562 cells transfected with CR3 confirm that CR3 ligation causes paxillin tyrosine phosphorylation. These data show that CR3 is required for paxillin phosphorylation in response to a broad range of adherent and soluble PMN activators and suggest that paxillin may be an important mediator of the many diverse functions of activated PMN for which CR3 is required. Materials and Methods