Beta 2 integrin engagement triggers actin polymerization and phosphatidylinositol trisphosphate formation in non-adherent human neutrophils.

Abstract

Abstract. /$2 integrins are involved in the adhesion of leukocytes to other cells and surfaces. Although adhesion is required for cell locomotion, little is known regarding the way/32 integrin-receptors affect the actin network in leukocytes. In the present study filamentous actin (F-actin) levels in non-adherent human neutrophils have been measured by phalloidin staining after antibody cross-linking of/$2 integrins. Antibody engagement of/$2 integrins resulted in a rapid and sustained (146 and 131% after 30 and 300 s, respectively) increase in the neutrophil F-actin content. This is in contrast to stimulation with N-formyl-l-methionyl-lleucyl-/-phenylalanine (fMLP), which causes a prompt and pronounced but rapidly declining rise in F-actin (214 and 127% after 15 and 300 s, respectively). Priming neutrophils with 1 nM PMA, a low concentration that did not influence the F-actin content per se, increased the magnitude of the/$2 integrin-induced response but had no effect on the kinetics (199% after 30 s and 169% after 300 s). Removal of extracellular Ca 2÷ only marginally affected the/$~ integrin-induced F-actin response for cells that were pretreated with PMA whereas the response for nonprimed cells was reduced by half. This suggests that even though extracellular Ca 2÷ has a modulatory effect it is not an absolute requirement for/$2 integrin-induced actin polymerization. /$2 integrin engagement did not affect the resting cellular level of cAMP arguing against a role of cAMP in/$2 integrin-induced actin assembly. The lack of a cAMP signal might instead explain, at least in part, the prolonged F-actin response triggered by/$2 integrins, since addition of cAMP and 1-isobutylmethylxanthine (IBMX) caused a prompt reversal of the/32 integrin-induced F-actin elevation in electropermeabilized neutrophils. Engagement of/32 integrins, as previously shown for activation of the chemotactic peptide receptor, resulted in a significant formation of PtdInsP3. The capacity of/$2 integrins and chemotactic peptide receptors to induce phosphatidylinositol trisphosphate (PtdlnsP3) formation correlated with their ability to induce actin polymerization.

Role of p190RhoGAP in β2 Integrin Regulation of RhoA in Human Neutrophils

“…The extensive changes in morphology exhibited by neutrophils during spreading and locomotion are associated with ␤ 2 integrininduced intracellular signals that, among other things, cause rearrangement of the actin-based cytoskeleton (10). In many cell types the small GTPases of the Rho subfamily have been implicated in cytoskeletal modulations generated by growth factors (11) and integrins (30).…”

“…First, we measured accumulation of radiolabeled guanine nucleotides bound to RhoA in neutrophils that had been labeled with 32 Pi and subjected to engagement of ␤ 2 integrins (Fig. 1) 10,20, and 30 min, respectively (n ϭ 4). We refrained from examining accumulation of 32 P-labeled guanine nucleotides bound to RhoA for longer than 30 min due to dilution of the labeled GDP/GTP pool.…”

“…These signals are associated with a profound alteration in cell morphology that leads to cell spreading and locomotion. It has been reported that these morphological changes are driven by dynamic ␤ 2 integrin-induced rearrangement of the actin cytoskeleton (10), but little is known about the nature of the signal(s) generated by ␤ 2 integrins regulating cytoskeletal rearrangements. However, in other cell types, small GTP-binding proteins that belong to the Rho subfamily, members of the Ras superfamily of small GTPases, have been strongly implicated (11).…”

Expression and Function of Formyl Peptide Receptors on Human Fibroblast Cells

“…Activation of neutrophil FPR with fMLP stimulates disassociation of G ␤␥ , signaling a phosphatidylinositol 3-kinase and Rac-dependent increase in F-actin and redistribution of the cytoskeleton leading to polarization and formation of lamellopodia and filopodia (3,4,27,28). To determine whether fMLP induces actin polymerization in fibroblasts, cells were treated with fMLP for 0 -60 s, fixed, stained for F-actin, and assayed by fluorescence spectrophotometer.…”

Mac-1-dependent tyrosine phosphorylation during neutrophil adhesion

“…Phosphorylation may depend on the ␣-subunit, as suggested by the observation that phosphorylation of paxillin in TNF-treated neutrophils is dependent on Mac-1 but not LFA-1 binding (26). Ligation of ␤ 2 -integrins on polymorphonuclear neutrophils (PMN) also induces an increase in the intracellular calcium concentration (31,36,58) and the formation of D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P 3 ], possibly by tyrosine phosphorylation of phospholipase C (PLC)-␥ and D-myoinositol-trisphosphate 3-kinase, respectively (31,48). Antibody cross-linking of ␤ 2 -integrins on adherent human neutrophils has been shown to trigger activation of p21 ras through tyrosine phosphorylation of the protooncogene product Vav (84).…”