Primary granule release from human neutrophils is potentiated by soluble fibrinogen through a mechanism depending on multiple intracellular signaling pathways

Abstract

. Primary granule release from human neutrophils is potentiated by soluble fibrinogen through a mechanism depending on multiple intracellular signaling pathways. Am J Physiol Cell Physiol 287: C1264 -C1272, 2004. First published June 30, 2004 doi:10.1152/ajpcell.00177.2004 is a potent activator of neutrophil degranulation. The intracellular signaling mechanisms involved in the potentiating effect of fibrinogen on fMLP-induced primary granule release from human neutrophils were investigated. Fibrinogen caused a significant leftward shift of the concentration-response curve of fMLP-induced elastase release. An antibody against Mac-1 (CD11b/CD18) prevented the potentiating effect of fibrinogen, suggesting that soluble fibrinogen potentiates fMLP-induced degranulating effect by a mechanism mediated by the integrin Mac-1. Fibrinogen enhanced fMLP-induced tyrosine phosphorylation in human neutrophils and markedly enhanced the phosphorylation of mitogen-activated protein kinases (MAPK) caused by fMLP. However, U0126, an inhibitor of p44/42 MAPK activation, or SB-203580, an inhibitor of p38 MAPK, did not alter the effect of fibrinogen on fMLP-induced elastase release. Wortmannin, a phosphatidylinositol 3-kinase (PI3K) kinase inhibitor, and genistein, a nonspecific tyrosine kinase inhibitor, strongly inhibited fMLP-induced elastase release both in the presence and in the absence of fibrinogen. An Akt/PKB inhibitor failed to alter the potentiating effect of fibrinogen, suggesting that the effect of fibrinogen is mediated by Akt-independent pathways. Gö6976, an inhibitor of classical PKC isoforms, caused a significant inhibition of fMLPinduced elastase release in the presence or absence of fibrinogen, while nonselective inhibitors of PKC, Ro 31-8220, GF-109203X, and staurosporine, caused potentiation of fMLP-induced elastase release. We conclude that fibrinogen potentiation of primary granule release induced by fMLP is mediated by the integrin CD11b/CD18 through pathways dependent on PI3K and tyrosine kinases, but other regulatory mechanisms may be also involved.

Response to Comment on “Neutrophil Apoptosis: Selective Regulation by Different Ligands of Integrin αMβ2”

“…However, the dose of PMA used itself also induces very little ERK1/2 and Akt/ PKB phosphorylation in the absence of fibrinogen. We believe that our observations are consistent with a well-established paradigm in integrin signaling: low concentrations of an agonist, in this case PMA, activate the integrin via PKC-dependent insideout signaling, allowing soluble fibrinogen to bind to ␣ M ␤ 2 (3,4,5). ERK1/2 and Akt/PKB phosphorylation depends on outside-in signaling arising from occupancy of the integrin since inhibitors of integrin clustering, a consequence of ligand binding, blocked PMN survival.…”

Neutrophils phagocytose activated platelets in vivo: a phosphatidylserine, P-selectin, and β2 integrin–dependent cell clearance program

“…Collagen and ADP directly activate neutrophils. 25 We therefore verified the down-regulation of the PSGL-1 receptor (a bona fide event associated to neutrophil activation) after 60 seconds; that is, before platelet P-selectin expression (Table S1). Indeed, both ADP and collagen, but not TRAP-6, significantly downregulated PSGL-1 expression as effectively as the positive control for neutrophil activation, fMLP (Table S1).…”

Involvement of SHIP in TLR2-Induced Neutrophil Activation and Acute Lung Injury

“…Bone marrow neutrophils (2 ϫ 10 6 /0.5 ml) were cultured in RPMI 1640/0.2% FCS. The p38 inhibitor SB203580 (0.4 and 12 M), the MEK 1/2 inhibitor U0126 (3 and 12 M), and the isoform nonspecific PI3K inhibitor LY294002 (5 and 25 M) were used at concentrations previously shown by our laboratory and others to inhibit selectively the kinase of interest (31,32).…”