The Src family kinases Hck and Fgr regulate neutrophil responses to N-formyl-methionyl-leucyl-phenylalanine

Abstract

The chemotactic peptide formyl-methionyl-leucyl-phenilalanine (fMLP) triggers intracellular protein tyrosine phosphorylation leading to neutrophil activation. Deficiency of the Src family kinases Hck and Fgr have previously been found to regulate fMLP-induced degranulation. In this study, we further investigate fMLP signaling in hck-/-fgr-/- neutrophils and find that they fail to activate a respiratory burst and display reduced F-actin polymerization in response to fMLP. Additionally, albeit migration of both hck-/-fgr-/-mouse neutrophils and human neutrophils incubated with the Src family kinase inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2) through 3-microm pore size Transwells was normal, deficiency, or inhibition, of Src kinases resulted in a failure of neutrophils to migrate through 1-microm pore size Transwells. Among MAPKs, phosphorylation of ERK1/2 was not different, phosphorylation of p38 was only partially affected, and phosphorylation of JNK was markedly decreased in fMLP-stimulated hck-/-fgr-/- neutrophils and in human neutrophils incubated with PP2. An increase in intracellular Ca(2+) concentration and phosphorylation of Akt/PKB occurred normally in fMLP-stimulated hck-/-fgr-/- neutrophils, indicating that activation of both phosphoinositide-specific phospholipase C and PI3K is independent of Hck and Fgr. In contrast, phosphorylation of the Rho/Rac guanine nucleotide exchange factor Vav1 and the Rac target p21-activated kinases were markedly reduced in both hck-/-fgr-/- neutrophils and human neutrophils incubated with a PP2. Consistent with these findings, PP2 inhibited Rac2 activation in human neutrophils. We suggest that Hck and Fgr act within a signaling pathway triggered by fMLP receptors that involves Vav1 and p21-activated kinases, leading to respiratory burst and F-actin polymerization.

FIGURE 1

Superoxide generation by hck^−/−fgr^−/− PMNs is defective in response to fMLP, but not PMA. WT or hck^−/−fgr^−/− (KO) bone marrow PMNs were isolated and assayed as described in Materials and Methods. A, Time-course of superoxide generation in response to 1 μM fMLP. Cells were preincubated for 10 min at 37°C in the absence or presence of 2 μg/ml CB before challenge with fMLP. Mean results of duplicate assays of five independent experiments ± SEM are reported. B, Superoxide generation by CB-treated PMNs in response to two different doses of fMLP after 2 min of stimulation. Mean results of duplicate assays of two independent experiments ± SEM are reported. C, Su-peroxide generation by CB-treated PMNs in response to 50 ng/ml PMA. Mean results of duplicate assays of five independent experiments ± SEM are reported. D, Su-peroxide generation by CB-treated, WT PMNs preincu-bated for 10 min at 37°C in the absence or presence of 10 μM PP2.

FIGURE 2

F-actin formation in hck^−/−fgr^−/− PMNs is defective in response to fMLP. WT or hck^−/− fgr^−/− (KO) bone marrow PMNs were isolated and assayed as described in Materials and Methods. A, F-actin formation after 25 s from stimulation with different doses of fMLP. B, F-actin formation at different times after stimulation with 1 μM fMLP. Mean results of duplicate assays of three independent experiments ± SEM are reported.

FIGURE 3

hck^−/−fgr^−/− mouse PMNs and human PMN treated with a Src family kinase inhibitor migrate normally in response to fMLP through 3-μm pore size Transwells, but are unable to migrate through 1-μm pore size Transwells. WT or hck^−/−fgr^−/− (KO) bone marrow PMNs and human PMNs were isolated and assayed as described in Materials and Methods. A, Mouse PMNs; B, human PMNs. PP2 refers to PMNs preincubated for 10 min at 37°C in the presence of 10 μM PP2. Mean results of duplicate assays of three independent experiments ± SEM are reported.

FIGURE 4

Decreased protein tyrosine phosphorylation in resting and fMLP-stimulated conditions in hck^−/−fgr^−/− bone marrow and PP2-treated human PMNs. A, WT or hck^−/−fgr^−/− (KO) bone marrow PMNs were stimulated with 1 μM fMLP for the times indicated after preincubation for 10 min at 37°C in the presence of CB (see Fig. 1 legend). At the end of the incubation, the reaction was terminated and proteins were extracted and subjected to immunoblot analysis with anti-phosphotyrosine Abs as described in Materials and Methods. Numbers at the left indicate migration of m.w. markers. Arrow points indicate major proteins whose tyrosine phosphorylation increased upon treatment with fMLP or whose extent of phosphorylation was markedly different in WT vs hck^−/−fgr^−/− PMNs. One of several experiments performed is reported. B, Human PMNs were preincubated with 10 μM PP2 for 10 min at 37°C and treated with CB as described in Fig. 1 legend for mouse PMNs. Cells were either left untreated or stimulated with 1 μM fMLP for the times indicated. Numbers at the left indicate migration of m.w. markers. One of three experiments performed is reported.

FIGURE 5

Increase in intracellular Ca²⁺ concentration ([Ca²⁺ ]_i) in response to fMLP is not different in WT and hck^−/−fgr^−/− PMNs. WT or hck^−/−fgr^−/− (KO) bone marrow PMNs were pretreated with CB as described in Fig. 1 legend before stimulation with 1 μM fMLP. One representative of three experiments performed is reported.

FIGURE 6

Phosphorylation of Akt/PKB in response to fMLP is not defective in murine hck^−/−fgr^−/− PMNs or human PMNs treated with the Src family inhibitor PP2. A, WT or hck^−/−fgr^−/− (KO) bone marrow PMNs were preincubated for 30 min at room T in the absence or presence of 100 nM wortmannin and stimulated with 1 μM fMLP for the times indicated after preincubation for 10 min at 37°C in the presence of CB (see Fig. 1 legend). B, Human PMNs were preincubated with 10 μM PP2 for 10 min at 37°C and stimulated as described in Fig. 4 legend. At the end of the incubation, the reaction was terminated and proteins were extracted and subjected to immunoblot analysis with anti-phospho-Akt (Ser⁴⁷³) Abs as described in Materials and Methods. After stripping, blots were incubated with anti-protein Abs. One representative experiment of three performed is reported.

FIGURE 7

Wortmannin inhibits superoxide generation in murine and human PMNs. WT or hck^−/−fgr^−/− (KO) bone marrow PMNs (A) or human PMNs (B) were preincubated for 30 min at room T in the absence or presence of 100 nM wortmannin and stimulated with 1 μM fMLP for the times indicated in the conditions described in Figs. 1 and 4 legend. Mean results ± SEM of one representative of three experiments performed is reported.

FIGURE 8

MAPK phosphorylation in response to fMLP in murine hck^−/−fgr^−/− PMNs or human PMNs treated with the Src family inhibitor PP2. A, WT or hck^−/−fgr^−/− (KO) bone marrow PMNs were stimulated with 1 μM fMLP for the times indicated after preincubation for 10 min at 37°C in the presence of CB (see Fig. 1 legend). B, Human PMNs were preincubated with 10 μM PP2 for 10 min at 37°C and stimulated as described in Fig. 4 legend. At the end of the incubation, the reaction was terminated and proteins were extracted and subjected to immunoblot analysis with anti-phosphospecific Abs as described in Materials and Methods. After stripping, blots were incubated with anti-protein Abs. One representative of three experiments performed is reported. C and D, Densitometric analysis of the immunoblots shown in A and B, respectively.

FIGURE 9

Fgr and Hck deficiency in murine or treatment with PP2 in human PMNs results in decreased phosphorylation of JNK. A, WT or hck^−/−fgr^−/− (KO) bone marrow PMNs were stimulated with 1 μM fMLP for the times indicated after preincubation for 10 min at 37°C in the presence of CB (see Fig. 1 legend). B, Human PMNs were preincubated with 10 μM PP2 for 10 min at 37°C and stimulated as described in Fig. 4 legend. At the end of the incubation, the reaction was terminated and proteins were extracted and subjected to immunoblot analysis with phosphospecific Abs directed agains JNK as described in Materials and Methods. After stripping, blots were incubated with anti-JNK Abs. One representative of three experiments performed is reported. Shown at the right are the densitometric analyses of the immunoblots.

FIGURE 10

PP2 inhibits fMLP-induced activation of Rac. Human PMNs were preincubated with 10 μM PP2 for 10 min at 37°C and stimulated as described in Fig. 4 legend. Cell lysates were incubated with GST-PBD and pull-down assays performed as described in Materials and Methods. A, Representative experiment showing the amount of GTP-bound Rac2 pulled down from the lysate or total Rac2. B, Mean results of densitometric analysis ± SEM of anti-Rac2 blots in three independent pull-down experiments.

FIGURE 11

Fgr and Hck deficiency in murine or treatment with PP2 in human PMNs results in decreased phosphorylation of Vav1. A, WT or hck^−/−fgr^−/− (KO) bone marrow PMNs were stimulated with 1 μM fMLP for the times indicated after preincubation for 10 min at 37°C in the presence of CB (see Fig. 1 legend). B, Human PMNs were preincubated with 10 μM PP2 for 10 min at 37°C and stimulated as described in Fig. 4 legend. At the end of the incubation, the reaction was terminated and proteins were extracted and subjected to immunoprecipitation with anti-Vav1 Abs followed by immunoblotting with anti-phosphotyrosine Abs as described in Materials and Methods. After stripping, blots were incubated with anti-Vav-1 Abs. One representative of three experiments performed is reported. Shown at the right are the densitometric analyses of the immunoblots. C, WT or hck^−/−fgr^−/− (KO) bone marrow PMNs or human PMNs were stimulated in the assay conditions described above. At the end of the incubation, the reaction was terminated and proteins were extracted and subjected to immunoblot analysis with anti-phosphospecific Abs directed against Vav as described in Materials and Methods. After stripping, blots were incubated with anti-Vav1 Abs. One representative of two experiments performed is reported.

FIGURE 12

Fgr and Hck deficiency in murine or treatment with PP2 in human PMNs results in decreased phosphorylation of PAKs. A, WT or hck^−/−fgr^−/− (KO) bone marrow PMNs were stimulated with 1 μM fMLP for the times indicated after preincubation for 10 min at 37°C in the presence of CB (see Fig. 1 legend). B, Human PMNs were preincubated with 10 μM PP2 for 10 min at 37°C and stimulated as described in Fig. 4 legend. At the end of the incubation, the reaction was terminated and proteins extracted and subjected to immunoblot analysis with anti-phosphospecific Abs directed against PAK1 and PAK2 as described in Materials and Methods. After stripping, blots were incubated with anti-PAK1/2 Abs. One representative of two experiments performed is reported. Shown at the right are the densitometric analyses of the immunoblots.

FIGURE 13

Model for fMLP-induced activation of NADPH oxidase in PMNs. fMLP receptor(s) trigger activation of Vav, P-Rex1, and DOCK2 GEFs. P-Rex1 requires the βγ subunits of trimeric G proteins and increased formation of phosphatidylinositol 3P by PI3K. Vav requires phosphorylation by Hck and Fgr and likely occurs downstream of G α_i. DOCK2 is also placed downstream of phosphatidylinositol 3P formation. P-Rex1-, Vav1-, and DOCK2-dependent Rac activation pathways are required to activate the NADPH oxidase. F-actin formation and cell migration are induced by either the Vav- or the P-Rex1-dependent pathway; deficiency of one of the two pathways results in a partial reduction in F-actin formation, which is however insufficient to decrease cell migration. In contrast, DOCK2 gives a major contribution to Rac activation, and its deficiency results in alteration of cell migration. (See the text for details and references.) Likely, via phosphorylation of additional substrates, Hck and Fgr regulate PMN deformability and possibly transmigration through cell barriers.