Class I phosphoinositide-3-kinases and SRC kinases play a nonredundant role in regulation of adhesion-independent and -dependent neutrophil reactive oxygen species generation

Abstract

Chemoattractant-induced reactive oxygen species (ROS) generation by adherent neutrophils occurs in two phases: the first is very rapid and transient, and the second one is delayed and lasts up to 30-40 min. We examined the role of phosphoinositide 3-kinases (PI3Ks) and Src-family kinases (SFKs) in these responses using human neutrophils treated with inhibitory compounds or murine neutrophils deficient of PI3Kγ or Hck, Fgr, and Lyn. Our studies show that PI3Kγ is indispensable for the early, fMLF-induced ROS generation and AKT and ERK phosphorylation, but is dispensable for the late response to fMLF. Additionally, the response to TNF, an agonist triggering only the delayed phase of ROS generation, was also unaffected in PI3Kγ-deficient neutrophils. In contrast, inhibition of SFKs by a selective inhibitor in human, or SFK deficiency in murine, neutrophils resulted in the inhibition of both the early and late phase of ROS generation, without affecting the early phase of AKT phosphorylation, but inhibiting the late one. Selective inhibitors of PI3Kα and PI3Kδ markedly reduced both the early and late response to fMLF and TNF in human neutrophils. These findings suggest that class IA PI3Ks may be activated by PI3Kγ via Ras in the early phase of the response and by SFKs in the late phase. The evidence that inhibition of SFKs in human, or SFK deficiency in murine, neutrophils results in suppression of Vav phosphorylation at all time points of the response to fMLF or TNF suggests that SFKs are indispensable for Vav phosphorylation.

Figure 1

Different roles of PI3K and SFK activities in fMLF-induced AKT phosphorylation. Human (A–C) or murine (D) neutrophils were plated in fibrinogen-coated wells and stimulated with 1 or 5 μM fMLF for human or mouse cells, respectively, in the presence or the absence of PP2 or wortmannin as described in Materials and Methods. After different times of incubation, cells were lysed and lysates subjected to immunoblot analysis with Abs of the indicated specificity as described in Materials and Methods. (C) reports densitometric analysis, expressed as ratio between the phosphoprotein versus the total specific protein signal, of AKT phosphorylation at 1 min after the addition of fMLF in neutrophils treated or not with 10 μM PP2. Mean results ± SD of three independent experiments are reported. In (E), histograms at the right of the immunoblot report densitometric analysis, expressed as ratio between the phosphoprotein versus the total specific protein signal, of AKT phosphorylation in wild-type versus hck^−/−fgr^−/−lyn^−/− neutrophils. Mean results ± SD of three independent experiments are reported. *p < 0.05.

Figure 2

Different roles of PI3K and SFK activities in fMLF-induced ERK phosphorylation. Human or murine neutrophils were stimulated in the presence or the absence of the indicated inhibitors and lysed as described in Fig. 1 and Materials and Methods. Lysates were subjected to immunoblot analysis with Abs of the indicated specificity as described in Materials and Methods. Human neutrophils stimulated with 1 μM fMLF in the presence or the absence of PP2 (A) or wortmannin (B). Graphs to the right of the immunoblots report densitometric analysis, expressed as ratio between the phosphoprotein versus the total ERK protein signal. One representative experiment of three to four performed with identical results is reported. (C and D) Wild-type or PI3Kγ^−/− murine neutrophils were left untreated or stimulated with 5 μM fMLF and lysed at the time points indicated. Graphs to the right of the immunoblots report densitometric analysis expressed as ratio between the phosphoprotein versus the total ERK (C) or AKT (D) protein signal. Mean results ± SD of three independent experiments are reported. (E) Wild-type or hck^−/−fgr^−/−lyn^−/− neutrophils were stimulated as described above for analysis of ERK phosphorylation. Graphs to the right of the immunoblots report densitometric analysis expressed as ratio between the phosphoprotein versus the total ERK protein signal. Mean results ± SD of three independent experiments are reported. (F and G) Human neutrophils were stimulated with 1 μM fMLF in the presence or the absence of 1 μM AS604850 for analysis of AKT and ERK phosphorylation. One representative experiment of two to three performed with identical results is reported. *p < 0.05, **p < 0.01.

Figure 3

PI3Kγ deficiency results in reduced Ras activation. Neutrophils were isolated from wild-type or PI3γ knockout mice and Ras activation after fMLF (5 μM) stimulation assayed as described in Materials and Methods. (A) Representative detection of active Ras and total Ras is shown. (B) Mean results of Ras activation obtained in four independent experiments. Values were normalized by setting the percentage stimulation of PI3Kγ^+/+ cells at time 0 s to 1. *p < 0.05.

Figure 4

Different roles of SFKs and PI3Kγ in regulation of the early and late phase of fMLF-induced ROS generation in murine neutrophils. (A and B) Neutrophils were isolated from wild-type, hck^−/−fgr^−/−lyn^−/− knockout or PI3γ knockout mice as described in Materials and Methods and plated in fibrinogen-coated wells. Cells were stimulated with 5 μM fMLF, and chemiluminescence was recorded every 1 min and up to 40 min. Mean results ± SD of three to four independent experiments are reported. Graphs in the right panel report the total chemiluminescence detected during the early (0–2 min) or the late (5–40 min) phase of ROS generation.(C) Human neutrophils were stimulated with 1 μM fMLF in the presence or the absence of 1 μM AS604850. Mean results ± SD of four independent experiments are reported. Graph in right panel reports the total chemiluminescence detected during the early (0–2 min) or the late (5–30 min) phase of ROS generation. *p < 0.05, **p < 0.01. KO, Knockout; WT, wild-type.

Figure 5

Late, TNF-induced ROS generation is independent of PI3Kγ. Neutrophils were isolated from wild-type or PI3γ knockout mice as described in Materials and Methods and plated in fibrinogen-coated wells in the presence of 5 ng/ml TNF (A) or in tissue-culture plastic wells in the absence of any stimulus (B). Mean results ± SD of four independent experiments are reported. (C–F) Neutrophils were isolated from wild-type, hck^−/−fgr^−/−lyn^−/−knockout, or PI3γ knockout mice as described in Materials and Methods and plated in tissue-culture plastic wells in the absence of any stimulus. Note that whereas both wild-type and PI3γ knockout neutrophils spread on plastic, either deficiency of Hck, Fgr, and Lyn or treatment with wortmannin totally suppressed spreading. Wild-type (G) or PI3γ knockout (H) neutrophils were plated in tissue-culture plastic wells in the absence of any stimulus and in the presence of 0.5 μM IC 87114. Photos were taken with a ×40 objective after 20 min. KO, Knockout; WT, wild-type.

Figure 6

Inhibitors of the α and δ isoforms of class IA PI3Ks inhibit the early and late phase of ROS generation by human neutrophils. Human neutrophils were plated in fibrinogen-coated wells and stimulated with 10 ng/ml TNF (A, D, G) or 1 μM fMLF (B, E, H) in the absence or the presence of 40 nM TGX-221 (A–C), 0.1 μM compound 15e (D–F), or 0.5 μM IC87114 (G–I). Chemiluminescence was recorded every 1 min and up to 40 min. Mean results ± SD of five experiments are reported. (C, F, and I) Histograms report the total chemiluminescence detected during the early (0–2 min) or late (5–30 min) phase of ROS generation in the absence or the presence of the indicated inhibitor. *p < 0.05, **p < 0.01.

Figure 7

SFKs are indispensable for phosphorylation of Vav during the early and the late phase of ROS generation. Human neutrophils were plated in fibrinogen-coated wells and stimulated with 1 μM fMLF (A) or 10 ng/ml TNF (B) in the presence or the absence of PP2 as described in Materials and Methods. After different times of incubation, cells were lysed and lysates subjected to immunoblot analysis with Abs of the indicated specificity as described in Materials and Methods. One representative experiment of three performed with identical results is reported. (C) Neutrophils were isolated from wild-type or hck^−/−fgr^−/−lyn^−/− knockout mice as described in Materials and Methods and plated in fibrinogen-coated wells. Cells were stimulated with 5 μM fMLF or 5 ng/ml TNF. After different times of incubation, cells were lysed and lysates subjected to immunoblot analysis with Abs of the indicated specificity. Graphs to the right of the immunoblot report densitometric analysis, expressed as ratio between the phosphoprotein versus the total Vav protein signal. Mean results ± SD of three independent experiments are reported. *p < 0.05, **p < 0.01, ***p < 0.001 (D–F). Human neutrophils were plated in fibrinogen-coated wells and stimulated with 10 ng/ml TNF or 1 μM fMLF in the absence or the presence of 100 nM wortmannin (D), 0.5 μM IC87114 alone, (E) or in combination with 0.1 μM compound 15e (F). After different times, cells were lysed and processed for immunoblot analysis as described in Materials and Methods. One representative experiment of two to three performed is reported.

Figure 8

Model for signal transduction mechanisms regulating early, adhesion-independent, and late, adhesion-dependent neutrophil ROS generation. Trimeric G proteins coupled to fMLF receptor(s) (fMLF-R) dissociate in a β/γ heterodimer and a Gαi subunit upon ligand–receptor interaction. The β/γ heterodimer activates PI3Kγ that promotes formation of PIP3. The evidence that Ras activation and phosphorylation of the Ras downstream target ERK is virtually absent in PI3Kγ-deficient murine neutrophils (Figs. 2, 3) suggests that activation of GEFs for Ras by PIP3 may result in activation of class IA PI3Ks. However, this scenario is complicated by the recent demonstration that G protein–coupled receptors impinge on PI3Kγ via a PLCβ/diacylglycerol/RasGRP4 pathway (blue arrows) that plays a key role in PI3K-dependent neutrophil responses (33). Taken together, these findings point to the existence of a positive-feedback circuit of PI3K activation implicating Ras/PI3Kγ/Ras. Gαi, β-arrestin bound to the receptor–G protein complex or the G protein– coupled receptor directly, impinges on SFKs (43, 44). For the sake of simplicity, only the first possibility is highlighted in the drawing. Although mechanism of activation of SFKs by trimeric G protein–coupled receptors in neutrophils is still unknown, several reports pointed to a role of SFKs in signal transduction by this class of receptors (, , –49). Activation of PI3Kγ and SFKs are both essential for the early activation of NADPH oxidase by fMLF, and either deficiency or inhibition of these kinases results in a marked or total suppression of ROS formation in murine or human neutrophils, respectively (Fig. 4, Supplemental Fig. 1). Because inhibitors of the class IA PI3K isoforms PI3Kα and PI3Kδ inhibit the early phase of ROS generation (Fig. 6), it is likely that a positive-feedback circuit triggered by PI3Kγ/Ras results in activation of class IA PI3Ks. Albeit, using selective inhibitors, we found that PI3Kβ plays a minor role in the early response to fMLF, this PI3K isoform has been implicated in the prolonged, late phase of ROS generation by neutrophils adhering to Aspergillus hyphae or immune complexes (26, 50). It is likely that class IA PI3Ks play a highly redundant role in regulation of this response. SFKs play a minor role in activation of class IA PI3Ks (broken arrow) in the early response to fMLF. In fact, inhibition of SFKs by PP2 in human, or deficiency of Hck, Fgr, and Lyn in murine, neutrophils only slightly inhibits, or has no effect on, phosphorylation of the PI3K downstream target AKT, respectively (Fig. 1). SFK inhibition (Supplemental Fig. 1) or deficiency (Fig. 4) result in a total or marked reduction of ROS generation. One important target of SFKs is the Vav family of GEFs for Rac. Inhibition of SFKs by PP2 in human, or deficiency of Hck, Fgr, and Lyn in murine, neutrophils results in a total inhibition of Vav phosphorylation (Fig. 7). Following interaction with chemoattractants, TNF, or other cytokines, neutrophils generate ROS in a fashion characterized by lateness and β₂ integrin dependency (6). This response is totally independent of PI3Kγ (Figs. 4, 5), and requires class IA PI3Ks and SFKs (Figs. 5, 6). SFKs regulate class IA PI3Ks in the course of this response (Fig. 1, Supplemental Fig. 3) and are essential for phosphorylation of Vav proteins (Fig. 7).