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.

Src Family Kinases and Syk Are Required for Neutrophil Extracellular Trap Formation in Response to β-Glucan Particles

“…Consistently with the evidence that Syk deficiency results in defective integrin-dependent ROS generation, but does not affect responses triggered by trimeric G protein-coupled receptor [38,39], we found that the early adhesion-independent fMLP-induced phase of ROS generation [see [26]] was equal in wild-type and Syk-deficient neutrophils (fig. 7g).…”

“…The plates were read in a fluorescence microplate reader (Victor 3, PerkinElmer; Waltham, Mass., USA) with a filter setting of 485/535 nm (excitation/emission). Coating of the wells with fibrinogen was performed because we noticed that on plain plastic the SYTOX green signal detected in unstimulated cells displayed quite a high variability with different cell preparations probably due to neutrophil spreading on plastic [see [26]].…”

“…As the latter mice showed similar results, for clarity, the data from these two groups were pooled and defined as the control group. Mouse neutrophils were isolated as described previously [26]. Briefly, bone marrow cells were flushed from the bones using HBSS without Ca 2+ and Mg 2+ , and containing 0.1% BSA.…”

Neutrophils promote Alzheimer's disease–like pathology and cognitive decline via LFA-1 integrin

“…Isoluminol-based chemiluminescence assays were prepared in Hank's balanced salt solution supplemented with 0.5 mM CaCl 2 and 5 mM d-glucose (HGCa) in the presence of horseradish peroxidase (HRP) and isoluminol in a temperature-controlled Multilabel Count Victor X5 (PerkinElmer). The assays were carried out in dark 96-well plates precoated with 250 µg/ml human fibrinogen in HGCa containing 100 µM isoluminol and 8 U/ml HRP 53,54 . Following the addition of 1 × 10 5 human neutrophils or 7.5 × 10 5 mouse neutrophils, the plates were left at 37 °C for 10 min and the reaction initiated by introducing the stimulus (see text for concentrations).…”

Photo-enhancement of macrophage phagocytic activity via Rac1-mediated signaling pathway: Implications for bacterial infection

“…Vav1 is a GEF that is primarily expressed in hematopoietic cells (Tong et al , 2013, Zenker et al , 2014. It has been reported that Vav1 can be phosphorylated in vivo by Src kinases (Fumagalli et al , 2013), suggesting that the guanine nucleotide-exchange activity of Vav1 can be modulated by tyrosine phosphorylation. Vav1 may also be regulated via the interaction of its PH domain with the lipid products of PI3K (Gambardella andVermeren, 2013, Navarro-Lerida et al , 2012).…”