Neutrophil-mediated oxidative burst and host defense are controlled by a Vav-PLCgamma2 signaling axis in mice

Abstract

Oxidative burst, a critical antimicrobial mechanism of neutrophils, involves the rapid generation and release of reactive oxygen intermediates (ROIs) by the NADPH oxidase complex. Genetic mutations in an NADPH oxidase subunit, gp91 (also referred to as NOX2), are associated with chronic granulomatous disease (CGD), which is characterized by recurrent and life-threatening microbial infections. To combat such infections, ROIs are produced by neutrophils after stimulation by integrin-dependent adhesion to the ECM in conjunction with stimulation from inflammatory mediators, or microbial components containing pathogen-associated molecular patterns. In this report, we provide genetic evidence that both the Vav family of Rho GTPase guanine nucleotide exchange factors (GEFs) and phospholipase C-gamma2 (PLC-gamma2) are critical mediators of adhesion-dependent ROI production by neutrophils in mice. We also demonstrated that Vav was critically required for neutrophil-dependent host defense against systemic infection by Staphylococcus aureus and Pseudomonas aeruginosa, 2 common pathogens associated with fatal cases of hospital-acquired pneumonia. We identified a molecular pathway in which Vav GEFs linked integrin-mediated signaling with PLC-gamma2 activation, release of intracellular Ca2+ cations, and generation of diacylglycerol to control assembly of the NADPH oxidase complex and ROI production by neutrophils. Taken together, our data indicate that integrin-dependent signals generated during neutrophil adhesion contribute to the activation of NADPH oxidase by a variety of distinct effector pathways, all of which require Vav.

Figure 1. Adherent neutrophils require Vav to undergo oxidative burst in response to integrin ligands.

ROI production in bone marrow–derived neutrophils was measured using lucigenin chemiluminescence. (A and B) WT and Vav^NULL neutrophils were stimulated in tubes coated with pRGD (A) or anti-CD18 (B). (C) Alternatively, neutrophils were stimulated in fibrinogen-coated tubes (100 μg/ml) with soluble TNF-α (50 ng/ml) (Fgn+TNF). (D) As a positive control, cells were stimulated with PMA (50 ng/ml) for 10–20 minutes or left untreated (Untx), and samples were performed in triplicate. Data shown are representative of at least 5 independent experiments. RLU, relative light units.

Figure 2. Vav^NULL mice exhibit severely compromised host defense against bacterial pneumonia.

(A and B) WT and Vav^NULL bone marrow–derived neutrophils were stimulated with P. aeruginosa (A) or S. aureus (B) and monitored for oxidative burst by lucigenin chemiluminescence. (C) WT and Vav^NULL mice were infected with P. aeruginosa by intratracheal instillation, and mouse viability was monitored over 7 days. n = 5 per group. P < 0.002. (D) After intratracheal infection with S. aureus, WT and Vav^NULL mice were sacrificed at 20 hours, and BAL was collected for quantitation of bacterial load in the lungs. n = 5 per group. P < 0.016.

Figure 3. Vav is required for NADPH oxidase assembly during adhesion.

(A) Bone marrow–derived neutrophils from WT and Vav^NULL mice were distributed onto untreated glass coverslips, coverslips coated with fibronectin (Fbn, 1 μg/ml), or uncoated coverslips with soluble PMA (50 ng/ml). After 30 minutes, cells were fixed, permeabilized, and stained with anti-gp91 (green) and anti-p40^phox (red). (B) Neutrophils were treated as in A and stained with anti-gp91 (green) and anti-p47 (red). Data shown are representative of at least 100 cells examined per condition.

Figure 4. Vav regulates PLC-γ2 signaling during neutrophil adhesion.

(A and B) WT and Vav^NULL neutrophils were stimulated on pRGD-coated tissue culture plates and lysed at the indicated time points. Lysates were resolved by SDS-PAGE and analyzed by Western blot for activated phosphorylated PLC-γ2 (pPLC-γ2) followed by total PLC-γ2 (A) and phosphorylated ERK (pERK) followed by total ERK2 (B). Data shown are representative of at least 3 independent experiments.

Figure 5. PLC-γ2 effector pathways through calcium and DAG are required for adhesion-dependent oxidative burst.

(A) Neutrophils were loaded for the indicated time periods in a 10-μM solution of the calcium chelator BAPTA and stimulated in luminometer tubes coated with pRGD (1 μg/ml). (B) Neutrophils were stimulated with the indicated concentrations of the PKC inhibitor calphostin C (Cal) and stimulated in luminometer tubes coated with pRGD (1 μg/ml). ROI production was measured using lucigenin chemiluminescence as in Figure 1. Data shown are representative of at least 3 independent experiments.

Figure 6. Adherent neutrophils require PLC-γ2 to undergo oxidative burst in response to integrin ligands.

ROI production in bone marrow–derived neutrophils was measured using lucigenin chemiluminescence. (A and B) WT and PLC-γ2^–/– neutrophils were stimulated in tubes coated with pRGD (A) or anti-CD18 (B). (C) Alternatively, neutrophils were stimulated in fibrinogen-coated tubes (100 μg/ml) with soluble TNF-α (50 ng/ml). (D) As a positive control, cells were stimulated with PMA (50 ng/ml) for 10–20 minutes or were left untreated, and samples were performed in triplicate. Data shown are representative of 3 independent experiments.