Activation of Human Neutrophils byMycobacterium tuberculosisH37Ra Involves Phospholipase Cγ2, Shc Adapter Protein, and p38 Mitogen-Activated Protein Kinase

Abstract

Recent studies have shown that human neutrophils play a significant protective role in mycobacteria infection. When encountered with mycobacteria, neutrophils exhibit the typical early bactericidal responses including phagocytosis and generation of reactive oxygen intermediates (ROI), but the underlying mechanisms are largely unknown. The present study shows that stimulation of neutrophils with an attenuated strain of Mycobacterium tuberculosis H37Ra (Mtb) led to a tyrosine kinase-dependent ROI production in these cells. Stimulation with Mtb induces a rapid and transient tyrosine phosphorylation of several proteins, one of which was identified as phospholipase C gamma 2 (PLC gamma 2). Several tyrosine-phosphorylated proteins were associated with the PLC gamma 2 precipitates from Mtb-stimulated neutrophils, of which pp46 was characterized as the Shc adapter protein. A role for PLC gamma 2-Shc association in the generation of ROI is supported by the observations that stimulation with Mtb causes the activation of p38 mitogen-activated protein kinase (MAPK), a downstream target of the Shc/Ras signaling cascade, and that the effect of genistein on ROI production coincided with its ability to inhibit both PLC gamma 2-Shc association and p38 MAPK activation. Moreover, pretreatment of neutrophils with a PLC inhibitor markedly suppresses the Mtb-stimulated ROI production as well as p38 MAPK activation in these cells. Taken together, these results indicate that stimulation of neutrophils with Mtb triggers the tyrosine phosphorylation of PLC gamma 2 and its association with Shc, and that such association is critical for the Mtb-stimulated ROI production through activating p38 MAPK.

Induction of p38 Mitogen-activated Protein Kinase Reduces Early Endosome Autoantigen 1 (EEA1) Recruitment to Phagosomal Membranes

“…Upon activation of p38, EEA1 is displaced from endocytic membranes (28). Interestingly, p38 MAPK is activated in cells infected with M. tuberculosis (29). In this work we examined whether the activation of p38 plays a role in EEA1 exclusion from M. tuberculosis phagosomes and in inhibiting the maturation of mycobacterial phagosomes.…”

Induction of apoptosis in human neutrophils by Mycobacterium tuberculosis is dependent on mature bacterial lipoproteins

“…This conclusion is supported by our observation that inhibition of Akt did not enhance PMN apoptosis. Phosphorylated p38 MAPK is involved in an abundance of cellular signalling cascades involving activation and regulation of ROS production by NADPH oxidase in PMNs [27,37,38]. We found that p38 MAPK was a key mediator in Mtb-induced apoptosis.…”

“…PMNs respond to mycobacteria by exhibiting characteristic bactericidal activity involving phagocytosis [24,25], activation of NADPH oxidase with subsequent ROS production [26,27], exocytosis of specific granules [13], and killing of mycobacteria through oxidative or non-…”

“…We found that p38 MAPK was a key mediator in Mtb-induced apoptosis. This finding may be explained by upstream modulation of NADPH oxidase activity since inhibition of p38 phosphorylation results in failed ROS production [27] whereas inhibition of NADPH oxidase does not affect p38 activity. The involvement of p38 is consistent with findings reported by Aleman et al (2004), who showed that p38 MAPK was activated in circulating PMNs from patients with tuberculosis, and that these PMNs showed accelerated apoptosis compared to uninfected control cells [31].…”

“…Human PMNs were isolated from heparinized peripheral blood from healthy donors by density-gradient separation on Polymorphprep® as described elsewhere [27]. RPMI 1640 supplemented with 2 mM L-glutamine and 10% heatinactivated FBS (RPMI) was used in PMN incubations and experiments.…”