C5a delays apoptosis of human neutrophils via an extracellular signal-regulated kinase and Bad-mediated signalling pathway

Aims We recently demonstrated that complement fragment C5a delays apoptosis of human neutrophils via induction of the phosphatidylinositol-3 kinase (PI 3-K) pathway. In the present study, we examined whether C5a modulates neutrophil survival through the extracellular signal-regulated kinase (ERK) and Bad-mediated signalling pathway. Methods Human neutrophils were isolated by percoll gradient and preincubated for 1 h with or without PD98059 (20 muM), a specific ERK inhibitor, followed by incubation with C5a (1 mug mL(-1)) for 24 h. Apoptosis was quantified by flow cytometry, using propidium iodide nuclear staining. Extracellular signal-regulated kinase downstream signalling events were evaluated by measuring the expression of cytosolic total and phosphorylated p44/p42 proteins, and Bad phosphorylation using immunoblot analyses. These time-dependent analyses were performed over a brief exposure to C5a (0-30 min). Modulation of cytosolic caspase-9 and caspase-3 activity was measured by Western blot analyses. Results C5a inhibited neutrophil apoptosis (P = 0.04), which was abrogated in the presence of PD98059 (P = 0.04). Time-dependent effect of C5a on p44/p42 phosphorylation was rapid, peaked at 5 min, and was abrogated by the ERK inhibitor (P = 0.04). In addition, brief stimulation of neutrophils with C5a induced phosphorylation of Bad, which was inhibited by the ERK inhibitor (P = 0.03). Further, C5a suppressed the proteolytic cleavage of caspase-9 and caspase-3, which was reversed by ERK inhibition. Finally, blockade of both the ERK (with PD98059) and PI 3-K (with wortmannin) pathways did not induce additive inhibition of neutrophil apoptosis by C5a. Conclusion This study demonstrates that in addition to the PI 3-K pathway, C5a also inhibits neutrophil apoptosis via an ERK-signalling pathway, resulting in phosphorylation of Bad and blockade of proteolytic cleavage of caspases. The activation of this additional survival-signalling pathway may be another important cellular mechanism that enhances neutrophil survival in inflammatory states.