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 µM), a specific ERK inhibitor, followed by incubation with C5a (1 µg 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.