Complement fragment C5a and neutrophils have been implicated in the pathogenesis of renal disease and C5a has also been shown to delay apoptosis of human neutrophils via a transcription-independent pathway. However, transcription-dependent pathways have not been well described. The present study examined whether activation of HL-60-derived neutrophils by C5a modulates the transcription of two members of the Bcl2 family, Bax (pro-apoptotic) and Bcl2 (anti-apoptotic) molecules, and whether the cAMP-response element-binding protein (CREB) transcription factor mediates these effects through the phosphatidylinositol 3-kinase (PI3K)/Akt and extra-cellular signal-regulated kinase (ERK) signalling pathways.Materials and methods
The human promyelocytic leukaemia HL-60 cell line was differentiated into neutrophils using 1·25% DMSO. Differentiated cells were incubated with recombinant human C5a for 30–120 min with, or without, pretreatment with wortmannin or PD98059. The cells were lysed and quantified for gene-specific Bax and Bcl2 mRNA. In separate experiments, cells were incubated with C5a for 5–30 min with, or without, pretreatment with wortmannin, PD98059, or alkaline phosphatase. Cells were then lysed and immunoblotted using antihuman phospho-CREB (Ser133) antibody. Apoptosis was assessed by measuring active caspase-3 in differentiated HL-60 cells.Results
C5a inhibited caspase-3 activation in HL-60-derived neutrophils (P = 0·003). C5a significantly increased the expression of Bcl2 mRNA (P = 0·028), which was time-dependent, peaking at 30 min, and was abrogated in the presence of either wortmannin or PD98059 (both P = 0·028). The C5a had no impact on Bax mRNA expression. The Bax: Bcl2 mRNA ratio markedly decreased at 30 min (P = 0·028). Time-dependent effect of C5a on CREB phosphorylation was demonstrable and rapid, peaking at 5 min, and was abrogated by either wortmannin or PD98059 (both P = 0·028). Phosphorylation of CREB, but not of Akt and ERK, was inhibited by alkaline phosphatase (P = 0·028). The effect of C5a on Bcl2 mRNA expression was abrogated by alkaline phosphatase (P = 0·028). The Bax: Bcl2 mRNA ratio markedly increased in the presence of alkaline phosphatase (P = 0·046).Conclusions
This study demonstrates that C5a induces Bcl2 mRNA transcription in HL-60-derived neutrophils, which is mediated in part by CREB through the convergence of the PI3K/Akt and ERK-signalling pathways.