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 microM), a specific ERK inhibitor, followed by incubation with C5a (1 microg 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.
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 microM), a specific ERK inhibitor, followed by incubation with C5a (1 microg 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.
Authors: Justin T Schwartz; Jason H Barker; Justin Kaufman; Drew C Fayram; Jenna M McCracken; Lee-Ann H Allen Journal: J Immunol Date: 2012-02-22 Impact factor: 5.422
Authors: Rebecca L McCullough; Megan R McMullen; Dola Das; Sanjoy Roychowdhury; Michael G Strainic; M Edward Medof; Laura E Nagy Journal: Mol Immunol Date: 2016-06-06 Impact factor: 4.407
Authors: Faikah Gueler; Song Rong; Wilfried Gwinner; Michael Mengel; Verena Bröcker; Sylvia Schön; Tim Friedrich Greten; Heiko Hawlisch; Thomas Polakowski; Karsten Schnatbaum; Jan Menne; Hermann Haller; Nelli Shushakova Journal: J Am Soc Nephrol Date: 2008-08-27 Impact factor: 10.121