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


CONCLUSIONS
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.
Perianayagam
M C
MC
Dialysis Research Laboratory, Division of Nephrology, Department of Medicine, Tufts-New England Medical Center, Boston, MA 02111, USA.
Balakrishnan
V S
VS
Pereira
B J G
BJ
Jaber
B L
BL
D017209:Apoptosis / Q000502:physiology*; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism; D002478:Cells, Cultured; D015936:Complement C5a / Q000276:immunology*; D003600:Cytosol / Q000378:metabolism; D005434:Flow Cytometry / Q000379:methods; D006801:Humans; D015151:Immunoblotting / Q000379:methods; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism*; D009504:Neutrophils / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D015398:Signal Transduction / Q000502:physiology; D051025:bcl-Associated Death Protein
C494819:BAD protein, human; D002352:Carrier Proteins; D051025:bcl-Associated Death Protein; D015936:Complement C5a; D019869:Phosphatidylinositol 3-Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases