Complement Factor C5a Inhibits Apoptosis of Neutrophils-A Mechanism in Polytrauma?

Abstract

Life-threatening polytrauma results in early activation of the complement and apoptotic system, as well as leukocytes, ultimately leading to the clearance of damaged cells. However, little is known about interactions between the complement and apoptotic systems in PMN (polymorphonuclear neutrophils) after multiple injuries. PMN from polytrauma patients and healthy volunteers were obtained and assessed for apoptotic events along the post-traumatic time course. In vitro studies simulated complement activation by the exposure of PMN to C3a or C5a and addressed both the intrinsic and extrinsic apoptotic pathway. Specific blockade of the C5a-receptor 1 (C5aR1) on PMN was evaluated for efficacy to reverse complement-driven alterations. PMN from polytrauma patients exhibited significantly reduced apoptotic rates up to 10 days post trauma compared to healthy controls. Polytrauma-induced resistance was associated with significantly reduced Fas-ligand (FasL) and Fas-receptor (FasR) on PMN and in contrast, significantly enhanced FasL and FasR in serum. Simulation of systemic complement activation revealed for C5a, but not for C3a, a dose-dependent abrogation of PMN apoptosis in both intrinsic and extrinsic pathways. Furthermore, specific blockade of the C5aR1 reversed C5a-induced PMN resistance to apoptosis. The data suggest an important regulatory and putative mechanistic and therapeutic role of the C5a/C5aR1 interaction on PMN apoptosis after polytrauma.

Keywords: C5a; apoptosis; neutrophils; polytrauma.

Figure 1

Mean fluorescence intensity (MFI) of C5aR (A) and C3aR (B) on PMN of polytraumatized patients (n = 10) in hours versus healthy controls (H). (n = 12). Results are shown as mean ± SEM. * = p < 0.05 vs. healthy volunteers.

Figure 2

Reduced PMN apoptosis after polytrauma. Time-course of apoptotic events in PMN (n = 10) post trauma vs. healthy donors (n = 12). (A): Annexin positive, 7AAD negative PMN of polytraumatized patients in percentage in the post-traumatic time course; (B,D): Mean fluorescence intensity (MFI) of FasL and FasR on PMN of polytraumatized patients in hours versus healthy controls (H). (C,E): Serum FasL/FasR in pg/mL in serum of polytraumatized patients. Results are shown as mean ± SEM. * = p < 0.05 vs. healthy volunteers.

Figure 3

Inhibition of the extrinsic, CH11 (CD95)-induced apoptosis rate of freshly isolated PMN from healthy volunteers by exposure to C5a (concentration–response curve) in the absence or presence C5aR1 antagonist (C5aRA) (n = 4) (A: original flowcytometric recording; B: summary). (C) Similar conditions under C3a exposure (n = 3). The percentage of apoptotic PMN was determined by Annexin-V/7-AAD staining and flow cytometry. Results are shown as mean ± SEM. * = p < 0.05.

Figure 4

C5a (n = 4) (A) but not C3a (n = 3) (B) inhibits the intrinsic, H₂O₂-induced apoptosis rate of freshly isolated PMN from healthy male volunteers in a concentration-dependent manner. The percentage of apoptotic PMN was determined by Annexin-V/7-AAD staining and flow cytometry. C5aRA = C5aR1 specific small peptide inhibitor PMX53. Results are shown as mean ± SEM. * = p < 0.05.

Figure 5

Caspase-3 activity measured by a chemiluminescence assay of (A) extrinsic CH11- (n = 3) as well as (B) intrinsic H₂O₂-induced apoptosis in freshly isolated PMN from healthy male volunteers (n = 3). C5aRA = C5aR1 specific small peptide inhibitor PMX53. Results are shown as mean ± SEM.

Figure 6

Inhibition of PI3-kinase by wortmannin (W) results in a partial reversal of the C5a-caused reduction of PMN apoptosis. Comparison of the apoptosis rate of freshly isolated PMN from healthy male volunteers (n = 5) in percent under influence of C5a and W before (transparent) as well as after induction of intrinsic, H₂O₂-induced (black) as well as extrinsic, CH11-induced apoptosis (gray). The percentage of apoptotic PMN was determined by Annexin-V/7-AAD staining and flow cytometry. Results are shown as mean ± SEM. * = p < 0.05.