Elsner J, Oppermann M, Czech W, Kapp A
Department of Dermatology, University of Freiburg, Germany.
Blood. 1994 Jun 1;83(11):3324-31.
In contrast to C5a, which represents a well-established potent activator of the respiratory burst in polymorphonuclear neutrophilic granulocytes (PMN), the functional role of C3a in the activation of PMN is, so far, poorly understood. Herein, the potential role of human C3a in the activation of the respiratory burst in human PMN was investigated. The release of reactive oxygen species (ROS) of PMN from healthy donors was measured by lucigenin-dependent chemiluminescence. C3a dose-dependently induced the production of ROS in human PMN in the range between 10 ng/mL and 1,000 ng/mL, whereas C3a-desArg was inactive. Flow cytometric measurement of H2O2 by dihydrorhodamine-123 labeling of anti-CD16-stained PMN showed that predominantly neutrophilic PMN are responsible for the C3a-induced activation of the respiratory burst. To exclude that C3a stimulation was caused by contamination with C5a, the specificity of C3a-induced activation of PMN was shown using monoclonal antibodies (MoAbs). Accordingly, the effect of C3a was completely abolished in the presence of Fab fragments of a blocking anti-C3a MoAb. In addition, blockade of the C5a receptor by the anti-C5a receptor (anti-C5aR) MoAb, S5/1, totally inhibited the C5a-induced production of ROS, whereas the C3a response in the presence of the anti-C5aR MoAb was unaffected. The specificity of the response was further confirmed by homologous desensitization after restimulation with C3a. In contrast, no cross-desensitization was observed upon stimulation with C5a. The C3a-induced ROS production by PMN was inhibited by pertussis toxin, indicating the involvement of guanine nucleotide-binding proteins (Gi proteins) in the signal transduction process initiated by C3a. In addition, stimulation of PMN by C3a resulted in a transient increase in the cytosolic free calcium concentration ([Ca2+]i) in a dose-dependent manner. In contrast to C3a-induced ROS production, C3a did not induce a chemotactic response in PMN, indicating functional qualitative differences as compared with C5a. In summary, these results show that C3a is a potent activator of the respiratory burst in human PMN. Therefore, these findings point to a novel role of C3a in the pathogenesis of inflammatory diseases associated with increased C3a levels and PMN activation.
与C5a（一种公认的多形核嗜中性粒细胞（PMN）呼吸爆发的强效激活剂）不同，C3a在PMN激活中的功能作用目前仍知之甚少。在此，研究了人C3a在人PMN呼吸爆发激活中的潜在作用。通过基于光泽精的化学发光法测量健康供体PMN中活性氧（ROS）的释放。C3a在10 ng/mL至1000 ng/mL范围内剂量依赖性地诱导人PMN产生ROS，而C3a去精氨酸则无活性。通过抗CD16染色的PMN的二氢罗丹明-123标记对H2O2进行流式细胞术测量表明，主要是嗜中性PMN负责C3a诱导的呼吸爆发激活。为了排除C3a刺激是由C5a污染引起的，使用单克隆抗体（MoAb）显示了C3a诱导的PMN激活的特异性。因此，在存在阻断抗C3a MoAb的Fab片段的情况下，C3a的作用完全被消除。此外，抗C5a受体（抗C5aR）MoAb S5/1对C5a受体的阻断完全抑制了C5a诱导的ROS产生，而在存在抗C5aR MoAb的情况下C3a反应不受影响。在用C3a重新刺激后的同源脱敏进一步证实了反应的特异性。相反，在用C5a刺激时未观察到交叉脱敏。C3a诱导的PMN产生ROS受到百日咳毒素的抑制，表明鸟嘌呤核苷酸结合蛋白（Gi蛋白）参与了由C3a引发的信号转导过程。此外，C3a对PMN的刺激导致胞质游离钙浓度（[Ca2+]i）以剂量依赖性方式短暂升高。与C3a诱导的ROS产生不同，C3a在PMN中未诱导趋化反应，表明与C5a相比存在功能上的质的差异。总之，这些结果表明C3a是人类PMN呼吸爆发的强效激活剂。因此，这些发现指出了C3a在与C3a水平升高和PMN激活相关的炎症性疾病发病机制中的新作用。
