Shutov Leonid P, Warwick Charles A, Shi Xiaoyu, Gnanasekaran Aswini, Shepherd Andrew J, Mohapatra Durga P, Woodruff Trent M, Clark J David, Usachev Yuriy M
Department of Pharmacology, University of Iowa, Iowa City, Iowa 52242.
Department of Anesthesia, Veterans Administration Palo Alto Healthcare System and Stanford University, Palo Alto, California 94305.
J Neurosci. 2016 May 4;36(18):5055-70. doi: 10.1523/JNEUROSCI.3249-15.2016.
The complement cascade is a principal component of innate immunity. Recent studies have underscored the importance of C5a and other components of the complement system in inflammatory and neuropathic pain, although the underlying mechanisms are largely unknown. In particular, it is unclear how the complement system communicates with nociceptors and which ion channels and receptors are involved. Here we demonstrate that inflammatory thermal and mechanical hyperalgesia induced by complete Freund's adjuvant was accompanied by C5a upregulation and was markedly reduced by C5a receptor (C5aR1) knock-out or treatment with the C5aR1 antagonist PMX53. Direct administration of C5a into the mouse hindpaw produced strong thermal hyperalgesia, an effect that was absent in TRPV1 knock-out mice, and was blocked by the TRPV1 antagonist AMG9810. Immunohistochemistry of mouse plantar skin showed prominent expression of C5aR1 in macrophages. Additionally, C5a evoked strong Ca(2+) mobilization in macrophages. Macrophage depletion in transgenic macrophage Fas-induced apoptosis mice abolished C5a-dependent thermal hyperalgesia. Examination of inflammatory mediators following C5a injection revealed a rapid upregulation of NGF, a mediator known to sensitize TRPV1. Preinjection of an NGF-neutralizing antibody or Trk inhibitor GNF-5837 prevented C5a-induced thermal hyperalgesia. Notably, NGF-induced thermal hyperalgesia was unaffected by macrophage depletion. Collectively, these results suggest that complement fragment C5a induces thermal hyperalgesia by triggering macrophage-dependent signaling that involves mobilization of NGF and NGF-dependent sensitization of TRPV1. Our findings highlight the importance of macrophage-to-neuron signaling in pain processing and identify C5a, NGF, and TRPV1 as key players in this cross-cellular communication.
This study provides mechanistic insight into how the complement system, a key component of innate immunity, regulates the development of pain hypersensitivity. We demonstrate a crucial role of the C5a receptor, C5aR1, in the development of inflammatory thermal and mechanical sensitization. By focusing on the mechanisms of C5a-induced thermal hyperalgesia, we show that this process requires recruitment of macrophages and initiation of macrophage-to-nociceptor signaling. At the molecular level, we demonstrate that this signaling depends on NGF and is mediated by the heat-sensitive nociceptive channel TRPV1. This deeper understanding of how immune cells and neurons interact to regulate pain processing is expected to facilitate mechanism-based approaches in the development of new analgesics.
补体级联反应是固有免疫的主要组成部分。最近的研究强调了C5a及补体系统其他成分在炎性疼痛和神经性疼痛中的重要性，尽管其潜在机制大多未知。特别是，尚不清楚补体系统如何与伤害感受器进行通讯以及涉及哪些离子通道和受体。在此，我们证明了完全弗氏佐剂诱导的炎性热痛觉过敏和机械性痛觉过敏伴随着C5a上调，并且通过C5a受体（C5aR1）基因敲除或用C5aR1拮抗剂PMX53治疗可显著减轻。将C5a直接注射到小鼠后爪可产生强烈的热痛觉过敏，该效应在TRPV1基因敲除小鼠中不存在，并且被TRPV1拮抗剂AMG9810阻断。小鼠足底皮肤的免疫组织化学显示巨噬细胞中C5aR1表达显著。此外，C5a在巨噬细胞中引起强烈的Ca(2+)动员。转基因巨噬细胞Fas诱导凋亡小鼠中的巨噬细胞耗竭消除了C5a依赖性热痛觉过敏。注射C5a后对炎性介质的检测显示NGF迅速上调，NGF是一种已知可使TRPV1敏感化的介质。预先注射NGF中和抗体或Trk抑制剂GNF-5837可预防C5a诱导的热痛觉过敏。值得注意的是，NGF诱导的热痛觉过敏不受巨噬细胞耗竭的影响。总体而言，这些结果表明补体片段C5a通过触发涉及NGF动员和NGF依赖性TRPV1敏感化的巨噬细胞依赖性信号传导来诱导热痛觉过敏。我们的发现突出了巨噬细胞与神经元之间的信号传导在疼痛处理中的重要性，并确定C5a、NGF和TRPV1是这种跨细胞通讯中的关键参与者。
本研究为固有免疫的关键组成部分补体系统如何调节疼痛超敏反应的发展提供了机制性见解。我们证明了C5a受体C5aR1在炎性热敏化和机械性敏化发展中的关键作用。通过关注C5a诱导热痛觉过敏的机制，我们表明该过程需要募集巨噬细胞并启动巨噬细胞与伤害感受器之间的信号传导。在分子水平上，我们证明该信号传导依赖于NGF，并由热敏感伤害性通道TRPV1介导。对免疫细胞和神经元如何相互作用以调节疼痛处理的这种更深入理解有望促进基于机制的新镇痛药开发方法。
