Autoimmune Mechanisms Supporting Chronic Pain after Limb Injury

 描述(由申请人提供)： 与战争有关的伤害、骨折和四肢手术都可能导致复杂的区域疼痛综合征(CRPS)，这是一种潜在的慢性和严重虚弱的状态。目前对这种神秘综合征的发病机制尚无共识，CRPS的治疗选择也非常有限。识别潜在的疾病机制和临床前评估潜在的治疗方法对于我们治疗甚至预防这种疾病的发生是至关重要的。最近的观察表明，CRPS可能是一种自身免疫性疾病。证据包括自身抗体的鉴定，静脉注射免疫球蛋白(IVIG)治疗的反应报告，与HLA基因座的遗传关联，以及CRPS患者皮肤中过多的朗格罕抗原提呈细胞。我们自己的研究表明，在经过充分验证的小鼠骨折/石膏模型中，CRPS样变化依赖于抗体的产生，并依赖于完整的神经肽信号。指导我们工作的中心假设是，肢体创伤导致神经调节的自身抗体的产生和自身抗原的局部表达，从而导致免疫复合体的形成、补体的结合，最终导致疼痛敏感化。这项建议的主要目标是定义自身免疫对CRPS的贡献，并确定针对这种疾病机制的潜在治疗方法。其具体目的是：1)阐明自身免疫在CRPS中的作用，了解自身抗体产生的调节。我们的多方面实验方法包括使用纵向免疫球蛋白同型分析、B细胞缺陷小鼠、化学和遗传神经肽耗竭、化学交感神经切除和血清转移实验。还将进行人体样本分析。2)明确自身免疫激活的补体级联在CRPS模型中支持疼痛相关改变和轴突丢失中的作用。在这些实验中，我们将使用药物和转基因小鼠来确定C5a在CRPS模型中支持疼痛的作用。我们将继续测量C5b-9膜攻击复合体的产生，以及它在破坏感觉神经突起中的作用。3)用小鼠和人CRPS患者标本鉴定与CRPS相关的自身免疫相关的自身抗原。我们假设自身免疫的靶点包括CRPS肢体中差异上调的蛋白质，神经肽和交感信号是自身抗原表达和自身抗体产生所必需的。我们将集中于用液相色谱/质谱仪初步鉴定的5种蛋白质的表达以及针对这些靶点的自身抗体产生的时间过程。这些实验将为进一步探索创伤后自身免疫的机制奠定坚实的基础，极大地提高我们对CRPS的理解，为未来的CRPS试验确定特定的分子靶点，并可能为其他神秘的慢性疼痛综合征提供新的机制支持。这项建议有可能确定新的药物治疗方法，可以减轻疼痛，提高CRPS患者的生活质量。

 DESCRIPTION (provided by applicant): War-related injuries, fractures and surgeries on the limbs can all lead to the development of complex regional pain syndrome (CRPS), a potentially chronic and profoundly debilitating state. Currently there is no consensus on the pathogenesis of this enigmatic syndrome, and the treatment options for CRPS are very limited. The identification of the underlying disease mechanisms and preclinical evaluation of potential approaches to therapy are essential in order for us to treat or even prevent this disorder from occurring. Recent observations suggest that CRPS may be an autoimmune disorder. Evidence includes the identification of autoantibodies, reports of response to intravenous immunoglobulin (IVIG) treatment, genetic associations with HLA loci and the overabundance of Langerhan's antigen presenting cells in the skin of CRPS patients. Our own studies indicate that CRPS-like changes in the well-validated mouse fracture/cast model are reliant upon antibody production, and rely on intact neuropeptide signaling. The central hypothesis guiding our work is that that limb trauma causes the neurally-regulated production of autoantibodies and the regional expression of autoantigens leading to immune complex formation, complement fixation and, ultimately, pain sensitization. The primary objective of this proposal is to define the autoimmune contribution to CRPS and identify potential treatments targeting this disease mechanism. The specific aims are; 1) To delineate the contribution of autoimmunity to CRPS and understand the regulation of autoantibody production. Our multi-faceted experimental approach includes the use of longitudinal immunoglobulin isotype analysis, B-cell deficient mice, chemical and genetic neuropeptide depletion, chemical sympathectomy and serum transfer experiments. Human sample analysis will be pursued as well. 2) To identify the role of autoimmune activation of the complement cascade in supporting pain-related changes and neurite loss in the CRPS model. In these experiments we will use pharmacological agents and transgenic mice to identify the roles of C5a in supporting pain in the CRPS model. We will go on to measure the production of the C5b-9 membrane attack complex, and its role in damaging sensory neurites. The regulation of complement activation by peptidergic and sympathetic signaling will be defined, and 3) To identify the autoantigens involved in CRPS-related autoimmunity using murine and human CRPS patient samples. We hypothesize that the targets of autoimmunity include proteins differentially up-regulated in CRPS limbs, and that neuropeptide and sympathetic signaling are required for autoantigen expression and autoantibody production. We will focus on the expression of 5 proteins tentatively identified using liquid chromatography/mass spectrometry and the time course of autoantibody production against these targets. These experiments will establish a rigorous foundation for further exploring the mechanisms of post- traumatic autoimmunity, greatly improve our understanding of CRPS, identify specific molecular targets for future CRPS trials, and potentially suggest novel mechanisms supporting other enigmatic chronic pain syndromes. This proposal has the potential to identify novel pharmacologic treatments that could reduce pain and enhance the quality of life of CRPS patients.