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区域疼痛综合征（CRPS）的发展，这是一种潜在的慢性和严重衰弱的状态。目前对这种神秘综合征的发病机制尚未达成共识，CRPS的治疗选择非常有限。潜在疾病机制的识别和潜在治疗方法的临床前评估对于我们治疗甚至预防这种疾病的发生至关重要。 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）治疗反应的报告，与HLA基因座的遗传相关性和CRPS患者皮肤中朗格罕抗原呈递细胞过多。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样变化依赖于抗体产生，并依赖于完整的神经肽信号传导。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产生和自身抗原的区域表达，导致免疫复合物形成，补体固定，并最终导致疼痛敏化。 该提案的主要目的是确定自身免疫对CRPS的贡献，并确定针对这种疾病机制的潜在治疗方法。具体目的是：1）描述自身免疫对CRPS的贡献，并了解自身抗体产生的调节。我们多方面的实验方法包括使用纵向免疫球蛋白同种型分析，B细胞缺陷小鼠，化学和遗传神经肽耗竭，化学交感神经切除术和血清转移实验。还将进行人体样本分析。2)确定补体级联的自身免疫激活在支持CRPS模型中疼痛相关变化和神经突丢失中的作用。在这些实验中，我们将使用药理学试剂和转基因小鼠来鉴定C5a在CRPS模型中支持疼痛的作用。我们将继续测量C5b-9膜攻击复合物的产生及其在损伤感觉神经突中的作用。将定义通过肽能和交感神经信号传导对补体激活的调节，和3）使用鼠和人CRPS患者样品鉴定参与CRPS相关自身免疫的自身抗原。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，以及自身抗原表达和自身抗体产生所需的神经肽和交感神经信号。我们将专注于使用液相色谱/质谱法初步鉴定的5种蛋白质的表达以及针对这些靶标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机制奠定坚实的基础，大大提高我们对CRPS的理解，为未来的CRPS试验确定特定的分子靶点，并可能提出支持其他神秘的慢性疼痛综合征的新机制。该建议有可能确定新的药物治疗，可以减少疼痛和提高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.