Innate Immunity in Experimental Arthritis of Kininogen

描述(由申请人提供)： 类风湿关节炎(RA)是一种免疫介导性、家族性、慢性复发性疾病，最终导致关节破坏。了解炎症介质是至关重要的，因为这些分子为特定的抑制剂和受体拮抗剂提供了药物阻断的途径。最近的啮齿动物关节炎模型表明，细菌细胞壁聚合物可以在遗传易感的大鼠品系中诱导急性和慢性关节炎。肠外注射多糖多糖复合体(PG-APS)可引起全身炎症反应，包括侵蚀性多发性关节炎、肝脏肉芽肿和贫血。我们已经证明实验性炎性关节炎与Lewis大鼠体内激肽释放酶-激肽释放酶系统(KKS)的激活有关，并且在这些易感的Lewis大鼠中选择性地被失活，而在耐药的Buffalo大鼠中不被激活。在PG-APS模型中，一种特定的激肽释放酶抑制剂既可以减少KKS的激活，又可以减少急性炎症变化(水肿、中性粒细胞浸润)，并预防关节炎和全身并发症(脾大、肝大、白细胞增多和急性时相反应)。我们现在集中于先天免疫机制在啮齿动物炎性关节炎模型中的作用，特别是KKS与单核细胞和中性粒细胞的相互作用导致细胞因子的释放。我们探索了两个主要的假设-使用本实验室新的方法。首先，我们假设缺乏高分子激肽原(HK)是释放缓激肽的底物，它是激活血浆激肽释放酶所必需的，并将调节PG-APS在遗传易感的Lewis大鼠中产生的急慢性关节炎和全身炎症。我们发现这些大鼠有一个单点突变$511N，导致N-糖基化，这增加了HK对蛋白质降解的易感性。我们将研究在Lewis大鼠体内加速释放缓激肽和裂解HK(Hka)将通过白细胞上的缓激肽和Hka的细胞受体刺激的先天性免疫系统，特别是巨噬细胞释放炎性细胞因子的机制。我们将使用激肽原基因单点突变导致肝脏分泌受阻和严重血浆缺陷的大鼠品系进一步探索这一假说，我们已将其置于易感的Lewis基因型。我们还将使用人类人类白细胞抗原-B27转基因大鼠作为“自发性”慢性关节炎的模型，我们将用抗HK的单抗进行治疗。其次，我们将比较Lewis和Fischer大鼠在实验性关节炎和PG-APS诱导的全身炎症期间不同程度的激肽原裂解I的炎症程度。我们将测试重组HK多肽、合成肽或该蛋白的抗体(已知可抑制HK或Hka与白细胞的结合)对炎症的调节是否可以防止许多炎症变化。我们将通过敲除它们的结合蛋白uPAR和B2R来研究Hka和缓激肽在小鼠关节炎和全身炎症中的作用。如果这些方法导致关节炎和全身炎症的大幅减少，它可能会导致风湿性疾病的控制，而副作用可能比目前的治疗方法更少。

DESCRIPTION (provided by applicant): Rheumatoid arthritis (RA) is an immtmologically mediated, familial, chronically relapsing disease eventually resulting in joint destruction. Understanding inflammatory mediators is critical because these molecules provide approaches for pharmacologic blockade by specific inhibitors and receptor antagonists. Recent rodent models of arthritis have shown that bacterial cell wall polymers can induce acute and chronic arthritis in genetically susceptible rat strains. Parenterally injected peptidoglycanpolysaccharide complexes (PG-APS) induce a systemic inflammatory response consisting of erosive polyarthritis, hepatic granulomas, and anemia. We have shown that experimental inflammatory arthritis is associated with in vivo activation of the kallikrein-kinln system (KKS) in Lewis rats and is selectively inactivated in these susceptible Lewis rats, but not in resistant Buffalo rats. A specific kallikrein inhibitor decreases both the KKS activation and acute inflammatory changes (edema, neutrophil infiltration) and prevented arthritis and the systemic complications (splenomegaly, hepatomegaly, leukocytosis and the acute phase reaction) in the PG-APS model. We now focus on the role of innate immune mechanisms in models of rodent inflammatory arthritis, specifically the interaction of the KKS with monocytes and neutrophils resulting in release of cytokines. We explore two major hypothesis-using approaches new to this laboratory. First, we hypothesize that the lack of high molecular weight kininogen (HK), which is required for activation of plasma kallikrein, is the substrate for release of bradykinin and will modulate the acute and chronic arthritis and systemic inflammation produced by PG-APS in genetically susceptible Lewis rats. We have found that these rats have a single point mutation $511N leading to N-glycosylation which increases the susceptibility of HK to proteolysis. We will examine the mechanisms by which accelerated release of bradykinin and cleaved HK (HKa) in Lewis rats will augment inflammatory cytokine release by the innate immune system, particularly macrophages, stimulated through cellular receptors for bradykinin and HKa on leukocytes. This hypothesis will be explored further using a rat line with a single point mutation in kininogen leading to a block in hepatic secretion and severe plasma deficiency, which we have placed onto the susceptible Lewis genotype. We will also use the human HLA-B27 transgenic rat as a model of "spontaneous" chronic arthritis, which we will treat with a monoclonal antibody to HK. Second, we will compare the degree of inflammation in Lewis vs. Fischer rats with variable degrees of kininogen cleavage I during experimental arthritis and systemic inflammation induced by PG-APS. We will test whether modulation of inflammation by] recombinant HK polypeptides, synthetic peptides, or antibodies to that protein, which are known to inhibit binding of HK or HKa to] leukocytes, can prevent many of the inflammatory changes. We will examine the role of HKa and bradykinin using knockouts of their binding proteins, uPAR and B2R, in murine arthritis and systemic inflammation. If these approaches result in a substantial decrease in arthritis and systemic inflammation, it could lead to the control of rheumatic diseases with potentially fewer side effects than current therapeutic approaches.