Cytokines And Growth Factors In Autoimmune Diseases

转化生长因子-β是一种多功能的免疫调节剂，具有免疫抑制和促炎作用。转化生长因子-β协调对炎症反应的进展和消退至关重要的事件。这种复杂的协同作用涉及炎症细胞的募集、淋巴细胞的激活、巨噬细胞功能的调节以及内皮细胞、单核细胞和淋巴细胞表达黏附分子。在哺乳动物中发现的三种亚型中，转化生长因子-β1是体内影响这些免疫功能的主要亚型。因此，在我们的小鼠中，转化生长因子-β1基因的靶向破坏导致以进行性多灶性炎症为特征的致命表型也就不足为奇了。我们之前已经确定了这种复杂的免疫失调背后的一些分子机制。我们进一步证明，超过一半的空白小鼠在子宫中死亡，这种胚胎致死性与早期造血和胚胎卵黄囊血管系统的缺陷有关。活体出生的转化生长因子-β1缺失小鼠在出生后第7天就表现出炎症反应，这与两类MHC分子的表达增强和白细胞与血管内皮细胞的粘附性增加是一致的。在随后的分析中，我们发现，基因缺失的小鼠还表现出自身免疫表现，如核抗原抗体水平升高和肾脏免疫复合物沉积。我们还证明，这种自身免疫表型可以通过骨髓移植将空白小鼠的骨髓细胞转移到受到致死照射的野生型小鼠身上。有趣的是，我们对与纤维连接蛋白的细胞结合序列和肝素结合序列相对应的合成肽的研究表明，它们对空小鼠有改善作用，特别是在减少淋巴细胞与血管内皮细胞的黏附以及减少唾液和泪腺的炎症方面。MHC-I和-II类抗原在转化生长因子-β1缺失小鼠体内的表达增强，提示这两种抗原在小鼠自身免疫反应的发生发展中起重要作用。随后，我们通过产生转化生长因子-β1/MHC-II双零小鼠来表征MHC-II类抗原的作用。这些小鼠没有任何炎性渗透、循环自身抗体或肾小球免疫复合体沉积的证据。相反，这些小鼠表现出髓外造血，存活时间略长于转化生长因子-β1基因缺失的小鼠。因此，MHC-II类抗原对转化生长因子-β1缺失小鼠的自身免疫表达是必不可少的，并可能与该生长因子共同调节造血。为了阐明MHC-I类分子在转化生长因子-β1缺失小鼠发病机制中的确切作用，我们在MHC-I/β2-微球蛋白(β2M)缺乏的遗传背景下产生了转化生长因子-β1缺失小鼠。？2M缺失小鼠缺乏MHC-I类抗原和外周CD8+T细胞的表达，这是因为？2M对于MHC-I类重链和轻链异源二聚体的正确组装、转运和细胞表面表达是必不可少的。Kaplan-Meier分析显示，转化生长因子-β1/β2M双缺失小鼠的存活率显著提高。虽然炎症病变的组织分布与转化生长因子-β1基因缺失小鼠相似，但双基因缺失小鼠的炎症严重程度显著减轻，尤其是心脏。流式细胞仪分析显示胸腺、脾和淋巴结中CD8+T细胞水平降低，表明胸腺中T细胞发育受损。这些小鼠还表现出骨髓生成增加，炎症减少。此外，这些小鼠的循环自身抗体和肾小球免疫复合体沉积水平显著降低。这些结果表明，MHC-I类分子影响转化生长因子-β1基因缺失小鼠的自身免疫和炎症的发生，CD8+T细胞可能参与了炎症的发生。为了评估循环中活跃的转化生长因子-β1水平的治疗潜力，我们通过将转化生长因子-β1(+/-)小鼠与在肝脏中表达转化生长因子-β1并在血液中分泌转化生长因子-β1的转基因小鼠(TG)杂交，在转化生长因子-β1缺失的背景(转化生长因子-β1(-/-/TG))上产生内分泌表达转化生长因子-β1的小鼠。转化生长因子-β1(-/-/TG)小鼠的存活曲线与转化生长因子-β1(-/-)小鼠相似，表明未能挽救致死表型。然而，转化生长因子-β1(-/-/TG)组小鼠的血清转化生长因子-β1水平恢复到正常水平，在所有组织中都有表达，尤其是在肾脏和脾组织中。组织病理学显示，所有靶组织的炎症反应都有所减轻，特别是心脏。有趣的是，与转化生长因子-b(-/-)小鼠不同的是，转化生长因子-β1(-/-/甘油三酯)小鼠的肾脏中的肾小球肾炎与转化生长因子-b(-/-)小鼠相似。因此，模型的表型提示了循环活性的转化生长因子-β1在抗炎中的潜在作用，但不能挽救转化生长因子-β1缺失小鼠的致死性，表明自分泌转化生长因子-β1的关键作用。 目前，正在研究转化生长因子-β亚型的特殊作用，并针对特定的细胞类型进行表达。

TGF-beta is a multifunctional immunomodulator with immunosuppressive as well as proinflammatory actions. TGF-beta coordinates events critical to the progression and resolution of inflammatory responses. This complex coordination involves recruitment of inflammatory cells, activation of lymphocytes, modulation of macrophage function, and expression of adhesion molecules by endothelial cells, monocytes, and lymphocytes. Of the three isoforms found in mammals, TGF-beta1 is the predominant isoform that affects these immune functions in vivo. Therefore, it was not unexpected that the targeted disruption of the TGF-beta1 gene in our mice resulted in a lethal phenotype characterized by progressive multifocal inflammation. We had previously identified some of the molecular mechanisms underlying this complex immune dysregulation. We further demonstrated that more than half of the null mice die in utero, and that this embryonic lethality is associated with defects in early hematopoiesis and vasculature in the embryonic yolk sac. Live-born TGF-beta1 null mice exhibit onset of inflammation as early as postnatal day 7, coincident with enhanced expression of both classes of MHC molecules and increased adhesion of leukocytes to the vascular endothelium. In subsequent analyses, we found that the null mice also exhibit autoimmune manifestations such as elevated levels of antibodies to nuclear antigens and kidney deposits of immune complexes. We have also demonstrated that this autoimmune phenotype can be transferred by bone marrow transplantation of bone marrow cells from the null mice to lethally-irradiated wild-type mice. Interestingly, our studies with the synthetic peptides corresponding to cell- and heparin-binding sequences of fibronectin indicate their ameliorating effects on the null mouse, especially in reducing the adhesion of lymphocytes to the vascular endothelium and reducing inflammation in the salivary and lachrymal glands. Enhanced expression of MHC class -I and- II antigens in TGF-beta1 null mice suggested that both of these antigens have important roles in the development of the autoimmune reactions in these mice. We subsequently characterized the role of MHC class II antigens by generating TGF-beta1/MHC-II double null mice. These mice are without any evidence of inflammatory infiltrates, circulating autoantibodies or glomerular immune complex deposits. Instead, these mice exhibit extramedullary hematopoiesis and survive slightly longer than the TGF-beta1 null mice. Thus, MHC class II antigens are essential for the expression of autoimmunity in the TGF-beta1 null mice and may normally cooperate with this growth factor to regulate hematopoiesis. To delineate the precise role of MHC class I molecules in the pathogenesis of TGF-beta1 null mice, we generated TGF-beta1 null mice in the genetic background of MHC class I/?2-microglobulin (?2M) deficiency. ?2M null mice lack expression of MHC class I antigen and peripheral CD8+ T cells because ?2M is essential for the proper assembly, transport and cell surface expression of the MHC class I heterodimers of heavy and light chains. Kaplan-Meier analysis showed a significant improvement in survival of the TGF-beta1/beta2M double null mice. While the tissue distribution of inflammatory lesions was similar to that found in TGF-beta1 null mice, the severity of inflammation was significantly reduced in the double null mice, especially in the heart. Flow cytometric analysis revealed reduced levels of CD8+ T cells in the thymus, spleen and lymph node, suggesting impaired T cell development in thymus. These mice also exhibited increased myelopoiesis with decreased inflammation. Moreover, these mice had significantly reduced levels of circulating autoantibodies and glomerular immune complex deposits. These results thus indicate that MHC class I molecules influence the development of the autoimmunity and inflammation seen in TGF-beta1 null mice, and CD8+ T cells may contribute to the inflammation in these mice. To assess the therapeutic potential of circulating levels of active TGF-b1, we generated mice with endocrine expression of active TGF-b1 on a TGF-b1 null background (TGF-b1(-/-/TG)) by crossing TGF-?1(+/-) mice with transgenic mice (TG) that express TGF-?1 in liver and secrete in the blood. The TGF-b1(-/-/TG) mice exhibit a survival profile similar to the TGF-b1 (-/-) mice indicating a failure to rescue lethal phenotype. However, serum TGF-?1 levels in theTGF-b1 (-/-/TG) mice were restored to normal level, with expression in all the tissues notably in the kidney and spleen. Histopathology showed reduced inflammation in all target tissues, especially in the heart. Interestingly unlike TGF-b (-/-) mice, the TGF-b1(-/-/TG) mice have glomerulonephritis in their kidneys similar to the TG mice. Thus, the phenotype of TGF-b1 (-/-/TG) animal model indicates the potential role of circulating active-TGF-?1 in reducing inflammation but it failure to rescue lethality in TGF-?1 null mice indicates critical role of autocrine TGF-?1. Currently, work is underway to study isform specific role of TGF-beta isoforms and also to target its expression to specific cell types.