Chemokine antagonists in a murine model for scleroderma

描述(由申请者提供)：我们正在研究一种小鼠模型 硬皮病，小鼠硬皮病移植物抗宿主病(Scl GVHD)。在……里面 经BALB/c(H-2d)照射的BALB/c(H-2d)小鼠移植Bl0.D2(H-2d)骨 骨髓和脾细胞发生自身免疫性疾病、皮肤增厚和肺 纤维化；移植了同基因细胞的对照动物不会发生 疾病。在SCL GVHD中，皮肤胶原产生的增加是由 CD11b激活的单核/巨噬细胞和CD3T细胞产生转化生长因子-βL 细胞。我们之前已经证明，用抗转化生长因子-β来抑制转化生长因子-β 静脉注射抗体可消除皮肤增厚和肺 骨髓移植后第21天的纤维化。患有SCL GVHD的小鼠 在上调前发生皮肤单个核细胞浸润 胶原合成、真皮致密增厚和肺纤维化。我们使用了RT/PCR 单个皮肤细胞制剂的皮肤总RNA和流式细胞术分析 并发现巨噬细胞趋化蛋白等趋化因子的mRNAs (MCP-1/JE)、巨噬细胞抑制蛋白(MIP-1α)和RANTES 在炎症细胞和皮肤聚集之前，SCL GVHD中表达上调 变厚了。假设：趋化因子吸引单核细胞、巨噬细胞(MCP-1， MIP-1a)和T细胞(RANTES)在纤维化的发病机制中起关键作用。 可能是硬皮病干预的靶点。具体目标一：第一和第二 年)研究C-C趋化因子MCP-1、MIP-1α和RANTES的作用 在导致SCL GVHD动物皮肤纤维化的早期危急事件中。 什么是趋化因子环境和趋化因子上调的时间进程 发生SCL GVHD的动物皮肤？在发生SCL GVHD的动物中，什么细胞 浸润性皮肤分泌趋化因子？它们是T细胞还是单核/巨噬细胞， 还是两者兼而有之？成纤维细胞和内皮细胞在SCL移植物抗宿主病中分泌趋化因子吗？ 免疫细胞上趋化因子受体的上调也存在吗？是一种细胞因子 与趋化因子上调相关的环境(Th1或Th2)？我们建议 描述细胞因子和趋化因子环境及其影响 单层皮肤免疫染色、流式细胞术对上述通路的干预 细胞悬液、RT/PCR和核糖核酸酶保护试验。评估蒙皮 对于增厚，我们使用常规的组织学和图像分析。特定目标II：(第2位 和3岁)使用体内干预措施抑制SCL GVHD中的趋化因子。 趋化因子抗体能抑制和逆转皮肤纤维化吗？中和 抗巨噬细胞单核细胞趋化蛋白-1的多克隆仓鼠抗体和大鼠抗鼠单抗 RANTES(T细胞)将在单独的实验中用于患有 SCL GVHD确定T细胞与巨噬细胞的相对重要性 SCL移植物抗宿主病中的化学吸引。小鼠SCL GVHD模型提供了一种理想的 测试硬皮病新干预措施的车辆。这些初步研究已经 通常与硬皮病和纤维性疾病高度相关，并可能导致 移植物抗宿主病的有效治疗也是如此。

DESCRIPTION (provided by applicant): We are studying a murine model for scleroderma, murine sclerodermatous graft versus host disease (Scl GVHD). In Scl GVHD, irradiated BALB/c (H-2d) mice transplanted with Bl0.D2 (H-2d ) bone marrow and spleen cells develop autoimmune disease, skin thickening and lung fibrosis; control animals transplanted with syngeneic cells do not develop disease. In Scl GVHD, increased cutaneous collagen production is driven by TGF-beta l, produced by activated CD11 b+ monocyte/macrophages and CD3+ T cells. We have previously shown that inhibiting TGF-beta with anti-TGF-beta antibodies administered intravenously abrogates skin thickening and lung fibrosis at day 21 after bone marrow transplantation (BMT). Mice with Scl GVHD develop cutaneous mononuclear cell infiltrates preceding upregulation of collagen synthesis, dense dermal thickening and lung fibrosis. We used RT/PCR analysis of total skin RNA and flow cytometry of single skin cell preparations and found that mRNAs for chemokines such as macrophage chemoattractant protein (MCP-1/JE), macrophage inhibitory protein (MIP-1alpha) and RANTES are upregulated in Scl GVHD before accumulation of inflammatory cells and skin thickening. Hypothesis: Chemokines that attract monocytes, macrophages (MCP-1, MIP-1 a) and T cells (RANTES) are critical in the pathogenesis of fibrosis, and may be targets for interventions in scleroderma. Specific Aim I: 1st and 2nd years) To investigate the role of C-C chemokines MCP-1, MIP- 1alpha, and RANTES in early critical events leading to skin fibrosis in animals with Scl GVHD. What is the chemokine environment and time course of chemokine upregulation in skin of animals developing Scl GVHD? In animals developing Scl GVHD, what cells infiltrating skin secrete chemokines? Are they T cells or monocyte/macrophages, or both? Do fibroblasts and endothelial cells secrete chemokines in Scl GVHD? Is chemokine receptor upregulation on immune cells also present? Is a cytokine milieu (Th1or Th2) associated with the chemokine upregulation? We propose to characterize the cytokine and chemokine environments and effects of intervention in those pathways by immunostaining, flow cytometry of single skin cell suspensions, RT/PCR and RNase protection assays. To evaluate skin thickening, we use routine histology and image analysis. Specific Aim II: (2nd and 3rd years) To use in vivo interventions to inhibit chemokines in Scl GVHD. Can antibodies to chemokines inhibit and reverse skin fibrosis? Neutralizing polyclonal hamster antibodies for MCP-1 (macrophages) and rat antimouse mAb for RANTES (T cells) will be administered in separate experiments to animals with Scl GVHD to determine the relative importance of T cell versus macrophage chemoattraction in Scl GVHD. The murine Scl GVHD model provides an ideal vehicle to test novel interventions for scleroderma. These pilot studies have high relevance to scleroderma and fibrosing diseases in general, and may lead to effective treatments for graft versus host disease as well.