Chemokine antagonists in a murine model for scleroderma

描述（由申请人提供）：我们正在研究一种小鼠模型， 硬皮病、鼠硬皮病移植物抗宿主病（Scl GVHD）。在 Scl GVHD，移植有B10.D2（H-2d）骨的辐照BALB/c（H-2d）小鼠 骨髓和脾细胞发展自身免疫性疾病，皮肤增厚和肺 纤维化;移植同基因细胞的对照动物未发生 疾病在Scl GVHD中，增加的皮肤胶原蛋白产生由以下因素驱动： TGF-β 1，由活化的CD 11 B+单核细胞/巨噬细胞和CD 3 + T细胞产生 细胞我们先前已经证明，用抗TGF-β抑制TGF-β， 静脉内施用的抗体消除了皮肤增厚和肺 在骨髓移植（BMT）后第21天观察到纤维化。Scl GVHD小鼠 出现皮肤单核细胞浸润， 胶原合成、致密真皮增厚和肺纤维化。我们用RT/PCR 单个皮肤细胞制备物的总皮肤RNA分析和流式细胞术 发现趋化因子的mRNA，如巨噬细胞趋化蛋白， MCP-1/JE、巨噬细胞抑制蛋白（MIP-1 α）和RANTES是 在炎性细胞和皮肤积聚之前在Scl GVHD中上调 增厚假设：趋化因子吸引单核细胞，巨噬细胞（MCP-1， MIP-1 a）和T细胞（RANTES）在纤维化的发病机制中是关键的， 可能是硬皮病干预的目标。具体目标一：第一和第二 研究C-C趋化因子MCP-1、MIP-1 α和RANTES在肺癌中的作用 导致Scl GVHD动物皮肤纤维化的早期关键事件。 什么是趋化因子的环境和时间过程中的趋化因子上调， 发生Scl GVHD的动物皮肤？在发生Scl GVHD的动物中， 浸润皮肤分泌趋化因子？它们是T细胞还是单核细胞/巨噬细胞， 还是两者都有Scl GVHD中成纤维细胞和内皮细胞分泌趋化因子吗？ 免疫细胞上的趋化因子受体上调也存在吗？是细胞因子 环境（Th 1或Th 2）与趋化因子的上调？我们建议 表征细胞因子和趋化因子环境以及 通过免疫染色、单个皮肤的流式细胞术 细胞悬浮液、RT/PCR和RNA酶保护测定。评估皮肤 增厚，我们使用常规组织学和图像分析。具体目标二：（第二 和第3年）使用体内干预来抑制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 MCP-1的多克隆仓鼠抗体（巨噬细胞）和MCP-1的大鼠抗小鼠mAb RANTES（T细胞）将在单独的实验中给予动物， Scl GVHD以确定T细胞相对于巨噬细胞的相对重要性 Scl GVHD中的化学吸引。鼠Scl GVHD模型提供了理想的 用于测试硬皮病的新干预措施的载体。这些试点研究 一般与硬皮病和纤维化疾病高度相关，并可能导致 移植物抗宿主病的有效治疗。

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.