REGULATION OF IMMUNE RESPONSES IN HUMANS AND NON-HUMAN PRIMATES

人类和非人类灵长类动物免疫反应的调节

• 批准号: 5200470
• 负责人: W STROBER
• 金额: --
• 依托单位: NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/5200470
• 关键词: B lymphocyte; CD3 molecule; T cell receptor; T lymphocyte; animal tissue; biological signal transduction; cell cell interaction; crosslink; cytokine; high endothelial venule; human subject; immunoprecipitation; immunoregulation; inflammation; interferon gamma; leukocyte activation /transformation; lymphocyte proliferation; phosphorylation; polymerase chain reaction; selectins

L-selectin (LAM-1/LECAM-1) the selectin expressed on leukocytes mediates a number of leukocyte-endothelial interactions, including the binding of lymphocytes to high endothelial venules (HEV) of peripheral lymph nodes and Peyer's patches, the binding of neutrophil to endothelial cells and the binding of all leukocytes to inflamed venules. Recently we showed that L-selectin ligation influences the T cell activation process: immobilized Leu-8 mAb influences the anti-CD3-mediated proliferation of T cells and co-immunoprecipitates molecules in the TCR/CD3 complex. In the present study, we provide further evidence of the involvement of L- selectin in signal transduction by showing that ligation of L-selectin augments phosphoinositol (PI) hydrolysis induced by anti-CD-3 and accelerates and enhances tyrosine phosphorylation induced by anti-CD3. In further studies, we examine the signaling function using immunoprecipitation techniques aimed at identifying molecules associated with L-selectin under various conditions. First, we showed that in resting cells--metabolically labeled with 35-S methionine and 35-S cysteine and then exposed to non-dissociating detergent lysing agents, anti-L-selectin coimmunoprecipitates both a 94 kD and a 210 kD molecule identified on SDS-PAGE. These molecules are likely to be intracytoplasmic (non-surface) molecules, since they are not detected in lysates derived from surface-labeled cells. Second, we showed that in metabolically labeled and anti-L-selectin clusterred cells, anti-L- selectin coimmunoprecipitates a 210 kD molecule which, in an in vitro kinase assay, takes up 32-P. Furthermore, in the same cells activated with Con A, anti-L-selectin coimmunoprecipitates a 150 kD molecule as well as the 210 kD molecule that take up 32-P. These data establish that L-selectin is a signal transduction molecule that associates with several other molecules, at least some of which have tyrosine kinase activity. We hypothesize that the latter are critical for the ability of L-selectin to signal cells directly or via TCR/CD3. In additional studies, we sought to determine if L-selectin-mediated cell signaling has functional consequences other than an effect on proliferation. In particular, we investigated the role of L-selectin in T cell cytokine production. We demonstrated utilizing quantitative RT-PCR, that L-selectin crosslinking of anti-CD3 activated CD4+ T cells leads to an approximately 10-fold higher steady state IFN-gamma mRNA levels at an early time point (6 hours), but not at later time points (12 and 24 hours) and this effect was seen when L-selectin and TCR/CD3 ligations were applied simultaneously or sequentially. L-selectin ligation alone, in the absence of anti-CD3 signalling did not induce an INF-gamma mRNA increase. The above results are relevant to T cell-endothelial interaction occurring in the vasculature at sites of inflammation, as well as to immune response at particular sites.

L-选择素（LAM-1/LECAM-1）是白细胞上表达的选择素， 许多白细胞-内皮细胞相互作用，包括 外周淋巴细胞高内皮微静脉（HEV） 淋巴结和Peyer集合淋巴结，中性粒细胞与内皮细胞的结合 以及所有白细胞与发炎的小静脉的结合。 最近我们 显示L-选择素连接影响T细胞活化过程： 固定化Leu-8 mAb影响抗CD 3介导的细胞增殖， TCR/CD 3复合物中的T细胞和免疫共沉淀分子。 在 在本研究中，我们提供了L- 通过显示L-选择素的连接， 增强抗CD-3诱导的磷酸肌醇（PI）水解， 加速和增强抗CD 3诱导的酪氨酸磷酸化。 在进一步的研究中，我们使用 免疫沉淀技术，旨在识别相关分子 与L-选择素在不同条件下的作用。 首先，我们展示了 静息细胞-用35-S蛋氨酸和35-S 半胱氨酸，然后暴露于非解离性去污剂裂解剂， 抗-L-选择素共免疫沉淀94 kD和210 kD分子 在SDS-PAGE上鉴定。 这些分子很可能是 胞质内（非表面）分子，因为它们在 来源于表面标记细胞的裂解物。 其次，我们发现， 代谢标记和抗-L-选择素聚集红细胞，抗-L- 选择素共免疫沉淀210 kD分子，在体外 激酶测定，占用32-P。此外，在相同的细胞激活 与ConA，抗-L-选择素共免疫沉淀150 kD分子， 以及吸收32-P的210 kD分子。这些数据证实， L-选择素是一种信号转导分子， 其它分子，其中至少一些具有酪氨酸激酶活性。 我们推测后者对L-选择素的能力至关重要。 直接或通过TCR/CD 3向细胞发出信号。 在其他研究中，我们 试图确定L-选择素介导的细胞信号传导是否具有功能性 除了对扩散的影响之外的其他后果。 我们尤其 研究了L-选择素在T细胞细胞因子产生中的作用。 我们 利用定量RT-PCR证明，L-选择素交联 抗CD 3激活的CD 4 + T细胞导致大约10倍的 在早期时间点较高的稳态IFN-γ mRNA水平（6 小时），但在随后的时间点（12和24小时）没有，并且这种效应 当应用L-选择素和TCR/CD 3连接时， 同时地或顺序地。 单独的L-选择素连接， 抗-CD 3信号传导的缺乏不诱导INF-γ mRNA的增加。 上述结果与T细胞-内皮细胞相互作用有关 发生在炎症部位的脉管系统中，以及 在特定部位的免疫反应。