REGULATION OF IMMUNE RESPONSES IN HUMANS AND NON-HUMAN PRIMATES

L-选择素(LAM-1/LECAM-1)白细胞表面表达的选择素介导 许多白细胞-内皮细胞的相互作用，包括结合 淋巴细胞向外周淋巴液高内皮微静脉(HEV)的转化 中性粒细胞与血管内皮细胞的结合 以及所有白细胞与发炎的小静脉的结合。最近我们 显示L-选择素结扎影响T细胞激活过程： 固定化Leu-8单抗对抗CD3介导的细胞增殖的影响 T细胞和免疫共沉淀物分子在TCR/CD3复合体中。在……里面 在本研究中，我们提供了L参与其中的进一步证据- L-选择素在信号转导中的连接 增强抗CD-3抗体诱导的磷脂酰肌醇(PI)水解 加速并增强抗CD3抗体诱导的酪氨酸磷酸化。 在进一步的研究中，我们使用 旨在识别相关分子的免疫沉淀技术 在各种条件下用L-选择素。首先，我们在 静息细胞--代谢性标记35-S蛋氨酸和35-S 半胱氨酸，然后暴露在不解离的洗涤剂裂解剂中， 抗L-选择素共沉淀94kD和210kD分子 已在十二烷基硫酸钠-PAGE上确认。这些分子很可能是 胞质内(非表面)分子，因为它们在 来自表面标记细胞的裂解物。其次，我们在 代谢性标记和抗L-选择素簇红细胞，抗L- 选择素免疫共沉淀210kD分子，在体外 蛋白水解酶测定，占32P。此外，在被激活的相同细胞中 在ConA的作用下，抗L-选择素共沉淀150kD的分子为 以及占据32-P的210kD分子。这些数据证实了 L-选择素是一种信号转导分子，与多个 其他分子，至少其中一些具有酪氨酸激酶活性。 我们假设后者对L-选择素的能力是至关重要的 直接或通过TCR/CD3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转导是否具有功能 除了对扩散的影响以外的其他后果。特别是，我们 探讨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法证明了L-选择素的交联性 抗CD3激活的CD4T细胞导致大约10倍 在早期时间点较高的稳态干扰素-γmRNA水平(6 小时)，但不是在以后的时间点(12小时和24小时)，并且这种效果 当应用L-选择素和TcR/CD3结扎时出现 同时或顺序地。L-选择素单独结扎，在 缺乏抗CD3信号并不能诱导干扰素-γ基因的表达增加。 上述结果与T细胞与内皮细胞的相互作用有关 发生在炎症部位的血管系统中，以及 特定部位的免疫反应。

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.