Molecular mechanisms underlying lymphocyte recirculation. III. Characterization of the LECAM-1 (L-selectin)-dependent adhesion pathway in rats.
Molecular mechanisms underlying lymphocyte recirculation. III. Characterization of the LECAM-1 (L-selectin)-dependent adhesion pathway in rats.
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淋巴细胞再循环的分子机制。
DOI：
10.4049/jimmunol.150.5.1735
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发表时间：
1993
影响因子：
4.4
通讯作者：
M. Miyasaka
中科院分区：
文献类型：
--
作者：
T. Tamatani;K. Kuida;T. Watanabe;S. Koike;M. Miyasaka
LECAM-1（L-selectin）被认为在淋巴细胞与外周淋巴结（LN）的高内皮微静脉（HEV）结合中起重要作用，这是淋巴细胞再循环的重要过程。在此之前，我们克隆了大鼠LECAM-1 cDNA。在这项研究中，通过使用这种探针，我们试图描述大鼠中LECAM-1依赖性粘附途径的特征。我们构建了大鼠LECAM-1-IgG嵌合体（rLEC-IgG）的cDNA，表达了该嵌合体，纯化了分泌的重组嵌合体分子，并利用该嵌合体分子制备了与大鼠LECAM-1同源物反应的mAb。rLEC-IgG的使用揭示了LECAM-1的配体选择性地积聚在LN的高内皮（HE）细胞、白色物质、神经元、小脑浦肯野细胞和中枢神经系统的脉络丛以及肾脏的远端小管和毛细血管中。冷冻切片上淋巴细胞与LN HEV的结合被rLEC-IgG或抗大鼠LECAM-1 mAb阻断。HEV衍生的细胞系Ax与固定在塑料板上的rLEC-IgG特异性结合。与LECAM-1的配体结合区域中存在C型凝集素结构域一致，结合是Ca 2+依赖性的，并且可通过富含甘露糖-6-磷酸的多糖聚磷酸甘露聚糖酯或抗大鼠LECAM-1 mAb来实现。这些结果表明，大鼠LECAM-1的特异性配体在Ax细胞上表达。rLEC-IgG从LN裂解物中沉淀55-、65-、120-、190-和> 250-kDa的硫酸化糖蛋白，从Ax细胞裂解物中沉淀190-、> 250-和> 500-kDa的蛋白。沉淀是Ca 2+依赖性的和LECAM-1特异性的。这些结果表明，LECAM-1识别的HE细胞上的碳水化合物结构可能是在有限数量的细胞表面硫酸化糖蛋白上产生的。还发现在LN培养上清液中分泌配体。rLEC-IgG和Ax细胞对于进一步研究LECAM-1在淋巴细胞和HE细胞之间的动态相互作用中的作用是有价值的。
LECAM-1 (L-selectin) is thought to play an important role in the binding of lymphocytes to high endothelial venules (HEV) of peripheral lymph nodes (LN), which is an essential process in lymphocyte recirculation. Previously we cloned the rat LECAM-1 cDNA. In this study, by using this probe we have sought to characterize a LECAM-1-dependent adhesion pathway in the rat. We have constructed a cDNA for rat LECAM-1-IgG chimera (rLEC-IgG), expressed it, purified the secreted recombinant chimera molecules, and produced mAb reactive with the rat homologue of LECAM-1 by using the chimera molecules. The use of rLEC-IgG revealed that ligands for LECAM-1 are selectively accumulated in high endothelial (HE) cells in LN, the white matter, neurons, cerebellar Purkinje cells, and choroid plexus of the central nervous system and also distal tubules and capillary blood vessels of the kidney. Binding of lymphocytes to LN HEV on frozen sections was blocked by either rLEC-IgG or the anti-rat LECAM-1 mAb. An HEV-derived cell line, Ax, specifically bound to rLEC-IgG fixed on plastic plate. Consistent with the presence of a C-type lectin domain in the ligand-binding region of LECAM-1, the binding was Ca2+ dependent and inhibitable by either the mannose-6-phosphate-rich polysaccharide polyphosphomannan ester or the anti-rat LECAM-1 mAb. These results indicate that the specific ligand for rat LECAM-1 is expressed on the Ax cells. rLEC-IgG precipitated 55-, 65-, 120-, 190-, and > 250-kDa sulfated glycoproteins from LN lysates and 190-, > 250-, and > 500-kDa proteins from Ax cell lysate. The precipitation was Ca2+ dependent and LECAM-1 specific. These results suggest that a carbohydrate structure on HE cells recognized by LECAM-1 is borne possibly on a limited number of cell surface-sulfated glycoproteins. The ligands were also found to be secreted in LN culture supernatants. rLEC-IgG and Ax cells should prove valuable for studying further the role of LECAM-1 in dynamic interactions between lymphocytes and HE cells.