Berg E L, Mullowney A T, Andrew D P, Goldberg J E, Butcher E C
Protein Design Labs, Inc., Mountain View, California 94043, USA.
Am J Pathol. 1998 Feb;152(2):469-77.
Carbohydrate ligands for lymphocyte L-selectin are expressed on high endothelial venules (HEVs) in peripheral lymph nodes and sites of chronic inflammation and mediate the recruitment of lymphocytes from the blood into these tissues. In the mouse, these ligands, collectively termed the peripheral lymph node addressin (PNAd), have been shown to contain fucose, sialic acid, and sulfate and to include several HEV glycoproteins including GlyCAM-1, CD34, and MAdCAM-1. Monoclonal antibody (MAb) MECA-79, which binds a sulfate-dependent epitope, recognizes PNAd in both mouse and man. In humans, only CD34 has been identified among the glycoprotein species that react with MECA-79. Although P-selectin is highly expressed in tonsil HEVs, it was not found to react with MECA-79 or to support L-selectin-mediated lymphocyte rolling. To further characterize human PNAd, MAbs were developed against purified PNAd immunoisolated from human tonsil. MAbs JG-1, JG-5, JG-9, and JG-10, like MECA-79, bind HEVs in human tonsil and react similarly in Western blots, and JG-9 and JG-10 also block lymphocyte rolling on purified PNAd. In addition, by competitive ELISA on purified tonsil PNAd, all MAbs were found to react with overlapping epitopes. However, JG-1, JG-5, JG-9, and JG-10 do not recognize mouse PNAd, and unlike MECA-79, they recognize determinants that are sensitive to neuraminidase. Strikingly, the epitope recognized by JG-1, although abundant in tonsil and peripheral lymph node, is absent from appendix HEVs or HEVs in some samples of chronically inflamed skin, even though these HEVs are MECA-79 reactive. Moreover, although JG-5 and JG-9 react well with tonsil, peripheral lymph node, and inflamed skin HEVs, they react only with occasional endothelial cells in appendix tissues. These findings point to significant diversity in the carbohydrate determinants expressed by HEVs and recognized by L-selectin and demonstrate their differential representation in different sites in vivo. These antibodies should be useful in probing the precise structure of human L-selectin ligands.
淋巴细胞L-选择素的碳水化合物配体在外周淋巴结和慢性炎症部位的高内皮微静脉（HEV）上表达，介导淋巴细胞从血液募集到这些组织中。在小鼠中，这些配体统称为外周淋巴结地址素（PNAd），已被证明含有岩藻糖、唾液酸和硫酸盐，并包括几种HEV糖蛋白，包括GlyCAM-1、CD34和MAdCAM-1。单克隆抗体（MAb）MECA-79可结合硫酸盐依赖性表位，在小鼠和人类中均可识别PNAd。在人类中，与MECA-79反应的糖蛋白种类中仅鉴定出CD34。虽然P-选择素在扁桃体HEV中高度表达，但未发现其与MECA-79反应或支持L-选择素介导的淋巴细胞滚动。为了进一步表征人类PNAd，制备了针对从人扁桃体免疫分离的纯化PNAd的单克隆抗体。单克隆抗体JG-1、JG-5、JG-9和JG-10与MECA-79一样，可结合人扁桃体中的HEV，在蛋白质印迹中反应相似，并且JG-9和JG-10还可阻断淋巴细胞在纯化的PNAd上滚动。此外，通过对纯化的扁桃体PNAd进行竞争性ELISA，发现所有单克隆抗体均与重叠表位反应。然而，JG-1、JG-5、JG-9和JG-10不识别小鼠PNAd，并且与MECA-79不同，它们识别对神经氨酸酶敏感的决定簇。引人注目的是，JG-1识别的表位虽然在扁桃体和外周淋巴结中丰富，但在阑尾HEV或某些慢性炎症皮肤样本的HEV中不存在，尽管这些HEV与MECA-79反应。此外，虽然JG-5和JG-9与扁桃体、外周淋巴结和炎症皮肤HEV反应良好，但它们仅与阑尾组织中的偶尔内皮细胞反应。这些发现表明HEV表达的、被L-选择素识别的碳水化合物决定簇存在显著差异，并证明它们在体内不同部位的表达不同。这些抗体应有助于探究人类L-选择素配体的精确结构。
