Meijne A M, Driessens M H, La Rivière G, Casey D, Feltkamp C A, Roos E
Division of Cell Biology, The Netherlands Cancer Institute, Amsterdam.
J Cell Sci. 1994 Sep;107 ( Pt 9):2557-66. doi: 10.1242/jcs.107.9.2557.
We have reported previously that the integrin LFA-1 is essential for metastasis of T-cell hybridomas to the liver. We show here that hepatocytes isolated from normal non-inflamed rat liver express intercellular adhesion molecule-1 (ICAM-1) at the dorsal surface and more prominently at the lateral and substratum-adherent surfaces. Anti-rat ICAM-1 mAb inhibited adhesion of TAM8C4 T-cell hybridoma cells to hepatocytes. Invasion between hepatocytes was not affected, but this is probably due to lack of penetration of the mAb between the hepatocytes. In all hepatocyte-adherent TAM8C4 cells, LFA-1 was concentrated at the adhesion site. Redistribution of ICAM-1 to the interacting hepatocyte membrane was also seen, but only for part of the adherent TAM8C4 cells. LFA-1 was highly concentrated on pseudopods of invading TAM8C4 cells inserted between hepatocytes, and on the upper surface of invaded TAM8C4 cells located under the hepatocytes. ICAM-1 was concentrated in the hepatocyte membrane overlying TAM8C4 cells located underneath the monolayer. These results suggests that ICAM-1 is of major importance for liver invasion by these lymphoma cells. For optimal adhesion to ICAM-1, LFA-1 on T-cell hybridomas requires activation, which apparently occurs upon contact with cell layers that are invaded (G. La Rivière et al., J. Cell Sci. 107, 551-559, 1994). LFA-1 can be activated artificially by Mn2+. To study LFA-1 redistribution upon ICAM-1 interaction with higher resolution, we performed immuno-EM on cells before and after Mn(2+)-induced adhesion and spreading on immobilized ICAM-1. By immune fluorescence, LFA-1 was observed to redistribute to the ICAM-1-adherent surface, and to be concentrated in lamellipodia of spreading TAM8C4 cells. By immuno-EM, LFA-1 was localized in microclusters of approximately 10 gold particles. This was seen in cells fixed in suspension, and the size of these clusters did not change upon adhesion to ICAM-1. LFA-1 was present at high density in thin filopodia, but again in microclusters of similar size. Comparable results were obtained with a cytotoxic T-cell clone. We conclude that Mn(2+)-induced activation of LFA-1 is not associated with the formation or enlargement of LFA-1 clusters.
我们之前曾报道，整合素淋巴细胞功能相关抗原-1（LFA-1）对于T细胞杂交瘤转移至肝脏至关重要。我们在此表明，从正常未发炎大鼠肝脏分离的肝细胞在背表面表达细胞间黏附分子-1（ICAM-1），在侧面和与基质黏附的表面表达更为显著。抗大鼠ICAM-1单克隆抗体抑制了TAM8C4 T细胞杂交瘤细胞与肝细胞的黏附。肝细胞之间的侵袭未受影响，但这可能是由于单克隆抗体无法穿透肝细胞之间。在所有黏附于肝细胞的TAM8C4细胞中，LFA-1集中于黏附位点。ICAM-1也重新分布至相互作用的肝细胞膜，但仅见于部分黏附的TAM8C4细胞。LFA-1高度集中于插入肝细胞之间的侵袭性TAM8C4细胞的伪足上，以及位于肝细胞下方的侵袭性TAM8C4细胞的上表面。ICAM-1集中于覆盖单层下方TAM8C4细胞的肝细胞膜中。这些结果表明，ICAM-1对于这些淋巴瘤细胞侵袭肝脏至关重要。为了与ICAM-1实现最佳黏附，T细胞杂交瘤上的LFA-1需要激活，这显然在与被侵袭的细胞层接触时发生（G. La Rivière等人，《细胞科学杂志》107卷，551 - 559页，1994年）。LFA-1可被锰离子人工激活。为了以更高分辨率研究ICAM-1相互作用时LFA-1的重新分布，我们对锰离子诱导黏附并铺展于固定化ICAM-1上的细胞进行了免疫电子显微镜观察。通过免疫荧光观察到，LFA-1重新分布至ICAM-1黏附表面，并集中于铺展的TAM8C4细胞的片状伪足中。通过免疫电子显微镜观察，LFA-1定位于约10个金颗粒的微簇中。在悬浮固定的细胞中可见此现象，并且这些簇的大小在黏附于ICAM-1后未发生变化。LFA-1以高密度存在于细丝状伪足中，但同样存在于类似大小的微簇中。用细胞毒性T细胞克隆也获得了类似结果。我们得出结论，锰离子诱导的LFA-1激活与LFA-1簇的形成或扩大无关。
