Iwata M, Sawada S, Sawa M, Thoft R A
Department of Ophthalmology, Hikarigaoka Hospital, Nihon University School of Medicine, Tokyo, Japan.
Curr Eye Res. 1997 Aug;16(8):751-60. doi: 10.1076/ceyr.16.8.751.8983.
The authors studied the adhesion mechanisms between peripheral blood lymphocytes (PBL) and cultured human corneal epithelial (HCE) cells to investigate the lymphocyte interaction with corneal epithelial cells in the corneal immune response.
First, the authors examined the expression of intercellular adhesion molecule (ICAM)-1 or lymphocyte function-associated antigen (LFA)-3 on the normal human corneal epithelium and cultured HCE cells by an immunostaining technique and flow cytometry. Effects of inflammatory cytokines such as interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha, on ICAM-1 or LFA-3 expression on cultured HCE cells were also examined. Second, the authors performed an adhesion assay with 51Cr-labeled monocyte-depleted PBL from normal, healthy volunteers and cultured HCE cells, with and without treatment of IFN-gamma or TNF-alpha in 96-well-plates for 1 hour at 37 degrees C in 5% CO2. After unbound PBL were removed, the radioactivity of the sample in each well was counted with a scintillation counter. In addition, the authors evaluated the blocking effects of monoclonal antibodies (mAbs) on the adhesion of PBL to the cultured HCE cells.
ICAM-1 expression was not detected in the normal human corneal epithelium. However, the expression of ICAM-1 was detected on the cultured HCE cells with Dulbecco's Modified Eagle Medium supplemented with 10% fetal bovine serum. In addition, both IFN-gamma and TNF-alpha increased ICAM-1 expression on the cultured HCE cells dramatically. LFA-3 expression was detected in all cell layers of the normal human corneal epithelia. Neither IFN-gamma nor TNF-alpha had any effect on LFA-3 expression on the cultured HCE cells. The PBL adhesion to the HCE cells with and without treatment of IFN-gamma or TNF-alpha was blocked dominantly by anti-ICAM-1 or anti-LFA-1 alpha mAb. Anti-LFA-3 mAb also blocked the PBL adhesion but had less blocking effect than anti-ICAM-1 or anti-LFA-1 alpha mAb. Anti-very late activation antigen beta, or anti-human leukocyte antigen (HLA)-class I or HLA-class II mAb had no effect on the PBL adhesion to the HCE cells. The adhesion percentile of the PBL applied to the HCE cells pretreated with IFN-gamma or TNF-alpha showed a dose-response curve dependent on the concentration of these cytokines.
The results in the present study demonstrate that (i) adhesion of lymphocytes to HCE cells could be mediated by the LFA-1-ICAM-1 pathway and/or the CD2-LFA-3 pathway; (ii) the LFA-1-ICAM-1 pathway could be crucial in lymphocyte adhesion to HCE cells; (iii) IFN-gamma or TNF-alpha exerts an enhancing effect not only on the ICAM-1 expression on HCE cells but also on the adhesion of lymphocytes to HCE cells.
作者研究外周血淋巴细胞（PBL）与培养的人角膜上皮（HCE）细胞之间的黏附机制，以探讨角膜免疫反应中淋巴细胞与角膜上皮细胞的相互作用。
首先，作者通过免疫染色技术和流式细胞术检测正常人角膜上皮和培养的HCE细胞上细胞间黏附分子（ICAM）-1或淋巴细胞功能相关抗原（LFA）-3的表达。还检测了炎性细胞因子如干扰素（IFN）-γ和肿瘤坏死因子（TNF）-α对培养的HCE细胞上ICAM-1或LFA-3表达的影响。其次，作者用来自正常健康志愿者的51Cr标记的单核细胞去除的PBL与培养的HCE细胞进行黏附试验，在96孔板中于37℃、5%二氧化碳条件下培养1小时，分别在有无IFN-γ或TNF-α处理的情况下进行。去除未结合的PBL后，用闪烁计数器计数各孔样品的放射性。此外，作者评估了单克隆抗体（mAb）对PBL与培养的HCE细胞黏附的阻断作用。
在正常人角膜上皮中未检测到ICAM-1表达。然而，在补充有10%胎牛血清的杜氏改良 Eagle 培养基培养的HCE细胞上检测到了ICAM-1表达。此外，IFN-γ和TNF-α均显著增加了培养的HCE细胞上ICAM-1的表达。在正常人角膜上皮的所有细胞层中均检测到LFA-3表达。IFN-γ和TNF-α对培养的HCE细胞上LFA-3的表达均无影响。抗ICAM-1或抗LFA-1α mAb主要阻断了经IFN-γ或TNF-α处理和未处理的HCE细胞与PBL的黏附。抗LFA-3 mAb也阻断了PBL的黏附，但阻断作用小于抗ICAM-1或抗LFA-1α mAb。抗极晚期活化抗原β、抗人白细胞抗原（HLA）-I类或HLA-II类mAb对PBL与HCE细胞的黏附无影响。应用于经IFN-γ或TNF-α预处理的HCE细胞的PBL黏附百分率呈现出依赖于这些细胞因子浓度的剂量反应曲线。
本研究结果表明：（i）淋巴细胞与HCE细胞的黏附可由LFA-1-ICAM-1途径和/或CD2-LFA-3途径介导；（ii）LFA-1-ICAM-1途径在淋巴细胞与HCE细胞的黏附中可能起关键作用；（iii）IFN-γ或TNF-α不仅对HCE细胞上ICAM-1的表达有增强作用，而且对淋巴细胞与HCE细胞的黏附也有增强作用。
