Yoshida Ayako, Elner Susan G, Bian Zong-Mei, Kindezelskii Andrei L, Petty Howard R, Elner Victor M
Department of Ophthalmology, University of Michigan, Ann Arbor, Michigan 48105, USA.
Lab Invest. 2003 Aug;83(8):1117-29. doi: 10.1097/01.lab.0000082393.02727.b5.
Dysfunction and loss of human retinal pigment epithelial (HRPE) cells is a significant component of many ocular diseases, in which mononuclear phagocyte infiltration at the HRPE-related interface is also observed. In this study, we investigated whether HRPE cell apoptosis may be induced by overlay of IFN-gamma-activated monocytes. Human monocytes primed with IFN-gamma overlaid directly onto HRPE cells elicited significant increases in terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)-positive HRPE cells (p < 0.0001) and decreases of proliferating cell nuclear antigen-positive (p < 0.0001) HRPE cells. The activated monocytes also induced HRPE cell caspase-3 activation, which was inhibited by the caspase-3 inhibitor, Z-DEVD-fmk. However, co-incubations in which activated monocytes were prevented from direct contact with HRPE cells or in which the monocytes were separated from the HRPE cells after 30 minutes of direct contact, did not induce significant HRPE cell apoptosis. Function-blocking anti-CD18 and anti-intercellular adhesion molecule-1 (ICAM-1) antibodies significantly reduced activated monocyte-induced TUNEL-positive HRPE cells by 48% (p = 0.0051) and 38% (p = 0.046), respectively. Anti-CD18 and anti-ICAM-1 antibodies significantly inhibited caspase-3 activity by 56% (p < 0.0001) and 45% (p < 0.0001), respectively. However, antibodies to vascular cell adhesion molecule-1, TNF-alpha, IL-1beta, or TNF-related apoptosis-inducing ligand did not inhibit apoptosis or caspase-3 activation. Direct overlay of monocytes also induced reactive oxygen metabolites (ROM) within HRPE cells. The intracellular HRPE cell ROM production was inhibited by the anti-CD18 and anti-ICAM-1 antibodies, but not by superoxide dismutase, presumably due to its failure to penetrate into HRPE cells. Accordingly, neither superoxide dismutase nor N(G)-monomethyl-L-arginine had significant effects on HRPE cell apoptosis or caspase-3 activation. Our results suggest that activated monocytes may induce ROM in HRPE cells through cell-to-cell contact, in part via CD18 and ICAM-1, and promote HRPE cell apoptosis. These mechanisms may compromise HRPE cell function and survival in a variety of retinal diseases.
人视网膜色素上皮（HRPE）细胞功能障碍和丧失是许多眼部疾病的重要组成部分，在这些疾病中还观察到HRPE相关界面处有单核吞噬细胞浸润。在本研究中，我们调查了IFN-γ激活的单核细胞覆盖是否可诱导HRPE细胞凋亡。用IFN-γ预处理的人单核细胞直接覆盖在HRPE细胞上，导致末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）阳性HRPE细胞显著增加（p < 0.0001），而增殖细胞核抗原阳性（p < 0.0001）的HRPE细胞减少。活化的单核细胞还诱导了HRPE细胞caspase-3的激活，这被caspase-3抑制剂Z-DEVD-fmk所抑制。然而，将活化的单核细胞与HRPE细胞直接接触的共培养，或在直接接触30分钟后将单核细胞与HRPE细胞分离的共培养，均未诱导显著的HRPE细胞凋亡。功能阻断性抗CD18和抗细胞间黏附分子-1（ICAM-1）抗体分别使活化单核细胞诱导的TUNEL阳性HRPE细胞显著减少48%（p = 0.0051）和38%（p = 0.046）。抗CD18和抗ICAM-1抗体分别使caspase-3活性显著抑制56%（p < 0.0001）和45%（p < 0.0001）。然而，针对血管细胞黏附分子-1、TNF-α、IL-1β或TNF相关凋亡诱导配体的抗体并未抑制凋亡或caspase-3激活。单核细胞的直接覆盖还诱导了HRPE细胞内的活性氧代谢产物（ROM）。细胞内HRPE细胞ROM的产生被抗CD18和抗ICAM-1抗体所抑制，但超氧化物歧化酶未起作用，推测是由于其未能穿透进入HRPE细胞。因此，超氧化物歧化酶和N(G)-单甲基-L-精氨酸对HRPE细胞凋亡或caspase-3激活均无显著影响。我们的结果表明，活化的单核细胞可能通过细胞间接触，部分经由CD18和ICAM-1，在HRPE细胞中诱导ROM，并促进HRPE细胞凋亡。这些机制可能在多种视网膜疾病中损害HRPE细胞的功能和存活。
