活化单核细胞诱导的人视网膜色素上皮细胞凋亡由半胱天冬酶-3激活介导。

Human RPE cell apoptosis induced by activated monocytes is mediated by caspase-3 activation.

作者信息

Elner Susan G, Yoshida Ayako, Bian Zong-Mei, Kindezelskii Andrei L, Petty Howard R, Elner Victor M

机构信息

Department of Ophthalmology, University of Michigan, Ann Arbor, USA.

出版信息

Trans Am Ophthalmol Soc. 2003;101:77-89; discussion 89-91.

PMID:14971566

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1358977/

Abstract

PURPOSE

To determine the effects of activated monocytes on the induction of human retinal pigment epithelial (HRPE) cell reactive oxygen metabolite (ROM) production and apoptosis.

METHODS

HRPE cells were co-cultured with interferon-gamma (IFN-gamma)-stimulated human monocytes. HRPE apoptosis was detected by propidium iodide, proliferating cell nuclear antigen (PCNA) and TdT-mediated dUTP nick end labeling (TUNEL) staining, caspase-3 activation, and Western blot analysis. HRPE cell ROMs were imaged using the fluorescent marker dihydrotetramethylrosamine (H2TMRos).

RESULTS

IFN-gamma-activated monocytes in direct contact with HRPE cells elicited significant increases in TUNEL-positive (P < .0001) and decreases in PCNA-positive (P < .0001) HRPE cells. The activated monocytes also induced HRPE cell caspase-3 activation, which was inhibited by inhibitor Z-DEVD-fmk. Co-incubations, in which monocytes were either prevented from direct contact with HRPE cells or separated from HRPE cells after 30 minutes of direct contact, did not induce significant HRPE cell apoptosis. Anti-CD18 and anti-ICAM-1 antibodies significantly reduced activated monocyte-induced TUNEL-positive HRPE cells, by 48% (P = .0051) and 38% (P = .046), respectively, and caspase-3 activity by 56% (P < .0001) and 45% (P < .0001), respectively. Overlay of monocytes induced HRPE cell ROM that was inhibited by anti-CD18 and anti-ICAM-1 antibodies, but not by superoxide dismutase (SOD) or nitric oxide (NO) inhibitors. Accordingly, neither SOD nor NO inhibitors had significant effects on HRPE cell apoptosis or caspase-3 activation.

CONCLUSIONS

We demonstrated that IFN-gamma-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 via caspase-3 activation. These mechanisms may compromise HRPE cell function and survival in retinal diseases in which mononuclear phagocyte infiltration at the HRPE interface is observed.

摘要

目的

确定活化的单核细胞对人视网膜色素上皮（HRPE）细胞活性氧代谢产物（ROM）生成及凋亡诱导的影响。

方法

将HRPE细胞与干扰素-γ（IFN-γ）刺激的人单核细胞共培养。通过碘化丙啶、增殖细胞核抗原（PCNA）和末端脱氧核苷酸转移酶介导的dUTP缺口末端标记（TUNEL）染色、半胱天冬酶-3激活及蛋白质免疫印迹分析检测HRPE细胞凋亡。使用荧光标记二氢四甲基罗丹明（H2TMRos）对HRPE细胞的ROM进行成像。

结果

与HRPE细胞直接接触的IFN-γ活化单核细胞使TUNEL阳性HRPE细胞显著增加（P <.0001），PCNA阳性HRPE细胞减少（P <.0001）。活化的单核细胞还诱导了HRPE细胞半胱天冬酶-3的激活，这被抑制剂Z-DEVD-fmk所抑制。在共培养中，若单核细胞被阻止与HRPE细胞直接接触，或在直接接触３０分钟后与HRPE细胞分离，则不会诱导显著的HRPE细胞凋亡。抗CD18和抗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），半胱天冬酶-3活性分别降低56%（P <.0001）和45%（P <.0001）。单核细胞覆盖诱导的HRPE细胞ROM被抗CD18和抗ICAM-1抗体抑制，但未被超氧化物歧化酶（SOD）或一氧化氮（NO）抑制剂抑制。因此，SOD和NO抑制剂对HRPE细胞凋亡或半胱天冬酶-3激活均无显著影响。