Elner S G, Elner V M, Bian Z M, Lukacs N W, Kurtz R M, Strieter R M, Kunkel S L
Department of Ophthalmology, University of Michigan, Ann Arbor 48105, USA.
Invest Ophthalmol Vis Sci. 1997 Feb;38(2):446-55.
The purpose of the study was to examine the effect of T-lymphocyte products on human retinal pigment epithelial (HRPE) cell interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) secretion and gene expression.
HRPE cells were stimulated for 2, 4, 8, or 24 hours with 20% conditioned media (CM) from T-lymphocytes stimulated with CD3 or CD28 monoclonal antibodies (mAbs) or phorbol myristic acid. In some experiments, CM from CD3 mAb-stimulated T-lymphocytes was preincubated with neutralizing anti-(alpha)-tumor necrosis factor (TNF), alpha-interferon-gamma (IFN-gamma), or alpha-interleukin-1 (IL-1) mAb (control) to determine the contributions of each of these cytokines to HRPE chemokine induction by stimulated T-lymphocyte CM. HRPE cells were stimulated for 8 and 24 hours with IL-1 beta (0.2 to 20.0 ng/ml) (positive control), TNF-alpha (0.2 to 20.0 ng/ml) (positive control), IFN-gamma (1 to 1000 U/ml), IFN-gamma + IL-1 beta, IFN-gamma + TNF-alpha. Interleukin-2 (IL-2; 100 ng/ml) alone or in combination with IL-1 beta, TNF-alpha, or IFN-gamma also was tested. Enzyme-linked immunosorbent assay (ELISA) and Northern blot analyses were performed to determine secreted IL-8 and MCP-1 and their steady state mRNA expression, respectively.
ELISA showed significant increases in HRPE IL-8 and MCP-1 secretion by CM from T-lymphocytes stimulated with CD3 or CD3 + CD28 mAb. Smaller, but significant, increases in IL-8 and MCP-1 resulted from CM phorbol myristic acid-stimulated T-lymphocytes. CM preincubated with neutralizing alpha-TNF or alpha-IFN-gamma mAb induced significantly less HRPE IL-8 and MCP-1, whereas preincubation of CM with neutralizing alpha-IL-1 mAb failed to inhibit CM-induced IL-8 or MCP-1. Northern blot analysis showed increased HRPE IL-8 and MCP-1 mRNA expression within 2 hours of stimulation and was maintained up to 24 hours. CM from T-lymphocytes stimulated with CD3 mAb or CD3 + CD28 mAb produced the greatest increases in IL-8 and MCP-1 mRNA. IFN-gamma induced dose-dependent increases in HRPE MCP-1, but not IL-8, IFN-gamma potentiated IL-1 beta and TNF-alpha-induced MCP-1 production, but showed little modulation of IL-1 beta and TNF-alpha-induced IL-8 production. IL-2 did not induce HRPE IL-8 or MCP-1, nor did it modulate the effects of the other cytokines. Northern blot analysis confirmed the ELISA results.
T-lymphocyte secretions induce HRPE IL-8 and MCP-1 gene expression and secretion. TNF and IFN-gamma appear to be necessary components of T-lymphocyte CM for the induction of HRPE IL-8 and MCP-1. IFN-gamma alone induces HRPE MCP-1, albeit to a lesser extent than would IL-1 beta or TNF-alpha, and potentiates IL-1 beta- and TNF-alpha-induced HRPE MCP-1. IL-2 does not appear to modulate cytokine-induced HRPE IL-8 or MCP-1.
本研究旨在探讨T淋巴细胞产物对人视网膜色素上皮（HRPE）细胞白细胞介素-8（IL-8）和单核细胞趋化蛋白-1（MCP-1）分泌及基因表达的影响。
用CD3或CD28单克隆抗体（mAb）或佛波醇肉豆蔻酸酯刺激T淋巴细胞产生的20%条件培养基（CM）刺激HRPE细胞2、4、8或24小时。在一些实验中，将CD3 mAb刺激的T淋巴细胞产生的CM与中和性抗α肿瘤坏死因子（TNF）、α干扰素-γ（IFN-γ）或α白细胞介素-1（IL-1）mAb（对照）预孵育，以确定这些细胞因子各自对受刺激的T淋巴细胞CM诱导HRPE趋化因子的作用。用IL-1β（0.2至20.0 ng/ml）（阳性对照）、TNF-α（0.2至20.0 ng/ml）（阳性对照）、IFN-γ（1至1000 U/ml）、IFN-γ + IL-1β、IFN-γ + TNF-α刺激HRPE细胞8和24小时。还检测了单独的白细胞介素-2（IL-2；100 ng/ml）或与IL-1β、TNF-α或IFN-γ联合使用的情况。分别进行酶联免疫吸附测定（ELISA）和Northern印迹分析，以确定分泌的IL-8和MCP-1及其稳态mRNA表达。
ELISA显示，CD3或CD3 + CD28 mAb刺激的T淋巴细胞产生的CM可显著增加HRPE细胞IL-8和MCP-1的分泌。佛波醇肉豆蔻酸酯刺激的T淋巴细胞产生的CM导致IL-8和MCP-1有较小但显著的增加。与中和性α-TNF或α-IFN-γ mAb预孵育的CM诱导的HRPE细胞IL-8和MCP-1显著减少，而与中和性α-IL-1 mAb预孵育的CM未能抑制CM诱导的IL-8或MCP-1。Northern印迹分析显示，刺激后2小时内HRPE细胞IL-8和MCP-1 mRNA表达增加，并持续至24小时。CD3 mAb或CD3 + CD28 mAb刺激的T淋巴细胞产生的CM使IL-8和MCP-1 mRNA增加最多。IFN-γ诱导HRPE细胞MCP-1呈剂量依赖性增加，但不诱导IL-8增加，IFN-γ增强IL-1β和TNF-α诱导的MCP-1产生，但对IL-1β和TNF-α诱导的IL-8产生几乎没有调节作用。IL-2不诱导HRPE细胞IL-8或MCP-1，也不调节其他细胞因子的作用。Northern印迹分析证实了ELISA结果。
T淋巴细胞分泌物可诱导HRPE细胞IL-8和MCP-1基因表达及分泌。TNF和IFN-γ似乎是T淋巴细胞CM诱导HRPE细胞IL-8和MCP-1所必需的成分。单独的IFN-γ可诱导HRPE细胞MCP-1，尽管程度低于IL-1β或TNF-α，并增强IL-1β和TNF-α诱导的HRPE细胞MCP-1产生。IL-2似乎不调节细胞因子诱导的HRPE细胞IL-8或MCP-1。
