Fleisher L N, McGahan M C, Ferrell J B, Pagan I
College of Veterinary Medicine, North Carolina State University, Raleigh 27606, USA.
Exp Eye Res. 1996 Jul;63(1):91-104. doi: 10.1006/exer.1996.0095.
This study was designed to determine the effects of interleukin-1 on basal and prostaglandin E2-stimulated adenosine 3',5'-cyclic monophosphate production by primary and first passage cultures of non-transformed rabbit pigmented and non-pigmented ciliary epithelial cells. Confluent cultures of rabbit pigmented and non-pigmented ciliary epithelial cells were incubated for varying periods of time in serum-free medium with or without interleukin-1 beta, tumor necrosis factor-alpha, bacterial lipopolysaccharide, transforming growth factor-beta 2, cycloheximide, indomethacin and combinations of these agents. Cells were then preincubated for 10 min with serum-free medium plus the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (for basal adenosine 3',5'-cyclic monophosphate production) or serum-free medium containing several concentrations of prostaglandin E2 and 3-isobutyl-1-methylxanthine. In certain experiments isoproterenol, vasoactive intestinal peptide, or forskolin was substituted for prostaglandin E2. Adenosine 3',5'-cyclic monophosphate was then extracted into ice-cold absolute ethanol and measured by radioimmunoassay. Prostaglandin E2 stimulated adenosine 3',5'-cyclic monophosphate production in pigmented and non-pigmented ciliary epithelial cells in a dose-dependent manner. Incubation with interleukin-1 beta (150 U ml-1) increased prostaglandin E2-stimulated, but not basal adenosine 3',5'-cyclic monophosphate production in pigmented ciliary epithelial cells. This interleukin-1 beta-induced enhancement of prostaglandin E2-stimulated adenosine 3',5'-cyclic monophosphate production, called the interleukin-1 effect, was not seen with non-pigmented ciliary epithelial cells. The interleukin-1 effect was dependent upon interleukin-1 beta concentration, time and de novo protein synthesis. The interleukin 1 effect could not be reproduced by replacing interleukin-1 beta with tumor necrosis factor-alpha or bacterial lipopolysaccharide and was specific for prostaglandin E2, since interleukin-1 beta did not enhance isoproterenol-, vasoactive intestinal peptide-, or forskolin-induced adenosine 3',5'-cyclic monophosphate production. Chronic exposure to prostaglandin E2 (during the 3 hr incubation period), with or without interleukin-1 beta in the incubation medium, reduced subsequent prostaglandin E2-stimulated adenosine 3',5'-cyclic monophosphate production. Inhibition of de novo prostaglandin synthesis with indomethacin increased the interleukin-1 effect. The interleukin-1 effect was inhibited by the immunosuppressive cytokine, transforming growth factor-beta 2, in a dose-dependent manner. This is the first report of prostaglandin E2-induced stimulation of adenosine 3',5'-cyclic monophosphate production by pigmented ciliary epithelial cells and of the unique ability of interleukin-1 to increase this effect. The results are consistent with interleukin-1-induced upregulation of prostaglandin E receptors. Since transforming growth factor-beta 2 inhibited this interleukin-1 effect, this immunosuppressive cytokine may exert negative feedback and thus regulate the physiological consequences of the interleukin-1 effect.
本研究旨在确定白细胞介素-1对未转化的兔色素性和非色素性睫状上皮细胞原代培养物及第一代传代培养物基础状态下以及前列腺素E2刺激的3',5'-环磷酸腺苷生成的影响。将兔色素性和非色素性睫状上皮细胞的汇合培养物在无血清培养基中培养不同时间，培养基中添加或不添加白细胞介素-1β、肿瘤坏死因子-α、细菌脂多糖、转化生长因子-β2、环己酰亚胺、吲哚美辛以及这些试剂的组合。然后，细胞先用无血清培养基加磷酸二酯酶抑制剂3-异丁基-1-甲基黄嘌呤预孵育10分钟（用于基础3',5'-环磷酸腺苷生成），或用含有几种浓度前列腺素E2和3-异丁基-1-甲基黄嘌呤的无血清培养基预孵育。在某些实验中，用异丙肾上腺素、血管活性肠肽或福斯高林替代前列腺素E2。然后将3',5'-环磷酸腺苷提取到冰冷的无水乙醇中，并用放射免疫分析法进行测定。前列腺素E2以剂量依赖的方式刺激色素性和非色素性睫状上皮细胞中3',5'-环磷酸腺苷的生成。用白细胞介素-1β（150 U/ml）孵育可增加色素性睫状上皮细胞中前列腺素E2刺激的3',5'-环磷酸腺苷生成，但不增加基础生成。这种白细胞介素-1β诱导的前列腺素E2刺激的3',5'-环磷酸腺苷生成增强，即白细胞介素-1效应，在非色素性睫状上皮细胞中未观察到。白细胞介素-1效应取决于白细胞介素-1β浓度、时间和从头蛋白质合成。用肿瘤坏死因子-α或细菌脂多糖替代白细胞介素-1β无法重现白细胞介素-1效应，并且该效应对前列腺素E2具有特异性，因为白细胞介素-1β不会增强异丙肾上腺素、血管活性肠肽或福斯高林诱导的3',5'-环磷酸腺苷生成。在孵育培养基中，无论有无白细胞介素-1β，长期暴露于前列腺素E2（在3小时孵育期内）都会降低随后前列腺素E2刺激的3',5'-环磷酸腺苷生成。用吲哚美辛抑制前列腺素的从头合成可增强白细胞介素-1效应。免疫抑制细胞因子转化生长因子-β2以剂量依赖的方式抑制白细胞介素-1效应。这是关于前列腺素E2诱导色素性睫状上皮细胞刺激3',5'-环磷酸腺苷生成以及白细胞介素-1增加该效应的独特能力的首次报道。这些结果与白细胞介素-1诱导前列腺素E受体上调一致。由于转化生长因子-β2抑制了这种白细胞介素-1效应，这种免疫抑制细胞因子可能发挥负反馈作用，从而调节白细胞介素-1效应的生理后果。
