Mühlberg T, Heberling H J, Joba W, Schworm H D, Heufelder A E
Department of Internal Medicine, Klinikum Innenstadt, Ludwig-Maximilians-Universität, München, Germany.
Invest Ophthalmol Vis Sci. 1997 Apr;38(5):1018-28.
To analyze the expression and regulation of intracellular and soluble interleukin-1 (IL-1) receptor antagonist (IL-1RA) in orbital fibroblasts and to determine whether a disbalance between IL-1 receptor agonist and antagonist may promote IL-1 mediated proinflammatory actions in Graves' ophthalmopathy (GO).
Early passages of cultured orbital fibroblasts (OFs) derived from four patients with active GO and six control subjects were examined for their baseline expression of the two variants of IL-1RA, intracellular IL-1RA (icIL-1RA) and soluble IL-1ra (sIL-1RA. In addition, modulation of icIL-1RA and sIL-1RA was studied after exposure of OF to a broad range of cytokines as well as to nonspecific stimulating agents such as lipopolysaccharide and phorbol-12-myristate 13-acetate. The IL-1RA gene and protein variants were analyzed by reverse transcriptase-polymerase chain reaction, immunocytochemical staining, immunoblotting, and enzyme-linked immunosorbent assay.
At baseline, both GO- and control OF showed low levels of constitutive icIL-1RA ribonucleic acid and absence of sIL-1RA ribonucleic acid expression. Exposure to various cytokines stimulated icIL-1RA and sIL-1RA gene expression in both groups, but generally to markedly lower levels in GO-OF compared to that of control OF (P < 0.01). Analysis of IL-1RA protein expression showed low levels of constitutive IL-1RA immunoreactivity (22 kDa) in cell lysates and absence of sIL-1RA immunoreactivity in culture supernatants derived from both GO-OF and control OF. Interleukin-1-alpha was capable of inducing expression of two variants (23 and 26 kDa) of IL-1RA immunoreactivity in supernatants, derived from control OF, and to a lesser degree, GO-OF (P < 0.01). Quantitative analysis showed markedly lower abundance of IL-1RA immunoreactivity in cell lysates and supernatants derived from GO-OF monolayers compared to that detected in control OF (P < 0.001).
Our results demonstrate differences in regulation of icIL-1RA and sIL-1RA and markedly lower levels of expression in cultured GO-OF compared to normal OF. Failure to generate, upon cytokine stimulation, sufficient quantities of icIL-1RA and sIL-1RA to balance agonist stimulation of the IL-1 receptor may facilitate IL-1-dependent proinflammatory and fibrogenic actions within the orbital tissue in GO.
分析眼眶成纤维细胞中细胞内及可溶性白细胞介素-1（IL-1）受体拮抗剂（IL-1RA）的表达及调控情况，并确定IL-1受体激动剂与拮抗剂之间的失衡是否会促进格雷夫斯眼病（GO）中IL-1介导的促炎作用。
对来自4例活动性GO患者和6例对照受试者的培养眼眶成纤维细胞（OFs）早期传代培养物进行检测，分析其IL-1RA两种变体，即细胞内IL-1RA（icIL-1RA）和可溶性IL-1ra（sIL-1RA）的基线表达。此外，在OF暴露于多种细胞因子以及非特异性刺激剂如脂多糖和佛波醇-12-肉豆蔻酸酯13-乙酸酯后，研究icIL-1RA和sIL-1RA的调节情况。通过逆转录-聚合酶链反应、免疫细胞化学染色、免疫印迹和酶联免疫吸附测定分析IL-1RA基因和蛋白质变体。
在基线时，GO组和对照组的OF均显示组成型icIL-1RA核糖核酸水平较低，且未检测到sIL-1RA核糖核酸表达。暴露于各种细胞因子后，两组中icIL-1RA和sIL-1RA基因表达均受到刺激，但与对照组OF相比，GO组OF中的表达水平普遍显著降低（P<0.01）。对IL-1RA蛋白表达的分析显示，细胞裂解物中组成型IL-1RA免疫反应性（22 kDa）水平较低，GO组OF和对照组OF培养上清液中均未检测到sIL-1RA免疫反应性。白细胞介素-1-α能够诱导对照组OF培养上清液中两种变体（23和26 kDa）的IL-1RA免疫反应性表达，而在GO组OF中诱导程度较低（P<0.01）。定量分析显示，与对照组OF相比，GO组OF单层细胞裂解物和上清液中IL-1RA免疫反应性丰度显著降低（P<0.001）。
我们的结果表明，icIL-1RA和sIL-1RA的调控存在差异，与正常OF相比，培养的GO组OF中其表达水平显著降低。细胞因子刺激后未能产生足够量的icIL-1RA和sIL-1RA来平衡IL-1受体激动剂的刺激，可能会促进GO眼眶组织中IL-1依赖性促炎和纤维化作用。
