Alvaro-Gracia J M, Zvaifler N J, Brown C B, Kaushansky K, Firestein G S
Division of Rheumatology, University of California, San Diego 92103.
J Immunol. 1991 May 15;146(10):3365-71.
Granulocyte-macrophage CSF (GM-CSF) is a potent stimulator of macrophages and neutrophils and is produced by rheumatoid arthritis (RA) synovium. We now report studies that identify some of the synovial cells and cytokines responsible for local GM-CSF production and gene expression in RA. GM-CSF was assayed by ELISA in supernatants from cultured RA fibroblast-like synoviocytes stimulated with various cytokines (IL-1 beta, TNF-alpha, macrophage-CSF, IFN-gamma, IL-6, and TGF-beta). Immunoreactive GM-CSF was detected in IL-1 beta and TNF-alpha-stimulated cultures, but not in cells cultured in medium or stimulated with any of the other cytokines. IL-1 and TNF-alpha had a synergistic effect on GM-CSF production. GM-CSF gene expression by fibroblast-like synoviocytes was analyzed by ribonuclease protection assay, Northern blot analysis, and in situ hybridization. Both IL-1 beta and TNF-alpha induced GM-CSF mRNA accumulation, with a maximum effect after 4 h of stimulation. We then studied GM-CSF production by macrophage-like synoviocytes (MLS) isolated from fresh synovial specimens by flow microfluorimetry. Fresh MLS spontaneously secreted the cytokine and exogenous IL-1 beta or TNF-alpha had no effect. After 1 wk in culture, additional stimulation with IL-1 beta or TNF-alpha was required for GM-CSF production. Finally, in situ hybridization performed on freshly isolated subpopulations of synovial cells, identified GM-CSF RNA transcripts in MLS.
粒细胞巨噬细胞集落刺激因子（GM-CSF）是巨噬细胞和中性粒细胞的强效刺激物，由类风湿性关节炎（RA）滑膜产生。我们现在报告的研究确定了一些负责局部GM-CSF产生以及RA中基因表达的滑膜细胞和细胞因子。通过ELISA法检测用各种细胞因子（IL-1β、TNF-α、巨噬细胞集落刺激因子、IFN-γ、IL-6和TGF-β）刺激的培养RA成纤维样滑膜细胞上清液中的GM-CSF。在IL-1β和TNF-α刺激的培养物中检测到免疫反应性GM-CSF，但在培养基中培养或用任何其他细胞因子刺激的细胞中未检测到。IL-1和TNF-α对GM-CSF的产生具有协同作用。通过核糖核酸酶保护试验、Northern印迹分析和原位杂交分析成纤维样滑膜细胞的GM-CSF基因表达。IL-1β和TNF-α均诱导GM-CSF mRNA积累，刺激4小时后效果最佳。然后，我们通过流式微荧光法研究了从新鲜滑膜标本中分离的巨噬样滑膜细胞（MLS）产生GM-CSF的情况。新鲜的MLS自发分泌细胞因子，外源性IL-1β或TNF-α无作用。培养1周后，GM-CSF的产生需要用IL-1β或TNF-α进行额外刺激。最后，对新鲜分离的滑膜细胞亚群进行原位杂交，在MLS中鉴定出GM-CSF RNA转录本。
