Regulation of expression of IL-1 receptor antagonist protein in human synovial and dermal fibroblasts.
Regulation of expression of IL-1 receptor antagonist protein in human synovial and dermal fibroblasts.
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人滑膜和真皮成纤维细胞中 IL-1 受体拮抗剂蛋白表达的调节。
作者：
R. Krzesicki;C. Hatfield;Michael J. Bienkowski;J. McGuire;G. Winterrowd;D. Chapman;Ann E. Berger;Robert N. McEwan;Donald B. Carter;J. Chosay
IL-1 R拮抗蛋白（IRAP）是IL-1的竞争性抑制剂，其主要由单核细胞合成。我们发现，这种分子也表达在人类滑膜成纤维细胞和真皮成纤维细胞（CRL 1445）。IL-1 α、TNF-α、LPS和PMA以时间和剂量依赖性方式调节IRAP mRNA。在用IL-1 α（1 U/ml）、TNF-α（10 U/ml）、LPS（50 ng/ml）和PMA（10 ng/ml）刺激后8 - 16 h观察到IRAP mRNA的最大诱导。其相对疗效为：PMA > LPS > IL-1 α> TNF-α。当用IL-1 α加碱性成纤维细胞生长因子和IL-1 α加血小板衍生生长因子-BB同型二聚体处理成纤维细胞时，观察到增强作用。虽然LPS和PMA是IRAP mRNA的最佳诱导剂，但IRAP蛋白的定量显示其合成和释放受到差异调节。免疫沉淀和SDS-PAGE的培养上清液从LPS处理的细胞和细胞裂解液的成纤维细胞用LPS或PMA处理显示一个单一的IRAP带的分子量约为22 kDa。在用PMA处理的细胞的培养上清液中检测到非常少的IRAP。IRAP的定量显示，LPS诱导分泌的IRAP的合成被释放，而PMA诱导的蛋白质的大部分保持细胞相关。逆转录-聚合酶链反应扩增表明，虽然LPS和PMA诱导两种转录，LPS优先诱导分泌IRAP，而PMA差异诱导细胞内IRAP mRNA。成纤维细胞根据诱导信号合成至少两种不同形式的IRAP，并且可以通过IRAP的负反馈机制抑制IL-1的促炎作用来调节炎症反应。成纤维细胞sIRAP与细胞内IRAP在调节结缔组织炎症反应中的相对重要性仍有待确定。
The IL-1R antagonist protein (IRAP) is a competitive inhibitor of IL-1, which is predominantly synthesized by monocytes. We show that this molecule is also expressed in human synovial fibroblasts and dermal fibroblasts (CRL 1445). IRAP mRNA was regulated in a time- and dose-dependent manner by IL-1 alpha, TNF-alpha, LPS, and PMA. Maximal induction of IRAP mRNA was observed between 8 and 16 h after stimulation with IL-1 alpha (1 U/ml), TNF-alpha (10 U/ml), LPS (50 ng/ml), and PMA (10 ng/ml). Their relative efficacy was as follows: PMA > LPS > IL-1 alpha > TNF-alpha. Potentiation was observed when fibroblasts were treated with IL-1 alpha plus basic fibroblast growth factor and IL-1 alpha plus platelet-derived growth factor-BB homodimer. Although LPS and PMA were the best inducers of IRAP mRNA, quantitation of the IRAP protein revealed that its synthesis and release were differentially regulated. Immunoprecipitation and SDS-PAGE of culture supernatant from LPS-treated cells and cell lysates of fibroblasts treated with LPS or PMA showed a single IRAP band with a molecular mass of approximately 22 kDa. Very little IRAP was detected in culture supernatants of cells treated with PMA. Quantitation of IRAP revealed that LPS induced the synthesis of secreted IRAP that was released, whereas the majority of the protein induced by PMA remained cell-associated. Reverse transcriptase-polymerase chain reaction amplification demonstrated that although LPS and PMA induced both transcripts, LPS preferentially induced secreted IRAP, whereas PMA differentially induced intracellular IRAP mRNA. Fibroblasts synthesize at least two different forms of IRAP depending on the inducing signal, and may regulate the inflammatory response by dampening the proinflammatory effects of IL-1 via a negative feedback mechanism with IRAP. The relative importance of fibroblast sIRAP vs intracellular IRAP in regulating the inflammatory response by the connective tissue remains to be determined.