Xiong Y, Hales D B
Department of Physiology and Biophysics, University of Illinois at Chicago 60612-7342.
Endocrinology. 1993 Dec;133(6):2568-73. doi: 10.1210/endo.133.6.8243279.
Tumor necrosis factor-alpha (TNF alpha) is a cytokine principally secreted from macrophages and monocytes activated by agents such as lipopolysaccharide (LPS). We have recently shown that TNF alpha inhibited mouse Leydig cell steroidogenesis in vitro. LPS injection has also been shown to repress Leydig cell function and induce TNF alpha messenger RNA (mRNA) expression in testicular interstitial macrophages in vivo. A paracrine regulation of Leydig cell testosterone synthesis by testicular interstitial macrophages via TNF alpha has been proposed. To further support this possibility, we examined whether LPS can induce TNF alpha mRNA expression and protein production in testicular interstitial macrophages in vitro. The regulation of LPS-stimulated TNF alpha mRNA expression in vitro was also investigated by employing the protein synthesis inhibitor cycloheximide (CHX). TNF alpha secretion into culture supernatants was examined by both bioassay and enzyme-linked immunosorbent assay. Isolated testicular interstitial macrophages were cultured for 24 h before the initiation of treatments. Cells were treated with or without LPS (1.0 micrograms/ml) and in the presence or absence of CHX (5.0 micrograms/ml) at different time points. Northern blot analysis showed that TNF alpha mRNA was rapidly and significantly induced by LPS in testicular interstitial macrophages. The peak expression was at 2 h after the treatment, which was 8.3 +/- 2.6-fold over the control (P < 0.05). TNF alpha mRNA then declined quickly and completely disappeared by 8 h after LPS treatment. In contrast to this rapid and transient induction of TNF alpha message by LPS alone, CHX extended the induction and caused a marked increase in LPS-induced TNF alpha mRNA at 2 and 6 h. CHX induced more LPS-stimulated TNF alpha mRNA at 6 h than that at 2 h. At 3 h after LPS treatment, TNF alpha secretion was significantly stimulated (5.6 +/- 1.2 U/micrograms macrophage DNA) measured by L929 tumor fibroblast cytotoxicity. TNF alpha was also detected by enzyme-linked immunosorbent assay in culture media of testicular interstitial macrophages treated with control medium or LPS for 1, 2, and 6 h. TNF alpha secretion was increased in a time-dependent way. There are significantly higher LPS-induced TNF alpha levels in culture media at 2 h (35.4 +/- 2.2 pg/micrograms macrophage DNA) and 6 h (85.5 +/- 11.1 pg/micrograms macrophage DNA) than those in control groups. The current study demonstrates that LPS activates testicular interstitial macrophages to express TNF alpha mRNA and secrete TNF alpha protein in vitro.
肿瘤坏死因子-α（TNFα）是一种主要由巨噬细胞和单核细胞分泌的细胞因子，这些细胞可被脂多糖（LPS）等物质激活。我们最近发现，TNFα在体外可抑制小鼠睾丸间质细胞的类固醇生成。体内实验也已表明，注射LPS可抑制睾丸间质细胞功能，并诱导睾丸间质巨噬细胞中TNFα信使核糖核酸（mRNA）的表达。有人提出，睾丸间质巨噬细胞可通过TNFα对睾丸间质细胞睾酮合成进行旁分泌调节。为进一步证实这种可能性，我们检测了LPS在体外能否诱导睾丸间质巨噬细胞表达TNFα mRNA并产生蛋白。我们还使用蛋白质合成抑制剂环己酰亚胺（CHX）研究了LPS刺激的TNFα mRNA在体外的表达调控。通过生物测定法和酶联免疫吸附测定法检测培养上清液中TNFα的分泌情况。在开始处理前，将分离的睾丸间质巨噬细胞培养24小时。在不同时间点，细胞分别用或不用LPS（1.0微克/毫升）处理，并在有或无CHX（5.0微克/毫升）存在的情况下培养。Northern印迹分析显示，LPS可在睾丸间质巨噬细胞中迅速且显著地诱导TNFα mRNA表达。处理后2小时达到表达峰值，比对照组高8.3±2.6倍（P<0.05）。之后，TNFα mRNA迅速下降，LPS处理8小时后完全消失。与LPS单独诱导的TNFα信息的快速短暂诱导不同，CHX延长了诱导时间，并导致LPS诱导的TNFα mRNA在2小时和6小时显著增加。CHX在6小时诱导的LPS刺激的TNFα mRNA比2小时更多。LPS处理3小时后，通过L929肿瘤成纤维细胞细胞毒性测定，TNFα分泌受到显著刺激（5.6±1.2单位/微克巨噬细胞DNA）。在用对照培养基或LPS处理1、2和6小时的睾丸间质巨噬细胞的培养基中，也通过酶联免疫吸附测定法检测到了TNFα。TNFα分泌呈时间依赖性增加。与对照组相比，LPS诱导的培养基中TNFα水平在2小时（35.4±2.2皮克/微克巨噬细胞DNA）和6小时（85.5±11.1皮克/微克巨噬细胞DNA）时显著更高。当前研究表明，LPS在体外可激活睾丸间质巨噬细胞表达TNFα mRNA并分泌TNFα蛋白。
