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 VSports手机版. 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. .