EXPRESSION, REGULATION, AND PRODUCTION OF TUMOR-NECROSIS-FACTOR-ALPHA IN MOUSE TESTICULAR INTERSTITIAL MACROPHAGES IN-VITRO

Tumor necrosis factor-alpha (TNFalpha) is a cytokine principally secreted from macrophages and monocytes activated by agents such as lipopolysaccharide (LPS). We have recently shown that TNFalpha inhibited mouse Leydig cell steroidogenesis in vitro. LPS injection has also been shown to repress Leydig cell function and induce TNFalpha messenger RNA (mRNA) expression in testicular interstitial macrophages in vivo. A paracrine regulation of Leydig cell testosterone synthesis by testicular interstitial macrophages via TNFalpha has been proposed. To further support this possibility, we examined whether LPS can induce TNFalpha mRNA expression and protein production in testicular interstitial macrophages in vitro. The regulation of LPS-stimulated TNFalpha mRNA expression in vitro was also investigated by employing the protein synthesis inhibitor cycloheximide (CHX). TNFalpha 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 mug/ml) and in the presence or absence of CHX (5.0 mug/ml) at different time points. Northern blot analysis showed that TNFalpha 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). TNFalpha mRNA then declined quickly and completely disappeared by 8 h after LPS treatment. In contrast to this rapid and transient induction of TNFalpha message by LPS alone, CHX extended the induction and caused a marked increase in LPS-induced TNFalpha mRNA at 2 and 6 h. CHX induced more LPS-stimulated TNFalpha mRNA at 6 h than that at 2 h. At 3 h after LPS treatment, TNFalpha secretion was significantly stimulated (5.6 +/- 1.2 U/mug macrophage DNA) measured by L929 tumor fibroblast cytotoxicity. TNFalpha 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. TNFalpha secretion was increased in a time-dependent way. There are significantly higher LPS-induced TNFalpha levels in culture media at 2 h (35.4 +/- 2.2 pg/mug macrophage DNA) and 6 h (85.5 +/- 11.1 pg/mug macrophage DNA) than those in control groups. The current study demonstrates that LPS activates testicular interstitial macrophages to express TNFalpha mRNA and secrete TNFalpha protein in vitro. CHX superstimulates and extends this LPS-induced TNFalpha mRNA expression, which indicates the existence of potential inhibitors in testicular interstitial macrophages that repress TNFalpha mRNA expression. These results thus provide additional evidence to support the hypothesis that TNFalpha secreted from testicular interstitial macrophages exerts a paracrine regulation of Leydig cell steroidogenesis in the testis.