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

Tumor necrosis factor-alpha (TNFalpha) is a cytokine principally secre ted from macrophages and monocytes activated by agents such as lipopol ysaccharide (LPS). We have recently shown that TNFalpha inhibited mous e Leydig cell steroidogenesis in vitro. LPS injection has also been sh own to repress Leydig cell function and induce TNFalpha messenger RNA (mRNA) expression in testicular interstitial macrophages in vivo. A pa racrine regulation of Leydig cell testosterone synthesis by testicular interstitial macrophages via TNFalpha has been proposed. To further s upport this possibility, we examined whether LPS can induce TNFalpha m RNA expression and protein production in testicular interstitial macro phages in vitro. The regulation of LPS-stimulated TNFalpha mRNA expres sion in vitro was also investigated by employing the protein synthesis inhibitor cycloheximide (CHX). TNFalpha secretion into culture supern atants was examined by both bioassay and enzyme-linked immunosorbent a ssay. Isolated testicular interstitial macrophages were cultured for 2 4 h before the initiation of treatments. Cells were treated with or wi thout 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 TNFal pha mRNA was rapidly and significantly induced by LPS in testicular in terstitial macrophages. The peak expression was at 2 h after the treat ment, 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 LP S treatment. In contrast to this rapid and transient induction of TNFa lpha message by LPS alone, CHX extended the induction and caused a mar ked increase in LPS-induced TNFalpha mRNA at 2 and 6 h. CHX induced mo re 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 cytotoxi city. TNFalpha was also detected by enzyme-linked immunosorbent assay in culture media of testicular interstitial macrophages treated with c ontrol medium or LPS for 1, 2, and 6 h. TNFalpha secretion was increas ed 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 macropha ge DNA) and 6 h (85.5 +/- 11.1 pg/mug macrophage DNA) than those in co ntrol groups. The current study demonstrates that LPS activates testic ular interstitial macrophages to express TNFalpha mRNA and secrete TNF alpha protein in vitro. CHX superstimulates and extends this LPS-induc ed TNFalpha mRNA expression, which indicates the existence of potentia l inhibitors in testicular interstitial macrophages that repress TNFal pha mRNA expression. These results thus provide additional evidence to support the hypothesis that TNFalpha secreted from testicular interst itial macrophages exerts a paracrine regulation of Leydig cell steroid ogenesis in the testis.