TNF induces the growth of thymocytes in rainbow trout

In order to investigate the effects of TNFalpha upon the growth of fish thymocytes, rainbow trout thymocytes were cultured in the conditioned medium (CM): the supernatants of the macrophage cultures stimulated with chitin derivative and LPS. Synthesis of TNFalpha by macrophages and subsequent secretion into CM were ascertained by RT-PCR and western blotting. While most of the thymocytes cultured in normal medium died within 7 days, the thymocytes cultured in CM exhibited markedly better growth as monitored by alamarBlue assay and BrdU assay. The proliferating cells appeared to be small lymphocytes. Since such activity in CM was significantly inhibited by an anti-trout TNF antibody, it was clearly evident that TNFalpha in the CM induced the proliferation of the thymocytes. Production of TNFalpha in the thymus of healthy fish was also demonstrated by RT-PCR. Collectively, this data suggest that TNFalpha is involved in T cell development in the trout thymus.