Expression profiles of cytokines released in intestinal epithelial cells of the rainbow trout, Oncorhynchus mykiss, in response to bacterial infection.

To determine whether fish intestinal epithelial cells (IECs) contribute to mucosal immunity, we established a method for isolating IECs from the rainbow trout Oncorhynchus mykiss and examined cytokine production in these cells. Components of the intestinal epithelium were released by incubation of intestinal pieces with 1mM dithiothreitol (DTT)/ethylenediamine tetraacetic acid (EDTA). The IEC-rich fraction (purity >90%; survival rate approximately 95%) was obtained by centrifugation on a 35%/40% Percoll gradient, followed by magnetic cell sorting using an anti-trout IgM antiserum. The gene expression profiles of 14 cytokines in trout IECs were investigated after culturing the cells for 6h with or without the pathogenic bacterium Aeromonas salmonicida. Trout IECs could produce several cytokines, of which IL-1beta and TNFalpha2 were upregulated when the cells were stimulated with live A. salmonicida. Immunohistochemical analyses with the anti-trout TNF antibody confirmed that the TNF protein was present in the IECs of trout that were intra-anally challenged with live A. salmonicida. These results show that trout IECs are an important trigger of the intestinal immune system. Further, formalin-killed A. salmonicida, conditioned medium of this bacterium, or live nonpathogenic Escherichia coli could not upregulate the expression of these cytokines. These results indicate that the production of inflammatory cytokines by IECs is caused by the adhesion of A. salmonicida, but is not due to only simple ligand-receptor interactions between the surface molecules of IECs and the bacterium or in response to bacterial secretions.

Enhancement effect of polyoxometalates on NGF-induced neurite-outgrowth of PC12 cells.

Nerve growth factor (NGF)-induced neurite-outgrowth of PC12 cells was enhanced by polyoxometalates such as Na9[SbW(9)O(33)].19.5H(2)O (SbW(9)) and (NH(3)Pr(i))6[Mo(7)O(24)].3H(2)O (Mo(7)). Western blotting analysis of polyoxometalate/NGF-treated PC12 cells showed a strong expression of growth-associated protein 43 (GAP-43), which is associated with the neurite outgrowth. Similar effects were observed for other polyoxometalates, K(11)H[(VO)3(SbW(9)O(33))2].27H(2)O ((VO)3(SbW(9))2), K6[P(2)W(18)O(62)].14H(2)O (P(2)W(18)), and K7[PTi(2)W(10)O(40)].6H2O (PTi(2)W(10)), in contrast with little effect for monomeric tungstate and molybdate. Of the polyoxometalates tested in this study, SbW(9) and Mo(7) were the most potent.

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.