| Summary: | 博士 === 國立中興大學 === 獸醫學系 === 91 === The aim of this study was to search the mechanism of non-specific immunity of nitric oxide and to investigate the macrophages—derived free radicals on apoptosis, particularly due to the pathogens infection in tilapia. The purified enzyme of gel filtration assays showed that the molecular weights of nNOS and iNOS were 178 kDa and 120 kDa, respectively. From the results on the SDS-PAGE gels, the molecular weights of nNOS and iNOS were 89 and 120 kDa. Therefore, our nNOS and iNOS appeared to be dimmer and monomer structure, respectively.
For activity assays, the results showed that two enzymes required L-arginine as the substrate and NADPH as the cofactor. The activities of purified enzymes were not increased following adding the amounts of Ca2+ or Mg2+. Nevertheless, our results of the competitive inhibition test of L-arginine-dependent NO biosynthesis by L-NMA showed that both NOSs were inhibited following increasing the amounts of L-NMA.
The results of immunoblot analyses suggested that nNOS was observed from the extracts of cerebrum. Inducible NOS was found in the extracts of kidney and spleen. The results of immunohistochemistry indicated that the nNOS existed in the cerebellum, olfactory bulb, and diencephalons. The nerve cell bodies and neuronal fibers of the spinal cord were positively stained. Interestingly, only macrophages from head kidney and spleen were iNOS positive reaction. By the same ways, the eNOS located in the endothelial cell of heart bloods and epithelium cells of vessels with the rabbit anti-bovine eNOS serum.
Because of the lack of specific phenotypic markers, the study of the tilapia cellular immune response needs the screening of both morphological characteristics and functional responses. Assays devoted to the study of immunoregulation of nitric oxide in tilapia were developed. The results showed that anterior kidney macrophage treated with interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), and IFN-γ＋LPS for 24h were expressed high rat of apoptosis. Paradoxically, low doses of NO donor protect from cytokines- induced macrophage apoptosis, whereas high levels of NO donor were associated with macrophage apoptosis. After adding high concentrations of NO donor, NO have been shown to induce cells from apoptosis. Therefore, it appears to be of considerable relevance that NO can regulate the pathways leading to apoptosis.
From the mechanisms of immunoregulation, the assessment of these defense mechanisms (phagocytic activity, super oxide and NO assays) was assayed. Our results indicated that macrophage treated with IFN-γ and TNF-α display a stronger phagocytic activity of Streptococcus iniae. Impairment of phagocytic activity occurred very early after treated with cytokines. In addition to, anterior kidney macrophage treated with IFN-γ, TNF-α, IL-1βand IL-2 expressed high bactericidal activity in vitro that correlated with increased super oxide and nitric oxide concentrations in culture supernatants. IFN-γ also induced high levels of apoptosis in macrophage. On the contrary, the superoxide, nitric oxide secretion and bacteria killing were inhibited, in a dose-dependent manner, by the IL-4 and IL-10 either separately or in combination. The rat of apoptosis was decrease by the IL-4 and IL-10 addition.
We studied the functions of macrophages met with various foreign bodies (yeast and latex bead) and pathogens (Vibrio sp., Aeromonas hydrophila, E. tarda, and Streptococcus iniae) by the phagocytotic activity, superoxide production, oxidative activity, nitric oxide production, and apoptosis assay, respectively. The results showed that the phagocytosis of macrophages could be induced and changed by different pathogens. The phagocytic activity of cells treated with yeast, latex bead, and Aeromonas hydrophila were significantly hight than other tested bacteria. The superoxide production of macrophages was measured in our study. The level of superoxide detected by lucigenin-derived chemiluminescence (LDCL) was markedly increased. It suggested that viable bacteria induced a high level of super oxide production by macrophage, whereas killed bacteria induced just a low level. Interestingly, we found the macrophage played the main role in the cellular immunity. That reasons might be that the induced macrophages activated the other macrophages to become apoptosis. Form the cytotoxic assay; the results indicated that the lactase dehydrogenase activity (LDH assay) was increased of nephro macrophages treated with bacteria. At the same time, we found the nitric oxide production was greatly enhanced. These findings demonstrated that the viable and killed bacteria were to enhance the phagocytosis of macrophages and the free radicals to stimulate macrophages apoptosis in fish.
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