br Discussion The use of
Discussion The use of carp primary cell cultures proved to be a valuable approach to evaluate the piscine immune response. A. hydrophila was a more potent stimulator of NO formation by carp leukocytes compared to commercial LPS often used for leukocyte stimulation (Mustafa and Olson, 1998, Chao et al., 2000, De Boer et al., 2001, Saeij et al., 2003). The moderate basal NO production in control measurements may be due to cytokines present in all cell cultures. Furthermore, the addition of BSA to cell cultures caused no induction of NO release. Obviously, the reaction of the immune Caspase-3/7 Inhibitor to the bacteria is a specific response upon recognition of specific pathogen patterns that have been described to be typical for outer membrane components of bacteria such as A. hydrophila (Maji et al., 2006). Immunosuppressive effects of progestogens on NO release by carp leukocytes were compared to the known immunosuppressive effects of glucocorticoids in mammals and fish (Simmons et al., 1996, Weyts et al., 1998, Wang et al., 2005). Similar to the known suppression of NO production by 1 µM cortisol in LPS-treated phagocytes derived from head kidneys of carp (Saeij et al., 2003), our experiments showed a reduction of NO release of leukocytes isolated from head and trunk kidneys with simultaneous stimulation by addition of A. hydrophila as well. The observed effects of cortisol on NO production were probably due to downregulation of iNOS which has been shown in LPS-stimulated carp phagocytes at similar concentrations of cortisol (Stolte et al., 2008). Moreover, the effects of synthetic glucocorticoids on carp leukocytes are more pronounced because the affinity of the synthetic glucocorticoid DEX to the glucocorticoid receptor (GR) was reported to be 4 times higher than for cortisol (Weyts et al., 1998). In our study, the glucocorticoids at 1 µM displayed immunosuppressive effects at least as strong as the exposure to progestogens at high concentrations, which was also described for mammalian leukocyte cultures (Ogawa et al., 1983). It has been shown that progesterone binding mainly takes place in gonads and PR in fish ovaries show a high affinity to DHP4 and P4, and no binding for cortisol (Pinter and Thomas, 1995, Todo et al., 2000). In fish, cytosolic and membrane bound receptors have been described (Pinter and Thomas, 1995, Todo et al., 2000, Kazeto et al., 2005b). DHP4 is especially important for oocyte maturation and possesses highest affinity to mPRs. Congruently, our findings revealed that DHP4 displayed more pronounced impact on immune function rather than P4. Besides the low affinity of immune cells to sexual steroids, the mechanisms by which progestogens influence leukocyte function remain unknown. A membrane-associated pathway is possible, because nuclear PR have not been found on mammalian macrophages (Miller and Hunt, 1996). Alternatively, progesterone displays affinity to glucocorticoid receptor, and thus progestogens could mediate their effects via the GR (Shyamala and McBlain, 1979). However, it has also been reported that the suppression of lymphocytes by P4 seems not to be mediated by glucocorticoid effects in mammals (Szekeres-Bartho et al., 1990) suggesting a similar mechanism for carp leukocytes. We also demonstrated that the use of trunk kidney derived leukocytes, in addition to commonly investigated head kidney cells in fish, yielded convenient results and that these cells are a useful tool for studies of immunity in fish. Without antigenic stimulation, steroid hormones had no effect on NO production in mammalian immune cells (Coughlan et al., 2005) which is in agreement with our findings upon progestogen treatment of carp leukocytes. This may be explained by studies on the mammalian iNOS gene promotor which was shown to be sensitive to P4, but shows no transactivation without addition of LPS/IFN γ (Miller et al., 1996). Obviously, the same may be true for the iNOS gene in fish.