Although in vitro and in
Although, in vitro and in vivo immunosuppressive effects of female steroids have been described in fish (Yamaguchi et al., 2001, Law et al., 2001), their mode of action was not evaluated in detail. Consequently, it is of considerable interest to investigate the intracellular pathways that regulate the expression of genes important for inflammatory reactions upon hormone exposure.
Materials and methods
Results Results from initial studies (Fig. 1) demonstrated that cultured unstimulated carp leukocytes exhibited a low basal level of NO production after 96 h of incubation. LPS-stimulated nitrite formation by leukocytes with more than 2.5-fold higher values compared to untreated cells. A. hydrophila proved to be an even more potent stimulator elevating NO production nearly 5-fold. In contrast, incubation of CK 666 formula with BSA at the same protein concentration as soluble proteins are present in A. hydrophila media preparations had no impact on NO release compared to untreated cells. Moreover, male and female fish showed no differences in NO production. The values of nitrite accumulation in head kidney leukocyte cultures from 12 different carps stimulated with A. hydrophila showed that natural progestogens displayed similar attenuating effects compared to the synthetic progestin MPA at 1 µM, whereas LEV had no significant impact on NO release (Fig. 2A). Moreover, similar results were obtained when bacterial-stimulated trunk kidney leukocytes were incubated with the same steroidal compounds. In contrast, basal NO levels of unstimulated trunk kidney leukocytes were significant lower than the NO formed by stimulated leukocytes in the previous experiment. All experiments were performed in phenolred-free RPMI containing charcoal-treated serum thus excluding the possibility that residual steroids in culture media could account for any of the observed effects. The addition of hormones alone to unstimulated leukocytes derived from trunk kidneys did not influence the basal NO level (Fig. 3). Nevertheless, we observed that isolated carp leukocytes stimulated with bacteria are sensitive to natural and synthetic glucocorticoids, which have already been reported to be immunosuppressive. Accordingly, it was noted that compared to control measurements the glucocorticoids cortisol (F), corticosterone (B), and dexamethasone (DEX) at 1 µM displayed a pronounced decrease of NO release which was comparable to the effects of the progestogens. Furthermore, especially leukocytes from trunk kidneys stimulated with A. hydrophila were more sensitive to glucocorticoid treatment compared to their counterparts derived from head kidneys (Fig. 4A vs. B). The strongest reduction of NO synthesis was observed upon treatment of stimulated leukocytes with 1 µM DEX in leukocytes derived from both, head kidneys and trunk kidneys. The mRNA expression of iNOS was specifically determined by semi-quantitative RT-PCR in head and trunk kidney leukocytes displaying similar expression patterns in these organs. When activated with A. hydrophila carp leukocytes presented significantly increased levels of iNOS mRNA in head kidney leukocytes after 96 h of bacterial stimulation. This expression was markedly attenuated by DHP4 at concentrations of either 10 nM or 1 µM, whereas P4 and MPA inhibited iNOS mRNA expression only at 1 µM (Fig. 5), and LEV was not effective at both concentrations used. A significant decrease of iNOS mRNA expression by treatment with 1 µM DHP4 or 1 µM MPA was also observed in leukocytes derived from trunk kidneys. To evaluate whether there is a specific molecular mechanism responsible for the immunosuppression by progestogens the expression of their receptor was analysed in head kidney tissue. Initial investigations revealed that nuclear receptors for progestogens could not be identified in kidneys of carp (data not shown). Further analysis showed that the distribution of mPR expression was not restricted to gonadal tissues (Fig. 6), although its sequence has mostly been characterized for reproductive organs in fish. In total, a partial sequence of 547 bp of this mPR α in kidneys of carps was obtained. Sequence alignment showed highest similarity with the described sequences for other cyprinids. The alignment of the deduced amino acid sequence showed the highest similarity to goldfish and zebrafish mPR α (Fig. 7), and therefore the newly identified receptor in head kidney tissue can be considered to be the α-isoform of this receptor. The calculation of molecular weight yielded a molecule of 20.7 kDa. Thus, the identified 182 amino acid residues accounted for approximately half of the putative functional protein. The deduced amino acid sequence for mPR α revealed a rich content of leucine, alanine, phenylalanine, serine, and valine in carp. The amino acid sequence alignment showed only 6 different sites of the carp sequence compared to the goldfish mPR α. However, comparing carp and zebrafish 14 different amino acids in this part of the sequence were identified.