Neuroendocrine-immune interactions in carp: a role for cortisol and interleukin-1

<font size="2"><p></font><FONT FACE="TIMES" SIZE=2>Maintaining a dynamic internal equilibrium, homeostasis, is crucial for survival of an organism. Disturbances in the environment may threaten the homeostasis and this will subsequently evoke an adaptive response in order to restore homeostasis. In vertebrates the adaptive response is mediated via the neuroendocrine system by adrenocortical and adrenergic activation. Glucocorticoids (GC) and catecholamines are the main actors in the response and can affect a whole range of processes, including those in the immune system. In response to pathogenic challenges the immune system is triggered, resulting in activation of components of innate and acquired immunity. Bi-directional communication between the Hypothalamus-Pituitary-Adrenal (HPA)-axis, sympathetic nervous system and the immune system is crucial to ensure homeostasis in mammals. Shared use of ligands and especially receptors forms a key component of this mutual interaction.</p><p> The Hypothalamus-Pituitary-Interrenal (HPI)-axis is the teleost equivalent of the HPA-axis. Stress induced immuno-suppression in fish is mostly attributed to actions of cortisol, major GC in fish and end-product of the HPI-axis. Stress in aquaculture is one of the potential factors causing increased susceptibility of fish to pathogens and subsequently considerable losses in production.</p><p> As part of a programme investigating adaptive strategies of carp ( <em>Cyprinus carpio</em> L.) after temperature stress, this study focuses on the possible neuroendocrine modulation of immune functioning during acute stress. We studied the effects of in vitro cortisol and in vivo acute temperature stress on carp leucocytes and functioning of these leucocytes. Moreover, the cortisol influence on gene expression of the cytokine interleukin-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>(IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>) was studied. IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>in mammals is part of the reciprocal signalling between neuroendocrine and immune system, therefore it may be an important candidate for modulating hormone secretion in carp.</p><p> Cortisol acts upon lymphocytes differentially; in previous research it was demonstrated that in carp, in particular the B lymphocytes are affected. In vertebrates B lymphocytes play an important role in acquired immunity as precursors of antibody producing cells. Maturation and activation state of B lymphocytes may have consequences for the influence cortisol has on these cells. Therefore, carp B lymphocytes were isolated from different tissues and compared with regard to their proliferation, apoptosis and the effects of cortisol on these processes. Head kidney and spleen B lymphocytes were characterised by high basal proliferation. Peripheral blood B lymphocytes showed a low basal proliferation which could be up-regulated by stimulation with lipopolysaccharide (LPS), a major constituent of the cell wall of gram-negative bacteria. LPS could not alter proliferation of head kidney B lymphocytes. In addition, Ig-crosslinking induced higher intracellular calcium responses in circulating B lymphocytes compared with B lymphocytes from head kidney or spleen origin. With respect to apoptosis, stimulation could enhance cell viability in all organs. However, in combination with cortisol high levels of apoptosis were induced. Especially activated peripheral blood B lymphocytes were sensitive to cortisol-induced apoptosis. Also head kidney and to a lesser extent spleen B lymphocytes, although less sensitive than their equivalent in circulation, underwent cortisol-induced apoptosis irrespective of extra stimulation. Proliferation was suppressed by cortisol in blood and spleen B lymphocytes and to a more limited extent in head kidney, regardless of LPS stimulation. It is suggested that cortisol may be important for immunoregulation in both stress and non-stress conditions, because the relatively modest concentration of cortisol used (compared to plasma values measured during stress conditions) could induce a significant increase in apoptosis in all three populations of B lymphocytes. This implies an impact of stress on B lymphocyte development and activity.</p><p> Stress-induced immunological changes that may contribute to a decreased disease resistance in carp were investigated. A 3 h drop in ambient water temperature was used as model for a relative mild and acute stressor for carp. After single or multiple temperature shocks, the relative number of circulating B lymphocytes decreased significantly within 4 h after the onset of the stressor, which was even more pronounced than after challenging the immune system. After a single temperature shock the relative number of B lymphocytes returned to control levels within 24 hours. In head kidney, an increase was measured in the relative number of B lymphocytes. Migration of B lymphocytes resulting in a redistribution of these cells to other body compartments may contribute to the relative drop in B lymphocytes in the circulation. Granulocyte numbers showed opposite reactions, doubling in circulation and decreasing significantly in head kidney. This demonstrates differential modulation of immune cells <em>in vivo</em> by a relative mild stressor. Freshly isolated blood lymphocytes from stressed carp showed a considerable higher number of apoptotic cells than lymphocytes from unstressed animals. Besides B lymphocytes, Ig <sup>-</SUP>lymphocytes contributed significantly to this stress-induced apoptosis. Glucocorticoid receptors could be detected in the vast majority of the B lymphocytes and also part of the Ig <sup>-</SUP>lymphocytes. As distribution of B lymphocytes was substantially affected by temperature stress, the effects of multiple temperature shocks on humoral antibody responses were determined. The kinetics of the antibody response to both, T lymphocyte independent (TI) antigens and T lymphocyte dependent (TD) antigens consistently showed a trend to decreased antibody response in stressed carp. In carp immunised with the TI-antigen TNP-LPS the antibody response was significantly slower in the stressed carp. These observations confirm the effect of temperature stress on the B lymphocyte population.</p><p> These results show that even a mild stressor can affect distribution of B lymphocyte and granulocyte cell populations reversibly with differential effects and thus can have implications for a subsequent immune response. However, during acute stress, the role of cortisol is most probably not purely immunosuppressive but more immunomodulatory. A stress-induced enhancement of an innate type of response could facilitate a fast and effective reaction of the immune system.</p><p> Cytokines, like IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>, play a pivotal role in the regulation of the immune system. Macrophages and a whole range of other cells release IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>as a response to infection or tissue damage. IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>has pleiotropic effects as an immune and inflammatory mediator. Furthermore, IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>is an important candidate able to affect the HPI-axis by altering the release of corticotropin releasing hormone (CRH) and adrenocorticotropic hormone (ACTH).</p><p> In fish, most interleukin molecules await identification but the IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>sequences of several teleost fishes were recently elucidated. In the tetraploid carp we describe gene organisation and expression of two IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>genes: IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1 and IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2. The two carp mRNA sequences share about 74% amino acid identity. The existence of two IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>copies in the carp genome probably originates from the tetraploid nature of the species. In contrast to carp IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1, the IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 locus is represented by multiple sequences with 95-99% identity. Detection of up to 6 distinct IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 sequences within single homozygous fish suggests the presence of multiple copies of the IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 gene in the carp genome. Both IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1 and IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 comprise seven exons with typical IL-1 characteristics as an IL-1 family motif and instability motifs in the 3'-untranslated region. A general discrepancy of teleost IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>sequences described thus far with mammalian IL-1b, is the lack of a clear caspase-1 (interleukin- 1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>-converting enzyme; ICE) cleavage site. Three IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1 RNA transcripts could be detected in carp: (1) a fully spliced product, (2) exon 1-7 with introns 5 and 6 and (3) exon 1-7 with intron 5 only. Intron-containing products were also detected for IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2. These intron-containing products probably represent partially spliced transcripts.</p><p> IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>mRNA expression in carp was semi-quantitatively analysed by RT-PCR in multiple organs, including brain and pituitary. <em>In vivo</em> , mRNA of both IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>sequences were constitutively expressed in healthy carp, for IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1 this was predominantly in the immune organs head kidney and spleen. Furthermore, a scattered distribution of IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1 producing cells was shown by <em>in situ</em> hybridisations of head kidney tissue. Administration of phorbol-myristate-acetate (PMA) or LPS to phagocytes isolated from the head kidney, resulted in up-regulation of IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1 expression. Also IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 transcripts could be up-regulated by <em>in vitro</em> LPS stimulation of head kidney phagocytes. Interestingly, by determining the ratio of expression it was demonstrated that IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 is expressed at a maximum of one tenth of the amount of the IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1 sequence. Together with the high number of amino acid substitutions in the IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 sequences this suggests either that IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 is approaching a pseudogene status or IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 is part of a complex receptor - ligand interaction network. The involvement of nuclear factor (NF)-</font><FONT FACE="Symbol" SIZE=2>k</font><FONT FACE="TIMES" SIZE=2>B in carp IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1 expression was shown with suppression of the LPS-induced IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>expression by the NF-</font><FONT FACE="Symbol" SIZE=2>k</font><FONT FACE="TIMES" SIZE=2>B inhibitor, pyrrolidine dithiocarbamate (PDTC). Data suggests also that carp IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 is regulated via NF-</font><FONT FACE="Symbol" SIZE=2>k</font><FONT FACE="TIMES" SIZE=2>B and consequently both IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>sequences appear to have similar promoter regions.</p><p> Cortisol, as endocrine-derived factor potentially mediating carp IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>expression, was able to inhibit constitutive expression of IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>1 as well as IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>2 transcripts in vitro. However, when cortisol was added in combination with LPS at a physiological dose, cortisol could not inhibit LPS-induced expression. Moreover, it appears that cortisol synergistically enhances LPS-induced IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>expression in carp. Probably LPS overrules the glucocorticoid receptor mediated inhibition via the NF-</font><FONT FACE="Symbol" SIZE=2>k</font><FONT FACE="TIMES" SIZE=2>B pathway. This might imply that cortisol can not suppress IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>activation during infection. At a tenfold higher cortisol dose, however, the expression is inhibited.</p><p> In conclusion, data presented in this thesis show that carp leucocytes are differentially sensitive to cortisol and in vivo stress, with regard to cell type, location and maturation or activation state. This affects cell viability, replication and migration with subsequent consequences for the immune status of carp. Also interaction of the neuroendocrine system with immune regulating factors was demonstrated: cortisol affects carp IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>mRNA expression. IL-1</font><FONT FACE="Symbol" SIZE=2>b</font><FONT FACE="TIMES" SIZE=2>in carp consists of multiple forms and is part of an immune regulating mechanism which probably matches that of mammals in complexity