B.M.L. Verburg-van Kemenade

Corticosteroid receptors involved in stress regulation in common carp, Cyprinus carpio

In higher vertebrates, mineralo- (aldosterone) and glucocorticoids (cortisol/corticosterone) exert their multiple actions via specific transcription factors, glucocorticoid (GR) and mineralocorticoid (MR) receptors. Teleostean fishes lack aldosterone and mineral regulatory processes seem under dominant control by cortisol. Despite the absence of the classical mineralocorticoid aldosterone, teleostean fishes do have an MR with cortisol and possibly 11-deoxycorticosterone (DOC) (as alternative for aldosterone) as predominant ligands. We studied corticoid receptors in common carp (Cyprinus carpio L). Through homology cloning and bioinformatic analysis, we found duplicated GR genes and a single MR gene. The GR genes likely result from a major genomic duplication event in the teleostean lineage; we propose that the gene for a second MR was lost. Transactivation studies show that the carp GRs and MR have comparable affinity for cortisol; the MR has significantly higher sensitivity to DOC, and this favours a role for DOC as MR ligand in fish physiology. mRNA of the GRs and the MR is expressed in forebrain (in pallial areas homologous to mammalian hippocampus), corticotrophin-releasing hormone (CRH) cells in the pre-optic nucleus (NPO) and pituitary pars distalis ACTH cells, three key neural/endocrine components of the stress axis. After exposure to prolonged and strong (not to mild acute) stressors, mRNA levels of both GRs and MR become down-regulated in the brain, but not in the NPO CRH cells or pituitary ACTH cells. Our data predicts a function in stress physiology for all CRs and suggest telencephalon as a first line cortisol target in stress.

Cortisol induces apoptosis in activated B cells, not in other lymphoid cells of the common carp, Cyprinus carpio L

In mammalian T and B cells glucocorticoids (GS) regulate development and selection through induction of apoptosis; more recently GS-induced apoptosis has also been implicated in the removal of circulating, activated T and B cells following an immune response. In an earlier report we have given the first evidence for cortisol-induced apoptosis as an immune regulator in an aquatic vertebrate, the common carp. Here we report on subpopulation-specific sensitivity of carp peripheral blood leukocytes (PBL) to cortisol-induced apoptosis. B cells, the most abundant leukocyte subpopulation in fish blood, are sensitised to cortisol-induced apoptosis by activation with the mitogens LPS or PHA. Cortisol-induced apoptosis in B cells is receptor mediated as it is blocked by the synthetic GS receptor blocker RU486. In contrast to what is known for mammalian lymphocytes, apoptosis in carp T cells is hardly affected by cortisol, both in unstimulated and in PHA-stimulated cell cultures. A culture supernatant of PHA-prestimulated PBL, containing IL-2-like activity, decreased spontaneous apoptosis in both T and B cells, but did not affect cortisol-induced apoptosis in B cells. Apoptosis in thrombocytes was unaffected by either mitogens, cortisol, or lymphocyte supernatant. The difference between mammalian and fish leukocyte sensitivity to cortisol is discussed in the light of differences in the immune response of mammals and fish.

Regulation of interleukin 1 beta RNA expression in the common carp, Cyprinus carpio L.

The intron-exon organisation of the carp IL-1beta gene consists of 2455bp and comprises seven exons. Three IL-1beta RNA transcripts have been found 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. The intron-containing products probably represent partially spliced transcripts. IL-1beta mRNA expression in carp was semi-quantitatively analysed by RT-PCR in multiple organs, including brain and pituitary. Constitutive expression of the IL-1beta mRNA was found in these organs with a predominant expression in the immune organs head kidney and spleen. Furthermore, a scattered distribution of IL-1beta producing cells was shown by in situ hybridisations of head kidney tissue. Administration of phorbol-myristate-acetate (PMA), lipopolysaccharide (LPS) or retinoic acid (RA), to phagocytes isolated from the head kidney, resulted in expression of IL-1beta intron-containing transcripts. Of these, only PMA and LPS were stimulators that induced the fully spliced transcript. A role for the nuclear factor (NF)-kappaB pathway in carp IL-1beta expression was shown with suppression of the LPS-induced IL-1beta expression by NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC). Cortisol was able to inhibit in vitro constitutive expression of IL-1beta transcripts. Addition of cortisol simultaneously with LPS could not substantially inhibit transcription.

Cortisol inhibits apoptosis in carp neutrophilic granulocytes

The direct effect of cortisol treatment on carp neutrophil viability was examined in vitro. Cortisol treatment caused an inhibition of neutrophil apoptosis. The effect was blocked by glucocorticoid receptor blocker RU486, showing that rescue from apoptosis was receptor mediated. Using binding studies with radioactive cortisol, a single class of glucocorticoid receptors was detected with high affinity (Kd = 2.6 nM) and low capacity (497 receptors/cell) for cortisol binding. Both in vitro and in vivo cortisol treatment did not affect neutrophil respiratory burst activity. These data indicate that cortisol can augment the supply of functional neutrophilic granulocytes in conditions of acute stress, which may be essential for survival, since phagocytes form the first line of defence against micro-organisms.

Multiple acute temperature stress affects leucocyte populations and antibody responses in common carp, Cyprinus carpio L

Stress is a potential factor causing increased susceptibility of fish to pathogens. In this study, stress-induced immunological changes that may contribute to a decreased immune status were investigated. A 3 h drop in ambient water temperature of 9 degrees C was used as a relative mild and acute stress model for carp. Effects of this stressor on the dynamics of leucocyte populations were determined with specific monoclonal antibodies. The relative number of circulating B-lymphocytes in the total leucocyte population decreased significantly within 4 h after the onset of single or multiple cold shocks. This decrease was reversible, as B-lymphocyte numbers were restored within 24 h. Most probably, a redistribution of B-lymphocytes contributed to this phenomenon. In head kidney, an increase was measured in the relative number of B-lymphocytes. Granulocyte numbers showed opposite reactions: the percentage of granulocytes in the total leucocyte population nearly doubled in circulation and decreased significantly in the head kidney. This demonstrates that in vivo, a mild stressor differentially alters the distribution of leucocytes. In stressed carp, the percentage of apoptotic lymphocytes in blood is significantly higher compared with the unstressed animals. B-lymphocytes as well as Ig- lymphoid cells contributed to this increased apoptosis. Labelling of blood lymphocytes with a polyclonal antiserum against the glucocorticoid receptor also showed, besides B-lymphocytes, part of the Ig- lymphoid cell population to be glucocorticoid receptor positive. As the distribution of B-lymphocytes was substantially affected, the effect of temperature stress on T-lymphocyte-independent (trinitrophenyl-lipopolysaccharide) and T-lymphocyte-dependent (dinitrophenyl-keyhole limpet hemocyanin) humoral antibody responses was determined. Kinetics of the primary antibody response to the T-lymphocyte-independent antigen showed lower antibody titres in stressed carp during the onset of the immune response, implying a slower development of the antibody response against the T-lymphocyte-independent antigen.

Carp macrophages and neutrophilic granulocytes secrete an interleukin-1-like factor.

Carp, Cyprinus carpio L, macrophages and neutrophilic granulocytes obtained from pronephros were cultured. Supernatant was harvested after 48 h and tested for interleukin-1 (IL-1) bioactivity. A concentration-dependent stimulation of proliferation was found of carp Ig- lymphocytes as well as of the murine IL-1 dependent D10 (N4) M cell line. A 4 h treatment of cells with phorbol myristate acetate prior to culture gave a two- to fourfold enhancement of the bioactivity in the supernatant. Antibodies raised in sheep against human recombinant IL-1 alpha or IL-1 beta added to the supernatant annulled the IL-1 bioactivity. Western blot analysis of supernatants with sheep or rabbit polyclonal antisera against human IL-1s revealed 22 kDa and 15 kDa protein species. The predominant newly synthesized protein that was immunoprecipitated with these antisera was a 15 kDa molecular species. We conclude that carp macrophages and neutrophilic granulocytes produce an IL-1-like molecule with T-cell proliferating potency that shares structural similarities with mammalian IL-1. This is the first evidence for the IL-1 signal protein in carp immunocompetent cells.

Increased efficacy of immersion vaccination in fish with hyperosmotic pretreatment

Immersion vaccination is common practice in aquaculture, because of its convenience for mass vaccination with sufficient protection. However, the mechanisms of antigen uptake and presentation, resulting in a protective immune response and the role of the innate immune system therein are largely unknown. The impact of immersion vaccination on fish physiology and on the ensuing innate and specific immune response was characterized with fluorescently labeled particulate and soluble model antigens. Vaccination of common carp by direct immersion (DI) or hyperosmotic immersion (HI; direct immersion, preceded by a brief immersion in a hypertonic solution) greatly enhanced the uptake of soluble, but not particulate antigen through temporary disruption of the integrity of the epithelia of gills and skin. Damage induced is mild and does not impose additional stress over the handling associated with immersion vaccination. Especially HI briefly but strongly activates the innate immune system. We conclude that HI more effectively increased the uptake of vaccine and enhanced the efficacy by which vaccine components are processed and presented by the innate immune system, dually enhancing the mucosal immune response. Understanding the mechanisms involved in uptake and processing of vaccine in the early phase of the immune response will greatly benefit the design of immersion vaccination.

Functional analysis of carp interferon-γ: evolutionary conservation of classical phagocyte activation.

In teleost fish two IFN-gamma gene sequences were found for which two phylogenetic clusters can be distinguished. Our previous analysis of expression of these in carp led us to hypothesize that a classical IFN-gamma function is associated with the IFN-gamma2 cluster. We investigated the evolutionary conservation of the IFN-gamma function, inducing classical activation of phagocytes, thus skewing towards a Th1-like profile of immune activation. Recombinant proteins for the carp IFN-gamma sequences of both clusters were made and we studied their effects on expression of proinflammatory mediators. Carp IFN-gamma2, in contrast to carp IFN-gamma1, was powerful in inducing a proinflammatory reaction in phagocytes: a classical synergistic response with lipopolysaccharide was observed for the induction of iNOS expression and NO release, for expression of CXCL9-11-like chemokines and the expression of proinflammatory cytokines IL-1beta, TNFalpha and the IL-12 subunits p35 and p40. In contrast, like in mammals, the CXCL8-like cytokines are LPS but not IFN-gamma sensitive. These results corroborate an evolutionary conserved nature of IFN-gamma function in lower vertebrates including classical activation of phagocytes.

Morphine affects the inflammatory response in carp by impairment of leukocyte migration

Opioid peptides are evolutionary conserved and in teleost fish their specific receptor types have been identified not only on neuroendocrine cells but also on immunocytes. In the present work we have studied the effects of morphine, ligand for the mu3 opioid receptor, on innate immune responses of common carp. Both in vitro and in vivo, during zymosan-induced peritonitis, morphine reduced gene expression of pro-inflammatory cytokines/chemokines and chemokine receptors. Furthermore, in vitro morphine administration also affects nitric oxide production, chemotaxis and apoptosis of head kidney leukocytes. These results provide evidence for an anti-inflammatory function of morphine and suggest an evolutionary conserved cross-talk between chemokines and opioids.

Neuroendocrine–immune interaction in fish: Differential regulation of phagocyte activity by neuroendocrine factors

Coping with physical, chemical and biological disturbances depends on an extensive repertoire of physiological, endocrinological and immunological responses. Fish provide intriguing models to study bi-directional interaction between the neuroendocrine and the immune systems. Macrophages and granulocytes are the main actors in the first and rapid innate immune response. They are resident in different organs and are moreover rapidly recruited and activated upon infection. They act in response to recognition of pathogen-associated molecular patterns (PAMPs) via a repertoire of surface and intracellular receptors by inducing a plethora of defense reactions aiming to eradicate the pathogen. Subsequent production of inflammatory mediators stimulates other leukocytes required to develop an adaptive and specific antibody response. The type of phagocyte reaction will therefore depend on their differentiation state, specific receptor repertoire and their specific location. Apart from these pathogen induced responses, immune reactivity may be modulated by neuroendocrine factors. Over the last years we extensively studied changes in carp stress axis activity and the effect of its end-products on the immune system in an acute stress paradigm. We focus on specific neuroendocrine receptors on leukocytes and their effect on crucial phagocyte activities. We performed identification and functional analyses of different glucocorticoid, opioid and adrenergic receptors on carp phagocytes. Results show that their ligands of neuroendocrine origin may have substantial impact on specific phagocyte functions in a differential way. Inflammatory and microbicidal responses fight pathogens but may be detrimental to the host tissue. Neuroendocrine modulation may regulate inflammation to reach an optimum defense while preventing excessive host cell damage.