Corticosteroids and interleukin-1, messengers for communication between the endocrine and immune system in carp.

<strong>Summary</strong>Stress-induced inummosuppression is a well known phenomenon and mostly attributed to actions of steroid hormones released upon activation of the hypothalamus-pituitary-adrenal (HPA)-axis. In mammals, this endocrine-immune interaction is part of a bidirectional communication network between the endocrine and the immune system. Until about 20 years ago, the two systems were thought to operate independently. Today we know that they communicate through shared receptors and signal molecules to ensure physiological homeostasis. Maintenance of physiological homeostasis in a changing and challenging environment is pivotal to the survival of every organism. Also in fish stress can have negative effects on immunity and again steroid hormones from the interrenal cells (the fish equivalent of the adrenals) are thought to be principle mediators. The mechanism by which these hormones exert their effect on the immune system, however, is unclear and it is not known whether these interactions are part of a bidirectional communication network like in mammals. Modem bony fish, teleosts, are representatives of the lowest vertebrates, which makes them interesting subjects for fundamental comparative studies. Moreover, they are the most successful group of vertebrates, as more than half of the vertebrate species living today are teleosts. Their capacity to adapt to diverse environments is reflected in the wide array of niches they occupy. Teleosts do have well-developed immune and endocrine systems. The peculiar Organisation of the head kidney in teleosts, where interrenal tissue is intermingled with haematopoietic cells, presents an unique extra argument to study immune-endocrine interactions in fish. This organ combines both important immunological functions with the production of cortisol, enabling direct, paracrine interactions.The studies presented in this thesis aim at elucidating mechanisms of immuneendocrine interactions involving the hypothalamus-pituitary-interrenaI (HPI)-axis in carp. The (bio)chemical messengers used by the immune system and their actions within the immune system require clarification, before possible interactions with the endocrine system can be studied. In mammals, IL- I is produced by macrophages as one of the factors that coordinate the immune response; moreover, IL-1 is an important modulator of HPA-axis activity. In order to investigate how the endocrine system conveys information to the immune system, we studied the mechanism of immune regulation by cortisol, the end product of the HPI-axis in fish.Carp macrophages and neutrophilic granulocytes secrete a factor into the culture medium with IL-1-like characteristics <strong>(chapter 2):</strong> 1) supernatants from these cells stimulate proliferation of both carp lymphocytes and a mouse IL-1-dependent T cell line; 2) this bioactivity can be blocked by polyclonal antibodies against recombinant human IL-1αand IL-1β; 3) Western blot analyses of culture supernatants revealed peptides similar in size to mammalian IL-1 (15 kDa) and its precursor (22 kDa); and 4) the predominant newlysynthesised protein precipitating with the antibodies against human IL-1 has an apparent molecular weight of 15 kDa. To obtain a reliable carp IL-1 source, the permanent carp leukocyte culture (CLC) (Faisal and Aline, 1990), was assayed for IL-1 production <strong>(chapter 3).</strong> The CLC cells did indeed produce a molecule which stimulated proliferation of both carp lymphocytes and IL-1-dependent mouse T lymphocytes. This cell line showed additional characteristics of macrophages, such as phagocytosis and mitogen-stimulated respiratory burst activity. Coculture of CLC cells with carp peripheral blood leukocytes also stimulated CLC respiratory burst activity, showing that lymphocyte derived signals stimulate the CLC cells.Subsequently, experiments were performed to study the mechanism by which corticosteroids affect the immune system. It was found that cortisol, and not its natural conversion product cortisone, decreased proliferation of carp peripheral blood leukocytes by induction of programmed cell death, or apoptosis <strong>(chapter 4).</strong> This is the first report to show that apoptosis as an immune regulatory mechanism originated from the early phase of vertebrate evolution, before the water-to-land transition in the Devonian period. Moreover, it was assessed that the conversion of cortisol into cortisone, which occurs in several fish tissues, represents a mode of inactivation of cortisol. The apoptosis inducing effect of cortisol on PBL was completely and dose-dependently abolished by the specific glucocorticoid receptor blocker RU486, demonstrating that cortisol-induced apoptosis is mediated by a glucocorticoid receptor <strong>(chapter 5).</strong> To characterise this receptor on carp peripheral blood leukocytes, binding studies with <sup>3</SUP>H-cortisol were performed. A single class of cortisol binding sites was found. These binding sites showed high affinity for cortisol (Kd of 3.8 nM), and a total of 490 binding sites per cell was calculated. The affinity of the receptor for cortisone was more than 250 times lower than the affinity for cortisol, which may explain the observation that cortisone does not affect lymphocyte apoptosis. Treatment of carp with cortisol in vivo, by adding cortisol to food pellets, decreased the average number of receptors per cell, which may be due to down-regulation of the receptor. However, at the same time a decrease in the percentage of circulating B cells was observed. If B cells constitute a glucocorticoid receptor (GR)-rich subpopulation, which is plausible since B cells are very sensitive to the effects of cortisol, the disappearance of this subpopulation from the circulation due to cortisol treatment may also be responsible for the decreased average number of receptors per blood lymphocyte.The decrease in the percentage of circulating B cells following in-vivo cortisol treatment implied leukocyte subpopulation-specific sensitivity to cortisol. Accordingly, in <strong>chapters 6 and 7</strong> it is shown that the sensitivity of carp leukocytes for cortisol are subtypeand differentiation state-specific. Cortisol did not affect apoptosis in the T cell fraction, in thrombocytes, and in non-stimulated B cells. However, apoptosis in activated B cells was increased by adding cortisol to the culture. In contrast, apoptosis in neutrophilic granulocytes, isolated from the head kidney, was inhibited by cortisol. Although cortisol is often considered to be a mere immune-suppressor, our results show that cortisol does not inhibit all aspects of the immune system, but specifically alters the viability of specific leukocyte (sub)populations. Additionally, cortisol did not affect neutrophil function, which was illustrated by normal respiratory burst activity of these cells even after 24h of cortisol treatment in vitro. One could imagine that in situations of stress this effect can augment the supply of functional neutrophils, which form the first line of defence against microorganisms. The cortisol-induced rescue of neutrophils from apoptosis could, like the induction of apoptosis in B cells, be completely blocked with RU486, demonstrating that this action of cortisol is also mediated by a glucocorticoid receptor. Analysis of the corticoid receptor on carp neutrophils revealed a single class of cortisol binding sites, with similar affinity and capacity as the receptors on carp peripheral blood leukocytes. Thus carp B cells and neutrophilic granulocytes probably share the same cortisol receptor.In mammals, cytokines (mainly IL-2) can regulate or modulate cortisol effects on the viability of leukocytes. <strong>Chapters 4 and 6</strong> report on the effects of lymphocyte culture supernatants, containing IL-2-like activity, on the viability of lymphocytes in culture. Addition of these supernatants to the cultures resulted in lower percentages of apoptotic B cells and putative T cells, showing that these supernatants indeed contain stimulating factors. The induction of apoptosis in activated B cells by cortisol, however, was not affected by these supernatants.The studies presented in this thesis describe the mechanism and the specificity of cortisol action on the immune system of a teleost fish. Binding of cortisol to its specific receptor in carp leukocytes leads to induction of apoptosis in B cells, which are involved in specific immunity. The same event leads to decreased apoptosis in neutrophilic granulocytes, which are involved in aspecific immunity. This may be important in situations of stress, and indicates a modulatory function for cortisol in the immune system. Although it remains to be elucidated whether immune-endocrine interactions in fish are bidirectional, IL-1-like signals of the immune system have been demonstrated here and provide an approach for studies on bidirectional communication