F.A.A. Weyts

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.

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.

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.

Conservation of Apoptosis as an Immune Regulatory Mechanism: Effects of Cortisol and Cortisone on Carp Lymphocytes

This is the first study to show that apoptosis as an immune regulatory mechanism is conserved in fish, demonstrating its importance in maintaining immunological homeostasis. The data further show that this mechanism is subject to control by glucocorticosteroids. Carp plasma cortisol concentrations increase from 20 to 434 ng/ml and cortisone from 5 to 50 ng/ml within 9 min of the onset of handling stress. At basal steroid concentrations in vitro, cortisol, but not its conversion product cortisone, inhibits proliferation of peripheral blood lymphocytes (PBL), as measured by [3H]thymidine incorporation. Induction of apoptosis in activated PBL is the apparent mechanism of cortisol action. In nonstimulated PBL cultures, apoptosis is induced by neglect (a lack of stimulating signals). Stimulation with LPS or PHA rescues lymphocytes from this type of apoptosis. Stimulated PBL populations, however, are sensitive to cortisol-induced apoptosis. Culture supernatants from activated PBL protect PBL from apoptosis by neglect, probably by supplying a growth signal. These supernatants, however, have no effect on cortisol-induced apoptosis.