Peter Felsner

Melatonin and the Immune System

In recent years, melatonin, i.e. the major endocrine product of the pineal gland, was investigated as to its possible regulatory role in the communication between the neuroendocrine and the immune systems. First indications that melatonin may be an endocrine immunomodulator came from early reports about antitumor effects in animals and humans. Since then evidence has accumulated suggesting that melatonin-as a well-preserved molecule during evolution-is indeed involved in the feedback between neuroendocrine and immune functions. At present we begin to discover molecular mechanisms, by which melatonin affects cellular functions in general, and from the variety of possible direct and indirect interactions it appears that melatonin may play a complex physiological role in neuroimmunomodulation. In this article we want to give a critical review of the numerous reports on melatonin influencing immune functions, and to discuss the possible different mechanisms of action, which were suggested recently.

Continuous in vivo treatment with catecholamines suppresses in vitro reactivity of rat peripheral blood T-lymphocytes via α-mediated mechanisms

A 20 h continuous treatment of rats with catecholamines, using subcutaneously implantable retard tablets, had either no (adrenaline, isoproterenol, midodrine) or a slight (noradrenaline) suppressive effect on the in vitro responsiveness of peripheral blood T-lymphocytes. A marked suppression of the mitogen response ensued when adrenaline, noradrenaline or midodrine, but not isoproterenol, was applied together with the beta-receptor blocker propranolol, whereas the combination with the alpha-receptor blocker phentolamine had no effect. The mitogen response of splenic lymphocytes was not affected by any of these treatments. This alpha-mediated adrenergic suppression of peripheral blood T-cells was not correlated with general metabolic alterations, shifts in white blood cell counts or CD4+/CD8+ subsets, or with elevated glucocorticoid levels. The data suggest that to consistently influence the reactivity of rat peripheral blood lymphocytes by chronic adrenergic stimuli in vivo requires both high catecholamine levels and a bias towards alpha-adrenergic receptivity.

Prolonged alpha-adrenergic stimulation causes changes in leukocyte distribution and lymphocyte apoptosis in the rat

We have previously shown in the rat model that acutely or chronically increased peripheral catecholamines lead to suppression of lymphocyte responsiveness via alpha(2)-adrenoceptor activation. Here we investigated the effects of alpha-adrenergic treatment on total leukocyte numbers and proportions of leukocyte subsets in peripheral blood and lymphoid tissues. It was found that a 12-h treatment with subcutaneously implanted tablets, one containing norepinephrine (NE) and one propranolol, leads to an increase in total blood leukocyte counts, due to a pronounced increase in granulocytes. In contrast, the numbers of all classes of lymphocytes other than NK cells were decreased. This decrease in blood lymphocytes is apparently not due to redistribution, since in the thymus, spleen, mesenteric and peripheral lymph nodes, the total numbers of lymphocytes were decreased as well, without any changes in subpopulations. Analogous results were obtained with rats adrenalectomized before the catecholamine treatment. Animals that received the alpha-adrenergic treatment displayed significantly more apoptotic cells in the lymphoid organs, as determined by the TUNEL technique. In the spleen, the enhanced rate of apoptosis was confined to the white pulp; red pulp areas exhibited significantly fewer apoptotic cells. Thus, an increased alpha-adrenergic tone in rats led to a general loss of lymphocytes due to lymphocyte directed apoptosis that was independent of glucocorticoids.

Adrenergic suppression of peripheral blood T cell reactivity in the rat is due to activation of peripheral α2-receptors

A 20-h treatment of rats with catecholamines using s.c.implantable retard tablets markedly suppresses the in vitro reactivity of peripheral blood (PBL) T lymphocytes, provided that beta-receptors are blocked with propranolol (Felsner et al., 1992). The results can be summarized as follows: (i) the suppressive effect of noradrenaline+propranolol to the concanavalin A (ConA) response of PBL was abolished by the simultaneous application of the alpha-blocker phentolamine. Using selective agonists, the relevant receptor was identified to belong to the alpha 2-subtype. (ii) The alpha-adrenergic suppression of the PBL T cell response was likewise observed in adrenalectomized animals, which rules out the participation of secondarily induced glucocorticoids. Furthermore, the combination of noradrenaline with the watersoluble beta-blocker nadolol was equally effective to suppress the ConA response of PBL. (iii) An analogous alpha-mediated suppression of T cell function of PBL, but not spleen cells, was observed 1 h after i.p. treatment with tyramine, which leads to the release of endogenous noradrenaline. From these results it is concluded that the adrenergic suppression of PBL T cell functions is primarily due to the activation of peripheral alpha 2-receptors and that it is likewise observed under acute indirect sympathomimetic treatment.

In vivo immunomodulation by peripheral adrenergic and cholinergic agonists/antagonists in rat and mouse models.

Abstract: Our work is devoted to defining relationships between the immune system and the adrenergic and cholinergic systems in vivo. In the rat model, we have shown that the cells of different immune compartments express the genes of a defined set of adrenergic/cholinergic receptors, and it was shown that lymphocytes are a site of non‐neuronal production of norepinephrine and acetylcholine. Furthermore, using implantable slow‐release tablets containing adrenergic or cholinergic agonists/antagonists, distinct and partly opposite effects were observed on peripheral immune functions. Concerning sympathetic immunoregulation, our data‐in contrast to those of other studies‐suggest that an enhanced adrenergic tonus leads to immunosuppression primarily via α2‐receptor‐mediated mechanisms. Beta‐blockade strongly enhances this effect, most likely by inhibition of pineal melatonin synthesis. In recent experiments on the kinetics it was found that the continuous α‐adrenergic treatment entails a strong suppression of cellular responsiveness during the first few hours, which is increasingly followed by a general loss of lymphocytes in blood and lymphoid organs most likely due to enhanced apoptosis. More recently, we have extended our studies to the mouse model. First data obtained with RNAse protection assays suggest a biphasic effect on the gene expression of several cytokines in spleen cells due to adrenergic in vivo treatment.

Characterization of the Spontaneous Apoptosis of Rat Thymocytes in vitro

Unlike splenic or blood lymphocytes, rat thymocytes spontaneously undergo continuously increasing apoptosis during culture. In this study we characterized apoptotic thymus cells of rats according to cell size, nuclear dye binding and surface marker expression. Furthermore, the effects of cell density in culture, the age of the donor animals, glucocorticoids, and inhibition of protein synthesis were studied. It was found that: (1) apoptotic rat thymocytes are recognized in flow cytometry as small, acridine orange low, CD4low, CD8high cells; (2) the rate of apoptosis is dependent on the cell density in the culture in a biphasic manner; (3) thymic apoptosis increases with age of the donor animal in fresh, as well as in 24-hour cultivated cell suspensions; (4) neither adrenalectomy nor in vivo or in vitro treatment with the glucocorticoid antagonist RU 38486 influenced spontaneous apoptosis of thymocytes, and (5) inhibition of protein synthesis, which decreases apoptosis induced by corticosterone, had no effect on spontaneous apoptosis of thymocytes.

Cholinergic signals to and from the immune system.

This article reviews recent data from our laboratory towards the impact of the autonomous nervous system on mutual interactions between the immune system and the central nervous system. Using a pharmacological approach in rats it is shown that shifts in the adrenergic/cholinergic balance in vivo affect in vitro functions of the non-specific and specific immune system, whereby adrenergic and cholinergic stimulation in general have opposite effects. A high degree of integration appears to exist between cells of the immune system with the cholinergic system. Lymphocytes were found to react to acetylcholine, but are also able to produce and to degradate this neurotransmitter. In addition, changes in the cholinergic tonus were found to affect immune signaling to the brain and to protect thymocytes from apoptosis, possibly via a direct effect on thymic epithelial cells.

Beta-blockade enhances adrenergic immunosuppression in rats via inhibition of melatonin release.

We have recently shown in rats that an in vivo treatment with catecholamines via alpha 2-receptors leads to a pronounced suppression of T- and B-cell mitogen responses of peripheral blood lymphocytes (PBL), provided that a beta-blocker is administered concomitantly. Since melatonin (MEL) reportedly has stress-protective effects on several immune functions, and since the release of MEL from the pineal gland is inhibited by beta-blockade, we tested the effect of MEL substitution on T- and B-cell mitogen responses of PBL in rats treated with two s.c. implanted retard tablets containing noradrenaline (NA) and propranolol. It was found that an oral treatment with MEL (about 40 micrograms/animal) abolished the adrenergic immunosuppression. Furthermore, functional pinealectomy induced by constant light had a similar enhancing effect on the alpha 2-adrenergic immunosuppression as observed with beta-blockers, whereas PBL from animals kept at the regular light/dark interval were resistant to the treatment with the selective alpha 2-agonist clonidine. It is concluded that endogenous MEL effectively protects rat PBL from adrenergic immunosuppression, and that beta-blockers enhance the immunosuppressive property of alpha 2-adrenergic agents via blocking the night-time release of MEL.

The dialogue between the brain and immune system involves not only the HPA-axis

Zusammenfassung Ausgehend von vorangegangenen Befunden, wonach Störungen des Feedbacks zwischen Immunsystem und Gehirn zu pathogenen Immunreaktionen prädisponieren, konzentriert sich unser Interesse auf die Rolle von adrenergen/cholinergen Neurotransmittern im Rahmen der Neuroimmunomodulation. Die Daten im Ratten- und Mausmodell zeigen, dass 1) sowohl Katecholamine als auch Azetylcholin potente immunregulatorische Eigenschaften besitzen, 2) cholinerge Mechanismen entscheidend an den afferenten Signalen des aktivierten Immunsystems beteiligt sind, und 3) Lymphozyten nicht nur funktionelle adrenerge/cholinerge Rezeptoren exprimieren, sondern auch in der Lage sind, Neurotransmitter, wie Azetylcholin, zu synthetisieren und in quantitativer Abhängigkeit des zellulären Aktivierungszustandes zu sezernieren. Laufende Untersuchungen haben zum Ziel, die Rolle dieser nicht neuronalen Neurotransmitter in Immungewegen, sowie die Relevanz exzitatorischer Aminosäuren als wichtige zentrale Neurotransmitter im Rahmen des Dialoges Gehirn-Immunsystem aufzuklären.Summary Starting out from our previous observations that defects in the immune system – brain feedback predispose to pathogenic immune responses, our interest focusses at the roles of adrenergic/cholinergic neurotransmitters in brain – immune interactions. We have shown in rodent models that 1) both catecholamines and acetylcholine are potent modulators of peripheral immune functions, 2) cholinergic signals are involved in the afferent signalling of the immune system, and 3) lymphocytes not only express functional adrenergic and cholinergic receptors, but synthesize and release neurotransmitters, such as acetylcholine, in quantitative dependence of differentiation and activation. Studies are presently being initiated to investigate the role(s) of these non-neural neurotransmitters within immune tissues, and to explore the relevance of excitatory amino acids as important central neurotransmitters in the brain – immune system dialogue.

Reactive disulfide bonds in immunoglobulin G. A unique feature in serum proteins of different species

A reactive disulfide bond (SS)* was detected and characterized in IgG of humans, rats and mice by virtue of disulfide interchange with dithionitrobenzoate. (SS)* was found exclusively in human IgG1 and rat IgG2b. In human IgG1 (SS)* was identified as the upper one of the two interheavy bridges in the hinge, where it appears to take part in complement activation. The biological significance of (SS)* in IgG was underlined by the fact that no other serum proteins were found to exhibit a similar reactivity.