I. Rinner

Gene expression of the key enzymes of melatonin synthesis in extrapineal tissues of the rat

Besides the pineal gland, melatonin is reported to be produced in a number of extrapineal sites, where it could act as an intracellular mediator or paracrine signal in addition to its endocrine effects. In view of the suggested immunoregulatory role of melatonin, we compared lymphoid organs and several other tissues of the rat for their potential to synthesize melatonin. Using the reverse transcription‐polymerase chain reaction (RT‐PCR) method, we determined the tissue‐specific expression of mRNAs encoding two key enzymes of the melatonin biosynthesis: serotonin‐N‐acetyltransferase (NAT) and hydroxyindole‐O‐methyltransferase (HIOMT). The minimal number of PCR cycles required to obtain a positive signal served as a measure for the abundance of a given mRNA. NAT and HIOMT mRNAs were detected in all tested tissues at high numbers of PCR cycles (40 and 45, respectively). At 35 cycles, only gut, testis, spinal cord, raphe nuclei, stomach fundus and striatum yielded positive signals for both enzymes. In conclusion, the presence of NAT and HIOMT mRNAs in a wide range of tissues corroborates and extends the notion of extrapineal melatonin synthesis. Comparatively low levels of the HIOMT messages in lymphoid organs, however, indicate a limited significance of melatonin synthesis within the immune system.

mRNA expression of serotonin receptors in cells of the immune tissues of the rat.

Serotonin (5-hydroxytryptamine, 5-HT) has been shown to play a role in immunoregulation; however, little is known about specific subtypes of 5-HT receptors involved in peripheral immunomodulation. In the present study we used RT-PCR methods to examine the mRNA expression of 5-HT receptors in the cells of lymphoid tissues of the rat. All 13 rat 5-HT receptor genes cloned so far were examined in ex vivo isolated spleen, thymus, and peripheral blood lymphocytes, as well as in mitogen-stimulated spleen cells. Positive signals were obtained for 5-HT1B, 5-HT1F, 5-HT2A, 5-HT2B, 5-HT6, and 5-HT7 receptor mRNAs in all three compartments. Mitogen (ConA and PWM) stimulated cells additionally expressed mRNA corresponding to the 5HT-3 receptor subtype. In contrast, 5-HT1A, 5-HT1D, 5-HT2C, 5-HT4, 5-HT5A, and 5-HT5B mRNAs were not detected in any of the examined cell populations. These results may be useful as a starting point for future functional studies on immunomodulatory effects of 5-HT and may help to understand conflicting serotonergic effects on immune functions as found in the literature.

Rat lymphocytes produce and secrete acetylcholine in dependence of differentiation and activation.

Previous data from this laboratory suggested for the first time that immune cells of the immune system of different species are capable to synthesize the neurotransmitter acetylcholine. In the present study we detected the RNA message for choline acetyltransferase in thymic, splenic and peripheral blood lymphocytes of rats using RT-PCR. Furthermore, using a sensitive radioimmunoassay, we measured acetylcholine in thymic, splenic and peripheral blood lymphocytes. T-cells were found to contain about three times the amount of acetylcholine as compared to B-cells, and CD4+ cells showed significantly higher levels as compared to CD8+ cells. Mitogenic stimulation with PHA increased the acetylcholine levels in lymphoid cells as well as the release into the supernatants.

Opposite effects of mild and severe stress on in vitro activation of rat peripheral blood lymphocytes

The effects of short-term handling and different durations of immobilization on serum levels of catecholamines, ACTH, prolactin, and corticosterone and in vitro functions of lymphocytes were examined in rats. The results show that changes in the immune response of peripheral blood lymphocytes (PBL) depend on the intensity of the stressor: Short (1 min) handling of cannulated rats induced an enhanced stimulation of PBL to respond to T and B cell mitogens, whereas immobilization of the same animals led to suppression, dependent on the time this stressor was applied. The decrease in the mitogen reactivity of PBL after 120 min of immobilization was reversible within 24 h, and could be largely prevented by adrenalectomy, confirming that factors released by this gland are mainly responsible for immunosuppression. In contrast to PBL, spleen cells showed an enhanced mitogen response to immobilization and adrenalectomy, indicating that the immune response is differently regulated in the various compartments of the immune system. Possible correlations of the various effects with changes in stress hormone levels are discussed.

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.

The parasympathetic nervous system takes part in the immuno-neuroendocrine dialogue.

In a series of experiments the communication between the parasympathetic nervous system and the immune system was examined in rats. The data are summarized as follows: (1) In vivo administration of physostigmine increased the number of plaque-forming cells whereas in vitro addition of cholinergic agonists decreased the specific antibody response. (2) Prolonged in vivo treatment with atropine or physostigmine influenced concanavalin A stimulation of lymphocytes of different compartments in different ways. (3) Immunization with sheep red blood cells changed the number and the affinity of muscarinic cholinergic receptors in the hippocampus. (4) Cholinergic stimulation in vivo inhibited the transient increase of plasma corticosterone following immunization. Our results provide evidence that the parasympathetic nervous system is included in the dialogue between the neuro-endocrine and the immune systems.

A Possible Role for Acetylcholine in the Dialogue between Thymocytes and Thymic Stroma

In this article we will review data suggesting that acetylcholine takes part in the mutual interplay between developing T cells and thymic epithelium, and thereby may influence the generation of the T-cell repertoire. In the first part we will recapitulate our findings according to which cholinergic agonists affect thymocyte apoptosis via a nicotinergic effect on thymic epithelial cells. In the second part we will present evidence that acetylcholine within the thymus is mainly derived from the thymocytes themselves, and that the production and release of this neurotransmitter is dependent on activation of thymic lymphocytes.

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.