Exocytotic exposure and retrieval of membrane antigens of chromaffin granules: quantitative evaluation of immunofluorescence on the surface of chromaffin cells.

The exocytotic exposure of antigens of chromaffin granule membranes was studied with chromaffin cells isolated from bovine adrenal medulla. Antigens on the cell surface were visualized by indirect membrane immunofluorescence employing antisera against glycoprotein III and dopamine beta-hydroxylase. With unstimulated cells, only weak immunofluorescence on the cell surface was observed, whereas stimulated cells (with carbachol or Ba2+) exhibited much stronger reactions. In all cases the staining appeared as dots and patches. To quantitatively prove these observations, we analyzed the immunostained cells using a fluorescence-activated cell sorter. After stimulation, the average fluorescence intensity of the cell population was enhanced. This increase correlated with the degree of catecholamine secretion. The fluorescence intensity of stimulated cells varied over a broad range indicating that individual cells reacted variably to the secretagogues. When stimulated cells were incubated at 37 degrees C for up to 45 min after stimulation, a decrease of membrane immunofluorescence approaching that of unstimulated control cells was observed. Apparently, the membranes of chromaffin granules, which had been incorporated into the plasma membrane, were retrieved by a specific and relatively fast process. This retrieval of the antigen from the cell surface was blocked by sodium azide, but not influenced by colchicine, cytochalasin B, and trifluoperazine. The quantitative methods established in this paper should prove useful for further study of the kinetics of the exo-endocytotic cycle in secretory tissues.

Neuroimmunomodulation via limbic structures--the neuroanatomy of psychoimmunology.

During the last 20 years, mutual communications between the immune, the endocrine and the nervous systems have been defined on the basis of physiological, cellular, and molecular data. Nevertheless, a major problem in the new discipline "Psychoneuroimmunology" is that controversial data and differences in the interpretation of the results make it difficult to obtain a comprehensive overview of the implications of immunoneuroendocrine interactions in the maintenance of physiological homeostasis, as well as in the initiation and the course of pathological conditions within these systems. In this article, we will first discuss the afferent pathways by which immune cells may affect CNS functions and, conversely, how neural tissues can influence the peripheral immune response. We will then review recent data, which emphasize the (patho)physiological roles of hippocampal-amygdala structures and the nucleus accumbens in neuroimmunomodulation. Neuronal activity within the hippocampal formation, the amygdaloid body, and the ventral parts of the basal ganglia has been examined most thoroughly in studies on neuroendocrine, autonomic and cognitive functions, or at the level of emotional and psychomotor behaviors. The interplay of these limbic structures with components of the immune system and vice versa, however, is still less defined. We will attempt to review and discuss this area of research taking into account recent evidences for neuroendocrine immunoregulation via limbic neuronal systems, as well as the influence of cytokines on synaptic transmission, neuronal growth and survival in these brain regions. Finally, the role of limbic structures in stress responses and conditioning of immune reactivity will be commented. Based on these data, we propose new directions of future research.

Temperature-dependent specificity of cis-trans isomeric fatty acid interaction with the erythrocyte membrane.

Stabilization of red cells against hypotonic haemolysis by cis-trans isomeric free C18 fatty acids occurs with pronounced specificity which is strongly temperature-dependent, but in a distinctly different manner for the two configurational isomers. Oleic acid (cis-18:1) stabilizes very efficiently at 0 degrees C, even at the highest concentrations. Elaidic acid (trans-18:1) causes neither stabilization nor haemolysis at this temperature. At room temperature (23 degrees C), elaidic acid acquires the ability to protect, without turning haemolytic at high concentrations. At 37 degrees C elaidic acid also becomes haemolytic. The protecting effect of oleic acid at 0 degrees C is the result of a rapid reaction. The characteristic, temperature-dependent specificity of cis-trans isomeric C18 fatty acid interaction with the red cell membrane appears to be a general phenomenon, since it was observed alike with erythrocytes of different species.

Structure- and configuration-dependent effects of C18 unsaturated fatty acids on the chicken and sheep erythrocyte membrane.

High concentrations of unsaturated fatty acids are known to cause hemolysis. At low concentrations, however, unsaturated cis fatty acids have been found to protect erythrocytes against hypotonic hemolysis. In the present experiments we examined the effect of oleic (18:1), linoleic (18:2), linolenic (18:3), and elaidic (18:1) acid on the osmotic fragility of chicken and sheep erythrocytes, which markedly differ in their resistance to osmotic rupture. The results are summarized as follows: (A) The phenomenon of stabilization was observed in both species alike. (B) Interaction of cells with the fatty acids under isotonic conditions led to a persistent stabilization, i.e., the cells remained more resistant against osmolysis even after several washings. (C) Oleic and elaidic acid protected against osmotic rupture with a high degree of specificity. Linoleic and linolenic acid were much less protective. Thus, this effect appears to be specific for one double bond. (D) Contrary to the unsaturated fatty acids with cis configuration, elaidic acid with the trans configuration showed no biphasic behaviour, and even at the highest concentrations applied no hemolysis was observed.

IgG-Fc and C3 receptors in the chicken: distribution, tissue localization and functional significance.

This paper summarizes data obtained in our laboratory on the demonstration of receptors for IgG-Fc (FcR) and complement (CR) on mononuclear cells of the fowl. A clearcut distinction of these two structures on spleen cells was achieved in rosette assays using SRBC coated with rabbit IgG as indicator cells which bind avian complement, but are not bound by the FcR. The tissue distribution and localization of FcR and CR positive cells was studied in mixed hemadsorption assays on sections of both central (bursa, thymus) and peripheral (spleen) lymphoid organs from normal chickens, as well as lymphocytic infiltrated thyroid glands from animals of the Obese strain (OS) afflicted with spontaneous autoimmune thyroiditis. The possible significance of both receptors in the pathogenesis of this autoimmune process is discussed.

Distribution and functional analysis of B-L/Ia-positive cells in the chicken: expression of B-L/Ia antigens on thyroid epithelial cells in spontaneous autoimmune thyroiditis.

The B-L region of the chicken major histocompatibility complex (MHC), the so-called B-locus, corresponds to the murine H-2 I-region. Using alloantibodies and monoclonal antibodies to B-L we analyzed: (a) the tissue distribution of B-L+ cells, (b) the function of B-L+ cells, and (c) the possible role of B-L+ cells in the development of spontaneous autoimmune thyroiditis (SAT) in Obese strain (OS) chickens. The tissue distribution of B-L+ cells in peripheral blood and various lymphoid and nonlymphoid organs corresponds to what is known for mammals. In the bursa of Fabricius most lymphoid cells and the dendritic cells carry the B-L antigen; B-L+ thymic nurse cells (TNC) first appear on day 17 of embryonic life; chickens possess dendritic B-L+ cells in the skin resembling mammalian Langerhans cells; in addition we found that the microglia is unequivocally B-L+. B-L+ peripheral blood lymphocytes (PBL) were separated with a fluorescence-activated cell sorter. Ten percent of unstimulated PBL and 60% of phytohemagglutinin (PHA) stimulated T-cell blasts are B-L+. In graft-vs-host (GvH) assays B-L- cells were identified as the effector cells. These cells respond to PHA and concanavalin A (Con A), but not to pokeweed mitogen (PWM). B-L+ cells cannot be stimulated by Con A and PHA, but respond to PWM. They possess only a very low activity in GvH assays which can be inhibited by anti-T-cell sera. In OS chickens B-L+/non-B/, non-T and B-L+ T (blasts?) cells are found in the "first line" of mononuclear cell infiltration in the thyroid glands. Most interesting, thyroid epithelial cells--which are normally B-L- -become B-L+ in the neighbourhood of B-L+ infiltrating mononuclear cells. This observation may be of significance for autoantigen presentation and perpetuation in autoimmune thyroiditis. Finally, OS thymuses contain significantly less TNC than normal controls.

Antioxidant role of melatonin in lipid peroxidation of human LDL.

The antioxidant effect of melatonin on LDL oxidation was studied in vitro using either a thermolabile initiator or copper ions to induce lipid peroxidation. Loading of LDL with melatonin showed only weak protection against oxidative damage as compared to alpha-tocopherol. In the presence of high concentrations of melatonin (1000 mol/mol LDL) in the medium a clear protective effect was found during lag- and propagation phase, albeit weaker than after loading with alpha-tocopherol. It is concluded that melatonin is not incorporated into LDL in sufficient concentrations to prevent lipid peroxidation effectively. When melatonin is present in the incubation medium during oxidation, a partitioning equilibrium between aqueous and lipid phase is established. Only under these conditions can melatonin act as a chain breaking antioxidant. The concentrations required, however, are far beyond those found in human plasma. Therefore, the data in this study do not support a direct physiological relevance of melatonin as an antioxidant in lipid peroxidation processes.

N-acetylserotonin is a better extra- and intracellular antioxidant than melatonin.

Both melatonin and its precursor N-acetylserotonin have been reported to exert antioxidant properties both in vitro and in vivo. Since little is known about their antioxidant activity in lymphocytes, we investigated their effects on spontaneous and on oxidant-induced reactive oxygen species formation in human peripheral blood lymphocytes in comparison to the antioxidant trolox, a water-soluble analogue of alpha-tocopherol. Both melatonin and N-acetylserotonin exhibited antioxidant properties against t-butylated hydroperoxide- and diamide-induced reactive oxygen species formation in peripheral blood lymphocytes. N-acetylserotonin turned out to be about three times more effective than melatonin. In resting cells, the intracellular reactive oxygen species concentration was only decreased by N-acetylserotonin and trolox, melatonin had no effect. In t-butylated hydroperoxide-mediated cell death, N-acetylserotonin was as effective as trolox in protecting peripheral blood lymphocytes from cell death and required 10-fold lower concentrations than melatonin. Furthermore, in an aqueous cell-free solution, the capacity of N-acetylserotonin to scavenge peroxyl radicals was much higher than that of melatonin. These results clearly indicate N-acetylserotonin to be a much better antioxidant than melatonin.

Prooxidant activity of melatonin promotes fas-induced cell death in human leukemic Jurkat cells.

The antioxidant activity of melatonin (MEL) has been considered to constitute part of its physiological as well as pharmacological effects. However, as described herein we found a profound prooxidant activity of micro- to millimolar concentrations of MEL in the human leukemic Jurkat cell line. This prooxidant effect was increased in glutathione-depleted cells and counteracted by antioxidants. As a consequence MEL promoted fas-induced cell death. These data therefore indicate that MEL may be a modulator of the cellular redox status, but does not necessarily act as an intracellular antioxidant.

IgG1--as the only subclass of human serum IgG--spontaneously undergoes O2(-)-induced, noncovalent self-aggregation upon storage at room temperature.

An apparent gradual decrease of IgG1 serum levels of up to 40% occurs within 48 h of storage at room temperature. The effect does not concern any other IgG subclass, and is more pronounced in sera of smokers. A linear correlation was found between the extent of this "storage effect" and the initial concentration of IgG1, which rules out an enzymatic process following Michaelis-Menten kinetics. PAGE and Western blots of density gradient separated serum proteins revealed the presence of noncovalent self aggregates of IgG1 in stored sera. Addition of superoxide dismutase prevented both the formation of aggregates as well as the decay of IgG1 values. It is concluded that the instability of IgG1 is due to an enhanced propensity of this molecule to form self-aggregates, whereby O2(-)-radicals play a functional role. This mechanism, however, is not relevant to a previously detected selective decrease of relative IgG1 levels in sera of patients afflicted with malignant diseases of various tissue origin.

Long-term follow-up of cytokines and soluble cytokine receptors in peripheral blood of patients with juvenile rheumatoid arthritis.

Plasma levels of interleukin-1beta (IL-1beta), IL-2, soluble IL-2 receptor (sIL-2R), IL-6, IL-8, tumor necrosis factor-alpha (TNF-alpha), and the p60 soluble TNF receptor (sTNFR) were repeatedly determined by enzyme-linked immunosorbent assays (ELISA) in 35 patients with different subtypes of juvenile rheumatoid arthritis (JRA) during an observation period of up to 36 months. The data were related to conventional inflammatory parameters and disease activity. Patients with systemic disease showed the most pronounced elevations of plasma cytokines, followed by polyarticular and pauciarticular JRA. Soluble receptors sIL-2R and sTNFR were consistently elevated in patients of all JRA subtypes and indicated disease activity even in patients with normal C-reactive protein (CRP). In contrast, the determination of IL-1beta, IL-2, IL-8, and TNF-alpha revealed strikingly different individual profiles in patients of the same clinical subtype of JRA and irrespective of disease activity. It is concluded that the determination of sIL-2R and sTNFR may be relevant for monitoring JRA, as they indicate disease activity also in cases with unaltered conventional inflammatory parameters. The different individual cytokine profiles of patients within identical subtypes of disease suggest JRA to be even more heterogeneous than hitherto assumed. The data should be considered in attempts to develop anticytokine strategies in the therapy of JRA.

Regulatory effects of thymus humoral factor on T cell growth factor in aging mice.

The effect of thymus humoral factor (THF) on T cell growth factor (TCGF) production by T cells and the association between splenic macrophages and T cells from young (2-3 months) and old (20-24 months) mice in this respect were studied. Splenocytes were divided into three groups: stimulated with concanavalin A (Con A); preincubated with THF and then stimulated with Con A; or stimulated with Con A and thereafter incubated with THF. These cells were then examined for production of TCGF. Cells treated with Con A and THF as described above were passed on nylon wool to enrich T cell populations and added to mitogen-sensitized (Con A or lipopolysaccharide) adherent splenocytes of old and young mice in the following combinations: young adherent and young T cells; young adherent and old T cells; old adherent and young T cells; and old adherent and old T cells. The results demonstrated that: (A) cells of old mice produced less TCGF than the young; (B) preincubation of splenocytes or nylon-wool enriched T cells with THF increased the production of TCGF consistently in young mice, whereas in the old a significant increase was observed only in some cases; (C) depressed TCGF activity was observed when treatment with THF to splenocytes or nylon-wool enriched T cells from young and old mice was performed after Con A stimulation, and this was also more pronounced in the young; (D) the reduced level of TCGF in the old seemed to be related to a lesion in the T cell compartment, since adherent cells from old and young mouse spleens could support TCGF production by T cells from young mice and not from old.

The micro-membrane-fluorescence test: a new semiautomated technique based on the microtiter system.

A semiautomated modification of the membrane immunofluorescence (MF) technique was developed employing units of the Microtiter system. This new rapid technique, which allows the performance of several hundred samples within a few hours is exemplified on a MF test proving the specific reaction to anti-chicken bursa cell sera (ABS) and antichicken thymus cell sera (ATS) on bursa and thymus cells respectively. The possible value of the new method for the further standardization of immunofluorescence is also emphasized.

Avian lymphokines: an improved method for chicken IL-2 production and assay. A Con A-erythrocyte complex induces higher T cell proliferation and IL-2 production than does free mitogen.

Optimized production conditions and a functional assay of avian T cell growth factor (TCGF) or interleukin 2 (IL-2) are described. Treatment of lymphocytes with mitogen (Con A)-coated chicken red blood cells (MRC) resulted in markedly enhanced mitogenic response and IL-2 secretion compared to stimulation with free Con A. A positive correlation (r = 0.89) was found between mitogenic response and IL-2 activity of conditioned media. Enrichment of target cells, i.e., Con A lymphoblasts, by Percoll consistently improved the sensitivity of the IL-2 assay. The half-life time of chicken IL-2 at 40 degrees C was 9.7 +/- 1.7 h, which was considerably shorter than the value obtained for murine IL-2, i.e., 53.1 +/- 8.5 h. High concentrations of conditioned media were found to contain a dialysable factor that suppressed IL-2 promoted blast proliferation. The relevance of the data for in vitro analysis of T cell function as well as for establishing T cell lines in the chicken system are discussed.

Investigation of the recovery phenomenon in immunofluorescence after laser excitation.

Laser excitation was applied in two standard immunopathological indirect immunoflorescence systems in order to investigate bleaching and recovery of fluorescence intensity upon repeated illuminations. Variation of illumination times and dark periods between these showed that the recovery phenomenon is a function of both the (time times energy) product of excitation and the length of dark intervals. The value of high energy excitation in immunofluorescence in providing insight into the mechanisms of bleaching and recovery is discussed.

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.

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.

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.

Failure to alter neonatal transplantation tolerance by the injection of interleukin 2.

It has been postulated that the establishment of acquired, neonatal immunologic tolerance is due to a "deficit" in interleukin 2 (IL-2). To test this hypothesis, chickens were made immunologically tolerant to both major and minor histocompatibility antigens by transplantation of skin grafts onto newly hatched recipients. In this study, we injected various doses of IL-2 and concanavalin A simultaneously with transplantation and in some cases, several days posttransplantation, and we failed to enhance graft rejection. These results may have practical importance in respect to the clinical use of recombinant IL-2. Injection of IL-2 in and around surviving skin grafts also failed to alter skin graft survival.

Adrenergic suppression of peripheral blood T cell reactivity in the rat is due to activation of peripheral alpha 2-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.