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Objective: Psychological stress has been implicated in the pathophysiology of both inflammatory and functional gastrointestinal (GI) diseases. The goal of this study was to address neuroendocrine modulation of cytokine production by peripheral blood cells in GI diseases., Methods: We analyzed the in vitro effects of the beta-adrenergic agonist terbutaline and the glucocorticoid agonist dexamethasone on TNF-alpha and IL-10 production by LPS-stimulated monocytes in whole cell blood cultures in patients with inflammatory bowel diseases in remission (N=10), diarrhoea-predominant irritable bowel syndrome (IBS, N=12), patients with a recent gastroenteritis (post-infectious group, N=10), and healthy controls (N=15)., Results: In response to terbutaline, there was a significant increase in IL-10 production (concentration effect: p<0.05), which was diminished in IBD (group effect: p<0.01), comparable in IBS and controls, but enhanced in the post-infectious group (group x concentration effect: p<0.05). In contrast, terbutaline resulted in a concentration-dependent suppression of TNF-alpha production, which was comparable in all groups. Dexamethasone suppressed TNF-alpha production in a dose-dependent manner in all groups, but this effect was significantly more pronounced in post-infectious subjects (group effect: p<0.05)., Conclusions: In IBD, disturbed adrenergic regulation of IL-10 could be part of the mechanism(s) underlying the modulation of disease activity by psychological stress. Diarrhoea-predominant IBS was not associated with altered adrenergic or glucocorticoid regulation of cytokine production by peripheral blood cells, whereas a recent history of gastroenteritis was associated with disturbed neuroendocrine modulation of cytokine production, which may play role in the pathophysiology of post-infectious IBS.

Background and Purpose: Production of nitric oxide is thought to play an important role in neuroinflammation. Previously, we have shown that combined inhibition of neuronal nitric oxide synthase (nNOS) and inducible NOS (iNOS) can reduce hypoxia-ischemia-induced brain injury in 12-day-old rats. The aim of this study was to analyze changes in expression of nNOS, iNOS and endothelial NOS (eNOS), and nitrotyrosine (NT) formation in proteins in neonatal rats up to 48 h after cerebral hypoxia-ischemia., Methods: Twelve-day-old rats were subjected to unilateral carotid artery occlusion and hypoxia, resulting in unilateral cerebral damage. NOS and nitrotyrosine expression were determined by immunohistochemistry and Western blot analysis at 30 min-48 h after hypoxia-ischemia., Results: nNOS was increased in both hemispheres from 30 min to 3 h after hypoxia-ischemia. In the contralateral hemisphere, eNOS was decreased 1-3 h after hypoxia-ischemia. In the ipsilateral hemisphere, eNOS was decreased at 0.5 h after hypoxia-ischemia, normalized at 1-3 h and was increased 6-12 h after hypoxia-ischemia. At 24 and 48 h after hypoxia-ischemia, eNOS levels normalized. Surprisingly, iNOS expression did not change from 30 min up to 48 h after hypoxia-ischemia in the ipsi- or contralateral hemisphere. In addition, the regional expression of iNOS in the brain as determined by immunohistochemistry did not change after hypoxia-ischemia. Expression of nitrotyrosine was slightly increased in both hemispheres only at 30 min after hypoxia-ischemia., Conclusion: In 12-day-old rat pups, cerebral hypoxia-ischemia induced a transient increase in nNOS, eNOS, and nitrotyrosine in proteins, but no change in iNOS expression up to 48 h after the insult.

The dopamine transporter (DAT) is responsible for the re-uptake of dopamine into presynaptic nerve terminals and thereby controls dopaminergic neurotransmission. Deletion of DAT results in a hyperdopaminergic phenotype and DAT(-/-) mice are characterized by pituitary hypoplasia, impaired maternal behavior, and increased locomotion. From earlier studies, we have evidence that the activity of the central dopaminergic system may play a role in determining immune reactivity and disease susceptibility. To further explore the functional relation between the dopaminergic system and the immune system, we investigated the activity of the immune system in DAT(-/-) mice. We show that in vitro, splenocytes from DAT(-/-) mice displayed reduced natural killer cell activity and reduced mitogen-induced cytokine responses. In contrast, LPS-induced cytokine production by macrophages was enhanced. In vivo, the cellular response to immunization with ovalbumine (OVA-induced delayed type hypersensitivity response) was significantly reduced. Interestingly, the OVA-induced humoral response (anti-OVA IgG) was increased in DAT(-/-) mice compared to wild-type animals. Plasma levels of catecholamines and corticosterone did not differ significantly between DAT(-/-) and wild-type animals. In conclusion, we show in the present study that interfering with the dopaminergic system has major consequences for both the acquired and the innate immune response.

Beta2-adrenergic agonists have immunomodulatory effects both in vitro and in vivo. We describe that oral salbutamol (beta-adrenergic agonist) administration has tissue-specific effects on cytokine production induced by intraperitoneal (i.p.) lipopolysaccharide (LPS) administration. Salbutamol reduced LPS-induced IFN-gamma levels at both mucosal and non-mucosal sites. However, salbutamol increased IL-10 levels in the peritoneal cavity, but decreased levels in terminal ileum and lung. Salbutamol did not alter LPS-induced TGF-beta levels in the terminal ileum, but increased levels in liver and peritoneal cavity. Thus, orally administered salbutamol decreases LPS-induced IFN-gamma levels in all tissues tested, but has tissue specific effects on IL-10 and TGF-beta levels.

Objective: To analyze the effects in vitro of alpha- and beta-adrenoceptor agonists on splenocyte proliferation and on proinflammatory cytokine production in splenocytes and peritoneal macrophages (MF) in different stages of EAE., Methods: Splenocytes and peritoneal macrophages were harvested in the acute phase of EAE and in remission, and from controls. The beta-agonist terbutaline, the alpha(1)-agonist methoxamine, and the alpha(2)-agonist UK-14304 were added with ConA or lipopolysaccharide (LPS). TNF-alpha and IFN-gamma contents in supernatant and splenocyte proliferation were determined., Results: Terbutaline and UK-14304 significantly suppressed TNF-alpha production by MF. However, EAE acute phase rats were resistant to the suppressive effect of UK-14304. Terbutaline significantly suppressed IFN-gamma and TNF-alpha production by splenocytes. EAE acute phase and remission animals showed reduced terbutaline-induced inhibition of IFN-gamma production., Conclusions: Disturbed sympathetic-immune communication in EAE is characterized by alterations in adrenergic sensitivity via both alpha- and beta-adrenergic pathways.

G-protein-coupled receptors (GPCR) play an important role in inflammation. Their responsiveness is regulated by G-protein-coupled receptor kinases (GRKs) and beta-arrestins. We show here that induction of experimental autoimmune encephalomyelitis (EAE) by myelin oligodendrocyte glycoprotein (MOG) resulted in a profound decrease in GRK2 and GRK6 protein in splenocytes during all phases of disease. GRK2 mRNA was also lower during EAE, although the decrease in mRNA was less pronounced than the decrease in GRK2 protein. Interestingly, beta-arrestin protein expression was significantly increased. Downregulation of GRK2 was restricted to the spleen and mesenteric lymph nodes and was not observed in peritoneal macrophages. Furthermore, EAE did not induce alterations in GRK2 expression in heart, liver and pituitary.

Maternal deprivation of neonatal animals has been shown to induce long-lasting changes in the reactivity of the neuroendocrine system. The aim of the present study was to investigate whether maternal deprivation also affects susceptibility to immune-mediated diseases such as experimental autoimmune encephalomyelitis (EAE) in adult life. To this end, 9-day-old rat pups were subjected to a short-lasting maternal deprivation for a period of 24 h. At the age of 8 weeks, we induced EAE in these rats by immunization with myelin basic protein (MBP) in complete Freund's adjuvant. Our data demonstrate that short-lasting maternal deprivation induces a marked increase in the severity of EAE in the animals in later life. The histopathological evaluation of spinal cord and cerebellum corresponded with the observed differences in clinical symptoms of EAE. Moreover, neonatal maternal deprivation affects macrophage functioning at adult age. In contrast, no differences were observed in in vitro mitogen- and MBP-induced cytokine production by splenocytes. LPS-induced corticosterone release did not differ either between maternally deprived and control animals. We conclude that short-lasting neonatal maternal deprivation of rat pups has long-lasting consequences for macrophage activity and for susceptibility to the inflammatory autoimmune disease EAE.

The family of adrenergic receptors (AR) plays a central role in regulation of the activity of many organ systems. Consequently, regulated expression of the various subtypes of AR is an important mechanism in maintaining homeostasis. Previously, we have shown that alpha(1)-AR triggering of peripheral blood mononuclear cells from patients with juvenile chronic arthritis results in increased IL-6 production. In contrast, alpha(1)-AR agonists do not alter cytokine production by cells of healthy individuals. The aim of the present study was to investigate whether pro-inflammatory cytokines can regulate the expression of mRNA encoding AR of the alpha(1)-family. We show that human THP-1 monocytic cells express mRNA encoding of two of the three cloned subtypes of alpha(1)-AR: alpha(1b)-AR and alpha(1d)-AR mRNA. The cytokines TNF-alpha and IL-1beta decrease level of mRNA for alpha(1d)-AR in THP-1 monocytic cells. In contrast, alpha(1b)-AR mRNA levels are not affected by these two cytokines. Interestingly, IL-1beta and TNF-alpha induce the expression of alpha(1a)-AR mRNA in THP-1 monocytic cells. In human umbilical vein endothelial cells (HUVEC), IL-1beta and TNF-alpha decrease both alpha(1b)-AR and alpha(1d)-AR mRNA levels in HUVEC. alpha(1a)-AR mRNA is not detectable in HUVEC.IL-6 and IL-8, two other pro-inflammatory cytokines tested in this study, do not change alpha(1)-AR subtype levels in HUVEC or monocytic cells. Our data demonstrate that TNF-alpha and IL-1beta can regulate expression of alpha(1)-AR mRNA and that cytokine regulation of alpha(1)-AR expression is subtype- and tissue-specific.

Glucocorticoids are frequently administered for the prevention of chronic lung disease in infants with respiratory distress syndrome. However, neonatal treatment may have consequences for immune functioning in the long-term. Here we demonstrate that neonatal glucocorticoid treatment has long-lasting effects on mRNA expression of several Vbeta genes within the CD4 and CD8 T cell subset in rats. Changes in the peripheral T cell Vbeta repertoire may be a consequence of altered intrathymic selection events in which corticosterone plays an important role. Indeed, here we show that neonatal glucocorticoid treatment affects corticosterone production by thymic epithelial cells during neonatal life. In conclusion, changes in T cell Vbeta repertoire after neonatal glucocorticoid treatment may contribute to altered immune reactivity in later life.

Juvenile rheumatoid arthritis (JRA) is characterized by chronic inflammation of the joints. In the present study we demonstrate that exposure of JRA patients to a noradrenergic stressor (cold pressor test) results in enhanced LPS-induced IL-6 production by peripheral blood cells of these patients. Healthy, age-matched controls had the same rise in norepinephrine, but do not respond with changes in IL-6 production after exposure to the cold pressor test. Moreover, PBMC of patients with JRA express mRNA encoding alpha(1)-adrenergic receptors (AR), predominantly of the alpha(1d)-AR subtype. In contrast, we could not detect mRNA encoding for alpha(1)-AR in PBMC of healthy controls. The results of this study suggest that expression of alpha(1)-AR mRNA in PBMC during chronic inflammation is associated with altered responses of the immune system to stress.

alpha(1)-Adrenergic receptors (ARs) are not expressed by peripheral blood mononuclear cells (PBMCs) of healthy human individuals. However, in the present study we show that alpha(1)-ARs can be induced in lymphocytes after culturing with either the mitogen PHA or the glucocorticoid dexamethasone. Moreover, incubation of these activated PBMCs with noradrenaline (NA) results in enhanced phosphorylation of ERK-2, a kinase involved in the activation of many immune functions. Similar induction of alpha(1)-AR mRNA with concomitant NA-induced activation of ERK-2 occurs in monocytes after culture with LPS. Our results demonstrate that functional alpha(1)-ARs can be induced on PBMCs and that these alpha(1)-ARs mediate NA-induced activation of ERK-2.

Neuroendocrine system and immune system can communicate via the use of soluble mediators like hormones, neurotransmitters and cytokines. The level of mediators secreted by either of these systems creates the milieu in which immune and neuroendocrine responses take place. For adequate communication between the systems, receptors for hormones, neurotransmitters and cytokines are required. This review describes the role of regulated expression and function of receptors for hormones and neurotransmitters within the immune system in neuroendocrine-immune communication.

Beta2- and alpha2-adrenergic receptors (AR) are thought to be the main AR subtypes to exert the effects of catecholamines on the immune system. However, in the present study, we demonstrate that another subtype of AR can be induced in human monocytes. Expression of alpha1b- and alpha1d-AR mRNA can be obtained by culturing freshly isolated human peripheral blood monocytes with the neuroendocrine mediators dexamethasone or the beta2-AR agonist terbutaline. Using the human monocytic cell line THP-1, we demonstrate that increased levels of alpha1b- and alpha1d-mRNA are accompanied by increased levels of receptor protein as determined by Western blot analysis and radioligand binding assays. This study describes for the first time regulated expression of alpha1-AR subtypes in human monocytes.

In this paper, we describe that met-enkephalin and/or enkephalin-containing intermediary peptides of the prohormone pro-enkephalin A are produced and secreted by human peripheral blood T cells and monocytes. The peptides are produced after stimulation with the mitogenic monoclonal antibodies anti-CD2.1/2.2 and anti-CD28. In monocytes, enkephalin synthesis was induced by stimulation with lipopolysaccharide. We demonstrate here that these immune cell-derived enkephalins play an important regulatory role in the immune response. By using an anti-sense oligonucleotide strategy we could block the production of enkephalins. Blockade of the production of met-enkephalin and enkephalin-containing intermediary peptides resulted in enhancement of the proliferative T cell response and inhibition of monocyte IL-6 secretion. In vitro reconstitution of the anti-sense treated cultures with synthetic met-enkephalin or the delta-type specific opioid receptor agonist deltorphin could reverse inhibition of monocyte IL-6 production, suggesting that endogenous enkephalins act via membrane opioid receptors. In contrast, addition of met-enkephalin or deltorphin to the anti-sense treated T cell cultures did not have any effect on T cell proliferation.

Exposure to synthetic glucocorticoids (GCs) or other stimuli around birth may affect neuroendocrine and immune responsiveness in the offspring. Experiments were conducted to investigate whether maternal manipulation with saline or with GCs alters the corticosterone (CORT) response to a mild stressor in the offspring, and whether maternal manipulation results in long-term altered in vivo humoral and cellular immune responsiveness in the offspring. Pregnant rats were given dexamethasone (DEX, 1.2 mg/kg body weight, i.p.) or saline (SAL) at day 17 and 19 of gestation. A third group of pregnant rats was left undisturbed (UNTR-group). After maternal DEX treatment, no altered CORT response was seen to a novel environment at 20 days of age, as compared to both the SAL-treated group and the UNTR-group. However, saline administration to pregnant rats caused an increased CORT response in female offspring, but not male offspring, as compared to the UNTR-group (P < or = 0.01). Furthermore, no effects of maternal DEX exposure were seen on IgG2a production after immunization with a conjugated pneumococcal polysaccharide (PPS-14-CRM197) at 6 weeks of age. However, maternal SAL treatment enhanced anti-PPS-14 IgG2a antibody levels in female offspring, but not in male offspring, as compared to the UNTR-group (P < or = 0.05). Cellular immune responses were measured by an oxazolone-induced contact hypersensitivity response (CHS-response), at 8 weeks of age. Maternal SAL treatment increased the CHS response in adult male rats, but not in female rats, as compared to both the UNTR-group and the DEX-group (P < or = 0.005). These data suggest that manipulations during late pregnancy not only affect endocrine responsiveness, but also influence immune responsiveness in the rat offspring. Furthermore, these effects may be long-term and gender-specific.

We investigated the regulatory influence of several cytokines on the expression of preproenkephalin (PPE) mRNA in human peripheral blood mononuclear cells (PBMC). By use of a quantitative reverse-transcriptase-polymerase chain reaction (RT-PCR), we demonstrate that the T helper 2 cytokines IL-4 and IL-10 are more potent in upregulating PPE mRNA expression in human PBMC than the T helper 1 cytokines IL-2 and gamma-IFN. In addition, TGF-beta is also an effective inducer of PPE mRNA. TGF-beta, IL-4 and IL-10 increase the cytoplasmatic concentration of met-enkephalin in PBMC. Secretion of met-enkephalin in the culture supernatant of IL-4- or IL-10-stimulated PBMC could not be observed, but proenkephalin A-derived met-enkephalin containing peptides could be demonstrated. IL-4 and IL-10 do not induce PPE mRNA via the same pathways. We could observe that PKA is involved in IL-4 mediated PPE mRNA induction, whereas IL-10 apparently uses another route.

Interleukin-8 (IL-8) is an important cytokine in inflammatory processes by functioning as a chemoattractant and as an activator of oxygen metabolism. In the present study we demonstrate that beta 2-adrenergic agonists potentiate the lipopolysaccharide (LPS) and IL-1 induced production of IL-8 by human monocytes. In addition, beta 2-adrenergic activation enhances IL-8 release and mRNA expression for IL-8 in the human monocytic cell line U937. beta 2-adrenergic activation of these cells also results in enhanced production of the anti-inflammatory cytokine IL-10. However, IL-10 is not involved in the regulation of IL-8 production. The effect of the beta 2-adrenergic agonist on IL-8 production is presumably mediated via increased cAMP formation, since it can be mimicked by the cAMP analogue dibutyryl-cAMP (db-cAMP). We conclude that enhancement of IL-8 production is one of the pathways via which beta 2-adrenergic agonists such as catecholamines can influence inflammatory responses.

Synthetic glucocorticoids (GC) are widely used drugs, also in the prevention of diseases that occur in the preterm newborn. Previously we have found that GC treatment of pregnant rats resulted in a persistent increase in the ratio of AVP over CRH in the mediobasal hypothalamus, and in an increased CD4/CD8 ratio in the thymus of the newborn. The objective of the present study was to investigate whether such effects were also seen after neonatal GC exposure, given in clinically-relevant doses. Dexamethasone-21-phosphate (DEX; 1.2 microg/g BW, i.p.) was given at day 5 and day 7 after birth. At day 18, 33, and 48 after birth effects of GC on the HPA-axis, and on CD4+ and CD8+ T cells in thymus and spleen were examined. Neonatal DEX treatment temporarily increased (p < 0.01) AVP stores in the external zone of the median eminence (ZEME) on day 18 after birth, and did not affect CRH stores. Resting plasma levels of ACTH and corticosterone remained unchanged after neonatal DEX treatment at any time interval studied. In the thymus and spleen, neonatal DEX treatment decreased (p < 0.0001) T cell numbers on day 18 after birth. Furthermore, neonatal DEX treatment increased (p < 0.01) the ratio of mature CD4-CD8+ over CD4-CD8+ thymocytes on day 18 after birth, but not on day 33 and day 48 after birth. In conclusion, neonatal DEX treatment has temporary effects on peptide expression in hypothalamic CRH neurons, and on thymocyte maturation. Apparently, neonatal exposure to GC affects potentially sensitive targets within the endocrine system and immune system but these alterations are reversible and readjusted during development.

During the last decade it has been shown that the central nervous system can influence the immune system. In healthy individuals, catecholamines can inhibit the production of pro-inflammatory cytokines like interleukin-6 (IL-6) and tumor necrosis factor alpha (TNF-alpha) via interaction with beta 2-adrenergic receptors. In contrast, we show here that catecholamines can stimulate the production of the interleukin-6 (IL-6) in children with the chronic inflammatory disease polyarticular juvenile rheumatoid arthritis (JRA). The induction of IL-6 is mediated by triggering of alpha 1-adrenergic receptors on peripheral blood leucocytes of the patients with polyarticular JRA. Functional alpha 1-adrenergic receptors are absent on leukocytes of normal donors and on leukocytes of patients with the oligoarticular form of the disease.

The effects of glucocorticoid (GC) treatment on the mature immune and neuroendocrine system are known to be reversible. However, prenatal GC exposure may have irreversible consequences on the development of the newborn. In this study, possible long-lasting effects of short-term prenatal GC treatment were examined on the developing thymus, spleen and hypothalamo-pituitary adrenal axis (HPA axis). Female rats were given dexamethasone (DEX, 400 micrograms, i.p.) on day 17 and 19 of pregnancy and offspring was studied at several time intervals (1-20 days) after birth, for examination of thymus, spleen, hypothalamus and blood plasma. Examination of thymus and spleen revealed that prenatal exposure to DEX resulted in decreased T cell numbers in thymus and spleen on day 1 after birth. Thymus regeneration after DEX exposure both during pregnancy and in adult life was completed after 24 days. However, the kinetics of regeneration of the thymi after prenatal DEX exposure were different from that seen after DEX in adult life. Whereas DEX treatment during pregnancy resulted in an increased ratio of CD4+/CD8- thymocytes over CD4-/CD8+ thymocytes compared to control groups on day 7 and day 20 after birth (time X treatment interaction; P < 0.05), DEX treatment in adult life did not change this ratio. T cell numbers in the spleen were significantly decreased at all neonatal ages studied. Regarding the hypothalamus, prenatal exposure to DEX altered the pattern of neonatal changes in peptide expression in corticotropin-releasing hormone neurons, with a selective reduction in CRH storage in the median eminence (7 and 9 days after birth) and an increase in AVP storage (9 and 20 days after birth). The ratio of AVP over CRH was significantly increased at all developmental ages studied. No effects were seen on basal ACTH and corticosterone levels in plasma. In conclusion, the kinetics of thymus regeneration after DEX exposure during pregnancy were different from that seen after DEX exposure in adult life. Prenatal DEX exposure also seemed to delay the migration of T cells into the spleen. Furthermore, prenatal DEX treatment exerted major effects on hypothalamic CRH neurons that maintained for at least 20 days after birth, which points towards an enhanced stress responsiveness of the HPA axis in later life.

The neuropeptide substance P (SP) has been shown to play an important role as a mediator of neurogenic inflammation. Moreover, in vitro SP is capable of modulating the activity of lymphocytes, monocytes and polymorphonuclear cells. We have examined one of the early events that occur after addition of SP to human peripheral blood mononuclear cells (PBMC). Addition of 10(-6)-10(-4) M SP to human peripheral blood mononuclear cells results in a dose-dependent rise in intracellular calcium concentration as determined by FACS analysis. We show that the effect of SP cannot be attenuated by the SP receptor antagonist [D-Pro4,D-Trp7,9]-SP(4-11), indicating that the response is not mediated via a SP receptor. Amphiphilic peptides like SP appear to have the capacity to insert themselves into the cell membrane and interact directly with intracellular proteins. This hypothesis is supported by the fact that the amphiphilic analogue of SP, [D-Pro2,D-Phe7,D-Trp9]-SP, is capable of inducing a calcium response in our system, although it is known as an SP receptor antagonist. Functionally, we show that SP increases the proliferative response of T cells induced by suboptimal concentrations of the mitogen PHA. These data provide evidence of a potential role of SP in the regulation of lymphocyte activation.

The insulin-like growth factors I and II (IGF-I and IGF-II) promote proliferation and differentiation of many cell types. We report that recombinant IGF-I and IGF-II augment both the lectin- and anti-CD3-induced proliferation of human peripheral blood mononuclear cells (PBMC) at concentrations proportional to their binding affinities. IGF-I and IGF-II also augmented the lectin-induced proliferation of purified T lymphocytes. Effects of IGF-I were found in cultures of T cells vigorously depleted for monocytes and supplemented with saturating concentrations of interleukin-1. The latter results indicate that the effect of IGF-I on the proliferation of T lymphocytes can occur independent of monocytes or monocyte-derived factors.