Maoto, a traditional Japanese medicine, controls acute systemic inflammation induced by polyI:C administration through noradrenergic function.

Maoto, a traditional Japanese medicine (Kampo), is widely used to treat upper respiratory tract infections, including influenza virus infection. Although maoto is known to inhibit pro-inflammatory responses in a rodent model of acute inflammation, its underlying mechanism remains to be determined. In this study, we investigated the involvement of immune responses and noradrenergic function in the inhibitory action of maoto. In a mouse model of polyI:C-induced acute inflammation, maoto was administered orally in conjunction with intraperitoneal injection of PolyI:C (6 mg/kg), and blood was collected after 2 h for measurement of plasma cytokines by ELISA. Maoto significantly decreased PolyI:C-induced TNF-α levels and increased IL-10 production. Neither pretreatment with IL-10 neutralizing antibodies nor T-cell deficiency using nude mice modified the inhibitory effect of maoto, indicating that the anti-inflammatory effects of maoto are independent of IL-10 and T cells. Furthermore, the inhibitory effects of maoto on PolyI:C-induced TNF-α production were not observed in ex vivo splenocytes, suggesting that maoto does not act directly on inflammatory cells. Lastly, pretreatment with a ß-adrenergic receptor antagonist partially cancelled the anti-inflammatory effects of maoto. Collectively, these results suggest that maoto mediates its anti-inflammatory effects via ß-adrenergic receptors in vivo.

Beta-adrenergic receptor stimulation influences the function of monocytes/macrophages in ayu (Plecoglossus altivelis).

Adrenergic receptors (ARs) are members of the G-protein-coupled receptor superfamily that can be categorized into αARs and ßARs. The specific function of ARs in teleost monocytes/macrophages (MO/MФ) remains unknown. We determined the cDNA sequence of ARs from ayu (Plecoglossus altivelis; PaαAR and PaßAR). Sequence comparisons showed that PaαAR was most closely related to the αAR of the Japanese flounder and Nile tilapia, while PaßAR was most closely related to the ßAR of Atlantic salmon. The AR transcripts were mainly expressed in the spleen, and their expression was altered in various tissues upon infection with Vibrio anguillarum. PaαAR and PaßAR proteins were upregulated in MO/MФ after infection, and PaßAR knockdown resulted in a pro-inflammatory status in ayu MO/MФ upon V. anguillarum infection and lowered the phagocytic activity of MO/MФ. Our results indicate that PaßAR plays the role of an anti-inflammatory mediator in the immune response of ayu against bacterial infection.

Adrenergic Modulation of Hematopoiesis.

Hematopoiesis produce every day billions of blood cells and takes place in the bone marrow (BM) by the proliferation and differentiation of hematopoietic stem cells (HSC). HSC are found mainly adjacent to the BM vascular sinusoids where endothelial cells and mesenchimal stromal cells promote HSC maintenance by producing a variety of factors. Other cell types that regulate HSC niches include sympathetic nerves, non-myelinating Schwann cells and a variety of mature hematopoietic cells such as macrophages, neutrophils, and megakaryocytes. This review will focus on the role of adrenergic signals, i.e. of catecholamines, in the regulation of the HSC niche. The available evidence is rather controversial possibly due to the fact that adrenergic receptors are expressed by many cellular components of the niche and also by the often neglected observation that catecholamines may be produced and released also by the BM cells themselves. In addition one has to consider that, physiologically, the sympathetic nervous system (SNS) activity follows a circadian rhythmicity as driven by the suprachiasmatic nucleus (SCN) of the hypothalamus but may be also activated by cognitive and non-cognitive environmental stimuli. The adrenergic modulation of hematopoiesis holds a considerable potential for pharmacological therapeutic approaches in a variety of hematopoietic disorders and for HSC transplantation however the complexity of the system demands further studies. Graphical Abstract Sympathetic nerve termini may release NE while mature BM cells may release norepinephrine (NE) and / or epinephrine (E). Both may bind to ß-adrenergic receptor (AR) expressed in nestin+MSC in the hematopoietic stem cell (HSC) niche and regulate the physiological trafficking of HSC by modulating the expression of CXCL12 and SCF. Both NE and E may also activate Lin - c-Kit+ Sca-1+ (LKS) cell via another AR. In addition, NE may also signal to α1-AR expressed in pre-B cells which by TGF-ß secretion might regulate proliferation of their lymphoid progenitors in an autocrine manner and/or inhibit myeloid progenitors.

Postural Orthostatic Tachycardia Syndrome Is Associated With Elevated G-Protein Coupled Receptor Autoantibodies.

Background The etiology of postural orthostatic tachycardia syndrome (POTS) is yet to be established. The disorder is often misdiagnosed as chronic anxiety or a panic disorder because the autonomic failure in these patients is not severe. A growing body of evidence suggests that POTS may be an autoimmune disorder. Antinuclear antibodies and elevations of ganglionic, adrenergic, and muscarinic acetylcholine receptor antibodies have all been reported. Methods and Results We collected detailed clinical symptoms of 55 patients diagnosed with POTS. We also evaluated serum levels of autoantibodies against 4 subtypes of G-protein coupled adrenergic receptors and 5 subtypes of G-protein coupled muscarinic acetylcholine receptors by ELISA. Our patients had a multitude of comorbidities, were predominantly young females, and reported viral-like symptoms preceding episodes of syncope. We detected a significant number of patients with elevated levels of autoantibodies against the adrenergic alpha 1 receptor (89%) and against the muscarinic acetylcholine M4 receptor (53%). Surprisingly, elevations of muscarinic receptor autoantibodies appeared to be dependent upon elevation of autoantibodies against the A1 adrenergic receptor! Four patients had elevations of G-protein coupled autoantibodies against all 9 receptor subtypes measured in our study. Five POTS patients had no elevation of any autoantibody; similarly, controls were also negative for autoantibody elevations. There was a weak correlation of clinical symptom severity with G-protein coupled autoantibodies. Conclusions Our observations provide further evidence that, in most cases, POTS patients have at least 1 elevated G-protein coupled adrenergic autoantibody and, in some instances, both adrenergic and muscarinic autoantibodies, supporting the hypothesis that POTS may be an autoimmune disorder.

Engineering of Nanobodies Recognizing the Human Chemokine Receptor CCR7.

The chemokine receptor CCR7 plays a pivotal role in health and disease. In particular, CCR7 controls homing of antigen-bearing dendritic cells and T cells to lymph nodes, where adaptive immune responses are initiated. However, CCR7 also guides T cells to inflamed synovium and thereby contributes to rheumatoid arthritis and promotes cancer cell migration and metastasis formation. Nanobodies have recently emerged as versatile tools to study G-protein-coupled receptor functions and are being tested in diagnostics and therapeutics. In this study, we designed a strategy to engineer novel nanobodies recognizing human CCR7. We generated a nanobody library based on a solved crystal structure of the nanobody Nb80 recognizing the ß2-adrenergic receptor (ß2AR) and by specifically randomizing two segments within complementarity determining region 1 (CDR1) and CDR3 of Nb80 known to interact with ß2AR. We fused the nanobody library to one half of split-YFP in order to identify individual nanobody clones interacting with CCR7 fused to the other half of split-YFP using bimolecular fluorescence complementation. We present three novel nanobodies, termed Nb1, Nb5, and Nb38, that recognize human CCR7 without interfering with G-protein-coupling and downstream signaling. Moreover, we were able to follow CCR7 trafficking upon CCL19 triggering using Nb1, Nb5, and Nb38.

Chronic psychosocial stress compromises the immune response and endochondral ossification during bone fracture healing via ß-AR signaling.

Chronic psychosocial stress/trauma represents an increasing burden in our modern society and a risk factor for the development of mental disorders, including posttraumatic stress disorder (PTSD). PTSD, in turn, is highly comorbid with a plethora of inflammatory disorders and has been associated with increased bone fracture risk. Since a balanced inflammatory response after fracture is crucial for successful bone healing, we hypothesize that stress/trauma alters the inflammatory response after fracture and, consequently, compromises fracture healing. Here we show, employing the chronic subordinate colony housing (CSC) paradigm as a clinically relevant mouse model for PTSD, that mice subjected to CSC displayed increased numbers of neutrophils in the early fracture hematoma, whereas T lymphocytes and markers for cartilage-to-bone transition and angiogenesis were reduced. At late stages of fracture healing, CSC mice were characterized by decreased bending stiffness and bony bridging of the fracture callus. Strikingly, a single systemic administration of the ß-adrenoreceptor (AR) blocker propranolol before femur osteotomy prevented bone marrow mobilization of neutrophils and invasion of neutrophils into the fracture hematoma, both seen in the early phase after fracture, as well as a compromised fracture healing in CSC mice. We conclude that chronic psychosocial stress leads to an imbalanced immune response after fracture via ß-AR signaling, accompanied by disturbed fracture healing. These findings offer possibilities for clinical translation in patients suffering from PTSD and fracture.

Agonistic autoantibodies against B2-adrenergic receptors correlating with macrovascular disease in longstanding diabetes type 2.

AIMS: Agonistic autoantibodies directed against adrenergic, endothelin, and angiotensin receptors are known as pathogenic factors in disease-causing vascular impairments such as Buergers' disease, dilatative cardiomyopathy, dementia, and preeclampsia. Diabetes mellitus also causes micro- and macrovascular damages, but pathogenesis is still not fully understood. Following indications for a pathogenic role of the mentioned antibodies from our preliminary investigations, we investigated the prevalence in a bigger cohort of patients with longstanding diabetes with or without diabetic complications. METHODS: We included 200 patients in four groups (grouping due to duration of diabetes and presence of complications) from our university polyclinic with longstanding diabetes mellitus type 2 and evaluated the prevalence of the agonistic autoantibodies using ELISA technique. RESULTS: Antibodies directed against the alpha1-(39%), the first extracellular loop of the beta2-(34,5%), and the first extracellular loop of the beta1-adrenergic receptor (29,0%) were the most often detectable. With progression of diabetes and its complications, we found a decrease in the prevalence of the antibodies. Regression analyses revealed a positive association of antibodies against the first loop of the beta2-receptor and the presence of macrovascular complications. CONCLUSIONS: This investigation found mid frequent prevalence of agonistic autoantibodies in patients with longstanding diabetes mellitus type 2. The association between an antibody against one epitope and the presence of macrovascular complications may indicates a pathogenic linkage. This finding is inconsistent with our preliminary data and needs further evaluation, maybe by follow-up.

Beta Adrenergic Receptors Stimulation Attenuates Phosphorylation of NF-κB and IκBα in Hyperglycemic Endothelial Cells.

BACKGROUND/AIMS: NF-κB induces transcription of a number of genes, associated with inflammation and apoptosis. In this study, we have investigated the effect of ß-adrenergic receptor stimulation on NF-κB and IκBα in HUVECs. METHODS: Human umbilical vein endothelial cells (HUVECs) were cultured in high and low glucose concentrations. All HUVECs were treated with different concentrations of isoproterenol and propranolol for different time periods. The analytical procedures consisted of Western Blot, ELISA, DCFH-DA and TUNEL assays. RESULTS: Isoproterenol (agonist of a beta-adrenergic receptor) significantly reduced phosphorylation at Ser-536 of NF-κB; and Ser-32 and Ser-36 of IκBα in hyperglycemic HUVECs. Isoproterenol also significantly reduced apoptosis and ROS generation. No effect of IκBα was observed on Tyr-42 phosphorylation. The effect of isoproterenol was reversed by the antagonist propranolol. We also checked if NF-κB inhibitor MG132 causes any change at the level of apoptosis. However, we observed an almost similar effect. CONCLUSION: Given data demonstrates that beta-adrenergic receptors stimulation has a protective effect on HUVECs that might be occuring via NF-κß and IκBα pathway.

Repetitive restraint stress changes spleen immune cell subsets through glucocorticoid receptor or ß-adrenergic receptor in a stage dependent manner.

Immune system is sensitive to stress. Spleen is the largest peripheral immune organ innervated with sympathetic nerves and controlled by adrenomedullary system in the body. However, the alterations and mechanism of spleen immune cell subsets caused by repetitive restraint stress (RRS) is poorly understood. In this study, we found that RRS reduced spleen index in mice, and induced an expansion of white pulp and involution of the red pulp. Meanwhile, the percentage of CD3+CD8+ T lymphocytes, CD11b+F4/80+ macrophages, CD11b+Ly-6G-Ly-6Chi monocytic myeloid derived suppressor cells (mMDSCs) and CD11b+Ly-6G+Ly-6Cint granulocytic myeloid derived suppressor cells (gMDSCs) in spleen were significantly changed by RRS. Mechanistically, we found that the expression of norepinephrine (NE) and ß-adrenergic receptor (ß-AR) in spleen were up-regulated after 21 days of RRS, but not 7 days. The expression of corticosterone (CORT) and glucocorticoid receptor (GR) in spleen were up-regulated after 7 days of RRS but were lower after 21 days of RRS, even though they were still higher than that in mice without stress. By treating the stressed mice with RU486 (antagonist of GR) or propranolol (antagonist of ß-AR), we demonstrated that GR was responsible for the changes of spleen induced by 7 days of RRS and ß-AR was for 21 days of RRS. Our data suggest that RRS changes spleen immune cell subsets through GR or ß-AR in a stage dependent manner.

Antibodies to ß adrenergic and muscarinic cholinergic receptors in patients with Chronic Fatigue Syndrome.

Infection-triggered disease onset, chronic immune activation and autonomic dysregulation in CFS point to an autoimmune disease directed against neurotransmitter receptors. Autoantibodies against G-protein coupled receptors were shown to play a pathogenic role in several autoimmune diseases. Here, serum samples from a patient cohort from Berlin (n=268) and from Bergen with pre- and post-treatment samples from 25 patients treated within the KTS-2 rituximab trial were analysed for IgG against human α and ß adrenergic, muscarinic (M) 1-5 acetylcholine, dopamine, serotonin, angiotensin, and endothelin receptors by ELISA and compared to a healthy control cohort (n=108). Antibodies against ß2, M3 and M4 receptors were significantly elevated in CFS patients compared to controls. In contrast, levels of antibodies against α adrenergic, dopamine, serotonin, angiotensin, and endothelin receptors were not different between patients and controls. A high correlation was found between levels of autoantibodies and elevated IgG1-3 subclasses, but not with IgG4. Further patients with high ß2 antibodies had significantly more frequently activated HLA-DR+ T cells and more frequently thyreoperoxidase and anti-nuclear antibodies. In patients receiving rituximab maintenance treatment achieving prolonged B-cell depletion, elevated ß2 and M4 receptor autoantibodies significantly declined in clinical responder, but not in non-responder. We provide evidence that 29.5% of patients with CFS had elevated antibodies against one or more M acetylcholine and ß adrenergic receptors which are potential biomarkers for response to B-cell depleting therapy. The association of autoantibodies with immune markers suggests that they activate B and T cells expressing ß adrenergic and M acetylcholine receptors. Dysregulation of acetylcholine and adrenergic signalling could also explain various clinical symptoms of CFS.

Sleep-deprivation reduces NK cell number and function mediated by ß-adrenergic signalling.

Reduction of sleep time triggers a stress response, leading to augmented levels of glucocorticoids and adrenaline. These hormones regulate components of the innate immune system such as natural killer (NK) and NKT cells. In the present study, we sought to investigate whether and how stress hormones could alter the population and function of NK and NKT cells of mice submitted to different lengths of paradoxical sleep deprivation (PSD, from 24 to 72 h). Results showed that 72h of PSD decreased not only NK and NKT cell counts, but also their cytotoxic activity against B16F10 melanoma cells in vitro. Propranolol treatment during PSD reversed these effects, indicating a major inhibitory role of beta-adrenergic receptors (ß-AR) on NK cells function. Moreover, both corticosterone plasma levels and expression of beta 2-adrenergic receptors (ß2-AR) in NK cells increased by 48 h of PSD. In vitro incubation of NK cells with dexamethasone augmented the level of ß2-AR in the cell surface, suggesting that glucocorticoids could induce ß2-AR expression. In summary, we propose that reduction of NK and NKT cell number and cytotoxic activity appears to be mediated by glucocorticoids-induced increased expression of ß2-AR in these cells.

Mutual modulation between norepinephrine and nitric oxide in haemocytes during the mollusc immune response.

Nitric oxide (NO) is one of the most important immune molecules in innate immunity of invertebrates, and it can be regulated by norepinephrine in ascidian haemocytes. In the present study, the mutual modulation and underlying mechanism between norepinephrine and NO were explored in haemocytes of the scallop Chlamys farreri. After lipopolysaccharide stimulation, NO production increased to a significant level at 24 h, and norepinephrine concentration rose to remarkable levels at 3 h and 12~48 h. A significant decrease of NO production was observed in the haemocytes concomitantly stimulated with lipopolysaccharide and α-adrenoceptor agonist, while a dramatic increase of NO production was observed in the haemocytes incubated with lipopolysaccharide and ß-adrenoceptor agonist. Meanwhile, the concentration of cyclic adenosine monophosphate (cAMP) decreased significantly in the haemocytes treated by lipopolysaccharide and α/ß-adrenoceptor agonist, while the content of Ca(2+) was elevated in those triggered by lipopolysaccharide and ß-adrenoceptor agonist. When the haemocytes was incubated with NO donor, norepinephrine concentration was significantly enhanced during 1~24 h. Collectively, these results suggested that norepinephrine exerted varied effects on NO production at different immune stages via a novel α/ß-adrenoceptor-cAMP/Ca(2+) regulatory pattern, and NO might have a feedback effect on the synthesis of norepinephrine in the scallop haemocytes.

Estrogen modulates ß2-adrenoceptor-induced cell-mediated and inflammatory immune responses through ER-α involving distinct intracellular signaling pathways, antioxidant enzymes, and nitric oxide.

Sympathetic noradrenergic neuronal activity in the lymphoid organs regulates immunity through the release and binding of norepinephrine to ß2-adrenergic receptors (AR) on lymphocytes. In women, estrogen modulates immune responses during menstrual cycles, and in aging and age-associated diseases. The intent of the present study is to characterize the extent of immunomodulation by ß2-AR in the presence of estrogen and the involvement of intracellular signaling mechanisms including the role of antioxidant enzymes (AOE) in lymphocytes. In vitro effects of terbutaline, ß2-AR agonist, either alone or in combination with 17ß-estradiol (E2) were examined on splenocyte proliferation, cytokine (IFN-γ, IL-2, and IL-6) production, intracellular signaling molecules (p-ERK, p-CREB, p-Akt, and p-NF-κB) expression, NO production, and AOE activities [superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx)]. The specificity of their actions was investigated using ß-AR antagonist, and inhibitors of signaling targets and inducible nitric oxide synthase (iNOS). Terbutaline suppressed T cell proliferation and IL-6 production and increased AOE activities involving ERK, PKA, PKC, and NF-κB pathways and NO production. E2 alone enhanced T cell proliferation and decreased IL-6 production and NF-κB expression through ER-α. E2 in the presence of terbutaline reversed terbutaline-induced effects on T cell proliferation, IL-6 production, p-ERK and p-CREB expression, AOE activities, NO production, and NF-κB expression. Estrogen through ER-α differentially modulates ß2-AR-induced immune responses involving ERK, PKA, PKC, and NF-κB pathways, and NO that may be responsible for estrogen-induced immunosenescence and development of female-specific diseases.

Montelukast prevents microparticle-induced inflammatory and functional alterations in human bronchial smooth muscle cells.

Microparticles (MPs) are membrane fragments that may play a role in the pathogenesis of chronic respiratory diseases. We aimed to investigate whether human monocytes/macrophage-derived MPs could induce a pro-inflammatory phenotype in human bronchial smooth muscle cells (BSMC) and the effect of montelukast in this setting. Experimental methods included isolation of human monocytes/macrophages and generation of monocyte-derived MPs, RT-PCR analysis of gene expression, immunoenzymatic determination of pro-inflammatory factor release, bioluminescent assay of intracellular cAMP levels and electromobility shift assay analysis of NF-κB nuclear translocation. Stimulation of human BSMC with monocyte-derived MPs induced a pro-inflammatory switch in human BSMC by inducing gene expression (COX-2 and IL-8), protein release in the supernatant (PGE2 and IL-8), and heterologous ß2-adrenoceptor desensitization. The latter effect was most likely related to autocrine PGE2 since pre-treatment with COX inhibitors restored the ability of salbutamol to induce cAMP synthesis in desensitized cells. Challenge with MPs induced nuclear translocation of NF-κB and selective NF-κB inhibition decreased MP-induced cytokine release in the supernatant. Montelukast treatment prevented IL-8 release and heterologous ß2-adrenoceptor desensitization in human BSMC exposed to monocyte-derived MPs by blocking NF-κB nuclear translocation. These findings provide evidence on the role of human monocyte-derived MPs in the airway smooth muscle phenotype switch as a novel potential mechanism in the progression of chronic respiratory diseases and on the protective effects by montelukast in this setting.

Activating autoantibodies and cardiovascular disease.

Stimulating antibodies against G-protein-coupled receptors, including the ß1- and ß2-adrenergic receptors, the α1-adrenergic receptor, and the angiotensin II AT1 receptor, have been described, as well as activating antibodies directed at the platelet-derived growth factor receptor tyrosine kinase. Their existence and actions appear to be established. Lacking are mechanistic studies of receptor activation and translational studies to document receptor-stimulating antibodies as worthwhile therapeutic targets.

Autoimmune myocarditis, valvulitis, and cardiomyopathy.

Myocarditis and valvulitis are inflammatory diseases affecting myocardium and valve. Myocarditis, a viral-induced disease of myocardium, may lead to dilated cardiomyopathy and loss of heart function. Valvulitis leads to deformed heart valves and altered blood flow in rheumatic heart disease. Animal models recapitulating these diseases are important in understanding the human condition. Cardiac myosin is a major autoantigen in heart, and antibodies and T cells to cardiac myosin are evident in inflammatory heart diseases. This unit is a practical guide to induction and evaluation of experimental autoimmune myocarditis (EAM) in several mouse strains and the Lewis rat. Purification protocols for cardiac myosin and protocols for induction of EAM by cardiac myosin and its myocarditis-producing peptides, and coxsackievirus CVB3, are defined. Protocols for assessment of myocarditis and valvulitis in humans and animal models provide methods to define functional autoantibodies targeting cardiac myosin, ß-adrenergic, and muscarinic receptors, and their deposition in tissues.

Repeated stressor exposure regionally enhances beta-adrenergic receptor-mediated brain IL-1ß production.

It has been proposed that increased brain cytokines during repeated stressor exposure can contribute to neuropathological changes that lead to the onset of depression. Previous studies demonstrate that norepinephrine acting via beta-adrenergic receptors (ß-ARs) mediate brain IL-1 production during acute stressor exposure. The aim of the current studies was to examine how the regulation of brain cytokines by adrenergic signaling might change following repeated stressor exposure. Fischer rats were exposed to four days of chronic mild stress and 24h after the last stressors ß-AR expression, norepinephrine turnover, and ß-AR-mediated induction of brain IL-1 were measured in limbic areas (e.g. hypothalamus, hippocampus, amygdala, and prefrontal cortex) and brainstem. Repeated stressor exposure resulted in decreases in ß-AR expression (B(max)) measured by saturation binding curves in many limbic brain areas, while an increase was observed in the brainstem. This coincided with significant increases in norepinephrine turnover in the prefrontal cortex, hypothalamus, and amygdala, a significant increase in norepinephrine turnover was not observed in the hippocampus or brainstem. Stress increased overall IL-1 production in the amygdala (both basal and stimulated). While stress did not affect basal IL-1 levels in any other brain area, central administration of isoproterenol (a ß-AR agonist) augmented IL-1 production in the hypothalamus of stressed animals. These data indicate that repeated stressor exposure results in brain area specific enhancements in ß-AR-mediated IL-1 production and extends current knowledge of stress-induced enhancement of brain cytokine beyond sensitized response to immunological stimuli.

Glucocorticoid-catecholamine interplay within the composite thymopoietic regulatory network.

This paper highlights the multiple putative thymic and extrathymic points of intersection and interaction between glucocorticoids (GCs) and catecholamines (CAs)--the end-point mediators of the major routes of communication between the brain and the immune system--in the context of intricate thymic T cell-developmental tuning. More specifically, we discuss in detail findings indicating that adrenal GCs can influence thymopoiesis by adjusting directly and/or indirectly (through modulation of pituitary and local ACTH synthesis) not only thymic GC synthesis, in a cell type-specific manner, but also thymic CA bioavailability (via altering CA outflow from sympathetic nerve endings and local CA synthesis), ß and α(1) -adrenoceptor (AR) expression, and/or AR-mediated intracellular signal transduction in thymic cells. In addition, this short review points to GC- and CA-sensitive stages along the multistep T cell-developmental journey and the possible effects of altered GC, and consequently CA signaling, on thymopoietic efficiency.