Chronic exposure to the anti-M3 muscarinic acetylcholine receptor autoantibody in pemphigus vulgaris contributes to disease pathophysiology.

Pemphigus vulgaris (PV) is a potentially lethal autoimmune mucocutaneous blistering disease characterized by binding of IgG autoantibodies (AuAbs) to keratinocytes (KCs). In addition to AuAbs against adhesion molecules desmogleins 1 and 3, PV patients also produce an AuAb against the M3 muscarinic acetylcholine (ACh) receptor (M3AR) that plays an important role in regulation of vital functions of KCs upon binding endogenous ACh. This anti-M3AR AuAb is pathogenic because its adsorption eliminates the acantholytic activity of PV IgG; however, the molecular mechanism of its action is unclear. In the present study, we sought to elucidate the mode of immunopharmacologic action of the anti-M3AR AuAb in PV. Short-term exposures of cultured KCs to PV IgG or the muscarinic agonist muscarine both induced changes in the expression of keratins 5 and 10, consistent with the inhibition of proliferation and upregulated differentiation and in keeping with the biological function of M3AR. In contrast, long-term incubations induced a keratin expression pattern consistent with upregulated proliferation and decreased differentiation, in keeping with the hyperproliferative state of KCs in PV. This change could result from desensitization of the M3AR, representing the net antagonist-like effect of the AuAb. Therefore, chronic exposure of KCs to the anti-M3AR AuAb interrupts the physiological regulation of KCs by endogenous ACh, contributing to the onset of acantholysis. Since cholinergic agents have already demonstrated antiacantholytic activity in a mouse model of PV and in PV patients, our results have translational significance and can guide future development of therapies for PV patients employing cholinergic drugs.

Retinoic acid attenuates nuclear factor kappaB mediated induction of NLRP3 inflammasome.

BACKGROUND: Acetylcholine (ACh), a neurotransmitter and a part of the cholinergic system, can modify immune responses. Expression of acetylcholine receptors (AChR) in immune cells, including macrophages, leads to modulation of their function. Inflammasomes are part of the innate immune system and have been linked to a variety of inflammatory diseases. The NLRP3/ASC/caspase-1/IL-1 axis has emerged as a critical signaling pathway in inflammation process initiation. The role of ACh in modulating inflammasomes in macrophages remains relatively under-explored. METHODS: The effect of AChR agonist carbachol on inflammasome expression was investigated using murine and human macrophages. Cell lysates were assessed by western blot for protein analysis. Immunofluorescence studies were used to study the translocation of p65. The experiments were conducted in the presence of NF-ĸB inhibitor, AChR antagonists, and retinoic acid (RA) to study the role of NF-ĸB, ACh receptors, and RA, respectively. RESULTS: We found that carbachol increased the expression of NLRP3 inflammasome (NLRP3, ASC, cleaved caspase-1, IL-1ß, and IL-18). The treated cells also showed an increase in NF-ĸB activation. The effect of carbachol was diminished by NF-ĸB inhibitor and atropine, a mAChR antagonist. The addition of RA also significantly reduced the effect of carbachol on NLRP3 inflammasomes. CONCLUSIONS: Our current study suggests that carbachol induces NLRP3 inflammasome activation through mAChR and NF-ĸB, and that RA abolishes the inflammatory response. It reveals the potentials of co-administration of RA with cholinergic drugs to prevent inflammatory responses during cholinergic medications.

Muscarinic receptors control markers of inflammation in the small intestine of BALB/c mice.

Muscarinic-acetylcholine-receptors (mAChRs) modulate intestinal homeostasis, but their role in inflammation is unclear; thus, this issue was the focus of this study. BALB/c mice were treated for 7 days with muscarine (mAChR/agonist), atropine (mAChR/antagonist) or saline. Small-intestine samples were collected for histology and cytofluorometric assays in Peyer's patches (PP) and lamina propria (LP) cell-suspensions. In LP, goblet-cells/leukocytes/neutrophils/MPO+ cells and MPO/activity were increased in the muscarine group. In PP, IFN-γ+/CD4+ T or IL-6+/CD4+ T cell numbers were higher in the muscarine or atropine groups, respectively. In LP, TNF-α+/CD4+ T cell number was higher in the muscarine group and lower in the atropine.

Altered Structural Brain Networks Related to Adrenergic/Muscarinic Receptor Autoantibodies in Chronic Fatigue Syndrome.

BACKGROUND AND PURPOSE: Recent studies suggest that the autoantibodies against adrenergic/muscarinic receptors might be one of the causes and potential markers of myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS). The purpose of this study was to investigate the structural network changes related to autoantibody titers against adrenergic/muscarinic receptors in ME/CFS by performing a single-subject gray matter similarity-based structural network analysis. METHODS: We prospectively examined 89 consecutive right-handed ME/CFS patients who underwent both brain MRI including 3D T1-wighted images and a blood analysis of autoantibodies titers against ß1 adrenergic receptor (ß1 AdR-Ab), ß2 AdR-Ab, M3 acetylcholine receptor (M3 AchR-Ab), and M4 AchR-Ab. Single-subject gray matter similarity-based structural networks were extracted from segmented gray matter images for each patient. We calculated local network properties (betweenness centrality, clustering coefficient, and characteristic path length) and global network properties (normalized path length λ, normalized clustering coefficient γ, and small-world network value δ). We investigated the correlations between the autoantibody titers and regional gray matter/white matter volumes, the local network properties, and the global network properties. RESULTS: Betweenness centrality showed a significant positive correlation with ß1-AdR-Ab in the right dorsolateral prefrontal cortex. The characteristic path length showed a significant negative correlation with ß2-AdR-Ab in the right precentral gyrus. There were no significant correlations between the antibody titers and the regional gray matter/white matter volumes, and the global network properties. CONCLUSIONS: Our findings suggest that ß1 AdR-Ab and ß2 AdR-Ab are potential markers of ME/CFS.

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.

Is there a role for antibodies targeting muscarinic acetylcholine receptors in the pathogenesis of schizophrenia?

OBJECTIVE: Muscarinic receptor dysfunction has been suggested to play an important role in the pathophysiology of schizophrenia. Recently, it has also become clear that immune reactivity directed against neurotransmitter receptors may play a pathogenic role in some cases of schizophrenia. The aim of this review is to summarize the case for muscarinic receptor dysfunction in schizophrenia and the evidence supporting the hypothesis that this dysfunction is related to the development of muscarinic receptor-targeting antibodies. METHOD: The article reviews studies of muscarinic receptors and the presence and potential role(s) of anti-muscarinic acetylcholine receptor antibodies in people with schizophrenia. RESULTS: There is accumulating evidence that altered or deficient muscarinic signalling underlies some of the key clinical features of schizophrenia. Although the number of studies investigating anti-muscarinic acetylcholine receptor antibodies in schizophrenia is relatively small, they consistently demonstrate that such antibodies are present in a proportion of patients. This evidence suggests that these antibodies could have pathogenic effects or exist as a biomarker to an unknown pathophysiological process in schizophrenia. CONCLUSION: The presence of elevated levels of anti-muscarinic acetylcholine receptor antibodies may identify a subgroup of people with schizophrenia, potentially informing aetiopathogenesis, clinical presentation and treatment. To date, all studies have examined antibodies in participants with chronic schizophrenia, who have likely received antipsychotic medication for many years. As these medications modulate immune functions and regulate receptor densities, it is recommended that future studies screen for the presence of anti-muscarinic antibodies in people experiencing their first episode of psychosis.

No evidence for humoral autoimmunity against cardiomyocytes, adrenergic or muscarinic receptors in patients with Tako-Tsubo cardiomyopathy.

BACKGROUND: An association between Tako-Tsubo cardiomyopathy (TTC) and underlying malignancies has been observed, suggesting that TTC might be the consequence of paraneoplastic phenomena. This study investigates the presence of autoantibodies against cardiomyocytes as well as adrenergic (ß1, ß2) and muscarinic (M2) receptors in patients with TTC. METHODS AND RESULTS: Serum from 20 TTC patients and 20 controls with ischemic heart disease was obtained. Indirect immunofluorescence testing for intracellular autoantibodies against cardiomyocytes showed a homogenous distribution, as in both groups 9 of 20 sera displayed a characteristic binding pattern of antibodies including vascular walls and intracellular structures. Flow cytometry analysis revealed no difference between TTC and controls in the binding of autoantibodies to the surface antigens of cardiomyocyte HL-1 cells (p = 0.569, t-test). Flow cytometry analysis of nontransfected wild type cells (p = 0.633, t-test), M2 receptor-transfected cells (p = 0.687, t-test), ß1 receptor-transfected cells (p = 0.444, t-test) and ß2 receptor-transfected cells (p = 0.632, t-test) showed similar results for control and TTC sera. Likewise, the binding pattern of TTC patients with a history of neoplasia compared to those without or to controls did not differ significantly (p > 0.05, u-test). CONCLUSION: Findings suggest that the presumed paraneoplastic etiology of TTC cannot be attributed to the formation of these antibodies.

Correlation of antimuscarinic acetylcholine receptor antibody titers and antidesmoglein antibody titers with the severity of disease in patients with pemphigus.

BACKGROUND: Acetylcholine receptor (AchR) antibody levels significantly correlate with disease severity at initial pemphigus diagnosis and during follow-up. However, it is not clear if they are just an epiphenomenon or a potential trigger of the known pathogenic process in pemphigus vulgaris. OBJECTIVE: We sought to assess the changes in anti-muscarinic (M3) AchR and anti-desmoglein (Dsg) antibody titers with therapy. METHODS: This was a hospital-based cohort study involving 45 patients with active pemphigus. Disease was graded clinically using Pemphigus Disease Area Index. Antibody titers were estimated using enzyme-linked immunosorbent assay at baseline, 3 months, and 15 months. RESULTS: All patients with pemphigus had significantly higher anti-M3 AchR titers when compared with a control group. Only 95.5% of patients had anti-Dsg1 antibodies and 84.4% of patients had anti-Dsg3 antibodies. A statistically significant reduction in all 3 antibody titers from baseline to follow-up with treatment was observed. There was a good correlation between all 3 antibody titer and Pemphigus Disease Area Index score at baseline and after therapy and between anti-M3 AchR and anti-Dsg1 antibody titers. LIMITATIONS: Sample size was small and follow-up period was short. CONCLUSIONS: Anti-M3 AchR antibodies are strongly associated with pemphigus. They significantly correlate with disease activity and their titers decline with therapy along with anti-Dsg antibodies.

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.

Role of ubiquitin ligase Cbl-b in B-cell activation of dendritic cells loaded with Tα146-162 in the treatment of experimental autoimmune myasthenia gravis in mice.

OBJECTIVE: This study aimed to examine the mechanism of Tα146-162-iMDC in the pathogenic intervention of mice with experimental autoimmune myasthenia gravis (EAMG) from the perspective of B-cell activation. MATERIALS AND METHODS: The mice were divided into three groups, model (A), intervention (B), and control (C), with the intervention of Tα146-162-iMDC. The expressions of Cbl-b mRNA, Syk, Lyn, Btk, and phospholipase C (PLC)-γ2 proteins and their phosphorylated proteins were detected. RESULTS: The Cbl-b mRNA expression in group A was lower than that in group C (p < 0.01) while that in group B increased compared with that in group A (p < 0.05), but was lower than that in group C (p < 0.05). The expression and phosphorylation of Syk and PLC-γ2 proteins in group A increased compared with those in group C (p < 0.01) while those in group B decreased compared with those in group A (p < 0.05), but were higher than those in group C (p < 0.05). The expression and phosphorylation of Lyn protein in group A decreased compared with those in group C (p < 0.01) while those in group B increased compared with those in group A (p < 0.05), but were lower than those in group C (p < 0.05). The Btk protein expression in group A increased compared with that in group C (p < 0.01) while that in group B decreased compared with that in group A (p < 0.05), but was still higher than that in group C (p < 0.05). However, no difference in phosphorylation levels among the three groups was observed (p > 0.05). CONCLUSIONS: Tα146-162-iMDC intervention can reduce the incidence of EAMG and may be associated with Cbl-b in the negative regulation of B-cell activation.

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.

Autoantibodies against muscarinic receptors in breast cancer: their role in tumor angiogenesis.

The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10(-8) M) and carbachol (10(-9) M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.

Immunoglobulin g from breast cancer patients regulates MCF-7 cells migration and MMP-9 activity by stimulating muscarinic acetylcholine receptors.

PURPOSE: We have previously reported the expression of muscarinic acetylcholine receptors (mAChR) in human breast tumors. The activation of these receptors triggered tumor cell proliferation. Considering that invasion and metastasis is the major cause of death in cancer, we investigated the action of autoantibodies against mAChR derived from breast cancer patients in stage I (T1N0Mx-IgG) on MCF-7 cells migration and metalloproteinase-9 (MMP-9) activity. We also analyzed the participation of phospholipase C/nitric oxide synthase/protein kinase C pathway. METHODS: Immunoglobulin G (IgG) was purified by chromatography in protein G-agarose from blood samples of breast cancer patients obtained under informed consent. Migration was assayed by an in vitro wound assay. MMP-9 activity was quantified by zymography. RESULTS: T1N0Mx-IgG promoted tumor cell migration and increased MMP9 activity mimicking the action of the muscarinic agonist carbachol. This effect was reduced not only by the presence of atropine but also by 4-DAMP or tropicamide, antagonists for M(3) and M(4) mAChR subtypes respectively. The actions of T1N0Mx-IgG and carbachol on MCF-7 cells, involved the participation of phospholipase C/nitric oxide synthase/protein kinase C pathway. CONCLUSIONS: IgG from breast cancer patients in stage I could be promoting tumor progression by regulating migration and MMP-9 activity in tumor cells via mAChR activation. The presence of these autoantibodies could be determining the prognosis of breast cancer in these patients.

Neuroimmune interactions in Sjögren's syndrome: relationship of exocrine gland dysfunction with autoantibodies to muscarinic acetylcholine receptor-3 and mental health status parameters.

OBJECTIVES: Antimuscarinic acetylcholine receptor-3 (m3AChR) autoantibodies have been described in primary Sjögren's syndrome (pSS). The aim of this study was to compare various methods for their detection and to assess the contributions of anti-m3AChR and other immunological and psychosocial factors to the pathomechanism of secondary SS (sSS). METHODS: Sixty-five rheumatoid arthritis (RA) patients, 103 systemic lupus erythematosus (SLE) patients, 76 pSS patients and 50 controls were compared. Three immunodominant epitopes of m3AChR were synthesized and used in ELISA. Two extracellular epitopes were also prepared in fusion with glutathione-S-transferase and one in conjugation with bovine serum albumin. Mental health status was assessed with the 36-item Short-Form Health Survey and Functional Assessment of Chronic Illness Therapy fatigue scale. Correlations were evaluated between glandular function and anti-m3AChR positivities and specificities, features of SLE and RA, and mental health parameters. RESULTS: Fourteen RA and 27 SLE patients had sSS. The autoantibody levels to all epitopes of m3AChR were significantly higher in pSS and SLE patients than in the controls. The fusion protein forms discriminated RA from pSS and SLE; furthermore, the YNIP fusion protein also distinguished pSS from SLE. The prevalence and the mean levels of all autoantibodies did not differ statistically between sicca and non-sicca SLE or RA patients. Glandular dysfunction correlated with higher age in SLE and RA and an impaired health-related quality of life in SLE. CONCLUSIONS: The second and third extracellular loops of m3AChR are antigenic in pSS. Immunoassays with antigens as fusion peptides demonstrate the best performance. Sicca SLE patients have worse mental health status. Anti-m3AChR antibodies represent a peculiar example of neuroimmune interactions.

Reduction of [11C](+)3-MPB binding in brain of chronic fatigue syndrome with serum autoantibody against muscarinic cholinergic receptor.

BACKGROUND: Numerous associations between brain-reactive antibodies and neurological or psychiatric symptoms have been proposed. Serum autoantibody against the muscarinic cholinergic receptor (mAChR) was increased in some patients with chronic fatigue syndrome (CFS) or psychiatric disease. We examined whether serum autoantibody against mAChR affected the central cholinergic system by measuring brain mAChR binding and acetylcholinesterase activity using positron emission tomography (PET) in CFS patients with positive [CFS(+)] and negative [CFS(-)] autoantibodies. METHODOLOGY: Five CFS(+) and six CFS(-) patients, as well as 11 normal control subjects underwent a series of PET measurements with N-[(11)C]methyl-3-piperidyl benzilate [(11)C](+)3-MPB for the mAChR binding and N-[(11)C]methyl-4-piperidyl acetate [(11)C]MP4A for acetylcholinesterase activity. Cognitive function of all subjects was assessed by neuropsychological tests. Although the brain [(11)C](+)3-MPB binding in CFS(-) patients did not differ from normal controls, CFS(+) patients showed significantly lower [(11)C](+)3-MPB binding than CFS(-) patients and normal controls. In contrast, the [(11)C]MP4A index showed no significant differences among these three groups. Neuropsychological measures were similar among groups. CONCLUSION: The present results demonstrate that serum autoantibody against the mAChR can affect the brain mAChR without altering acetylcholinesterase activity and cognitive functions in CFS patients.

The non-neuronal cholinergic system as novel drug target in the airways.

The parasympathetic nervous system is a key regulator of the human organism involved in the pathophysiology of various disorders through cholinergic mechanisms. In the lungs, acetylcholine (ACh) released by vagal nerve endings stimulates muscarinic receptors thereby increasing airway smooth muscle tone. Contraction of airway smooth muscle cells leads to increased respiratory resistance and dyspnea. An additional branch of the cholinergic system is the non-neuronal cholinergic system expressed in nearly all cell types present in the airways. Activation of this system may contribute to an increased cholinergic tone in the lungs, inducing pathophysiological processes like inflammation, remodeling, mucus hypersecretion and chronic cough. Selective muscarinic receptor antagonists specifically inhibit acetylcholine at the receptor inducing bronchodilation in patients with obstructive airway diseases. This paper reviews preclinical pharmacological research activities on anticholinergics including experimental models of asthma and chronic obstructive pulmonary disease, COPD. It discloses various options to follow up the non-neuronal cholinergic system as a novel drug target for the treatment of key aspects of obstructive airway diseases, in particular those of a chronic nature.

Critical roles of acetylcholine and the muscarinic and nicotinic acetylcholine receptors in the regulation of immune function.

Lymphocytes express both muscarinic and nicotinic acetylcholine (ACh) receptors (mAChRs and nAChRs, respectively), and stimulation of mAChRs and nAChRs produces various biochemical and functional changes. Although it has been postulated that parasympathetic cholinergic nerves directly innervate immune cells, no evidence has supported this hypothesis. We measured ACh in the blood of various animal species and determined its localization in T cells using a sensitive and specific radioimmunoassay. Furthermore, we showed that T cells express choline acetyltransferase (ChAT), an ACh synthesizing enzyme. Immunological T cell activation enhances ACh synthesis through the up-regulation of ChAT expression, suggesting lymphocytic cholinergic activity is related to immunological activity. Most immune cells such as T cells, B cells, and monocytes express all five subtypes of mAChRs (M(1)-M(5)), and various subunits of the nAChR, such as α3, α5, α7, α9, and α10. Studies on serum antibody production in M(1) and M(5) combined mAChR gene knockout (KO) mice immunized with ovalbumin (OVA) revealed that M(1)/M(5) mAChRs up-regulate TNF-α, IFN-γ and IL-6 production in spleen cells, leading to an elevation of serum anti-OVA specific IgG(1). In contrast, studies of nAChR α7 subunit gene KO mice immunized with OVA show that α7 nAChRs down-regulate these proinflammatory cytokines, thereby leading to a reduction of anti-OVA specific IgG(1). Taken together, these findings demonstrate that both mAChRs and nAChRs modulate production of cytokines, such as TNF-α, resulting in a modification of antibody production. These findings support the notion that a non-neuronal cholinergic system is involved in the regulation of immune cell function.

The role of muscarinic acetylcholine receptor type 3 polypeptide (M3RP205-220) antibody in the saliva of patients with primary Sjögren's syndrome.

BACKGROUND: Sjögren's syndrome (SS) is a chronic autoimmune disorder of unknown cause. Recent studies have shown that antimuscarinic acetylcholine type 3 receptor (M3R) antibodies can be detected in patients with Sjögren's syndrome (SS), but little is known about the diagnostic value of this antibody. OBJECTIVES: To assess the clinical correlations of anti-M3R (muscarinic acetylcholine receptor type 3) polypeptide (M3RP205-220) antibodies in saliva from patients of primary Sjögren's syndrome (pSS). METHODS: Serum samples and unstimulated mixed saliva from 100 patients with SS were collected and examined. Their mean (SD) age was 54.2 (13.4) years, and the mean (SD) disease duration was 6.2 (3.8) years. Serum samples from 40 patients with systemic lupus erythematosus (SLE), 40 with rheumatoid arthritis (RA), and 60 healthy subjects were analysed as controls. All the patients with SS were carefully evaluated according to European and American criteria. A circular M3RP205-220 peptide sequence was synthesized using solid-phase techniques on an applied biosytems peptide synthesizer. The correlation between anti-M3RP205-220 antibodies and clinical manifestations of pSS was analysed. RESULTS: The IgG of anti-M3RP205-220 antibodies was present in 69% of patients with pSS, 27.5% with SLE, 22.5% with RA, and 23.3% of normal saliva donors. The prevalence of anti-M3RP205-220 antibodies in pSS was significantly higher than in SLE, RA, and normal controls. The specificity of anti-M3RP205-220 antibodies in pSS was 75%. The salivary flow rate in the group positive for anti-M3RP205-220 was 436 µl/10 min, compared to a rate of 658 µl/10 min for the negative group (p<0.05). CONCLUSIONS: The anti-M3RP205-220 antibody was detected in most patients with pSS. The presence of the antibody was closely associated with the salivary flow rate. This indicated that it may act as an autoantigen, with a role in the pathogenesis of pSS.