Hiroto Tsuboi

Analysis of IgG4 class switch-related molecules in IgG4-related disease

IntroductionImmunoglobulin G4 (IgG4)-related disease (IgG4-RD) is a new disease entity characterized by high serum IgG4 levels, IgG4-positive plasmacytic infiltration, and fibrosis in various organs. The purpose of this study was to determine the mechanism of upregulation of IgG4 class switch recombination in IgG4-RD.MethodsWe extracted RNA from peripheral blood mononuclear cells (PBMCs) of patients with IgG4-RD (n = 6), Sjögren syndrome (SS) (n = 6), and healthy controls (n = 8), from CD3-positive T cells and CD20-positive B cells sorted from PBMCs of patients with IgG4-RD (n = 3), SS (n = 4), and healthy controls (n = 4), as well as from labial salivary glands (LSGs) of patients with IgG4-RD (n = 11), SS (n = 13), and healthy controls (n = 3). The mRNA expression levels of IgG4-specific class switch-related molecules, such as Th2 cytokines (IL-4 and IL-13), Treg cytokines (IL-10 and TGF-β), and transcriptional factors (GATA3 and Foxp3) were examined with quantitative polymerase chain reaction (PCR). IgG4-nonspecific class switch-related molecules, such as CD40, CD154, BAFF, APRIL, IRF4, and AID, were also examined.ResultsThe expression levels of Treg cytokines (IL-10 and TGF-β) and AID were significantly higher in LSGs of IgG4-RD than in SS and the controls (P < 0.05, each). In contrast, those of CD40 and CD154 were significantly lower in PBMCs of IgG4-RD than in SS (P < 0.05, each), whereas CD40 in CD20-positive B cells and CD154 in CD3-positive T cells were comparable in the three groups.ConclusionOverexpression of IL-10, TGF-β, and AID in LSGs might play important roles in the pathogenesis of IgG4-RD, such as IgG4-specific class-switch recombination and fibrosis. IgG4 class-switch recombination seems to be mainly upregulated in affected organs.

Pathogenic role of immune response to M3 muscarinic acetylcholine receptor in Sjögren's syndrome-like sialoadenitis

The aim of this study was to clarify the role of the immune response to muscarinic type 3 receptor (M3R) in the pathogenesis of Sjögrens syndrome (SS). M3R(-/-) mice were immunized with murine M3R peptides and their splenocytes were inoculated into Rag1(-/-) (M3R(-/-)→Rag1(-/-)) mice. M3R(-/-)→Rag1(-/-) mice had high serum levels of anti-M3R antibodies and low saliva volume. Histological examination showed marked infiltration of mononuclear cells in the salivary glands and immunohistochemistry demonstrated that the majority of these cells were CD4(+) T cells with a few B cells and several IFN-γ- and IL-17-producing cells. Apoptotic cells were present in the salivary glands of M3R(-/-)→Rag1(-/-) mice. Moreover, transfer of only CD3(+) T cells from M3R(-/-) immunized with M3R peptides into Rag1(-/-) mice resulted in cell infiltration and destruction of epithelial cells in the salivary glands, suggesting that M3R reactive CD3(+) T cells play a pathogenic role in the development of autoimmune sialoadenitis. Our findings support the notion that the immune response to M3R plays a crucial role in the pathogenesis of SS-like autoimmune sialoadenitis.

New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjögren's syndrome

M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjögrens syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti‐M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human‐M3R including the N‐terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme‐linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti‐M3R antibody‐positive SS, ‐negative SS and controls for 12 h. After loading with Fluo‐3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca2+ concentrations [(Ca2+)i] were measured. Antibodies to the N‐terminal, first, second and third loops were detected in 42·9% (18 of 42), 47·6% (20 of 42), 54·8% (23 of 42) and 45·2% (19 of 42) of SS, while in 4·8% (two of 42), 7·1% (three of 42), 2·4% (one of 42) and 2·4% (one of 42) of controls, respectively. Antibodies to the second loop positive SS‐IgG inhibited the increase of (Ca2+)i induced by cevimeline hydrochloride. Antibodies to the N‐terminal positive SS‐IgG and antibodies to the first loop positive SS‐IgG enhanced it, while antibodies to the third loop positive SS‐IgG showed no effect on (Ca2+)i as well as anti‐M3R antibody‐negative SS‐IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti‐M3R antibodies on salivary secretion might differ based on these epitopes.

Preferential M2 macrophages contribute to fibrosis in IgG4-related dacryoadenitis and sialoadenitis, so-called Mikulicz's disease

IgG4-related dacryoadenitis and sialoadenitis (IgG4-DS) is characterized by bilateral swelling of glandular tissues with extensive fibrosis, and is immunologically considered a Th2-predominant disease. Recent studies reported that alternatively activated (M2) macrophages enhanced Th2 immune responses and fibrosis by production of pro-fibrotic factors (IL-10, IL-13 and CCL18). Therefore, we examined the association between M2 macrophages and fibrosis in submandibular glands from 7 patients with IgG4-DS, 10 patients with chronic sialoadenitis, 10 patients with Sjögrens syndrome, and 10 healthy subjects. The number of M2 macrophages in SMGs from patients with IgG4-DS was also significantly higher than in the other groups. Double immunofluorescence staining showed that IL-10 and CCL18 expression co-localized with M2 macrophage-marker (CD163). Furthermore, the SMG fibrosis score was positively correlated with the frequency of M2 macrophages in only IgG4-DS. These results indicate that IL-10 and CCL18 secreted by preferential M2 macrophages possibly play a key role in the development of severe fibrosis in IgG4-DS.

The role of M3 muscarinic acetylcholine receptor reactive T cells in Sjögren's syndrome: A critical review

CD4+ T cells constitute the majority of infiltrating cells in salivary glands and lachrymal glands of patients with Sjögrens syndrome (SS). The pathophysiology of SS involves T cell recognition of antigens through the T cell antigen receptor, which triggers cytokine production and chronic inflammation. The M3 muscarinic acetylcholine receptor (M3R) molecule is expressed in exocrine glands, such as salivary glands and lachrymal glands, and plays an important role in exocrine secretion. Previous studies indicated the presence of M3R reactive T cells in peripheral blood of 40% of patients with SS and autoantibodies against M3R in sera of 9-100% of the same patients. Thus, M3R is considered a candidate receptor for autoantigen recognition by T and B cells. The relationship between B cell epitopes and the function of anti-M3R antibodies has been reported, suggesting the pathogenic role of anti-M3R antibodies in xerostomia commonly seen in SS patients. We generated new experimental mouse model, M3R-induced sialadenitis (MIS), using Rag1(-/-) mice inoculated with splenocytes from M3R(-/-) mice immunized with M3R synthetic peptides. Mice with MIS developed severe SS-like sialadenitis. Cell transfer experiments using M3R(-/-)xIFNγ(-/-) mice and M3R(-/-)xIL-17(-/-) mice showed that IFNγ and IL-17 are key cytokines in the pathogenesis of sialadenitis. These findings indicate the crucial roles of M3R-reactive Th1 and Th17 cells in autoimmune sialadenitis, and suggest that these cells, in addition to anti-M3R antibodies, are potential targets in new treatments for SS.

Efficacy and safety of abatacept for patients with Sjögren's syndrome associated with rheumatoid arthritis: Rheumatoid Arthritis with Orencia Trial toward Sjögren's syndrome Endocrinopathy (ROSE) trial—an open-label, one-year, prospective study—Interim analysis of 32 patients for 24 weeks

Abstract Objective. To assess the efficacy and safety of abatacept for secondary Sjögrens syndrome (SS) associated with rheumatoid arthritis (RA). Methods. The primary endpoint of this 1-year, open-labeled, prospective, observational multicenter study of RA-associated secondary SS was the rate of SDAI remission at 52 weeks after initiation of abatacept therapy. The secondary endpoints included that of Saxsons test and Schirmers test. Adverse events during the study period were also analyzed. Results. Thirty-two patients (all females) were enrolled in this study. Interim analysis at 24 weeks included assessment of efficacy (n = 31) and safety (n = 32). The mean SDAI decreased from 19.8 ± 11.0 (± SD) at baseline to 9.9 ± 9.9 at 24 weeks (P < 0.05). Patients with clinical remission, as assessed by SDAI, increased from 0 patient (0 week) to 8 patients (25.8%) at 24 weeks. Saliva volume (assessed by Saxsons test) increased slightly from 2232 ± 1908 (0 week) to 2424 ± 2004 (24 weeks) mg/2 min (n = 29). In 11 patients with Greenspan grading 1/2 of labial salivary glands biopsy, saliva volume increased from 2945 ± 2090 (0 week) to 3419 ± 2121 (24 weeks) mg/2 min (P < 0.05). Schirmers test for tear volume showed increase from 3.6 ± 4.6 (0 week) to 5.5 ± 7.1 (24 weeks) mm/5 min (n = 25; P < 0.05). Five adverse events occurred in five of 32 patients (15.6%), and three of these events were infections. Conclusion. Abatacept seems to be effective for both RA and RA-related secondary SS.

Functional role of M3 muscarinic acetylcholine receptor (M3R) reactive T cells and anti-M3R autoantibodies in patients with Sjögren's syndrome.

Sjögrens syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration into the lachrymal and salivary glands, leading to dry eyes and mouth. Infiltration is also found in the kidneys, lungs, thyroid, and liver. Immunohistochemical studies have shown that most infiltrating lymphocytes are CD4(+) T cell receptor (TCR) alphabeta T cells. The antigen specificity of T cells is decided by TCR expressed on T cells. The usage of TCRalpha and TCRbeta genes have been examined by immunological and molecular biological methods. Autoantigens recognized by T cells infiltrating into salivary glands have been analyzed and several candidates for autoantigens have been clarified. In the present study, we focused on M3 muscarinic acetylcholine receptor (M3R) as a salivary gland-specific autoantigen and clarified T cell epitopes and B cell epitopes on M3R. The functions of anti-M3R antibodies and M3R reactive T cells were also carried out. To clarify whether M3R reactive T cells play a crucial role in the generation of autoimmune sialoadenitis, splenic CD3+T cells form M3R(-/-) mice immunized by M3R peptides were transferred into Rag-1(-/-) mice and sialoadenitis analyzed. The functional role of M3R reactive T cells in the generation of SS was also discussed.

Primary and secondary surveys on epidemiology of Sjögren's syndrome in Japan

Abstract Objective. To characterize the epidemiology of Sjögrens syndrome (SS), including prevalence, disease type, extra-glandular involvement, satisfaction of diagnostic criteria sets, and treatment used in Japan. Methods. The Research Team for Autoimmune Diseases, the Research Program for Intractable Disease by the Ministry of Health, Labor and Welfare conducted primary and secondary surveys on epidemiology of SS in 2011. The primary survey covered 4,729 out of 14,095 Japan-wide Hospital Departments to investigate the prevalence of SS. The secondary survey encompassed 214 Hospital Departments that agreed to the survey, to characterize disease type, extra-glandular involvement, satisfaction of diagnostic criteria sets, and treatments. Results. The number of patients with SS in Japan estimated by the primary survey was 68,483. The secondary survey involving data collected from 2,195 SS patients from 98 Hospital Departments showed that the mean age of patients was 60.8 ± 15.2 years, male/female ratio was 1/17.4, primary/secondary SS was about 60%/40% and glandular/extra-glandular form in primary SS was about 70%/25%. The satisfaction rate was 53.8% for the 1999 revised Japanese Ministry of Health criteria for the diagnosis of SS, 47.7% for the 2002 American–European Consensus Group classification criteria for SS and 49.6% for 2012 American College of Rheumatology classification criteria for SS. Corticosteroids were used by 752 of 2,195 patients (34%), immunosuppressants by 358 patients (16%), biologics by 68 patients (3%) and secretagogues by 695 patients (32%). Conclusion. The surveys provided valuable information on the epidemiology of SS including prevalence, disease type, extra-glandular involvement, satisfaction of diagnostic criteria sets and treatments used today in Japan.

Overexpression of T‐bet gene regulates murine autoimmune arthritis

OBJECTIVE To clarify the role of T-bet in the pathogenesis of collagen-induced arthritis (CIA). METHODS T-bet-transgenic (Tg) mice under the control of the CD2 promoter were generated. CIA was induced in T-bet-Tg mice and wild-type C57BL/6 (B6) mice. Levels of type II collagen (CII)-reactive T-bet and retinoic acid receptor-related orphan nuclear receptor γt (RORγt) messenger RNA expression were analyzed by real-time polymerase chain reaction. Criss-cross experiments using CD4+ T cells from B6 and T-bet-Tg mice, as well as CD11c+ splenic dendritic cells (DCs) from B6 and T-bet-Tg mice with CII were performed, and interleukin-17 (IL-17) and interferon-γ (IFNγ) in the supernatants were measured by enzyme-linked immunosorbent assay. CD4+ T cells from B6, T-bet-Tg, or T-bet-Tg/IFNγ-/- mice were cultured for Th17 cell differentiation, then the proportions of cells producing IFNγ and IL-17 were analyzed by fluorescence-activated cell sorting. RESULTS Unlike the B6 mice, the T-bet-Tg mice did not develop CIA. T-bet-Tg mice showed overexpression of Tbx21 and down-regulation of Rorc in CII-reactive T cells. Criss-cross experiments with CD4+ T cells and splenic DCs showed a significant reduction in IL-17 production by CII-reactive CD4+ T cells in T-bet-Tg mice, even upon coculture with DCs from B6 mice, indicating dysfunction of IL-17-producing CD4+ T cells. Inhibition of Th17 cell differentiation under an in vitro condition favoring Th17 cell differentiation was observed in both T-bet-Tg mice and T-bet-Tg/IFNγ-/- mice. CONCLUSION Overexpression of T-bet in T cells suppressed the development of autoimmune arthritis. The regulatory mechanism of arthritis might involve dysfunction of CII-reactive Th17 cell differentiation by overexpression of T-bet via IFNγ-independent pathways.

Nationwide epidemiological survey of 169 patients with adult Still's disease in Japan.

Abstract Objectives. A nationwide survey was conducted to assess the number of patients, clinical aspects, treatment, and prognosis of adult Stills disease (ASD) in Japan. Methods. A primary questionnaire was sent to randomly selected medical institutions in order to estimate the number of patients. We sent a secondary questionnaire to the same institutions to characterize the clinical manifestations and treatment of ASD. Results. The estimated prevalence of ASD was 3.9 per 100,000. Analysis of 169 patients showed a mean age at onset of 46 years. The main clinical symptoms were fever, arthritis, and typical rash in agreement with previous surveys. Oral glucocorticoids were used to treat 96% of the patients, while methotrexate was used in 41% and biological agents were used in 16%. Lymphadenopathy and macrophage activation syndrome were significantly associated with increased risk of relapse (P < 0.05, each). Patients who achieved remission after tocilizumab therapy had significantly longer disease duration (6.2 years) than patients who did not (1.9 years) (p < 0.05). Conclusions. The 2010–2011 nationwide survey of ASD identified important changes in treatment and improvement of prognosis compared with previous surveys.