須藤 明

Objective. Helios+FoxP3+CD4+ (Helios+) Treg cells are believed to be involved in the regulation of various autoimmune diseases; however, the regulatory mechanisms underlying the development of Helios+ Treg cells remain uncertain. This study was undertaken to elucidate the regulatory mechanisms of Helios expression in CD4+ T cells and its roles in transforming growth factor beta (TGF)-induced Treg cell function. Methods. We examined the expression of Helios in CD4+ T cells in patients with rheumatoid arthritis by DNA microarray analysis before and after treatment with biologic agents. We also examined the effect of interleukin-6 (IL-6) and TGF beta on Helios expression in CD4+ T cells in humans and mice. The effect of forced expression of Helios on murine induced Treg cell function was also examined. The role of FoxP3 in the induction and function of Helios was assessed by using CD4+ T cells from FoxP3-deficient scurfy mice. Results. Tocilizumab, but not tumor necrosis factor (TNF) inhibitors or abatacept, increased Helios expression in CD4+ T cells in patients with a good response. IL-6 inhibited the TGF beta-induced development of Helios+ induced Treg cells in both humans and mice. Both cell-intrinsic FoxP3 expression and TGF beta signaling were required for Helios induction in murine induced Treg cells. The forced expression of Helios enhanced the expression of various Treg cell-related molecules and the suppressive function in murine induced Treg cells. Helios-mediated enhancement of the suppressive function of induced Treg cells was obvious in FoxP3-sufficient CD4+ T cells but not in FoxP3-deficient CD4+ T cells. Conclusion. Our findings indicate that Helios enhances induced Treg cell function in cooperation with FoxP3.

Objective. FoxP3 induces Treg cells and prevents autoimmune diseases. However, the precise mechanisms of FoxP3 in the prevention of autoimmune diseases remain unknown. We undertook this study to determine the regulatory roles of FoxP3 in autoimmune inflammation by using FoxP3-mutant sf mice. Methods. We characterized interleukin-21 (IL-21)-producing cells in sf mice. We examined the underlying mechanisms of enhanced IL-21 production in sf mouse CD4+ T cells. We examined the roles of IL-21 and CD8+ T cells in autoimmune inflammation in sf mice using IL-21 receptor (IL-21R)-deficient sf mice. Results. IL-21-producing c-Maf+CD4+ T cells, which were distinct from Th17 cells, were increased in sf mice. Increased c-Maf expression was involved in enhanced IL-21 production in sf mouse CD4+ T cells. Experiments using bone marrow chimeric mice showed that lack of cell-extrinsic suppression by FoxP3+ Treg cells, but not cell-intrinsic defects in FoxP3 in sf mouse CD4+ T cells, was mainly involved in the development of IL-21-producing c-Maf+CD4+ T cells in sf mice. IL-21R deficiency prolonged survival and reduced multiorgan autoimmune inflammation in sf mice. Moreover, IL-21R deficiency decreased short-lived effector CD8+ T cells in the lung in sf mice. Furthermore, depletion of CD8+ T cells inhibited lung inflammation in sf mice, suggesting that CD8+ T cells are critical for inducing lung inflammation in sf mice. Conclusion. Unique IL-21-producing c-Maf+CD4+ T cells develop in the absence of FoxP3+ Treg cells, induce short-lived effector CD8+ T cells, and enhance multiorgan autoimmune inflammation in sf mice.

Inflammatory DCs are crucial for the development of airway hyper-reactivity.Dendritic cells (DCs) play critical roles in determining the fate of CD4 T cells. Among DC sub-populations, monocyte-derived inflammatory DCs (iDCs) have been shown to play an important role in the induction of adaptive immune responses under inflammatory conditions. Although previous studies have shown that DCs have an indispensable role in the induction of allergic airway inflammation and airway hyperreactivity (AHR) in murine asthma models, the precise roles of iDCs in the asthmatic responses remain largely unknown. We show here that T(h)2 cell-mediated inflammation in murine asthma models induces the expression of some markers of alternatively activated macrophage such as arginase 1 and resistin-like molecule- in iDCs by a mechanism depending on the intrinsic expression of STAT6. In contrast, T(h)1 cell-mediated inflammation induces iDCs to express TNF- and inducible nitric oxide synthase (iNOS), markers of TNF-- and iNOS-producing DCs. Moreover, we show that iDCs under a T(h)2 environment play an important role in the induction of AHR, independently of allergic airway inflammation. Our results thus indicate the importance of iDCs in the induction of AHR as downstream effector cells in T(h)2 cell-mediated asthmatic responses.

The tumor suppressor p53 plays a central role in tumor suppression by inducing apoptosis, cell cycle arrest, senescence, and DNA repair. In addition to the antitumor functions of p53, accumulating evidence using systemic p53-deficient mice suggests that p53 suppresses autoimmunity. However, it remains unknown how p53 suppresses autoimmunity. In this study, we generated T cell-specific p53-deficient mice (CD4-Cre p53(fl/fl) mice, or p53 conditional knockout [cKO] mice) and found that aged p53-cKO mice spontaneously developed inflammatory lesions in various organs, including lung, liver, stomach, thyroid gland, submandibular gland, and kidney. Additionally, anti-nuclear Abs and autoantibodies against gastric parietal cells were detected in p53-cKO mice but not in control p53(fl/fl) mice (p53 wild-type mice). Importantly, the number of Foxp3(+)CD4(+) regulatory T cells (Tregs) in the spleen and lung as well as in vitro differentiation of induced Tregs was significantly reduced in p53-cKO mice as compared with that in p53 wild-type mice. Regarding the mechanisms underlying p53-mediated Treg induction, p53 enhanced the transcription of Foxp3 by binding to the promoter and the conserved noncoding DNA sequence-2 of the Foxp3 gene. Taken together, these results suggest that p53 expressed in T cells functions as a suppressor for autoimmunity by inducing Treg differentiation.

Yellow nail syndrome is an idiopathic condition characterized by a triad consisting of yellow nail, lymphedema, and pulmonary manifestations. Thiol compounds such as D-penicillamine have been reported to be the major cause of drug-induced yellow nail syndrome in patients with rheumatoid arthritis (RA). We recently experienced two Japanese cases with RA who developed yellow nail under treatment with bucillamine, a thiol-containing anti-rheumatic drug developed and approved in Japan. We reviewed the literature for similar cases and identified 36 RA cases with bucillamine-induced yellow nail, mostly in Japanese medical journals. Most of these cases (90.3%) showed improvement of yellow nail after discontinuation of bucillamine, whereas lymphedema and pulmonary manifestations improved only in 30.8 and 35.0% of the patients, respectively. © 2011 Springer-Verlag.

A significant proportion of patients with connective tissue disease (CTD) have gastric esophageal reflux disease (GERD) symptoms despite receiving proton pump inhibitors (PPIs). Although pre-meal administration of PPIs is recommended in Western countries, the benefit of this administration timing in Japanese CTD patients with refractory GERD symptoms has not been proven. To determine whether pre-dinner administration of PPIs is more efficacious for refractory GERD symptoms in Japanese CTD patients. CTD patients receiving oral PPIs were instructed to take PPIs 1 h before dinner. Gastrointestinal symptoms were evaluated with frequency scale for the symptoms of GERD (FSSG) and gastrointestinal symptom rating scale (GSRS) before and after the intervention. Pre-dinner administration of PPIs significantly improved FSSG total score, from a median of 8 to 6.5 (P = 0.005). Pre-dinner administration was more effective in patients with overt GERD symptoms (from median 18 to 10, P < 0.001) than in those with mild GERD symptoms (from median 2 to 2, P = 0.201). In addition to reflux syndrome, pre-dinner administration of PPIs significantly decreased abdominal pain syndrome and constipation syndrome of GSRS. Pre-dinner administration of PPIs may increase their efficacy in Japanese CTD patients with GERD, especially those with overt symptoms.

In the past decade, mechanisms underlying allergic contact dermatitis have been intensively investigated by using contact hypersensitivity (CHS) models in mice. However, the regulatory mechanisms, which could be applicable for the treatment of allergic contact dermatitis, are still largely unknown. To determine the roles of B and T lymphocyte attenuator (BTLA), a CD28 family coinhibitory receptor, in hapten-induced CHS, BTLA-deficient (BTLA(-/-)) mice and littermate wild-type (WT) mice were subjected to DNFB-induced CHS, severe combined immunodeficient (SCID) mice were injected with CD4(+) T cells, and CD8(+) T cells from either WT mice or BTLA(-/-) mice were subjected to CHS. BTLA(-/-) mice showed enhanced DNFB-induced CHS and proliferation and IFN-gamma production of CD8(+) T cells as compared with WT mice. SOD mice injected with WT CD4(+) T cells and BTLA(-/-) CD8(+) T cells exhibited more severe CHS as compared with those injected with WT CD4(+) T cells and WT CD8(+) T cells. On the other hand, SCID mice injected with BTLA(-/-) CD4(+) T cells and WT CD8(+) T cells exhibited similar CHS to those injected with WT CD4(+) T cells and WT CD8(+) T cells. Finally, to evaluate the therapeutic potential of an agonistic agent for BTLA on CHS, the effects of an agonistic anti-BTLA antibody (6A6) on CHS were examined. In vivo injection of 6A6 suppressed DNFB-induced CHS and IFN-gamma production of CD8(+) T cells. Taken together, these results suggest that stimulation of BTLA with agonistic agents has therapeutic potential in CHS. Journal of Investigative Dermatology (2013) 133, 702-711; doi:10.1038/jid.2012.396; published online 29 November 2012

Objective: This study aimed to determine whether [18F]fluorodeoxyglucose-PET/CT ([18F]FDG-PET/CT) discriminates PM/DM from non-muscular diseases and also whether FDG uptake in proximal muscles reflects the activity and severity of muscular inflammation in PM/DM. Methods: Twenty treatment-naïve PM/DM patients who underwent [18F]FDG-PET/CT were retrospectively identified by reviewing medical records. The same number of age- and sex-matched control patients with non-muscular diseases were also identified. Standardized uptake value (SUV) was calculated for each of the seven proximal muscles. For patient-based assessment, mean proximal muscle SUV was calculated by averaging the SUVs for these proximal muscles bilaterally. Results: Mean proximal muscle SUVs were significantly greater in PM/DM patients than in control patients (median 1.05 vs 0.69, P&lt 0.001). Mean proximal muscle SUVs significantly correlated with mean proximal manual muscle test scores (B=0.49, P= 0.028) and serum levels of creatine kinase (B=0.54, P=0.015) and aldolase (B = 0.64, P= 0.002). Furthermore, SUVs in proximal muscles from which biopsy specimens were obtained significantly correlated with histological grade for inflammatory cell infiltration (B=0.66, P= 0.002). Conclusion: Our results suggest that [18F]FDG-PET/CT is useful in the diagnosis of PM/DM when inflammation in proximal muscles is globally assessed with quantitative measurements. Our results also indicate that local FDG uptake in a proximal muscle reflects the activity of inflammation in the same muscle and provides useful information in determining the region for muscle biopsy. © The Author 2013. Published by Oxford University Press on behalf of the British Society for Rheumatology. All rights reserved.

Background: Mast cells are known to play a pivotal role in allergic diseases by releasing granules containing histamine and other preformed chemical mediators. Cross-linking of high-affinity receptors for IgE (Fc epsilon RI) on mast cells results in rapid increases in intracellular free calcium concentration [Ca2+](i) and consequent activation of many transcription factors, including NFAT, NF-kB, JNK and CREB. Ca2+ signaling is essential for many cellular activities such as proliferation, gene expression and degranulation in mast cells. In addition to Ca2+ signaling, previous reports have shown that IkappaB kinase 2 (IKK2 or IKK beta), a central component of the IKK complex mediating NF-kB activation, also plays a crucial role in Fc epsilon RI-mediated degranulation and cytokine production. Moreover, it has been demonstrated that activation of PKC beta, a calcium-dependent PKC isoform, leads to IKK2 activation in many cell types. However, the roles of Ca 2+ signaling and PKC beta in the activation of IKK2 in mast cells remain largely unknown. Methods: We investigated the effect of PKC inhibitor Go6976 on calcium ionophore A23187-induced activation of IKK2 in mast cells. We also examined the role of IKK2 in A23187-induced NF-kB-dependent gene induction, degranulation, proinflammatory cytokine production and extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation by using IKK2-deficient (IKK2(-/-)) fetal liver-derived mast cells (FLMCs). Results: A23187 activated IKK2 and NF-kB even in the presence of Go6976 in mast cells. A23187-induced degranulation, cytokine production and activation of ERK1/2 were diminished in IKK2(-/-)FLMCs compared to those in wild-type FLMCs. Conclusions: Ca2+-IKK2 signaling is involved in the degranulation and cytokine production in activated mast cells by a mechanism independent of PKC beta. Copyright (C) 2013 S. Karger AG, Basel

MRL/Mp-Fas (lpr) (MRL-lpr) mice develop a systemic autoimmune disease and are considered to be a good model for systemic lupus erythematosus in humans. We have recently shown that mice lacking B and T lymphocyte attenuator (BTLA), an inhibitory co-receptor expressed mainly on lymphocytes, on a 129SvEv background spontaneously develop lymphocytic infiltration in multiple organs and an autoimmune hepatitis (AIH)-like disease. In this study, we investigated the role of BTLA in the pathogenesis of autoimmune diseases in MRL-lpr mice. We found that BTLA-deficient (BTLA(-/-)) MRL-lpr/lpr mice developed severe lymphocytic infiltration in salivary glands, lungs, pancreas, kidneys and joints as compared with BTLA-sufficient (BTLA(+/+)) MRL-lpr/lpr mice. In addition, although AIH-like disease was not found in BTLA(+/+) MRL-lpr/lpr mice, AIH-like disease was exacerbated in BTLA(-/-) MRL-lpr/lpr mice as compared with that in BTLA(-/-) 129SvEv mice. These results suggest that BTLA plays a protective role in autoimmune diseases in MRL-lpr mice and that AIH-like disease develops in BTLA(-/-) mice even in the absence of Fas-dependent signaling.

We recently showed that mice lacking B and T lymphocyte attenuator (BTLA), a third inhibitory coreceptor expressed on B cells and T cells, exhibit an increased Ag-specific IgG response and gradually develop hyper-gamma-globulinemia and autoantibody production. Recent studies revealed that follicular Th (Tfh) cells, which are non-Th1, non-Th2 effector T cells that express CXCR5 and provide help for B cells to produce Ig, also express BTLA. However, the role of BTLA in Tfh cell function remains unknown. In this study, we examined the regulatory role of BTLA in the development and function of Tfh cells. We found that CXCR5(+) Tfh cells expressed higher levels of BTLA than did CXCR5(-) conventional CD4(+) T cells. We also found that adoptive transfer of BTLA(-/-) CD4(+) T cells, stimulated under Tfh cell-inducing conditions (Tfh-like cells), to wild-type (WT) mice induced more Ag-specific IgG2a and IgG2b production compared with that of WT Tfh-like cells. By contrast, another adoptive-transfer experiment using BTLA(-/-) mice as recipients showed that the expression of BTLA on B cells was not involved in the regulation of Tfh-like cell-mediated Ag-specific IgG responses. Moreover, the development of IL-21-producing CXCR5+ Tfh-like cells was significantly increased in BTLA(-/-) CD4(+) T cells compared with WT CD4(+) T cells. Furthermore, Tfh-like cell-mediated IgG responses were abolished when IL-21R(-/-) mice were used as recipients. These results suggest that BTLA signaling suppresses IL-21 production from Tfh cells and subsequent Tfh cell-mediated IgG responses. The Journal of Immunology, 2010, 185: 2730-2736.

Previous studies have shown that IL-6 potently induces IL-21 production in CD4(+) T cells, whereas TGF-beta inhibits IL-6-induced IL-21 production in CD4(+) T cells. In this study, we addressed the mechanisms underlying the transcriptional regulation of IL-21 production in CD4(+) T cells. We found that IL-6 induced c-Maf expression in CD4(+) T cells and that the enforced expression of c-Maf induced IL-21 production in CD4(+) T cells without IL-6, IL-4/STAT6 signaling, or an autocrine effect of IL-21. Moreover, we found that c-Maf directly bound to and activated IL-21P and the CNS-2 enhancer through MARE sites. On the other hand, we also found that although TGF-beta up-regulated IL-6-induced c-Maf expression in CD4(+) T cells, TGF-beta inhibited c-Maf-induced IL-21 production in CD4(+) T cells. Finally, we found that Foxp3 bound to IL-21P and the CNS-2 enhancer and inhibited c-Maf-induced IL-21 production modestly but significantly in CD4(+) T cells. Taken together, these results suggest that c-Maf induces IL-21 production directly in CD4(+) T cells by activating IL-21P and the CNS-2 enhancer and that TGF-beta suppresses c-Maf-induced IL-21 production in CD4(+) T cells. J. Leukoc. Biol. 87: 703-712; 2010.

Although B and T lymphocyte attenuator (BTLA) was originally identified as an inhibitory coreceptor selectively expressed on Th1 cells and B cells, recent studies have revealed that BTLA is expressed on a variety of cells, including macrophages, dendritic cells, and NK cells, and modulates their functions. However, the role of BTLA in the regulation of NKT cell function remains unknown. In this study, we found that BTLA was expressed on NKT cells at the levels similar to those on T cells and that BTLA-deficient (BTLA(-/-)) NKT cells produced larger amounts of IL-4 and IFN-gamma upon alpha-glactosylceramide stimulation as compared with wild-type (WT) NKT cells. In vivo, BTLA(-/-) mice produced larger amounts of IL-4 and IFN-gamma upon Con A injection and were more susceptible to Con A-induced hepatitis than WT mice. In addition, the augmentation of Con A-induced hepatitis in BTLA(-/-) mice was not observed in BTLA/NKT-double deficient mice. Moreover, NKT(-/-) mice reconstituted with BTLA(-/-) NKT cells were significantly more susceptible to Con A-induced hepatitis as compared with NKT(-/-) mice reconstituted with WT NKT cells. These results suggest that BTLA functions as the inhibitory coreceptor of NKT cells and plays a critical role in the prevention of NKT cell-mediated liver injury. The Journal of Immunology, 2010,184: 127-133.

Background: Lacrimal gland enlargement (LGE) is one of the characteristics of Mikulicz's disease (MD). Recently, marked serum IgG4 elevation and infiltration of IgG4-positive plasmacytes in the enlarged exocrine glands have been reported in MD patients. However, little is known about the role of CD4+ T cells and their cytokines in IgG4-related diseases. The aim of this study was to evaluate the characteristics of CD4+ T cells in patients with IgG4-related diseases. Methods: We investigated the clinical characteristics of 9 patients with LGE and elevated serum IgG4 levels (named IgG4-related LGE). We also examined mRNA expression of cytokines and transcription factors of peripheral blood CD4+ T cells in patients with IgG4-related LGE. Results: All patients with IgG4-related LGE showed elevated serum IgE levels. In addition, 5 of 9 patients with IgG4-related LGE exhibited eosinophilia and asthma-like symptoms. In patients with IgG4-related LGE, mRNA expression of IL-4, IL-5, IL-10 and GATA-3 but not IFN-gamma or T-bet was enhanced on CD4+ T cells compared with that in healthy controls. Conclusions: Th2 cells may be involved in the pathogenesis of IgG4-related diseases. Copyright (C) 2010 S. Karger AG, Basel

Recent studies have suggested that statins, the inhibitors for 3-hydroxy-3-methyglutaryl (HMG)-CoA reductase in the mevalonate pathway, exhibit anti-inflammatory effects. However, the immune modulatory effects of statins on the differentiation of CD4(+) T cells and their underlying mechanisms are still largely unknown. To address these issues, we examined the effect of simvastatin and inhibitors for protein farnesylation and geranylgeranylation on the differentiation of IL-17-producing T cells (T(h)17 cells) and Foxp3(+) CD4(+) T cells. Simvastatin inhibited the differentiation of T(h)17 cells through the inhibition of HMG-CoA reductase activity but enhanced the differentiation of Foxp3(+) CD4(+) T cells. Geranylgeranyltransferase I inhibitor, GGTI-298, but not farnesyltransferase inhibitor, FTI-277, mimicked the effects of simvastatin, indicating that the inhibition of protein geranylgeranylation is responsible for the effects. Moreover, Foxp3(+) CD4(+) T cells developed in the presence of transforming growth factor-beta and GGTI-298 functioned as regulatory T cells (Tregs) in in vitro T cell proliferation assay as well as in an autoimmune colitis model. Finally, GGTI-298 induced SOCS3 expression and inhibited IL-6-induced signal transducers and activators of transcription3 phosphorylation in CD4(+) T cells. Taken together, these results indicate that protein geranylgeranylation enhances the differentiation of T(h)17 cells and inhibits the differentiation of Foxp3(+) Tregs partly via the inhibition of SOCS3 expression.

OBJECTIVE-Interleukin (IL)-21 is a type 1 cytokine that has been implicated in the pathogenesis of type 1 diabetes via the unique biology of the nonobese diabetic (NOD) mouse strain. The aim of this study was to investigate a causal role for IL-21 in type 1 diabetes. RESEARCH DESIGN AND METHODS-We generated IL-21R-deficient NOD mice and C57Bl/6 mice expressing IL-21 in pancreatic beta-cells, allowing the determination of the role of insufficient and excessive IL-21 signaling in type 1 diabetes. RESULTS-Deficiency in IL-21R expression renders NOD mice resistant to insulitis, production of insulin autoantibodies, and onset of type 1 diabetes. The lymphoid compartment in IL-21R(-/-) NOD is normal and does not contain an increased regulatory T-cell fraction or diminished effector cytokine responses. However, we observed a clear defect in autoreactive effector T-cells in IL-21R(-/-) NOD by transfer experiments. Conversely, overexpression of IL-21 in pancreatic beta-cells induced inflammatory cytokine and chemokines, including IL-17A, IL17F, IFN-gamma, monocyte chemoattractant protein (MCP)-1, MCP-2, and interferon-inducible protein-10 in the pancreas. The ensuing leukocytic infiltration in the islets resulted in destruction of beta-cells and spontaneous type 1 diabetes in the normally diabetes-resistant C57Bl/6 and NOD X C57Bl/6 backgrounds. CONCLUSIONS-This work provides demonstration of the essential prodiabetogenic activities of 11,21 on diverse genetic backgrounds (NOD and C57BL/6) and indicates that IL-21 blockade could be a promising strategy for interventions in human type 1 diabetes. Diabetes 58:1144-1155, 2009

STAT6 plays critical roles in Th2 cell differentiation, whereas STAT4 and T-bet are important for Th1 cell differentiation. However, it is still largely unknown about the cross talk of these transcription factors during Th1/Th2 cell differentiation. To further address the regulatory mechanisms underlying Th1/Th2 cell differentiation, we generated the mice lacking both STAT6 and T-bet (STAT(-/-) T-bet(-/-) mice). Importantly, although Th2 cell differentiation was severely and similarly decreased in STAT6(-/-) T-bet(-/-) mice and STAT6(-/-) mice. Th1 cell differentiation was rescued in part in STAT6(-/-) T-bet(-/-) mice as compared with that in T-bet(-/-). While no significant difference was observed in the expression of IL-12R beta 2 and STAT4 between STAT6(-/-) T-bet(-/-) CD4(+) T cells and T-bet(-/-) CD4(+) T cells, IL-12-induced STAT4 phosphorylation was increased in STAT6(-/-) T-bet(-/-) CD4(+) T cells as compared with that in T-bet(-/-) CD4(+) T cells. These results indicate that STAT6 inhibits T-bet-independent Th1 cell differentiation by suppressing IL-12-STAT4 signaling. (C) 2009 Elsevier Inc. All rights reserved.

Rationale The IL-23-IL-17A-producing CD4(+) T-cell (Th17 cell) axis plays an important role in the development of chronic inflammatory diseases, including autoimmune diseases. However, the role of the IL-23-Th17 cell axis in the regulation of allergic airway inflammation is still largely unknown. Objectives: To determine the role of IL-23 and Th17 cells in allergic airway inflammation. Methods: We examined the effect of anti-IL-23 antibody on antigen-induced airway inflammation. We also investigated the effect of enforced expression of IL-23 on allergic airway inflammation by generating lung-specific IL-23 transgenic mice. Moreover, we examined the effect of adoptive transfer of antigen-specific Th17 cells on allergic airway inflammation. Measurements and Main Results: IL-23 mRNA was expressed in the lung of sensitized mice upon antigen inhalation, and the neutralization of IL-23 decreased antigen-induced eosinophil recruitment and Th2 cytokine production in the airways. The enforced expression of IL-23 in the airways significantly enhanced antigen-induced eosinophil and neutrophil recruitment into the airways; Th2 cytokine, IL-17A, and tumor necrosis factor (TNF)-alpha production in the airways; goblet cell hyperplasia; and airway hyperresponsiveness. Moreover, IL-23-mediated enhancement of antigen-induced Th2 cytokine production and eosinophil recruitment in the airways was still observed in the mice lacking IL-17A. Furthermore, although adoptive transfer of antigen-specific Th17 cells alone induced neutrophil but not eosinophil recruitment into the airways upon antigen inhalation, cotransfer of Th17 cells with Th2 cells significantly enhanced antigen-induced Th2-cell-mediated eosinophil recruitment into the airways and airway hyperresponsiveiness. Conclusions: IL-23 and Th17 cells not only induce Th17-cell-mediated neutrophilic airway inflammation but also up-regulate Th2-cell-mediated eosinophilic airway inflammation.

Asthma is characterized by airway inflammation with intense eosinophil infiltration and MUCUS hyperproduction, in which antigen-specific Th2 cells play critical roles. Nuclear factor-kappa B (NF-kappa B) pathway has been demonstrated to be essential for the production of Th2 cytokines and chemokines in the airways in murine asthma models. in the present study, we examined the effect of GS 143, a novel small-molecule inhibitor of I kappa B ubiquitination, On antigen-induced airway inflammation and Th2 cytokine production in mice. Intranasal administration of GS143 prior to antigen challenge Suppressed antigen-induced NF-kappa B activation in the lung of sensitized mice. Intranasal administration of GS143 also inhibited antigen-induced eosinophil and lymphocyte recruitment into the airways as well as the expression of Th2 cytokines and eotaxin in the airways. Moreover, GS143 inhibited antigen-induced differentiation of Th2 cells but not of Th1 cells in vitro. Taken together, these results suggest that I kappa B ubiquitination inhibitor may have therapeutic potential against asthma. (C) 2008 Elsevier Inc. All rights reserved.

Objective. B and T lymphocyte attenuator (BTLA), a coreceptor expressed on lymphocytes, was recently described as an inhibitory coreceptor that negatively regulates lymphocyte activation. The purpose of this study was to investigate the role of BTLA in the regulation of immune homeostasis and the pathogenesis of autoimmunity. Methods. We examined the levels of immunoglobulins and autoantibodies to nuclear antigens and the activation status of T cells in BTLA(-/-) mice. We also examined histopathologic changes in the organs of BTLA(-/-) mice. Results. We observed that BTLA(-/-) mice gradually developed hypergammaglobulinemia, antinuclear antibodies, anti-SSA antibodies, anti-double-stranded DNA antibodies, and an increased number of activated CD4+ T cells in the periphery with age. Lack of BTLA led to spontaneous development of autoimmune hepatitis-like disease characterized by an elevation in the level of transaminases, interface hepatitis, and spotty necrosis of the liver. BTLA(-/-) mice also showed inflammatory cell infiltration of multiple organs, including the salivary glands, lungs, and pancreas; these features are similar to those of Sjogren's syndrome, which is a frequent complication of autoimmune hepatitis. Furthermore, the survival rate of BTLA(-/-) mice was significantly reduced after the age of 7 months. Conclusion. Our results indicate that BTLA plays an important role in the maintenance of immune tolerance and the prevention of autoimmune diseases.

It has recently been shown that interleukin (IL)-21 is produced by Th17 cells, functions as an autocrine growth factor for Th17 cells, and plays critical roles in autoimmune diseases. In this study, we investigated the differentiation and characteristics of IL-21-producing CD4(+) T cells by intracellular staining. Unexpectedly, we found that under Th17-polarizing conditions, the majority of IL-21-producing CD4(+) T cells did not produce IL-17A and -17F. We also found that IL-6 and -21 potently induced the development of IL-21-producing CD4(+) T cells without the induction of IL-4, IFN-gamma, IL-17A, or IL-17F production. On the other hand, TGF-beta inhibited IL-6- and IL-21-induced development of IL-21-producing CD4(+) T cells. IL-2 enhanced the development of IL-21-producing CD4(+) T cells under Th17-polarizing conditions. Finally, IL-21-producing CD4(+) T cells exhibited a stable phenotype of IL-21 production in the presence of IL-6, but retained the potential to produce IL-4 under Th2-polarizing conditions and IL-17A under Th17-polarizing conditions. These results suggest that IL-21-producing CD4(+) T cells exhibit distinct characteristics from Th17 cells and develop preferentially in an IL-6-rich environment devoid of TGF-beta, and that IL-21 functions as an autocrine growth factor for IL-21-producing CD4(+) T cells.

T-bet and STAT4 play critical roles in helper T cell differentiation, especially for Th1 cells. However, it is still unknown about the relative importance and redundancy of T-bet and STAT4 for Th1 differentiation. It is also unknown about their independent role of T-bet and STAT4 in the regulation of allergic airway inflammation. In this study, we addressed these issues by comparing T-bet-deficient (T-bet(-/-)) mice, STAT4(-/-) mice, and T-bet- and STAT4-double-deficient (T-bet(-/-)STAT4(-/-)) mice on the same genetic background. Th1 differentiation was severely decreased in T-bet(-/-) mice and STAT4(-/-) mice as compared with that in wild-type mice, but Th1 differentiation was still observed in T-bet(-/-) mice and STAT4(-/-) mice. However, Th1 cells were hardly detected in T-bet(-/-)STAT4(-/-) mice. In contrast, the maintenance of Th17 cells was enhanced in T-bet(-/-) mice but was reduced in STAT4(-/-) mice and T-bet(-/-)STAT4(-/-) mice. In vivo, Ag-induced eosinophil and neutrophil recruitment into the airways was enhanced in T-bet(-/-) mice but was attenuated in STAT4(-/-) mice and T-bet(-/-)STAT4(-/-) mice. Ag-induced IL-17 production in the airways was also diminished in STAT4(-/-) mice and T-bet(-/-)STAT4(-/-) mice. These results indicate that STAT4 not only plays an indispensable role in T-bet-independent Th1 differentiation but also is involved in the maintenance of Th17 cells and the enhancement of allergic airway inflammation.

Background: Statins inhibit 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, a key rate-limiting enzyme in the mevalonate pathway. Accumulating data suggest that statins exhibit anti-inflammatory effects on a number of experimental models including experimental autoimmune encephalomyelitis and antigen-induced allergic airway inflammation. However, the mechanism underlying the antiinflammatory effect of statins is still largely unknown. In this study, we examined the effect of a representative statin, simvastatin, on proinflammatory cytokine production from murine mast cells. Methods: Bone marrow-derived mast cells (BMMCs) were stimulated with lipopolysaccharide (LPS) in the presence or absence of simvastatin, and TNF-alpha and IL-6 production from BMMCs was evaluated at mRNA and protein levels. The effect of simvastatin on the expression of tristetraprolin, an RNA-binding protein that promotes decay of TNF-alpha mRNA, was evaluated. Results: Incubation of BMMCs with simvastatin resulted in the inhibition of LPS-induced TNF-alpha production at both mRNA and protein levels. Simvastatin also inhibited IL-6 production from LPS-stimulated BMMCs. However, simvastatin did not enhance the expression of tristetraprolin. Conclusions: Simvastatin inhibits the production of TNF-alpha and IL-6 from activated mast cells in part by inhibiting de novo synthesis of their transcripts and the inhibition may account for the anti-inflammatory effect of simvastatin. Copyright (C) 2008 S. Karger AG, Basel.

It is well documented that dendritic cells (DCs), representative antigen-presenting cells, are important sources of Th1-promoting cytokines and are actively involved in the regulation of T-helper-cell differentiation. However, the intracellular event that regulates this process is still largely unknown. In this study, we examined the role of Tyk2, a JAK kinase that is involved in the signaling pathway under IL-12 and IL-23, in DC functions. While the differentiation and maturation of DCs was normal in Tyk2-deficient (Tyk2(-/-)) mice, IL-12-induced Stat4 phosphorylation was diminished in Tyk2(-/-) DCs. IL-12-induced IFN-T production was also significantly diminished in Tyk2(-/-) DCs to levels similar to those in Stat4(-/-) DCs. Interestingly, Tyk2(-/-) DCs were defective in IL-12 and IL-23 production upon stimulation with CpG ODN. Furthermore, Tyk2(-/-) DCs were impaired in their ability to induce Th1 -cell differentiation but not Th2-cell differentiation. Taken together, these results indicate that the expression of Tyk2 in DCs is crucial for the production of Th1-promoting cytokines such as IL-12 and IFN-gamma from DCs and thereby for the induction of antigen-specific Th1-cell differentiation.

Background: A novel IL-17 family cytokine, IL-25, has been reported to induce IL-4, IL-5, and IL-13 production from undefined non-T/non-B cells and then induce T(H)2-type immune responses. However, the roles of IL-25 in inducing allergic airway inflammation remain unknown. Objective: We sought to determine whether IL-25 is involved in causing allergic airway inflammation. Methods: We examined the expression of IL-25 mRNA in the lungs of sensitized mice on antigen inhalation. We also examined the effect of IL-25 neutralization by soluble IL-25 receptor on antigen-induced airway inflammation. We then generated IL-25 transgenic mice that express IL-25 specifically in the lung under the control of the Clara cells-10-kd protein promoter and investigated the effect of enforced IL-25 expression on antigen-induced airway inflammation. Results: IL-25 mRNA was expressed in the lungs of sensitized mice on antigen inhalation, and the neutralization of IL-25 by soluble IL-25 receptor decreased antigen-induced eosinophil and CD4(+) T-cell recruitment into the airways. The enforced expression of IL-25 in the lung itself failed to induce allergic airway inflammation, whereas the expression of IL-25 significantly enhanced antigen-induced T(H)2 cytokine production, eosinophil and CD4(+) T cell recruitment, and goblet cell hyperplasia in the airways. Moreover, IL-25-induced enhancement of allergic airway inflammation was inhibited by the depletion of CD4(+) T cells or by the absence of signal transducer and activator of transcription 6. Conclusion: IL-25 enhances antigen-induced allergic airway inflammation by amplifying a T(H)2 cell-dependent pathway. Clinical implications: IL-25 might be involved in the enhancement, prolongation, or both of T(H)2 cell-mediated allergic diseases, such as asthma.

Exposure of naive Th cell precursors (Thp) to IL-21 inhibits IFN-gamma production from developing Th1 cells. The inhibition of IFN-gamma seen in IL-21-treated Thp cells is specific as the expression of other Th1 cytokines is unaffected. Recently, it has been reported that Eomesodermin (Eomes), a member of the T-box gene family, is expressed in developing CD8(+) T cells and plays an important role in regulating IFN-gamma production and cytolytic effector function. In this study, we show that Eomes mRNA and protein are also expressed in developing Th1 cells, and exposure of naive Thp cells to IL-21 results in a decrease in Eomes expression. Moreover, the repression of Eomes expression by IL-21 is not due to an indirect effect of IL-21 on the expression of IFN-gamma or STAT4 and is independent of STAT1 and T-bet expression. Finally, we show that ectopic expression of Eomes prevents the inhibition of IFN-gamma production from IL-21-treated Thp cells. Taken together, these results demonstrate that Eomes plays a role in regulating IFN-gamma production in CD4(+) T cells and IL-21 inhibits IFN-gamma production in developing Th1 cells through the repression of Eomes expression.

IL-4 plays a key role in inducing IL-4 production in CD4(+) T cells, functioning as an important determinant for Th2 cell differentiation. We show here that IL-4 induces IFN-gamma production in B220(+) plasmacytoid dendritic cells (PDCs). By searching for cell populations that produce IFN-gamma upon IL-4 stimulation, we found that PDCs were a major IFN-gamma-producing cell upon IL-4 stimulation in wild-type and Rag-2(-/-) splenocytes. Isolated PDCs, but not CD11b(+) DCs or CD8(+) DCs, produced IFN-gamma upon IL-4 stimulation. In vivo, the depletion of PDCs by anti-Ly6G/C Ab prevented IFN-gamma production induced by IL-4 administration. We also found that IL-4 induced IFN-gamma production, but not IL-12 or IFN-alpha production, in PDCs and also strongly enhanced CpG oligodeoxynucleotide-induced IFN-gamma production, but not CpG oligodeoxynucleotide-induced IL-12 or IFN-alpha production. However, IL-4 did not induce IFN-gamma production in Stat6(-/-) PDCs. Moreover, IL-4 induced Stat4 expression in PDCs through a Stat6-dependent mechanism, and only the Stat4-expressing PDCs produced IFN-gamma. Furthermore, IL-4 did not induce IFN-gamma production in Stat4(-/-) PDCs. These results indicate that PDCs preferentially produce IFN-gamma upon IL-4 stimulation by Stat6- and Stat4-dependent mechanisms.

In previous studies, we have shown that Th2 cell differentiation is diminished but Th1 cell differentiation is increased in Stat5a-deficient (Stat5a(-/-)) CD4(+) T cells. In the present study, we clarified the molecular mechanisms of Stat5a-mediated Th cell differentiation. We found that enhanced Th1 cell differentiation and the resultant IFN-gamma production played a dominant inhibitory role in the down-regulation of IL-4-induced Th2 cell differentiation of Stat5a(-/-) CD4(+) T cells. We also found that IL-12-induced Stat4 phosphorylation and Th1 cell differentiation were augmented in Stat5a(-/-) CD4(+) T cells. Importantly, the expression of suppressor of cytokine signaling (SOCS)3, a potent inhibitor of IL-12-induced Stat4 activation, was decreased in Stat5a(-/-) CD4(+) T cells. Moreover, a reporter assay showed that a constitutively active form of Stat5a but not Stat6 activated the SOCS3 promoter. Furthermore, chromatin immunoprecipitation assays revealed that Stat5a binds to the SOCS3 promoter in CD4(+) T cells. Finally, the retrovirus-mediated expression of SOCS3 restored the impaired Th cell differentiation of Stat5a(-/-) CD4(+) T cells. These results suggest that Stat5a forces the Th1/Th2 balance toward a Th2-type by preventing IL-12-induced Th1 cell differentiation through the induction of SOCS3.

It is well-recognized that Stat6 plays a critical role in Th2 cell differentiation and the induction of allergic inflammation. We have previously shown that Stat5a is also required for Th2 cell differentiation and allergic airway inflammation. However, it is the relative importance and redundancy of Stat6 and Stat5a in Th2 cell differentiation and allergic airway inflammation are unknown. In this study we addressed these issues by comparing Stat5a-deficient (Stat5a(-/-)) mice, Stat6(-/-) mice, and Stat5a- and Stat6 double-deficient (Stat5a(-/-) Stat6(-/-)) mice on the same genetic background. Th2 cell differentiation was severely decreased in Stat6(-/-)CD4(+) T cells, but Stat6-independent Th2 cell differentiation was still significantly observed in Stat6(-/-)CD4(+) T cells. However, even in the Th2-polarizing condition (IL-4 plus anti-IFN-gamma mAb), no Th2 cells developed in Stat5a(-/-)Stat6(-/-) CD4(+) T cells. Moreover, Ag-induced eosinophil and lymphocyte recruitment in the airways was severely decreased in Stat5a(-/-)Stat6(-/-)mice compared with that in Stat6(-/-) mice. These results indicate that Stat5a plays an indispensable role in Stat6-independent Th2 cell differentiation and subsequent Th2 cell-mediated allergic airway inflammation.

Increasing evidence has revealed that mast cell-derived tumor necrosis factor alpha (TNF-alpha) plays a critical role in a number of inflammatory responses by recruiting inflammatory leukocytes. In this paper, we investigated the regulatory role of interleukin 4 (IL-4) in TNF-alpha production in mast cells. IL-4 inhibited immunoglobulin E-induced TNF-alpha production and neutrophil recruitment in the peritoneal cavity in wild-type mice but not in signal transducers and activators of transcription 6 (Stat6)-deficient mice. IL-4 also inhibited TNF-alpha production in cultured mast cells by a Stat6-dependent mechanism. IL-4-Stat6 signaling induced TNF-alpha mRNA destabilization in an AU-rich element (ARE)-dependent manner, but did not affect TNF-alpha promoter activity. Furthermore, IL-4 induced the expression of tristetraprolin (TTP), an RNA-binding protein that promotes decay of ARE-containing mRNA, in mast cells by a Stat6-dependent mechanism, and the depletion of TTP expression by RNA interference prevented IL-4-induced down-regulation of TNF-alpha production in mast cells. These results suggest that IL-4-Stat6 signaling induces TTP expression and, thus, destabilizes TNF-alpha mRNA in an ARE-dependent manner.

Interleulkin-25 (IL-25) is a recently described T helper 2 (T(H)2) cell-derived cytokine that belongs to the IL-17 family and induces the production of IL-4, IL-5, and IL-13 from an unidentified non-T-cell population. Here, we show that mast cells are also potent IL-25-producing cells. When bone marrow-derived mast cells were stimulated by immunoglobulin E cross-linking, IL-25 mRNA was induced within 30 minutes in a calcineurin-dependent manner, and the levels of IL-25 mRNA were comparable with those of ctivated T(H)2 cells. Production of IL-25 by mast cells was also detected at protein levels by immunoblotting. These results suggest that mast cells may enhance T(H)2-type immune response by producing IL-25. (C) 2003 by The American Society of Hematology.