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DNase 1–like 3 (DNase1L3), which belongs to DNase1 family, was originally identified as one of apoptosis- and necrosis-related endonucleases that fragmentate intranucleosomal DNA. A loss-of-function mutation has been reported in murine models of systemic lupus erythematosus (SLE) and in familial SLE patients. These reports suggest DNase1L3 plays an important role in the prevention of developing SLE; however, expression and function of DNase1L3 in human immune systems have been largely unclarified. As previous reports showed DNase1L3 is expressed in hematopoietic organs, we first analyzed expression levels of DNase1L3 in each subset of human peripheral blood cells by quantitative real-time PCR. Plasmacytoid dendritic cells showed the highest expression levels of DNase1L3 mRNA among peripheral blood cells. IL-4 enhanced DNase1L3 expression in monocytes, monocyte-derived dendritic cells, and monocyte-derived macrophages (MDMs), but not in T cells, B cells, or plasmacytoid dendritic cells. Together with IL-4, all-trans retinoic acid and apoptotic cells efficiently upregulated expression of DNalse1L3 in MDMs. As a result of intracellular signaling analysis, Jak1-IRS2-ERK/PI3K pathway was essential for IL-4–induced DNase1L3 expression. IL-4–treated monocyte-derived dendritic cells and MDMs secreted active DNase1L3 protein that could degrade liposome–DNA complexes, which were resistant to DNase1. Our results indicate DNase1L3 is secreted by innate immune cells and may play a critical role in the tissue homeostasis and on prevention of developing autoimmunity by degrading self-DNA.

Summary Systemic sclerosis (SSc) is a T helper type 2 (Th2)-associated autoimmune disease characterized by vasculopathy and fibrosis. Efficacy of B cell depletion therapy underscores antibody-independent functions of B cells in SSc. A recent study showed that the Th2 cytokine interleukin (IL)-4 induces granulocyte–macrophage colony-stimulating factor (GM-CSF)-producing effector B cells (GM-Beffs) in humans. In this study, we sought to elucidate the generation mechanism of GM-Beffs and also determine a role of this subset in SSc. Among Th-associated cytokines, IL-4 most significantly facilitated the generation of GM-Beffs within memory B cells in healthy controls (HCs). In addition, the profibrotic cytokine transforming growth factor (TGF)-β further potentiated IL-4- and IL-13-induced GM-Beffs. Of note, tofacitinib, a Janus kinase (JAK) inhibitor, inhibited the expression of GM-CSF mRNA and protein in memory B cells induced by IL-4, but not by TGF-β. GM-Beffs were enriched within CD20+CD30+CD38−/low cells, a distinct population from plasmablasts, suggesting that GM-Beffs exert antibody-independent functions. GM-Beffs were also enriched in a CD30+ fraction of freshly isolated B cells. GM-Beffs generated under Th2 conditions facilitated the differentiation from CD14+ monocytes to DC-SIGN+CD1a+CD14−CD86+ cells, which significantly promoted the proliferation of naive T cells. CD30+ GM-Beffs were more pronounced in patients with SSc than in HCs. A subpopulation of SSc patients with the diffuse type and concomitant interstitial lung disease exhibited high numbers of GM-Beffs. Together, these findings suggest that human GM-Beffs are enriched in a CD30+ B cell subset and play a role in the pathogenesis of SSc.

An association between immunoglobulin G4-related disease (IgG4-RD) and malignancy has been suggested. We report two cases of IgG4-RD with suspected paraneoplastic syndrome. In both patients, malignancy was observed immediately after diagnosis of IgG4-RD, and surgical resection resulted in spontaneous regression of IgG4-RD. We review the reports on IgG4-RD associated with malignancy, including these two cases, and discuss their relevance.

Background Follicular helper CD4+ T (Tfh) cells have a critical role in IgG4 production by B cells in IgG4-related disease (IgG4-RD). Recent studies including ours showed that SLAMF7+CD4+ T cells are an important pathological driver of IgG4-RD. In this study, we have sought to elucidate a relationship between helper CD4+ T (Th), particularly Tfh, cells and SLAMF7+ CD4+ T cells in IgG4-RD. Results The patients with IgG4-RD enrolled in this study were aged 66 ± 12 years and their titers of serum IgG4 were 372 ± 336 mg/dl. Th1 cells, activated circulating Tfh1 (cTfh1), and activated cTfh2 cells increased in IgG4-RD. SLAMF7 was mainly expressed on Th1 and cTfh1, but not cTfh2, cells in the patients. SLAMF7+ cTfh1 cells were PD-1/CD28 double-positive, whereas SLAMF7+ Th1 cells were CD28 negative. Positive correlations were noted between serum IgG4 levels and the number of activated cTfh2 cells and SLAMF7+ cTfh1 cells, but not SLAMF7+ Th1 cells. Intriguingly, among cTfh1 cells, activated SLAMF7+ cTfh1 cells were high producers of IL-10 along with IL-21. Blimp-1, but not Bcl-6, mRNA was expressed at high levels in activated SLAMF7+ cTfh1 cells. In addition to CD4+ T cells, the frequency of SLAMF7+ fraction was higher in memory B cells than naïve B cells in patients with IgG4RD. Finally, upon stimulation via B-cell receptor and CD40, Tfh1-associated cytokines, IL-21 and IFN-γ, most significantly induced SLAMF7 expression in memory B cells. Conclusions Together, these results suggest that circulating SLAMF7+ Tfh1 cells, along with Tfh2 cells, play a pathologic role in IgG4 production in IgG4-RD.

Background Currently, although several categories of biological disease-modifying antirheumatic drugs (bDMARDs) are available, there are few data informing selection of initial treatment for individual patients with rheumatoid arthritis (RA). Therefore, tumor necrosis factor inhibitor (TNF-i) and tocilizumab (TCZ) are treated as equivalent treatments in the recent disease management recommendations. We focused on two anticytokine therapies, TCZ and TNF-i, and aimed to develop a scoring system that predicts a better treatment for each RA patient before starting an IL-6 or a TNF-i. Methods The expression of IL-6 and TNF-α mRNA in peripheral blood from 45 newly diagnosed RA patients was measured by DNA microarrays to evaluate cytokine activation. Next, laboratory indices immediately before commencing treatment and disease activity score improvement ratio after 6 months in 98 patients treated with TCZ or TNF-i were retrospectively analyzed. Some indices correlated with TCZ efficacy were selected and their cutoff values were defined by receiver operating characteristic (ROC) analysis to develop a scoring system to discriminate between individuals more likely to respond to TCZ or TNF-i. The validity of the scoring system was verified in these 98 patients and an additional 228 patients. Results There was significant inverse correlation between the expression of IL-6 and TNF-α mRNA in newly diagnosed RA patients. The analysis of 98 patients revealed significant correlation between TCZ efficacy and platelet counts, hemoglobin, aspartate aminotransferase, and alanine aminotransferase; in contrast, there was no similar correlation in the TNF-i group. The cutoff values were defined by ROC analysis to develop a scoring system (1 point/item, maximum of 4 points). A good TCZ response was predicted if the score was ≥2; in contrast, TNF-i seemed to be preferable if the score was ≤1. Similar results were obtained in a validation study of an additional 228 patients. If the case scored ≥3, the good responder rates of TCZ/TNF-i were 75.0%/37.9% (p

Objective: In the present work, we investigate the role of interleukin (IL)27/IL27 receptor a (Ra) (WSX-1) in the development of autoimmune disorders in the MRL/lpr mouse, which is considered as an experimental model of systemic lupus erythaematosus (SLE) in humans. Methods: We generated two strains of WSX-1 transgenic mice in the MRL/lpr background with different expression levels of WSX-1, and investigated the effect of WSX-1 overexpression on survival, glomerulonephritis and immunological properties. Results: In comparison with wild type (WT) MRL/lpr and transgenic (Tg) low (TgL) mice, Tg high (TgH) mice exhibited a prolonged lifespan and no apparent development of autoimmune nephritis. Production of anti-dsDNA antibody and total IgG and IgG2a were significantly lower in TgH mice than those of TgL and WT mice. The expressed amounts of interferon (IFN)c and IL4 mRNA by CD4 + T cells from Tg mice decreased in a dose-dependent fashion. CD4 + splenic lymphocytes in TgH mice were more subject to the IL27-mediated suppression of cytokine production. In vitro stimulation of CD4 + T cells by IL27 resulted in over phosphorylation of STAT3 in TgH cells than in WT cells. Conclusion: WSX-1 overexpression in the MRL/lpr background rendered the autoimmune prone mice protected from the development of autoimmune diseases. Our results suggest that IL27 signalling may be a therapeutic target against autoimmune diseases, including human SLE.

MRL/lpr mice develop spontaneous glomerulonephritis that is essentially identical with diffuse proliferative glomerulonephritis (World Health Organization class IV) in human lupus nephritis. Lupus nephritis is one of the most serious complications of systemic lupus erythematosus. Diffuse proliferative glomerulonephritis is associated with autoimmune responses dominated by Th1 cells producing high levels of IFN-γ. The initial mounting of Th1 responses depends on the function of the WSX-1 gene, which encodes a subunit of the IL-27R with homology to IL-12R. In mice deficient for the WSX-1 gene, proper Th1 differentiation was impaired and abnormal Th2 skewing was observed during infection with some intracellular pathogens. Disruption of the WSX-1 gene dramatically changed the pathophysiology of glomerulonephritis developing in MRL/lpr mice. WSX-1−/− MRL/lpr mice developed disease resembling human membranous glomerulonephritis (World Health Organization class V) with a predominance of IgG1 in glomerular deposits, accompanied by increased IgG1 and IgE in the sera. T cells in WSX-1−/− MRL/lpr mice displayed significantly reduced IFN-γ production along with elevated IL-4 expression. Loss of WSX-1 thus favors Th2-type autoimmune responses, suggesting that the Th1/Th2 balance may be a pivotal determinant of human lupus nephritis development.

Background IL-12 is a heterodimeric proinflammatory cytokine that forms a link between innate and adaptive immunity. Although associations between polymorphisms of IL-12B on chromosome 5q31-33 and asthma have been reported, the genetic influences of the polymorphisms and haplotype of IL-12B are unclear. Objective To examine whether polymorphisms in IL-12B are associated with childhood atopic asthma in a Japanese population. Methods We identified a total of 13 polymorphisms and characterized the linkage disequilibrium mapping of the gene. Three variants in the promoter and 3′ untranslated region were genotyped, and we conducted case-control and case-only association studies between those variants and asthma-related phenotypes (childhood atopic asthma, n=297; normal controls, n=555). Haplotype association analysis and functional analysis of these variants were also performed. Results 3′ Untranslated region 10841C>A was significantly associated with the risk of childhood atopic asthma ( P = .00062). The −6415 promoter variant, in linkage disequilibrium with the 10841C>A (D′ = 0.78 and r 2 = 0.61), was also marginally associated with childhood atopic asthma ( P = .051). We analyzed the 2-locus haplotype by using these 2 polymorphisms and found a positive association with haplotype CTCTAA-C (−6415 CTCTAA and 10841C; P = .00078). Furthermore, 10841C>A affects the stability of transcripts, and promoter variant −6415GC enhances the transcriptional level of IL-12B . Conclusion Our results imply that functional haplotype CTCTAA-C, which affects the instability of transcripts and the lower transcriptional level of IL-12B , has a protective effect in childhood atopic asthma. On the basis of these findings, the IL-12B gene might be involved in the development of atopic asthma through functional genetic polymorphisms.

Atopic dermatitis (AD) is frequently associated with eosinophilia, highly elevated immunoglobulin E (IgE) levels and increased levels of T-helper 2-type (Th2) cytokines in skin lesions due to infiltrating T cells. Interleukin-12 (IL-12), in combination with interferon-gamma (IFN-gamma), inhibits IgE synthesis and Th2 cell function. As the IFN-gamma-inducing cytokines IL-12 and IL-23 utilize IL-12Rbeta1 as part of their receptors, it is possible that polymorphic variants of the IL-12Rbeta1 (IL12RB1) gene might determine an individual's susceptibility to AD. Here, we carried out a systemic search for genetic variants of the human IL12RB1 in Japanese subjects and identified 48 genetic variants. In a case-control association study, we found that promoter polymorphisms -111A/T and -2C/T were significantly associated with an increased risk of AD under a recessive model. The -111T-allele frequency in the independent population of child asthmatics was also much higher than that in the control group. In addition, the -111T/T genotype was progressively more common in AD with high total serum IgE levels in an IgE-level-dependent manner. Deletion analysis of the IL12RB1 promoter suggested that the -265 to -104 region that contained the -111A/T polymorphic site harbored an important regulatory element. Furthermore, we showed that the -111A/T substitution appeared to cause decreased gene transcriptional activity such that cells from -111A/A individuals exhibited higher IL12RB1 mRNA levels than those from -111T allele carriers. Our results suggested that in individuals with the -111T/T genotype, reduced IL-12Rbeta1 expression may lead to increased Th2 cytokine production in the skin and contribute to the development of AD and other subsequent allergic diseases.

Asthma is a phenotypically heterogeneous disorder with many etiologic factors and clinical characteristics. T-bet, a Th1-specific transcription factor of T-box family, has been found to control interferon-gamma (IFN-gamma) expression in T cells. Mice lacking the T-bet gene (tbx21) demonstrate multiple physiological and inflammatory features reminiscent of human asthma. In order to examine whether polymorphisms in the candidate gene, TBX21, located on chromosome 17q21.32, are related to the risk of human asthma phenotypes, we have searched for genetic variations in the human TBX21 gene and identified 24 single nucleotide polymorphisms (SNPs), including five novel SNPs, by direct sequencing in Japanese subjects. Among asthma phenotypes, a promoter -1993T--C SNP, which is in linkage disequilibrium with a synonymous coding 390A--G SNP in exon 1, is significantly associated with a risk of aspirin-induced asthma (AIA; P = 0.004, P(c) = 0.016). This association has also been confirmed in additional independent samples of asthma with nasal polyposis (P = 0.008), regardless of aspirin hypersensitivity. Furthermore, our data indicate that the -1993T--C substitution increases the affinity of a particular nuclear protein to the binding site of TBX21 covering the -1993 position, resulting in increased transcriptional activity of the TBX21 gene. Thus, in addition to the antigen-driven excess Th2 response, increased T-bet (and subsequent IFN-gamma) production in human airways of individuals with the -1993T--C polymorphism could contribute to the development of certain asthma-related phenotypes, such as AIA.

We describe a case of hypertrophic cranial pachymeningitis (HCP) associated with Wegener’s granulomatosis (WG) in a 60-year-old man presenting with chronic headache and multiple cranial nerve neuropathies. A test for antibodies to the neutrophil cytoplasmic protein myeloperoxidase (MPO-ANCA) was positive in this case. We review the literature on perinuclear (p)-ANCA-related HCP, including our case. This case indicates the link between MPO-ANCA-positive WG and HCP.

Sarcoidosis is known to be a systemic granulomatous disorder characterized by a cell-mediated Th1-type inflammatory response. To identify a key genetic factor in the pathogenesis of sarcoidosis, we investigated single nucleotide polymorphisms within 10 candidate genes involved in type 1 immune process ( IFNA17, IFNB, IFNG, IFNGR1, IFNGR2, IL12B, IL12RB1, IL12RB2, ETA-1, and NRAMP1) in an association-based study of 102 Japanese patients with sarcoidosis, 114 with tuberculosis, and 110 control subjects. After correction for multiple testing, an IFNA17 polymorphism (551T--G) was found to be associated with susceptibility to sarcoidosis (odds ratio 3.27 [95% CI: 1.44-7.46], P=0.004, P(c)=0.04), but not to tuberculosis. We observed no significant associations with the other polymorphisms of the Th1-related genes. We further typed another IFNA polymorphism ( IFNA10 60T--A) and confirmed two major haplotypes of the IFNA gene, viz., allele 1: IFNA10 [60T]- IFNA17 [551T] and allele 2: IFNA10 [60A]- IFNA17 [551G], in the Japanese population. In healthy subjects, IFNA allele 2, which is over-represented in patients with sarcoidosis, was significantly associated with increased IFN-alpha and IL-12p70 production induced by Sendai virus in vitro. This study suggests that possession of the IFNA allele with higher levels of IFN-alpha significantly increases the risk of sarcoidosis.

Host genetic factors may be important determinants of susceptibility to tuberculosis, and several candidate gene polymorphisms have been shown to date. A series of recent reports concerning rare human deficiencies in the type-1 cytokine pathway suggest that more subtle variants of relevant genes may also contribute to susceptibility to tuberculosis at the general population level. To investigate whether polymorphisms in the interleukin-12 receptor (IL-12R) gene predispose individuals to tuberculosis, we studied these genes by single-strand conformational polymorphism analysis and direct sequencing. Although no common polymorphisms could be identified in the IL-12R beta 2 gene ( IL-12RB2), we confirmed four single nucleotide polymorphisms (SNPs; 641A--G, 684C--T, 1094T--C, and 1132G--C) causing three missense variants (Q214R, M365T, G378R) and one synonymous substitution in the extracellular domain of the IL-12R beta 1 gene ( IL12RB1). All SNPs were in almost perfect linkage disequilibrium (D'=0.98), and two common haplotypes of IL12RB1(allele 1: Q214-M365-G378; allele 2: R214-T365-R378) were revealed. Polymerase chain reaction/restriction fragment length polymorphism and sequence analyses were used to type IL12RB1polymorphisms in 98 patients with tuberculosis and 197 healthy controls in Japanese populations. In our case-control association study of tuberculosis, the R214-T365-R378 allele (allele 2) was over-represented in patients with tuberculosis, and homozygosity for R214-T365-R378 (the 2/2 genotype) was significantly associated with tuberculosis (odds ratio: 2.45; 95% CI: 1.20-4.99; P=0.013). In healthy subjects, homozygotes for R214-T365-R378 had lower levels of IL-12-induced signaling, according to differences in cellular responses to IL-12 between two haplotypes. These data suggest that the R214-T365-R378 allele, i.e., variation in IL12RB1, contribute to tuberculosis susceptibility in the Japanese population. This genetic variation may predispose individuals to tuberculosis infection by diminishing receptor responsiveness to IL-12 and to IL-23, leading to partial dysfunction of interferon-gamma-mediated immunity.

Systemic sclerosis (SSc) is an autoimmune disease characterized by vascular damage and fibrosis of the skin and internal organs. Because activated and oligoclonally expanded CD8+ T cells can be detected in peripheral blood and lungs of SSc patients, effector memory CD8+ T cells may play a critical role for organ involvement in SSc; however, the pathogenic functions of effector memory CD8+ T cells remain incompletely understood. In this study, we performed DNA microarray analysis of the sort-purified effector memory CD8+ T cells from SSc patients and healthy controls, and showed that the expression of genes related to immune response and cell adhesion, including CD226 (also known as DNAX accessory molecule-1 [DNAM-1]), was significantly altered. Moreover, detailed analysis of CD226 revealed that CD226highCD8+ T cells were increased in SSc patients (mean, 50.7%) compared with healthy controls (32.9%) and were appreciably associated with the severity of skin sclerosis and interstitial lung disease. Furthermore, CD226+CD8+ T cells produced higher amount of various cytokines than CD226− ones, and CD226highCD8+ T cells from SSc patients showed upregulated IL-13 production and positive correlation with the cytotoxic capacity of CD8+ T cells against HUVECs. Finally, the neutralization of CD226 in CD8+ T cells impaired costimulation, cytokine productions, and cytolysis against HUVECs. These findings indicate that upregulated CD226 expression on CD8+ T cells reflects disease severity and is involved in SSc pathogenesis via the production of various cytokines, including profibrotic IL-13 and endothelial cell injury, and that CD226 may be a useful target in the treatment of SSc.

Mitogen-activated protein kinases (MAPKs) are activated by various extracellular stimuli and play an important role in regulating the expression of proinflammatory molecules in monocytes/macrophages. We first questioned whether MAPK activation in involved in cyclooxygenase (COX)-2 expression in lipopolysaccharide (LPS)-stimulated human monocytes. LPS induced the expression of COX-2 protein and COX-2 mRNA as well as the phosphorylation and activation of extracellular signal-regulated protein kinase (ERK)2 and p38 MAPK in monocytes. The induction of COX-2 mRNA, COX-2 protein, and prostaglandin (PG)E2 by LPS was inhibited by the specific inhibitors of ERK and p38 MAPK, suggesting that the activation of ERK2 and p38 MAPK is involved in COX-2 expression in LPS-stimulated monocytes. Since we previously showed that interleukin (IL)-10 and IL-4 similarly inhibited COX-2 expression in LPS-stimulated monocytes, we next questioned whether these cytokines regulate the phosphorylation and activation of ERK2 and p38 MAPK in LPS-stimulated monocytes. Interestingly, LPS-induced phosphorylation and activation of ERK2 was significantly inhibited by IL-4 and IL-10, while that of p38 MAPK was inhibited by IL-10, but not IL-4. These results suggest that the mechanisms of inhibition by IL-10 and IL-4 of the LPS-induced expression of proinflammatory molecules could be ascribed to the regulatory effects of both cytokines on MAPK activation.

Hirofumi Tsuzuki, Yojiro Arinobu, Kohta Miyawaki, Ayako Takaki, Shun-ichiro Ota, Naoko Ueki, Yuri Ota, Siamak Jabbarzadeh Tabrizi, Mitsuteru Akahoshi, Hiroaki Niiro, Hiroshi Tsukamoto, Takahiko Horiuchi, Shoichiro Ohta, Kenji Izuhara, MD, PhD, Hiroyuki Fukui, Koichi Akashi; Department of Medicine and Biosystemic Science, Kyushu University Graduate School of Medical Sciences, Fukuoka City, Japan, Clinical Education Center, Kyushu University Hospital, Fukuoka City, Japan, Department of Rheumatology, Internal medicine and connective tissue disorders, Shimonoseki City Hospital, Shimonoseki City, Japan, Division of Nephrology and Rheumatology, Fukuoka University Hospital, Fukuoka City, Japan, Department of Internal Medicine, Kyushu University Beppu Hospital, Beppu City, Japan, Division of Medical Biochemistry, Department of Biomolecular Sciences, Saga Medical School, Saga City, Japan, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima City, Japan. RATIONALE: A dried root of Sophora flavescens (S. flavescens) is a traditional Chinese herbal medicine used for the treatment of allergic diseases. Previous studies have demonstrated that S. flavescens possesses antiinflammatory activities and suppresses allergic inflammation such as allergic rhinitis. However, the mechanism of anti-asthmatic effect of S. flavescens is not yet clarified. METHODS: To clarify the effect of S. flavescens on asthma, we analyzed OVA-induced asthma mice treated with S. flavescens extract. We also conducted flow cytometry, cell count assay and gene expression analysis to evaluate the effect of S. flavescens on eosinophil hematopoiesis. To reveal the effect of S. flavescens on eosinophil chemotaxis, we performed transwell migration assay. RESULTS: Lung eosinophilic inflammation inOVA-induced asthmamice was suppressed by the treatment of S. flavescens. In addition, S. flavescens inhibited allergen-induced increment of eosinophil progenitors (EoPs) in the bonemarrow. In vitro, S. flavescens suppressed IL-5-induced expansion of EoPs and eosinophil lineage specification directly. Moreover, IL-5 expression was suppressed in CD4 T cells isolated from asthmatic mice treatedwith Sophora flavescens. In terms of eosinophil migration, S. flavescens suppressed eosinophil chemotaxis for eotaxin-1, one of themajor chemokines for eosinophil migration. CONCLUSIONS: S. flavescens suppressed eosinophil accumulation in the lung of asthmatic mice by inhibiting both eosinophil hematopoiesis and migration. S. flavescens might be valuable for the treatment of eosinophil-related diseases such as asthma.

We have identified that there are only two IL-4 gene haplotypes (I and II) in the Japanese population. There are significant differences among three genotypes (I/I, I/II and II/II) in the IL-4 producing proportion of peripheral Th cells using intracellular cytokine detection assay. These results make it likely that IL-4 genotype could influence the type of immune response.

Mast cell degranulation is important in the pathogenesis of anaphylaxis and allergic disorders. Many animal venoms contain components that can induce mast cell degranulation, and this has been thought to contribute to the pathology and mortality caused by envenomation. However, we recently reported evidence that mast cells can enhance the resistance of mice to the venoms of certain snakes and that mouse mast cell–derived carboxypeptidase A3 (CPA3) can contribute to this effect. Here, we investigated whether mast cells can enhance resistance to the venom of the Gila monster, a toxic component of that venom (helodermin), and the structurally similar mammalian peptide, vasoactive intestinal polypeptide (VIP). Using 2 types of mast cell–deficient mice, as well as mice selectively lacking CPA3 activity or the chymase mouse mast cell protease-4 (MCPT4), we found that mast cells and MCPT4, which can degrade helodermin, can enhance host resistance to the toxicity of Gila monster venom. Mast cells and MCPT4 also can limit the toxicity associated with high concentrations of VIP and can reduce the morbidity and mortality induced by venoms from 2 species of scorpions. Our findings support the notion that mast cells can enhance innate defense by degradation of diverse animal toxins and that release of MCPT4, in addition to CPA3, can contribute to this mast cell function.

Two alleles of IL-3 have been reported to GenBank (GenBank M14743, M20137). The sequence difference between these two alleles is at the first nucleotide of the 27th codon (the 131st nucleotide from the initiation site): thymine and cytosine, and leading the amino acid difference: proline and serine (Pro27Ser). The other allelism, thymine and cytosine, was also observed at position -16 of the IL-3 upstream promotor region (GenBank L10616, M60870). We clarified that these substitutions were frequent polymorphisms in the Japanese population by using the mismatch-PCR (polymerase chain reaction)/RFLP (restriction fragment length polymorphism) method.

Summary Background ADAM33, a member of the ADAM (a disintegrin and metalloprotease) family, is a putative asthma susceptibility gene recently identified by positional cloning. It is important to know whether the association exists in ethnically diverse populations. Objective To assess whether genetic functional variants of ADAM33 relate to the susceptibility or some phenotypes in adult patients with bronchial asthma in a Japanese population. Methods We searched for single nucleotide polymorphisms (SNPs) in ADAM33 by PCR-directed sequencing and identified 48 SNPs. Fourteen SNPs were selected with regard to the LD pattern, and genotyped by Taq-Man and PCR–RFLP methods. We conducted an association study of ADAM33 with 504 adult asthmatic patients and 651 controls, and haplotype analyses of related variants were performed. Results Significant associations with asthma were found for the SNPs T1 (Met764Thr), T2 (Pro774Ser), S2 and V−3 (with the lowest P-value for T1, P=0.0015; OR 0.63). We analysed the haplotype using these four polymorphisms, and found a positive association with haplotype CCTG (P=0.0024). Conclusion Our results replicate associations reported recently in other ethnic populations, and suggest that the ADAM33 gene is involved in the development of asthma through genetic polymorphisms.

Systemic lupus erythematosus (SLE) is a systemic autoimmune disease with a complex genetic basis that includes many susceptibility genes on multiple chromosomes. As complex human diseases like SLE involve multiple, interacting genetic and environmental determinants, identifying genes for complex traits is challenging and has had limited success so far. However, recent advances in genetic resources and technology have been providing new tools to identify the novel pathways or the sequence variants that contribute to autoimmune diseases. During the past several years, several new candidate genes have been implicated in development of SLE though association studies. In this article we describe an overview of the latest findings in the genetics of SLE, especially focusing on the genetic variations in the signalling or immunoregulatory molecules including CD28 and IRF family members.

Atopic dermatitis (AD) is a common inflammatory skin disease associated with the local infiltration of T helper type 2 (Th2) cells. The ST2 gene encodes both membrane-bound ST2L and soluble ST2 (sST2) proteins by alternative splicing. The orphan receptor ST2L is functionally indispensable for Th2 cells. We found a significant genetic association between AD and the -26999G/A single nucleotide polymorphism (SNP) (X 2 -test, raw P-value = 0.000007, odds ratio 1.86) in the distal promoter region of the ST2 gene (chromosome 2q12) in a study of 452 AD patients and 636 healthy controls. The -26999A allele common among AD patients positively regulates the transcriptional activity of the ST2 gene. In addition, having at least one -26999A allele correlated with high sST2 concentrations and high total IgE levels in the sera from AD patients. Thus, the - 26999A allele is correlated with an increased risk for AD. We also found that the -26999G/A SNP predominantly affected the transcriptional activity of hematopoietic cells. Immunohistochemical staining of a skin biopsy specimen from an AD patient in the acute stage showed ST2 staining in the keratinocytes as well as in the infiltrating cells in the dermal layer. Our data show that functional SNPs in the ST2 distal promoter region regulate ST2 expression which induces preferential activation of the Th2 response. Our findings will contribute to the evaluation of one of the genetic risk factors for AD.

The extracellular matrix glycoprotein tenascin-C (TNC) has been accepted as a valuable histopathological subepithelial marker for evaluating the severity of asthmatic disease and the therapeutic response to drugs. We found an association between an adult asthma and an SNP encoding TNC fibronectin type III-D (Fn-lll-D) domain in a case-control study between a Japanese population including 446 adult asthmatic patients and 658 normal healthy controls. The SNP (44513A/T in exon 17) strongly associates with adult bronchial asthma (X 2 test, P = 0.00019, Odds ratio = 1.76, 95% confidence interval = 1.31-2.36). This coding SNP induces an amino acid substitution (Leu1677lle) within the Fn-lll-D domain of the alternative splicing region. Computer-assisted protein structure modeling suggests that the substituted amino acid locates at the outer edge of the beta-sheet in Fn-III-D domain and causes instability of this beta-sheet. As the TNC fibronectin-lll domain has molecular elasticity, the structural change may affect the integrity and stiffness of asthmatic airways. In addition, TNC expression in lung fibroblasts increases with Th2 immune cytokine stimulation. Thus, Leu1677lle may be valuable marker for evaluating the risk for developing asthma and plays a role in its pathogenesis.

Summary Background A recent report provided evidence that a disintegrin and metalloprotease domain 33 (ADAM33), a member of the ADAM family, is a novel susceptibility gene in asthma linked to bronchial hyper-responsiveness. However, there has been no investigation of the genetic role of ADAM33 variants in nasal allergy. Objective The purpose of this study was to test the association between ADAM33 polymorphisms and Japanese cedar pollinosis (JCPsis), a most common seasonal allergic rhinitis in Japan. Methods We conducted a case–control association study among a Japanese population, involving 95 adult individuals with JCPsis and 95 normal healthy controls. A total of 22 single-nucleotide polymorphisms (SNPs) in ADAM33 were genotyped using PCR-based molecular methods. Results Six SNPs of ADAM33 gene, three in introns (7575G/A, 9073G/A and 12540C/T) and three in the coding region (10918G/C, 12433T/C and 12462C/T), were strongly associated with JCPsis (P=0.0002−0.022 for absolute allele frequencies) and most of the SNPs were in linkage disequilibrium with each other. A higher frequency of the common alleles of these SNPs was noted for the subjects with JCPsis in comparison with healthy controls. We also identified a haplotype associated with the disease susceptibility. In addition, associations were found between ADAM33 polymorphisms and various cedar pollinosis phenotypes including clinical severity, eosinophil counts in nasal secretion and allergen-specific IgE levels in sera, but not total serum IgE levels. Conclusion These results indicate that polymorphisms in the ADAM33 gene are associated with susceptibility to allergic rhinitis due to Japanese cedar pollen, but the functional relationship still needs clarification.

Interleukins IL-4 and IL-10 are considered to be central regulators for the limitation and eventual termination of inflammatory responses in vivo, based on their potent anti-inflammatory effects toward LPS-stimulated monocytes/macrophages and neutrophils. However, their role in T cell-dependent inflammatory responses has not been fully elucidated. In this study, we investigated the effects of both cytokines on the production of PGE2, a key molecule of various inflammatory conditions, in CD40-stimulated human peripheral blood monocytes. CD40 ligation of monocytes induced the synthesis of a significant amount of PGE2 via inducible expression of the cyclooxygenase (COX)-2 gene. Both IL-10 and IL-4 significantly inhibited PGE2 production and COX-2 expression in CD40-stimulated monocytes. Using specific inhibitors for extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), we found that both kinase pathways are involved in CD40-induced COX-2 expression. CD40 ligation also resulted in the activation of NF-κB. Additional experiments exhibited that CD40 clearly induced the activation of the upstream kinases MAPK/ERK kinase 1/2, MAPK kinase 3/6, and I-κB in monocytes. IL-10 significantly inhibited CD40-induced activation of the ERK, p38 MAPK, and NF-κB pathways; however, inhibition by IL-4 was limited to the ERK pathway in monocytes. Neither IL-10 nor IL-4 affected the recruitment of TNFR-associated factors 2 and 3 to CD40 in monocytes. Collectively, IL-10 and IL-4 use novel regulatory mechanisms for CD40-induced prostanoid synthesis in monocytes, thus suggesting a potential role for these cytokines in regulating T cell-induced inflammatory responses, including autoimmune diseases.

Lung epithelium plays a central role in modulation of the lung inflammatory response, and lung repair and airway epithelial cells are targets in asthma and viral infection. Activated T lymphocytes release cytokines such as interferon-gamma (IFN-gamma) that induce apoptosis, or programmed cell death, of damaged epithelial cells. Death-associated protein-3 (DAP3) is involved in mediating IFN-gamma-induced cell death. To assess the possible involvement of genetic variants of DAP3 with asthma, we searched for single-nucleotide polymorphisms (SNPs) in the gene and conducted a case-control study with 1,341 subjects. We found a strong association between bronchial asthma (BA) in adults (P=0.0051, odds ratio=1.87, 95% CI=1.20-2.92), whereas no association was found with childhood asthma. The tendency was more prominent in patients with higher serum total immunoglobulin E (IgE) (250 IU/ml) (P=0.00061, odds ratio=2.40, 95% CI=1.44-4.00). DAP3 was expressed in normal bronchial epithelial cells, and the expression was induced by IFN-gamma. These results indicated that specific variants of the DAP3 gene might be associated with the mechanisms responsible for adult BA and contribute to airway inflammation and remodeling.

Neutrophils are an important cellular source of proinflammatory mediators, whose regulation may be of potential benefit for the treatment of a number of inflammatory diseases. However, the mechanisms of lipopolysaccharide (LPS)-induced neutrophil activation and its regulation by anti-inflammatory cytokines have not yet been fully elucidated. Recent studies have revealed that mitogen-activated protein kinases (MAPK) play a crucial role in the generation of proinflammatory mediators in some cell types. Therefore, we conducted this study to determine whether MAPK activation could be involved in prostaglandin E(2) (PGE(2)) production and cyclooxygenase (COX)-2 expression in LPS-stimulated human neutrophils. PD98059 (MEK1 inhibitor) and SB203580 (p38(MAPK) inhibitor) reduced PGE(2) production as well as COX-2 expression in LPS-stimulated neutrophils. In addition, both extracellular signal-regulated protein kinase (ERK) and p38(MAPK) were phosphorylated and activated in time- and dose-dependent manners. Since we previously showed that IL-10 and IL-4 similarly inhibited COX-2 expression in LPS-stimulated neutrophils, we next tested the effects of IL-10 and IL-4 on the phosphorylation and activation of both kinases. IL-10 inhibited the phosphorylation and activation of p38(MAPK), but not ERK. In addition, IL-4 caused a marginal inhibition in the activation of p38(MAPK). Taken together, these results suggest that both ERK and p38(MAPK) pathways are involved in LPS-induced COX-2 expression and PGE(2) production in neutrophils, and IL-10 and IL-4 inhibit neutrophil prostanoid synthesis by down-regulating the activation of p38(MAPK).

Objective To clarify whether the interferon-gamma (IFN-gamma) gene (IFNG) is associated with the histological phenotype of lupus nephritis. Method We analysed microsatellite polymorphisms located within the first intron of the IFNG gene to determine the genotypes of patients with lupus nephritis WHO class IV (n=24), patients with WHO class V (n=12) and healthy controls (n=61). We used flow cytometric detection of intracellular cytokines to identify CD4(+) T cells producing IFN-gamma. Production of IFN-gamma by peripheral blood mononuclear cells after stimulation with phytohaemagglutinin was evaluated with an enzyme-linked immunosorbent assay. Result The frequency of the IFNG allele 114 was significantly greater in WHO class V patients than in WHO class IV patients. Furthermore, the IFNG 114 +/+ genotype was more frequent in WHO class V than in WHO class IV patients. The level of IFN-gamma and the percentage of IFN-gamma-producing CD4(+) T cells were lower in individuals with genotype 114 +/+ than in individuals with genotype 114 -/-. Conclusion The IFN-gamma gene is associated with the histological phenotype in lupus nephritis.

Background DNAM-1 (CD226) is well known as a genetic risk factor of many autoimmune diseases such as systemic sclerosis (SSc). More recently, Avouac et al. have reported that mice deficient for DNAM-1 are protected from bleomycin-induced dermal fibrosis. In human, however, the roles of DNAM-1 in SSc are still unknown. Objectives To evaluate cell surface expression of DNAM-1 on peripheral blood lymphocytes from SSc patients and to identify the roles of DNAM-1 in the pathogenesis of SSc. Methods The expression of DNAM-1 was analyzed by flow cytometry (FCM) on surface of CD4+ T cells, CD8+ T cells and CD19+ B cells from 41 SSc patients (median age, 59 years; female, 37) and 23 healthy controls (HCs). We evaluated the relationship between DNAM-1 expression level on CD8+ T cells and clinical manifestations of SSc. Intracellular and extracellular cytokine production in CD8+ T cells with or without DNAM-1 was measured by FCM (intracellular staining and multiplex bead array, respectively) after stimulation with PMA/Ionomycin. The cytotoxic capacity of CD8+ T cells against human umbilical vein endothelial cells (HUVECs) was assessed in the presence or absence of anti-DNAM-1 neutralizing antibody in SSc patients and HCs. Results The proportion of DNAM-1 high CD8+ T cells was increased in SSc patients (median, 52.5%) compared to HCs (29.7%, P=0.0002). We found no significant difference between SSc patients and HCs in CD4+ T cells and CD19+ B cells. The expression of DNAM-1 on CD8+ T cells was higher in patients with diffuse cutaneous subtype than those with limited cutaneous subtype (P=0.0009) and was higher in those with interstitial pneumonia than those without (P=0.0016). The percentage of DNAM-1 high CD8+ T cells was significantly correlated with modified Rodnan skin thickness score (r =0.4295, P=0.0071). The production of IL-13 was significantly increased in DNAM-1 positive CD8+ T cells compared with DNAM-1 negative ones and the neutralization of DNAM-1 impaired the IL-13 production from CD8+ T cells. The cytotoxic ability of CD8+ T cells was significantly elevated in SSc patients and positively correlated with the percentage of DNAM-1 high CD8+ T cells. The cytotoxicity against HUVECs was partially inhibited in the presence of anti-DNAM-1 neutralizing antibody. Conclusions These findings indicate that DNAM-1 high CD8+ T cells are involved in the pathogenesis of SSc via the production of profibrotic IL-13 and endothelial cell injury. References Avouac J, Elhai M, Tomcik M, et al. Critical role of the adhesion receptor DNAX accessory molecule-1 (DNAM-1) in the development of inflammation-driven dermal fibrosis in a mouse model of systemic sclerosis. Ann Rheum Dis. 2013;72:1089-98. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3217

Background Receptor-interacting protein (RIP)-2 is a serine/threonine kinase containing a caspase recruitment domain (CARD) that is involved in the Toll-like receptor-signaling pathway. Although associations between endotoxin exposure or respiratory infection and asthma have been recognized, the genetic influences in these conditions are unclear. The aim of our study was to examine whether polymorphisms or haplotypes in RIP2 were associated with childhood atopic asthma in a Japanese population. Methods We screened the RIP2 gene for polymorphisms by direct sequencing and characterized the linkage disequilibrium (LD) mapping of the gene. Seven variants were genotyped in childhood atopic asthma ( n = 300) and normal controls ( n = 637). We conducted case-control and case-only association studies between the variants and asthma-related phenotypes. Haplotype association analyses were also performed. Results A total of 31 variants were identified and none of the alleles or haplotypes of RIP2 were associated with asthma susceptibility. In the case-only study, an association between an RIP2 promoter polymorphism and childhood severe asthma (P=0.0032;odds ratio (OR) 3.37, 95% confidence interval (CI) 1.45-7.87) was observed. Conclusions Although polymorphisms in RIP2 are not likely to be associated with the development of asthma, the genetic variants might contribute to asthma severity in the Japanese population.

Background The efficacy of B cell-depleting therapies for rheumatoid arthritis underscores antibody-independent functions of effector B cells such as cognate T–B interactions and production of pro-inflammatory cytokines. Receptor activator of nuclear factor κB ligand (RANKL) is a key cytokine involved in bone destruction and is highly expressed in synovial fluid B cells in patients with rheumatoid arthritis. In this study we sought to clarify the generation mechanism of RANKL+ effector B cells and their impacts on osteoclast differentiation. Methods Peripheral blood and synovial fluid B cells from healthy controls and patients with rheumatoid arthritis were isolated using cell sorter. mRNA expression of RANKL, osteoprotegerin, tumor necrosis factor (TNF)-α, and Blimp-1 was analyzed by quantitative real-time polymerase chain reaction. Levels of RANKL, CD80, CD86, and CXCR3 were analyzed using flow cytometry. Functional analysis of osteoclastogenesis was carried out in the co-culture system using macrophage RAW264 reporter cells. Results RANKL expression was accentuated in CD80+CD86+ B cells, a highly activated B-cell subset more abundantly observed in patients with rheumatoid arthritis. Upon activation via B-cell receptor and CD40, switched-memory B cells predominantly expressed RANKL, which was further augmented by interferon-γ (IFN-γ) but suppressed by interleukin-21. Strikingly, IFN-γ also enhanced TNF-α expression, while it strongly suppressed osteoprotegerin expression in B cells. IFN-γ increased the generation of CXCR3+RANKL+ effector B cells, mimicking the synovial B cell phenotype in patients with rheumatoid arthritis. Finally, RANKL+ effector B cells in concert with TNF-α facilitated osteoclast differentiation in vitro. Conclusions Our current findings have shed light on the generation mechanism of pathogenic RANKL+ effector B cells that would be an ideal therapeutic target for rheumatoid arthritis in the future. Electronic supplementary material The online version of this article (doi:10.1186/s13075-016-0957-6) contains supplementary material, which is available to authorized users.