Tomoko Sugiura

Increased CD40 Expression on Muscle Cells of Polymyositis and Dermatomyositis: Role of CD40-CD40 Ligand Interaction in IL-6, IL-8, IL-15, and Monocyte Chemoattractant Protein-1 Production

In polymyositis (PM)/dermatomyositis (DM), T cells infiltrate the muscle tissues and interact with muscle cells via cell surface molecules. Recently, myoblasts have been reported to express CD40, but little is known about the role of CD40 in myoblasts. In the present study we examined the expression and involvement of CD40 and CD40 ligand (CD40L) in the interaction between muscle cells and T cells in PM/DM. Immunohistochemical staining revealed that CD40 was expressed on muscle cells in five of five PM and four of five DM patients, and that infiltrating mononuclear cells (MNCs) expressed CD40L in all cases of PM/DM. These CD40L-expressing MNCs were primarily CD4+ T cells. IFN-γ, which is known to induce CD40 expression on various types of cells, was also expressed on the MNCs in four of the PM and four of the DM patients. Although cultured human myoblasts (SkMC 2859) did not express CD40 constitutively, IFN-γ induced CD40 expression in a dose-dependent manner. To clarify the functional roles of CD40-mediated signals, the effects of a trimeric form of recombinant human CD40L on cytokine production were studied in SkMC 2859 that were prestimulated with IFN-γ to express CD40. Recombinant human CD40L markedly increased the production of IL-6, IL-8, IL-15, and monocyte chemoattractant protein-1 of SkMC 2859. The expression of these humoral factors in muscle cells of PM and DM was demonstrated by immunohistochemistry. These results suggest that interaction between T cells and muscle cells via the CD40-CD40L system contributes to the immunopathogenesis of PM/DM by augmenting inflammation via cytokine production by the muscle cells.

NOS2 polymorphisms associated with the susceptibility to pulmonary arterial hypertension with systemic sclerosis: contribution to the transcriptional activity.

Systemic sclerosis (SSc) is a connective tissue disease characterized by tissue fibrosis. One of several complications of SSc, pulmonary arterial hypertension (PAH) can be refractory to treatment, both novel and established. In the present study we investigated the ratio of circulating nitric oxide to endothelin-1 in patients with both SSc and PAH, and determined whether polymorphisms in NOS2 (the nitric oxide synthase 2 gene) are associated with susceptibility to PAH. Endothelin-1 in plasma and nitric oxide metabolites (nitrate and nitrite) in serum were measured. The nitric oxide/endothelin-1 ratio was significantly lower in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. We confirmed the presence of two single nucleotide polymorphisms at positions -1,026 and -277 and a pentanucleotide repeat (CCTTT) at -2.5 kilobases. There were significant differences in single nucleotide polymorphisms between patients with SSc who had PAH and those who did not, and between patients with both SSc and PAH and healthy control individuals. The CCTTT repeat was significantly shorter in patients with both SSc and PAH than in patients with SSc only or in healthy control individuals. Transcriptional activity were analyzed using the luciferase reporter assay. The transcriptional activity of NOS2 was much greater in fibroblasts transfected by a vector with a long allele of the CCTTT repeat than in those transfected by a vector with a short allele. Polymorphisms in the NOS2 gene are associated with transcriptional activity of the NOS2 gene and with susceptibility to SSc-related PAH.

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-γ and anti-double-stranded DNA antibody production

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4+CD45RO+ T cells from peripheral blood, the percentage of ICOS+ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [3H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-γ, IL-4 and IL-10 in both SLE and normal T cells. IFN-γ in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.

A promoter haplotype of the interleukin-18 gene is associated with juvenile idiopathic arthritis in the Japanese population.

Recently, we reported that genetic polymorphisms within the human IL18 gene were associated with disease susceptibility to adult-onset Stills disease (AOSD), which is characterized by extraordinarily high serum levels of IL-18. Because high serum IL-18 induction has also been observed in the systemic type of juvenile idiopathic arthritis (JIA), we investigated whether similar genetic skewing is present in this disease. Three haplotypes, S01, S02, and S03, composed of 13 genetic polymorphisms covering two distinct promoter regions, were determined for 33 JIA patients, including 17 with systemic JIA, 10 with polyarthritis, and 6 with oligoarthritis. Haplotypes were also analyzed for 28 AOSD patients, 164 rheumatoid arthritis (RA) patients, 102 patients with collagen diseases, and 173 healthy control subjects. The frequency of individuals carrying a diplotype configuration (a combination of two haplotypes) of S01/S01 was significantly higher in the JIA patients, including all subgroups, than in the healthy controls (P = 0.0045, Fischer exact probability test; odds ratio (OR) = 3.55, 95% confidence interval (CI) = 1.55–8.14). In patients with systemic JIA, its frequency did not differ statistically from that of normal controls. Nevertheless, it is possible that haplotype S01 is associated with the phenotype of high IL-18 production in systemic JIA because the patients carrying S01/S01 showed significantly higher serum IL-18 levels compared with patients with other diplotype configurations (P = 0.017, Mann-Whitney U test). We confirmed that the frequency of the diplotype configuration of S01/S01 was significantly higher in AOSD patients than in healthy control subjects (P = 0.011, OR = 3.45, 95% CI = 1.42–8.36). Furthermore, the RA patients were also more predisposed to have S01/S01 (P = 0.018, OR = 2.00, 95% CI = 1.14–3.50) than the healthy control subjects, whereas the patients with collagen diseases did not. In summary, the diplotype configuration of S01/S01 was associated with susceptibility to JIA as well as AOSD and RA, and linked to significantly higher IL-18 production in systemic JIA. Possession of the diplotype configuration of S01/S01 would be one of the genetic risk factors for susceptibility to arthritis in the Japanese population.

Increased CD40 expression in skin fibroblasts from patients with systemic sclerosis (SSc): role of CD40‐CD154 in the phenotype of SSc fibroblasts

Systemic sclerosis (SSc) is a connective tissue disease of unknown etiology that is characterized by tissue fibrosis, which may result from the activation of lesional fibroblasts exhibiting excessive production of extracellular matrix components. However, it has yet to be determined how SSc fibroblasts are activated. CD40 is a cell surface molecule expressed on various cells that is important for the response to activated T cells through CD154. CD40 mRNA was found to be constitutively expressed in both SSc and normal fibroblasts by reverse transcription PCR. Expression of CD40 protein was increased on SSc fibroblasts compared to normal fibroblasts as measured by flow cytometry. Ligation of CD40 by recombinant human CD154 (0.5–2 μg/ml) resulted in increased production of IL‐6, IL‐8, and monocyte chemoattractant protein‐1 in SSc fibroblasts in a dose‐dependent manner, whereas these phenomena were not shown in normal fibroblasts even with the addition of CD154. CD80, a costimulatory molecule, was also induced on SSc fibroblasts by CD40 ligation. In the present study, our findings suggest the possibility of a cell‐mediated response between fibroblasts and T cells in the lesional skin of SSc patients. Since it is suggested that the CD40‐CD154 system may play a crucial role in the aberrant production of immune mediators by SSc fibroblasts, blockage of CD40‐CD154 may be a novel therapeutic strategy for SSc.

Clinical Manifestations of Adult‐Onset Still's Disease Presenting With Erosive Arthritis: Association With Low Levels of Ferritin and Interleukin‐18

Adult‐onset Stills disease (AOSD) is a clinical entity with a heterogeneous etiology. We have encountered patients with AOSD who had severe polyarthritis and who fulfilled the classification criteria for rheumatoid arthritis (RA); however, most patients with AOSD typically exhibit mild arthritis. In this study, we proposed 2 clinical subsets of AOSD and investigated the clinically significant characteristics of the 2 subtypes.

Association study of a polymorphism of the CTGF gene and susceptibility to systemic sclerosis in the Japanese population

Objectives: To validate the association of a single nucleotide polymorphism (SNP) of the connective tissue growth factor gene (CTGF) with susceptibility to systemic sclerosis (SSc) in the Japanese population. Methods: 395 Japanese patients with SSc, 115 patients with rheumatoid arthritis and 269 healthy Japanese volunteers were enrolled in the study. An SNP (rs6918698) at –945 bp from the start codon in the promoter region of the CTGF gene was determined by allelic discrimination with the use of a specific TaqMan probe. Results: The G allele showed a significantly higher frequency in patients with SSc than in controls (p<0.001; odds ratio 1.5; 95% confidence interval 1.2 to 1.9). In particular, the clinical subsets of SSc showed a more significant association between the G allele and diffuse cutaneous SSc (p<0.001) and the presence of interstitial lung disease (p<0.001), the presence of anti-topoisomerase I antibody (p<0.001) and anti-U1RNP antibody (p = 0.010). Association analyses using the genotype of the SNP yielded results similar to those of analyses using the allele. Conclusions: This study confirms the association between an SNP in the CTGF gene and susceptibility to SSc, especially in the presence of diffuse cutaneous SSc, interstitial lung disease and anti-topoisomerase I antibody. The results strongly suggest that this SNP may be a powerful indicator of severe skin and lung involvement in patients with SSc.

Brief Report: Association of HLA–DRB1*0101/*0405 with susceptibility to anti–melanoma differentiation–associated gene 5 antibody–positive dermatomyositis in the Japanese population

OBJECTIVE The complication of interstitial lung disease (ILD) in polymyositis/dermatomyositis (PM/DM) is associated with anti-aminoacyl-transfer RNA synthetase (anti-aaRS) antibody or anti-melanoma differentiation-associated gene 5 (anti-MDA-5) antibody positivity. Anti-MDA-5 antibody is associated with clinically amyopathic DM and fatal outcome due to rapidly progressive ILD in Asian populations. The association between genetic factors and anti-MDA-5 antibody-positive DM is unclear. This study was undertaken to investigate the HLA-DRB1 genotype in patients with anti-MDA-5 antibody-positive DM. METHODS We examined genetic differences among 17 patients with anti-MDA-5 antibody-positive DM, 33 patients with anti-aaRS antibody-positive PM/DM, 33 patients with PM/DM without anti-aaRS antibody or ILD, and 265 healthy controls. RESULTS The frequencies of HLA-DRB1*0101 and DRB1*0405 were 29% and 71%, respectively, in patients with anti-MDA-5 antibody-positive DM, which were higher than the frequencies in healthy controls (10% and 25%, respectively). Among the 17 patients with anti-MDA-5 antibody-positive DM, 16 (94%) harbored either the DRB1*0101 or DRB1*0405 allele. The combined frequency of the DRB1*0101 allele and the DRB1*0405 allele was significantly higher in patients with anti-MDA-5 antibody-positive DM than in patients with PM/DM without anti-aaRS antibody or ILD, with an odds ratio (OR) of 42.7 (95% confidence interval [95% CI] 4.9-370.2) (P = 1.1 × 10(-5)), or in patients with anti-aaRS antibody-positive PM/DM (OR 13.3 [95% CI 1.6-112.6], P = 4.5 × 10(-3)). CONCLUSION Our findings indicate that HLA-DRB1*0101/*0405 is associated with susceptibility to anti-MDA-5 antibody-positive DM in the Japanese population.

Contribution of single nucleotide polymorphisms of the IL1A gene to the cleavage of precursor IL-1α and its transcription activity

We previously demonstrated the association of IL1A gene single nucleotide polymorphisms (SNPs) with susceptibility to systemic sclerosis (SSc) patients. In this study, we explored the effects of SNP on the transcriptional activity and processing of the precursor IL-1α (pre-IL-1α) in skin fibroblasts. Two kinds of promoter regions of the IL1A gene were prepared including C or T at −889, referred to C/IL1A and T/IL1A, and inserted into a luciferase reporter vector (pGL3). Skin fibroblasts were explanted from two SSc patients whose genomic DNA contained GG and TT genotypes at +4845 of the IL1A gene, respectively. Cell lysates were collected and reacted with various concentrations of calpain, and then the processing of pre-IL-1α was analyzed by Western blotting using monoclonal anti-IL-1α antibody. A SNP was determined by the allelic discrimination method using fluorescence-labeled Taq-Man probes. Significant differences in the luciferase activities were not detected between pGL3 (C/IL1A) and pGL3 (T/IL1A) in SSc fibroblasts. Calpain required a 100-fold higher concentration to process the pre-IL-1α containing Ala at the 114th amino acid than that to do containing Ser. The frequency of the GG genotype was significantly higher in SSc patients than that in healthy donors, whereas the frequency of TT genotype was significantly higher in RA patients than that in healthy donors. Our observation showed that the SNP at +4845 affected the enzymatic efficiency of the protease in cleaving pre-IL-1α. Pre-IL-1α with Ala, which was high in frequency in SSc patients, was more resistant to be cleaved by proteases in human sera than pro-IL-1α with Ser.

Interleukin-18 is a key mediator in dermatomyositis: potential contribution to development of interstitial lung disease

OBJECTIVE To determine whether IL-18 is involved in the inflammation of DM and PM. METHODS Thirty-three patients with DM were enrolled in this study, including 25 with interstitial lung disease (ILD). In addition, 16 patients with PM were enrolled, including 6 with ILD. All patients were admitted to our hospital as a result of their condition requiring treatment, and clinical laboratory data including serum IL-18 were recorded on admission. RESULTS Serum IL-18 was significantly (P < 0.0001) higher in both DM and PM patients than in healthy controls (n = 30). Serum ferritin and IL-18 were significantly (P = 0.003 and 0.0044, respectively) higher in DM than in PM patients. Additionally, ferritin and IL-18 were significantly (P = 0.023 and 0.034, respectively) higher in DM patients with ILD than in DM patients without ILD. Significant positive correlations were found between creatine kinase (CK) and ferritin (r(s) = 0.39, P = 0.024); CK and IL-18 (r(s) = 0.48, P = 0.005); and IL-18 and ferritin (r(s) = 0.54, P = 0.0012) in the DM group as a whole. These findings were different for the DM plus ILD subgroup: significant positive correlations were found between CK and ferritin (r(s) = 0.40, P = 0.047); CK and IL-18 (r(s) = 0.63, P = 0.0008); and IL-18 and ferritin (r(s) = 0.41, P = 0.042). CONCLUSION Serum IL-18 was strikingly elevated in patients with DM and was associated particularly with disease activity and ILD complication in DM.