Eiji Muroi

Clinical Correlations With Dermatomyositis-Specific Autoantibodies in Adult Japanese Patients With Dermatomyositis: A Multicenter Cross-sectional Study

OBJECTIVE To clarify the association of clinical and prognostic features with dermatomyositis (DM)-specific autoantibodies (Abs) in adult Japanese patients with DM. DESIGN Retrospective study. SETTING Kanazawa University Graduate School of Medical Science Department of Dermatology and collaborating medical centers. Patients A total of 376 consecutive adult Japanese patients with DM who visited our hospital or collaborating medical centers between 2003 and 2008. MAIN OUTCOME MEASURES Clinical and laboratory characteristics of adult Japanese patients with DM and DM-specific Abs that include Abs against Mi-2, 155/140, and CADM-140. RESULTS In patients with DM, anti-Mi-2, anti-155/140, and anti-CADM-140 were detected in 9 (2%), 25 (7%), and 43 (11%), respectively. These DM-specific Abs were mutually exclusive and were detected in none of 34 patients with polymyositis, 326 with systemic sclerosis, and 97 with systemic lupus erythematosus. Anti-Mi-2 was associated with classical DM without interstitial lung disease or malignancy, whereas anti-155/140 was associated with malignancy. Patients with anti-CADM-140 frequently had clinically amyopathic DM and rapidly progressive interstitial lung disease. Cumulative survival rates were more favorable in patients with anti-Mi-2 compared with those with anti-155/140 or anti-CADM-140 (P < .01 for both comparisons). Nearly all deaths occurred within 1 year after diagnosis in patients with anti-CADM-140. Conclusion Dermatomyositis-specific Abs define clinically distinct subsets and are useful for predicting clinical outcomes in patients with DM.

Common and Distinct Clinical Features in Adult Patients with Anti-Aminoacyl-tRNA Synthetase Antibodies: Heterogeneity within the Syndrome

Objective To identify similarities and differences in the clinical features of adult Japanese patients with individual anti-aminoacyl-tRNA synthetase antibodies (anti-ARS Abs). Methods This was a retrospective analysis of 166 adult Japanese patients with anti-ARS Abs detected by immunoprecipitation assays. These patients had visited Kanazawa University Hospital or collaborating medical centers from 2003 to 2009. Results Anti-ARS Ab specificity included anti-Jo-1 (36%), anti-EJ (23%), anti-PL-7 (18%), anti-PL-12 (11%), anti-KS (8%), and anti-OJ (5%). These anti-ARS Abs were mutually exclusive, except for one serum Ab that had both anti-PL-7 and PL-12 reactivity. Myositis was closely associated with anti-Jo-1, anti-EJ, and anti-PL-7, while interstitial lung disease (ILD) was correlated with all 6 anti-ARS Abs. Dermatomyositis (DM)-specific skin manifestations (heliotrope rash and Gottron’s sign) were frequently observed in patients with anti-Jo-1, anti-EJ, anti-PL-7, and anti-PL-12. Therefore, most clinical diagnoses were polymyositis or DM for anti-Jo-1, anti-EJ, and anti-PL-7; clinically amyopathic DM or ILD for anti-PL-12; and ILD for anti-KS and anti-OJ. Patients with anti-Jo-1, anti-EJ, and anti-PL-7 developed myositis later if they had ILD alone at the time of disease onset, and most patients with anti-ARS Abs eventually developed ILD if they did not have ILD at disease onset. Conclusion Patients with anti-ARS Abs are relatively homogeneous. However, the distribution and timing of myositis, ILD, and rashes differ among patients with individual anti-ARS Abs. Thus, identification of individual anti-ARS Abs is beneficial to define this rather homogeneous subset and to predict clinical outcomes within the “anti-synthetase syndrome.”

Correlation of IgE Autoantibody to BP180 With a Severe Form of Bullous Pemphigoid

OBJECTIVE To determine the prevalence, immunoglobulin subclass distribution, and clinical correlation of antibodies (Abs), especially of IgE Abs, to BP180 and BP230 in patients with bullous pemphigoid (BP). DESIGN Retrospective case series analysis. SETTING Department of Dermatology, Nagasaki University Graduate School of Biomedical Science. PATIENTS Serum samples from 37 patients with BP, 6 with pemphigus vulgaris, 5 with pemphigus foliaceus, and 26 healthy controls (n = 26) were examined by enzyme-linked immunosorbent assay. MAIN OUTCOME MEASURES Prevalence, immunoglobulin subclass distribution, and clinical correlation of Abs, especially of IgE Abs, to BP180 and BP230. RESULTS IgG anti-BP180 and anti-BP230 Abs were detected in 35 (95%) and 26 (70%) of the 37 BP serum samples, respectively. IgG1 and IgG4 isotypes were positive in 32 (87%) and 25 (68%), respectively, of the BP serum samples for anti-BP180 Abs, while they were detected in 16 (44%) and 26 (70%), respectively, for anti-BP230 Abs. IgE anti-BP180 and anti-BP230 Abs were equally detected in 8 (22%) of the BP serum samples. Similar to IgG anti-BP180 Abs, the presence or levels of IgE anti-BP180 Abs was associated with broader skin lesions. Furthermore, patients with BP positive for IgE anti-BP180 Abs required longer duration for remission, higher dosage of prednisolone, and more intensive therapies for remission. By contrast, this was not true for those with of IgE anti-BP230 Abs. Remarkably, when analyzed in patients with BP who had a high titer of IgG anti-BP180 Abs, the presence or levels of IgE anti-BP180 Abs, but not IgG anti-BP180 Abs, were associated with a more severe form. CONCLUSIONS The present study suggests that IgE anti-BP180 Abs are related to the disease severity and activity of BP. Moreover, it may be possible to identify treatment-refractory patients with BP more specifically by assessing the presence or levels of IgE anti-BP180 Abs in those with a high IgG anti-BP180 Ab titer.

Cell Adhesion Molecules Regulate Fibrotic Process via Th1/Th2/Th17 Cell Balance in a Bleomycin-Induced Scleroderma Model

Mice s.c. injected with bleomycin, an experimental model for human systemic sclerosis, develop skin and lung fibrosis, which is mediated by inflammatory cell infiltration. This process is highly regulated by multiple adhesion molecules and does not require Ag sensitization. To assess the role of adhesion molecules in this pathogenetic process, bleomycin-induced fibrosis was examined in mice lacking adhesion molecules. L-selectin and/or ICAM-1 deficiency inhibited skin and lung fibrosis with decreased Th2 and Th17 cytokines and increased Th1 cytokines. In contrast, P-selectin deficiency, E-selectin deficiency with or without P-selectin blockade, or P-selectin glycoprotein ligand 1 (PSGL-1) deficiency augmented the fibrosis in parallel with increased Th2 and Th17 cytokines and decreased Th1 cytokines. Furthermore, loss of L-selectin and/or ICAM-1 reduced Th2 and Th17 cell numbers in bronchoalveolar lavage fluid, whereas loss of P-selectin, E-selectin, or PSGL-1 reduced Th1 cell numbers. Moreover, Th1 cells exhibited higher PSGL-1 expression and lower expression of LFA-1, a ligand for ICAM-1, whereas Th2 and Th17 cells showed higher LFA-1 and lower PSGL-1 expression. This study suggests that L-selectin and ICAM-1 regulate Th2 and Th17 cell accumulation into the skin and lung, leading to the development of fibrosis, and that P-selectin, E-selectin, and PSGL-1 regulate Th1 cell infiltration, resulting in the inhibition of fibrosis.

Clinical Significance of Serum HMGB-1 and sRAGE Levels in Systemic Sclerosis: Association with Disease Severity

IntroductionThe high mobility group box 1 protein (HMGB-1)/advanced glycation end products (RAGE) system is recently shown to play an important part in immune/inflammatory disorders. However, the association of this system in systemic sclerosis (SSc) remains unknown.Materials and MethodsTo determine clinical association of serum levels of HMGB-1 and soluble RAGE (sRAGE) in patients with SSc, sera from 70 patients with SSc and 25 healthy controls were examined by enzyme-linked immunosorbent assay. Sera from tight-skin mice and bleomycin-induced scleroderma mice, animal models for SSc, were also examined. Skin HMGB-1 and RAGE expression was assessed by immunohistochemistry.Results and DiscussionSerum HMGB-1 and sRAGE levels in SSc were higher than those in controls. Similarly, HMGB-1 and sRAGE levels in animal SSc models were higher than those in control mice. SSc patients with elevated HMGB-1 and sRAGE levels had more frequent involvement of several organs and immunological abnormalities compared to those with normal levels. Furthermore, HMGB-1 and sRAGE levels correlated positively with modified Rodnan total skin thickness score and negatively with pulmonary function test.ConclusionsHMGB-1 and sRAGE expression in the sclerotic skin was more intense than normal skin. These results suggest that elevated serum HMGB-1 and sRAGE levels are associated with the disease severity and immunological abnormalities in SSc.

Platelets control leukocyte recruitment in a murine model of cutaneous arthus reaction.

Platelets have been shown to be important in inflammation, but their role in the cutaneous Arthus reaction remains unclear. To assess the role of platelets in this pathogenetic process, the cutaneous Arthus reaction was examined in wild-type mice and mice lacking E-selectin, P-selectin, or P-selectin glycoprotein ligand-1 (PSGL-1) with or without platelet depletion by busulfan, a bone marrow precursor cell-specific toxin. Edema and hemorrhage induced by immune complex challenge significantly decreased in busulfan-treated wild-type mice compared with untreated mice. Busulfan treatment did not affect edema and hemorrhage in P-selectin- or PSGL-1-deficient mice, suggesting that the effect by busulfan is dependent on P-selectin and PSGL-1 expression. The inhibited edema and hemorrhage paralleled reduced infiltration of neutrophils and mast cells and reduced levels of circulating platelets. Increased cutaneous production of interleukin-6, tumor necrosis factor-alpha, and platelet-derived chemokines during Arthus reaction was inhibited in busulfan-treated wild-type mice relative to untreated mice, which paralleled the reduction in cutaneous inflammation. Flow cytometric analysis showed that immune complex challenge generated blood platelet-leukocyte aggregates that decreased by busulfan treatment. In thrombocytopenic mice, the cutaneous inflammation after immune complex challenge was restored by platelet infusion. These results suggest that platelets induce leukocyte recruitment into skin by forming platelet-leukocyte aggregates and secreting chemokines at inflamed sites, mainly through the interaction of P-selectin on platelets with PSGL-1 on leukocytes.

CD19, a Response Regulator of B Lymphocytes, Regulates Wound Healing through Hyaluronan-Induced TLR4 Signaling

Immune cells are critical to the wound-healing process, through both cytokine and growth factor secretion. Although previous studies have revealed that B cells are present within wound tissue, little is known about the role of B cells in wound healing. To clarify this, we investigated cutaneous wound healing in mice either lacking or overexpressing CD19, a critical positive-response regulator of B cells. CD19 deficiency inhibited wound healing, infiltration of neutrophils and macrophages, and cytokine expression, including basic and acidic fibroblast growth factor, interleukin-6, platelet-derived growth factor, and transforming growth factor-beta. By contrast, CD19 overexpression enhanced wound healing and cytokine expression. Hyaluronan (HA), an endogenous ligand for toll-like receptor (TLR)-4, stimulated B cells, which infiltrates into wounds to produce interleukin-6 and transforming growth factor-beta through TLR4 in a CD19-dependent manner. CD19 expression regulated TLR4 signaling through p38 activation. HA accumulation was increased in injured skin tissue relative to normal skin, and exogenous application of HA promoted wound repair in wild-type but not CD19-deficient mice, suggesting that the beneficial effects of HA to the wound-healing process are CD19-dependent. Collectively, these results suggest that increased HA accumulation in injured skin induces cytokine production by stimulating B cells through TLR4 in a CD19-dependent manner. Thus, this study is the first to reveal a critical role of B cells and novel mechanisms in wound healing.

Elevated serum interleukin-27 levels in patients with systemic sclerosis: association with T cell, B cell and fibroblast activation

Objective To determine serum levels of interleukin 27 (IL-27) in patients with systemic sclerosis (SSc) and relate the results to the clinical features of SSc. Methods Serum levels of IL-27 in 91 patients with SSc and the production of IL-27 by isolated monocytes were examined by ELISA. The expression of IL-27 receptor in the skin fibroblasts, B cells and T cells was quantified by real-time PCR. The effect of IL-27 on immunoglobulin G (IgG) production of B cells, IL-17 production of CD4 T cells and proliferation and collagen synthesis of fibroblasts was also analysed. Results Serum IL-27 levels were raised in patients with SSc compared with healthy controls and correlated positively with the extent of skin and pulmonary fibrosis and immunological abnormalities. IL-27 levels also correlated positively with serum levels of hyaluronan, recently identified as an endogenous ligand for Toll-like receptors. The retrospective longitudinal analysis showed a tendency for serum IL-27 levels to be attenuated during the follow-up period. IL-27 production by cultured monocytes was increased by hyaluronan stimulation. IL-27 receptor expression was upregulated in the affected skin fibroblasts, B cells and CD4 T cells of patients with SSc. Moreover, IL-27 stimulation increased IgG production of B cells, IL-17 production of CD4 T cells and proliferation and collagen synthesis of fibroblasts in patients with SSc compared with those in healthy controls. Conclusion These results suggest that IL-27 and its signalling in B cells, T cells and fibroblasts contributes to disease development in patients with SSc.

Increased serum pentraxin 3 in patients with systemic sclerosis.

Objective. To determine serum concentrations of pentraxin 3 (PTX3) and its clinical associations in patients with systemic sclerosis (SSc). Methods. Serum PTX3 levels from 45 patients with diffuse cutaneous SSc (dSSc), 46 with limited cutaneous SSc (lSSc), and 20 healthy controls were examined by ELISA. PTX3 expression in the sclerotic skin from SSc patients was evaluated immunohistochemically. Normal and SSc fibroblasts were cultured and PTX3 levels in the culture medium were also examined by ELISA. Results. Serum PTX3 levels were elevated in patients with SSc relative to controls. PTX3 levels in dSSc patients were significantly higher than in controls and lSSc patients. PTX3 expression in the sclerotic skin from SSc patients was more intense relative to normal skin. Elevation of serum PTX3 levels was associated with more frequent presence of pulmonary fibrosis, cardiac disease, and pitting scar/ulcer and increased serum immunoglobulin levels and erythrocyte sedimentation rates. PTX3 levels correlated positively with modified Rodnan total skin thickness score, and negatively with percentage vital capacity and percentage DLCO in patients with SSc. PTX3 levels also correlated positively with serum levels of 8-isoprostane, a marker of oxidative stress, and hyaluronan, recently identified as an endogenous ligand for Toll-like receptors. PTX3 production from cultured SSc fibroblasts was increased by stimulation with hyaluronan. Conclusion These results suggest that elevated serum PTX3 levels are associated with the disease severity of SSc.

Calreticulin Represses E-cadherin Gene Expression in Madin-Darby Canine Kidney Cells via Slug

Calreticulin (CRT) is a multifunctional Ca2+-binding molecular chaperone in the endoplasmic reticulum. In mammals, the expression level of CRT differs markedly in a variety of organs and tissues, suggesting that CRT plays a specific role in each cell type. In the present study, we focused on CRT functions in the kidney, where overall expression of CRT is quite low, and established CRT-overexpressing kidney epithelial cell-derived Madin-Darby canine kidney cells by gene transfection. We demonstrated that, in CRT-overexpressing cells, the morphology was apparently changed, and the original polarized epithelial cell phenotype was destroyed. Furthermore, CRT-overexpressing cells showed enhanced migration through Matrigel®-coated Boyden chamber wells, compared with controls. E-cadherin expression was significantly suppressed at the protein and transcriptional levels in CRT-overexpressing cells compared with controls. On the other hand, the expression of mesenchymal protein markers, such as N-cadherin and fibronectin, was up-regulated. We also found that the expression of Slug, a repressor of the E-cadherin promoter, was up-regulated by overexpression of CRT through altered Ca2+ homeostasis, and this led to enhanced binding of Slug to the E-box element in the E-cadherin promoter. Thus, we conclude that CRT regulates the epithelial-mesenchymal transition-like change of cellular phenotype by modulating the Slug/E-cadherin pathway through altered Ca2+ homeostasis in cells, suggesting a novel function of CRT in cell-cell interaction of epithelial cells.