Norihito Yazawa

Characterization of autoantibodies to endothelial cells in systemic sclerosis (SSc): association with pulmonary fibrosis

To determine the prevalence and the characterization of antibodies to endothelial cells in patients with SSc, serum samples from 80 patients with SSc, 20 patients with systemic lupus erythematosus (SLE), and 20 healthy control subjects were examined by ELISA using cultured human umbilical vein endothelial cells (HUVEC), indirect immunofluorescence analysis (IIF), and immunoblotting using cytoplasmic extract of HUVEC. IgG and/or IgM isotype anti‐endothelial cell antibodies (AECA) were demonstrated by ELISA in 43 of 80 patients with SSc (54%), in 15 of 20 patients with SLE (75%), and in none of 20 healthy control subjects. Immunofluorescence analysis on HUVEC substrate showed homogeneous cytoplasmic staining. Immunoblotting demonstrated that these patients had antibodies directed to one or several antigens of approximately 60, 90, 110 and 140 kD, and the most common responses were to the 90‐kD antigen. By the immunofluorescence method using HUVEC, affinity‐purified anti‐90‐kD antibodies showed identical cytoplasmic staining to that produced by sera positive for AECA. Furthermore, AECA were closely correlated with pulmonary fibrosis in patients with SSc. These findings suggest that patients with SSc have abnormal antibodies to endothelial cell antigens, and support the hypothesis that endothelial dysfunction is involved in the development of this disease.

Clinical significance of surfactant protein D as a serum marker for evaluating pulmonary fibrosis in patients with systemic sclerosis.

OBJECTIVE To determine the clinical significance of surfactant protein D (SP-D), a useful marker for evaluating various lung diseases, in patients with systemic sclerosis (SSc) and to clarify any clinical significance between SP-D and KL-6, which is known to be correlated with pulmonary fibrosis (PF) in SSc patients. METHODS We used a specific enzyme-linked immunosorbent assay to measure serum SP-D levels in 83 patients with SSc and 31 healthy control subjects. RESULTS The serum levels of SP-D were significantly higher in patients with SSc than in healthy controls (mean +/- SD 81.9+/-59.2 versus 34.8+/-13.7 ng/ml). Serum SP-D levels in patients with diffuse cutaneous SSc were significantly higher than those in patients with limited cutaneous SSc (98.8+/-72.1 versus 66.8+/-40.0 ng/ml). Serum SP-D levels in patients with PF were significantly elevated compared with those in patients without PF (99.7+/-64.1 versus 65.3+/-49.4 ng/ml). Moreover, the incidences of decreased percentage diffusing capacity for carbon monoxide and decreased percentage vital capacity were also significantly greater in patients with elevated SP-D levels than in those with normal levels (67% versus 43% and 36% versus 17%, respectively). There was a significant positive correlation between serum levels of SP-D and KL-6. Serum SP-D and KL-6 levels showed almost the same sensitivities and specificities in the diagnosis of PF (68% versus 73% and 70% versus 74%, respectively). These two markers also predicted PF to almost the same degree (31% versus 33%, respectively). CONCLUSION These results suggest that SP-D, as well as KL-6, may be a useful serum marker for evaluating PF in patients with SSc.

B Lymphocyte signaling established by the CD19/CD22 loop regulates autoimmunity in the tight-skin mouse.

Systemic sclerosis (SSc) is characterized by fibrosis and autoimmmunity. Peripheral blood B cells from SSc patients specifically overexpress CD19, a critical cell-surface signal transduction molecule in B cells. CD19 deficiency in B cells also attenuates skin fibrosis in the tight-skin (TSK/+) mouse, a genetic model for SSc. Herein we analyzed two transgenic mouse lines that overexpress CD19. Remarkably, 20% increase of CD19 expression in mice spontaneously induced SSc-specific anti-DNA topoisomerase I (topo I) antibody (Ab) production, which was further augmented by 200% overexpression. In TSK/+ mice overexpressing CD19, skin thickness did not increase, although anti-topo I Ab levels were significantly augmented, indicating that abnormal CD19 signaling influences autoimmunity in TSK/+ mice and also that anti-topo I Ab does not have a pathogenic role. The molecular mechanisms for abnormal CD19 signaling were further assessed. B-cell antigen receptor crosslinking induced exaggerated calcium responses and augmented activation of extracellular signal-regulated kinase in TSK/+ B cells. CD22 function was specifically impaired in TSK/+ B cells. Consistently, CD19, a major target of CD22-negative regulation, was hyperphosphorylated in TSK/+ B cells. These findings indicate that reduced inhibitory signal provided by CD22 results in abnormal activation of signaling pathways including CD19 in TSK/+ mice and also suggest that this disrupted B cell signaling contribute to specific autoantibody production.

CD83 expression is a sensitive marker of activation required for b cell and CD4+ T cell longevity in vivo

CD83 is a surface marker that differentiates immature and mature human dendritic cell populations. Thymic epithelial cell expression of CD83 is also necessary for efficient CD4+ T cell development in mice. The altered phenotypes of peripheral B and CD4+ T cells, and the reduction of peripheral CD4+ T cells in CD83−/− mice, suggest additional functions for CD83. To assess this, a panel of mAbs was generated to characterize mouse CD83 expression by peripheral leukocytes. As in humans, activation of conventional and plasmacytoid murine dendritic cell subsets led to rapid up-regulation of CD83 surface expression in mice. In primary and secondary lymphoid compartments, a subset of B cells expressed low-level CD83, while CD83 was not detected on resting T cells. However, CD83 was prominently up-regulated on the majority of spleen B and T cells within hours of activation in vitro. In vivo, a low dose of hen egg lysozyme (1 μg) induced significant CD83 but not CD69 expression by Ag-specific B cells within 4 h of Ag challenge. Although B cell development appeared normal in CD83−/− mice, B and CD4+ T cell expression of CD83 was required for lymphocyte longevity in adoptive transfer experiments. Thus, the restricted expression pattern of CD83, its rapid induction following B cell and T cell activation, and its requirement for B cell and CD4+ T cell longevity demonstrate that CD83 is a functionally significant and sensitive marker of early lymphocyte activation in vivo.

Prevalence and clinical relevance of 52-kDa and 60-kDa Ro/SS-A autoantibodies in Japanese patients with systemic sclerosis

OBJECTIVE To determine the prevalence of 52-Kda and 60-Kda Ro/SS-A autoantibodies in serum samples from Japanese patients with systemic sclerosis (SSc). METHODS Serum samples from 263 Japanese patients with SSc were examined by double immunodiffusion and enzyme linked immunosorbent assay (ELISA). RESULTS By double immunodiffusion, 29 serum samples from patients with SSc were found to possess anti-Ro/SS-A antibodies. By ELISA, 31 serum samples were positive for anti-Ro52 and/or anti-Ro60. Of 27 serum samples that were positive by both methods, 15 reacted with both Ro52 and Ro60, five with Ro52 alone, and seven with Ro60 alone. Eleven of 12 patients with both anti-Ro52 and anti-Ro60 and all five patients with anti-Ro52 alone had Sjögren’s syndrome, while only one of six patients with anti-Ro60 alone had this disorder. CONCLUSIONS Anti-Ro52 may be a serological marker for the presence of Sjögren’s syndrome in anti-Ro/SS-A-positive patients with SSc.

Distribution and antigen specificity of anti-U1RNP antibodies in patients with systemic sclerosis

Systemic sclerosis (SSc) is a generalized connective tissue disease which is characterized by the presence of several autoantibodies. To determine the prevalence and antigen specificity of anti‐U1RNP antibodies (anti‐U1RNP) in patients with SSc, serum samples from 223 patients with SSc, 117 patients with systemic lupus erythematosus (SLE), 18 patients with mixed connective tissue disease (MCTD) and 40 healthy control subjects were examined by indirect immunofluorescent analysis (IIF), double immunodiffusion, and immunoblotting using nuclear extract of HeLa cells. Eighteen of the 223 (8%) serum samples from patients with SSc were shown to be positive for anti‐U1RNP. The frequency of anti‐U1RNP positivity in limited cutaneous SSc (14%) was significantly higher than that in those with diffuse cutaneous SSc (3%). Anti‐Sm antibodies were detected in patients with SLE positive for anti‐U1RNP, but not in those with SSc positive for anti‐U1RNP or those with MCTD. Immunoblotting demonstrated that anti‐70‐kD antibodies were detected more often in patients with SSc positive for anti‐U1RNP and in those with MCTD than in those with SLE. Furthermore, anti‐U1RNP was closely correlated with pulmonary fibrosis and joint involvement in patients with SSc. These results suggest that anti‐70‐kD antibodies are useful in the classification of patients with anti‐U1RNP.

Clinical usefulness of anti-RNA polymerase III antibody measurement by enzyme-linked immunosorbent assay

OBJECTIVE To evaluate the clinical usefulness of measuring anti-RNA polymerase (RNAP) III antibody with a commercially available ELISA in Japanese patients with SSc. METHODS This multicentre study involved 354 patients with SSc, 245 with non-SSc CTDs and 102 healthy controls. ELISAs were used to detect anti-RNAP III antibody, anti-topo I antibody and ACA. The presence of anti-RNAP III antibody in selected serum samples was confirmed by immunoprecipitation (IP) assay. RESULTS By ELISA, anti-RNAP III antibody was detected in 38 (10.7%) patients with SSc, 3 (1.2%) with non-SSc CTD and no healthy controls. The clinical specificity for SSc was excellent (98.8%), although a small number of false positives occurred. The sensitivity of the anti-topo I and ACA ELISAs for SSc was 59.9%, which increased to 68.2% without a reduction in specificity when the anti-RNAP III measurement was added. Clinical features associated with positivity for the anti-RNAP III antibody include dcSSc, a high total skin score and a trend towards high prevalence of renal crisis, consistent with previous studies that used an IP assay. Furthermore, on clinical severity scales, SSc patients with anti-RNAP III antibody scored highest for skin and renal involvement among patients subgrouped by the presence of individual SSc-related antibodies. CONCLUSIONS The measurement of anti-RNAP III antibody by ELISA is useful in routine clinical practice, because it helps diagnose SSc and identify a disease subset with severe skin and renal involvement.

Serum levels of BAFF are increased in bullous pemphigoid but not in pemphigus vulgaris

Background  BAFF [B‐cell activating factor belonging to the tumour necrosis factor (TNF) family] is a member of the TNF superfamily that regulates B‐lymphocyte proliferation and survival. It has been demonstrated that increased levels of soluble BAFF are associated with systemic autoimmunity in patients with systemic lupus erythematosus, rheumatoid arthritis and Sjögrens syndrome, and in animal models of spontaneous autoimmune diseases. However, the significance of circulating BAFF in autoimmune bullous diseases is unknown.

Serum levels of tissue inhibitor of metalloproteinase-1 and 2 in patients with eosinophilic fasciitis.

Background  Serum levels of tissue inhibitor of metalloproteinases (TIMPs) have been reported to be elevated in patients with various connective tissue diseases. However, there has been no report that evaluates TIMPs in patients with eosinophilic fasciitis (EF).

Serum chemokine profile in patients with bullous pemphigoid

Background  Bullous pemphigoid (BP) is an autoimmune inflammatory disease causing blister formation at the dermoepidermal junction. Cutaneous infiltration of activated CD4+ T cells and eosinophils is an early event in blister formation during the disease process, suggesting that the trafficking of circulating leucocytes through the sites of inflammation is crucial in the pathogenesis of the disease. While the accumulated evidence suggests that some cytokines are involved in the pathogenesis, there have been few reports about serum chemokine profiles in patients with BP.