Masahiro Sekiguchi

Hepatocyte growth factor prevents lupus nephritis in a murine lupus model of chronic graft-versus-host disease

Chronic graft-versus-host disease (GVHD) induced in (C57BL/6 × DBA/2) F1 (BDF1) mice by the injection of DBA/2 mouse spleen cells represents histopathological changes associated with systemic lupus erythematosus (SLE), primary biliary cirrhosis (PBC) and Sjogrens syndrome (SS), as indicated by glomerulonephritis, lymphocyte infiltration into the periportal area of the liver and salivary glands. We determined the therapeutic effect of hepatocyte growth factor (HGF) gene transfection on lupus using this chronic GVHD model. Chronic GVHD mice were injected in the gluteal muscle with either HVJ liposomes containing 8 μg of the human HGF expression vector (HGF-HVJ liposomes) or mock vector (untreated control). Gene transfer was repeated at 2-week intervals during 12 weeks. HGF gene transfection effectively prevented the proteinuria and histopathological changes associated with glomerulonephritis. While liver and salivary gland sections from untreated GVHD mice showed prominent PBC- and SS-like changes, HGF gene transfection reduced these histopathological changes. HGF gene transfection greatly reduced the number of splenic B cells, host B cell major histocompatibility complex class II expression, and serum levels of IgG and anti-DNA antibodies. IL-4 mRNA expression in the spleen, liver, and kidneys was significantly decreased by HGF gene transfection. CD28 expression on DBA/2 CD4+ T cells was decreased by the addition of recombinant HGF in vitro. Furthermore, IL-4 production by DBA/2 CD4+ T cells stimulated by irradiated BDF1 dendritic cells was significantly inhibited by the addition of recombinant HGF in vitro. These results suggest that HGF gene transfection inhibited T helper 2 immune responses and reduced lupus nephritis, autoimmune sialoadenitis, and cholangitis in chronic GVHD mice. HGF may represent a novel strategy for the treatment of SLE, SS and PBC.

Role of Sphingosine 1-Phosphate in the Pathogenesis of Sjögren’s Syndrome

Primary Sjögren’s syndrome (SS) is an autoimmune disease characterized by inflammatory mononuclear cell infiltration and destruction of epithelial cells of lacrimal and salivary glands. Sphingosine 1-phosphate (S1P) and signaling through its receptor S1P1 have been implicated in many critical cellular events including inflammation, cancer, and angiogenesis. This study was undertaken to examine the role of S1P1 signaling in the pathogenesis of primary SS. S1P1 and sphingosine kinase 1, which converts sphingosine to S1P, were detected in the cytoplasm of inflammatory mononuclear cells, vascular endothelial cells, and epithelial cells in all labial salivary glands by immunohistochemistry. The expression of S1P1 in inflammatory mononuclear cells was enhanced in advanced stages of primary SS. S1P enhanced proliferation and IFN-γ production by CD4+ T cells. The enhancing effect of S1P on IFN-γ production by CD4+ T cells was stronger in patients with primary SS than in healthy controls. S1P also enhanced Fas expression and Fas-mediated caspase-3 induction in salivary gland epithelial cells. IL-6 expression was detected in the cytoplasm of inflammatory mononuclear cells and ductal epithelial cells and was enhanced in advanced stages of primary SS. Furthermore, both IFN-γ and S1P augmented IL-6 secretion by salivary gland epithelial cells. These effects of S1P were inhibited by pretreatment of pertussis toxin. Our data reveal that S1P1 signaling may modulate the autoimmune phenotype of primary SS by the action of immune as well as epithelial cells.

Sphingosine 1-phosphate (S1P)/S1P receptor 1 signaling regulates receptor activator of NF-κB ligand (RANKL) expression in rheumatoid arthritis.

Sphingosine 1-phosphate (S1P)/S1P receptor 1 (S1P1) signaling plays an important role in synovial cell proliferation and inflammatory gene expression by rheumatoid arthritis (RA) synoviocytes. The purpose of this study is to clarify the role of S1P/S1P1 signaling in the expression of receptor activator of NF-κB ligand (RANKL) in RA synoviocytes and CD4(+) T cells. We demonstrated MH7A cells, a human RA synovial cell line, and CD4(+) T cells expressed S1P1 and RANKL. Surprisingly, S1P increased RANKL expression in MH7A cells and CD4(+) T cells in a dose-dependent manner. Moreover, S1P enhanced RANKL expression induced by stimulation with TNF-α in MH7A cells and CD4(+) T cells. These effects of S1P in MH7A cells were inhibited by pretreatment with PTX, a specific Gi/Go inhibitor. These findings suggest that S1P/S1P1 signaling may play an important role in RANKL expression by MH7A cells and CD4(+) T cells. S1P/S1P1 signaling of RA synoviocytes is closely connected with synovial hyperplasia, inflammation, and RANKL-induced osteoclastogenesis in RA. Thus, regulation of S1P/S1P1 signaling may become a novel therapeutic target for RA.

Biphasic anti-osteoclastic action of intravenous alendronate therapy in multiple myeloma bone disease

Multiple myeloma is a malignancy of plasma cells with osteolytic bone destruction. Bisphosphonates inhibit osteoclast activity and are widely used for the treatment of myeloma bone disease. We analyzed the changes in urinary cross-linked N-telopeptides of collagen (u-NTx) and urinary calcium (u-Ca) after bisphosphonate alendronate therapy in ten patients with myeloma bone disease. In all patients, the levels of u-Ca and u-NTx decreased within a week. After the maximum decrease of u-NTx, u-NTx started increasing in half of the patients. However, this further increase in u-NTx decreased again without any additional therapy. Disease severity and pretreatment u-NTx concentrations did not differ between patients with and without the rebound. Patients who did not have rebound had decreased bone marrow monocytes and decreased serum concentrations of interleukin 18, which is produced by monocytes. Our results suggest that impaired activity of monocytes, which are possible osteoclast precursors, is related to reduced bone destruction in multiple myeloma.

Differences in Predictive Factors for Sustained Clinical Remission with Abatacept Between Younger and Elderly Patients with Biologic-naive Rheumatoid Arthritis: Results from the ABROAD Study

Objective. To differentiate predictive factors for sustained clinical remission between elderly and younger patients with rheumatoid arthritis (RA) receiving abatacept (ABA) as an initial biological disease-modifying antirheumatic drug. Methods. The study involved 277 biologic-naive patients with RA with high or moderate disease activity, who were treated with intravenous ABA and evaluated for 48 weeks in 43 Japanese hospitals and rheumatology clinics (the ABatacept Research Outcomes as a First-line Biological Agent in the Real WorlD study: UMIN000004651). Predictive factors associated with sustained clinical remission defined by the 28-joint Disease Activity Score with C-reactive protein (DAS28-CRP) during the 24–48–week or 36–48–week periods were determined in elderly (≥ 65 yrs, n = 148) and younger patient groups (< 65 yrs, n = 129) using logistic regression analysis. Results. Clinical remission was achieved at 24 and 48 weeks in 35.1% and 36.5% of patients in the elderly group and 34.9% and 43.4% in the younger group, respectively. In elderly patients, anticitrullinated protein antibody (ACPA) positivity and a lower DAS28-CRP score were significantly associated with sustained clinical remission; however, a lower Health Assessment Questionnaire-Disability Index (HAQ-DI) score was not related to sustained clinical remission. In younger patients, lower DAS28-CRP and HAQ-DI scores were predictive factors for sustained clinical remission, whereas ACPA positivity was not a useful predictive factor for sustained clinical remission. Conclusion. Although the effectiveness of ABA in biologic-naive patients with RA was equally recognized in elderly and younger patients, the baseline clinical characteristics associated with sustained clinical remission were substantially different.

Correlation between salivary epidermal growth factor levels and refractory intraoral manifestations in patients with Sjögren's syndrome

Abstract Objective. To assess changes in salivary epidermal growth factor (EGF) levels and the correlation between these levels and the severity of intraoral manifestations in Sjögrens syndrome (SS). Methods. Forty SS patients and 23 controls were enrolled. Salivary EGF concentration was measured using an enzyme-linked immunosorbent assay, and intraoral manifestations were evaluated using a short version of the Oral Health Impact Profile (OHIP-14). The associations among salivary flow rate, EGF levels and the severity of intraoral manifestations were analyzed. Results. The total salivary EGF output was significantly decreased in the SS patients compared with the controls (9237.6 ± 8447.0 vs. 13296.9 ± 7907.1 pg/10 min, respectively, p = 0.033). In the SS patients, total EGF output and salivary flow rate showed a strong positive correlation (rs = 0.824, p = 0.0005), while total EGF output and disease duration showed a negative correlation (rs = −0.484, p = 0.008). Further, total EGF output was significantly correlated with the OHIP-14 score (rs = −0.721, p = 0.012). Conclusions. The salivary flow rate and EGF levels are decreased in SS, and this deterioration in saliva quality causes refractory intraoral manifestations. Our findings have provided new therapeutic targets for SS.

Differential regulation of c-Met signaling pathways for synovial cell function

We previously demonstrated that blocking the hepatocyte growth factor (HGF) receptor, c-Met, using a HGF antagonist, NK4, inhibited arthritis in a rheumatoid arthritis (RA) model mice. In the present study, we investigated the role of c-Met signaling in synovial cell function. We demonstrated that synovial tissues from RA patients and MH7A cells, a human RA synovial cell line, expressed HGF and c-Met. HGF and c-Met expression in RA synovium was increased compared to osteoarthritis synovium suggesting increased c-Met signaling in RA synovial cells. The c-Met inhibitor, SU11274, inhibited ERK1/2 and AKT phosphorylation in HGF-stimulated MH7A cells. MEK and PI3K inhibitors suppressed production of matrix metalloproteinase-3 (MMP-3), vascular endothelial growth factor (VEGF) and prostaglandin E2 (PGE2) by MH7A cells, suggesting that c-Met-MEK-ERK and c-Met-PI3K-AKT pathways are involved positively regulating MH7A cell function. Although SU11274 suppressed MMP-3 and VEGF production it enhanced PGE2 production by MH7A cells suggesting that negative regulation by c-Met signaling, independent of the MEK-ERK and PI3K-AKT pathways, is involved in PGE2 production. Blocking c-Met signaling may be therapeutically useful to inhibit angiogenesis and cartilage and bone destruction by inhibiting VEGF and MMP-3 production, while enhancing PGE2 production in synovial cells in RA.

Rapid decrease in salivary epidermal growth factor levels in patients with Sjögren's syndrome: A 3-year follow-up study

Objectives. To assess changes in salivary epidermal growth factor (EGF) levels within three years and investigate the correlation between these changes and the severity of intraoral manifestations in patients with Sjögrens syndrome (SS). Methods. Twenty-three SS patients (14 primary SS and 9 secondary SS) and 14 controls were followed up for three years. Salivary EGF concentration was measured using an enzyme-linked immunosorbent assay, and intraoral manifestations were evaluated using a short version of the Oral Health Impact Profile (OHIP-14). Changes in salivary flow rate, EGF level, and severity of intraoral manifestations were analyzed, along with associations among them. Results. The OHIP-14 score significantly increased and the total salivary EGF output significantly decreased after three years in the SS group (10.2 ± 8.8 vs. 12.6 ± 9.2, p = 0.040; 10158.4 ± 9820.9 vs. 8352.8 ± 7813.3 pg/10 min, p = 0.032), though the salivary flow rate did not change. The decrease in total EGF output was especially high in patients with long disease duration and poor oral health-related quality of life (OHRQoL). In patients with poor OHRQoL, the change in total EGF output significantly correlated with the OHIP-14 score (r = − 0.847, p = 0.008). However, there was no correlation between the change in salivary flow rate and the OHIP-14 score. Conclusions. The rapid decrease in salivary EGF level contributes to the progression of intraoral manifestations of SS.

Reliability and clinical utility of Enzyme-linked immunosorbent assay for detection of anti-aminoacyl-tRNA synthetase antibody.

Anti-aminoacyl-tRNA synthetase (ARS) antibody is one of the myositis-specific autoantibodies to make a diagnosis of polymyositis (PM) and dermatomyositis (DM). Recently a new enzyme-linked immunosorbent assay (ELISA) kit of concurrently detected anti-ARS antibodies (anti-Jo-1, anti-PL-7, anti-PL-12, anti-EJ and anti-KS) have become to measure in the clinical setting. To evaluate the reliability of this ELISA kit, we measured anti-ARS antibodies in 75 PM and DM patients using by this ELISA assay and compared them with the results by RNA immunoprecipitation assay. Between the measurements of anti-PL-7, anti-PL-12, anti-EJ and anti-KS autoantibodies by ELISA assay and RNA-IP assay, the concordance rate of reproducibility is 95.1% and the positive agreement rate is 90.9% and negative agreement rate is 96.0% and kappa statistic is 0.841. Between the measurements of existing anti-Jo-1 antibody ELISA kit and anti-ARS antibody ELISA kit, the concordance rate of reproducibility is 96.9%, the positive agreement rate is 100%, negative agreement rate is 96.1% and kappa statistic is 0.909. The lung involvement in patients with PM and DM patients are positive of anti-ARS antibodies and anti-melanoma differentiation associated gene5 (MDA5) antibody at a rate around 70%. Then most life-threatening ILD with anti-MDA5 positive clinically amyopathic dermatomyositis patients could be highly guessed when anti-ARS antibodies are negative.

Anticentromere antibody-positive primary Sjögren's syndrome: Epitope analysis of a subset of anticentromere antibody-positive patients

Objectives: Anticentromere antibody (ACA) is generally considered to be a serological marker for systemic sclerosis (SSc). ACA-positive patients with primary Sjögrens syndrome (pSS) have also been reported. ACA often recognizes centromere proteins (CENPs): CENP-A, CENP-B, and CENP-C, and sometimes reacts to heterochromatin protein 1 (HP1)α. We compared the reactivity against six different epitopes for three ACA-positive clinical subgroups: 29 patients with pSS, 36 SSc patients with sicca symptoms, and 28 SSc patients without sicca symptoms. Methods: We utilized enzyme-linked immunosorbent assays (ELISAs) with recombinant proteins covering six different epitope regions of ACA (the amino terminus (Nt) of CENP-A, CENP-B, and CENP-C, the carboxyl terminus (Ct) of CENP-B and CENP-C, and HP1α). Results: The patients with pSS were found to have IgG-class autoantibodies against CENP-C-Nt and HP1α, and IgA-class autoantibodies against CENP-C-Ct with significantly higher frequencies than the SSc patients with or without sicca symptoms. The positive predictive value and the negative predictive value of the combination of these three autoantibodies for pSS were 73% and 82%, respectively, for pSS. Conclusions: Based on the result that reactivities against CENP-C and HP1α in patients with pSS differ from those in patients with SSc, we propose ACA-positive pSS as a clinical subset of SS that is independent of SSc.