Kotaro Shikano

The growth factor midkine may play a pathophysiological role in rheumatoid arthritis

Abstract Objectives: Midkine (MK) is involved in cell proliferation, differentiation, migration, and survival. In this study, we measured serum MK levels in rheumatoid arthritis (RA) and investigated the correlation of serum MK with RA disease activity. Expression and effect of MK in RA synovial tissue were also examined. Methods: Serum MK and production of inflammatory mediators by rheumatoid synovial fibroblasts (RSFs) were measured by enzyme-linked immunosorbent assay. MK expression in synovial tissue was examined by immunohistochemistry. MK receptor expression was analyzed by RT-PCR and Western blotting. Results: RA patients had a significantly higher serum MK level than healthy controls. In RA patients, the MK level was correlated with DAS28-ESR, disability index of the Health Assessment Questionnaire, and rheumatoid factor level. The serum MK level tended to be decreased by anti-TNF therapy. MK was expressed by synovial lining cells in RA synovial tissues and it enhanced the production of IL-6, IL-8, and CCL2 by RSFs. RSFs expressed LDL receptor-related protein 1, candidate receptor for MK. Conclusions: The serum MK level could be a marker of disease activity in RA and an indicator of a poor prognosis. MK may have a role in the pathogenesis of RA via induction of inflammatory mediators.

Efficacy of Vitamin K2 for Glucocorticoid-induced Osteoporosis in Patients with Systemic Autoimmune Diseases

Objective Vitamin K2 (menatetrenone) is an effective treatment for patients with postmenopausal osteoporosis. We herein performed a subanalysis of patients with systemic autoimmune diseases undergoing glucocorticoid therapy in our previous prospective study. Methods Sixty patients were categorized into a group with vitamin K2 treatment (n=20, Group A) and a group without vitamin K2 treatment (n=40, Group B). All patients were treated with bisphosphonates. Results Serum levels of osteocalcin and undercarboxylated osteocalcin decreased significantly after the start of glucocorticoid therapy in both groups, while the serum osteocalcin level was significantly higher in Group A than Group B during the third (p=0.0250) and fourth weeks (p=0.0155). The serum level of the N-terminal peptide of type I procollagen, a bone formation marker, decreased during glucocorticoid therapy, but was significantly higher in Group A than Group B during the fourth week (p=0.0400). The bone mineral density and fracture rate showed no significant differences between the two groups. Conclusion Although vitamin K2 improves bone turnover markers in patients with osteoporosis on glucocorticoid therapy, it has no significant effect on the bone mineral density and fracture rate after 1.5 years of treatment.

Folylpolyglutamate synthase is a major determinant of intracellular methotrexate polyglutamates in patients with rheumatoid arthritis

We investigated major determinants of the intracellular concentrations of methotrexate polyglutamates (MTXPGs) in patients with rheumatoid arthritis (RA). In 271 RA patients on stable oral low dose weekly pulse MTX therapy, the concentrations of MTXPGs in red blood cells (RBCs) were measured by liquid chromatography-electrospray ionization-tandem mass spectrometry. Polymerase chain reaction-restriction fragment length polymorphism analysis was performed to determine the genotypes of solute carrier family 19 member 1 (SLC19A1), folylpolyglutamate synthase (FPGS), and gamma-glutamyl hydrolase (GGH). The mean total MTXPG concentration and the concentrations of individual MTXPGs increased dose-dependently, but reached a plateau at MTX doses >10 mg weekly. The MTXPG3-5/1-2 ratio was lower in patients with adverse events related to MTX than in patients without adverse events. Three polymorphisms of FPGS significantly influenced the MTXPG3-5/1-2 ratio in RBCs, while polymorphisms of SLC19A1 and GGH had no impact. The minor allele frequencies of 2 FPGS genotypes were significantly increased in our patients compared with a Caucasian population. FPGS may have a major role in regulating intracellular polyglutamation of MTX in RA patients receiving low-dose weekly MTX therapy.

Apparent Hypothalamic-Pituitary-Adrenal Axis Suppression via Reduction of Interleukin-6 by Glucocorticoid Therapy in Systemic Autoimmune Diseases.

Context Suppression of the hypothalamic-pituitary-adrenal (HPA) axis is a serious complication of systemic glucocorticoid therapy. Objective To clarify the influence of proinflammatory cytokines on the HPA axis after onset of glucocorticoid therapy in patients with systemic autoimmune diseases. Patients and Methods Forty-eight glucocorticoid-naïve patients with systemic autoimmune diseases (28 women) who were starting prednisolone therapy according to our standard regimens were prospectively observed. Patients were classified into high-dose and low-dose groups depending on the dose of prednisolone administered as indicated for their diseases. Plasma adrenocorticotropic hormone (ACTH) and serum cortisol levels were measured by electrochemiluminescence immunoassay. The corticotropin-releasing hormone (CRH) test was performed at baseline and second and forth weeks after starting glucocorticoid therapy. The increased levels of ACTH (ΔACTH) and cortisol (Δcortisol) were investigated. Serum levels of 10 proinflammatory cytokines were measured simultaneously by a multi-spot assay system. Results In the high-dose group, both basal and stimulated levels of ACTH and cortisol were significantly decreased by glucocorticoid therapy. In the low-dose group, basal ACTH and cortisol levels were also significantly decreased by glucocorticoid therapy, but ΔACTH and Δcortisol were unchanged. Among 10 cytokines, only interleukin (IL)-6 was significantly decreased by glucocorticoid therapy in both groups and was more closely correlated with cortisol than ACTH. Basal cortisol level was positively correlated with serum IL-6 level in all patients before glucocorticoid therapy. Conclusion In patients with systemic autoimmune diseases, apparent suppression of cortisol during glucocorticoid therapy may be partly mediated by reduced production of IL-6.

SAT0026 Suppression of Adrenal Function is Obvious when Compared to that of Pituitary Function in Patients with Systemic Autoimmune Diseases After Glucocorticoid Therapy

Background Glucocorticoids are widely used to treat a variety of diseases, including systemic autoimmune disease. Although glucocorticoids can improve the outcome for patients with these diseases, various side effects, such as suppression of the hypothalamic-pituitary-adrenal (HPA) axis are important problems. On the other hand, pro-inflammatory cytokines, such as IL-6 have been reported as stimulators of HPA axis in patients with systemic autoimmune diseases [1]. However, the influence of glucocorticoid therapy on HPA axis of inflammatory diseases has not be studied yet. Objectives The aim of this study is to examine the process of suppression of HPA axis after glucocorticoid therapy using CRH test in patients with systemic autoimmune diseases. Methods Thirty-five patients with systemic autoimmune diseases (64.4±16.1 y.o., M±SD), including 5 patients with systemic lupus erythematosus, 6 patients with polymyositis/dermatomyositis, 4 patients with vasculitis syndrome, 10 patients with polymyalgia rheumatica, 3 patients with rheumatoid arthritis, and 7 patients with other systemic autoimmune diseases, who received initial glucocorticoid therapy were gave written informed consent and enrolled. HPA axis was assessed by plasma adrenocorticotropin (ACTH) and serum cortisol levels of pre-CRH treatment, 30 and 60 minutes after administration of 100 μg of exogenous human CRH, and that was performed at 0, 2, and 4 weeks after starting glucocorticoid therapy. Patients were divided into two groups with prednisolone daily dose: 30 mg or more (18 patients, high-dose group), and 15mg or less (17 patients, low-dose group). The basal concentrations of plasma ACTH and serum cortisol, and their increased values after CRH stimulation (ΔACTH and Δcortisol, respectively) were evaluated. C-reactive protein (CRP) was also measured. This study was approved by the Ethics Committee at Toho University Omori Medical Center (approval no.25-215). Results As shown in Table1, basal ACTH and ΔACTH levels were significantly decreased after glucocorticoid therapy in the high-dose group, while those in the low-dose group were not suppressed. Basal cortisol levels in high and low dose groups were significantly suppressed by glucocorticoid therapy. Δcortisol level in high dose group was significantly suppressed, however, that in low dose group had a trend to decrease. CRP was significantly decreased after glucocorticoid therapy in both groups. Conclusions In the low-dose group, the pituitary function was not suppressed by glucocorticoid therapy, however, the adrenal function was significantly suppressed. It is suggested that adrenal suppression by glucocorticoid therapy might be caused not only by feedback regulation via pituitary suppression, but also suppression of systemic inflammation, as possible activating factors of adrenal function, in patients with systemic autoimmune diseases. References Mastorakos G, Chrousos GP, Weber JS. Recombinant interleukin-6 activates the hypothalamic-pituitary-adrenal axis in humans. J Clin Endocrinol Metab. 1993 Dec;77(6):1690-4. Disclosure of Interest None declared

SAT0007 Resistin Directly Stimulates Chemokine Gene Expressions in Rheumatoid Synovial Fibroblasts: DNA Microarray Analysis

Background Adipose tissue synthesizes and releases physiologically active molecules that are known as adipokines. Although the most widely discussed biological effects of resistin has been the regulation of glucose homeostasis and insulin sensitivity, resistin is now considered to represent a new family of compounds that act as mediators of immune and inflammatory processes [1]. We previously reported that serum resistin concentration was elevated in patients with rheumatoid arthritis (RA) and it was associated with serum C-reactive protein concentration [2]. However, the direct effects of resistin on rheumatoid synovial fibroblasts (RSFs) have not been clarified yet. Objectives The aim of this study is to determine the direct effects of resistin on RSFs by DNA microarray analysis. Methods Synovial tissues were obtained at total knee replacement operation from patients with RA who gave consent to use their tissue for research. This study was approved by the Ethics Committee of Toho University School of Medicine. RSFs were harvested from the synovial tissues of RA patients. After 18h incubation with different concentrations of resistin (0, 100 and 1000 ng/mL), total RNA was extracted from the cells and the gene expression profile of RSFs was analyzed by DNA microarray (Agilent Technologies, CA, USA). In addition, reverse transcription-polymerase chain reaction (RT-PCR) was performed to confirm the result of microarray analysis. Results The DNA microarray analysis revealed that 45 inflammation-related genes were up-regulated in RSFs in concentration dependent manners when we applied equal to or more than a 3 fold increase to cut-off value for gene expression. We picked up 13 chemokine (CCL2, CCL3, CCL4, CCL7, CCL20, CCL3L3, CXCL1, CXCL2, CXCL3, CXCL6, CXCL8, CXCL10, and CXCR4) genes out of these 45 candidates. Concentration-dependent up-regulation of only CXCL8, CXCL1, CXCL6, CCL2 and CCL7 gene expressions were confirmed by RT-PCR. Conclusions In this study, we demonstrated that resistin increased gene expressions of 45 inflammation-related molecules in RSFs, when screened by DNA microarray analysis. Resistin-induced chemokines were identified as CXCL8, CXCL1, CXCL6, CCL2 and CCL7. Since they are known as essential mediators in synovial inflammation of RA, resistin may possibly play an important role in pathogenesis of RA. References Gόmez R, Conde J, Scotece M, Gόmez-Reino JJ, Lago F, Gualillo O. Whats new in our understanding of the role of adipokines in rheumatic diseases? Nat Rev Rheumatol. 2011 Aug 2;7(9):528-36. Yoshino T, Kusunoki N, Tanaka N, Kaneko K, Kusunoki Y, Endo H, Hasunuma T, Kawai S. Elevated serum levels of resistin, leptin, and adiponectin are associated with C-reactive protein and also other clinical conditions in rheumatoid arthritis. Intern Med. 2011;50(4):269-75. Disclosure of Interest None declared

FRI0030 Jak2/stat3 is a major pathway of leptin-induced interleukin-6 production by rheumatoid synovial fibroblasts

Background Since proinflammatory cytokines, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-6, play major pathophysiological roles in rheumatoid arthritis (RA), their inhibitors have recently been developed as potent biological anti-rheumatic drugs. However, they are not curative and the effects are still partial, with many patients failing to respond. Leptin is the product of the ob gene, and is a peptide hormone synthesized almost exclusively by adipocytes that regulates appetite and energy expenditure. It is also suggested that leptin may contribute to inflammation and autoimmunity. We previously reported that serum leptin level was elevated in patients with RA [1]. Accordingly, we examined the direct effects of leptin on cultured rheumatoid synovial fibroblasts (RSFs) in the present study. Objectives To determine the effects of leptin on the production of proinflammatory cytokines by RSFs. Methods Synovial tissue was obtained at total knee replacement operation from patients with RA who gave consent. RSFs were harvested from the synovial tissues of these patients. Leptin receptor mRNAs were detected by reverse transcription–polymerase chain reaction. Productions of mRNA by RSFs and protein concentrations of TNF-α, IL-1β, and IL-6 in the culture medium were detected by real-time PCR and ELISA kit, respectively. Small interfering RNA (siRNA) was transfected into RSFs to down-regulate the expression of leptin receptor. Effects of inhibitors of janus kinase 2 (JAK2), phosphatidylinositol 3-kinase (PI3K), and mitogen-activated protein kinase (MAPK) on IL-6 production were evaluated. Phosphorylation of signal transducer and activator of transcription 3 (STAT3) in RSFs were determined by Western blot analysis. This study was approved (No. 19021) by Ethics Committee of Toho University School of Medicine. Results We detected leptin receptor mRNAs in RSFs. Expressions of IL-1β and IL-6 mRNAs were enhanced in a concentration-dependent manner by addition of leptin to RSFs. IL-6 secretion by RSFs showed an increase after leptin stimulation, while IL-1β did not increase in the culture media. Leptin-induced production of IL-6 by RSFs was decreased after exposure to siRNA targeting leptin receptor (Ob-Rb). A JAK2 inhibitor, but not PI3K and MAPK inhibitors, decreased leptin-induced IL-6 production. Enhanced phosphorylation of STAT3 was observed in RSFs after stimulation by leptin. Conclusions Leptin possibly acts as a proinflammatory cytokine that up-regulates IL-6 production in RSFs via activation of JAK2/STAT3 in RSFs. Leptin and JAK/STAT pathway may represent a new alternative therapeutic target in the treatment of RA. References Yoshino T, Kusunoki N, Tanaka N, Kaneko K, Kusunoki Y, Endo H, Hasunuma T, Kawai S. Elevated serum levels of resistin, leptin, and adiponectin are associated with C-reactive protein and also other clinical conditions in rheumatoid arthritis. Intern Med. 2011;50:269. Disclosure of Interest None Declared

THU0388 Menatetrenone (Vitamin K2) Partially Restores the Suppression of Bone Formation by Glucocorticoid Therapy in Patients with Systemic Autoimmune Diseases

Background Osteoporosis is a serious complication of systemic glucocorticoid (GC) therapy. Bisphosphonate is recommended, however insufficient result have been reported in some cases. Menatetrenone (vitamin K2;VK) is a stimulator of bone formation in vitro and in vivo. We recently have reported that severity of GC-induced osteoporosis might be determined by serum basal sRANKL levels in patients with systemic autoimmune disease [1]. In the present study, we conducted post-hoc analysis to determine whether VK influenced bone metabolism markers in these patients. Objectives To study the efficacy of VK on serum bone metabolism markers and on bone mineral density (BMD) in patients under GC therapy. Methods This study was approved by the Ethics Committee at Toho University Omori Hospital (No. 21-61). Detailed study design was previously shown elsewhere [1]. Briefly, sixty patients (40 women) with systemic lupus erythematosus (n=21), vasculitis syndrome (n=19), polymyositis/dermatomyositis (n=15), and adult-onset Still’s disease (n=5) who were started to received prednisolone (30-60mg daily) were enrolled. Serum samples were obtained just before and 1 to 4 weeks after the start of GC therapy. All patients received bisphosphonate for our regimen of GC therapy. Twenty patients received VK 45 mg daily from 2 weeks after GC therapy. As bone formation markers, serum levels of osteocalcin (OC), procollagen type I N-terminal peptide (PINP), undercarboxylated OC (ucOC), and bone alkaline phosphatase (BAP) were measured. As bone resorption markers, serum levels of type I collagen cross-linked N-telopeptide (NTx) and tartrate resistant acid phosphatase isoform 5b (TRACP-5b) were measured. The BMD of lumbar spine was measured before and after (at 15 months) GC therapy. Results Serum levels of OC and ucOC were significantly (P<0.01) decreased at 1 to 4 weeks after GC therapy. Serum PINP level was also significantly (P<0.01) decreased after GC therapy, while the serum BAP level was not changed. Serum levels of NTx and TRACP-5b did not change during 4 weeks after GC therapy. Additional use of VK restored serum OC level to the half levels of the baseline. In contrast, serum ucOC level was decreased in patients both with and without administration of VK. The percentage of BMD was not changed from baseline in the VK group, whereas it was decreased in the bisphosphonate monotherapy group. Conclusions Additional use of VK partially restored serum OC level from the suppressed level by GC therapy. In addition, the BMD value in the VK group was not changed after GC therapy. These results suggest that VK may possibly provide some effects on bisphosphonate therapy to prevent from GC-induced osteoporosis. References Kaneko K, et al. Changes of serum soluble receptor activator for nuclear factor-κB ligand after glucocorticoid therapy reflect regulation of its expression by osteoblast. J Clin Endocrinol Metab. 2012; 97:E1909-1917. Disclosure of Interest None Declared