Ken Kayakabe

Interleukin-6 promotes destabilized angiogenesis by modulating angiopoietin expression in rheumatoid arthritis

OBJECTIVE To examine whether IL-6 promotes angiogenesis by modulating angiopoietin (Ang) expression in RA. METHODS Synovial fibroblasts derived from RA patients (RASFs) and human umbilical vein endothelial cells (HUVECs) were co-cultured for 6 days with or without recombinant IL-6, VEGF or Ang-1. HUVECs were stained with anti-CD31 antibody and their growth was determined by quantifying the CD31-positive area. SFs were collected from RA (n = 25) and OA (n = 7) patients. RESULTS In the co-culture system, IL-6 and VEGF significantly enhanced HUVEC growth to a similar extent. However, the morphology of proliferating cells was distinct between IL-6- and VEGF-stimulated HUVEC. HUVEC stimulated with IL-6 exhibited small, loose clusters surrounded by dispersed single cells, suggesting destabilized angiogenesis by IL-6. In the supernatants, IL-6 up-regulated VEGF compared with controls and Ang-2, while it down-regulated Ang-1. In contrast, down-regulation of Ang-1 was not observed with VEGF stimulation. Consistent with the destabilized morphology, stimulation with IL-6 decreased cell surface expression of vascular endothelial cadherin (VE-cadherin) on HUVEC, presumably by inducing internalization. Interestingly, adding recombinant Ang-1 partially inhibited IL-6-induced morphological changes in HUVEC including a destabilized morphology with small, loose clusters and internalization of VE-cadherin. In SFs from RA patients, VEGF was negatively correlated with Ang-1 (r = -0.559, P=0.004). CONCLUSION IL-6 not only enhances VEGF expression but also inhibits Ang-1 signalling by directly down-regulating Ang-1 expression and up-regulating Ang-2, an antagonist of Ang-1. These synergistic effects may play a critical role in destabilized angiogenesis in RA.

Interleukin-1β measurement in stimulated whole blood cultures is useful to predict response to anti-TNF therapies in rheumatoid arthritis

OBJECTIVE In RA, response to TNF blockers may be associated with a profile of cytokine production unique to each patient. This study sought to predict the response to biologic agents by examining pro-inflammatory cytokine synthesis in stimulated whole blood cultures (WBCs). METHODS We measured the concentration of TNF-α, IL-1β and IL-6 in supernatants of lipopolysaccharide (LPS)-stimulated WBCs obtained from RA patients (n = 41) before anti-TNF therapy (infliximab, 13; etanercept, 26; and adalimumab, 2) and from healthy controls (n = 12). At 24 weeks after biologics, whole bloods were again drawn from 14 of 41 patients. Response was defined by the European League Against Rheumatism response criteria after 24 weeks of therapy. RESULTS Among 41 patients, 32 were responders (good 14/moderate 18), while 9 were non-responders. All cytokines measured were significantly lower in RA patients than in controls. In RA, IL-1β production was lower in non-responders than in responders [median (interquartile range): 3.5 (1.5-9.4) vs 10.0 (5.1-93.1) pg/ml, P = 0.048]. The area under the curve from a receiver operating characteristic curve analysis for the prediction of response using IL-1β was 0.717 (95% CI 0.520, 0.914). The sensitivity and specificity of IL-1β (cut-off value 4.84 pg/ml) was 78.1 and 77.8%, respectively. All cytokines were significantly higher 6 months later compared with their respective baseline. CONCLUSION IL-1β measurement in LPS-stimulated WBC is useful to predict responsiveness to anti-TNF agents. Cytokine production capacities in LPS-stimulated WBCs are up-regulated by biologics.

Renal outcomes in mixed proliferative and membranous lupus nephritis (Class III/IV + V): A long-term observational study.

Abstract Objectives: In this study, we aimed to assess the effect of combination of proliferative and membranous lesions (Class III + V or IV + V) on renal outcomes as an independent category distinct from Class III and IV. Methods: We retrospectively analyzed 103 Japanese patients (14 male and 89 female) with Class III/IV LN, with or without Class V, who underwent renal biopsy and were treated at our institution. Renal endpoint was defined as doubling of serum creatinine or end-stage renal disease (ESRD). Results: The number of patients in each group was as follows: pure Class III/IV, 81 patients and mixed Class III/IV + V, 22 patients. During a median follow-up period of 125.0 months, 10 patients developed renal endpoint: five had Class III/IV LN and five had a combination of Class III/IV + V. Kaplan–Meier analyses demonstrated that patients with mixed Class III/IV + V LN had significantly poorer renal outcomes than patients with Class III/IV LN. Multivariate Cox regression analyses identified serum creatinine, active and chronic lesions (A/C), and mixed Class III/IV + V) as independent risk factors for poor renal outcomes. Conclusions: This study demonstrated a combination of proliferative and membranous LN (ISN/RPS Class III/IV + V) predicts poor renal outcomes.

Involvement of Infiltrating Macrophage-derived Activin A in the Progression of Renal Damage in MRL-lpr Mice.

Lupus nephritis is a life-threatening complication of systemic lupus erythematosus (SLE). Various growth factors, cytokines, and chemokines are implicated in the development of SLE. However, the pathophysiological processes involved in the development of lupus nephritis still remain unclear. In this study, we examined the involvement of activin A, a member of the transforming growth factor β (TGF-β) superfamily, in the progression of renal damage in lupus-prone MRL-lpr mice. Activin A was not expressed in the kidneys of control MRL-MpJ mice but was detectable in perivascular infiltrating cluster of differentiation 68 (CD68)-positive cells in the kidneys of MRL-lpr mice. Urinary activin A, which was also absent in MRL-MpJ mice, was detectable in MRL-lpr mice from 16 wk onward. Urinary activin A levels were significantly correlated with the number of perivascular inflammatory cell layers, the number of crescentic glomeruli, and the percentage of Elastica van Gieson (EVG)-positive fibrotic areas, but not with urinary protein levels or serum activin A. When activin action was blocked in vivo by the intraperitoneal administration of an activin antagonist, follistatin, the number of crescentic glomeruli, percentage of EVG-positive fibrotic areas, CD68-positive cell infiltration, and proteinuria were significantly reduced in a dose-dependent manner. These data suggest that infiltrating macrophage-derived activin A is involved in the progression of renal damage in MRL-lpr mice.

Clinical and histological features of lupus nephritis in Japan: a cross‐sectional analysis of the Japan Renal Biopsy Registry (J‐RBR)

The clinical and histological features of lupus nephritis (LN) are highly variable, depending on race and ethnicity. The Japan Renal Biopsy Registry (J‐RBR) is a nationwide registry of renal biopsies. Here, we report a cross‐sectional analysis of Japanese LN using the J‐RBR database.

AB0521 Repeat Biopsy in Lupus Nephritis: A Single Center Analysis in Japan

Background Repeat renal biopsy is occasionally performed in lupus nephritis (LN) patients in case of flare or worsening renal function. However, limited data are available regarding the histological changes of LN at repeat biopsy, especially by ISN/RPS 2003 classification. Objectives In the present study, we sought to determine characteristics of patients who received repeat renal biopsy and to elucidate histological changes. Methods We retrospectively analyzed LN patients with repeat renal biopsy among 150 biopsy-proven Japanese LN patients (19 males and 131 females with mean age of 36.8±13.8 years) at our department between 1976 and 2012. Renal histology was categorized by ISN/RPS 2003 classification. The chi-square test was used to compare the percentages between groups. Results Nineteen patients (6 males and 13 females with mean age of 31.5±10.2 years) received repeat renal biopsy. The mean intervals between initial and repeat biopsy was 6.4±2.6 years. The reasons for the repeat biopsy were flare of LN in 18 patients and persistent proteinuria despite treatment in 1 patient. Among 79 patients who had class IV or III/IV+V (mixed type) at initial biopsy, 16 patients (20.3%) received repeat biopsy, whereas only 3 patients (4.2%) underwent it among patients with class II, III, or V at initial biopsy (p=0.003). A repeat biopsy was more frequently performed in male (31.6%) than in female (10.3%, p=0.008). The histological changes who underwent repeat biopsy was shown in Table. The class switches were observed in 7 patients (37%). Six patients changed to mixed type, and 1 patent with mixed type transformed to class III. In addition, chronic lesions (A/C or C by ISN/RPS) were increased from 4 patients at initial biopsy to 12 patients in repeat biopsy (p=0.020). Repeat biopsy Initial biopsy II III IV V Mixed Total II 0 0 0 0 0 0 III 0 0 0 0 1 1 (5%) IV 0 0 7 0 0 7 (37%) V 0 0 0 1 0 1 (5%) Mixed 1 1 4 0 4 10 (53%) Total 1 (5%) 1 (5%) 11 (58%) 1 (5%) 5 (26%) 19 (100%) Conclusions Repeat biopsy was more frequently performed in male and in patients who had class IV or mixed type at initial biopsy. Approximately 40% patients showed class switches on repeat biopsy during a flare and most of them were a transition to mixed type. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4586

AB0934 Clinical Features and Prognosis of Adult-Onset Still's Disease: S Single Center Study in Japan

Background Adult-onset Stills disease (AOSD) is an inflammatory diseases characterized by spiking fever, arthritis and evanescent rash. Although AOSD is considered as a benign disease, relapses are common and life-threatening severe complications occur in some patients. Objectives In this study, we reviewed the clinical features and outcomes of AOSD patients to seek out risk factors for unfavorable outcomes. Methods We retrospectively reviewed medical charts of AOSD patients who were admitted to our department between 1997 and 2013. For the statistical analyses, Mann Whitney U test and Fishers exact test were carried out. Multivariate logistic analysis was used to identify factors independently associated with death. It was considered statistically significant if p value was less than 0.05. Results A total of 27 patients (8 male and 19 female) were available to analysis. The median age was 33 years (range, 15-64) and the median follow-up period was 2.2 years (range, 0.1-9.1). Corticosteroids and immunosuppressants were used in 92.6% and 33.3% of the patients, respectively. Methylprednisolone pulse therapy was applied in 18.5% of the patients and the median initial dose of steroids was 50 mg/day (range, 30-70; equivalent dose of prednisolone, excluding the dose of pulse therapy). All patients responded to the initial therapy, including 2 patients treated with nonsteroidal anti-inflammatory drugs (NSAIDs) alone. Twelve patients (44.4%) continued remission, whereas 15 patients (55.6%) had at least one relapse (median 2 times; range, 1-6). Five patients (18.5%) died from multiple organ failure in 3, sepsis in 1 and respiratory failure in 1 patient. The comparison of baseline characteristics between dead and alive patients was shown in Table. The complications of hemophagocytic syndrome (HPS) and disseminated intravascular coagulation (DIC) were significantly more frequent, and the hemoglobin level was significantly lower in dead patients than patients who survived. Multivariate logistic analysis showed a presence of DIC was an independent risk factor for death (OR 20.0, 95%CI 1.613-247.981, p=0.020). Table 1. Comparison of baseline characteristics between dead and alive patients Dead patients (n=5) Alive patients (n=22) P value Age (year) 46.0 (31.0–64.0) 29.0 (15.0–63.0) 0.1112 Time to diagnosis (days) 77.0 (0–498.0) 29.0 (0–120.0) 0.0802 HPS (patients) 3 (60.0%) 1 (4.5%) 0.0008 DIC (patients) 4 (80.0%) 4 (18.2%) 0.0048 Serum ferritin (ng/ml) 3889 (1039.9–89242.5) 3984.6 (158.0–100000.0) 0.6621 CRP (mg/dl) 7.0 (0.8–24.1) 7.6 (0.1–44.5) 0.8452 White blood cells (/μl) 10900 (8200–26700) 9300 (4600–32500) 0.5365 Lymphocytes (/μl) 620 (380–984) 860 (280–5525) 0.0699 Hemoglobin (g/dl) 8.5 (8.0–11.3) 11.4 (8.2–14.7) 0.0076 Platelets (/103) 27.4 (9.4–34.8) 24.4 (9.9–71.6) 0.5365 LDH (U/ml) 555.0 (330.0–1908.0) 564.0 (170.0–2530.0) 0.4945 Data expressed as the median with range or number with percentage. Conclusions Relapse rate was relatively high and death occurred not rarely in our cohort. The complication of DIC at the initial presentation was associated with death. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.4939

SAT0072 Frequency of interferon-gamma-positive TH17 (TH17-1) cells is up-regulated in synovial fluids in patients with rheumatoid arthritis

Background Th17 cells are a recently identified CD4+ CD45RO+helper T cell subset that produce IL-17 with proinflammatory actions and are thought to play a critical role in the pathogenesis of collagen-induced arthritis (CIA), a murine model of rheumatoid arthritis (RA) (1). Recently, Nistala et al. showed that Th17 cells obtained from the synovial fluid (SF) of juvenile idiopathic arthritis (JIA) patients co-expressed IFN-γ (2). Moreover, they found that Th17-1 cells (Th17 cells co-expressing IFN-γ) were enriched in SF compared to peripheral blood mononuclear cells (PBMC) in patients with JIA. However Th17-1 has not been investigated in patients with RA. Objectives To explore the significance of Th17-1 in human RA, we examined the frequency of Th1, Th17, and Th17-1 cells in PBMC and SF derived from RA patients by flow cytometry and compared them with those in PBMC from healthy donors. Methods PBMC from 14 healthy donors (Control-PBMC) and 33 RA patients (RA-PBMC), and SF from 17 RA patients (RA-SF) were obtained and used in this experiment. Isolated mononuclear cells were stimulated with PMA and ionomycin for 5 hours, and then the expression of intracellular cytokines and cell surface markers were analyzed by flow cytometry. Among double positive cells for CD4 and CD45RO, single positive cells for IFN-γ or IL-17, and double positive cells for both IFN-γ and IL-17 were defined as Th1, Th17, and Th17-1 cells, respectively. Results The frequency of Th17 and Th17-1 cells in PBMC from RA patients were significantly lower than those from controls, whereas the frequency of Th1 cells were similar between them (Th17, 2.2±1.1% vs. 3.0±1.1%, P=0.006; Th17-1, 0.4±0.3% vs. 1.0±0.5%, P=0.0002; Th1, 24±11% vs. 28±5.6%, P=0.0624; RA-PBMC vs. Control-PBMC). Among RA patients, the frequency of Th17 cells in SF was significantly lower than that of PBMC, whereas Th1 and Th17-1 cells in SF were significantly higher than those in PBMC (Th17, 2.2±1.1% vs. 1.8±1.4%, P=0.0381; Th1, 24±11% vs. 38±14%, P=0.0004; Th17-1, 0.4±0.3% vs. 1.1±0.9%, P=0.0006; RA-PBMC vs. RA-SF). Conclusions In patients with RA, the frequency of both Th1 and Th17-1 cells but not of Th17 in SF were significantly up-regulated, suggesting that a local inflammation in RA joints is dominantly mediated by IFN-γ. References Korn T, Bettelli E, Oukka M, Kuchroo VK. IL-17 and Th17 Cells. Annu Rev Immunol. 2009. 27:485–517. Nistala K, Adams S, Cambrook H, Ursu S, Olivito B, de Jager W, Evans JG, Cimaz R, Bajaj-Elliott M, Wedderburn LR. Th17 plasticity in human autoimmune arthritis is driven by the inflammatory environment. Proc Natl Acad Sci U S A. 2010. Aug 17;107(33):14751-6. Disclosure of Interest None Declared

Insulin-like growth factor binding protein-related protein 1 is expressed in rheumatoid synovium and regulates synovial fibroblast proliferation

Insulin-like growth factor binding protein-related protein 1 (IGFBP-rP1) is a secretory protein that shares a structural similarity with IGFBP. Studies have shown that IGFBP-rP1 synergistically increases fibroblast growth with insulin and stimulates angiogenesis in tumor tissues. In this report, we examined the expression and function of IGFBP-rP1 in rheumatoid arthritis (RA). IGFBP-rP1 expression in synovial tissues was examined by reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, and immunohistochemical analysis. In vitro, IGFBP-rP1 expression was examined in synovial fibroblasts established from rheumatoid synovium (RASFs) by RT-PCR, Western blot, and immunostaining. The effect of IGFBP-rP1 small interfering RNA (siRNA) on RASF proliferation was assessed by alamarBlue assay. IGFBP-rP1 mRNA was detected by RT-PCR in all synovial tissues from RA and OA patients. In immunohistochemical analysis, IGFBP-rP1 was mainly expressed in synovial cells in the lining layers and endothelial cells in the sublining layers of RA synovium. In vitro, constitutive expression of IGFBP-rP1 in RASFs was detected by RT-PCR, Western blot, and immunostaining. Treatment with IGFBP-rP1 siRNA induced a 26% decrease in RASF growth compared to control siRNA. A similar extent of growth-suppressive effect by IGFBP-rP1 siRNA was also observed when RASF proliferation was induced by TNF-α. Collectively, these data suggest that IGFBP-rP1 may regulate synovial fibroblast proliferation in RA.