Y. Arinobu

Novel Regulatory Mechanisms of CD40-Induced Prostanoid Synthesis by IL-4 and IL-10 in Human Monocytes

Interleukins IL-4 and IL-10 are considered to be central regulators for the limitation and eventual termination of inflammatory responses in vivo, based on their potent anti-inflammatory effects toward LPS-stimulated monocytes/macrophages and neutrophils. However, their role in T cell-dependent inflammatory responses has not been fully elucidated. In this study, we investigated the effects of both cytokines on the production of PGE2, a key molecule of various inflammatory conditions, in CD40-stimulated human peripheral blood monocytes. CD40 ligation of monocytes induced the synthesis of a significant amount of PGE2 via inducible expression of the cyclooxygenase (COX)-2 gene. Both IL-10 and IL-4 significantly inhibited PGE2 production and COX-2 expression in CD40-stimulated monocytes. Using specific inhibitors for extracellular signal-related kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), we found that both kinase pathways are involved in CD40-induced COX-2 expression. CD40 ligation also resulted in the activation of NF-κB. Additional experiments exhibited that CD40 clearly induced the activation of the upstream kinases MAPK/ERK kinase 1/2, MAPK kinase 3/6, and I-κB in monocytes. IL-10 significantly inhibited CD40-induced activation of the ERK, p38 MAPK, and NF-κB pathways; however, inhibition by IL-4 was limited to the ERK pathway in monocytes. Neither IL-10 nor IL-4 affected the recruitment of TNFR-associated factors 2 and 3 to CD40 in monocytes. Collectively, IL-10 and IL-4 use novel regulatory mechanisms for CD40-induced prostanoid synthesis in monocytes, thus suggesting a potential role for these cytokines in regulating T cell-induced inflammatory responses, including autoimmune diseases.

Two polymorphisms within interleukin-3 (hIL3) gene detected by mismatch PCR/RFLP.

Two alleles of IL-3 have been reported to GenBank (GenBank M14743, M20137). The sequence difference between these two alleles is at the first nucleotide of the 27th codon (the 131st nucleotide from the initiation site): thymine and cytosine, and leading the amino acid difference: proline and serine (Pro27Ser). The other allelism, thymine and cytosine, was also observed at position −16 of the IL-3 upstream promotor region (GenBank L10616, M60870). We clarified that these substitutions were frequent polymorphisms in the Japanese population by using the mismatch-PCR (polymerase chain reaction)/RFLP (restriction fragment length polymorphism) method.

OP0093 Comparison of CD34-Selected and Unmanipulated Autologous Hematopoietic Stem Cell Transplantation for Systemic Sclerosis: Four-Year Follow-Up Results

Background The superior efficacy of autologous hematopoietic stem cell transplantation (auto-HSCT) versus intravenous pulses cyclophosphamide was demonstrated in a randomized phase II trial in 19 severe systemic sclerosis (SSc) patients and a large randomized phase III trial in 156 patients. However, the usefulness of CD34-selection in auto-HSCT for SSc has not been investigated. Objectives The aim of this study is to compare the safety and the efficacy of CD34-selected auto-HSCT with those of unmanipulated auto-HSCT for severe SSc during a four-year follow-up period. Methods Nineteen patients (4 male and 15 female) with severe SSc were enrolled. Peripheral blood stem cells (PBSCs) were mobilized with cyclophosphamide (CY, 4 g/m2) and G-CSF. After collecting PBSC more than 2×106CD34+ cells/kg by apheresis, CD34+ cells were immunologically selected in 11 patients. All of the patients were treated with high-dose CY (200 mg/kg) and received CD34-selected (n=11; group A) or unmanipulated (n=8; a group B) auto-HSCT. Immune reconstitution was evaluated serially by analyzing lymphocyte subpopulations for 48 months after HSCT. Results The age of the patients was 52.3±7.5 years in group A and 55.1±5.5 years in group B. The modified Rodnans skin score was 21.5±14.0 in group A and 22.9±9.0 in group B. Numbers of the infused CD34+ and CD3+ cells were 4.7±2.6×106 and 0.011±0.012×106/kg, respectively, in group A and 7.6±10.7×106 and 45.6±28.1×106/kg, respectively, in group B. The time to engraftment of neutrophil and platelet was not different between two groups. There was no treatment-related mortality in both groups. As toxicity, 2, 5 and 6 out of 11 patients had adenoviral hemorrhagic cystitis, herpes zoster and cytomegaloviral antigenemia, respectively, in the group A, on the other hand, no patients had adenoviral hemorrhagic cystitis, herpes zoster and only 2 out of 8 patients had cytomegaloviral antigenemia, respectively, in group B. All of the patients in both groups had marked improvement of skin sclerosis within 3 months and the improvement was sustained for 48 months after auto-HSCT. Surprisingly, the reduction of the skin scores was significantly greater at 1 and 12-48 months after auto-HSCT in group A than in group B. About the effect on interstitial pneumonia, %VC increased continuously until 48 months after auto-HSCT in group A, on the other hand, it once increased but decreased after 12 months in group B. The recovery of lymphocyte subpopulations after auto-HSCT was not significantly different between two groups. Conclusions CD34-selected auto-HSCT had greater effects but more susceptiblity to viral infections than unmanipulated auto-HSCT. References Tsukamoto H, Nagafuji K, Horiuchi T, et al. A Phase I-II Trial of Autologous Peripheral Blood Stem Cell Transplantation in the Treatment of Refractory Autoimmune Disease. Ann Rheum Dis. 65: 508-14, 2006. Tsukamoto H, Nagafuji K, Horiuchi T, et al. Analysis of immune reconstitution after autologous CD34+ stem/progenitor cell transplantation for systemic sclerosis: predominant reconstitution of Th1 CD4+ T cells. Rheumatology (Oxford) 50: 944-52, 2011. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3166

AB0201 Cell Surface Expression of DNAM-1 (CD226) on CD8+ T Cells is Increased in Patients with Systemic Sclerosis

Background DNAM-1 (CD226) is well known as a genetic risk factor of many autoimmune diseases such as systemic sclerosis (SSc). More recently, Avouac et al. have reported that mice deficient for DNAM-1 are protected from bleomycin-induced dermal fibrosis. In human, however, the roles of DNAM-1 in SSc are still unknown. Objectives To evaluate cell surface expression of DNAM-1 on peripheral blood lymphocytes from SSc patients and to identify the roles of DNAM-1 in the pathogenesis of SSc. Methods The expression of DNAM-1 was analyzed by flow cytometry (FCM) on surface of CD4+ T cells, CD8+ T cells and CD19+ B cells from 41 SSc patients (median age, 59 years; female, 37) and 23 healthy controls (HCs). We evaluated the relationship between DNAM-1 expression level on CD8+ T cells and clinical manifestations of SSc. Intracellular and extracellular cytokine production in CD8+ T cells with or without DNAM-1 was measured by FCM (intracellular staining and multiplex bead array, respectively) after stimulation with PMA/Ionomycin. The cytotoxic capacity of CD8+ T cells against human umbilical vein endothelial cells (HUVECs) was assessed in the presence or absence of anti-DNAM-1 neutralizing antibody in SSc patients and HCs. Results The proportion of DNAM-1high CD8+ T cells was increased in SSc patients (median, 52.5%) compared to HCs (29.7%, P=0.0002). We found no significant difference between SSc patients and HCs in CD4+ T cells and CD19+ B cells. The expression of DNAM-1 on CD8+ T cells was higher in patients with diffuse cutaneous subtype than those with limited cutaneous subtype (P=0.0009) and was higher in those with interstitial pneumonia than those without (P=0.0016). The percentage of DNAM-1high CD8+ T cells was significantly correlated with modified Rodnan skin thickness score (r =0.4295, P=0.0071). The production of IL-13 was significantly increased in DNAM-1 positive CD8+ T cells compared with DNAM-1 negative ones and the neutralization of DNAM-1 impaired the IL-13 production from CD8+ T cells. The cytotoxic ability of CD8+ T cells was significantly elevated in SSc patients and positively correlated with the percentage of DNAM-1high CD8+ T cells. The cytotoxicity against HUVECs was partially inhibited in the presence of anti-DNAM-1 neutralizing antibody. Conclusions These findings indicate that DNAM-1high CD8+ T cells are involved in the pathogenesis of SSc via the production of profibrotic IL-13 and endothelial cell injury. References Avouac J, Elhai M, Tomcik M, et al. Critical role of the adhesion receptor DNAX accessory molecule-1 (DNAM-1) in the development of inflammation-driven dermal fibrosis in a mouse model of systemic sclerosis. Ann Rheum Dis. 2013;72:1089-98. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3217

THU0063 Serum Progranulin Levels are Elevated in Dermatomyositis Patients with Acute Interstitial Lung Disease, Reflecting Severity and Prognosis

Background Recently, it has been reported that granulin (GRN) and/or progranulin (PGRN), precursor of GRN, is a soluble cofactor for TLR9 signaling [1]. We reported that serum PGRN levels are associated with SLE global activity and PGRN may have a role in the pathogenesis via increased cytokine production [2]. TLR9 is also involved in the pathological condition of dermatomyositis (DM). Moreover, DM is occasionally complicated with interstitial lung disease (ILD). However, the roles of PGRN and GRN in DM are still unknown. Objectives To determine if serum PGRN levels are elevated in DM patients, in particular, complicated with ILD and are associated with severity and prognosis. Methods The serum levels of PGRN were measured by ELISA in patients with DM (n=50; acute/subacute interstitial pneumonia (A/SIP), defined as a rapidly progressive ILD within 3 months from the onset of symptoms: n=13, chronic interstitial pneumonia (CIP): n=20, without ILD: n=17), polymyositis (PM, n=21) and normal healthy controls (NHCs, n=60). We assessed the correlation between the serum PGRN levels and the activity indexes of ILD. The sera from some of the patients were reevaluated after the disease was ameliorated by treatment (n=6). Moreover, we calculated the cumulative survival rate for 6 months in DM patients with ILD, which is classified two groups, serum PGRN levels > or =200 ng/ml and < 200 ng/ml. Results The serum PGRN levels were significantly higher in the DM patients (median: 100 ng/ml) than in the PM patients (60.4 ng/ml, P=0.0028) and NHCs (48.3 ng/ml, P<0.0001). Of the total DM patients, the levels were significantly higher in DM with A/SIP than that in DM with CIP (P<0.0001) or without ILD (P=0.0002). Proportion of clinically amyopathic DM is higher in DM with A/SIP than in DM with CIP or without ILD (76.9%, 40%, and 5.9%, respectively). The serum PGRN levels correlated significantly with serum ferritin (rs=0.71, P=0.0001), LDH (rs=0.59, P=0.0003), and CRP (rs=0.57, P=0.0005) levels. They significantly decreased following successful treatment of DM (P=0.0313). Moreover, the cumulative survival rate for 6 months was significantly lower in the group with serum PGRN levels > or =200 ng/ml (60%) than that in the group with serum PGRN levels < 200 ng/ml (P=0.001). Conclusions These findings indicate that PGRN is associated with severity and prognosis of DM with ILD. PGRN may play a role in the pathogenesis of DM by affecting the TLR9 signaling and could be a useful biomarker. References Park B, Buti L, Lee S, et al. Granulin is a soluble cofactor for toll-like receptor 9 signaling. Immunity 2011;34:505-513. Tanaka A, Tsukamoto H, Mitoma H, et al. Serum progranulin levels are elevated in patients with systemic lupus erythematosus, reflecting disease activity. Arthritis Res Ther 2012;14(6):R244. Disclosure of Interest None Declared

THU0238 Long-term follow-up of autologous hematopoietic stem cell transplantation for severe systemic sclerosis

Background Recent phase II study demonstrated that autologous hematopoietic stem cell transplantation (auto-HSCT) is more effective than conventional intravenous cyclophosphamide in the treatment of severe systemic sclerosis (SSc). However, the long-term results of CD34-selectiod or unmanipulated auto-HSCT for SSc has not been investigated. Objectives The aim of this study is to investigate the long-term results of auto-HSCT for severe SSc and to compare efficacy of CD34-selected auto-HSCT with that of unmanipulated auto-HSCT. Methods Nineteen patients (4 male and 15 female) with severe SSc were enrolled. Peripheral blood stem cells (PBSCs) were mobilized with cyclophosphamide (CY, 4 g/m2) and G-CSF. After collecting PBSCs more than 2×106CD34+cells/kg by apheresis, CD34+ cells were immunologically selected in 11 patients. All of the patients were treated with high-dose CY (200 mg/kg) and received CD34-selected (n=11; a group with CD34-selection) or unmanipulated (n=8; a group without CD34-selection) auto-HSCT. Immune reconstitution was evaluated serially by analyzing lymphocyte subpopulations for 36 months after HSCT. Results There was no treatment-related mortality. As toxicity, there were a variety of post-transplant infections such as adenoviral hemorrhagic cystitis, herpes zoster and cytomegaloviral antigenemia. Sclerosis of the skin was markedly improved in all of the patients within 6 months and the improvement was sustained for 60 months after auto-HSCT. Vital capacity was significantly increased at 48 months after HSCT and KL-6, a marker for interstitial pneumonia (IP), was significantly decreased in patients with IP during 12-60 months after HSCT. A titer of anti-Scl-70 was significantly decreased during 1-60 months after HSCT. Progression-free and overall 5-year survivals were 68 and 95%, respectively. Interestingly, the reduction of the skin scores was significantly greater and viral infections were more frequent in the patients with CD34-selection than those without. Effect of auto-HSCT on interstitial pneumonia, and the recovery of lymphocyte subpopulations after auto-HSCT were not significantly different between two groups. Conclusions Auto-HSCT had long-term effects on skin sclerosis and IP, resulting in improved prognosis in patients with severe SSc. CD34-selected auto-HSCT had greater effect on skin sclerosis than unmanipulated auto-HSCT. References Tsukamoto H, Nagafuji K, Horiuchi T, et al. A Phase I-II Trial of Autologous Peripheral Blood Stem Cell Transplantation in the Treatment of Refractory Autoimmune Disease. Ann Rheum Dis. 65: 508-14, 2006. Tsukamoto H, Nagafuji K, Horiuchi T, et al. Analysis of immune reconstitution after autologous CD34+ stem/progenitor cell transplantation for systemic sclerosis: predominant reconstitution of Th1 CD4+ T cells. Rheumatology 2011; 50: 944-52. Disclosure of Interest None Declared

THU0168 Serum progranulin levels are elevated in patients with systemic lupus erythematosus, reflecting disease activity

Background Granulin (GRN) is a soluble cofactor for TLR9 signaling and enhances the interaction between TLR9 and CpG-DNA. TLR9 is involved in the pathogenesis of systemic lupus erythematosus (SLE) via this DNA-containing immune complex. The roles of progranulin (PGRN) and GRN in SLE are still unknown. Objectives To determine if serum PGRN levels are elevated in patients with SLE and are correlated with disease activity and to investigate the role of PGRN in the pathogenesis of SLE. Methods The serum levels of PGRN and IL-6 were measured by ELISA in patients with SLE (n=68; active (SLEDAI score ≥6): n=46, inactive: n=22; female: n=58, male: n=10, median age=37 years), age- and sex-matched rheumatoid arthritis (RA, n=20) and normal healthy controls (NHCs, n=32). We assessed the correlation between the serum PGRN levels and an established disease activity index. The sera from the patients with high PGRN titers (>80 ng/ml) at the initial evaluation were reevaluated after the disease was ameliorated by treatment (n=15). We also measured the IL-6 secreted by peripheral blood mononuclear cells (PBMCs) from healthy donors stimulated with 1) CpG-B in the presence or absence of recombinant human PGRN (rhPGRN) 2) lupus serum in the presence or absence of an anti-PGRN antibody. Results The serum PGRN levels were significantly higher in the SLE patients (median: 87.6 ng/ml) than in the RA patients (54.2 ng/ml, P=0.0082) and NHCs (44.5 ng/ml, P<0.0001). The levels significantly correlated with the presence of malar rash/discoid rash (P=0.0021), alopecia (P=0.026), serositis (P=0.0038), arthritis (P=0.0003), thrombocytopenia (P=0.026), leucopenia (P=0.0004), anemia (P=0.0071), anti-Sm (P=0.03), or anti-nRNP (P=0.0028) antibody. They also correlated significantly with SLEDAI (r=0.53, P=0.0003) and anti-double stranded DNA antibody titers (r=0.45, P=0.0023) and were inversely associated with CH50 (r=-0.47, P=0.01), C3 (r=-0.41, P=0.002), and C4 (r=-0.37, P=0.0035) levels. Moreover, serum PGRN levels significantly decreased following successful treatment of SLE (P<0.0001). The rhPGRN significantly upregulated the production of IL-6 by PBMCs stimulated with CpG-B and neutralizing PGRN in the lupus serum significantly attenuated the production of IL-6 by PBMCs in vitro (respectively, P<0.05). The serum PGRN levels significantly correlated with the serum IL-6 levels (r=0.47, P<0.0001). Conclusions These findings indicate that PGRN is associated with global activities of lupus. PGRN may play a role in the pathogenesis of SLE by affecting the TLR9 signaling elicited by immune complexes and could therefore be a therapeutic target for SLE. References Park B, Buti L, Lee S, et al. Granulin is a soluble cofactor for toll-like receptor 9 signaling. Immunity 2011;34:505-513. Disclosure of Interest None Declared

FRI0406 Seven-year follow-up of autologous hematopoietic stem cell transplantation for severe systemic sclerosis

Background Autologous stem cell transplantation for international scleroderma (ASTIS) trial demonstrated that autologous hematopoietic stem cell transplantation (auto-HSCT) is more effective than conventional intravenous cyclophosphamide in the treatment of severe systemic sclerosis (SSc). However, there is paucity of the study showing the long-term efficacy of auto-HSCT for severe SSc. Objectives The aim of this study is to investigate seven-year follow-up results of auto-HSCT for severe SSc. Methods Nineteen patients (4 male and 15 female) with severe SSc were enrolled. Peripheral blood stem cells (PBSCs) were mobilized with cyclophosphamide (CY, 4 g/m2) and G-CSF. After collecting PBSCs more than 2x 106CD34+cells/kg by apheresis, CD34+ cells were immunologically selected in 11 patients. All of the patients were treated with high-dose CY (200 mg/kg) and received CD34-selected (n=11; a group with CD34-selection) or unmanipulated (n=8; a group without CD34-selection) auto-HSCT. Immune reconstitution was evaluated serially by analyzing lymphocyte subpopulations for 36 months after HSCT. Results There was no treatment-related mortality. As toxicity, there were a variety of post-transplant infections such as adenoviral hemorrhagic cystitis, herpes zoster and cytomegaloviral antigenemia. Sclerosis of the skin was markedly improved in all of the patients within 6 months and the improvement was sustained for 84 months after auto-HSCT. Vital capacity was significantly increased at 48 and 60 months after HSCT and KL-6, a marker for interstitial pneumonia (IP), was significantly decreased in patients with IP during 12-84 months after HSCT. A titer of anti-Scl-70 was significantly decreased during 1-84 months after HSCT. Five and seven years after auto-HSCT, the overall survival was 89 and 78%, and the progression-free survival was 65 and 57%, respectively. Interestingly, the reduction of the skin scores was significantly greater and viral infections were more frequent in the patients with CD34-selection than those without. Effect of auto-HSCT on interstitial pneumonia, and the recovery of lymphocyte subpopulations after auto-HSCT were not significantly different between two groups. Conclusions Auto-HSCT was effective in the treatment of SSc and the effect was sustained for 7 years. References Tsukamoto H, Nagafuji K, Horiuchi T, et al. A Phase I-II Trial of Autologous Peripheral Blood Stem Cell Transplantation in the Treatment of Refractory Autoimmune Disease. Ann Rheum Dis. 65: 508-14, 2006. Tsukamoto H, Nagafuji K, Horiuchi T, et al. Analysis of immune reconstitution after autologous CD34+ stem/progenitor cell transplantation for systemic sclerosis: predominant reconstitution of Th1 CD4+ T cells. Rheumatology 2011; 50: 944-52. Disclosure of Interest None Declared