Chieko Aoki

Interleukin-6 induces both cell growth and VEGF production in malignant mesotheliomas.

Malignant mesothelioma (MM), an incurable tumor, is reportedly an interleukin‐6 (IL‐6) secreting tumor. The pathological significance of IL‐6 overexpression in this tumor, however, has remained unclear. We investigated the biological functions of IL‐6 in mesotheliomas. Five mesothelioma cell lines were analyzed for IL‐6 production and IL‐6 receptor (IL‐6R) expression. Of them, 2 produced high levels of IL‐6, 2 produced intermediate levels and 1 cell line showed no secretion. All mesothelioma cell lines used in this study expressed very small amounts of IL‐6R mRNA. We compensated for this low level of IL‐6R expression in mesotheliomas by adding recombinant soluble IL‐6R (sIL‐6R) to mediate the IL‐6 signal. IL‐6 together with sIL‐6R was found to promote cell growth of H2052 and H226 MMs classified as high‐level IL‐6 producers in a dose‐dependent manner. Moreover, a humanized anti‐IL‐6R antibody (MRA) capable of blocking IL‐6 signaling suppressed the cell growth of mesotheliomas induced by IL‐6/sIL‐6R. These findings demonstrate that IL‐6 serves as an autocrine growth factor in the development of mesothelioma. In addition, IL‐6/sIL‐6R stimulation increased the expression of vascular endothelial growth factor (VEGF) in 4 out of 5 cell lines, and this induction was inhibited by MRA treatment. The involvement of the signal transducer and activator of transcription 3 (STAT3) pathway in both cell growth and VEGF induction by IL‐6/sIL‐6R was verified by dominant negative STAT3 transduction combined with adenovirus gene‐delivery methods. Although IL‐6 induces VEGF through the JAK2/STAT3 pathway, anti‐VEGF antibody could not inhibit the IL‐6‐induced cell growth observed in H2052 and H226. We concluded that IL‐6‐dependent growth does not occur via VEGF induction. These results suggest that treatment with anti‐IL‐6R antibody may constitute a potential molecular targeting therapy for MMs.

Establishment of a New Interleukin-6 (IL-6) Receptor Inhibitor Applicable to the Gene Therapy for IL-6-Dependent Tumor

Interleukin-6 (IL-6) is a key molecule involved in the pathogenesis of several inflammatory diseases and malignancies. Treatments that inhibit IL-6 mitigate the clinical conditions of such diseases. Here, we report on the development of a new receptor inhibitor of IL-6 (NRI) by genetically engineering tocilizumab, a humanized anti-IL-6 receptor monoclonal antibody which specifically blocks IL-6 signaling. This NRI consists of VH and VL of tocilizumab in a single-chain fragment format dimerized by fusing to the Fc portion of human immunoglobulin G(1). The binding activity to IL-6 receptor and the biological activity of the purified NRI were found to be similar to those of parental tocilizumab. Because NRI is encoded on a single gene, it is easily applicable to a gene delivery system using virus vehicles. We administered an adenovirus vector encoding NRI to mouse i.p. and monitored the serum NRI level and growth reduction property on S6B45, an IL-6-dependent multiple myeloma cell line, in vivo. Adequate amount of the serum NRI level to exert anti-IL-6 action could be obtained by the NRI gene introduction combined with adenovirus gene delivery, and this treatment inhibited the in vivo S6B45 cell growth significantly. These findings indicate that NRI is a promising agent applicable to the therapeutic gene delivery approach for IL-6-driven diseases.

Underexpression of mitochondrial-DNA encoded ATP synthesis-related genes and DNA repair genes in systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by various systemic symptoms and multiple organ damage. We clarify biological and functional abnormalities in SLE by comparing the gene expression profiles of SLE patients with those of healthy individuals.MethodsGene expression profiles from the peripheral blood of 21 SLE patients and 45 healthy individuals were obtained using a DNA microarray. Gene ontology analysis and network pathway analysis were performed on the genes differentially expressed between SLE and healthy individuals.ResultsA total of 2,329 upregulated genes and 1,884 downregulated genes were differentially expressed. Gene ontology analysis revealed that the upregulated genes were classified as response to biotic stimulus genes, which mainly includes genes related to immune response. Abnormalities in other categories such as cell motility and regulation of apoptosis were also revealed. Downregulated genes were mainly sorted into two gene categories, sensory perception and response to radiation/light. The sensory perception genes included ATPase/ATPase domain-containing genes, myosin-related genes, and two excision repair cross-complementing genes, which are involved in DNA repair. Other genes in this group - including three crystallin genes, genes encoding the receptor protein for melanocyte-stimulating hormone, and six mitochondrial-DNA encoded genes, which are involved in ATP synthesis - were also categorized as response to radiation genes. Using network pathway analysis, IL-6, transforming growth factor beta 1, TNF, and hepatocyte nuclear factor 4α were found to play central roles in the networks of sensory perception-related molecules.ConclusionsFunctional abnormalities in ATP synthesis and DNA repair are implicated in peripheral blood cells from SLE patients.

Abnormal expression of the genes involved in cytokine networks and mitochondrial function in systemic juvenile idiopathic arthritis identified by DNA microarray analysis.

Objectives: Systemic juvenile idiopathic arthritis (sJIA) is a rheumatic disease in childhood characterised by systemic symptoms and a relatively poor prognosis. Peripheral leukocytes are thought to play a pathological role in sJIA although the exact cause of the disease is still obscure. In this study, we aimed to clarify cellular functional abnormalities in sJIA. Methods: We analysed the gene expression profile in peripheral leukocytes from 51 patients with sJIA, 6 patients with polyarticular type JIA (polyJIA) and 8 healthy children utilising DNA microarrays. Gene ontology analysis and network analysis were performed on the genes differentially expressed in sJIA to clarify the cellular functional abnormalities. Result: A total of 3491 genes were differentially expressed in patients with sJIA compared to healthy individuals. They were functionally categorised mainly into a defence response group and a metabolism group according to gene ontology, suggesting the possible abnormalities in these functions. In the defence response group, molecules predominantly constituting interferon (IFN)γ and tumour necrosis factor (TNF) network cascades were upregulated. In the metabolism group, oxidative phosphorylation-related genes were downregulated, suggesting a mitochondrial disorder. Expression of mitochondrial DNA-encoded genes including cytochrome c oxidase subunit 1(MT-CO1) and MT-CO2 were suppressed in patients with sJIA but not in patients with polyJIA or healthy children. However, nuclear DNA-encoded cytochrome c oxidases were intact. Conclusion: Our findings suggest that sJIA is not only an immunological disease but also a metabolic disease involving mitochondria disorder.

Interactions among type I and type II interferon, tumor necrosis factor, and β-estradiol in the regulation of immune response-related gene expressions in systemic lupus erythematosus

IntroductionSystemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by various clinical manifestations. Several cytokines interact and play pathological roles in SLE, although the etiopathology is still obscure. In the present study we investigated the network of immune response-related molecules expressed in the peripheral blood of SLE patients, and the effects of cytokine interactions on the regulation of these molecules.MethodsGene expression profiles of peripheral blood from SLE patients and from healthy women were analyzed using DNA microarray analysis. Differentially expressed genes classified into the immune response category were selected and analyzed using bioinformatics tools. Since interactions among TNF, IFNγ, β-estradiol (E2), and IFNα may regulate the expression of interferon-inducible (IFI) genes, stimulating and co-stimulating experiments were carried out on peripheral blood mononuclear cells followed by analysis using quantitative RT-PCR.ResultsThirty-eight downregulated genes and 68 upregulated genes were identified in the functional category of immune response. Overexpressed IFI genes were confirmed in SLE patient peripheral bloods. Using network-based analysis on these genes, several networks including cytokines – such as TNF and IFNγ – and E2 were constructed. TNF-regulated genes were dominant in these networks, but in vitro TNF stimulation on peripheral blood mononuclear cells showed no differences in the above gene expressions between SLE and healthy individuals. Co-stimulating with IFNα and one of TNF, IFNγ, or E2 revealed that TNF has repressive effects while IFNγ essentially has synergistic effects on IFI gene expressions in vitro. E2 showed variable effects on IFI gene expressions among three individuals.ConclusionsTNF may repress the abnormal regulation by IFNα in SLE while IFNγ may have a synergistic effect. Interactions between IFNα and one of TNF, IFNγ, or E2 appear to be involved in the pathogenesis of SLE.

Abnormal networks of immune response-related molecules in bone marrow cells from patients with rheumatoid arthritis as revealed by DNA microarray analysis.

IntroductionRheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic synovitis that progresses to destruction of cartilage and bone. Bone marrow (BM) cells have been shown to contribute to this pathogenesis. In this study, we compared differentially expressed molecules in BM cells from RA and osteoarthritis (OA) patients and analyzed abnormal regulatory networks to identify the role of BM cells in RA.MethodsGene expression profiles (GEPs) in BM-derived mononuclear cells from 9 RA and 10 OA patients were obtained by DNA microarray. Up- and down-regulated genes were identified by comparing the GEPs from the two patient groups. Bioinformatics was performed by Expression Analysis Systemic Explorer (EASE) 2.0 based on gene ontology, followed by network pathway analysis with Ingenuity Pathways Analysis (IPA) 7.5.ResultsThe BM mononuclear cells showed 764 up-regulated and 1,910 down-regulated genes in RA patients relative to the OA group. EASE revealed that the gene category response to external stimulus, which included the gene category immune response, was overrepresented by the up-regulated genes. So too were the gene categories signal transduction and phosphate metabolism. Down-regulated genes were dominantly classified in three gene categories: cell proliferation, which included mitotic cell cycle, DNA replication and chromosome cycle, and DNA metabolism. Most genes in these categories overlapped with each other. IPA analysis showed that the up-regulated genes in immune response were highly relevant to the antigen presentation pathway and to interferon signaling. The major histocompatibility complex (MHC) class I molecules, human leukocyte antigen (HLA)-E, HLA-F, and HLA-G, tapasin (TAP) and TAP binding protein, both of which are involved in peptide antigen binding and presentation via MHC class I molecules, are depicted in the immune response molecule networks. Interferon gamma and interleukin 8 were overexpressed and found to play central roles in these networks.ConclusionsAbnormal regulatory networks in the immune response and cell cycle categories were identified in BM mononuclear cells from RA patients, indicating that the BM is pathologically involved in RA.

Interleukin 11 and paired immunoglobulin-like type 2 receptor α expression correlates with the number of joints with active arthritis in systemic juvenile idiopathic arthritis

Systemic juvenile idiopathic arthritis (sJIA) is characterised by systemic inflammatory symptoms such as spiking fever, skin rash, pericarditis and hepatosplenomegaly, along with arthritis.1 We reported the abnormal expression of genes involved in cytokine networks and mitochondrial function in patients with sJIA identified by DNA microarray.2 The current study was performed to extend these results. To identify genes that correlate with arthritis severity or systemic inflammation in patients with sJIA, we further analysed the 3491 genes identified by prior microarray analysis to be abnormally expressed in the peripheral blood of 51 patients with sJIA.2 They all fulfilled International League of Associations for Rheumatology (ILAR) criteria.3 Of these …

FRI0198 Expressions of immune response related genes were normalised after tocilizumab treatment in rheumatoid arthritis (RA) patients

Background Tocilizumab (TCZ), a humanised anti-interleukin (IL)6 receptor antibody, was shown to be therapeutically effective in RA patients but the biological effect of TCZ on the immune response was not fully understood. DNA microarray can be amenable to exhaustively analyse the gene expression (GE) including immune response related genes. Objectives To investigate the effect of TCZ on the immune response related GE profiles and to elucidate the changes of immune response functions in RA patients before and after treatments. Methods Two-color microarray was performed by using whole blood samples here. The baseline GE profiles of the 112 RA patients in the Japanese phase III SATORI study (1), in which active RA patients were allocated to receive either TCZ 8mg/kg every 4 weeks (TCZ group: 54 patients) or MTX 8mg/week (MTX/control group: 58 patients) for 24 weeks, and 45 healthy individuals (HI) were obtained. The effect of treatments was investigated by directly comparing the samples before versus after the treatment. Each investigation was performed in duplicate with DNA labeling color reversal (dye swap). The average values of log2 (after/before) were used for further analysis. The genes with median expression levels of 1.2-fold changed after TCZ treatment were functionally categorized using Expression Analysis Systematic Explorer (EASE) version 2.0 bioinformatics software. Significant differences of GE levels between 112 RA at baseline and 45 HI, and between 54 TCZ and 58 MTX treated RA patients were defined by Mann-Whitney test. Results The expressions of 491 genes were identified to have 1.2-fold changed in TCZ group. EASE analysis revealed that these genes mainly related to protein biosynthesis (EASE Score =1.64E-41), ribosome biogenesis (4.23E-10), and response to biotic stimulus (4.19E-04). 44 genes out of the 45 genes categorized to response to biotic stimulus were included in defense response category, in which 36 genes also belonged to immune response category. Arachidonate 5-lipoxygenase-activating protein, defensin alpha (DEFA)3, DEFA4, complement component 8 gamma polypeptide, platelet factor 4 (chemokine (C-X-C motif) ligand 4), pro-platelet basic protein (chemokine (C-X-C motif) ligand 7), S100A9, S100A12, IL-1 receptor type II, peptidoglycan recognition protein 1, and C-type lectin domain family 4 member E in this category were found significantly overexpressed in RA patients before TCZ treatment when compared to HI. Most remaining other genes such as major histocompatibility complex (MHC) class II molecules (HLA-DPA1, HLA-DPB1, HLA-DPQA1, HLA-DPQB1, HLA-DRA, and HLA-DMA), killer cell lectin-like receptor subfamily members (KLRB1, KLRD1, KLRK1, and KLRF1), granulysin, immunoglobulin J, and chemokine (C-C motif) ligand 5 were found significantly underexpressed. All the GEs were normalised after TCZ treatment whereas these were unchanged in MTX group. Conclusions TCZ treatment normalised the expressions of immune response related genes in RA patients. The therapeutic effect of TCZ may be partly contributed by normalisation of these genes. References Nishimoto N, et al. Mod Rheumatol. 2009;19:12-9 Disclosure of Interest N. Nishimoto Grant/Research support from: Chugai Pharamaceutical Co. Ltd., Consultant for: Chugai Pharamaceutical Co. Ltd., Roche group, H.-M. Lee: None Declared, M. Murakami: None Declared, C. Aoki: None Declared, Y. Li: None Declared, T. Matsutani: None Declared

THU0378 Interleukin-6 blocking therapy by tocilizumab in patients with multicentric castleman’s disease results in a significant decrease in serum levels of IgG4 and IGE

Background Multicentric Castleman disease (MCD) is a rare lymphoproliferative disorder characterized by systemic lymphadenopathy, constitutional inflammatory symptoms, and abnormal laboratories such as hyper-γ-globulinemia, increases in various auto-antibodies as well as acute phase proteins. While the symptoms are closely associated with dysregulated overproduction of interleukin (IL)-6, an increase in serum IgG4 and/or IgE levels in MCD patients is frequently observed. Blocking IL-6 with anti-IL-6 receptor antibody, tocilizumab (TCZ), ameliorates not only systemic inflammatory symptoms but also abnormal laboratories including hyper-γ-globulinemia. Objectives 1) To clarify differences in clinical features between MCD patients with high serum IgG4 and those with normal serum IgG4. 2) To analyze changes in serum IgG subclasses as well as IgE levels of MCD patients with TCZ treatment. Methods Serum levels of total IgG and four IgG subclasses as well as IgE of 18 MCD patients were measured. Eleven of 18 patients were treated with TCZ. Serum IgG subclass levels (n=11) and serum IgE levels (n=9) were compared before and after the treatment. Results Serum IgG4 levels at baseline were elevated in 12 of 18 MCD patients (542.2±387.7 mg/dl, n=12 vs. 45.2±39.8 mg/dl, n=6; the normal range is less than 105 mg/dl). Clinical features between MCD patients with high serum IgG4 and those with normal serum IgG4 were not distinct. Serum IgE levels at baseline were elevated in 12 of 16 MCD patients (3414.0±7198.2 IU/ml, n=12 vs. 95.8±66.4 IU/ml, n=4). All the 12 patients with high serum IgG4 showed elevated levels of IgE while only 2 out of 4 patients with normal IgG4 showed high IgE levels. IgG4 and IgE levels were significantly correlated. The mean absolute numbers of all IgG subclasses were significantly decreased after TCZ treatment. Furthermore, the mean ratio of IgG4 to total IgG was significantly decreased (7.4% vs. 5.5%, p<0.05, n=11), while those of the other subclasses did not change. The mean absolute number of IgE levels was also significantly decreased by TCZ treatment (1316.7 IU/ml vs. 481.0 IU/ml, p<0.05, n=9). Conclusions Decreases in the serum IgG4/IgG ratios and IgE levels after TCZ treatment suggests that IL-6 may be involved in the class switch to IgG4 and IgE. Disclosure of Interest M. Murakami: None Declared, T. Matsutani: None Declared, C. Aoki: None Declared, H.-M. Lee: None Declared, Y. Li: None Declared, N. Nishimoto Grant/Research support from: Chugai and Roche group, the product company of TCZ., Consultant for: Chugai and Roche group, the product company of TCZ.

FRI0206 Abatacept (CTLA4-IG) suppresses T cell activation and reduces TH17 cells as well as plasma IL-6 in patients with rheumatoid arthritis

Background Abatacept (CTLA4-Ig) controls RA via competitive inhibition of CD28 binding to CD80/CD86 ligands. However, the mechanism of action of abatacept in human remains inadequately understood. Objectives To elucidate how abatacept treatment achieves therapeutic effect on RA, we examined alterations of T cell phonotypes, T cell subsets, and plasma cytokine profiles in RA patients during abatacept treatment. Methods PBMCs and plasmas were collected from healthy individuals (HI, n=15) and RA patients before (RA-0M, n=45) and 6 months after treatment (RA-6M, n=25). Paired data from identical patients were included (n=24). Surface phenotypes (CD3, CD4, CD8, CD28, CD45RO) and activation markers (CD25, CD69, CD62L-) of T cells were analyzed with FACS. Treg cells (CD4+CD25+Foxp3+) were measured with intracellular staining with anti-Foxp3 antibody. After in vitro stimulation with PMA/Ionomycin, cells were intracellularly stained with anti-IFN-γ, anti-IL-4 or anti-IL-17A antibodies. The plasma levels of IL-2, IL-4, IL-6, IL-10, IFN-γ, TNF were measured using bead-based cytometric bead array. Changes in the proportions of T cell subsets and the plasma cytokine levels were examined between before and 6 months after abatacept treatment. Statistical analyses were done using one-way ANOVA (multiple tests) or Wilcoxon matched paired test (paired data). Results There were little differences of proportion of CD25+ on CD4+ between RA and HI. However, the proportion and the mean fluorescent intensity of CD25 significantly decreased and became significantly lower than in HI after abatacept treatment. Proportions of Treg cells (CD25+Foxp3+) in CD4+ T cells and their absolute numbers were not significantly different among HI, RA-0M and RA-6M. Th1 (CD4+IFN-γ+) and Th2 (CD4+IL-4+) cells significantly increased in RA-0M compared with HI and didn’t change during treatment. Meanwhile, Th17 (CD4+IL-17A+) cells were significantly abundant in RA-0M than in HI and significantly reduced at 6 months after treatment. Similarly, high levels of plasma IL-6 were detected in RA-0M and significantly decreased after treatment. Conclusions Abatacept is suggested to inhibit IL-2 dependent CD4+ T cell proliferation by decreasing the expression of CD25 (IL-2Rα). Given that naïve CD4+ cells differentiate into IL-17 producing Th17 in the presence of IL-6 and that IL-17 induces RANKL expression in osteoblast/synovial fibroblast, these results suggest the possibility that abatacept has suppressive effect on IL-6 production and Th17 differentiation, resulting in reduction of synovial inflammation and osteoclastogenesis in rheumatoid arthritic joint. Disclosure of Interest T. Matsutani: None Declared, Y. Li: None Declared, M. Murakami: None Declared, H.-M. Lee: None Declared, C. Aoki: None Declared, M. Sekiguchi Grant/Research support from: Bristol-Myers Squibb Japan, K. Matsui: None Declared, M. Kitano Grant/Research support from: Bristol-Myers Squibb Japan, M. Namiki: None Declared, K. Ohmura Grant/Research support from: Bristol-Myers Squibb Japan, K. Murakami: None Declared, T. Fujii Grant/Research support from: Bristol-Myers Squibb Japan, T. Kuroiwa: None Declared, Y. Shimaoka: None Declared, H. Nakahara: None Declared, K. Maeda: None Declared, S. Irimajiri Grant/Research support from: Bristol-Myers Squibb Japan, M. Funauchi Grant/Research support from: Bristol-Myers Squibb Japan, Y. Imura: None Declared, T. Ikawa: None Declared, A. Nanpei: None Declared, T. Azuma: None Declared, T. Sasaki: None Declared, A. Yokota: None Declared, Y. Kawahito Grant/Research support from: Bristol-Myers Squibb Japan, T. Mimori Grant/Research support from: Bristol-Myers Squibb Japan, H. Sano Grant/Research support from: Bristol-Myers Squibb Japan, N. Nishimoto Grant/Research support from: Bristol-Myers Squibb Japan