DEK-targeting DNA aptamers as therapeutics for inflammatory arthritis.

Novel therapeutics are required for improving the management of chronic inflammatory diseases. Aptamers are single-stranded RNA or DNA molecules that have recently shown utility in a clinical setting, as they can specifically neutralize biomedically relevant proteins, particularly cell surface and extracellular proteins. The nuclear chromatin protein DEK is a secreted chemoattractant that is abundant in the synovia of patients with juvenile idiopathic arthritis (JIA). Here, we show that DEK is crucial to the development of arthritis in mouse models, thus making it an appropriate target for aptamer-based therapy. Genetic depletion of DEK or treatment with DEK-targeted aptamers significantly reduces joint inflammation in vivo and greatly impairs the ability of neutrophils to form neutrophil extracellular traps (NETs). DEK is detected in spontaneously forming NETs from JIA patient synovial neutrophils, and DEK-targeted aptamers reduce NET formation. DEK is thus key to joint inflammation, and anti-DEK aptamers hold promise for the treatment of JIA and other types of arthritis.

Inflammation-Induced Oxidative Stress Mediates Gene Fusion Formation in Prostate Cancer.

Approximately 50% of prostate cancers are associated with gene fusions of the androgen-regulated gene TMPRSS2 to the oncogenic erythroblast transformation-specific (ETS) transcription factor ERG. The three-dimensional proximity of TMPRSS2 and ERG genes, in combination with DNA breaks, facilitates the formation of TMPRSS2-ERG gene fusions. However, the origins of DNA breaks that underlie gene fusion formation in prostate cancers are far from clear. We demonstrate a role for inflammation-induced oxidative stress in the formation of DNA breaks leading to recurrent TMPRSS2-ERG gene fusions. The transcriptional status and epigenetic features of the target genes influence this effect. Importantly, inflammation-induced de novo genomic rearrangements are blocked by homologous recombination (HR) and promoted by non-homologous end-joining (NHEJ) pathways. In conjunction with the association of proliferative inflammatory atrophy (PIA) with human prostate cancer, our results support a working model in which recurrent genomic rearrangements induced by inflammatory stimuli lead to the development of prostate cancer.

Scleroderma dermal microvascular endothelial cells exhibit defective response to pro-angiogenic chemokines.

OBJECTIVES: Angiogenesis plays a critical role in SSc (scleroderma). The aim of this study was to examine the expression of growth-regulated protein-γ (Gro-γ/CXCL3), granulocyte chemotactic protein 2 (GCP-2/CXCL6) and their receptor CXCR2 in endothelial cells (ECs) isolated from SSc skin and determine whether these cells mount an angiogenic response towards pro-angiogenic chemokines. The downstream signalling pathways as well as the pro-angiogenic transcription factor inhibitor of DNA-binding protein 1 (Id-1) were also examined. METHODS: Skin biopsies were obtained from patients with dcSSc. ECs were isolated via magnetic positive selection. Angiogenesis was measured by EC chemotaxis assay. RESULTS: Gro-γ/CXCL3 and GCP-2/CXCL6 were minimally expressed in both skin types but elevated in SSc serum. Pro-angiogenic chemokine mRNA was greater in SSc ECs than in normal ECs. SSc ECs did not migrate to vascular endothelial growth factor (VEGF), Gro-γ/CXCL3, GCP-2/CXCL6 or CXCL16. The signalling pathways stimulated by these chemokines were also dysregulated. Id-1 mRNA in SSc ECs was lower compared with normal ECs, and overexpression of Id-1 in SSc ECs increased their ability to migrate towards VEGF and CXCL16. CONCLUSION: Our results show that SSc ECs are unable to respond to pro-angiogenic chemokines despite their increased expression in serum and ECs. This might be due to the differences in the signalling pathways activated by these chemokines in normal vs SSc ECs. In addition, the lower expression of Id-1 also decreases the angiogenic response. The inability of pro-angiogenic chemokines to promote EC migration provides an additional mechanism for the impaired angiogenesis that characterizes SSc.

A key role for Fut1-regulated angiogenesis and ICAM-1 expression in K/BxN arthritis.

OBJECTIVES: Angiogenesis contributes to the pathogenesis of rheumatoid arthritis. Fucosyltransferases (Futs) are involved in angiogenesis and tumour growth. Here, we examined the role of Fut1 in angiogenesis and K/BxN serum transfer arthritis. METHODS: We examined Fut1 expression in human dermal microvascular endothelial cells (HMVECs) by quantitative PCR. We performed a number of angiogenesis assays to determine the role of Fut1 using HMVECs, Fut1 null (Fut1(-/-)), and wild type (wt) endothelial cells (ECs) and mice. K/BxN serum transfer arthritis was performed to determine the contribution of Fut1-mediated angiogenesis in Fut1(-/-) and wt mice. A static adhesion assay was implemented with RAW264.7 (mouse macrophage cell line) and mouse ECs. Quantitative PCR, immunofluorescence and flow cytometry were performed with Fut1(-/-) and wt ECs for adhesion molecule expression. RESULTS: Tumour necrosis factor-α induced Fut1 mRNA and protein expression in HMVECs. HMVECs transfected with Fut1 antisense oligodeoxynucleotide and Fut1(-/-) ECs formed significantly fewer tubes on Matrigel. Fut1(-/-) mice had reduced angiogenesis in Matrigel plug and sponge granuloma angiogenesis assays compared with wt mice. Fut1(-/-) mice were resistant to K/BxN serum transfer arthritis and had decreased angiogenesis and leucocyte ingress into inflamed joints. Adhesion of RAW264.7 cells to wt mouse ECs was significantly reduced when Fut1 was lacking. Fut1(-/-) ECs had decreased intercellular adhesion molecule-1 (ICAM-1) expression at mRNA and protein levels compared with wt ECs. ICAM-1 was also decreased in Fut1(-/-) arthritic ankle cryosections compared with wt ankles. CONCLUSIONS: Fut1 plays an important role in regulating angiogenesis and ICAM-1 expression in inflammatory arthritis.

NK4 therapy: a new approach to target angiogenesis and inflammation in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a progressive autoimmune disease characterized by synovial membrane hyperplasia, inflammation, and angiogenesis. Hepatocyte growth factor (HGF) and its receptor, c-Met, are both overexpressed in the RA synovium. NK4 is an antagonist of HGF which has been shown to inhibit tumor growth, metastasis, and angiogenesis. In an experimental model of RA, NK4 gene therapy inhibited joint damage and inflammation in both preventative and therapeutic models. NK4 treatment therefore represents a possible therapeutic option in combating RA.

ADAM-10 is overexpressed in rheumatoid arthritis synovial tissue and mediates angiogenesis.

OBJECTIVE: To examine the expression of ADAM-10 in rheumatoid arthritis (RA) synovial tissue (ST) and the role it plays in angiogenesis. METHODS: ADAM-10 expression was determined using immunohistology, Western blotting, and quantitative polymerase chain reaction. In order to examine the role of ADAM-10 in angiogenesis, we performed in vitro Matrigel tube formation and chemotaxis assays using human microvascular endothelial cells (HMVECs) transfected with control or ADAM-10 small interfering RNA (siRNA). To determine whether ADAM-10 plays a role in angiogenesis in the context of RA, we performed Matrigel assays using a coculture system of HMVECs and RA synovial fibroblasts. RESULTS: Endothelial cells and lining cells within RA ST expressed high levels of ADAM-10 compared with cells within osteoarthritis ST and normal ST. ADAM-10 expression was significantly elevated at the protein and messenger RNA levels in HMVECs and RA synovial fibroblasts stimulated with proinflammatory mediators compared with unstimulated cells. ADAM-10 siRNA-treated HMVECs had decreased endothelial cell tube formation and migration compared with control siRNA-treated HMVECs. In addition, ADAM-10 siRNA-treated HMVECs from the RA synovial fibroblast coculture system had decreased endothelial cell tube formation compared with control siRNA-treated HMVECs. CONCLUSION: These data show that ADAM-10 is overexpressed in RA and suggest that ADAM-10 may play a role in RA angiogenesis. ADAM-10 may be a potential therapeutic target in inflammatory angiogenic diseases such as RA.

H-2g, a glucose analog of blood group H antigen, mediates monocyte recruitment in vitro and in vivo via IL-8/CXCL8.

OBJECTIVE: Monocyte (MN) recruitment is an essential inflammatory component of many autoimmune diseases, including rheumatoid arthritis (RA). In this study we investigated the ability of 2-fucosyllactose (H-2g), a glucose analog of blood group H antigen to induce MN migration in vivo and determined if H-2g-induced interleukin-8 (IL-8/CXCL8) plays a role in MN ingress in RA. METHODS: Sponge granuloma and intravital microscopy assays were performed to examine H-2g-induced in vivo MN migration and rolling, respectively. MNs were stimulated with H-2g, and the production of IL-8/CXCL8 was assessed by enzyme-linked immunosorbent assay and quantitative polymerase chain reaction. Lastly, in vitro MN migration assays and an in vivo RA synovial tissue severe combined immunodeficiency mouse model were used to determine the role of IL-8/CXCL8 in H-2g-induced MN migration. RESULTS: In vivo, H-2g induced significantly greater MN migration compared to phosphate buffered saline. Intravital microscopy revealed that H-2g mediates MN migration in vivo by inducing MN rolling. In addition, H-2g induced MN production of IL-8/CXCL8, a process that was dependent on Src kinase. Moreover, we found that H-2g mediated MN migration in vitro, and in vivo migration was inhibited by a neutralizing anti-IL-8/CXCL8 antibody. CONCLUSION: These findings suggest that H-2g mediates MN recruitment in vitro and in vivo (in part) via IL-8/CXCL8.

Type I interferons modulate vascular function, repair, thrombosis, and plaque progression in murine models of lupus and atherosclerosis.

OBJECTIVE: Patients with systemic lupus erythematosus (SLE) have a notable increase in atherothrombotic cardiovascular disease (CVD) which is not explained by the Framingham risk equation. In vitro studies indicate that type I interferons (IFNs) may play prominent roles in increased CV risk in SLE. However, the in vivo relevance of these findings, with regard to the development of CVD, has not been characterized. This study was undertaken to examine the role of type I IFNs in endothelial dysfunction, aberrant vascular repair, and atherothrombosis in murine models of lupus and atherosclerosis. METHODS: Lupus-prone New Zealand mixed 2328 (NZM) mice and atherosclerosis-prone apolipoprotein E- knockout (apoE(-/-) ) mice were compared to mice lacking type I IFN receptor (INZM and apoE(-/-) IFNAR(-/-) mice, respectively) with regard to endothelial vasodilatory function, endothelial progenitor cell (EPC) function, in vivo neoangiogenesis, plaque development, and occlusive thrombosis. Similar experiments were performed using NZM and apoE(-/-) mice exposed to an IFNα-containing or empty adenovirus. RESULTS: Loss of type I IFN receptor signaling improved endothelium-dependent vasorelaxation, lipoprotein parameters, EPC numbers and function, and neoangiogenesis in lupus-prone mice, independent of disease activity or sex. Further, acute exposure to IFNα impaired endothelial vasorelaxation and EPC function in lupus-prone and non-lupus-prone mice. Decreased atherosclerosis severity and arterial inflammatory infiltrates and increased neoangiogenesis were observed in apoE(-/-) IFNAR(-/-) mice, compared to apoE(-/-) mice, while NZM and apoE(-/-) mice exposed to IFNα developed accelerated thrombosis and platelet activation. CONCLUSION: These results support the hypothesis that type I IFNs play key roles in the development of premature CVD in SLE and, potentially, in the general population, through pleiotropic deleterious effects on the vasculature.

Angiogenesis and vasculopathy in systemic sclerosis: evolving concepts.

Systemic sclerosis (scleroderma [SSc]) is a multifactorial disease characterized by inflammation, extensive and progressive fibrosis, and multiple vasculopathies. The vascular manifestations can be seen early in the pathogenesis of the disease and include malformed capillaries, Raynaud's phenomenon, and digital ulcers. As the disease progresses, the vasculopathy proceeds to significant clinical manifestations, including renal crisis and pulmonary arterial hypertension. Moreover, later stages of the disease are marked by increasingly avascular areas. Despite the obliteration of microvascular structures, compensatory vasculogenesis and angiogenesis do not occur normally. This is in spite of a general increase in many potent angiogenic factors. Recent studies are beginning to examine this paradox and subsequent paucity of an angiogenic response in SSc. In this review, we discuss these findings and examine the role that chemokine and growth factor receptors, proteases, adhesion molecules, and transcription factors play in the dysregulation of angiogenesis in SSc.

Blocking of interferon regulatory factor 1 reduces tumor necrosis factor α-induced interleukin-18 bioactivity in rheumatoid arthritis synovial fibroblasts by induction of interleukin-18 binding protein a: role of the nuclear interferon regulatory factor 1-NF-κB-c-jun complex.

OBJECTIVE: To examine the role of interferon regulatory factor 1 (IRF-1) in tumor necrosis factor α (TNFα)-induced interleukin-18 binding protein a (IL-18BPa) expression in rheumatoid arthritis synovial fibroblasts (RASFs). METHODS: TNFα-induced IRF-1 expression was assessed by real-time quantitative polymerase chain reaction and Western blotting. The effect of TNFα on IRF-1 was assessed using nuclear and cytoplasmic extracts, Western blots, and immunofluorescence. Chemical inhibitors of NF-κB or MAP kinases were used to analyze the signaling pathways of TNFα-induced IRF-1 expression and IRF-1 nuclear translocation. Control and IRF-1 small interfering RNA (siRNA) were used to analyze the effect of IRF-1 down-regulation on TNFα-induced IL-18BP expression. IL-18BPa expression was assessed by enzyme-linked immunosorbent assay, and IL-18 was assessed at the transcription and bioactivity levels using KG-1 cells. RESULTS: TNFα induced RASF IRF-1 expression at the messenger RNA and protein levels, with a maximal effect at 2 hours (P < 0.05; n ≥ 3). Furthermore, TNFα induced nuclear translocation of IRF-1, with maximal translocation at 2 hours (∼6 fold-induction) (P < 0.05; n = 4). Blocking of the NF-κB or JNK-2 pathways reduced TNFα-induced IRF-1 nuclear translocation by 35% and 50%, respectively (P < 0.05; n ≥ 4). Using siRNA to knock down IRF-1, we observed reduced IL-18BPa expression. Additionally, IL-18 bioactivity was higher when siRNA was used to knock down IRF-1 expression. CONCLUSION: These results show that IRF-1 is a key regulator of IL-18BPa expression and IL-18 bioactivity in RASFs. Regulation of IRF-1 will be a new therapeutic target in RA.

Dysregulated expression of MIG/CXCL9, IP-10/CXCL10 and CXCL16 and their receptors in systemic sclerosis.

INTRODUCTION: Systemic sclerosis (SSc) is characterized by fibrosis and microvascular abnormalities including dysregulated angiogenesis. Chemokines, in addition to their chemoattractant properties, have the ability to modulate angiogenesis. Chemokines lacking the enzyme-linked receptor (ELR) motif, such as monokine induced by interferon-γ (IFN-γ) (MIG/CXCL9) and IFN-inducible protein 10 (IP-10/CXCL10), inhibit angiogenesis by binding CXCR3. In addition, CXCL16 promotes angiogenesis by binding its unique receptor CXCR6. In this study, we determined the expression of these chemokines and receptors in SSc skin and serum. METHODS: Immunohistology and enzyme-linked immunosorbent assays (ELISAs) were used to determine chemokine and chemokine receptor expression in the skin and serum, respectively, of SSc and normal patients. Endothelial cells (ECs) were isolated from SSc skin biopsies and chemokine and chemokine receptor expression was determined by quantitative PCR and immunofluorescence staining. RESULTS: Antiangiogenic IP-10/CXCL10 and MIG/CXCL9 were elevated in SSc serum and highly expressed in SSc skin. However, CXCR3, the receptor for these chemokines, was decreased on ECs in SSc vs. normal skin. CXCL16 was elevated in SSc serum and increased in SSc patients with early disease, pulmonary arterial hypertension, and those that died during the 36 months of the study. In addition, its receptor CXCR6 was overexpressed on ECs in SSc skin. At the mRNA and protein levels, CXCR3 was decreased while CXCR6 was increased on SSc ECs vs. human microvascular endothelial cells (HMVECs). CONCLUSIONS: These results show that while the expression of MIG/CXCL9 and IP-10/CXCL10 are elevated in SSc serum, the expression of CXCR3 is downregulated on SSc dermal ECs. In contrast, CXCL16 and CXCR6 are elevated in SSc serum and on SSc dermal ECs, respectively. In all, these findings suggest angiogenic chemokine receptor expression is likely regulated in an effort to promote angiogenesis in SSc skin.

Interleukin 18 induces angiogenesis in vitro and in vivo via Src and Jnk kinases.

BACKGROUND: Interleukin 18 (IL-18) is a novel mediator of angiogenesis in rheumatoid arthritis (RA). OBJECTIVE: To examine the role of IL-18 in RA angiogenesis and the signalling mechanisms involved. METHODS: Human dermal microvascular endothelial cell (HMVEC) chemotaxis, capillary morphogenesis assays and Matrigel plug angiogenesis assays were performed in vivo using IL-18 with or without signalling inhibitors. A novel model of angiogenesis was devised using dye-tagged HMVECs to study their homing into RA and normal (NL) synovial tissues (STs) engrafted in severe combined immunodeficient (SCID) mice. RESULTS: IL-18-mediated angiogenesis depended on Src and Jnk, as the inhibitors of Src and Jnk blocked IL-18-induced HMVEC chemotaxis, tube formation and angiogenesis in Matrigel plugs. However, inhibitors of Janus kinase 2, p38, MEK, phosphatidylinositol-3-kinase and neutralising antibodies to vascular endothelial growth factor or stromal derived factor-1α did not alter IL-18-induced HMVEC migration. These results were confirmed with Jnk or Src sense or antisense oligodeoxynucleotides. Moreover, IL-18 induced phosphorylation of Src and Jnk in HMVECs. As proof of principle, IL-18 null mice had a significantly decreased angiogenesis compared with wild-type mice in Matrigel plug angiogenesis assays in vivo. IL-18 markedly enhanced mature HMVEC homing to human RA ST compared with NL ST in SCID mice, confirming the role of IL-18-induced angiogenesis in RA ST in vivo. CONCLUSION: Targeting IL-18 or its signalling intermediates may prove to be a potentially novel therapeutic strategy for angiogenesis-dependent diseases, such as RA.

Junctional adhesion molecule-C is a soluble mediator of angiogenesis.

Junctional adhesion molecule-C (JAM-C) is an adhesion molecule expressed by endothelial cells (ECs) that plays a role in tight junction formation, leukocyte adhesion, and transendothelial migration. In the current study, we investigated whether JAM-C is found in soluble form and whether soluble JAM-C (sJAM-C) mediates angiogenesis. We found that JAM-C is present in soluble form in normal serum and elevated in rheumatoid arthritis (RA) serum. The concentration of sJAM-C is also elevated locally in RA synovial fluid compared with RA serum or osteoarthritis synovial fluid. sJAM-C was also present in the culture supernatant of human microvascular ECs (HMVECs) and immortalized human dermal microvascular ECs, and its concentration was increased following cytokine stimulation. In addition, sJAM-C cleavage from the cell surface was mediated in part by a disintegrin and metalloproteinases 10 and 17. In functional assays, sJAM-C was both chemotactic and chemokinetic for HMVECs and induced HMVEC tube formation on Matrigel in vitro. Neutralizing anti-JAM-C Abs inhibited RA synovial fluid-induced HMVEC chemotaxis and sJAM-C-induced HMVEC tube formation on Matrigel. sJAM-C also induced angiogenesis in vivo in the Matrigel plug and sponge granuloma models. Moreover, sJAM-C-mediated HMVEC chemotaxis was dependent on Src, p38, and PI3K. Our results show that JAM-C exists in soluble form and suggest that modulation of sJAM-C may provide a novel route for controlling pathological angiogenesis.

Expression and function of CXCL16 in a novel model of gout.

OBJECTIVE: To better define the activity of soluble CXCL16 in the recruitment of polymorphonuclear neutrophils (PMNs) in vivo, utilizing a novel animal model of gout involving engraftment of SCID mice with normal human synovial tissue (ST) injected intragraft with gouty human synovial fluid (SF). METHODS: For in vitro studies, a modified Boyden chemotaxis system was used to identify CXCL16 as an active recruitment factor for PMNs in gouty SF. Migration of PMNs could be reduced by neutralization of CXCL16 activity in gouty SF. For in vivo analyses, fluorescent dye-tagged PMNs were injected intravenously into SCID mice while, simultaneously, diluted gouty SF containing CXCL16, or depleted of CXCL16 by antibody blocking, was administered intragraft. In addition, the receptor for CXCL16, CXCR6, was inhibited by incubating PMNs with a neutralizing anti-CXCR6 antibody prior to injection into the mouse chimeras. Recruitment of PMNs to the gouty SF-injected normal human ST was then examined in this SCID mouse chimera system. RESULTS: CXCL16 concentrations were highly elevated in gouty SF, and PMNs were observed to migrate in response to CXCL16 in vitro. Normal human ST-SCID mouse chimeras injected intragraft with gouty SF that had been depleted of CXCL16 during PMN transfer showed a significant reduction of 50% in PMN recruitment to engrafted tissue as compared with that after administration of sham-depleted gouty SF. Similar findings were achieved when PMNs were incubated with a neutralizing anti-CXCR6 antibody before injection into chimeras. CONCLUSION: Overall, the results of this study outline the effectiveness of the human-SCID mouse chimera system as a viable animal model of gout, serving to identify the primary function of CXCL16 as a significant mediator of in vivo recruitment of PMNs to gouty SF.

The proadhesive phenotype of systemic sclerosis skin promotes myeloid cell adhesion via ICAM-1 and VCAM-1.

OBJECTIVE: SSc is characterized by microvascular abnormalities and leucocyte infiltration. Previous studies have suggested a proadhesive phenotype in SSc skin, but the functional consequences of this phenotype are not fully understood. Molecules known to mediate leucocyte adhesion include those present at intracellular junctions, such as junctional adhesion molecule-B (JAM-B), JAM-C and CD99, as well as intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1). The aim of this study was to examine adhesive interactions in SSc skin. METHODS: The expression of JAM-B, JAM-C, CD99, ICAM-1 and VCAM-1 in SSc skin was determined by immunohistology and cell surface ELISA. Myeloid U937 cell-SSc dermal fibroblast adhesion assays or in situ adhesion assays to SSc skin were performed. RESULTS: JAM-C and CD99 expression on endothelial cells (ECs) in SSc skin was decreased compared with expression on normal ECs. CD99 was overexpressed on mononuclear cells in SSc skin and on SSc dermal fibroblasts. Neutralizing ICAM-1 inhibited the binding of U937 cells to SSc dermal fibroblasts. In addition, blocking both ICAM-1 and VCAM-1 inhibited U937 cell adhesion to either proximal (less involved) or distal (more involved) SSc skin. CONCLUSIONS: These studies show that JAM-C and CD99 are aberrantly expressed in SSc skin. However, these adhesion molecules do not mediate myeloid cell-SSc skin adhesion. In contrast, we demonstrate an important role for ICAM-1 and VCAM-1 in the retention of myeloid cells in SSc skin, suggesting that targeting these molecules may be useful SSc therapies.

Synovial inflammation in patients with osteonecrosis of the femoral head.

Much of the work aimed at elucidating the pathogenesis of osteonecrosis (ON) of the femoral head has focused on bone blood supply, with little attention to the surrounding synovial tissue (ST). We hypothesized that patients with ON exhibit synovial inflammation. Using immunohistological techniques, we found that a large population of patients with ON had synovial inflammation. Moreover, a population of ON patients had inflamed ST without having an inflammatory disease co-morbidity. The inflammatory infiltrate in these patients comprised primarily CD4(+) T cells and CD68(+) macrophages, the latter of which expressed increased levels of cellular adhesion molecules. Our results suggest the presence of a previously unrecognized population of ON patients without a diagnosed inflammatory co-morbidity and a highly inflammed synovium consisting primarily of a macrophage and CD4(+) T-cell infiltrate.

Junctional adhesion molecule C mediates leukocyte adhesion to rheumatoid arthritis synovium.

OBJECTIVE: Leukocyte infiltration into the rheumatoid arthritis (RA) synovium is a multistep process in which leukocytes leave the bloodstream and invade the synovial tissue (ST). Leukocyte transendothelial migration and adhesion to RA ST requires adhesion molecules on the surface of endothelial cells and RA ST fibroblasts. This study was undertaken to investigate the role of junctional adhesion molecule C (JAM-C) in mediating leukocyte recruitment and retention in the RA joint. METHODS: Immunohistologic analysis was performed on RA, osteoarthritis (OA), and normal ST samples to quantify JAM-C expression. Fibroblast JAM-C expression was also analyzed using Western blotting, cell surface enzyme-linked immunosorbent assay, and immunofluorescence. To determine the role of JAM-C in leukocyte retention in the RA synovium, in vitro and in situ adhesion assays and RA ST fibroblast transmigration assays were performed. RESULTS: JAM-C was highly expressed by RA ST lining cells, and its expression was increased in OA ST and RA ST endothelial cells compared with normal ST endothelial cells. JAM-C was also expressed on the surface of OA ST and RA ST fibroblasts. Furthermore, we demonstrated that myeloid U937 cell adhesion to both OA ST and RA ST fibroblasts and to RA ST was dependent on JAM-C. U937 cell migration through an RA ST fibroblast monolayer was enhanced in the presence of neutralizing antibodies against JAM-C. CONCLUSION: Our results highlight the novel role of JAM-C in recruiting and retaining leukocytes in the RA synovium and suggest that targeting JAM-C may be important in combating inflammatory diseases such as RA.

Epigallocatechin-3-gallate inhibits IL-6 synthesis and suppresses transsignaling by enhancing soluble gp130 production.

Regulation of IL-6 transsignaling by the administration of soluble gp130 (sgp130) receptor to capture the IL-6/soluble IL-6R complex has shown promise for the treatment of rheumatoid arthritis (RA). However, enhancing endogenous sgp130 via alternative splicing of the gp130 gene has not yet been tested. We found that epigallocatechin-3-gallate (EGCG), an anti-inflammatory compound found in green tea, inhibits IL-1beta-induced IL-6 production and transsignaling in RA synovial fibroblasts by inducing alternative splicing of gp130 mRNA, resulting in enhanced sgp130 production. Results from in vivo studies using a rat adjuvant-induced arthritis model showed specific inhibition of IL-6 levels in the serum and joints of EGCG-treated rats by 28% and 40%, respectively, with concomitant amelioration of rat adjuvant-induced arthritis. We also observed a marked decrease in membrane-bound gp130 protein expression in the joint homogenates of the EGCG-treated group. In contrast, quantitative RT-PCR showed that the gp130/IL-6Ralpha mRNA ratio increased by approximately 2-fold, suggesting a possible mechanism of sgp130 activation by EGCG. Gelatin zymography results showed EGCG inhibits IL-6/soluble IL-6R-induced matrix metalloproteinase-2 activity in RA synovial fibroblasts and in joint homogenates, possibly via up-regulation of sgp130 synthesis. The results of these studies provide previously undescribed evidence of IL-6 synthesis and transsignaling inhibition by EGCG with a unique mechanism of sgp130 up-regulation, and thus hold promise as a potential therapeutic agent for RA.