Wan Uk Kim

CD40 Engagement on Synovial Fibroblast Up-Regulates Production of Vascular Endothelial Growth Factor

We tested the impact of CD40 engagement on the production of vascular endothelial growth factor (VEGF) from rheumatoid synovial fibroblasts. Fibroblast-like synovial cells (FLS) were prepared from the synovial tissues of rheumatoid arthritis patients and cultured in the presence of CD40 ligand-transfected (CD40L+) L cells. VEGF levels were determined in the culture supernatants by ELISA. Stimulation of FLS by CD40L+ L cells increased the production of VEGF by 4.1-fold over the constitutive levels of unstimulated FLS. The CD40L on activated T cells from rheumatoid synovial fluid also up-regulated VEGF production from FLS. Neither indomethacin nor Abs to IL-1β, TNF-α, and TGF-β did affect CD40L-induced VEGF production. Stimulation of FLS with TNF-α, IL-1β, and TGF-β increased VEGF production by 1.6-, 2.0-, and 5.2-fold, respectively, and displayed an additive effect on the production of VEGF by CD40L. VEGF mRNA expression was also up-regulated by the stimulation of FLS with membranes from the CD40L+ L cells. Dexamethasone completely abrogated CD40L-induced VEGF production. In addition, pyrrolidine dithiocarbamate partially down-regulated CD40L-induced VEGF production, showing that the NF-κB pathway was partly involved in the signaling of CD40L leading to VEGF production. Collectively, these results suggest that the interaction between CD40 on synovial fibroblasts and CD40L expressed on activated T lymphocytes may be directly involved in the neovascularization in rheumatoid synovitis by enhancing the production of VEGF.

Elevated matrix metalloproteinase-9 in patients with systemic sclerosis.

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of cancer, autoimmune disease, and various pathologic conditions characterized by excessive fibrosis. In this study, we investigated the expression of MMP-9 and its clinical significance in systemic sclerosis (SSc). The patients (n = 42) with SSc had higher concentrations of MMP-9 and of tissue inhibitor of metalloproteinase-1 (TIMP-1) and a higher ratio of MMP-9 to TIMP-1 in sera than healthy controls (n = 32). Serum MMP-9 concentrations were significantly higher in the diffuse type (n = 23) than the limited type of SSc (n = 19). Serum concentrations of MMP-9 correlated well with the degree of skin involvement, as determined by the Rodnan score and with serum concentrations of transforming growth factor β. Moreover, dermal fibroblasts from patients with SSc produced more MMP-9 than those from healthy controls when they were stimulated with IL-1β, tumor necrosis factor α, or transforming growth factor β. Such an increase in MMP-9 production was partially blocked by treatment with cyclosporin A. In summary, the serum MMP-9 concentrations were elevated in SSc patients and correlated well with skin scores. The increased MMP-9 concentrations may be attributable to overproduction by dermal fibroblasts in SSc. These findings suggest that the enhanced production of MMP-9 may contribute to fibrogenic remodeling during the progression of skin sclerosis in SSc.

Type II collagen autoimmunity in rheumatoid arthritis

&NA; This review summarizes the autoimmune reaction to type II collagen (CII) autoimmunity with regard not only to antibody response to CII but also to the clinical significance or biological characteristics of the CII‐reactive T cell, focusing on studies of human RA rather than on animal models. The authors investigated the effect of the interaction between CII‐reactive T cells and fibroblast‐like synoviocytes (FLSs) on the production of inflammatory cytokines. When the CII‐reactive T cells were co‐cultured with FLS, the production of interleukin‐15 and tumor necrosis factor‐alpha from FLSs were significantly increased, and this increase was clearly presented in accord with the expansion of CII‐reactive T cells. In addition, the production of interferon‐&ggr; and interleukin‐17, T cell–derived cytokines, was increased by the co‐incubation of CII‐reactive T cells with FLSs. When FLSs were co‐cultured with CII‐stimulated T cells, the production of interleukin‐8, monocyte chemoattractant protein‐1, and macrophage inflammatory protein‐1&agr; was significantly enhanced. The increased production of these chemokines was strongly correlated with an increase in T‐cell response to CII. Conclusively, high reactivity to CII was frequently found in RA patients. Enhanced T‐cell responses to CII were associated with increased production of proinflammatory cytokines and chemokines, which were critical for inflammatory responses in RA. Interaction of CII‐reactive T cells with FLS further augmented this phenomenon. Taken together, the authors’ recent studies have suggested that autoimmunity to CII could play a crucial role not only in the initiation but also in the amplification and perpetuation of the inflammatory process in RA.

Cyclosporine differentially regulates interleukin-10, interleukin-15, and tumor necrosis factor α production by rheumatoid synoviocytes

OBJECTIVE To determine the direct effect of cyclosporin A (CSA) on the production of cytokines by rheumatoid synovial fibroblasts. METHODS Fibroblast-like synoviocytes (FLS) were prepared from the synovial tissues of patients with rheumatoid arthritis and cultured in the presence of CSA. The production of interleukin-10 (IL-10), IL-15, and tumor necrosis factor a (TNFalpha) by FLS was measured in culture supernatants by enzyme-linked immunosorbent assay. The expression of IL-10, IL-15, and TNFalpha messenger RNA (mRNA) in FLS was determined by polymerase chain reaction (PCR). RESULTS CSA (1-1,000 ng/ml) increased the production of IL-10, but decreased in a dose-dependent manner the levels of IL-15 and TNFalpha that were spontaneously secreted from FLS. CSA also potently inhibited the production of IL-15 and TNFalpha stimulated with interferon-gamma, IL-1beta, or lipopolysaccharide. The inhibitory effect of CSA on IL-15 and TNFalpha production depended on the increase in IL-10, since neutralizing anti-IL-10 antibodies were able to partially reverse this inhibition. In a semiquantitative PCR, CSA increased IL-10 mRNA expression but strongly suppressed IL-1beta-induced IL-15 and TNFalpha mRNA expression, indicating that the production of these cytokines by CSA was regulated at the transcriptional level. Results with the calcineurin inhibitor FK-506, but not with the immunosuppressant rapamycin, were similar to those with CSA. Agonists of cAMP displayed an additive effect on the changes produced in the IL-10, IL-15, and TNFalpha levels by CSA, while a cAMP antagonist almost completely abrogated the effect of CSA, suggesting that cAMP is the major intracellular signal that mediates cytokine regulation by CSA. CONCLUSION These results suggest that CSA differentially regulates the production of cytokines by rheumatoid synoviocytes via a cAMP-dependent pathway.

Role of placenta growth factor in cancer and inflammation

Accumulating evidences have documented that angiogenesis is closely linked to inflammation and regulators of angiogenesis play key roles in various inflammatory conditions. PlGF is an angiogenic protein belonging to the VEGF family and is upregulated mainly in pathologic conditions. Recently, PlGF was discovered having a proinflammatory role in inflammatory arthritis and its serum level drew attention not only as a useful surrogate biomarker but also a potential therapeutic target in atherosclerosis and various cancers. Particularly, PlGF has attractive clinical values because endogenous PlGF is redundant for vascular development and physiological vessel maintenance in healthy adults. However, there have been conflicting results about the efficacy of PlGF inhibition depending on the experimental and clinical settings. Further close investigations for resolving the puzzle of PlGF biology are required.

Antiphospholipid Antibodies Induce Monocyte Chemoattractant Protein-1 in Endothelial Cells

The presence of antiphospholipid Ab is associated with increased risk of thrombosis. The monocyte-endothelial cell interaction has been suggested to play a key role at the site of vascular injury during thrombosis. Therefore, we tested the effect of anticardiolipin Abs (aCL) on the production of monocyte chemoattractant protein-1 (MCP-1) in HUVEC. We found that monoclonal aCL as well as IgG fractions from patients with antiphospholipid syndrome (APS-IgG) could induce the production of MCP-1 in HUVEC. The ability of IgG aCL to induce MCP-1 production could be abrogated by preabsorption with cardiolipin liposomes. Simultaneous addition of either monoclonal aCL or APS-IgG with IL-1β resulted in synergistic increase in MCP-1 production, whereas the addition of control IgG lacking aCL activity did not alter IL-1β-induced levels of MCP-1. MCP-1 mRNA expression was also up-regulated when HUVEC were incubated with either APS-IgG or monoclonal aCL, and down-regulated by the treatment of dexamethasone. In addition, we found that serum levels of MCP-1 in 76 systemic lupus erythematosus patients correlated well with the titers of IgG aCL. Collectively, these results indicate that aCL could promote endothelial cell-monocyte cross-talk by enhancing the endothelial production of MCP-1, thereby shifting the hemostatic balance toward the prothrombotic state of APS.

Decreased production of interleukin-12 and interferon-gamma is associated with renal involvement in systemic lupus erythematosus.

Objective. To analyze the type 1 / type 2 cytokine balance in patients with systemic lupus erythematosus (SLE) according to the presence of renal disorder and activity status. Methods. We measured the serum levels of type 1 (IFN-γ, IL-12) and type 2 cytokines (IL-4, IL-10) as well as spontaneous and stimulated cytokine production from peripheral blood mononuclear cells (PBMC) in 40 patients with SLE. Results. Patients with lupus nephritis (LN) showed significantly lower levels of serum IL-12 and IFN-γ than those without LN. Production of IL-12 and IFN-γ by stimulated PBMC were also decreased in patients with LN. The circulating IL-12 correlated positively with IFN-γ, but inversely with IL-10. The SLEDAI scores correlated well with the ratio of IL-4/IFN-γ levels. Conclusion. The reduced production of IL-12 and IFN-γ and the resultant shift towards the type 2 cytokine phenotype may be associated with LN.OBJECTIVE To analyze the type 1/type 2 cytokine balance in patients with systemic lupus erythematosus (SLE) according to the presence of renal disorder and activity status. METHODS We measured the serum levels of type 1 (IFN-gamma, IL-12) and type 2 cytokines (IL-4, IL-10) as well as spontaneous and stimulated cytokine production from peripheral blood mononuclear cells (PBMC) in 40 patients with SLE. RESULTS Patients with lupus nephritis (LN) showed significantly lower levels of serum IL-12 and IFN-gamma than those without LN. Production of IL-12 and IFN-gamma by stimulated PBMC were also decreased in patients with LN. The circulating IL-12 correlated positively with IFN-gamma, but inversely with IL-10. The SLEDAI scores correlated well with the ratio of IL-4/IFN-gamma levels. CONCLUSION The reduced production of IL-12 and IFN-gamma and the resultant shift towards the type 2 cytokine phenotype may be associated with LN.

Identification of key regulators for the migration and invasion of rheumatoid synoviocytes through a systems approach

Significance Rheumatoid arthritis (RA) is an autoimmune disorder that affects ∼1% of the population. Macrophage- and fibroblast-like synoviocytes are thought to play central roles in the pathogenesis of RA. They have shared and distinct features defining RA pathology. However, these features have not been systematically explored. Using global gene expression profiling, we identified molecular signature and biological network models underlying the pathologic features of macrophage-like vs. fibroblast-like synoviocytes. Based on the network models, we selected key regulators, including POSTN and TWIST1, responsible for synoviocyte migration/invasion causing joint destruction, and their validity was experimentally confirmed. Our findings provide a comprehensive and systematic basis for mechanisms explaining RA pathogenesis and also for identification of therapeutic targets for RA. Rheumatoid synoviocytes, which consist of fibroblast-like synoviocytes (FLSs) and synovial macrophages (SMs), are crucial for the progression of rheumatoid arthritis (RA). Particularly, FLSs of RA patients (RA-FLSs) exhibit invasive characteristics reminiscent of cancer cells, destroying cartilage and bone. RA-FLSs and SMs originate differently from mesenchymal and myeloid cells, respectively, but share many pathologic functions. However, the molecular signatures and biological networks representing the distinct and shared features of the two cell types are unknown. We performed global transcriptome profiling of FLSs and SMs obtained from RA and osteoarthritis patients. By comparing the transcriptomes, we identified distinct molecular signatures and cellular processes defining invasiveness of RA-FLSs and proinflammatory properties of RA-SMs, respectively. Interestingly, under the interleukin-1β (IL-1β)–stimulated condition, the RA-FLSs newly acquired proinflammatory signature dominant in RA-SMs without losing invasive properties. We next reconstructed a network model that delineates the shared, RA-FLS–dominant (invasive), and RA-SM–dominant (inflammatory) processes. From the network model, we selected 13 genes, including periostin, osteoblast-specific factor (POSTN) and twist basic helix–loop–helix transcription factor 1 (TWIST1), as key regulator candidates responsible for FLS invasiveness. Of note, POSTN and TWIST1 expressions were elevated in independent RA-FLSs and further instigated by IL-1β. Functional assays demonstrated the requirement of POSTN and TWIST1 for migration and invasion of RA-FLSs stimulated with IL-1β. Together, our systems approach to rheumatoid synovitis provides a basis for identifying key regulators responsible for pathological features of RA-FLSs and -SMs, demonstrating how a certain type of cells acquires functional redundancy under chronic inflammatory conditions.

Role of Endoplasmic Reticulum Stress in Rheumatoid Arthritis Pathogenesis

Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by abnormal proliferation of synoviocytes, leukocyte infiltration, and angiogenesis. The endoplasmic reticulum (ER) is the site of biosynthesis for all secreted and membrane proteins. The accumulation of unfolded proteins in the ER leads to a condition known as ER stress. Failure of the ERs adaptive capacity results in abnormal activation of the unfolded protein response. Recently, we have demonstrated that ER stress-associated gene signatures are highly expressed in RA synovium and synovial cells. Mice with Grp78 haploinsufficiency exhibit the suppression of experimentally induced arthritis, suggesting that the ER chaperone GRP78 is crucial for RA pathogenesis. Moreover, increasing evidence has suggested that GRP78 participates in antibody generation, T cell proliferation, and pro-inflammatory cytokine production, and is therefore one of the potential therapeutic targets for RA. In this review, we discuss the putative, pathophysiological roles of ER stress and GRP78 in RA pathogenesis.

Proteomics in Rheumatoid Arthritis Research

Although rheumatoid arthritis (RA) is the most common chronic inflammatory autoimmune disease, diagnosis of RA is currently based on clinical manifestations, and there is no simple, practical assessment tool in the clinical field to assess disease activity and severity. Recently, there has been increasing interest in the discovery of new diagnostic RA biomarkers that can assist in evaluating disease activity, severity, and treatment response. Proteomics, the large-scale study of the proteome, has emerged as a powerful technique for protein identification and characterization. For the past 10 years, proteomic techniques have been applied to different biological samples (synovial tissue/fluid, blood, and urine) from RA patients and experimental animal models. In this review, we summarize the current state of the application of proteomics in RA and its importance in identifying biomarkers and treatment targets.