Impaired plasmacytoid dendritic cell maturation and differential chemotaxis in chronic hepatitis C virus: associations with antiviral treatment outcomes.

BACKGROUND: Dendritic cell (DC) defects may contribute to chronicity in hepatitis C virus (HCV) infection and determine response to PEG-interferon and ribavirin therapy via poor T cell stimulation. Studies to date have produced inconsistent results regarding DC maturation and function: no large study has examined DCs before and after therapy. AIMS: We examined if DC defects in maturation and chemotaxis are present by comparing therapeutic responders to non-responders. METHODS: We analysed peripheral DCs of 64 HCV genotype 1-infected patients from the Virahep-C study 2 weeks before and 24 weeks after therapy. We used flow cytometry to enumerate plasmacytoid DC (pDC) and myeloid DCs (mDC) and quantify expression of chemokine receptors and maturation markers. Chemotaxis was measured with an in vitro assay. RESULTS: Pre-treatment frequencies of pDCs and mDCs were significantly lower in HCV patients than controls and successful therapy normalised pDCs. Levels of CXCR3 and CXCR4 on pDCs were higher at baseline compared to normal controls and decreased with therapy. Pre-therapy levels of co-stimulatory marker CD40 and the maturation marker CD83 were higher in pDCs of patients chronically infected with HCV compared to normal patients, and levels of both markers dropped significantly with therapy in the SVR+ group only. Other maturation markers (CD86 and CCR7) were not elevated suggesting a partially activated phenotype. Baseline chemotaxis of pDCs to CXCL12 and CXCL10 predicted failure of antiviral response and correlated with the histological activity index inflammation score. CONCLUSIONS: Plasmacytoid DC defects exist in chronic HCV and successful antiviral therapy normalises many phenotypic and functional abnormalities.

IL-1 induces collagenase-3 (MMP-13) promoter activity in stably transfected chondrocytic cells: requirement for Runx-2 and activation by p38 MAPK and JNK pathways.

Osteoarthritic chondrocytes secrete matrix metalloproteinase-13 (MMP-13) in response to interleukin-1 (IL-1), causing digestion of type II collagen in cartilage. Using chondrocytic cells, we previously determined that IL-1 induced a strong MMP-13 transcriptional response that requires p38 MAPK, JNK and the transcription factor NF-kappaB. Now, we have studied the tissue-specific transcriptional regulation of MMP-13. Constitutive expression of the transcription factor Runx-2 correlated with the ability of a cell type to express MMP-13 and was required for IL-1 induction; moreover, Runx-2 enhanced IL-1 induction of MMP-13 transcription by synergizing with the p38 MAPK signaling pathway. Transiently transfected MMP-13 promoters were not IL-1 inducible. However, -405 bp of stably integrated promoter was sufficient for 5- to 6-fold IL-1 induction of reporter activity and this integrated reporter required the same p38 MAPK pathway as the endogenous gene. Finally, mutation of the proximal Runx binding site and the proximal AP-1 site blunted the transcriptional response to IL-1, and double mutation synergistically decreased reporter activity. In summary, our data suggest that the transcriptional MMP-13 response to IL-1 is controlled by the p38 pathway interacting at the MMP-13 promoter through the tissue-specific transcription factor Runx-2 and the ubiquitous AP-1 transcription factor.

Interleukin-1 induction of collagenase 3 (matrix metalloproteinase 13) gene expression in chondrocytes requires p38, c-Jun N-terminal kinase, and nuclear factor kappaB: differential regulation of collagenase 1 and collagenase 3.

OBJECTIVE: To examine the mechanism of interleukin-1 (IL-1)-induced collagenase 3 (matrix metalloproteinase 13 [MMP-13]) gene expression in cultured chondrocytes for the purpose of better understanding how the gene is induced in these cells, and how it contributes to cartilage degradation in osteoarthritis. METHODS: The transcriptional and posttranscriptional responses of the MMP-13 gene to IL-1 were assessed first. Then, direct inhibitors of mitogen-activated protein kinase (MAPK) signaling pathways and a constitutive repressor of nuclear factor kappaB (NF-kappaB) were used to assess the role of each pathway in IL-1-mediated induction of MMP-13. RESULTS: We found that IL-1 induction of MMP-13 requires p38 activity, c-Jun N-terminal kinase (JNK) activity and NF-kappaB translocation. These results suggest that both NF-kappaB and activator protein 1 transcription factors are necessary for IL-1 induction of MMP-13. We also compared the signaling pathways necessary for IL-1 to stimulate collagenase 1 (MMP-1) in articular chondrocytes and chondrosarcoma cells and found that IL-1 induction of MMP-1 requires different pathways from those required by MMP-13. In chondrosarcoma cells, MMP-1 induction depends on p38 and MEK (an MAPK kinase of the extracellular signal-regulated kinase pathway) and does not require JNK or NF-kappaB. In articular chondrocytes, inhibition of MEK had no effect, while inhibition of p38 gave variable results. CONCLUSION: These studies demonstrate, for the first time, that p38, JNK, and NF-kappaB are required for IL-1 induction of MMP-13. The results also highlight the differential requirements for signaling pathways in the induction of MMP-1 and MMP-13. Additionally, they demonstrate that induction of MMP-1 by IL-1 in chondrocytic cells depends on unique combinations of signaling pathways that are cell type-specific.

A synthetic triterpenoid selectively inhibits the induction of matrix metalloproteinases 1 and 13 by inflammatory cytokines.

OBJECTIVE: To address the effects of a novel synthetic triterpenoid, 2-cyano-3,12-dioxoolean-1,9-dien-28-oic acid (CDDO), on the induction of matrix metalloproteinases 1 and 13 (MMP-1, MMP-13) by inflammatory cytokines. METHODS: Human chondrosarcoma cells stimulated with inflammatory cytokines (interleukin-1beta [IL-1beta], tumor necrosis factor alpha) were used to study the effects of CDDO on the induction of MMPs and the invasion of cells through a collagen matrix. RESULTS: CDDO selectively reduced the induction of MMP-1 and MMP-13 at the levels of messenger RNA and protein. Treatment with CDDO prior to cytokine stimulation enhanced this inhibition, and we demonstrated that CDDO functions at the level of transcription. Additionally, CDDO reduced IL-1beta-mediated invasion of cells through a collagen matrix. CONCLUSION: This study demonstrates that CDDO is a novel inhibitor of MMP-1 and MMP-13 gene expression mediated by inflammatory cytokines. Thus, CDDO may have therapeutic potential for the inhibition of joint degradation in osteoarthritis.

Cloning of the gene for interstitial collagenase-3 (matrix metalloproteinase-13) from rabbit synovial fibroblasts: differential expression with collagenase-1 (matrix metalloproteinase-1).

Cartilage, bone and the interstitial stroma, composed largely of the interstitial collagens, types I, II and III, are remodelled by three members of the metalloproteinase (MMP) family, collagenase-1 (MMP-1), collagenase-2 (MMP-8) and collagenase-3 (MMP-13). MMP-1 and MMP-13 may contribute directly to disease progression, since they are induced in patients with rheumatoid arthritis and osteoarthritis. The study of MMP-1 and MMP-13 gene regulation in models of arthritic disease has been problematic because mice and rats, which are typically used, only possess a homologue of MMP-13. Here we show that in contrast with mice and rats, rabbits possess distinct genes homologous to human MMP-1 and MMP-13. Furthermore, rabbit MMP-13 is expressed simultaneously with MMP-1 in chondrocytes and synovial fibroblasts in response to the cytokines interleukin-1 and tumour necrosis factor-alpha, or the phorbol ester PMA. The time course of MMP-13 induction is more rapid and transient than that of MMP-1, suggesting that distinct mechanisms regulate the expression of these two collagenases. We have cloned the rabbit MMP-13 gene from synovial fibroblasts and demonstrated that the rabbit gene shares greater homology with human MMP-13 than does the mouse interstitial collagenase. Together with the fact that mice and rats do not possess a homologue to human MMP-1, our data suggest that the rabbit provides an appropriate model for studying the roles of interstitial collagenases in connective-tissue diseases, such as rheumatoid arthritis and osteoarthritis.