sq-23377 and Osteoarthritis

The aim of this study was to quantify the number of T-helper (TH)-17 cells present in rheumatoid arthritis (RA) synovial fluid (SF) and to determine the level of interleukin (IL)-17 cytokine in RA, osteoarthritis (OA) and normal synovial tissue, as well as to examine SF macrophages for the presence of IL-23, IL-27 and interferon (IFN)-gamma.. Peripheral blood (PB) mononuclear cells from normal and RA donors and mononuclear cells from RA SF were examined either without stimulation or after pretreatment with IL-23 followed by stimulation with phorbol myristate acetate (PMA) plus ionomycin (P/I). The abundance of TH-17 cells in RA SF was determined by flow cytometry. IL-17 levels were quantified in synovial tissue from RA, OA and normal individuals by ELISA and IL-23 was identified in SFs by ELISA. RA SF and control in vitro differentiated macrophages were either untreated or treated with the toll-like receptor (TLR) 2 ligand peptidoglycan, and then IL-23, IL-27 and IFN-gamma mRNA levels were quantified by real-time polymerase chain reaction (RT-PCR).. Treatment with P/I alone or combined with IL-23 significantly increased the number of TH-17 cells in normal, RA PB and RA SF. With or without P/I plus IL-23, the percentage of TH-17 cells was higher in RA SF compared with normal and RA PB. IL-17 levels were comparable in OA and normal synovial tissues, and these values were significantly increased in RA synovial tissue. Although IL-17 was readily detected in RA SFs, IL-23 was rarely identified in RA SF. However, IL-23 mRNA was significantly increased in RA SF macrophages compared with control macrophages, with or without TLR2 ligation. IL-27 mRNA was also significantly higher in RA SF compared with control macrophages, but there was no difference in IL-27 levels between RA and control macrophages after TLR2 ligation. IFN-gamma mRNA was also detectable in RA SF macrophages but not control macrophages and the increase of IFN-gamma mRNA following TLR2 ligation was greater in RA SF macrophages compared with control macrophages.. These observations support a role for TH-17 cells in RA. Our observations do not strongly support a role for IL-23 in the generation of TH-17 cells in the RA joint, however, they suggest strategies that enhance IL-27 or IFN-gamma might modulate the presence of TH-17 cells in RA.

Topics: Arthritis, Rheumatoid; Carcinogens; Case-Control Studies; Cells, Cultured; Female; Humans; Interferon-gamma; Interleukin-17; Interleukin-23; Interleukins; Ionomycin; Ionophores; Leukocytes, Mononuclear; Macrophages; Male; Middle Aged; Osteoarthritis; RNA, Messenger; Synovial Fluid; T-Lymphocytes, Helper-Inducer; Tetradecanoylphorbol Acetate

We investigated the energy-adaptive potential of human CD4(+) T cells under conditions of impaired oxidative phosphorylation (OXPHOS) and/or low glucose (inhibiting glycolysis). These cells often encounter these conditions when executing their functions in injured/inflamed tissues, even though T cells themselves require constant and adequate energy supply via ATP. We assessed two specific functions, cytokine synthesis and proliferation, and addressed whether adaptive characteristics also emerged in vivo. In glucose-containing medium, both cytokine production and proliferation were unaffected, even under complete OXPHOS suppression. Only when glucose was also absent were these functions significantly decreased. Partial recovery of OXPHOS and induced glycolysis were crucial for the maintenance of cellular energy supply. Adaptive regulatory mechanisms are clinically relevant because hypoxia up-regulates glycolytic genes but down-regulates OXPHOS genes in vivo. Our data demonstrate an unexpectedly high, clinically relevant adaptive potential of human CD4(+) T cells to maintain specific functions even under severely impaired bioenergetic conditions.

Topics: Adenosine Triphosphate; Arthritis, Rheumatoid; Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone; CD4-Positive T-Lymphocytes; Cell Proliferation; Cytochromes b; Cytokines; Electron Transport Complex III; Gene Expression; Gene Expression Profiling; Glucose; Glycolysis; Humans; Hypoxia; Ionomycin; Joint Capsule; Lymphocyte Activation; Methacrylates; Osteoarthritis; Oxidative Phosphorylation; Oxygen Consumption; Tetradecanoylphorbol Acetate; Thiazoles

Interleukin-18 (IL-18) is a member of the IL-1 cytokine family and has proinflammatory activity. It has been detected in osteoarthritic (OA) and at higher levels in rheumatoid arthritic (RA) synovial tissue. Therefore we investigated major signal transduction pathways for their contribution to IL-18 expression. Here we report that cyclic adenosine monophosphate reduced and ionomycin increased IL-18 mRNA in RA synovial fibroblasts (SF) but not in OA SF. Moreover, activation of G-proteins by Mas-7 augmented IL-18 reverse transcriptase polymerase chain reaction signals in OA SF but not in RA SF. Specific protein kinase C activator phorbol myristate acetate reduced transcription and secretion of IL-18 in RA SF and OA SF. Staurosporine changed spontaneous IL-18 mRNA levels and increased the secretion of IL-18 protein. We conclude that G-protein activation and protein kinase C activation might partially be responsible for elevated IL-18 levels during RA.

Topics: Arthritis, Rheumatoid; Cell Line, Transformed; Cells, Cultured; Cyclic AMP; Enzyme Inhibitors; Fibroblasts; GTP-Binding Proteins; Humans; Intercellular Signaling Peptides and Proteins; Interleukin-18; Ionomycin; Microscopy, Electron, Scanning; Osteoarthritis; Peptides; Protein Kinase C; RNA, Messenger; Signal Transduction; Synovial Membrane; Tetradecanoylphorbol Acetate; Up-Regulation

Recent data suggest that IL-15 plays an important role in the pathogenesis of rheumatoid arthritis. In the present study, we hypothesized that elevated in the joints of rheumatoid arthritis, but not osteoarthritis, patients, IL-15 may exert its proinflammatory properties via the induction of IL-17, a cytokine known to stimulate synoviocytes to release several mediators of inflammation including IL-6, IL-8, GM-CSF and PGE2. To test this hypothesis, we first measured the levels of IL-17 and IL-15 using specific ELISA and found that synovial fluids of patients with rheumatoid arthritis, but not with osteoarthritis, contain high levels of these cytokines. A strong correlation between IL-15 and IL-17 levels in synovial fluids was observed. Among tested factors, LPS and TNF-alpha failed, IL-15 and IL-2 were equipotent, and PMA + ionomycin was far more efficient in the induction of IL-17 secretion by PBMCs isolated from healthy blood donors. Interestingly, synovial fluid cells, in contrast to PBMCs isolated from patients with rheumatoid arthritis, but not osteoarthritis, respond to PMA + ionomycin with much lower, comparable to IL-15-triggered IL-17 secretion. Moreover, PMA + ionomycin-triggered IL-17 secretion is completely or partially blocked in the presence of low doses of cyclosporin A or high doses of methylprednisolone, respectively. IL-15-triggered IL-17 secretion by PBMCs was completely inhibited by these drugs. Thus, our results suggest for the first time that IL-15 may represent a physiological trigger that via cyclosporin A and steroid sensitive pathways leads to the overproduction of IL-17 in the joints of rheumatoid arthritis patients.

Topics: Adult; Aged; Arthritis, Rheumatoid; Cell Separation; Cells, Cultured; Cyclosporine; Humans; Interleukin-15; Interleukin-17; Interleukin-2; Ionomycin; Leukocytes, Mononuclear; Methylprednisolone; Middle Aged; Osteoarthritis; Phytohemagglutinins; Synovial Fluid; Tetradecanoylphorbol Acetate