sq-23377 and Arthritis--Rheumatoid

In rheumatoid arthritis, an autoimmune inflammatory arthritis, citrullinated proteins are targeted by autoantibodies and thus thought to drive disease. Neutrophil extracellular traps (NETs) are a source of citrullinated proteins and are increased in rheumatoid arthritis and therefore also implicated in disease pathogenesis. However, not all NETs are citrullinated. One theory aiming to clarify the intersection of citrullination, NETs, and rheumatoid arthritis suggests that specific stimuli induce different types of NETs defined by citrullination status. However, most studies do not evaluate uncitrullinated NETs, only citrullinated or total NETs. Further, the requirement for peptidylarginine deiminase (PAD) 2 and 4, two important citrullinating enzymes in neutrophils and rheumatoid arthritis, in the formation of different NETs has not been clearly defined. To determine if specific stimulants induce citrullinated or uncitrullinated NETs and if those structures require PAD2 or PAD4, human and murine neutrophils, including from PAD4

Topics: Animals; Arthritis, Rheumatoid; Citrullination; Citrulline; Extracellular Traps; Humans; Ionomycin; Mice; Mice, Inbred DBA; Mice, Knockout; Neutrophils; Protein-Arginine Deiminase Type 2; Protein-Arginine Deiminases; Tetradecanoylphorbol Acetate; Uric Acid

Autoantibodies against citrullinated proteins are diagnostic for rheumatoid arthritis. However, the molecular mechanisms driving protein citrullination in patients with rheumatoid arthritis remain poorly understood. Using two independent western blotting methods, we report that agents that trigger a sufficiently large influx of extracellular calcium ions induced a marked citrullination of multiple proteins in human neutrophils, monocytes, and, to a lesser extent, T lymphocytes and natural killer cells, but not B lymphocytes or dendritic cells. This response required 250-1,000 μM extracellular calcium and was prevented by EDTA. Other neutrophil activating stimuli, such as formyl-peptides, GM-CSF, IL-6, IL8, TNFα, or phorbol ester, did not induce any detectable increase in protein citrullination, suggesting that receptor-induced calcium mobilization is insufficient to trigger hypercitrullination. We conclude that loss of membrane integrity and subsequent influx of high levels of calcium, which can be triggered by perforin released from cytotoxic cells or complement mediated formation of membrane attack complexes in the joints of rheumatoid arthritis patients, are sufficient to induce extensive protein citrullination in immune cells, notably neutrophils. This mechanism may provide the citrullinated autoantigens that drive autoimmunity in this devastating disease.

Topics: Arthritis, Rheumatoid; Blotting, Western; Cells, Cultured; Citrulline; Humans; Ionomycin; Leukocytes; Neutrophils; Perforin

In the present study, the frequency and function of B10 cells in patients with rheumatoid arthritis (RA) was examined. A total of 24 healthy controls and 97 patients with RA were enrolled in the present study. Among the 75 patients with an active disease status, 51 patients received either no treatment or were treated with non‑steroidal anti‑inflammatory drugs (NSAIDs) only, while 24 patients underwent a disease relapse. Flow cytometry was used to assess the frequency of CD19(+)CD24(hi)CD38(hi) interleukin (IL)‑10(+) cells stimulated by lipopolysaccharide plus CD40L for 48 h, followed by re‑stimulation with phorbol myristate acetate and ionomycin for 5 h. The correlation of CD19(+)CD24(hi)CD38(hi)IL‑10(+)‑cell frequency with clinical/laboratory characteristics and with levels of inflammatory cytokines were assessed along with the effects of CD19(+)CD24(hi)CD38(hi) cells on the proliferation and tumor necrosis factor α expression of CD3+ T cells. The median frequency of IL‑10‑competent cells among the CD19+ B cells was significantly increased among patients with RA with active disease. However, a sub‑group of patients with a high disease status that received no treatment/NSAIDs exhibited a significantly lower frequency (≤1% IL‑10+ B cells). These patients exhibited longer symptom duration, a greater number of tender and swollen joints and a higher patient global visual analogue scale and disease activity score in 28 joints‑C reactive protein. Functional assays further demonstrated that B10 cells from the sub‑group with ≤1% IL‑10+ B cells secreted significantly lower levels of IL‑10 and exerted a significantly decreased suppressive effect on CD3+ T-cell proliferation and tumor necrosis factor‑α production. The frequency and functional heterogeneity of B10 cells in patients with RA at different disease stage suggested that further investigation on the underlying mechanism of the generation and function of B10 cells in patients with RA is required prior to use in clinical practice.

Topics: Adolescent; Adult; Anti-Inflammatory Agents, Non-Steroidal; Antigens, CD; Arthritis, Rheumatoid; B-Lymphocytes, Regulatory; Case-Control Studies; CD40 Ligand; Cell Proliferation; Female; Gene Expression; Humans; Interleukin-10; Ionomycin; Lipopolysaccharides; Lymphocyte Activation; Lymphocyte Count; Male; Middle Aged; Primary Cell Culture; Recurrence; Severity of Illness Index; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha

Circulating monocytes contain a subpopulation of CD14+CD16+ cells; this subpopulation of cells has been described to be proinflammatory and to have an increased frequency in rheumatoid arthritis (RA). New evidence suggests that this subpopulation can be further subdivided into CD14(dim) CD16+ and CD14(bright) CD16+ cells. The aim of this study was to determine which of the two CD16+ monocyte subpopulations is expanded in patients with RA and to investigate their possible role in disease pathogenesis.. The frequencies of monocyte subpopulations in the peripheral blood of healthy donors and patients with RA were determined by flow cytometry. Monocyte subpopulations were sorted and cocultured with CD4+ T cells. Cytokines were determined in the supernatant, and Th17 cell frequencies were measured by flow cytometry.. In comparison with the other monocyte subpopulations, CD14(bright) CD16+ cells showed higher HLA-DR and CCR5 expression and responded with higher tumor necrosis factor production to direct cell contact with preactivated T cells. They were observed at increased frequencies in the peripheral blood of patients with RA, while CD14(dim) CD16+ monocyte frequencies were not increased. CD14(bright) CD16+ cells were extremely potent inducers of Th17 cell expansion in vitro. Their frequency in the peripheral blood of patients with RA correlated closely with Th17 cell frequencies determined ex vivo.. This study is the first to provide a link between the increased frequency of the CD14(bright) CD16+ monocyte subpopulation in RA and the expansion of Th17 cells, which are likely to have a role in the pathogenesis of autoimmunity.

Topics: Arthritis, Rheumatoid; Cell Differentiation; Cell Separation; Coculture Techniques; Cytokines; Female; Flow Cytometry; HLA-DR Antigens; Humans; Ionomycin; Leukocyte Count; Lipopolysaccharide Receptors; Male; Middle Aged; Monocytes; Receptors, CCR5; Receptors, IgG; Tetradecanoylphorbol Acetate; Th17 Cells

To analyze the role of interleukin 22 (IL-22) in rheumatoid arthritis (RA).. IL-22 serum levels were measured in 83 patients with established RA under treatment with disease-modifying antirheumatic drugs and in 30 healthy controls matched for age and sex. Patients were assessed for clinical and laboratory variables. Correlations of IL-22 serum levels with disease activity measures [Clinical Disease Activity Index (CDAI) and Disease Activity Score for 28 joints (DAS28)], serological markers, bone erosions, and demographic factors were assessed. Peripheral blood mononuclear cells (PBMC) from 30 patients with RA and 14 controls were purified and stimulated in vitro with phorbol myristate acetate (PMA)/ionomycin. IL-22 production by PBMC and in serum was investigated by ELISA.. IL-22 levels were increased in patients with RA compared with controls (mean 432.37 pg/ml and 67.45 pg/ml, respectively; p < 0.001). Levels of IL-22 correlated with DAS28 and CDAI measures. Rheumatoid factor (RF) positivity was correlated with higher levels of IL-22 in patients with RA (mean 575.08 pg/ml; p = 0.001). The presence of bone erosions was associated with high IL-22 levels (p = 0.0001). PBMC stimulated with PMA/ionomycin expressed higher levels of IL-22 in patients with RA than controls but this was not significant (mean 584.75 pg/ml and 295.57 pg/ml; p = 0.553).. IL-22 is elevated in the serum of patients with established RA. Elevated serum IL-22 allows discrimination between patients with different clinical and laboratory measures and indicates the potential of IL-22 as an additional tool for assessment of activity in RA, particularly in patients with RF antibodies and longterm disease. IL-22 is associated with bone-destructive disease.

Topics: Adult; Aged; Antirheumatic Agents; Arthritis, Rheumatoid; Calcium Ionophores; Carcinogens; Cells, Cultured; Disease Progression; Female; Humans; Interleukin-22; Interleukins; Ionomycin; Leukocytes, Mononuclear; Male; Middle Aged; Osteolysis; Rheumatoid Factor; Severity of Illness Index; Tetradecanoylphorbol Acetate

LIGHT (TNFSF 14) belongs to the tumor necrosis factor superfamily and is expressed by activated T cells as well as various types of antigen presenting cells. LIGHT binds to its cellular receptors TR2 and LTbetaR and has a co-stimulatory role in T cell activation. Here, we compared the relative expression of LIGHT in different immune cells and the biological activity of immune cell-derived LIGHT on endothelial cells.. Surface expression of LIGHT and mRNA production by PBMC and isolated T cells (CD4+ or CD8+) significantly increased after stimulation with PMA (Phorbolester-12- Myristat-13-Acetat)+ionomycin. No LIGHT expression on PMA stimulated monocytes or monocytic-like THP-1 cells could be detected; differentiation of monocytes and THP-1 cells into macrophages, however, resulted in up-regulation of LIGHT. Supernatants of stimulated T cells contained higher concentrations of soluble LIGHT than macrophage supernatants normalized to cell numbers; release of soluble LIGHT was found to be dependent on metalloproteinase activity. Size determination of released soluble LIGHT by size exclusion chromatography revealed a molecular mass of approximately 60 kDa, suggesting a trimeric form. Released soluble LIGHT induced expression of proinflammatory antigens ICAM-1, tissue factor and IL-8 in human endothelial cells and caused apoptosis of IFN-g pretreated endothelial cells. Soluble LIGHT was detected at low levels in sera of healthy controls and was significantly enhanced in sera of patients with chronic hepatitis C and rheumatoid arthritis (24.93+/-9.41 vs. 129.53+/-49.14 and 172.13+/-77.64; p<0.0005).. These findings suggest that among immune cells activated T lymphocytes are the main source of soluble LIGHT with released amounts of soluble LIGHT markedly higher compared to platelets. Immune cell-derived membrane-bound and soluble trimeric LIGHT is biologically active, inducing proinflammatory changes in endothelial cells. Enhanced plasma levels of soluble LIGHT in patients with chronic infections suggest a role of LIGHT in systemic inflammatory activation.

Topics: Adolescent; Adult; Aged; Arthritis, Rheumatoid; Cell Differentiation; Cells, Cultured; Drug Combinations; Endothelium, Vascular; Enzyme-Linked Immunosorbent Assay; Female; Gene Expression; Hepatitis C, Chronic; Humans; Intercellular Adhesion Molecule-1; Interleukin-8; Ionomycin; Leukocytes, Mononuclear; Macrophages; Middle Aged; Molecular Weight; RNA, Messenger; T-Lymphocytes; Tetradecanoylphorbol Acetate; Thromboplastin; Tumor Necrosis Factor Ligand Superfamily Member 14; Umbilical Veins; Up-Regulation; Young Adult

Although interleukin-17 (IL-17)-producing gamma/delta T cells were reported to play pathogenic roles in collagen-induced arthritis (CIA), their characteristics remain unknown. The aim of this study was to clarify whether gamma/delta T cells or CD4+ T cells are the predominant IL-17-producing cells, and to determine what stimulates gamma/delta T cells to secret IL-17 in mice with CIA. The involvement of IL-17-producing gamma/delta T cells in SKG mice with autoimmune arthritis and patients with rheumatoid arthritis (RA) was also investigated.. IL-17-producing cells in the affected joints of mice with CIA were counted by intracellular cytokine staining during 6 distinct disease phases, and these cells were stimulated with various combinations of cytokines or specific antigens to determine the signaling requirements. Similar studies were performed using SKG mice with arthritis and patients with RA.. Gamma/delta T cells were the predominant population in IL-17-producing cells in the swollen joints of mice with CIA, and the absolute numbers of these cells increased in parallel with disease activity. IL-17-producing gamma/delta T cells expressed CC chemokine receptor 6, were maintained by IL-23 but not by type II collagen in vitro, and were induced antigen independently in vivo. Furthermore, IL-17 production by gamma/delta T cells was induced by IL-1beta plus IL-23 independently of T cell receptor. In contrast to what was observed in mice with CIA, IL-17-producing gamma/delta T cells were nearly absent in the affected joints of SKG mice and patients with RA, and Th1 cells were predominant in the joints of patients with RA.. Gamma/delta T cells were antigen independently stimulated by inflammation at affected joints and produced enhanced amounts of IL-17 to exacerbate arthritis in mice with CIA but not in SKG mice with arthritis or patients with RA.

Topics: Adult; Aged; Aged, 80 and over; Animals; Arthritis, Experimental; Arthritis, Rheumatoid; CD4-Positive T-Lymphocytes; Cells, Cultured; Female; Hindlimb; Humans; Interleukin-17; Ionomycin; Joints; Mice; Mice, Inbred BALB C; Mice, Inbred DBA; Middle Aged; Receptors, Antigen, T-Cell, gamma-delta; Tetradecanoylphorbol Acetate

Here we studied and characterized different peripheral blood (PB) regulatory T cell (Treg) subsets in rheumatoid arthritis (RA) patients and tested the hypothesis that changes in these cells can be linked to the degree of inflammation and relapsing/remission periods. PB cells were examined from RA subjects (n = 60) with different disease activity score-28 (DAS28) and from healthy controls (n = 40). Frequencies of Treg subsets expressing characteristic membrane antigens, FoxP3 or intracellular cytokines were quantified by flow cytometry. We observed a decrease in the percentages of CD4(+)CD25(high), CD4(+)CD25(int), CD4(+)CD25(int/high)FoxP3(+), CD4(+)CD38(+), CD4(+)CD62L(+), CD8(+)CD25(high)CD45RA(+) and CD8(+)CD25(int)CD45RA(+) T cells in PB of RA patients compared to healthy controls. In addition, we found increased percentages of cells expressing membrane/intracellular regulatory antigens such as OX40 (CD134), CD45RB(low) or CTLA-4 (CD152), and a higher proportion of other T cell subsets including CD4(+)CTLA-4(+), CD4(+)IL10(+), CD4(+)CD25(int)IL10(+), CD4(+)CD25(int) TGFbeta(+), CD4(+)CD25(low) TGFbeta(+) and CD8(+)CD28(- ). We show that most of these changes parallel the intensity of inflammation, with lowest or highest values in patients with moderately/very active disease compared to healthy controls and at times to patients with inactive RA. The balance between these cell subsets and their antigen expression would determine the inflammation levels and could thus be linked to the relapsing/remission periods of the disease.

Topics: Aged; Antigens, CD; Arthritis, Rheumatoid; CD4 Lymphocyte Count; CTLA-4 Antigen; Female; Forkhead Transcription Factors; Humans; Immunophenotyping; Inflammation; Interferon-gamma; Interleukin-10; Interleukin-2 Receptor alpha Subunit; Ionomycin; Male; Middle Aged; Recurrence; T-Lymphocyte Subsets; T-Lymphocytes, Regulatory; Tetradecanoylphorbol Acetate; Transforming Growth Factor beta

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

Vasoactive intestinal peptide (VIP) is an anti-inflammatory immunomodulatory neuropeptide with therapeutic potential demonstrated for collagen-induced arthritis. The aim of this study was to characterise its potential anti-arthritic effect on human monocytes, macrophages, T cells, and rheumatoid arthritis synovial membrane cells. Monocytes, macrophages, and T cells derived from human peripheral blood were treated with VIP and compared with other cAMP-elevating drugs for a range of activating stimuli. Cytokine production was assessed for cell cultures and, in addition, the ability of VIPs to activate cAMP response element binding protein. VIP partially suppressed monocyte- and macrophage-derived tumour necrosis factor alpha (TNF-alpha) with no effect on IL-10, whereas VIP fails to regulate IL-10 and TNF-alpha production by T lymphocytes. No such modulation of cytokine profile was observed for rheumatoid arthritis synovial membrane cells. Elevation of intracellular cAMP, on the other hand, potently suppressed macrophage TNF-alpha production and modulated T-cell response by inhibiting TNF-alpha and IFN-gamma. VIP's lack of effect on IL-10 and its slight effect on TNF-alpha results from cAMP being rapidly degraded as the phosphodiesterase IV inhibitor, rolipram, rescues cAMP-dependent activation of cAMP response element binding protein. Interestingly, macrophages stimulated with phorbol 12-myristate 13-acetate/ionomycin displayed an augmented IL-10 response upon addition of dibutyryl cAMP, with corresponding downregulation in TNF-alpha, suggesting a complex interaction between protein kinase C and protein kinase A in cytokine regulation. In conclusion, VIP may represent an efficaceous anti-arthritic treatment modulating macrophage and T-cell cytokine profiles when used alongside a phosphodiesterase inhibitor.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Arthritis, Rheumatoid; Bucladesine; Cells, Cultured; Colforsin; Cyclic AMP; Cyclic AMP-Dependent Protein Kinase Type II; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 4; Gene Expression Regulation; Humans; Interferon-gamma; Interleukin-10; Ionomycin; Lipopolysaccharides; Macrophages; Monocytes; Phosphodiesterase Inhibitors; Protein Kinase C; Rolipram; Synovial Membrane; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tumor Necrosis Factor-alpha; Vasoactive Intestinal Peptide

To provide a comprehensive understanding of the humoral immune response that takes place at the site of inflammation in rheumatoid arthritis (RA), we studied the functional properties of synovial B cells. In particular, the response to various modes of mitogen stimulation was investigated.. Purified synovial fluid (SF) B cells were cultured in the presence of CD40 ligand (CD40L)-expressing fibroblasts and cytokines, activated T cells, or phorbol myristate acetate (PMA)/ionomycin. Proliferation was determined by 3H-thymidine incorporation. Release of intracellular calcium was studied by flow cytometry.. The inflamed joints of RA patients contained a population of CD20+,CD38- B cells with dramatically impaired mitogen responsiveness. Although the Ig-producing capacity was intact, these cells failed to proliferate in response to (a) CD40 in the presence of interleukin-2 (IL-2) and IL-10, (b) activated T cells, or (c) stimulation via the B cell receptor. Moreover, SF CD20+,CD38- B cells revealed a defective B cell receptor-induced Ca2+ influx, reminiscent of anergic B cells. Release of intracellular Ca2+ by ionomycin in the presence of the protein kinase C activator PMA did not restore the proliferative capacity. These findings indicate blockades in the proximal and distal intermediates involved in mitogen signaling.. SF CD20+,CD38- B cells have functionally impaired proliferative responsiveness. The capacity of these cells to respond to activation by the production of Ig supports the notion that these cells might serve as Ig-producing effector cells and, as such, play a role in the pathophysiology of RA.

Topics: ADP-ribosyl Cyclase; ADP-ribosyl Cyclase 1; Adult; Aged; Antigens, CD; Antigens, CD20; Antigens, Differentiation; Arthritis, Rheumatoid; B-Lymphocytes; Calcium Signaling; Carcinogens; CD40 Antigens; CD40 Ligand; Cell Division; Cells, Cultured; Female; Humans; Immunoglobulins; Interleukin-10; Interleukin-2; Ionomycin; Ionophores; Male; Membrane Glycoproteins; Middle Aged; NAD+ Nucleosidase; Oxidation-Reduction; Synovial Fluid; Synovial Membrane; T-Lymphocytes; Tetradecanoylphorbol Acetate

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

Topics: Arthritis, Rheumatoid; Blotting, Western; CD13 Antigens; Cell Communication; Cell Movement; Coculture Techniques; Collagen; Cytochalasin B; Fibroblasts; Fluorescent Antibody Technique, Indirect; Gels; Gene Expression Profiling; Humans; Ionomycin; Jurkat Cells; Lymphocyte Activation; MAP Kinase Signaling System; Microscopy, Confocal; Promoter Regions, Genetic; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Stromal Cells; Synovial Fluid; T-Lymphocytes; Tetradecanoylphorbol Acetate

Peripheral blood (PB) T cells from rheumatoid arthritis (RA) patients proliferate poorly to mitogen, a change that is related to decreased intracellular Ca2+ ([Ca2+]i) signaling after T cell receptor (TCR) stimulation. We hypothesized that this was, in part, due to the effect of mediators of inflammation and predicted that greater changes in [Ca2+]i signaling would be seen in synovial fluid (SF) T cells. We also examined the mechanisms underlying the altered [Ca2+]i signals.. Paired PB and SF T cells from patients with chronic inflammatory arthritis were stimulated with mitogen to assess the magnitude of the [Ca2+]i signal in cell populations by fluorometry, the pattern of the [Ca2+]i signal in individual cells in a single-cell ion-imaging system, and the spatial distribution of Ca2+ within intracellular organelles.. There was a significantly smaller [Ca2+]i signal after phytohemagglutinin protein stimulation of SF T cells (peak rise in [Ca2+]i signal PB versus SF 200 nM versus 180 nM; P < 0.05). In single SF T cells, a change in the pattern of the [Ca2+]i signal and a reduction in the number of responding cells was seen. These changes were a magnification of those seen in RA PB compared with control PB T cells. The contribution of Ca2+ release from intracellular stores to the final [Ca2+]i signal in PB and SF T cells was equal, but there was a significant increase in the Ca2+ remaining in the endoplasmic reticulum (ER) in SF T cells after TCR activation (PB versus SF 6 nM versus 19 nM; P < 0.05). Non-ER Ca2+ stores were not similarly affected.. We found abnormalities in the magnitude, pattern, and spatial distribution of [Ca2+]i signaling in T cells from SF of patients with chronic inflammatory arthritis. A reduction in the number of responding SF T cells may partly explain some of our observations. However, we propose that the observed redistribution of SF Ca2+ stores may underlie the altered [Ca2+]i signaling, thus making these cells hyporesponsive to mitogen. The inflammatory environment of the joint and the late stage of differentiation of SF T cells are both likely to contribute to these changes in [Ca2+]i signaling, resulting in aberrant T cell function and promotion of disease chronicity.

Topics: Arthritis, Rheumatoid; Calcium; Calcium Signaling; Chronic Disease; Endoplasmic Reticulum; Enzyme Inhibitors; Humans; Inositol 1,4,5-Trisphosphate; Intracellular Membranes; Ionomycin; Ionophores; Organelles; Reference Values; Synovial Fluid; T-Lymphocytes; Thapsigargin

The analysis of cytokine production is increasingly important in defining the course of an immune response and in evaluating specific therapies of immune diseases. In rheumatoid arthritis (RA), a dysregulation in T1/T2 cell balance, as defined by the production of their specific cytokines, IFN-gamma and IL-4, respectively, is suggested. A predominance of T1-cell mediated macrophage activity in the joint plays a key role in the destruction of articular cartilage and subchondral bone, whereas local T2 cell activity, mitigating disease, fails. However, analysis of the cytokines defining both T cell subsets is difficult and spontaneous production is often below detection limits. Several stimuli are therefore used to increase cytokine production. In the present study we examined whether stimulation of peripheral blood T cells in the context of mononuclear cells (PB MNC) by CD3-CD28 is a reliable method for assessing IFN-gamma and IL-4 production and is representative for the spontaneous production of these cytokines. The production of IFN-gamma and IL-4 following CD3-CD28 stimulation of RA PB MNC correlated significantly in a ratio 1 : 1 with production following ionomycin-PMA stimulation. In samples with detectable spontaneous production of IFNgamma and IL-4, production following CD3-CD28 stimulation was significantly higher than in stimulated samples with undetectable spontaneous production. Moreover, in the case of spontaneous production there was a significant positive linear correlation between the CD3-CD28 stimulated and spontaneous IFNgamma and IL-4 production, although production of both cytokines was not equally enhanced. Serial sampling did not show significant daily or weekly variation in stimulated cytokine production. The results demonstrate that a pecific T-cell stimulation by CD3-CD28 is a reliable way to enhance IFN-gamma and IL-4 production above the detection limit and so measure the T1/T2 cell balance in RA.

Topics: Arthritis, Rheumatoid; CD28 Antigens; CD3 Complex; Humans; Interferon-gamma; Interleukin-4; Ionomycin; Lymphocyte Activation; Statistics, Nonparametric; T-Lymphocytes; Tetradecanoylphorbol Acetate; Th1 Cells; Th2 Cells

To investigate the mechanisms of the deficient proliferative responses by rheumatoid arthritis (RA) peripheral blood T cells to the recall antigen tuberculin purified protein derivative (PPD).. The concomitant production of interleukin (IL)-2, IL-10 and lymphocyte proliferation were studied by enzyme-linked immunosorbent assay and [(3)H]thymidine uptake, respectively, in 12 normal controls and eight RA patients.. An inverse correlation was found between IL-10 production and proliferation to PPD. The proliferative response was shown to be critically affected by the IL-2:IL-10 ratio so that absolute levels of secreted IL-2 or IL-10 correlated non-significantly with lymphocyte proliferation.. The deficient T-cell proliferation in RA peripheral blood mononuclear cells is related to the relative proportions of IL-2:IL-10 rather than the absolute amounts secreted.

Topics: Adult; Aged; Arthritis, Rheumatoid; Cell Division; Female; Humans; Interleukin-10; Interleukin-2; Ionomycin; Leukocytes, Mononuclear; Male; Middle Aged; T-Lymphocytes; Tetradecanoylphorbol Acetate; Tuberculin

The effects of non-lethal amounts of a variety of pore-forming agents on cultured human rheumatoid synovial cells (HRSC) have been investigated. Non-lethal complement membrane attack and non-lethal amounts of melittin, perforin and ionomycin all caused a biphasic release of prostaglandin E2 (PGE2) from HRSC, an early phase of release occurring within 1 hr and a second larger phase commencing after 4 hr and continuing over the 24-hr time-course. Removal of extracellular calcium abolished the release of PGE2 under all conditions of non-lethal attack. Modulation of G-protein activity reduced the second phase of release caused by non-lethal doses of the membrane-attack complex (MAC) from 800 ng/10(6) cells PGE2 to around 300 ng/10(6) cells. Non-lethal levels of the MAC also caused release of interleukin-6 (IL-6) from HRSC over the 24-hr time-course, with levels reaching 550 ng/10(6) cells at 24 hr compared to background levels of 200 ng/10(6) cells. No detectable release of IL-1 alpha could be measured at any time following non-lethal complement membrane attack. These results suggest a role for the MAC as an initiating mediator inducing the inflammation associated with rheumatoid arthritis.

Topics: Arthritis, Rheumatoid; Calcium; Cells, Cultured; Complement Membrane Attack Complex; Dinoprostone; Humans; Interleukins; Ionomycin; Melitten; Membrane Glycoproteins; Membrane Proteins; Perforin; Pore Forming Cytotoxic Proteins; Synovial Membrane; Virulence Factors, Bordetella