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T528 0-149 Sentence denotes Increased interleukin-17 production via a phosphoinositide 3-kinase/Akt and nuclear factor κB-dependent pathway in patients with rheumatoid arthritis
T1 0-149 Sentence denotes Increased interleukin-17 production via a phosphoinositide 3-kinase/Akt and nuclear factor κB-dependent pathway in patients with rheumatoid arthritis
T2 151-159 Sentence denotes Abstract
T529 160-272 Sentence denotes Inflammatory mediators have been recognized as being important in the pathogenesis of rheumatoid arthritis (RA).
T3 160-272 Sentence denotes Inflammatory mediators have been recognized as being important in the pathogenesis of rheumatoid arthritis (RA).
T530 273-443 Sentence denotes Interleukin (IL)-17 is an important regulator of immune and inflammatory responses, including the induction of proinflammatory cytokines and osteoclastic bone resorption.
T4 273-443 Sentence denotes Interleukin (IL)-17 is an important regulator of immune and inflammatory responses, including the induction of proinflammatory cytokines and osteoclastic bone resorption.
T531 444-574 Sentence denotes Evidence for the expression and proinflammatory activity of IL-17 has been demonstrated in RA synovium and in animal models of RA.
T5 444-574 Sentence denotes Evidence for the expression and proinflammatory activity of IL-17 has been demonstrated in RA synovium and in animal models of RA.
T532 575-749 Sentence denotes Although some cytokines (IL-15 and IL-23) have been reported to regulate IL-17 production, the intracellular signaling pathways that regulate IL-17 production remain unknown.
T6 575-749 Sentence denotes Although some cytokines (IL-15 and IL-23) have been reported to regulate IL-17 production, the intracellular signaling pathways that regulate IL-17 production remain unknown.
T533 750-893 Sentence denotes In the present study, we investigated the role of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the regulation of IL-17 production in RA.
T7 750-893 Sentence denotes In the present study, we investigated the role of the phosphoinositide 3-kinase (PI3K)/Akt pathway in the regulation of IL-17 production in RA.
T534 894-1125 Sentence denotes Peripheral blood mononuclear cells (PBMC) from patients with RA (n = 24) were separated, then stimulated with various agents including anti-CD3, anti-CD28, phytohemagglutinin (PHA) and several inflammatory cytokines and chemokines.
T8 894-1125 Sentence denotes Peripheral blood mononuclear cells (PBMC) from patients with RA (n = 24) were separated, then stimulated with various agents including anti-CD3, anti-CD28, phytohemagglutinin (PHA) and several inflammatory cytokines and chemokines.
T535 1126-1253 Sentence denotes IL-17 levels were determined by sandwich enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction.
T9 1126-1253 Sentence denotes IL-17 levels were determined by sandwich enzyme-linked immunosorbent assay and reverse transcription–polymerase chain reaction.
T536 1254-1391 Sentence denotes The production of IL-17 was significantly increased in cells treated with anti-CD3 antibody with or without anti-CD28 and PHA (P < 0.05).
T10 1254-1391 Sentence denotes The production of IL-17 was significantly increased in cells treated with anti-CD3 antibody with or without anti-CD28 and PHA (P < 0.05).
T537 1392-1609 Sentence denotes Among tested cytokines and chemokines, IL-15, monocyte chemoattractant protein-1 and IL-6 upregulated IL-17 production (P < 0.05), whereas tumor necrosis factor-α, IL-1β, IL-18 or transforming growth factor-β did not.
T11 1392-1609 Sentence denotes Among tested cytokines and chemokines, IL-15, monocyte chemoattractant protein-1 and IL-6 upregulated IL-17 production (P < 0.05), whereas tumor necrosis factor-α, IL-1β, IL-18 or transforming growth factor-β did not.
T538 1610-1745 Sentence denotes IL-17 was also detected in the PBMC of patients with osteoarthritis, but their expression levels were much lower than those of RA PBMC.
T12 1610-1745 Sentence denotes IL-17 was also detected in the PBMC of patients with osteoarthritis, but their expression levels were much lower than those of RA PBMC.
T539 1746-1911 Sentence denotes Anti-CD3 antibody activated the PI3K/Akt pathway; activation of this pathway resulted in a pronounced augmentation of nuclear factor κB (NF-κB) DNA-binding activity.
T13 1746-1911 Sentence denotes Anti-CD3 antibody activated the PI3K/Akt pathway; activation of this pathway resulted in a pronounced augmentation of nuclear factor κB (NF-κB) DNA-binding activity.
T540 1912-2114 Sentence denotes IL-17 production by activated RA PBMC is completely or partly blocked in the presence of the NF-κB inhibitor pyrrolidine dithiocarbamate and the PI3K/Akt inhibitor wortmannin and LY294002, respectively.
T14 1912-2114 Sentence denotes IL-17 production by activated RA PBMC is completely or partly blocked in the presence of the NF-κB inhibitor pyrrolidine dithiocarbamate and the PI3K/Akt inhibitor wortmannin and LY294002, respectively.
T541 2115-2236 Sentence denotes However, inhibition of activator protein-1 and extracellular signal-regulated kinase 1/2 did not affect IL-17 production.
T15 2115-2236 Sentence denotes However, inhibition of activator protein-1 and extracellular signal-regulated kinase 1/2 did not affect IL-17 production.
T542 2237-2405 Sentence denotes These results suggest that signal transduction pathways dependent on PI3K/Akt and NF-κB are involved in the overproduction of the key inflammatory cytokine IL-17 in RA.
T16 2237-2405 Sentence denotes These results suggest that signal transduction pathways dependent on PI3K/Akt and NF-κB are involved in the overproduction of the key inflammatory cytokine IL-17 in RA.
T17 2407-2419 Sentence denotes Introduction
T2748 2420-2548 Sentence denotes Rheumatoid arthritis (RA) is characterized by infiltrations of macrophages and T cells into the joint, and synovial hyperplasia.
T18 2420-2548 Sentence denotes Rheumatoid arthritis (RA) is characterized by infiltrations of macrophages and T cells into the joint, and synovial hyperplasia.
T2749 2549-2666 Sentence denotes Proinflammatory cytokines released from these cells are known to be important in the destruction of joints in RA [1].
T19 2549-2666 Sentence denotes Proinflammatory cytokines released from these cells are known to be important in the destruction of joints in RA [1].
T2750 2667-2921 Sentence denotes The favorable clinical benefits obtained with inhibitors of tumor necrosis factor (TNF)-α) and interleukin (IL)-1 suggest that the blockade of key inflammatory cytokines has been the important issue in the development of new therapeutic applications [2].
T20 2667-2921 Sentence denotes The favorable clinical benefits obtained with inhibitors of tumor necrosis factor (TNF)-α) and interleukin (IL)-1 suggest that the blockade of key inflammatory cytokines has been the important issue in the development of new therapeutic applications [2].
T2751 2922-3112 Sentence denotes A little over a decade ago, the primacy of T cells in the pathogenesis of autoimmune disease such as RA was undisputed because they are the largest cell population infiltrating the synovium.
T21 2922-3112 Sentence denotes A little over a decade ago, the primacy of T cells in the pathogenesis of autoimmune disease such as RA was undisputed because they are the largest cell population infiltrating the synovium.
T2752 3113-3368 Sentence denotes However, a series of studies demonstrated paucity of T cell-derived cytokines such as IL-2 and interferon-γ in the joints of RA, whereas macrophage and fibroblast cytokines including IL-1, IL-6, IL-15, IL-18 and TNF-α were abundant in rheumatoid synovium.
T22 3113-3368 Sentence denotes However, a series of studies demonstrated paucity of T cell-derived cytokines such as IL-2 and interferon-γ in the joints of RA, whereas macrophage and fibroblast cytokines including IL-1, IL-6, IL-15, IL-18 and TNF-α were abundant in rheumatoid synovium.
T2753 3369-3447 Sentence denotes This paradox has questioned the role of T cells in the pathogenesis of RA [3].
T23 3369-3447 Sentence denotes This paradox has questioned the role of T cells in the pathogenesis of RA [3].
T2754 3448-3699 Sentence denotes Because we have already demonstrated the enhanced proliferation of antigen specific T cells, especially to type II collagen, and the skewing of T helper type 1 (Th1) cytokines in RA [4], the role of T cells needs to be elucidated in different aspects.
T24 3448-3699 Sentence denotes Because we have already demonstrated the enhanced proliferation of antigen specific T cells, especially to type II collagen, and the skewing of T helper type 1 (Th1) cytokines in RA [4], the role of T cells needs to be elucidated in different aspects.
T2755 3700-3831 Sentence denotes IL-17 is one of the inflammatory cytokines secreted mainly by activated T cells, which can induce IL-6 and IL-8 by fibroblasts [5].
T25 3700-3831 Sentence denotes IL-17 is one of the inflammatory cytokines secreted mainly by activated T cells, which can induce IL-6 and IL-8 by fibroblasts [5].
T2756 3832-3996 Sentence denotes This cytokine is of interest for two major reasons: first, similarly to TNF-α and IL-1, IL-17 has proinflammatory properties; second, it is produced by T cells [6].
T26 3832-3996 Sentence denotes This cytokine is of interest for two major reasons: first, similarly to TNF-α and IL-1, IL-17 has proinflammatory properties; second, it is produced by T cells [6].
T2757 3997-4216 Sentence denotes Recent observations demonstrated that IL-17 can also activate osteoclastic bone resorption by the induction of RANKL (receptor activator of nuclear factor κB [NF-κB] ligand), which is involved in bony erosion in RA [7].
T27 3997-4216 Sentence denotes Recent observations demonstrated that IL-17 can also activate osteoclastic bone resorption by the induction of RANKL (receptor activator of nuclear factor κB [NF-κB] ligand), which is involved in bony erosion in RA [7].
T2758 4217-4442 Sentence denotes It also stimulates the production of IL-6 and leukemia inhibitory factor by synoviocytes, and of prostaglandin E2 and nitric oxide by chondrocytes, and has the ability to differentiate and activate the dendritic cells [8-10].
T28 4217-4442 Sentence denotes It also stimulates the production of IL-6 and leukemia inhibitory factor by synoviocytes, and of prostaglandin E2 and nitric oxide by chondrocytes, and has the ability to differentiate and activate the dendritic cells [8-10].
T2759 4443-4627 Sentence denotes Levels of IL-17 in synovial fluids were significantly higher in patients with RA than in patients with osteoarthritis (OA), and it was produced by CD4+ T cells in the synovium [11,12].
T29 4443-4627 Sentence denotes Levels of IL-17 in synovial fluids were significantly higher in patients with RA than in patients with osteoarthritis (OA), and it was produced by CD4+ T cells in the synovium [11,12].
T2760 4628-4837 Sentence denotes IL-15, secreted from activated macrophages, has been reported to be an important trigger of IL-17 production in RA peripheral blood mononuclear cells (PBMC) by cyclosporine and steroid sensitive pathways [13].
T30 4628-4837 Sentence denotes IL-15, secreted from activated macrophages, has been reported to be an important trigger of IL-17 production in RA peripheral blood mononuclear cells (PBMC) by cyclosporine and steroid sensitive pathways [13].
T2761 4838-4980 Sentence denotes Recently, Happel and colleagues also showed that IL-23 could be an efficient trigger of IL-17 production from both CD4+ and CD8+ T cells [14].
T31 4838-4980 Sentence denotes Recently, Happel and colleagues also showed that IL-23 could be an efficient trigger of IL-17 production from both CD4+ and CD8+ T cells [14].
T2762 4981-5180 Sentence denotes Although the contribution of IL-17 in joint inflammation in RA has been documented in earlier studies [12,15,16], the intracellular signal transduction pathway for IL-17 production remains uncertain.
T32 4981-5180 Sentence denotes Although the contribution of IL-17 in joint inflammation in RA has been documented in earlier studies [12,15,16], the intracellular signal transduction pathway for IL-17 production remains uncertain.
T2763 5181-5325 Sentence denotes In the present study we used various stimuli to investigate IL-17 production in PBMC of patients with RA and its signaling transduction pathway.
T33 5181-5325 Sentence denotes In the present study we used various stimuli to investigate IL-17 production in PBMC of patients with RA and its signaling transduction pathway.
T2764 5326-5518 Sentence denotes We found that the intracellular signaling pathway involving phosphoinositide 3-kinase (PI3K)/Akt and NF-κB might be involved in the overproduction of the key inflammatory cytokine IL-17 in RA.
T34 5326-5518 Sentence denotes We found that the intracellular signaling pathway involving phosphoinositide 3-kinase (PI3K)/Akt and NF-κB might be involved in the overproduction of the key inflammatory cytokine IL-17 in RA.
T2765 5519-5647 Sentence denotes These results might provide new insights into the pathogenesis of RA and future directions for new therapeutic strategies in RA.
T35 5519-5647 Sentence denotes These results might provide new insights into the pathogenesis of RA and future directions for new therapeutic strategies in RA.
T36 5649-5670 Sentence denotes Materials and methods
T4872 5672-5680 Sentence denotes Patients
T37 5672-5680 Sentence denotes Patients
T4873 5681-5889 Sentence denotes Informed consent was obtained from 24 patients (5 men and 19 women) with RA who fulfilled the 1987 revised criteria of the American College of Rheumatology (formerly the American Rheumatism Association) [17].
T38 5681-5889 Sentence denotes Informed consent was obtained from 24 patients (5 men and 19 women) with RA who fulfilled the 1987 revised criteria of the American College of Rheumatology (formerly the American Rheumatism Association) [17].
T4874 5890-5972 Sentence denotes The age of the patients with RA was 50 ± 8 (mean ± SEM) years (range 23–71 years).
T39 5890-5972 Sentence denotes The age of the patients with RA was 50 ± 8 (mean ± SEM) years (range 23–71 years).
T4875 5973-6037 Sentence denotes All medications were stopped 48 hours before entry to the study.
T40 5973-6037 Sentence denotes All medications were stopped 48 hours before entry to the study.
T4876 6038-6186 Sentence denotes Comparisons were made with 14 patients with OA (3 men and 11 women) and with 14 healthy controls (3 men and 11 women) who had no rheumatic diseases.
T41 6038-6186 Sentence denotes Comparisons were made with 14 patients with OA (3 men and 11 women) and with 14 healthy controls (3 men and 11 women) who had no rheumatic diseases.
T4877 6187-6325 Sentence denotes The mean ages of the patients with OA and the healthy controls were 50 ± 8 years (range 34–68 years) and 30 ± 6 years (range 24–57 years).
T42 6187-6325 Sentence denotes The mean ages of the patients with OA and the healthy controls were 50 ± 8 years (range 34–68 years) and 30 ± 6 years (range 24–57 years).
T4878 6326-6455 Sentence denotes Informed consent was obtained, and the protocol was approved by the Catholic University of Korea Human Research Ethics Committee.
T43 6326-6455 Sentence denotes Informed consent was obtained, and the protocol was approved by the Catholic University of Korea Human Research Ethics Committee.
T5348 6457-6465 Sentence denotes Reagents
T44 6457-6465 Sentence denotes Reagents
T5349 6466-6664 Sentence denotes Recombinant IL-17, IL-18, IL-15, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, IL-6 and IL-8 were purchased from R & D systems (Minneapolis, MN, USA).
T45 6466-6664 Sentence denotes Recombinant IL-17, IL-18, IL-15, monocyte chemoattractant protein-1 (MCP-1), macrophage inflammatory protein (MIP)-1α, MIP-1β, IL-6 and IL-8 were purchased from R & D systems (Minneapolis, MN, USA).
T5350 6665-6754 Sentence denotes Recombinant transforming growth factor (TGF)-β was purchased from Peprotech (London, UK).
T46 6665-6754 Sentence denotes Recombinant transforming growth factor (TGF)-β was purchased from Peprotech (London, UK).
T5351 6755-6836 Sentence denotes Recombinant TNF-α and IL-1 were purchased from Endogen Inc. (Cambridge, MA, USA).
T47 6755-6836 Sentence denotes Recombinant TNF-α and IL-1 were purchased from Endogen Inc. (Cambridge, MA, USA).
T5352 6837-6900 Sentence denotes Cyclosporin A was provided by Sandos Ltd. (Basel, Switzerland).
T48 6837-6900 Sentence denotes Cyclosporin A was provided by Sandos Ltd. (Basel, Switzerland).
T5353 6901-7067 Sentence denotes Phytohemagglutinin (PHA), pyrrolidine dithiocarbamate (PDTC), rapamycin, dexamethasone and curcumin were all obtained from the Sigma Chemical Co. (St Louis, MA, USA).
T49 6901-7067 Sentence denotes Phytohemagglutinin (PHA), pyrrolidine dithiocarbamate (PDTC), rapamycin, dexamethasone and curcumin were all obtained from the Sigma Chemical Co. (St Louis, MA, USA).
T5354 7068-7186 Sentence denotes Anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody were obtained from BD Biosciences (San Diego, CA, USA).
T50 7068-7186 Sentence denotes Anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody were obtained from BD Biosciences (San Diego, CA, USA).
T5355 7187-7289 Sentence denotes LY294002, SB203580, FK506, wortmannin and PD98059 were obtained from Calbiochem (Schwalbach, Germany).
T51 7187-7289 Sentence denotes LY294002, SB203580, FK506, wortmannin and PD98059 were obtained from Calbiochem (Schwalbach, Germany).
T6081 7291-7365 Sentence denotes Production of IL-17 by T cell receptor activation, cytokines or chemokines
T52 7291-7365 Sentence denotes Production of IL-17 by T cell receptor activation, cytokines or chemokines
T6082 7366-7467 Sentence denotes PBMC were prepared from heparinized blood by Ficoll-Hypaque (SG1077) density-gradient centrifugation.
T53 7366-7467 Sentence denotes PBMC were prepared from heparinized blood by Ficoll-Hypaque (SG1077) density-gradient centrifugation.
T6083 7468-7526 Sentence denotes Cell cultures were performed as described previously [18].
T54 7468-7526 Sentence denotes Cell cultures were performed as described previously [18].
T6084 7527-7726 Sentence denotes In brief, the cell suspensions were adjusted to a concentration of 106/ml in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine.
T55 7527-7726 Sentence denotes In brief, the cell suspensions were adjusted to a concentration of 106/ml in RPMI 1640 medium supplemented with 10% fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 2 mM L-glutamine.
T6085 7727-7867 Sentence denotes Cell suspension (1 ml) was dispensed into 24-well multi-well plates (Nunc, Roskilde, Denmark), and incubated for 24 hours at 37°C in 5% CO2.
T56 7727-7867 Sentence denotes Cell suspension (1 ml) was dispensed into 24-well multi-well plates (Nunc, Roskilde, Denmark), and incubated for 24 hours at 37°C in 5% CO2.
T6086 7868-8000 Sentence denotes Subsequently, various concentrations of cyclosporin A (10–500 ng/ml) were added to the medium and cells were incubated for 24 hours.
T57 7868-8000 Sentence denotes Subsequently, various concentrations of cyclosporin A (10–500 ng/ml) were added to the medium and cells were incubated for 24 hours.
T6087 8001-8115 Sentence denotes To each well was added FK506, rapamycin, curcumin, PDTC, LY294002, SB203580, PD98059, dexamethasone or wortmannin.
T58 8001-8115 Sentence denotes To each well was added FK506, rapamycin, curcumin, PDTC, LY294002, SB203580, PD98059, dexamethasone or wortmannin.
T6088 8116-8238 Sentence denotes After incubation for 24 hours (unless otherwise stated), cell-free media were collected and stored at -20°C until assayed.
T59 8116-8238 Sentence denotes After incubation for 24 hours (unless otherwise stated), cell-free media were collected and stored at -20°C until assayed.
T6089 8239-8320 Sentence denotes All cultures were set up in triplicate, and results are expressed as means ± SEM.
T60 8239-8320 Sentence denotes All cultures were set up in triplicate, and results are expressed as means ± SEM.
T6724 8322-8351 Sentence denotes CD4+ T-cell isolation by MACS
T61 8322-8351 Sentence denotes CD4+ T-cell isolation by MACS
T6725 8352-8445 Sentence denotes Anti-CD4 microbeads were used essentially as recommended by the manufacturer (Miltenyi) [19].
T62 8352-8445 Sentence denotes Anti-CD4 microbeads were used essentially as recommended by the manufacturer (Miltenyi) [19].
T6726 8446-8500 Sentence denotes PBMC were resuspended in 80 μl of FBS staining buffer.
T63 8446-8500 Sentence denotes PBMC were resuspended in 80 μl of FBS staining buffer.
T64 8501-8575 Sentence denotes Anti-CD4 microbeads (20 μl) were added and incubated for 15 min at 6–12°C.
T6727 8501-8665 Sentence denotes Anti-CD4 microbeads (20 μl) were added and incubated for 15 min at 6–12°C. Saturating amounts of fluorochrome-conjugated antibodies were added for a further 10 min.
T65 8576-8665 Sentence denotes Saturating amounts of fluorochrome-conjugated antibodies were added for a further 10 min.
T6728 8666-8838 Sentence denotes Cells were diluted in 2.5 ml of FBS staining buffer, pelleted, resuspended in 500 μl and magnetically separated, usually on an AutoMACS magnet fitted with a MACS MS column.
T66 8666-8838 Sentence denotes Cells were diluted in 2.5 ml of FBS staining buffer, pelleted, resuspended in 500 μl and magnetically separated, usually on an AutoMACS magnet fitted with a MACS MS column.
T6729 8839-8912 Sentence denotes Flow-through and two 1 ml washes were collected as the negative fraction.
T67 8839-8912 Sentence denotes Flow-through and two 1 ml washes were collected as the negative fraction.
T6730 8913-9012 Sentence denotes Enriched cells were collected in two 0.5 ml aliquots from the column after removal from the magnet.
T68 8913-9012 Sentence denotes Enriched cells were collected in two 0.5 ml aliquots from the column after removal from the magnet.
T6731 9013-9244 Sentence denotes Alternatively, cells stained with anti-CD4–phycoerythrin were washed, magnetically labeled with anti-phycoerythrin microbeads (20 μl added to 80 μl of cell suspension; 15 min, 6–12°C), and magnetically separated as described above.
T69 9013-9244 Sentence denotes Alternatively, cells stained with anti-CD4–phycoerythrin were washed, magnetically labeled with anti-phycoerythrin microbeads (20 μl added to 80 μl of cell suspension; 15 min, 6–12°C), and magnetically separated as described above.
T6732 9245-9348 Sentence denotes The purity of cells was assessed by flow cytometric analysis of stained cells on a FACS Vantage sorter.
T70 9245-9348 Sentence denotes The purity of cells was assessed by flow cytometric analysis of stained cells on a FACS Vantage sorter.
T6733 9349-9418 Sentence denotes Most (more than 97%) of the isolated cells had the CD4 T cell marker.
T71 9349-9418 Sentence denotes Most (more than 97%) of the isolated cells had the CD4 T cell marker.
T7492 9420-9462 Sentence denotes Enzyme-linked immunosorbent assay of IL-17
T72 9420-9462 Sentence denotes Enzyme-linked immunosorbent assay of IL-17
T7493 9463-9581 Sentence denotes IL-17 in culture supernatants was measured by sandwich enzyme-linked immunosorbent assay as described previously [20].
T73 9463-9581 Sentence denotes IL-17 in culture supernatants was measured by sandwich enzyme-linked immunosorbent assay as described previously [20].
T7494 9582-9708 Sentence denotes In brief, a 96-well plate (Nunc) was coated with 4 μg/ml monoclonal antibodies against IL-17 (R & D Systems) at 4°C overnight.
T74 9582-9708 Sentence denotes In brief, a 96-well plate (Nunc) was coated with 4 μg/ml monoclonal antibodies against IL-17 (R & D Systems) at 4°C overnight.
T7495 9709-9985 Sentence denotes After blocking with phosphate-buffered saline/1% bovine serum albumin (BSA)/0.05% Tween 20 for 2 hours at room temperature (22–25°C), test samples and the standard recombinant IL-17 (R & D Systems) were added to the 96-well plate and incubated at room temperature for 2 hours.
T75 9709-9985 Sentence denotes After blocking with phosphate-buffered saline/1% bovine serum albumin (BSA)/0.05% Tween 20 for 2 hours at room temperature (22–25°C), test samples and the standard recombinant IL-17 (R & D Systems) were added to the 96-well plate and incubated at room temperature for 2 hours.
T7496 9986-10194 Sentence denotes Plates were washed four times with phosphate-buffered saline/Tween 20, and then incubated with 500 ng/ml biotinylated mouse monoclonal antibodies against IL-17 (R & D Systems) for 2 hours at room temperature.
T76 9986-10194 Sentence denotes Plates were washed four times with phosphate-buffered saline/Tween 20, and then incubated with 500 ng/ml biotinylated mouse monoclonal antibodies against IL-17 (R & D Systems) for 2 hours at room temperature.
T7497 10195-10453 Sentence denotes After washing, streptavidin–alkaline phosphate–horseradish peroxidase conjugate (Sigma) was incubated for 2 hours, then washed again and incubated with 1 mg/ml p-nitrophenyl phosphate (Sigma) dissolved in diethanolamine (Sigma) to develop the color reaction.
T77 10195-10453 Sentence denotes After washing, streptavidin–alkaline phosphate–horseradish peroxidase conjugate (Sigma) was incubated for 2 hours, then washed again and incubated with 1 mg/ml p-nitrophenyl phosphate (Sigma) dissolved in diethanolamine (Sigma) to develop the color reaction.
T7498 10454-10563 Sentence denotes The reaction was stopped by the addition of 1 M NaOH and the optical density of each well was read at 405 nm.
T78 10454-10563 Sentence denotes The reaction was stopped by the addition of 1 M NaOH and the optical density of each well was read at 405 nm.
T7499 10564-10612 Sentence denotes The lower limit of IL-17 detection was 10 pg/ml.
T79 10564-10612 Sentence denotes The lower limit of IL-17 detection was 10 pg/ml.
T7500 10613-10729 Sentence denotes Recombinant human IL-17 diluted in culture medium was used as a calibration standard, ranging from 10 to 2000 pg/ml.
T80 10613-10729 Sentence denotes Recombinant human IL-17 diluted in culture medium was used as a calibration standard, ranging from 10 to 2000 pg/ml.
T7501 10730-10900 Sentence denotes A standard curve was drawn by plotting optical density against the log of the concentration of recombinant cytokines, and used for determination of IL-17 in test samples.
T81 10730-10900 Sentence denotes A standard curve was drawn by plotting optical density against the log of the concentration of recombinant cytokines, and used for determination of IL-17 in test samples.
T8623 10902-10998 Sentence denotes Quantification of IL-17 mRNA by semiquantitative reverse transcription–polymerase chain reaction
T82 10902-10998 Sentence denotes Quantification of IL-17 mRNA by semiquantitative reverse transcription–polymerase chain reaction
T8624 10999-11117 Sentence denotes PBMC were incubated with various concentrations of anti-CD3 in the presence or absence of inhibitors (LY294002, PDTC).
T83 10999-11117 Sentence denotes PBMC were incubated with various concentrations of anti-CD3 in the presence or absence of inhibitors (LY294002, PDTC).
T8625 11118-11272 Sentence denotes After 16 hours of incubation, mRNA was extracted with RNAzol B (Biotex Laboratories, Houston, TX, USA) in accordance with the manufacturer's instructions.
T84 11118-11272 Sentence denotes After 16 hours of incubation, mRNA was extracted with RNAzol B (Biotex Laboratories, Houston, TX, USA) in accordance with the manufacturer's instructions.
T8626 11273-11414 Sentence denotes Reverse transcription of 2 μg of total mRNA was performed at 42°C using the Superscript™ reverse transcription system (Takara, Shiga, Japan).
T85 11273-11414 Sentence denotes Reverse transcription of 2 μg of total mRNA was performed at 42°C using the Superscript™ reverse transcription system (Takara, Shiga, Japan).
T8627 11415-11568 Sentence denotes PCR amplification of cDNA aliquots was performed by adding 2.5 mM dNTPs, 2.5 U of Taq DNA polymerase (Takara) and 0.25 μM of sense and antisense primers.
T86 11415-11568 Sentence denotes PCR amplification of cDNA aliquots was performed by adding 2.5 mM dNTPs, 2.5 U of Taq DNA polymerase (Takara) and 0.25 μM of sense and antisense primers.
T8628 11569-11687 Sentence denotes The reaction was performed in PCR buffer (1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3) in a total volume of 25 μl.
T87 11569-11687 Sentence denotes The reaction was performed in PCR buffer (1.5 mM MgCl2, 50 mM KCl, 10 mM Tris-HCl, pH 8.3) in a total volume of 25 μl.
T8629 11688-12007 Sentence denotes The following sense and antisense primers for each molecules were used: IL-17 sense, 5'-ATG ACT CCT GGG AAG ACC TCA TTG-3'; IL-17 antisense, 5'-TTA GGC CAC ATG GTG GAC AAT CGG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense, 5'-CGA TGC TGG GCG TGA GTA C-3'; GAPDH antisense, 5'-CGT TCA GCT CAG GGA TGA CC-3'.
T88 11688-12007 Sentence denotes The following sense and antisense primers for each molecules were used: IL-17 sense, 5'-ATG ACT CCT GGG AAG ACC TCA TTG-3'; IL-17 antisense, 5'-TTA GGC CAC ATG GTG GAC AAT CGG-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense, 5'-CGA TGC TGG GCG TGA GTA C-3'; GAPDH antisense, 5'-CGT TCA GCT CAG GGA TGA CC-3'.
T89 12008-12233 Sentence denotes Reactions were processed in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA) through cycles for 30 s of denaturation at 94°C, 1 min of annealing at 56°C for GAPDH and IL-17, followed by 1 min of elongation at 72°C.
T8630 12008-12399 Sentence denotes Reactions were processed in a DNA thermal cycler (Perkin-Elmer Cetus, Norwalk, CT, USA) through cycles for 30 s of denaturation at 94°C, 1 min of annealing at 56°C for GAPDH and IL-17, followed by 1 min of elongation at 72°C. PCR rounds were repeated for 25 cycles each for both GAPDH and IL-17; this was determined as falling within the exponential phase of amplification for each molecule.
T90 12234-12399 Sentence denotes PCR rounds were repeated for 25 cycles each for both GAPDH and IL-17; this was determined as falling within the exponential phase of amplification for each molecule.
T8631 12400-12494 Sentence denotes The level of mRNA expression was presented as a ratio of IL-17 PCR product over GAPDH product.
T91 12400-12494 Sentence denotes The level of mRNA expression was presented as a ratio of IL-17 PCR product over GAPDH product.
T9362 12496-12554 Sentence denotes Western blot analysis of Akt, phosphorylated Akt and IκB-α
T92 12496-12554 Sentence denotes Western blot analysis of Akt, phosphorylated Akt and IκB-α
T9363 12555-12647 Sentence denotes PBMC were incubated with anti-CD3 (10 μg/ml) in the presence or absence of LY294002 (20 μM).
T93 12555-12647 Sentence denotes PBMC were incubated with anti-CD3 (10 μg/ml) in the presence or absence of LY294002 (20 μM).
T9364 12648-12825 Sentence denotes After incubation for 1 hour, whole cell lysates were prepared from about 107 cells by homogenization in the lysis buffer, and centrifuged at 14,000 r.p.m. (19,000 g) for 15 min.
T94 12648-12825 Sentence denotes After incubation for 1 hour, whole cell lysates were prepared from about 107 cells by homogenization in the lysis buffer, and centrifuged at 14,000 r.p.m. (19,000 g) for 15 min.
T9365 12826-12939 Sentence denotes Protein concentrations in the supernatants were determined with the Bradford method (Bio-Rad, Hercules, CA, USA).
T95 12826-12939 Sentence denotes Protein concentrations in the supernatants were determined with the Bradford method (Bio-Rad, Hercules, CA, USA).
T9366 12940-13078 Sentence denotes Protein samples were separated by 10% SDS–PAGE and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden).
T96 12940-13078 Sentence denotes Protein samples were separated by 10% SDS–PAGE and transferred to a nitrocellulose membrane (Amersham Pharmacia Biotech, Uppsala, Sweden).
T97 13079-13436 Sentence denotes For western hybridization, membrane was preincubated with 0.1% skimmed milk in TBS-T buffer (0.1% Tween 20 in Tris-buffered saline) at room temperature for 2 hours, then primary antibodies against Akt, phosphorylated Akt and IκB-α (Cell Signaling Technology Inc., Beverly, MA, USA), diluted 1:1000 in 5% BSA/TBS-T, were added and incubated overnight at 4°C.
T9367 13079-13595 Sentence denotes For western hybridization, membrane was preincubated with 0.1% skimmed milk in TBS-T buffer (0.1% Tween 20 in Tris-buffered saline) at room temperature for 2 hours, then primary antibodies against Akt, phosphorylated Akt and IκB-α (Cell Signaling Technology Inc., Beverly, MA, USA), diluted 1:1000 in 5% BSA/TBS-T, were added and incubated overnight at 4°C. After washing four times with TBS-T, horseradish peroxidase-conjugated secondary antibodies were added and allowed to incubate for 1 hour at room temperature.
T98 13437-13595 Sentence denotes After washing four times with TBS-T, horseradish peroxidase-conjugated secondary antibodies were added and allowed to incubate for 1 hour at room temperature.
T9368 13596-13752 Sentence denotes After TBS-T washing, hybridized bands were detected with the enhanced chemiluminescence (ECL) detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).
T99 13596-13752 Sentence denotes After TBS-T washing, hybridized bands were detected with the enhanced chemiluminescence (ECL) detection kit and Hyperfilm-ECL reagents (Amersham Pharmacia).
T10177 13754-13800 Sentence denotes Gel mobility-shift assay of NF-κB binding site
T100 13754-13800 Sentence denotes Gel mobility-shift assay of NF-κB binding site
T10178 13801-13857 Sentence denotes Nuclear proteins were extracted from about 5 × 106 PBMC.
T101 13801-13857 Sentence denotes Nuclear proteins were extracted from about 5 × 106 PBMC.
T10179 13858-14115 Sentence denotes Oligonucleotide probes encompassing the NF-κB binding site of the human IL-17 promoter (5'-ATG ACC TGG AAA TAC CCA AAA TTC-3') were generated by 5'-end labeling of the sense strand with [γ-32P]dATP (Amersham Pharmacia) and T4 polynucleotide kinase (TaKaRa).
T102 13858-14115 Sentence denotes Oligonucleotide probes encompassing the NF-κB binding site of the human IL-17 promoter (5'-ATG ACC TGG AAA TAC CCA AAA TTC-3') were generated by 5'-end labeling of the sense strand with [γ-32P]dATP (Amersham Pharmacia) and T4 polynucleotide kinase (TaKaRa).
T10180 14116-14226 Sentence denotes Unincorporated nucleotides were removed by NucTrap probe purification columns (Stratagene, La Jolla, CA, USA).
T103 14116-14226 Sentence denotes Unincorporated nucleotides were removed by NucTrap probe purification columns (Stratagene, La Jolla, CA, USA).
T10181 14227-14539 Sentence denotes Nuclear extracts (2 μg of protein) were incubated with radiolabeled DNA probes (10 ng; 100,000 c.p.m.) for 30 min at room temperature in 20 μl of binding buffer consisting of 20 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 5% glycerol, 1 mg/ml BSA, 0.2% Nonidet P40 and 50 ng/μl poly(dI-dC).
T104 14227-14539 Sentence denotes Nuclear extracts (2 μg of protein) were incubated with radiolabeled DNA probes (10 ng; 100,000 c.p.m.) for 30 min at room temperature in 20 μl of binding buffer consisting of 20 mM Tris-HCl, pH 7.9, 50 mM KCl, 1 mM dithiothreitol, 0.5 mM EDTA, 5% glycerol, 1 mg/ml BSA, 0.2% Nonidet P40 and 50 ng/μl poly(dI-dC).
T105 14540-14673 Sentence denotes Samples were subjected to electrophoresis on nondenaturing 5% polyacrylamide gels in 0.5 × Tris-borate-EDTA buffer (pH 8.0) at 100 V.
T10182 14540-14771 Sentence denotes Samples were subjected to electrophoresis on nondenaturing 5% polyacrylamide gels in 0.5 × Tris-borate-EDTA buffer (pH 8.0) at 100 V. Gels were dried under vacuum and exposed to Kodak X-OMAT film at -70°C with intensifying screens.
T106 14674-14771 Sentence denotes Gels were dried under vacuum and exposed to Kodak X-OMAT film at -70°C with intensifying screens.
T10183 14772-14900 Sentence denotes Rabbit polyclonal antibodies against NF-κB subunits p50, p65 and c-Rel were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
T107 14772-14900 Sentence denotes Rabbit polyclonal antibodies against NF-κB subunits p50, p65 and c-Rel were from Santa Cruz Biotechnology (Santa Cruz, CA, USA).
T10822 14902-14950 Sentence denotes Cell viability (Trypan blue dye exclusion assay)
T108 14902-14950 Sentence denotes Cell viability (Trypan blue dye exclusion assay)
T10823 14951-15100 Sentence denotes For cell viability assays, the trypan blue dye exclusion method was used to evaluate the potential of direct cytotoxic effect of inhibitors on cells.
T109 14951-15100 Sentence denotes For cell viability assays, the trypan blue dye exclusion method was used to evaluate the potential of direct cytotoxic effect of inhibitors on cells.
T10824 15101-15296 Sentence denotes After incubation for 24 hours, the cells were harvested and the percentage cell viability was calculated with the formula 100 × (number of viable cells/number of both viable and dead cells) [21].
T110 15101-15296 Sentence denotes After incubation for 24 hours, the cells were harvested and the percentage cell viability was calculated with the formula 100 × (number of viable cells/number of both viable and dead cells) [21].
T11014 15298-15318 Sentence denotes Statistical analysis
T111 15298-15318 Sentence denotes Statistical analysis
T11015 15319-15353 Sentence denotes Data are expressed as means ± SEM.
T112 15319-15353 Sentence denotes Data are expressed as means ± SEM.
T11016 15354-15429 Sentence denotes Statistical analysis was performed with Student's t-test for matched pairs.
T113 15354-15429 Sentence denotes Statistical analysis was performed with Student's t-test for matched pairs.
T11017 15430-15482 Sentence denotes P values less than 0.05 were considered significant.
T114 15430-15482 Sentence denotes P values less than 0.05 were considered significant.
T115 15484-15491 Sentence denotes Results
T11220 15493-15580 Sentence denotes IL-17 production in PBMC from patients with RA, patients with OA and normal individuals
T116 15493-15580 Sentence denotes IL-17 production in PBMC from patients with RA, patients with OA and normal individuals
T11221 15581-15778 Sentence denotes PBMC were separated and cultured with PHA (5 μg/ml) from patients with RA, patients with OA, and age-matched normal controls; IL-17 levels were then determined in the culture supernatants (Fig. 1).
T117 15581-15778 Sentence denotes PBMC were separated and cultured with PHA (5 μg/ml) from patients with RA, patients with OA, and age-matched normal controls; IL-17 levels were then determined in the culture supernatants (Fig. 1).
T11222 15779-16118 Sentence denotes Although the amounts of basal IL-17 secretion were not different between RA, OA and normal controls (62 ± 31, 43 ± 19 and 43 ± 10 pg/ml, respectively), the IL-17 production stimulated by PHA was significantly higher in RA PBMC than in those from OA and controls (768 ± 295 versus 463 ± 211 pg/ml [P < 0.05] and 241 ± 29 pg/ml [P < 0.001]).
T118 15779-16118 Sentence denotes Although the amounts of basal IL-17 secretion were not different between RA, OA and normal controls (62 ± 31, 43 ± 19 and 43 ± 10 pg/ml, respectively), the IL-17 production stimulated by PHA was significantly higher in RA PBMC than in those from OA and controls (768 ± 295 versus 463 ± 211 pg/ml [P < 0.05] and 241 ± 29 pg/ml [P < 0.001]).
T11761 16120-16212 Sentence denotes Increased IL-17 production in PBMC of patients with RA by anti-CD3 and/or anti-CD28, and PHA
T119 16120-16212 Sentence denotes Increased IL-17 production in PBMC of patients with RA by anti-CD3 and/or anti-CD28, and PHA
T11762 16213-16493 Sentence denotes Because IL-17 was already known from earlier reports to be produced mainly by activated T cells, we investigated the effect of different concentrations of anti-CD3 (1, 5 and 10 μg/ml) as a T cell activation, which showed a dose-dependent increase in IL-17 levels (data not shown).
T120 16213-16493 Sentence denotes Because IL-17 was already known from earlier reports to be produced mainly by activated T cells, we investigated the effect of different concentrations of anti-CD3 (1, 5 and 10 μg/ml) as a T cell activation, which showed a dose-dependent increase in IL-17 levels (data not shown).
T11763 16494-16578 Sentence denotes On the basis of this, we chose 10 μg/ml as a stimulation concentration for anti-CD3.
T121 16494-16578 Sentence denotes On the basis of this, we chose 10 μg/ml as a stimulation concentration for anti-CD3.
T11764 16579-16783 Sentence denotes As shown in Table 1, anti-CD3 significantly upregulated IL-17 production up to 3.7-fold, and the combination of anti-CD28 and anti-CD3 produced more IL-17 (approximately 1.3-1.5-fold) than anti-CD3 alone.
T122 16579-16783 Sentence denotes As shown in Table 1, anti-CD3 significantly upregulated IL-17 production up to 3.7-fold, and the combination of anti-CD28 and anti-CD3 produced more IL-17 (approximately 1.3-1.5-fold) than anti-CD3 alone.
T11765 16784-16968 Sentence denotes Furthermore, when incubated with T cell mitogens such as PHA, increased IL-17 production was more pronounced than with anti-CD3 and anti-CD28 (588 ± 85 versus 211 ± 1 pg/ml; P < 0.05).
T123 16784-16968 Sentence denotes Furthermore, when incubated with T cell mitogens such as PHA, increased IL-17 production was more pronounced than with anti-CD3 and anti-CD28 (588 ± 85 versus 211 ± 1 pg/ml; P < 0.05).
T12597 16970-17052 Sentence denotes Regulation of IL-17 production in RA PBMC by inflammatory cytokines and chemokines
T124 16970-17052 Sentence denotes Regulation of IL-17 production in RA PBMC by inflammatory cytokines and chemokines
T12598 17053-17253 Sentence denotes Because RA PBMC include several cell types in addition to T cells, some inflammatory cytokines released from macrophages and other lymphocytes might have affected the production of IL-17 from T cells.
T125 17053-17253 Sentence denotes Because RA PBMC include several cell types in addition to T cells, some inflammatory cytokines released from macrophages and other lymphocytes might have affected the production of IL-17 from T cells.
T12599 17254-17410 Sentence denotes To evaluate the effects of inflammatory cytokines released by activated PBMC, we tested the effects of several cytokines and chemokines on IL-17 production.
T126 17254-17410 Sentence denotes To evaluate the effects of inflammatory cytokines released by activated PBMC, we tested the effects of several cytokines and chemokines on IL-17 production.
T12600 17411-17622 Sentence denotes We detected an increase in IL-17 level after stimulation with IL-15 (10 ng/ml), whereas with IL-1β (10 ng/ml), TNF-α (10 ng/ml), IL-18 (10 ng/ml) or TGF-β (10 ng/ml) the levels in IL-17 were unchanged (Fig. 2a).
T127 17411-17622 Sentence denotes We detected an increase in IL-17 level after stimulation with IL-15 (10 ng/ml), whereas with IL-1β (10 ng/ml), TNF-α (10 ng/ml), IL-18 (10 ng/ml) or TGF-β (10 ng/ml) the levels in IL-17 were unchanged (Fig. 2a).
T12601 17623-17901 Sentence denotes When treated with MCP-1 (10 ng/ml) or IL-6 (10 ng/ml), significant upregulations of IL-17 proteins were observed (62 ± 42 and 50 ± 10 versus 31 ± 11 pg/ml, respectively; P < 0.05), whereas none was observed with IL-8 (10 ng/ml), MIP-1α (10 ng/ml) or MIP-1β (10 ng/ml) (Fig. 2b).
T128 17623-17901 Sentence denotes When treated with MCP-1 (10 ng/ml) or IL-6 (10 ng/ml), significant upregulations of IL-17 proteins were observed (62 ± 42 and 50 ± 10 versus 31 ± 11 pg/ml, respectively; P < 0.05), whereas none was observed with IL-8 (10 ng/ml), MIP-1α (10 ng/ml) or MIP-1β (10 ng/ml) (Fig. 2b).
T13528 17903-17992 Sentence denotes Inhibition of IL-17 production by signal transduction inhibitors and anti-rheumatic drugs
T129 17903-17992 Sentence denotes Inhibition of IL-17 production by signal transduction inhibitors and anti-rheumatic drugs
T13529 17993-18126 Sentence denotes Having observed the increased IL-17 production in RA PBMC, it was important to know which signal transduction pathways were involved.
T130 17993-18126 Sentence denotes Having observed the increased IL-17 production in RA PBMC, it was important to know which signal transduction pathways were involved.
T13530 18127-18390 Sentence denotes As illustrated in Fig. 3, an significant decrease in anti-CD3-induced IL-17 production was observed when co-incubated with NF-κB inhibitor, PDTC and dexamethasone in comparison with anti-CD3 alone (38 ± 5 and 54 ± 11 versus 98 ± 19 pg/ml, respectively; P < 0.05).
T131 18127-18390 Sentence denotes As illustrated in Fig. 3, an significant decrease in anti-CD3-induced IL-17 production was observed when co-incubated with NF-κB inhibitor, PDTC and dexamethasone in comparison with anti-CD3 alone (38 ± 5 and 54 ± 11 versus 98 ± 19 pg/ml, respectively; P < 0.05).
T13531 18391-18599 Sentence denotes LY294002 and wortmannin, as an inhibitor of PI3K, also markedly inhibited the anti-CD3-induced IL-17 production in RA PBMC (98 ± 19 versus 38 ± 10 pg/ml [P < 0.005] and 48 ± 4 pg/ml [P < 0.05], respectively).
T132 18391-18599 Sentence denotes LY294002 and wortmannin, as an inhibitor of PI3K, also markedly inhibited the anti-CD3-induced IL-17 production in RA PBMC (98 ± 19 versus 38 ± 10 pg/ml [P < 0.005] and 48 ± 4 pg/ml [P < 0.05], respectively).
T13532 18600-18842 Sentence denotes The calcineurin inhibitors cyclosporin A and FK506 also downregulated the IL-17 secretion as well as the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 did, whereas rapamycin and PD98059 had no effect on IL-17 levels (Fig. 3).
T133 18600-18842 Sentence denotes The calcineurin inhibitors cyclosporin A and FK506 also downregulated the IL-17 secretion as well as the mitogen-activated protein kinase (MAPK) p38 inhibitor SB203580 did, whereas rapamycin and PD98059 had no effect on IL-17 levels (Fig. 3).
T13533 18843-19032 Sentence denotes To evaluate the possibility of non-specific inhibition by the drug at high concentrations, we observed the dose response of PDTC and LY294002 for the inhibition of IL-17 production in PBMC.
T134 18843-19032 Sentence denotes To evaluate the possibility of non-specific inhibition by the drug at high concentrations, we observed the dose response of PDTC and LY294002 for the inhibition of IL-17 production in PBMC.
T13534 19033-19126 Sentence denotes There were dose-dependent inhibitions of IL-17 production with chemical inhibitors (Fig. 4a).
T135 19033-19126 Sentence denotes There were dose-dependent inhibitions of IL-17 production with chemical inhibitors (Fig. 4a).
T13535 19127-19219 Sentence denotes The other inhibitors in addition to PDTC and LY294002 showed the same pattern of inhibition.
T136 19127-19219 Sentence denotes The other inhibitors in addition to PDTC and LY294002 showed the same pattern of inhibition.
T13536 19220-19332 Sentence denotes Cytotoxic effects on PBMC by the chemical inhibitors at experimental concentrations were not observed (Fig. 4b).
T137 19220-19332 Sentence denotes Cytotoxic effects on PBMC by the chemical inhibitors at experimental concentrations were not observed (Fig. 4b).
T14487 19334-19366 Sentence denotes IL-17 mRNA expression in RA PBMC
T138 19334-19366 Sentence denotes IL-17 mRNA expression in RA PBMC
T14488 19367-19535 Sentence denotes To see whether enhanced IL-17 production could be regulated at a transcriptional level, semi-quantatitive reverse transcription–polymerase chain reaction was performed.
T139 19367-19535 Sentence denotes To see whether enhanced IL-17 production could be regulated at a transcriptional level, semi-quantatitive reverse transcription–polymerase chain reaction was performed.
T14489 19536-19724 Sentence denotes We observed a dose-dependent increase in IL-17 mRNA transcripts after stimulation with anti-CD3; this was inhibited by the PI3K inhibitor LY294002 and by the NF-κB inhibitor PDTC (Fig. 5).
T140 19536-19724 Sentence denotes We observed a dose-dependent increase in IL-17 mRNA transcripts after stimulation with anti-CD3; this was inhibited by the PI3K inhibitor LY294002 and by the NF-κB inhibitor PDTC (Fig. 5).
T14928 19726-19808 Sentence denotes Activation of PI3K/Akt signal transduction pathway on IL-17 production by anti-CD3
T141 19726-19808 Sentence denotes Activation of PI3K/Akt signal transduction pathway on IL-17 production by anti-CD3
T14929 19809-19928 Sentence denotes To determine downstream effector molecules of the PI3K pathway, we evaluated the activation of Akt by western blotting.
T142 19809-19928 Sentence denotes To determine downstream effector molecules of the PI3K pathway, we evaluated the activation of Akt by western blotting.
T14930 19929-20083 Sentence denotes As shown in Fig. 6, at 10 min of incubation with anti-CD3 (10 μg/ml) or LY294002 (20 μM), no difference in the amounts of phosphorylated Akt was observed.
T143 19929-20083 Sentence denotes As shown in Fig. 6, at 10 min of incubation with anti-CD3 (10 μg/ml) or LY294002 (20 μM), no difference in the amounts of phosphorylated Akt was observed.
T14931 20084-20259 Sentence denotes However, after 30 min of incubation, phosphorylated Akt increased (lane 2), and the effect of inhibition by LY294002 (lane 3) reached a peak at 60 min, lasting to 120–240 min.
T144 20084-20259 Sentence denotes However, after 30 min of incubation, phosphorylated Akt increased (lane 2), and the effect of inhibition by LY294002 (lane 3) reached a peak at 60 min, lasting to 120–240 min.
T14932 20260-20357 Sentence denotes In contrast, non-phosphorylated Akt and β-actin remained unchanged regardless of incubation time.
T145 20260-20357 Sentence denotes In contrast, non-phosphorylated Akt and β-actin remained unchanged regardless of incubation time.
T14933 20358-20555 Sentence denotes PHA, concanavalin A and IL-15 also demonstrated the same effect on phosphorylated Akt as shown with anti-CD3, which was an inhibition by wortmannin and PDTC as well as by LY294002 (data not shown).
T146 20358-20555 Sentence denotes PHA, concanavalin A and IL-15 also demonstrated the same effect on phosphorylated Akt as shown with anti-CD3, which was an inhibition by wortmannin and PDTC as well as by LY294002 (data not shown).
T15817 20557-20648 Sentence denotes Activation of the NF-κB and activator protein-1 (AP-1) pathway in the IL-17 promoter region
T147 20557-20648 Sentence denotes Activation of the NF-κB and activator protein-1 (AP-1) pathway in the IL-17 promoter region
T15818 20649-20937 Sentence denotes To investigate further the intracellular signaling pathway activated by anti-CD3 plus anti-CD28, concanavalin A, PHA and IL-15, and responsible for inducing IL-17 expression, we performed an electrophoretic mobility-shift assay (EMSA) of NF-κB recognition sites in the promoters of IL-17.
T148 20649-20937 Sentence denotes To investigate further the intracellular signaling pathway activated by anti-CD3 plus anti-CD28, concanavalin A, PHA and IL-15, and responsible for inducing IL-17 expression, we performed an electrophoretic mobility-shift assay (EMSA) of NF-κB recognition sites in the promoters of IL-17.
T15819 20938-21142 Sentence denotes As shown in Fig. 7a, nuclear extracts from RA PBMC stimulated with anti-CD3 plus anti-CD28 (lane 2) demonstrated increased binding of NF-κB to IL-17 promoters in comparison with that of controls (lane 1).
T149 20938-21142 Sentence denotes As shown in Fig. 7a, nuclear extracts from RA PBMC stimulated with anti-CD3 plus anti-CD28 (lane 2) demonstrated increased binding of NF-κB to IL-17 promoters in comparison with that of controls (lane 1).
T15820 21143-21242 Sentence denotes A supershift assay demonstrated shifted bands in p65 and p50 (lanes 3 and 4) not in c-Rel (lane 5).
T150 21143-21242 Sentence denotes A supershift assay demonstrated shifted bands in p65 and p50 (lanes 3 and 4) not in c-Rel (lane 5).
T15821 21243-21399 Sentence denotes In normal PBMC the same pattern was observed, but the degree of NF-κB activation by anti-CD3 plus anti-CD28 was less intense than that in RA PBMC (Fig. 7b).
T151 21243-21399 Sentence denotes In normal PBMC the same pattern was observed, but the degree of NF-κB activation by anti-CD3 plus anti-CD28 was less intense than that in RA PBMC (Fig. 7b).
T15822 21400-21555 Sentence denotes To confirm the link between PI3K activity and NF-κB, we performed EMSA to determine the NF-κB binding activity after treatment with both LY294002 and PDTC.
T152 21400-21555 Sentence denotes To confirm the link between PI3K activity and NF-κB, we performed EMSA to determine the NF-κB binding activity after treatment with both LY294002 and PDTC.
T15823 21556-21633 Sentence denotes Both agents block NF-κB DNA-binding activity in the IL-17 promoter (Fig. 7c).
T153 21556-21633 Sentence denotes Both agents block NF-κB DNA-binding activity in the IL-17 promoter (Fig. 7c).
T15824 21634-21751 Sentence denotes Western blotting for IκB-α showed inhibition of degradation of IκB-α by LY294002 and PDTC at the same time (Fig. 7c).
T154 21634-21751 Sentence denotes Western blotting for IκB-α showed inhibition of degradation of IκB-α by LY294002 and PDTC at the same time (Fig. 7c).
T15825 21752-21991 Sentence denotes In contrast, the AP-1 pathway was not activated by stimulation with anti-CD3 plus anti-CD28 (data not shown), demonstrating that NF-κB is the main intracellular signaling pathway in IL-17 production by activated PBMC from patients with RA.
T155 21752-21991 Sentence denotes In contrast, the AP-1 pathway was not activated by stimulation with anti-CD3 plus anti-CD28 (data not shown), demonstrating that NF-κB is the main intracellular signaling pathway in IL-17 production by activated PBMC from patients with RA.
T156 21993-22003 Sentence denotes Discussion
T18135 22004-22089 Sentence denotes IL-17 was first described as a T cell product with proinflammatory properties [5,22].
T157 22004-22089 Sentence denotes IL-17 was first described as a T cell product with proinflammatory properties [5,22].
T18136 22090-22204 Sentence denotes RA is characterized by hyperplasia of synovial lining cells and an intense infiltration by mononuclear cells [23].
T158 22090-22204 Sentence denotes RA is characterized by hyperplasia of synovial lining cells and an intense infiltration by mononuclear cells [23].
T18137 22205-22421 Sentence denotes Proinflammatory cytokines such as IL-1 and TNF-α are abundant in rheumatoid synovium, whereas the T cell-derived cytokines, especially IL-4 and interferon-γ, have often proved difficult to detect in RA synovium [24].
T159 22205-22421 Sentence denotes Proinflammatory cytokines such as IL-1 and TNF-α are abundant in rheumatoid synovium, whereas the T cell-derived cytokines, especially IL-4 and interferon-γ, have often proved difficult to detect in RA synovium [24].
T18138 22422-22576 Sentence denotes Although T cells may have a role in the augmentation of rheumatoid synovial inflammation, the lack of T cell-derived cytokines has limited its importance.
T160 22422-22576 Sentence denotes Although T cells may have a role in the augmentation of rheumatoid synovial inflammation, the lack of T cell-derived cytokines has limited its importance.
T18139 22577-22704 Sentence denotes In this respect, IL-17 is appealing because it has been described as a T cell-derived cytokine with proinflammatory properties.
T161 22577-22704 Sentence denotes In this respect, IL-17 is appealing because it has been described as a T cell-derived cytokine with proinflammatory properties.
T18140 22705-22824 Sentence denotes In our studies, we tried to evaluate how IL-17 production is regulated in RA PBMC, and which signaling pathway it used.
T162 22705-22824 Sentence denotes In our studies, we tried to evaluate how IL-17 production is regulated in RA PBMC, and which signaling pathway it used.
T18141 22825-22917 Sentence denotes Levels of IL-17 were found to be higher in RA synovial fluid than in OA synovial fluid [15].
T163 22825-22917 Sentence denotes Levels of IL-17 were found to be higher in RA synovial fluid than in OA synovial fluid [15].
T18142 22918-23134 Sentence denotes However, there are few data available on the agents that stimulate IL-17 production in RA, although the highest level of IL-17 production can be achieved by anti-CD3/anti-CD28 stimulation in healthy individuals [25].
T164 22918-23134 Sentence denotes However, there are few data available on the agents that stimulate IL-17 production in RA, although the highest level of IL-17 production can be achieved by anti-CD3/anti-CD28 stimulation in healthy individuals [25].
T18143 23135-23312 Sentence denotes In our experiments, PHA as mitogens, as well as anti-CD3/anti-CD28 for signaling through the T cell receptor, increased IL-17 production from RA PBMC in a dose-dependent manner.
T165 23135-23312 Sentence denotes In our experiments, PHA as mitogens, as well as anti-CD3/anti-CD28 for signaling through the T cell receptor, increased IL-17 production from RA PBMC in a dose-dependent manner.
T18144 23313-23485 Sentence denotes We found, by a cell proliferation assay (data not shown), that this upregulation of IL-17 might be due to increased cellular activity rather than to cellular proliferation.
T166 23313-23485 Sentence denotes We found, by a cell proliferation assay (data not shown), that this upregulation of IL-17 might be due to increased cellular activity rather than to cellular proliferation.
T18145 23486-23595 Sentence denotes IL-17 is produced mainly by activated CD4+ T cells, especially for Th1/Th0 cells, not the Th2 phenotype [26].
T167 23486-23595 Sentence denotes IL-17 is produced mainly by activated CD4+ T cells, especially for Th1/Th0 cells, not the Th2 phenotype [26].
T18146 23596-23721 Sentence denotes However, it can also be produced by CD8+ T cells via an IL-23 triggering mechanism in Gram-negative pulmonary infection [14].
T168 23596-23721 Sentence denotes However, it can also be produced by CD8+ T cells via an IL-23 triggering mechanism in Gram-negative pulmonary infection [14].
T18147 23722-23863 Sentence denotes In addition, IL-17 production was significantly augmented by T cells recognizing type II collagen in a collagen-induced arthritis model [27].
T169 23722-23863 Sentence denotes In addition, IL-17 production was significantly augmented by T cells recognizing type II collagen in a collagen-induced arthritis model [27].
T18148 23864-24037 Sentence denotes A complex interaction between cells in inflamed RA joints might produce a variety of proinflammatory cytokines and chemokines, which also activate other cells in the joints.
T170 23864-24037 Sentence denotes A complex interaction between cells in inflamed RA joints might produce a variety of proinflammatory cytokines and chemokines, which also activate other cells in the joints.
T18149 24038-24232 Sentence denotes For example, IL-17 stimulates rheumatoid synoviocytes to secrete several cytokines such as IL-6, IL-8 and tumor necrosis factor-stimulated gene 6 as well as prostaglandin E2 in vitro [12,28,29].
T171 24038-24232 Sentence denotes For example, IL-17 stimulates rheumatoid synoviocytes to secrete several cytokines such as IL-6, IL-8 and tumor necrosis factor-stimulated gene 6 as well as prostaglandin E2 in vitro [12,28,29].
T18150 24233-24417 Sentence denotes There are as yet few data available on the agents that stimulate IL-17 production in RA, although some cytokines (IL-15 and IL-23) have been known to regulate IL-17 production [13,14].
T172 24233-24417 Sentence denotes There are as yet few data available on the agents that stimulate IL-17 production in RA, although some cytokines (IL-15 and IL-23) have been known to regulate IL-17 production [13,14].
T18151 24418-24616 Sentence denotes We therefore investigated the in vitro production of IL-17 in RA PBMC responding to a variety of cytokines/chemokines and mitogens as well as T cell receptor (TCR) ligation using anti-CD3/anti-CD28.
T173 24418-24616 Sentence denotes We therefore investigated the in vitro production of IL-17 in RA PBMC responding to a variety of cytokines/chemokines and mitogens as well as T cell receptor (TCR) ligation using anti-CD3/anti-CD28.
T18152 24617-24746 Sentence denotes Our studies demonstrated that IL-15 and MCP-1 as well as TCR ligation significantly increased the production of IL-17 in RA PBMC.
T174 24617-24746 Sentence denotes Our studies demonstrated that IL-15 and MCP-1 as well as TCR ligation significantly increased the production of IL-17 in RA PBMC.
T18153 24747-24826 Sentence denotes Adding IL-15 or MCP-1 to TCR ligation augmented IL-17 production more markedly.
T175 24747-24826 Sentence denotes Adding IL-15 or MCP-1 to TCR ligation augmented IL-17 production more markedly.
T18154 24827-24973 Sentence denotes In contrast, IL-1 and TNF-α, which are known to have proinflammatory properties and to be increased in RA joints, did not affect IL-17 production.
T176 24827-24973 Sentence denotes In contrast, IL-1 and TNF-α, which are known to have proinflammatory properties and to be increased in RA joints, did not affect IL-17 production.
T18155 24974-25105 Sentence denotes Our data were consistent with a recent report that IL-15 triggered in vitro IL-17 production in PBMC, but TNF-α did not do so [13].
T177 24974-25105 Sentence denotes Our data were consistent with a recent report that IL-15 triggered in vitro IL-17 production in PBMC, but TNF-α did not do so [13].
T18156 25106-25291 Sentence denotes Although there were no data that MCP-1 directly induces T cell activation, it might exert effects indirectly on T cells through the activation of monocytes/macrophages in PBMC cultures.
T178 25106-25291 Sentence denotes Although there were no data that MCP-1 directly induces T cell activation, it might exert effects indirectly on T cells through the activation of monocytes/macrophages in PBMC cultures.
T18157 25292-25420 Sentence denotes As reported for normal individuals [25], T cell activation through anti-CD3/anti-CD28 also increases IL-17 induction in RA PBMC.
T179 25292-25420 Sentence denotes As reported for normal individuals [25], T cell activation through anti-CD3/anti-CD28 also increases IL-17 induction in RA PBMC.
T18158 25421-25640 Sentence denotes Although the signaling pathway for the induction of cytokines/chemokines by IL-17 has been documented widely [8,30,31], no data have been available on how IL-17 production can be regulated by certain signaling pathways.
T180 25421-25640 Sentence denotes Although the signaling pathway for the induction of cytokines/chemokines by IL-17 has been documented widely [8,30,31], no data have been available on how IL-17 production can be regulated by certain signaling pathways.
T18159 25641-25785 Sentence denotes By using signal transduction inhibitors, we therefore examined which signaling pathway was mainly involved in the induction of IL-17 in RA PBMC.
T181 25641-25785 Sentence denotes By using signal transduction inhibitors, we therefore examined which signaling pathway was mainly involved in the induction of IL-17 in RA PBMC.
T18160 25786-25998 Sentence denotes We identified that anti-CD3-induced IL-17 production in RA PBMC was significantly hampered by the PI3K inhibitor LY294002 and the NF-κB inhibitor PDTC to comparable levels of basal production without stimulation.
T182 25786-25998 Sentence denotes We identified that anti-CD3-induced IL-17 production in RA PBMC was significantly hampered by the PI3K inhibitor LY294002 and the NF-κB inhibitor PDTC to comparable levels of basal production without stimulation.
T18161 25999-26120 Sentence denotes We also found that anti-CD3-induced IL-17 production was downregulated by the addition of SB203580, a p38 MAPK inhibitor.
T183 25999-26120 Sentence denotes We also found that anti-CD3-induced IL-17 production was downregulated by the addition of SB203580, a p38 MAPK inhibitor.
T18162 26121-26206 Sentence denotes It is interesting that a series of evidence supports crosstalk between NF-κB and p38.
T184 26121-26206 Sentence denotes It is interesting that a series of evidence supports crosstalk between NF-κB and p38.
T18163 26207-26375 Sentence denotes In myocytes, IκB kinase-β is activated by p38 [32], and the activated p38 can stimulate NF-κB by a mechanism involving histone acetylase p300/CREB-binding protein [33].
T185 26207-26375 Sentence denotes In myocytes, IκB kinase-β is activated by p38 [32], and the activated p38 can stimulate NF-κB by a mechanism involving histone acetylase p300/CREB-binding protein [33].
T18164 26376-26592 Sentence denotes Our results revealed that p38 MAPK activation was not affected by LY294002, whereas NF-κB binding activity was decreased by LY294002, which provided the evidence for a p38 MAPK pathway independent of PI3K activation.
T186 26376-26592 Sentence denotes Our results revealed that p38 MAPK activation was not affected by LY294002, whereas NF-κB binding activity was decreased by LY294002, which provided the evidence for a p38 MAPK pathway independent of PI3K activation.
T18165 26593-26702 Sentence denotes The direct relationship between p38 and NF-κB for IL-17 production needs to be studied in future experiments.
T187 26593-26702 Sentence denotes The direct relationship between p38 and NF-κB for IL-17 production needs to be studied in future experiments.
T18166 26703-26851 Sentence denotes The search for a downstream pathway of PI3K seemed to have a maximal response of Akt activation at 1 hour and a gradual loss of activity at 2 hours.
T188 26703-26851 Sentence denotes The search for a downstream pathway of PI3K seemed to have a maximal response of Akt activation at 1 hour and a gradual loss of activity at 2 hours.
T18167 26852-26988 Sentence denotes The fact that Akt is phosphorylated upon anti-CD3 stimulation suggests the possible involvement of PI3K in the induction of IL-17 in RA.
T189 26852-26988 Sentence denotes The fact that Akt is phosphorylated upon anti-CD3 stimulation suggests the possible involvement of PI3K in the induction of IL-17 in RA.
T18168 26989-27204 Sentence denotes In view of the fact that NF-κB was also activated by anti-CD3/anti-CD28, IL-15 or mitogens in our experiments, it is most likely that the NF-κB pathway is also actively involved in the induction of IL-17 in RA PBMC.
T190 26989-27204 Sentence denotes In view of the fact that NF-κB was also activated by anti-CD3/anti-CD28, IL-15 or mitogens in our experiments, it is most likely that the NF-κB pathway is also actively involved in the induction of IL-17 in RA PBMC.
T18169 27205-27372 Sentence denotes In contrast, the AP-1 signal transduction pathway, another important signaling pathway for cytokines/chemokines, was not activated in our experiments (data not shown).
T191 27205-27372 Sentence denotes In contrast, the AP-1 signal transduction pathway, another important signaling pathway for cytokines/chemokines, was not activated in our experiments (data not shown).
T18170 27373-27562 Sentence denotes Although PI3K and its downstream kinase Akt in association with NF-κB have been reported to deliver activating signals in many cell types, the data on the signal inducing IL-17 are lacking.
T192 27373-27562 Sentence denotes Although PI3K and its downstream kinase Akt in association with NF-κB have been reported to deliver activating signals in many cell types, the data on the signal inducing IL-17 are lacking.
T18171 27563-27838 Sentence denotes Our data clearly demonstrated that PI3K/Akt and resultant NF-κB activation could be an important arbitrator of the upregulation of IL-17 in RA, on the basis of our experiments showing simultaneous blocking of NF-κB binding activity in the IL-17 promoter by PDTC and LY294002.
T193 27563-27838 Sentence denotes Our data clearly demonstrated that PI3K/Akt and resultant NF-κB activation could be an important arbitrator of the upregulation of IL-17 in RA, on the basis of our experiments showing simultaneous blocking of NF-κB binding activity in the IL-17 promoter by PDTC and LY294002.
T18172 27839-28087 Sentence denotes Considering its proinflammatory activities and successful induction of anti-IL-17 for ameliorating arthritis in animal models [2,6,34-36], understanding the IL-17 signaling pathway is an important element of developing new targeted therapies in RA.
T194 27839-28087 Sentence denotes Considering its proinflammatory activities and successful induction of anti-IL-17 for ameliorating arthritis in animal models [2,6,34-36], understanding the IL-17 signaling pathway is an important element of developing new targeted therapies in RA.
T195 28089-28100 Sentence denotes Conclusions
T22197 28101-28245 Sentence denotes We have detected a more pronounced production of IL-17 from RA PBMC in response to IL-15 and MCP-1 as well as stimulation by anti-CD3/anti-CD28.
T196 28101-28245 Sentence denotes We have detected a more pronounced production of IL-17 from RA PBMC in response to IL-15 and MCP-1 as well as stimulation by anti-CD3/anti-CD28.
T22198 28246-28424 Sentence denotes We have also shown that upregulation of IL-17 by activated T cells in patients with RA could be the result of activation via the PI3K/Akt pathway with resultant NF-κB activation.
T197 28246-28424 Sentence denotes We have also shown that upregulation of IL-17 by activated T cells in patients with RA could be the result of activation via the PI3K/Akt pathway with resultant NF-κB activation.
T22199 28425-28609 Sentence denotes Our data provide insights into cellular mechanisms of the regulation of IL-17 production in RA, and highlight the role of T cells, which has hitherto been neglected in RA pathogenesis.
T198 28425-28609 Sentence denotes Our data provide insights into cellular mechanisms of the regulation of IL-17 production in RA, and highlight the role of T cells, which has hitherto been neglected in RA pathogenesis.
T22200 28610-28796 Sentence denotes Together with recent data on the successful introduction of anti-IL-17 in RA, our results have added information for the future molecular targeting of new therapeutic applications in RA.
T199 28610-28796 Sentence denotes Together with recent data on the successful introduction of anti-IL-17 in RA, our results have added information for the future molecular targeting of new therapeutic applications in RA.
T200 28798-28811 Sentence denotes Abbreviations
T201 28812-29395 Sentence denotes AP-1, activator protein-1; BSA = bovine serum albumin; EMSA = electrophoretic mobility-shift assay; GAPDH = glyceraldehyde-3-phosphate dehydrogenase; IL = interleukin; MAPK = mitogen-activated protein kinase; MCP-1 = monocyte chemoattractant protein-1; MIP = macrophage inflammatory protein; NF-κB = nuclear factor κB; OA = osteoarthritis; PBMC = peripheral blood mononuclear cells; PDTC = pyrrolidine dithiocarbamate; PHA = phytohemagglutinin; PI3K = phosphoinositide 3-kinase; RA = rheumatoid arthritis; TGF = transforming growth factor; Th = T helper; TNF = tumor necrosis factor.
T202 29397-29416 Sentence denotes Competing interests
T203 29417-29477 Sentence denotes The author(s) declare that they have no competing interests.
T204 29479-29501 Sentence denotes Authors' contributions
T205 29502-29590 Sentence denotes KWK performed the cellular immune response studies and participated in the immunoassays.
T206 29591-29674 Sentence denotes MLC participated in the design of the study and performed the statistical analysis.
T207 29675-29722 Sentence denotes MKP participated in the isolation of the cells.
T208 29723-29750 Sentence denotes CHY drafted the manuscript.
T209 29751-29808 Sentence denotes SHP participated in the molecular biology and in the PCR.
T210 29809-29913 Sentence denotes SHL conceived the study, participated in its design and coordination and helped to draft the manuscript.
T211 29914-29949 Sentence denotes HYK helped to draft the manuscript.
T212 29950-30001 Sentence denotes All authors read and approved the final manuscript.