OBJECTIVE: To study the diagnostic value of P2X7 receptor for rheumatoid arthritis (RA) and its role in the inflammatory response. METHODS: With the synovial tissues from 25 patients with bone and joint replacement as the control,the synovial tissues of 25 RA patients were examined for the relative expression of P2X7 receptor mRNA using qRT-PCR.In an immortalized RA synovial cell line (MH7A),the effect of P2X7 receptor knockdown via a small interfering RNA were examined on the productions of the inflammatory cytokines including interleukin-1β(IL-1β),IL-6,and IL-8 using ELISA. RESULTS: The RA patients showed significantly higher levels of P2X7 receptor mRNA expression in the synovial tissue than the control patients.P2X7 receptor had a good diagnostic value for RA.The expression levels of IL-1β,IL-6,and IL-8 were positively correlated with the levels of P2X7 receptor in the synovial tissues of RA patients (<0.001).In MH7A cells,P2X7 receptor knockdown obviously reduced the secretion of IL-1β and IL-6. CONCLUSIONS RA patients show elevated P2X7 receptor level in the synovial tissue, which has a good diagnostic value for RA.Blocking P2X7 receptor can inhibit inflammatory factor secretion and suppress inflammatory reactions.
Arthritis, Rheumatoid ; diagnosis ; physiopathology ; Case-Control Studies ; Cell Line ; Gene Knockdown Techniques ; Humans ; Inflammation ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Purinergic P2X Receptor Antagonists ; RNA, Messenger ; metabolism ; Receptors, Purinergic P2X7 ; physiology ; Synovial Membrane ; metabolism

This study was purposed to explore the changes of possible angiogenetic factors other than VEGF after inhibition of NHE1 and their related mechanisms. The K562 cells were treated by NHE1 specific inhibitor cariporide, the angiogenesis factors after inhibition of NHE1 were screened by using protein chip, the IL-8 expression level after cariporide treatment was detected by real-time quantitative PCR; the K562 cells with stable interference of NHE1 were constructed, the IL-8 expression level after interference of NHE1 was detected by real-time quantitative PCR; the p38 phosphorylation level in K562 cells treated with cariporide was detected by Western blot. After treatment of K562 cells with p38 inhibitor SB203580, the IL-8 expression level was decreased by real-time quantitative PCR. The results of protein chip showed that IL-8 expression decreased after cariporide treatment. Real-time quantitative PCR confirmed this inhibitory effect. The p38 phosphorylation level increased after cariporide treatment. The down-regulation of IL-8 expression induced by cariporide treatment was partially restored after K562 cells were treated with p38 inhibitor SB203580. It is concluded that the inhibition of NHE1 can inhibit IL-8 expression through up-regulation of p38 phosphorylation.
Cation Transport Proteins ; antagonists & inhibitors ; Down-Regulation ; Guanidines ; pharmacology ; Humans ; Imidazoles ; pharmacology ; Interleukin-8 ; metabolism ; K562 Cells ; Phosphorylation ; drug effects ; Pyridines ; pharmacology ; Sodium-Hydrogen Exchanger 1 ; Sodium-Hydrogen Exchangers ; antagonists & inhibitors ; Sulfones ; pharmacology ; p38 Mitogen-Activated Protein Kinases ; metabolism

OBJECTIVETo explore the roles of NF-κB in factor VIIa-induced proliferation and migration of a colon cancer cell line (SW620) in vitro and its possible mechanism.

METHODSThe expression levels of NF-κB (p65), inhibitory protein of NF-κB (IκB-α), caspase-7, interleukin 8 (IL-8) and tissue factor (TF) in SW620 cells treated with factor VIIa, PDTC (an inhibitor of NF-κB) and other factors were measured by Western-blotting and real-time PCR. Proliferation and migration of the cells were analyzed by flow cytometry and Transwell assay, respectively.

RESULTSFactor VIIa down-regulated the IκB-α level in SW620 cells and increased the intranuclear level of NF-κB. Those effects of factor VIIa were blocked by anti-TF or anti-PAR2 antibodies. The effects of factor VIIa on proliferation and migration of SW620 cells, expression of IL-8, TF as well as caspase-7, were interfered by PDTC (the inhibitor of NF-κB).

CONCLUSIONSTF/VIIa complex activates NF-κB pathway via PAR2, thereby up-regulates IL-8 and down-regulates caspase-7 expression in SW620 cells, finally promotes proliferation and migration of colon cancer cells. In addition, TF/VIIa/PAR2/NF-κB pathway also upregulates TF expression, thus to create a positive feedback loop of TF/VIIa/PAR2/NF-κB/TF.

Antineoplastic Agents ; pharmacology ; Caspase 7 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Movement ; drug effects ; Cell Proliferation ; drug effects ; Colonic Neoplasms ; metabolism ; pathology ; Factor VIIa ; pharmacology ; Humans ; I-kappa B Proteins ; metabolism ; Interleukin-8 ; genetics ; metabolism ; NF-KappaB Inhibitor alpha ; Proline ; analogs & derivatives ; pharmacology ; RNA, Messenger ; metabolism ; Receptor, PAR-2 ; metabolism ; Thiocarbamates ; pharmacology ; Thromboplastin ; genetics ; metabolism ; Transcription Factor RelA ; antagonists & inhibitors ; metabolism

BACKGROUNDC-reactive protein (CRP) has been reported to damage the vascular wall by inducing endothelial dysfunction and inflammation, and it is also speculated to have a role in attenuating angiogenic functions of human endothelial progenitor cells (EPCs). Interleukin-8 (IL-8) is an important mediator of the paracrine mitogenic effect of EPCs, which has direct angiogenic effects on mature endothelial cells. We, herein, investigated the direct effect of CRP on IL-8 production and gene expression in cultured human EPCs.

METHODSEPCs were isolated from the peripheral venous blood of healthy male volunteers. Cells were cultured in EndoCult liquid medium in the absence and presence of CRP at clinically relevant concentrations (5 to 25 microg/ml) for different durations (3 to 48 hours). IL-8 protein and mRNA of cultured EPCs were evaluated using ELISA and real-time PCR.

RESULTSThe results showed that CRP at a concentration of 10 microg/ml significantly reduced IL-8 secretion of cultured EPCs with a peak at 25 microg/ml, and also decreased mRNA expression in EPCs with a peak at 12 hours. In addition, preincubation of EPCs with SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK) decreased CRP inhibition of IL-8 mRNA expression at 12 hours in EPCs.

CONCLUSIONSOur study, for the first time, demonstrates that CRP directly inhibits EPCs IL-8 secretion, a key cytokine player of angiogenesis induced by EPCs. Inhibition occurred in part via an effect of CRP to active the p38 MAPK signal transduction pathway in EPC. The ability of CRP to inhibit EPCs IL-8 secretion may represent an important mechanism that further links inflammation to cardiovascular disease.

C-Reactive Protein ; pharmacology ; Cells, Cultured ; Endothelial Cells ; drug effects ; metabolism ; Humans ; Imidazoles ; pharmacology ; Interleukin-8 ; genetics ; Male ; Pyridines ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; Stem Cells ; drug effects ; metabolism ; p38 Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism

OBJECTIVETo examine the synergistic effect of recombinant human high mobility group box 1 (HMGB1) protein and lipopolysaccharides (LPS) on the release of interleukin-8 (IL-8) and monocyte chemotactic protein 1 (MCP-1) in human umbilic vein endothelial cells (HUVECs), and explore the role of mitogen-activated protein kinases (MAPK) signal transduction in cytokine release.

METHODSHUVECs were incubated with recombinant HMGB1 (0-75 ng/ml) for 24 h and the culture medium supernatant was harvested for detection of IL-8 and MCP-1 with LiquiChip system. At 0, 1, 3, 6, 12 and 24 h after stimulation with 15 ng/ml HMGB1 or 15 ng/ml HMGB1 plus 10 ng/ml LPS, the levels of IL-8 and MCP-1 in the HUVECs were examined. To test the effect of MAPK inhibitors, HUVCs were pretreated with the inhibitors SB203580 (20 mol/L), PD98059 (20 mol/L), and JNK inhibitor II (50 nmol/L) 1 h before HMGB1 and LPS stimulation.

RESULTSThe levels of IL-8 and MCP-1 were significantly increased in the HUVECs stimulated with HMGB1 protein at the concentrations of 3, 15 and 75 ng/ml in comparison with the control levels (P<0.01). Since 3-6 h after the stimulation with HMGB1, the levels of IL-8 and MCP-1 began to increase gradually, and steadily increased at 12 and 24 h, all significantly higher than those of the control group (P<0.01). Stimulation of the HUVECs with LPS (10 ng8 and MCP-1 (P<0.01), which were further increased after costimulation with LPS and HMGB1, suggesting a synergistic effect between HMGB1 and LPS (P<0.01). This synergistic effect was significantly inhibited by pretreatment with MAPK signaling kinases inhibitors, especially the p38 MAP kinase inhibitor SB203580, and the cocktail of MAP kinase inhibitors almost totally blocked the expression of these chemokines in HUVECs treated with HMGB1 and LPS.

CONCLUSIONHMGB1 protein can activate HUVECs to produce the chemokines IL-8 and MCP-1 in a dose- and time-dependent manner. HMGB1 also acts synergistically with LPS to induce IL-8 and MCP-1 release, which might play an important role in the development of sepsis. MAPK signal transduction plays an important role in HMGB1 and LPS-induced IL-8 and MCP-1 release.

Cell Line ; Chemokine CCL2 ; blood ; metabolism ; Dose-Response Relationship, Drug ; Endothelial Cells ; drug effects ; metabolism ; HMGB1 Protein ; pharmacology ; Humans ; Interleukin-8 ; blood ; metabolism ; Mitogen-Activated Protein Kinases ; antagonists & inhibitors ; metabolism ; Protein Kinase Inhibitors ; pharmacology ; Time Factors

Serum amyloid A (SAA) has been regarded as an important mediator of inflammatory responses. The effect of several formyl peptide receptor-like 1 (FPRL1) ligands on the production of IL-8 by SAA was investigated in human neutrophils. Among the ligands tested, LL-37 was found to specifically inhibit SAA-induced IL-8 production in transcriptional and post-transcriptional levels. Since SAA stimulated IL-8 production via ERK and p38 MAPK in human neutrophils, we tested the effect of LL-37 on SAA induction for these two MAPKs. LL-37 caused a dramatic inhibition of ERK and p38 MAPK activity, which is induced by SAA. LL-37 was also found to inhibit SAA-stimulated neutrophil chemotactic migration. Further, the LL-37-induced inhibitory effect was mediated by FPRL1. Our findings indicate that LL-37 is expected to be useful in the inhibition of SAA signaling and for the development of drugs against SAA-related inflammatory diseases.
Animals ; Antimicrobial Cationic Peptides/*pharmacology ; Cell Line, Tumor ; Cell Movement ; Chemotaxis, Leukocyte ; Humans ; Interleukin-8/*biosynthesis ; MAP Kinase Kinase Kinases/metabolism ; Neutrophils/drug effects/*immunology ; Proto-Oncogene Proteins/metabolism ; Rats ; Receptors, Formyl Peptide/metabolism ; Receptors, Lipoxin/metabolism ; Serum Amyloid A Protein/*antagonists & inhibitors ; Signal Transduction ; Transcription, Genetic

Serum amyloid A (SAA) has been regarded as an important mediator of inflammatory responses. The effect of several formyl peptide receptor-like 1 (FPRL1) ligands on the production of IL-8 by SAA was investigated in human neutrophils. Among the ligands tested, LL-37 was found to specifically inhibit SAA-induced IL-8 production in transcriptional and post-transcriptional levels. Since SAA stimulated IL-8 production via ERK and p38 MAPK in human neutrophils, we tested the effect of LL-37 on SAA induction for these two MAPKs. LL-37 caused a dramatic inhibition of ERK and p38 MAPK activity, which is induced by SAA. LL-37 was also found to inhibit SAA-stimulated neutrophil chemotactic migration. Further, the LL-37-induced inhibitory effect was mediated by FPRL1. Our findings indicate that LL-37 is expected to be useful in the inhibition of SAA signaling and for the development of drugs against SAA-related inflammatory diseases.
Animals ; Antimicrobial Cationic Peptides/*pharmacology ; Cell Line, Tumor ; Cell Movement ; Chemotaxis, Leukocyte ; Humans ; Interleukin-8/*biosynthesis ; MAP Kinase Kinase Kinases/metabolism ; Neutrophils/drug effects/*immunology ; Proto-Oncogene Proteins/metabolism ; Rats ; Receptors, Formyl Peptide/metabolism ; Receptors, Lipoxin/metabolism ; Serum Amyloid A Protein/*antagonists & inhibitors ; Signal Transduction ; Transcription, Genetic

Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)beta, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRalpha. However, recent data suggest that GRbeta is not a dominant negative inhibitor of GRalpha in the transrepressive process and has its own functional role. We investigated the functional role of GRbeta expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-alpha-induced IL-8 release in an airway epithelial cell line. GRbeta expression was induced by treatment of epithelial cells with either dexamethasone or TNF-alpha. GRbeta was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-alpha-induced IL-8 transcription was not affected by GRbeta overexpression, rather GRbeta had its own weak suppressive activity on TNF-alpha-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRbeta overexpression, but TNF-alpha-induced histone H4 acetylation at the IL-8 promoter was decreased with GRbeta overexpression. This study suggests that GRbeta overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRbeta induces its own histone deacetylase activity around IL-8 promoter site.
Acetylation ; Cell Line, Tumor ; Dexamethasone/pharmacology ; Epithelial Cells/metabolism ; *Gene Expression Regulation ; Histones/*metabolism ; Humans ; Interleukin-8/*genetics/metabolism ; Receptors, Glucocorticoid/genetics/*metabolism ; Transcriptional Activation ; Transfection ; Tumor Necrosis Factor-alpha/*antagonists & inhibitors/pharmacology

Increased expression of a number of proinflammatory genes, including IL-8, is associated with inflammatory conditions such as asthma. Glucocorticoid receptor (GR)beta, one of the GR isoforms, has been suggested to be upregulated in asthma associated with glucocorticoid insensitivity and to work as a dominant negative inhibitor of wild type GRalpha. However, recent data suggest that GRbeta is not a dominant negative inhibitor of GRalpha in the transrepressive process and has its own functional role. We investigated the functional role of GRbeta expression in the suppressive effect of glucocorticoids on tumor necrosis factor (TNF)-alpha-induced IL-8 release in an airway epithelial cell line. GRbeta expression was induced by treatment of epithelial cells with either dexamethasone or TNF-alpha. GRbeta was able to inhibit glucocorticoid-induced transcriptional activation mediated by binding to glucocorticoid response elements (GREs). The suppressive effect of dexamethasone on TNF-alpha-induced IL-8 transcription was not affected by GRbeta overexpression, rather GRbeta had its own weak suppressive activity on TNF-alpha-induced IL-8 expression. Overall histone deacetylase activity and histone acetyltransferase activity were not changed by GRbeta overexpression, but TNF-alpha-induced histone H4 acetylation at the IL-8 promoter was decreased with GRbeta overexpression. This study suggests that GRbeta overexpression does not affect glucocorticoid-induced suppression of IL-8 expression in airway epithelial cells and GRbeta induces its own histone deacetylase activity around IL-8 promoter site.
Acetylation ; Cell Line, Tumor ; Dexamethasone/pharmacology ; Epithelial Cells/metabolism ; *Gene Expression Regulation ; Histones/*metabolism ; Humans ; Interleukin-8/*genetics/metabolism ; Receptors, Glucocorticoid/genetics/*metabolism ; Transcriptional Activation ; Transfection ; Tumor Necrosis Factor-alpha/*antagonists & inhibitors/pharmacology

To investigate the effect of cetirizine hydrochloride on the expression of neurokinin 1 receptor (NK-1R) and cytokines production induced by substance P (SP) in HaCaT cells (a human epidermal keratinocyte cell line) and dermal fibroblasts. The effect of cetirizine on the expression of NK-1R protein was detected by flow cytometry and Western blotting analysis. The modulation of cetirizine on the production of interferon (IFN)-gamma, interleukin (IL)-1beta, IL-6 and IL-8 in HaCaT cells and fibroblasts was measured by ELISA. The results showed that cetirizine significantly inhibited the expression of NK-1R in HaCaT cells and fibroblasts. SP induced the production of IFN-gamma, IL-1beta and IL-8 in both cell types. Cetirizine 1-100 micromol x L(-1) inhibited SP-induced IL-1beta and IL-8 production in HaCaT cells and fibroblasts, while had no effect on the production of IFN-gamma in both cells. Both SP and cetirizine had no effect on the secretion of IL-6 in HaCaT cells and fibroblasts. These findings suggest that cetirizine may be involved in the treatment of SP-induced skin inflammation by inhibiting the expression of substance P receptor and regulation the production of IL-1beta and IL-8 in epidermal keratinocyte and dermal fibroblasts.
Anti-Allergic Agents ; pharmacology ; Cell Line ; Cetirizine ; pharmacology ; Fibroblasts ; cytology ; metabolism ; Histamine H1 Antagonists, Non-Sedating ; pharmacology ; Humans ; Interferon-gamma ; metabolism ; Interleukin-1beta ; metabolism ; Interleukin-8 ; metabolism ; Keratinocytes ; cytology ; metabolism ; Receptors, Neurokinin-1 ; metabolism ; Substance P ; pharmacology