OBJECTIVEKawasaki disease (KD) is an acute febrile, multi-system endangeitis, which is mainly found in early childhood. Its etiology is still unknown. A great deal of clinical evidence and epidemiologic data suggest that KD is correlated with an acute immune dysfunction caused by infection. Many evidences in the past suggested that over-expression of proinflammatory cytokines, co-stimulatory molecules and chemokines, which were observed in KD, may contribute to the pathologic lesion of vascular endothelial cells. But the causative factors are still unknown. Toll-like receptor is a type I trans-membrane protein which could recognize ligands of pathogenic microbes, induce interferon beta (IFN-beta) and promote gene transcription of proinflammatory cytokines, co-stimulatory molecules and chemokines. This study was designed to investigate the role of MyD88-independent signal transduction of Toll-like receptor 4 in immunological pathogenesis of KD.

METHODSThirty-two children with KD and 16 age-matched healthy children were studied. Reverse-transcription PCR (RT-PCR) and real-time PCR were used to evaluate the mRNA levels of Toll-like receptor 4 and the molecules such as Toll-IL-1-receptor domain containing adaptor inducing IFN-beta (TRIF), TRIF-related adaptor molecule (TRAM), TANK-binding kinase 1 (TBK-1), IFN-beta, interferon-gamma-inducible protein 10 (IP-10), regulated on activation normal T cells expressed and secreted (RANTES), inducible nitric oxide synthase (iNOS) and suppressor of cytokine signaling 1 (SOCS-1) in monocytes/macrophages (MC), which participate in MyD88-independent signal transduction of toll-like receptors. Expression of costimulatory molecules such as CD40 in MC was analyzed by flow cytometry. Methylation-specific PCR was performed to analyze the methylation status of cytosine-phosphate-guanine (CpG) motif in SOCS-1 gene.

RESULTS(1) Compared with healthy controls, transcription levels of the molecules such as TLR4, TRIF, TRAM, TBK-1 and IFN-beta, were significantly up-regulated during acute phase of KD (P < 0.05), and down-regulated after treatment with intravenous immunoglobulin therapy. (2) Expression of iNOS and chemokines such as IP10 and RANTES in MC during acute phase of KD was remarkably elevated (P < 0.05), and down-regulated to some extents after treatment with intravenous immunoglobulin therapy. (3) Expression of costimulatory molecule CD40 in MC increased significantly during acute phase of KD [(6.19 +/- 2.25)% vs. (2.00 +/- 1.37)%, P < 0.05], while the protein levels of CD40 in KD-coronary artery lesion (CAL)(+) group was found to be significantly higher than that of KD-CAL-group [KD-CAL, (9.63 +/- 2.96)% vs. (4.12 +/- 1.91)%, P < 0.05]. (4) Expression levels of SOCS-1 mRNA were significantly up-regulated during acute phase of KD [(4.31 +/- 0.83) x 10(-3) vs. (1.09 +/- 0.23) x 10(-3), P < 0.05], and the levels of SOCS-1 gene in KD-CAL(+) group was found to be significantly lower than that of KD-CAL(-) group [(5.73 +/- 1.04) x 10(-3) vs (1.94 +/- 0.46) x 10(-3), P < 0.05]. (5) The CpG island of SOCS-1 DNA in KD patients was remarkably demethylated [(26.9 +/- 8.6)% vs (5.9 +/- 1.4)%, P < 0.05], and demethylation levels of SOCS-1 in KD-CAL(-) group were higher than that in KD-CAL+ group [(35.1 +/- 10.3)% vs. (13.2 +/- 3.7)%, P < 0.05].

CONCLUSIONAberrant activation of MyD88-independent pathways of Toll-like receptor 4 may be one of the factors causing disturbed immunological function in KD.

Child ; Humans ; Interleukin-1 ; metabolism ; Macrophages ; drug effects ; pathology ; Nitric Oxide Synthase Type II ; metabolism ; Pyrimidinones ; pharmacology ; Signal Transduction ; drug effects ; physiology ; Thiazoles ; pharmacology ; Toll-Like Receptor 4 ; metabolism ; Toll-Like Receptors ; deficiency ; drug effects ; metabolism

This study used in vivo microdialysis to examine the effects of intragingival application of lipopolysaccharide (LPS) derived from Porphyromonas gingivalis (Pg-LPS) on gingival tumour necrosis factor (TNF)-α and interleukin (IL)-6 levels in rats. A microdialysis probe with an injection needle attached to the surface of the dialysis membrane was implanted into the gingiva of the upper incisor. For comparison, the effects of LPS derived from Escherichia coli (Ec-LPS) on IL-6 and TNF-α levels were also analysed. Pg-LPS (1 μg/1 μL) or Ec-LPS (1 or 6 μg/1 μL) was applied by microsyringe, with gingival dialysates collected every hour. Enzyme-linked immunosorbent assay (ELISA) revealed that gingival dialysates contained approximately 389 pg·mL⁻¹ of IL-6 basally; basal TNF-α levels were lower than the detection limit of the ELISA. Pg-LPS failed to alter IL-6 levels but markedly increased TNF-α levels, which remained elevated for 2 h after treatment. Neither IL-6 nor TNF-α were affected by Ec-LPS. Reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed that the gingiva expresses Toll-like receptor (TLR) 2 and TLR4 mRNA. Immunohistochemical examination showed that TLR2 and TLR4 are expressed by gingival epithelial cells. The present study provides in vivo evidence that locally applied Pg-LPS, but not Ec-LPS, into the gingiva transiently increases gingival TNF-α without affecting IL-6. The present results suggest that TLR2 but not TLR4 expressed on gingival epithelial cells may mediate the Pg-LPS-induced increase in gingival TNF-α in rats.
Animals ; Gingiva ; drug effects ; metabolism ; Interleukin-6 ; metabolism ; Lipopolysaccharides ; administration & dosage ; Male ; Porphyromonas gingivalis ; metabolism ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

OBJECTIVETo investigate the effect of compound qingqin liquid (CQL) on Toll-like receptor 2 (TLR2) and toll-like receptor 4 (TLR4) in rats with urate nephropathy, and to explore its renal protection mechanism.

METHODSTotally 55 SD rats were randomly divided into 5 groups, i.e., the normal control group (n =5), the model group (n =10), the positive drug group (n=10), and the high-, medium-, low-dose CQL groups (n=10) respectively. The urate nephropathy model was induced by intragastrically administering adenine and feeding yeast. Distilled water was intragastrically administered at the daily dose of 10 mL/kg to rats in the normal control group and the model group. Allopurinol was intragastrically administered at the daily dose of 9.33 mg/kg to rats in the positive control group. CQL was intragastrically administered at the daily dose of 3.77, 1.89, 0.94 g/kg to rats in the high-, medium-, and low-dose CQL groups. Rats of each group were executed in batches at the 4th and 6th week respectively. Their kidney tissues were taken out to determine the mRNA transcription level of TLR2 and TLR4 by reverse transcription-polymerase chain reaction (RT-PCR). The protein expression level of TLR2 and TLR4 were determined by Western blot. The protein expression level of TLR4 was also detected by immunohistochemical assay.

RESULTSAt week 4 and 6, the protein expression of TLR2 and TLR4 as well as the mRNA transcription of TLR4 increased in the model group, when compared with the control group (P < 0.05, P < 0.01). Compared with the model group, there was no statistical difference in the transcription level of TLR2 mRNA or TLR4 mRNA among the 3 CQL groups (P > 0.05) at week 4 and 6. Additionally, at week 6, the protein expression of TLR4 and TLR2 could be reduced by CQL (P < 0.05, P < 0.01).

CONCLUSIONCQL might protect kidney tissue against inflammatory injury by inhibiting the protein expression levels of TLR2 and TLR4.

Animals ; Disease Models, Animal ; Drugs, Chinese Herbal ; pharmacology ; Kidney ; drug effects ; metabolism ; Kidney Diseases ; drug therapy ; metabolism ; Male ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 2 ; genetics ; metabolism ; Toll-Like Receptor 4 ; genetics ; metabolism ; Uric Acid

To study the effect and underlying mechanism of Mahuang Tang against influenza A virus ， the influenza virus-infected Madin-Darby canine kidney(MDCK) cells were used as the carrier in this study to detect the median tissue culture-infective dose(TCID₅₀) of influenza A virus strains(A/PR8/34) on MDCK cells with cytopathic effect(CPE) assay. Blocking influenza virus invading host cells and anti-influenza virus biosynthesis were used as two different administration methods， and then the methyl thiazolyl tetrazolium(MTT) assay was utilized to determine the antiviral effective rate(ER)， median efficacious concentration(EC₅₀) and therapeutic index(TI) of Mahuang Tang. The quantitative Real-time polymerase chain reaction(RT-PCR) was used to measure virus load and the mRNA expression levels of TLR4， TLR7， MyD88 and TRAF6 in MDCK cells at 24， 48 h after the treatment. The experiment results indicated that TCID₅₀ of A/PR8/34 for MDCK cells was 1×10-4.32/mL. The EC₅₀ values of two different treatment methods were 4.92，1.59 g·L⁻¹ respectively， the TI values were 12.53， 38.78 respectively， and when the concentration of Mahuang Tang was 5.00 g·L⁻¹， ER values were 50.21%， 98.41% respectively， showing that Mahuang Tang can block influenza virus into the host cells and significantly inhibit their biosynthesis. Meanwhile， as compared with the virus group， the virus load was significantly inhibited in Mahuang Tang groups， and Mahuang Tang high and middle doses had the significant effect on decreasing the mRNA expression of TLR4， TLR7，MyD88 and TRAF6 at 24， 48 h after the treatment. It can be demonstrated that the mechanisms of Mahuang Tang against influenza A virus are related to the inhibition of influenza virus replication and the mRNA expression of correlative genes in TLR4 and TLR7 signaling pathways.
Animals ; Antiviral Agents ; pharmacology ; Dogs ; Drugs, Chinese Herbal ; pharmacology ; Influenza A Virus, H1N1 Subtype ; drug effects ; physiology ; Madin Darby Canine Kidney Cells ; Orthomyxoviridae Infections ; Toll-Like Receptor 4 ; metabolism ; Toll-Like Receptor 7 ; metabolism ; Virus Replication ; drug effects

Arthritis, Rheumatoid ; metabolism ; Cells, Cultured ; HSP72 Heat-Shock Proteins ; pharmacology ; Humans ; Interleukin-10 ; metabolism ; Synovial Membrane ; cytology ; drug effects ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo investigate whether Candida albicans-native phospholipomannan (PLM) induce an inflammation response through Toll-like receptor(TLRé2 in human acute monocytic leukemia cell line (THP-1) cells.

METHODSHuman THP-1 monocytes were challenged with PLM in vitro. The mRNA expressions of TLR2, TLR4, proinflammatory cytokine [interleukin(IL)-6], and chemokine (IL-8) were assayed by real time reverse transcription polymerase chain reaction. The secretions of IL-6 and IL-8 were measured by enzyme-linked immunosorbent assay. The expression of TLR2 was analyzed with Western blot.

RESULTSPLM increased the mRNA expressions and secretions of proinflammatory cytokines (IL-6) and chemokines (IL-8) in THP-1 cells (all P=0.0000). PLM up-regulated the mRNA and protein levels of TLR2 (P=0.0000), whereas the mRNA level of TLR4 was not altered. PLM hydrolyzed with β-D-mannoside manno hydrolase failed to induce gene and protein expressions of TLR2, IL-6, and IL-8. Anti-TLRS-neutralizing antibody blocked the PLM-induced secretions of IL-6 and IL-8 in THP-1 cells (P = 0.0003, P = 0.0010).

CONCLUSIONCanidada albicans-native PLM may contribute to the inflammatory responses during Candida infection in a TLR2-dependent manner.

Candida albicans ; chemistry ; Cells, Cultured ; Glycolipids ; pharmacology ; Humans ; Interleukin-6 ; metabolism ; Interleukin-8 ; metabolism ; Monocytes ; drug effects ; immunology ; metabolism ; Toll-Like Receptor 2 ; metabolism ; Toll-Like Receptor 4 ; metabolism

The study was aimed to investigate the mechanism of mannan-binding lectin (MBL) on bacterial lipopolysaccharide (LPS)-induced human peripheral blood monocyte-derived dendritic cell (DC) maturation. The monocytes were prepared from the peripheral blood of healthy adult volunteers. The immature dendritic cells (imDC) were induced by 5-day-culture in medium supplemented with rhGM-CSF and rhIL-4. FACS was used to investigate the interaction of MBL with imDC and the impact of MBL on LPS binding to imDC. ELISA and Western blot was used to analyze the interaction of MBL with soluble TLR4 ectodomain protein (sTLR4); Western blot was used to detect LPS-induced NF-κB translocation in imDC. The results showed that MBL could directly bind to imDC in the presence of calcium. sTLR4 protein or LPS could competitively inhibit the binding of MBL to imDC. ELISA and Western blot showed that MBL could evidently bind to sTLR4 protein in a concentration-dependent manner. FACS showed that MBL could competitively inhibit the binding of LPS to imDC by binding to imDC directly. Western blot showed that MBL decreased LPS-induced NF-κB translocation in imDC. It is concluded that MBL may competitively inhibit the binding of LPS to imDC by binding to TLR4 expressed on imDC, resulted in inhibition of LPS-induced DC maturation, suggesting that MBL can regulate DC maturation through ligand-binding. This study provides the good foundation to clarify the mechanism of MBL inhibiting the LPS-induced DC maturation.
Cell Differentiation ; Cells, Cultured ; Dendritic Cells ; cytology ; drug effects ; metabolism ; Humans ; Ligands ; Lipopolysaccharides ; adverse effects ; Mannose-Binding Lectin ; pharmacology ; Monocytes ; cytology ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo investigate the expression of Toll-like receptor 2 (TLR2) and Toll-like receptor 4 (TLR4) and the effect of lipopolysaccharide (LPS) on their expression in cultured endothelial cells.

METHODSTotal RNA was extracted from ECV304 cells and isolated human umbilical vein endothelial cells (HUVECs) exposed to LPS, respectively. The quantification of TLR2 and TLR4 mRNA in HUVECs and EVC304 cells was carried out by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTSECV304 cells and HUVECs were able to express TLR2 and TLR4 mRNA, but the expression levels of TLR4 appeared to be stronger than those of TLR2. LPS could upregulate the expression levels of TLR4 obviously, whereas it had no effect on the expression level of TLR2.

CONCLUSIONSOur data indicate that TLR4 may be the LPS signal transducer in endothelial cells and plays important roles in the cell activation of LPS. The ECV304 cell line is a better experimental model than isolated HUVECs in the research of endothelial cells.

Cell Line ; Dose-Response Relationship, Drug ; Drosophila Proteins ; Endothelium, Vascular ; cytology ; drug effects ; metabolism ; Gene Expression Regulation ; drug effects ; Humans ; Lipopolysaccharides ; pharmacology ; Membrane Glycoproteins ; genetics ; RNA, Messenger ; drug effects ; genetics ; metabolism ; Receptors, Cell Surface ; genetics ; Time Factors ; Toll-Like Receptor 2 ; Toll-Like Receptor 4 ; Toll-Like Receptors ; Umbilical Veins ; cytology ; drug effects ; metabolism ; Up-Regulation

BACKGROUNDIncreased levels of plasma lipopolysaccharide (LPS) have been found in obesity and diabetes patients. This study was to investigate the effect of LPS on pancreatic beta-cell viability and the involvement of caspase 3 in NIT-1 cell line.

METHODSMouse insulinoma NIT-1 cells were treated with LPS for the indicated time and dose. Cell viability was measured by cell counting kit-8 reagent. Toll-like receptor 4 (TLR4), caspase 3 and cleaved caspase 3 were detected by Western blotting. Insulin was determined by radioimmunoassay (RIA).

RESULTSLPS promoted NIT-1 cell proliferation at 1 µg/ml, peaked at 72 hours of incubation. A reduction in cleavage of caspase 3 was observed upon LPS treatment. Bay11-7082, a specific inhibitor of nuclear factor (NF)-κB, blunted LPS-induced inhibition of caspase 3 cleavage. Reduction in chronic insulin secretion was observed after treatment with LPS at 1 µg/ml for 48 and 72 hours, not for 24 hours. TLR4 protein was upregulated when NIT-1 cells were treated with LPS at 1 µg/ml for 24 hours.

CONCLUSIONSLPS promotes early NIT-1 cell proliferation in association with NF-κB-mediated inhibition of caspase 3 cleavage. LPS exerts a time-dependent inhibitory effect on chronic insulin secretion from NIT-1 cells.

Animals ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Insulin ; secretion ; Insulinoma ; metabolism ; Lipopolysaccharides ; pharmacology ; Mice ; NF-kappa B ; metabolism ; Toll-Like Receptor 4 ; metabolism

OBJECTIVETo observe the effect of Qingchang Huashi Recipe (QHR) on the activation and expressions of nuclear factor kappaB (NF-kappaB), Toll-like receptors (TLRs), and contents of interleukin-8 (IL-8), thus exploring its possible mechanisms for treating ulcerative colitis (UC).

METHODSThe HT-29 cells were induced to inflammation model by tumor necrosis factor-alpha (TNF-alpha) and lipopolysaccharides (LPS). HT-29 cells were divided into 6 groups, i.e., the vehicle control group, the model control group, the sulfasalazine (SASP) group, the high dose QHR group, the middle dose QHR group, the low dose QHR group. Effects on the cell growth were detected by MTT. The chemoattractant of macrophages was observed using Transwell. The expressions of NF-kappaB and TLR4 protein were detected using immune cell fluorescence method. The content of IL-8 was detected by ELISA.

RESULTSThe growth of cells were not inhibited in each group. Statistical difference existed in each dose QHR group in inhibiting the chemoattractant of macrophages, reducing activation of NF-kappaB, lowing expressions of TLR4 protein, and decreasing the secretion of IL-8, when compared with the model control group (P < 0.05). No statistical difference existed in inhibiting the chemoattractant of macrophages between the high dose QHR group and the vehicle control group (P > 0.05). But its inhibition on NF-kappaB activation was higher in the high dose QHR group than in the SASP group (P < 0.05).

CONCLUSIONQHR could obviously attenuate the inflammatory reaction of HT-29 cells, inhibit the chemoattractant of macrophages, reduce the activation of NF-kappaB, lower expressions of TLR-4, and attenuate the secretion of IL-8, which might be one of its mechanisms for treating UC.

Colitis, Ulcerative ; metabolism ; pathology ; Drugs, Chinese Herbal ; pharmacology ; HT29 Cells ; Humans ; Inflammation ; Interleukin-8 ; metabolism ; NF-kappa B ; metabolism ; Signal Transduction ; drug effects ; Toll-Like Receptor 4 ; metabolism