The Expression Of Tlr2,4 In Pbmc From Severe Pneumonia And Correlated With The Levels Of Tnf-α, Il-1β, Il-8 In Plasma

BackgroundSevere pneumonia are bacteria, viruses microbial invasion of the body as the main cause of the inflammation after the signal transduction of immune disorders, induced by TNF-α, IL-1βand IL-8 and other pro- inflammatory cytokine over-activation, trigger inflammation ultimately trigger a cascade of inflammatory cytokines and systemic inflammatory response syndrome (SIRS). What causes the over expression of inflammatory cytokines? There is no good explanation. Recently,the discovery of TLRs have a significant development in rnatural immunity research, has proved that the receptor plays an important role in the immune infection [1]. TLR2,4 in the TLR family occupies an important position, it was found that infectious disease known as immune mechanisms important milestone in the study. TLRs recognize their ligands, the activation of NF-ΚB such as transcription factors and protein kinases, release of inflammatory mediators, activation of innate immunity; but also to antigen-presenting cells of costimulatory molecules CD80, CD86, and eventually start the follow-up of T cells, B cell-mediated specific immune [2] TLR2, 4 as an important inflammatory receptors, mainly distributed in the marrow- derived mononuclear cells in patients with clinical severe pneumonia expressed in monocytes and the expression of inflammatory cytokines, has not been reported to be studied further.ObjectiveAt the protein level and mRNA molecules of severe pneumonia in peripheral blood mononuclear cells, preliminary study TLR2, 4 expression in the inflammatory response induced by severe pneumonia,observed the expression of TLR2,4 in PBMC from Severe Pneumonia and Correlated with the levels of TNF-α,IL-1β, IL-8 in Plasma and PSI score.Materials and Methods1.Collection of specimens Specimens were collected from patients with severe pneumonia in Respiratory Department of Guangzhou First People's Hospital from August 2008 to May 2009.All patients meet the diagnostic criteria for sever pneumonia that is established by the American Thoracic Society (AST) in 2007. Pneumonia group were in line with our diagnosis of community- acquired pneumonia and control group is from healthy physical examinees in the same period. 12 patients with severe pneumonia, pneumonia group, 18 cases and control group ,12 cases,After patents themseleves and their relatives assent,we collect their peripheral blood, 11mL, EDTAK3 anti- coagulant.2.Methods2.1 The expression of TLR2,4 in the monocyte surface Using flow cytometry with three colors direct immunological fluorescence analytics.Taking 1ml blood(EDTAK3 anticoagulant), each sample loaded two points, test tubes and control tubes. With FITC,PE and APC fluorescein direct labeled monoclonal antibody to detect single cells with the three epitopes (TLR2,TLR4,CD14). With a forward scattering angle (FSC) and side scatter angle (SSC) to determine mononuclear cell , then FSC/CD14 an APC double Parametric doors, with BD FACSDivaTM software acquisition and analysis of each tube gate 10000 cells, CellQuest ModFitTM software to analyze monocyte TLR2, 4 positive rate.2.2 The expression of TLR2,4 mRNA in the monocyte2.2.1 Separation of human peripheral blood mononuclear cells: Ficoll-Hypaque density gradient centrifugation, extraction of severe pneumonia group, pneumonia, and human peripheral blood mononuclear cells samples (PBMC), using isolated and purified by adhesion of mononuclear cells.2.2.2 monocyte TLR2, 4mRNA relative quantitative Reference TRIzol (U.S. invitrogen) instructions to extract total RNA, detected from total RNA quality and concentration. severe pneumonia, pneumonia patients and healthy controls all of the extracted total RNA reverse transcription into cDNA. Using Power SYBR Green PCR Master Mix (ABI) kit for quantitative PCR, the TLR2, 4 andβ-actin positive and healthy control template cDNA at the same time quantitative PCR instrument amplification. After, the computer Sequence Detection System will sample amplification curve and standard curve are compared and automatically calculate the internal reference sample of TLR2, 4 gene copy number.2.3 The expression of TNF-α, IL-1β, IL-8 in the plasma . Using double antibody enzyme-linked immunosorbent assay (ELISA), determine the Concentration of TNF-α, IL-1β, IL-8 in three group .2.4 Statistical MethodsAll experimental data were analyzed using SPSS 13.0 software, measurement data were presented as mean±standard deviation (±S) . experimental data was normally distributed, between group and group comparison of these two single factor analysis of variance, homogeneity of variance method used LSD; variance missing by Tamhane, s T2 multiple comparison;, PSI score and plasma TNF-α, IL-1β, IL-8 concentration of TLR2, TLR4 between correlation using Pearson correlation analysis.In allexperiments,P<0.05 was considered to indicate a statistically significant difference. Result3.1 TLR-2, 4-positive in Peripheral blood mononuclear cellsCompared with the healthy group, severe pneumonia group, pneumonia, peripheral blood mononuclear cells of TLR2, 4-positive rate was sign- ificantly higher (P <0.01); severe pneumonia peripheral blood mononuclear cells of TLR4-positive rate of pneumonia was significantly higher than that (P <0.01). but the two groups showed no significant difference in TLR2-positive (P> 0.05).3.2 the expression of TLR-2, 4 mRNA in peripheral blood mononuclear cellsThe peripheral blood mononuclear cells of TLR2, 4mRNA expression in Pneumonia, severe pneumonia were significantly higher than those in healthy controls (P <0.01); severe pneumonia, peripheral blood mono- nuclear cells of TLR4mRNA expression was significantly higher than pneumonia increased (P <0.01); but the two groups of expression TLR2mRNA no significant difference (P> 0.05).3.3 The concentration of TNF-α, IL-1β, IL-8 in plasmaPneumonia and severe pneumonia patients in the TNF-α, IL-1β, IL-8 levels were significantly higher than in healthy (P <0.01), and severe pneumonia, plasma TNF-α, IL-1β, IL-8 levels than pneumonia was signi- ficantly higher (P <0.01), and comparisons between the three groups was statistically significant.3.4 The expression of TNF-α, IL-1β, IL-8 in plasma and PSI score in patients with TLR2, 4 positive correlation. Three samples of peripheral blood mononuclear cells in TLR2, 4 positive rate and the concentrations of plasma TNF-α, IL-1β, IL-8 in plasma were positively correlated, while TLR4 and the PSI score is a positive correlation, determination coefficient R = 0.517, P = 0.003.Conclusion1. Severe pneumonia in patients with peripheral blood mononuclear cells in TLR2, 4 protein increased, probably due to peripheral Monocyte TLR2, 4mRNA upregulation caused.2. Severe pneumonia of TNF-α, IL-1β, IL-8 in plasma than the pneumonia group, healthy group was significantly higher, and TLR2, 4 all have a positive correlation, indicating severe pneumonia in plasma TNF-α, IL-1β, IL-8 concentration in peripheral blood mononuclear cells with TLR2, 4 regulated gene expression is associated.2. Severe pneumonia, peripheral blood mononuclear cells of TLR4 expression was significantly higher than the pneumonia group, and a positive correlation with the PSI score, indicating TLR4 may be closely related to the severity of pneumonia.