Effects Of Tim-3 Regulation Of The Th1/Th2 Cells Balance In Asthma

Part 1 Effect of Tim-3 regulation of the Th1/Th2 cells balance inasthmatic miceChapter 1Expression of Tim-3 in CD4⁺T Cells and its relationship with Th1/Th2cytokines in peripheral blood in asthmatic murineObjective: To detect the expression of Th1-specific cell surface protein Tcell immunoglobulin mucin 3(Tim-3) gene in CD4⁺T cells and its relationshipwith Th1/Th2 cytokines isolated from peripheral blood in asthmatic murinemodel and explore the elementarily role of Tim-3 in the occurrence anddevelopment of asthmatic inflammation.Methods: The asthmatic murine model were established by ovalbumin(OVA) injection and inhalation. The mice were randomly divided into 3groups: normal control group, asthma 1 day group and asthma 7 day group.Peripheral blood T lymphocytes were collected. Expression of Tim-3 mRNAin peripheral blood T lymphocytes was detected by RT-PCR, and the ratio ofTim-3⁺/CD4⁺ cells in total CD4⁺cells was detected by flow cytometry to reflect the level of Tim-3. The IL-4 and IFN-γlevel was determined withELISA. The correlation between Tim-3 and IL-4, IFN-γlevel was analyzed.Results: Contrast with the normal control group, the level of Tim-3mRNA and the ratio of CD4⁺Tim-3⁺/CD4⁺ in peripheral blood T lymphocytesin the asthma 1 day group and asthma 7 day group increased significantly(P＜0.01), and those in the asthma 7 day group were higher than asthma 1 daygroup(P＜0.05). The level of IL-4 in the asthma 1 day group and asthma 7 daygroup were higher than those in normal control group (P＜0.05), and those inthe asthma 7 day group were higher than asthma 1 day group (P＜0.05). Thelevel of IFN-γin the asthma 1 day group and asthma 7 day group were lowerthan those in normal control group (P＜0.05), and those in the asthma 7 daygroup were lower than asthma 1 day group (P＜0.05); The level of IL-4 wascorrelated positively with the Tim-3mRNA expression and the ratio of CD4⁺Tim-3⁺/CD4⁺ in asthma 7 day group (r=0.78, r=0.81 both P＜0.05), The levelof IFN-γwas correlated negatively with the Tim-3mRNA expression and theratio of CD4⁺ Tim-3⁺/CD4⁺ (r=-0.82, r=-0.89 both P＜0.05).Conclusions: The level of Tim-3 mRNA and protein in asthma werehigher than those in normal control group, and those in asthma 7 day groupwere higher than those in asthma 1 day group; meanwhile, the expression ofTim-3 has significantly positively correlated with the level of IL-4 and negatively correlated with the level of IFN-γ, which were suggested thatTim-3 might take part in the occurrence and development of asthmaticinflammation.Chapter 2Expression of Tim-3 in the inflammation cells in alveolar lavage ofasthmatic murineObjective: To investigate the expression of Tim-3 and the level of IL-4and IFN-7 of airway T lymphocytes in asthmatic murine and explore theeffects of Tim-3 in immunologic mechanism of bronchial asthma.Methods: Twenty-four Kunming mice were classified randomly 3groups; there were normal control group, asthma 1 day group and asthma 7day group. Bronchoalveolar lavage fluid (BALF) cells were collected and thenumbers of EOS were calculated after cells stained with Wright's staining.The expression of Tim-3 mRNA in BALF cells was detected using RT-PCR,and the ratio of Tim-3⁺/CD4⁺ cells in total CD4⁺cells was detected usingflow cytometry. IL-4 and IFN-γin alveolar lavage supernatants were detectedusing ELISA. The correlation between EOS and Tim-3, IL-4, IFN-γlevel was analyzed.Results: Contrast with the asthma 1 day group and normal control group,the amount of total leucocytes, EOS in BALF cells and the level of IL-4 inalveolar lavage supernatants in the asthma 7 day group increased significantly(P＜0.01), and those in the asthma 1 day group were higher than normalcontrol group (P＜0.05). The level of IFN-γin alveolar lavage supernatants inthe asthma 7 day group were lower than those in asthma 1 day group andnormal control group (P＜0.01), and those in the asthma 1 day group werelower than normal control group (P＜0.05). Contrast with the normal controlgroup, the level of Tim-3 mRNA and the ratio of CD4⁺Tim-3⁺/CD4⁺ in BALFcells in the asthma 1 day group and asthma 7 day group increasedsignificantly (P＜0.01), and those in the asthma 7 day group were higher thanasthma 1 day group(P＜0.05). The amount of EOS was correlated positivelywith the Tim-3mRNA expression, the ratio of CD4⁺Tim-3⁺/CD4⁺ and the levelof IL-4 in asthma 7 day group (r=0.73, r=0.79, r=0.86 both P＜0.05), and wascorrelated negatively with the level of IFN-γ(r=-0.81, P＜0.05).Conclusions: Compared with normal control group, the level of Tim-3mRNA and protein in asthmatic mice increased significantly in BALF cells. Itwas suggested that Tim-3 might participate the pathogenesis of asthma; andthe levels of Tim-3 in asthma 7 day group were higher than those in asthma 1 day group which was suggested that Tim-3 might also play a certain role inthe persistent attack of asthma. In addition, there were a positive correlationbetween the amount of EOS and the Tim-3mRNA expression, the ratio ofCD4⁺Tim-3⁺/CD4⁺, suggesting a close relationship between Tim-3 and airwayEOS inflammation in asthmatic mice.Chapter 3Effects of Tim-3 Neutralization Antibody on T Lymphocytes Functionin Asthmatic miceObjective: To investigate the effect of Tim-3 neutralization antibody onproliferation and cytokine synthesis of T lymphocytes from asthmatic mice,and explore its mechanism of asthma.Methods: Sixteen kunming mice were randomly divided into two groups,8 mice were sensitized as the asthmatic model group and the others taken asthe normal control group. T lymphocytes were isolated from peripheral bloodmononuclear cells and bronchoalveolar lavage fluid cells of the mice, andwere cultured in vitro with Tim-3 neutralization antibody in differentconcentrations or without any of them (control group). T lymphocytes proliferation in groups was measured by using MTT assay. The expressionlevels of interleukin4 and interfen-γwere detected with enzyme-linkedimmunosorbent assay.Results: Compared with control group, Tim-3 neutralization antibodycould significantly inhibit the proliferation of T lymphocytes in both controland asthmatic mice in vitro(P＜0.05). The effects were enhanced as the doseincreasing and the time prolonging, the effects to the latter were higher thanthose to the former, there were significant difference between them(P＜0.05).In either blood or BALF, the IFN-γlevel was significantly elevated butthe IL-4 level was much lower in the intervention group compared with theblank group.Results: Tim-3 neutralization antibody can reduce the abnormallyincreased proliferation of T lymphocytes in asthma, while decrease the levelof IL-4 and increase the level of IFN-γ. There may be the mechanismsunderlying its potential therapeutic effects in asthma. Part 2 Effect of Tim-3 regulation of the Th1/Th2 cellsbalance in asthmatic patientsChapter 1A correlative study of Tim-3mRNA expression in peripheral bloodlymphocytes and asthmatic pathogenesis and pulmonary function inasthmatic patientsObjective: To detect the expression of Th1-specific cell surface proteinTim-3 mRNA in peripheral blood lymphocyte isolated from asthmatic patients,and analysis the correlation among Tim-3mRNA, IL-4, IFN-γlevel andpulmonary ventilatory capacity, explore the elementarily role of Tim-3 in theoccurrence and development of asthmatic inflammation.Methods: There were 44 asthma patients who were classified into twogroups: 23 patients with acute asthma exacerbation and 21 patients withasthma remission. 17 health volunteers were as control. The Tim-3mRNAexpression was measured by Reverse Transcription-polymerase ChainReaction method. The IL-4 and IFN-γlevel were determined with ELISA. Thecorrelation among Tim-3mRNA, IL-4, IFN-γlevel and pulmonary ventilatorycapacity was analyzed.Results: The expression of Tim-3mRNA and the level of IL-4 inperipheral blood lymphocytes in patients with acute asthma exacerbation were significantly higher than those in remission stage and control group (P＜0.05),and those in remission stage was higher than control group (P＜0.05); the levelof IFN-γin patients with acute asthma exacerbation was significantly lowerthan those in remission stage and control group (P＜0.05), and those inremission stage was lower than control group (P＜0.05). The Tim-3mRNAexpression correlated positively with the level of IL-4(r=0.68, P＜0.05), andcorrelated negatively with the level of IFN-γand pulmonary ventilatorycapacity (r=-0.85, r=-0.76, both P＜0.01).Conclusions: The level of Tim-3mRNA in peripheral blood lymphocytesin asthma patients was significantly higher than that in normal group, and thelevel of Tim-3 in asthma patients with acute asthma exacerbation was higherthan that in remission stage which was suggested that Tim-3 might participatein the occurrence and development of asthmatic inflammation. There was anegative correlation between the level of Tim-3mRNA and FEV₁,FEV₁/FVC(%) in peripheral blood lymphocytes in asthma patients, both inacute asthma exacerbation and remission stage, suggested that Tim-3 might beused as an index to indirectly reflect the severity of asthma. Chapter 2Tim-3mRNA expression in induced sputum from asthmatic patientsObjective: To investigate the expression of Tim-3mRNA in theinflammation cells and the producing of IL-4, IFN-γin induced sputum inasthma patients, and analyze their correlation with pulmonary ventilatorycapacity and explore their relationship with airway inflammation.Methods: There were 53 asthma patients who were classified into twogroups: 28 patients with acute asthma exacerbation and 25 patients withasthma remission. 19 health volunteers were as control. The numbers ofeosinophils in induced sputum were calculated after cells stained withWright's staining. The level of Tim-3mRNA was detected in each group withReverse Transcription-polymerase Chain Reaction, IL-4 and IFN-γin inducedsputum supernatants were detected with enzyme-linked immunosorbent assay.Lung ventilatory function and forced expiratory volume in one second (FEV₁)were measured to all individuals.Results: The level of Tim-3mRNA,IL-4 and the amount of EOS ininduced sputum in patients with acute asthma exacerbation were significantlyhigher than those in control group (P＜0.05), and those in remission stage werehigher than control group (P＜0.05); the level of IFN-γand FEV₁,FEV₁/FVC(%) in patients with acute asthma exacerbation were significantly lower than those in control group (P＜0.05), and those in remission stage werelower than control group (P＜0.05). The Tim-3mRNA expression in inducedsputum was correlated positively with the amount of EOS and the level ofIL-4 in the asthmatic patients with acute asthma exacerbation (r=0.76,r=0.65,both P＜0.05), and correlated negatively with the level of IFN-γandpulmonary ventilatory capacity (r=-0.89, r=-0.78, both P＜0.01).Conclusions: The expression of Tim-3mRNA in airway inflammationcells in asthma patients was significantly higher than that in normal group, andthat with acute asthma exacerbation was higher than in remission stage,suggested that Tim-3 might take part in the occurrence and development ofasthma. The level of Tim-3mRNA was correlated positively with the level ofIL-4 and EOS in induced sputum and correlated negatively with the level ofIFN-7. So detection the level of Tim-3 in induced sputum not only reflectsasthmatic airway inflammation but also can help us comprehensivelyunderstand it combined with the amounts of EOS in induced sputum, andprovide objective basis for long-term anti-inflammation therapy.