Th17 Cells And Related Factors Expression And Significance In The Pulmonary Of Stable Chronic Obstructive Pulmonary Disease Patients

PARTⅠInterleukin-17 Expression and Significance in Normal Lung Function Smokers and Chronic Obstructive Pulmonary Disease PatientsObjective To study the pulmonary inflammatory mechanism of interleukin 17 (IL-17) in the normal lung function smokers and chronic obstructive pulmonary disease (COPD) patients.Methods The peripheral lung cancer patients which need surgical treatment were divided into normal lung function non-somking group (NS group, 10 cases), normal lung function and smoking group (S group,13 cases), smoking with stable COPD group (COPD group,10 cases). Select the fresh normal lung tissue from the surgical specimens whice is 5cm over from the lung cancer resection, then detected the lung tissue IL-17 levels with Enzyme-linked immunosorbent assay (ELISA), the average alveolar area, the total integration of small airway pathologyacinar, and the pulmonary muscular artery (MA) wall thickness by HE and Victoria blue-van Gieson's stain, the IL-17+cells and CD4+, CD8+lymphocytes in the alveolar walls, small airways and lung muscular artery (MA) by immunohistochemistry. Analyze the relationships between IL-17 level, pathological morphology of lung parenchyma, small airway, pulmonary artery reconstruction and pulmonary function.Results The IL-17 levels in lung tissue of NS group, S group and COPD group were 6.1 (3.7～12.4),9.7 (3.5～69.7) and 22.7 (7.0～114.4) pg/mg respectively. The COPD group was significantly higher than the NS group (P =0.000) and the S group (P=0.036), the S group was significantly higher than the NS group (P=0.042). The average alveolar area were (50708±14125), (106517±13851) and (152344±43783)μm2,the total integration of small airway pathology were(49±10), (101±34) and (163±36), the MA wall thickness were (119±11),(139±25) and (172±28)μm. The S group and the COPD group were significantly higher than the NS group (P<0.05, P<0.01), and the COPD group was significantly higher than the S group (P<0.05, P<0.01).IL-17 was mainly expressed in the lung infiltration of inflammatory cells, IL-17 of the S group and COPD group in the alveolar walls, small airway wall and the MA wall were significantly higher than the NS group, and the COPD group was significantly higher than NS group (P<0.05). IL-17+cells were positively correlated with the average alveolar area in the lung parenchyma (r=0.561, P<0.01), the pulmonary artery wall thickness in the MA(r=0.682, P<0.01), and the pathological score in the small airways (r=0.425, P<0.05). IL-17+cells of the lung parenchyma, small airways and the MA, were positively correlated with CD4+and CD8+ lymphocytes in the lung(P<0.05, P<0.01). The levels of IL-17 in lung homogenate tissue showed a negative correlation with the FEV1 percentage of predicted value (r=-0.471, P=0.006).Conclusions The IL-17 was increased in the lung tissues of normal lung function smokers and COPD patients, and was closely related to CD4+and CD8+lymphocytes in the lung, lung parenchyma destruction, pulmonary inflammation, pulmonary artery reconstruction and airflow limitation. All of these suggested that IL-17 playing an important pro-inflammatory role in COPD.PARTⅡThe Activation, Distribution and Contribution of Th17 cell in Normal Lung Function Smokers and Chronic Obstructive Pulmonary Disease PatientsObjective To observe the normal lung function smokers and COPD patients lung tissue whether they are supporting the existence of activated Th 17 cells, and the activation relationship with IL-17,then explore the lung parenchyma IL-17 whether it derived from Thl7 cells, and analyzed CD4+IL-17+cells expression and distribution in the lung, the correlation with the pulmonary pathology and lung function changes, explored the role that Th17 cell play on the COPD Pulmonary inflammation.Methods Test cases selected and grouped as the first chapter. Measured lung homogenates RORγt (retinoic acid receptor-related orphanγt) mRNA expression levels by Quantitative PCR, detected lung tissue ROR gamma t protein expression levels by Western blot, detected Th17 cells expression in the alveolar walls, small airway wall, pulmonary arterioles by double immunofluorescence. The relevance of ROR gammat RNA expression in the lung tissues was analyzed between IL-17 protein levels and FEV1 percentage of predicted value. The correlationship of Th17 cells in lung tissue was Analyzed with emphysema, small airway pathology total score, pulmonary artery wall thickness, IL-17+cells and CD4+T lymphocytes.Results (1)The results of Quantitative PCR showed that, the mRNA expression level of lung tissue ROR gammat in the NS group was (2.685±0.886), the S group was (8.564±1.419), the COPD group was (10.158±1.574), compared with each group, the three groups had significant difference (P<0.01 or P<0.05), and the COPD was the most significant. (2) The result of Western blot showed that the ROR gammat protein expression levels in the lung tissue of the NS group was (0.369±0.094), the S group was (0.614±0.241), the COPD group was (90.886±0.184), compared with each other the three groups had significant differences (P<0.01 or P<0.05), and the COPD group was the most significant. (3)The result of Immunofluorescence double labeling showed that the expression of CD4+IL-17+cells in the alveolar wall, the S group was (25±7) cells/cm,the COPD group was (27±4) cells/cm compared with NS group (14±5) cells/cm, the COPD group had a significantly increased (all P<0.01), but the COPD group and the S group showed no significant difference (P> 0.05).In small airway wall the expression of COPD group (220 [81,422])cells/ mm2 was stronger than the expression of S group (121 [22,170]) cells/mm2 and NS group (54 [16,258]) cells/mm2 (P<0.05), the tendency which the expression of CD4+IL-17+cells was more in S group than that in NS group exist but without significant difference (P>0.05). (4) The ROR gammat RNA in the Lung tissues had a significant negative correlation with FEV1 percentage of the predicted value (r=-0.643, p= 0.000), the ROR gammat protein content and the FEV1 percentage of the predicted value was significant negatively correlated (r =-0.539, p= 0.002),but the lung tissue IL-17 protein and the ROR gammat RNA content was significant positively correlated (r=0.678,p=0.000). (5) The CD4+IL-17+cells in the alveolar wall was significant positively correlated with the mean alveolar area (r= 0.738, p= 0.000), in the airway wall the Th17 cells and airway total pathology score was significantly correlated (r= 0.476, p= 0.034), but the Th17 cells of MA wall showed no significant correlation with vascular wall thickness (r=0.355, p=0.125).(6) In alveolar wall, the CD4+IL-17+cells and the IL-17+lymphocyte cells show positive correlation (r= 0.584, P<0.01), and the CD4+lymphocyte cells (r= 0.646, P<0.01) In small airway wall, the CD4+IL-17+cells and the IL-17+lymphocyte cells show positive correlation (r= 0.510, P<0.05), and the CD4+lymphocyte cells (r= 0.518, P<0.05). In the pulmonary artery wall, the CD4+IL-17+cells and the IL-17+lymphocyte cells show positive correlation (r= 0.517,P<0.05).but no significant correlation with the CD4+lymphocyte cells (r= 0.200, p= 0.399)Conclusions The Th17 cell-specific transcription factor ROR gammat mRNA and protein levels in lung tissue of normal smokers has begun to increase,and the COPD patients was more pronounced and closely related with IL-17 protein.Th17 cells and IL-17+cells, CD4+T lymphocytes were closely related to lung tissue destruction, airway and pulmonary inflammation and remodeling, airflow limitation. The results suggest that Th17 in the lung tissue of smokers and COPD patients was in the proliferation and activation state, which secreted the IL-17 participate and promote the lung inflammation. PART IIIThe Pulmonary Inflammation Mechanism of Th17 Cells and Related Factors Involved in Normal Lung Function Smokers and COPD PatientsObjective To investigate the expression and significance of upstream factor IL-23R and CCR6 which was activated by the Thl7 cells, and related factors IL-21 and IL-17 in lung tissue, and explored Thl7 activation and the possible mechanism of inflammatory amplification.Methods Test cases selected and grouped as the first chapter. Detected the expression of alveolar wall IL-17, IL-23R and CCR6 by immunohistochemical, analyzed each other relationship among IL-17, IL-23R and CCR6, and their correlation between Th17 cells. Detected the IL-21 mRNA, 1L-17mRNA content in the lung tissue by quantitative PCR, and analyzed the relationship between the two, as well as the correlation between RORγtmRNA and FEV1. Analyzed the correlation between Thl7 cells and CD8+T cells in alveolar wall, small airway wall and pneumono-arteriola wall.Results (1) IL-23R+cells in alveolar wall of the NS group, S group and COPD groups were (46±7), (60±15) and (65±12) cells/cm respectively, the S group and the COPD group compared with the NS group, there were significant differences (P<0.05 or P<0.01), COPD group tended to increase when compared with S group, but had no significant difference (P> 0.05). In the alveolar wall, the CCR6+cells of the NS group, S group and COPD group were (42±6), (56±8) and (62±8) cells/cm respectively, and there were significant differences when the S group and the COPD group compared with the NS group (P<0.05 or P<0.01), the COPD group were in the trend of increasing than the S group, but had no significant difference (P>0.05). (2) Quantitative PCR results showed that, IL-21mRNA in NS group, S group, COPD group were respectively (4.784±2.282), (46.312±15.463) and (84.804±21.884) in lung homogenates, compared with each other in the three groups,there were significant difference (all P<0.01), and the expression of COPD group increased most obvious.The IL-17mRNA in NS Group, S Group and COPD group were respectively (3.121±1.262), (26.434±7.529), (44.929±15.303), compared with each among the three groups there were significantly different (all P<0.01), and the expression of COPD group increased most obvious. (3)In the alveolar wall, CD4+IL-17+cells had a significant positive correlation with IL-17+cells, IL-23R+cells and CCR6+cells (r=0.584,0.422, 0.670, P<0.01 or P<0.05). IL-17+cells had no correlation with IL-23R+cells (r =0.302, p=0.105). IL-23R+cells and CCR6+cells had a significant positive correlation (r= 0.572, p= 0.001),and IL-17+cells and CCR6+cells had a significant positive correlation too (r=0.508, p=0.004). (4) In the alveolar wall, the correlation coefficient between CD4+IL-17+cells and CD8+T lymphocytes was (r=0.664, P<0.01); in the airway wall, the correlation coefficient between CD4+IL-17+cells and CD8+T lymphocytes was (r= 0.491, P<0.05); in the pulmonary artery wall, the correlation coefficient between CD4+ IL-17+cells and CD8+T lymphocytes was (r= 0.518, P<0.05). (5)In the lung homogenate, IL-21mRNA and RORγt mRNA content had a significant positive correlation (r=0.757,p=0.000), RORγt mRNA and IL-17mRNA content had a significant positive correlation (r=0.802,p=0.000), IL-21mRNA and IL-17mRNA content had a significant positive correlation (r= 0.746, p= 0.000). (6) In the lung tissue, IL-21mRNA, IL-17mRNA and FEV1 showed a significant negative correlation (r=-0.694,-0.725, respectively, all p=0.000).Conclusions In the alveolar wall of normal lung function smokers and COPD patients, the expression of IL17, IL-23R and CCR6 were significantly increased, and showed a significant positively relationship with Th17 cells. In the alveolar wall, airway and pulmonary artery, there was alse a significant positive correlation between Th17 cells and CD8+lymphocytes.The RNA transcription of Th17 cell-related factor IL-21, IL-17 in lung homogenates showed a significant positive correlation between RORγtmRNA, and was closely related with airflow limitation. The results suggest that in the normal lung function smokers and COPD patients, Th17 chemotactically invaded the lung parenchyma to proliferated and activated through CCR6 and IL-23,secreted IL-17 and IL-21 cytokines, regulating neutrophils and CD8+T lymphocytes, cause and enlarge the inflammation.