Role Of T Lymphocyte-associated Costimulatory Molecules In The Pathogenesis Of Severe Pneumonia

BackgroundAt present, little is known about the definite pathogenesis of severe pneumonia. Studies have found that lymphocytes in patients with severe pneumonia had been significantly reduced, which was closely related to the prognosis of patients. Then what are the reasons for the reduction of lymphocytes? Studies have showed that expression of co-stimulatory molecules in the peripheral blood had been abnormal in patients with severe pneumonia, which may lead to T lymphocytes in a state of " anergy". However it's not clear yet whether it was due to the abnormal expression of T lymphocyte-associated co-stimulatory molecules such as CD28, CD152 and CD86, or resulted from unbalanced expression of mutual antagonism between signal molecules CD28 and CD152. Researches have found that thymosin-α1 (Tα1) played an important role in the course of immune responses, and it may regulate immune system through enhancing the T cell functions. In addition, thymosin-α1 was found to be could improve T cell proliferation in patients with septic shock and regulate the body's cellular immunity. It has been clear that thymosin-α1 has ability to regulate body's immune abnormality. If it's through regulating co-stimulatory molecules signals to affect T cell proliferation still needs to be further studied.AimTo study the relationship between T cell-associated co-stimulatory molecules and T cell proliferation ability in severe pneumonia, and explore the pathogenesis of this disease. Moreover, on this basis, we interfere the thymosin-α1 by immune regulation in vitro to study its effects on T lymphocytes-associated co-stimulatory molecules and cell proliferation in severe pneumonia. These will provide theoretical basis for treatment of thymosin-α1 to severe pneumonia, and further clarify the relationship between the severe pneumonia and T lymphocyte-associated co-stimulatory molecules.Materials and methods1 Collection of specimensSpecimens were collected from patients with severe pneumonia in Respiratory Department of Guangzhou First Municipal People's Hospital from August 2007 to June 2008. All patients meet the diagnostic criteria for severe pneumonia that is established by the American Thoracic Society (ATS) in 2007. Healthy control group is selected from healthy physical examinees from clinic of the same period. After patents themselves and their relatives signed the informed consents, peripheral venous blood was collected. In 21 cases of patients with severe pneumonia, 2ml of peripheral venous blood was collected with EDTA-K3 anticoagulation; among these, 11 exceptions to the peripheral blood was pumped more than 13ml, with heparin anticoagulation. In addition, 11 cases of patients were drawn blood for 2ml again, after hospitalization for 10 days (among whom 8 cases were improved and 3 dead after 28-day treatment). In 12 cases of healthy people, 2ml of peripheral venous blood was collected with EDTA-K3 anticoagulation; among these, 10 cases was drawn blood for more than 8 ml with heparin anticoagulation.2 Methods2.1 Detection of T lymphocyte-associated co-stimulatory moleculesWhole blood was drawn to EDTA-K3 anticoagulant vacutainer blood collection tubes. Testing tubes and isotypic control tubes were used for each sample. Four epitope parameters (CD4, CD8, CD28, CD152) for T lymphocyte population and two epitope parameters (CD86, HLA-DR) for associated monocytes population were detected. Studies were performed according to the manufacture's instructions. The flow cytometer was calibrated and controlled according to the standard procedures before detect. Acquisition and analysis were performed using FACScan flow cytometer and CELLQuest software.A biparametric gate in the forward scatter-side scatter dot plot was drawn around two cell population. Fluorescein isothiocyanate (FITC), phycoerythrin (PE), Phycoerythrin and cyanine dye (PC5), Allophycocyanin (APC) and Allophycocyanin and cyanine dye (APCcy7) directly labeled monoclonal antibodies were used for fluorescence measurement. Total 10000 events were counted. All gated cells were expressed in the percentage of the cells analyzed.2.2 T lymphocyte proliferation assays2.2.1 Extraction of PBMCPeripheral blood mononuclear cells (PBMCs) from patients and healthy control were prepared by Ficoll density gradient centrifugation of peripheral blood. Cells were used either fresh or frozen before use.2.2.2 PKH26 staining and PBMC cultruePBMCs were stained by PKH26 according to the manufacture's instruction and then diluted into 1×106/ml. An amount of 0.6 ml of stained cell suspensions were seeded into 24-well culture plate and incubated with PBS (as negative control group), PHA (as positive control group), thymosin-α1 plus endotoxin, or endotoxin alone respectively in a humidified CO2 incubator at 37℃for five days.2.2.3 Dection of T lymphocyte proliferationAfter incubation, cells were collected and washed by centrifugation, then incubated with CD3-PC5 at the concentration of 1μg/ 106 cells at room temperature in the dark. After washing, cells were fixed with 2g/l of paraformaldehyde. Data were collected and analyzed by CELLQuest software, and T lymphocyte proliferation factor (PF) and proliferation index (PI) analysis was performed with ModFitTM software.2.2.4 Detection of T lymphocyte cultured in vitro and associated moleculesPBMCs isolated from peripheral blood were cultured in complete RPMI-1640 medium supplemented with 10% fetal bovine serum, 100U/ml penicillin and 100μg/ml streptomycin at a density of 1×106/ml. Total 0.6 ml of cell suspensions were seeded into 24-well culture plate and incubated with PBS (as negative control group), PHA (as positive control group), thymosin-α1 plus endotoxin, or endotoxin alone respectively at 37℃in 5% CO2 for five days. After incubation, cells were collected, washed by centrifugation and then incubated with FITC-CD28, PE-CD152 and PC5-CD3 at the concentration of 1μg/ 106 cells at room temperature avoid of light. Subsequently, cells were washed and then fixed with 2g/l of paraformaldehyde. The expressions of co-stimulatory molecules CD28 and CD152 were quantificationally monitored using FACScan flow cytometry and analyzed with CELLQuest software.2.3 Statistical analysisStatistical analyses were carried out with SPSS 11.5 software. Quantitative data were presented as mean values±SD (±SD) and compared using the student's t-test. The relative difference between random groups was compared by analysis of variance, and multiple comparisons were performed using LSD method. In all experiments, p < 0.05 was considered to indicate a statistically significant difference.Results1 T lymphocyte, its subsets and associated co-stimulatory molecules in severe pneumoniaIn 21 cases with severe pneumonia, T lymphocytes expressed with CD3, CD4, CD86, HLA-DR, CD28/CD152 and CD4/CD8 were reduced compared to healthy control group (p < 0.05). Expression of CD8, CD28 and CD152 was increased compared to healthy control group (p < 0.05).2 T lymphocyte, its subsets and associated co-stimulatory molecules of survived patients after 28-day treatmentOf eight survived patients, the APACHEⅡscore was decreased significantly after treatment for 10 days (p = 0.000). The expression of CD28, CD152, CD8 and HLA-DR was increased significantly (p < 0.05). CD3 T cell was increased obviously (p = 0.030), while CD8CD3 T cell and CD4CD3 T cell showed no significant change (p > 0.05).3 PMBC labeled by PHK26 of patient with severe pneumoniaThe fluorescence histogram representing PHK26 labeled PMBC revealed that the fluorescence density showed a significant right shift, single peak up to 104, and covering 96.48% of total cells counted. Observed under fluorescent microscope, the cells gave out red fluorescence.4 Proliferation ability of T lymphocyte in peripheral blood of patient to antigen stimulationPeripheral blood T cells have lower PI (1.55±0.42) and PF (0.02±0.01) to endotoxin ( LPS )stimulation compared to healthy control whose PI is 2.15±0.29 and PF is 0.04±0.02 in 10 cases with severe pneumonia. The difference had statistical significance (p< 0.05).5 Effect of Thymosin-α1 on T cell proliferation in peripheral blood of severe pneumonia5.1 Effect of Thymosin-α1 on T cell proliferation in peripheral blood of severe pneumonia observed under light microscopyThe peripheral blood T cell of severe pneumonia cultured in vitro for five days with different intervention factors were observed under light microscopy (10×magnification). The cells cultured with PHA or thymosin-α1 plus endotoxin appeared significant lymphocyte blastogenesis, while cells of other two groups didn't show such change.5.2 Effect of Thymosin-α1 on T cell proliferation in peripheral blood of severe pneumoniaThe PF of T lymphocyte treated with PHA, thymosin-α1 plus endotoxin or endotoxin alone groups from ten cases with severe pneumonia are 0.03±0.03, 0.03±0.02 and 0.02±0.01 respectively, and PI are 1.68±0.49, 1.84±0.53 and 1.55±0.42 respectively. The PF and PI values of T cells treated with thymosin-α1 plus endotoxin are higher than that of endotoxin alone treatment group (p < 0.05). While the difference of PF and PI between thymosin-α1 plus endotoxin treatment group and PHA treatment group has no significance (p > 0.05). 6 Effect of thymosin-α1 on T cell-associated co-stimulatory molecules in peripheral blood of severe pneumonia6.1 Effect of thymosin-α1 on T cell-associated co-stimulatory molecules CD28 in peripheral blood of severe pneumoniaCalculated from the FACS analysis, the percentages of peripheral blood T cells expressed co-stimulatory molecule CD28 of group PBS, PHA, thymosin-α1 plus endotoxin or endotoxin alone are (67.40±6.45)%, (72.21±7.05)%, (73.70±8.66)% and (66.94±11.85)% respectively. The expression of CD28 of thymosin-α1 plus endotoxin group was higher than that of endotoxin alone group (p < 0.05), and has no significant difference with PHA group (p = 0.479).6.2 Effect of thymosin-α1 on T cell-associated co-stimulatory molecules CD152 in peripheral blood of severe pneumoniaThe percentages of peripheral blood T cells expressed co-stimulatory molecule CD152 of group PBS, PHA, thymosin-α1 plus endotoxin or endotoxin alone are (3.01±1.08)%, (2.69±1.23)%, (2.41±0.77)% and (2.86±1.03)% respectively. The expression of CD152 of thymosin-α1 plus endotoxin group was lower than that of PBS group (p < 0.05), and has no significant difference with PHA group (p = 0.306).6.3 Effect of thymosin-α1 on expression ratio of co-stimulatory molecules CD28/CD152 on peripheral T cellsThe expression ratio of co-stimulatory molecules CD28/CD152 on peripheral T cells of group PBS, PHA, thymosin-α1 plus endotoxin or endotoxin alone are 25.26±9.57, 31.38±11.67, 33.30±9.96 and 21.13±5.39 respectively. The ratio of CD28/CD152 of thymosin-α1 plus endotoxin group is bigger than that of PBS group (p < 0.05), and has no significant difference compared to PHA group (p = 0.490).Conclusion1. T lymphocytes exist "anergy" in the peripheral blood of patients with severe pneumonia, which cause reduction of T lymphocyte and CD4 subset in patient and suppress the body's immune function.2. The abnormal expression of T lymphocyte-associated co-stimulatory molecules such as CD28, CD152 and CD86 involved in the pathogenesis of severe pneumonia.3. In vitro thymosin-α1 can regulate peripheral T lymphocyte co-stimulatory molecules CD28, CD152 expression in the peripheral blood of patients with severe pneumonia , it also can improve antigen stimulation of T lymphocyte proliferation.