CD4~+CD25~+ Regulatory T Lymphocytes In Tuberculous And Malignant Pleural Effusions

The excretion and absorption of fluid are kept in balance in thoracic cavity. Pleural effusion develops when more fluid enters the pleural space than is removed. Although many different diseases may cause a pleural effusion of exudates, the most common causes in adults are tuberculosis and malignant pleural effusion. The malignant pleural effusion are caused by malignancy pleural involvement and malignant pleural mesotheliomas, the former account for 90% of cases, lung cancer are in common. Tuberculous pleural effusion(TPE) is caused by a severeⅣ-type hypersensitivity reaction in response to the rupture of a subpleural focus of Mycobacterium tuberculosis infection. The inflammatory process results in an increased pleural vascular permeability leading to the accumulation of protei-richnfluid and the recruitment of specific leukocytes into the pleural space. CD4~+CD25~+ regulatory T cells is a distinctpopulation of CD4~+ T cells that constitutively express the interleukin-2 (IL-2) receptor (R)α-chain (CD25), This subgroup cell posses the characteristic of immunosuppressant. It was defined as "professional" regulatory/suppressor T cells. Following T cell receptor (TCR) engagement, CD4~+CD25~+ T cells can suppress the activation and proliferation of other CD4~+ and CD8~+ T cells in an antigen-nonspecific manner. CD4~+CD25~+ T cells mediate the suppression of effectors T cell function both in vitro and in vivo via several mechanisms requiring either cell-cell contact or the production of immunosuppressive cytokines. Several studies have suggested that the escape of tumors from immune surveillance may contribute to malignancy development. CD4~+CD25~+ regulatory T cells can against anti-tumor immunity. The involvement of CD4~+CD25~+ regulatory T cells in human tuberculosis has been documented recently. Based on the theory above, our study will research on the quantity and function of CD4~+CD25~+ regulatory T cells in tuberculosis and malignant pleural effusion, and investigate the probable mechanisms about which CD4~+CD25~+ regulatory T cells infiltrate into thoracic cavity. The purpose is to help to find a new way for lung cancer treatment.PARTⅠ: DETECTION OF CD4~+CD25~+ REGULATORY T CELL OF MALIGNANT PLEURAL EFFUSIONOBJECTIVES: To detect the proportion of CD4~+CD25~+ regulatory T lymphocyte in malignant pleural effusion and to determine whether their function are normal.METHODS: The percentages of CD4~+CD25~+ T lymphocytes in pleural effusion and peripheral blood from patients with lung cancer with malignant pleural effusion (15patients), pleural lavage and peripheral blood from patients with lung cancer without effusion (13 patients), and peripheral blood from healthy control (14 subjects) were determined by flow cytometry. The expressions of forkhead transcription factor Foxp3 and cytotoxic lymphocyte-associated antigen-4 were also examined. CD4~+CD25~+ and CD4~+CD25~- T cells from pleural effusion and peripheral blood were isolated, and were cultured to investigate the effects of CD4~+CD25~+ cells on proliferation response of CD4~+CD25~- T cells in vitro.RESULTS: There were increased numbers of CD4~+CD25~+ T cells in malignant pleural effusion (18.9±1.7%) from patients with lung cancer compared with pleural lavage (10.7±0.6%) from patients with lung cancer without pleural effusion (P< 0.001), and that these cells have constitutive high-level expression of Foxp3 and cytotoxic lymphocyte-associated antigen-4. Furthermore, CD4~+CD25~+ T cells mediate potent inhibition of proliferationresponse of CD4~+CD25~- T cells, and anticytotoxic lymphocyte-associated antigen-4 monoclonal antibody could reduce the inhibitory activity of CD4~+CD25~+ T cells.CONCLUSIONS: The increased CD4~+CD25~+ T cells found in malignant pleural effusion express high levels of Foxp3 transcription factor, suggest these CD4~+CD25~+ T cells are regulatory T lymphocytes, They can potently suppress the proliferation of CD4~+CD25~-T cells, and cytotoxic lymphocyte-associated antigen-4 is involved in the suppressive activity of pleural CD4~+CD25~+ T cells.PARTⅡ: DETECTION OF CD4CD25~+ REGULATORY T CELL OF TUBERCULOUS PLEURAL EFFUSIONOBJECTIVES: To detect the proportion of CD4~+CD25~+ regulatory T lymphocyte in tuberculous pleural effusion and to determine whether their function are normal.METHODS: The percentages of CD4~+CD25~+ T cells in pleural effusion and peripheral blood from patients with tuberculous pleurisy (15patients), and peripheral blood from healthy control (15 subjects) were determined by flow cytometry. The expression of forkhead transcription factor Foxp3 was also examined. CD4~+CD25~+ and CD4~+CD25~- T cells from pleural effusion and blood were isolated, and were cultured to study the effects of CD4~+CD25~+ T cells on proliferation response of CD4~+CD25~- T cells in vitro.RESULTS: There were increased numbers of CD4~+CD25~+ T cells in tuberculous pleural effusion(19.2±1.5 %) compared with peripheral blood from both patients with tuberculous pleurisy(12.1±0.8%) and normal subjects(7.7 ±0.6%)(P<0.001), and that these cells had constitutive high-level expression of Foxp3. It was also noted that CD4~+CD25~+ T cells mediated potent inhibition of proliferation response of CD4~+CD25~- T cells.CONCLUSIONS: The increased CD4~+CD25~+ T cells found in tuberculous pleural effusion express high level of Foxp3 transcription factor, potently suppress the proliferation of CD4~+CD25~- T cells. The study suggest these CD4~+CD25~+ T cells are regulatory T lymphocytes.PARTⅢ: INTERLEUKIN-16 IN TUBERCULOUS AND MALIGNANT PLEURAL EFFUSIONOBJECTIVES: to explore the presence of interleukin-16 (IL-16) in pleural effusions, the correlation between IL-16 levels and cytological parameters, as well as the chemoattractant activity of IL-16 on CD4~+ T-lymphocytes.METHODS: Total nucleated cell and differential counts, and IL-16 concentrations in the pleural effusion from 32 patients with tuberculous pleurisy and 30 patients with lung cancer were determined. Threecolour flow cytometry was performed to determine T-lymphocyte subsets in cell pellets of pleural effusion. The chemoattractant activity of IL-16 for CD4~+ T-lymphocytes was also analyzed.RESULTS: The levels of IL-16 were significantly higher in tuberculous(2118.1±227.0 ng/L) than that in malignant effusions(1068.7±101.3 ng/L)(P<0.001). However, IL-16 levels could not be used for diagnostic purposes due to significant overlap between the two groups. Positive correlations were found between the IL-16 levels and CD4~+ Tcells(r=0.701, P< 0.001), and pleural fluid was chemotactic for CD4~+ T-cells in vitro. Intrapleural administration of IL-16 to patients produced a marked progressive influx of CD4~+ T-cells into the pleural space. CONCLUSIONS: Compared with malignant pleural effusion, IL-16 appeare to be increased in tuberculous pleural effusion. IL-16 levels are positively related to the numbers of CD4~+ T-cells, and IL-16 could directly induce CD4~+ T-cell infiltration into the pleural space.PARTⅣ: THE MECHANISMS ABOUTHOW CD4CD25~+ REGULATORY T CELLTO INFILTRATE INTO THORACIC CAVITYOBJECTIVES: To determine chemotatic factor CCL17 and CCL22 level, and CCR4, CCR8 on CD4~+CD25~+ T cells in tuberculous and malignant pleuraleffusion, explore the probable mechanisms about how CD4~+CD25~+ regulatory T cells to infiltrate into thoracic cavity.METHODS: The CCL17 and CCL22 level in pleural effusion and peripheral blood from patients with tuberculous pleurisy (33patients) and malignant pleural effusion (33 patients) and in blood from 6 controls were detect by ELISA. Double immunofluorescence staining was performed on cell pellets of PE to study the distribution of CCL22 in T lymphocytes, macrophages and malignant cells; CCR4, CCR8 on CD4~+CD25~+T cells were determined by flow cytometry. The chemoattractant activity of CCL22 and CCL17 for CD4~+ CD25~+T lymphocytes was also analyzed.RESULTS: The levels of CCL22 were significantly higher in tuberculous than that in malignant effusions. CCR4 on CD4~+CD25~+ T cells were increased. CD4~+ CD25~+ T cells were attracted dy fluid pleural in vitro, and monoclonal antibody to CCL22 can block this chemotactic activity. T lymphocytes, macrophages and malignant cells was all express CCL22.CONCLUSIONS: Compared with malignant pleural effusion, CCL22 and CCR4 appeare to be increased in tuberculous pleural effusion. And CCL22 could directly induce CD4~+ T-cell infiltration into the pleural space. CCL22 is generated by T lymphocytes, macrophages and malignant cells.