ABSTRACT
BACKGROUND: We have previously described the increased apoptosis rate in smokers alveolar lymphocytes (AL) that was independent from the FASL/ FAS system activation. Consequently, the role of intrinsic apoptosis pathway and other ligand/death receptor pairs as TNFalpha/TNFR1 and TRAIL/DR4 important for apoptosis regulation should be considered in this phenomenon. The purpose of the study was to evaluate the impact of tobacco consumption on expression of selected BCL-2 family members and ligand/receptors pairs in bronchoalveolar lavage (BAL) harvested from patients with pulmonary sarcoidosis (PS), idiopathic pulmonary fibrosis (IPF) and healthy volunteers. The results were analyzed in the context of AL apoptosis rate. METHODS: AL apoptosis from PS (n=36, incl. 22 smokers), IPF (11, incl. 5 smokers) and controls (n=17, incl. 9 smokers) was evaluated by flow cytometry (sub-G1 of cell cycle). AL were stained for BCL-2, BCL-xL, BAK, TNFR1 (CD120A) TNFR2 (CD120B) and DR4. ELISA assay was used to evaluate the BAL supernatant levels of TNFalpha and TRAIL. RESULTS: According to previous observations, AL apoptosis rate was significantly higher in smoker subgroups as compared to nonsmoking counterparts. Decreased AL BCI-2+ relative number was observed in smoking PS (80.5 +/- 6.2 vs 91 +/- 9.8% in nonsmokers) and controls (59 +/- 14.1% vs 75 +/- 16.1%, p<0.05). TNFalpha concentration in BAL supernatant was significantly higher only in healthy smokers (2.32 +/- 0.77 vs 0.42 +/- 0.27 pg/ml, p<0.05), whereas TRAIL levels were remarkably enhanced in IPF smokers (44.8 +/- 12.8 vs 13.5 +/- 5.0 pg/ml, p<0.05) only. However, TUNEL. detected AL apoptosis positively correlated with TNFalpha. in smokers (p<0.05) and negatively with AL CD120B:CD120A expression ratio. Paradoxically, TNFalpha levels were positively correlated with AL BCL-2 expression in nonsmokers (Rs +0.58, p<0.01), but not in smokers. No differences were observed in all subgroups in respect to AL expression of DR4, BCL-xL or BAK. CONCLUSIONS: 1. AL were not sufficiently protected against apoptosis in smokers. 2. The most likely mechanisms involve down-regulation of BCL-2 expression and altered AL susceptibility to TNFalpha, mediated by imbalance between AL membrane expression of TNF receptor type 1 (death receptor) and type 2 (survival mediator). 3. Mechanisms regulating the increased AL apoptosis in smokers seem to be different in each tested group.
Subject(s)
Apoptosis , Idiopathic Pulmonary Fibrosis/metabolism , Lymphocytes/metabolism , Sarcoidosis, Pulmonary/metabolism , Smoking/adverse effects , Tumor Necrosis Factor-alpha/metabolism , bcl-2-Associated X Protein/metabolism , Bronchoalveolar Lavage Fluid/cytology , Down-Regulation , Flow Cytometry , Humans , Idiopathic Pulmonary Fibrosis/etiology , Idiopathic Pulmonary Fibrosis/pathology , Lymphocytes/pathology , Reference Values , Sarcoidosis, Pulmonary/etiology , Sarcoidosis, Pulmonary/pathology , Smoking/metabolism , Smoking/pathologyABSTRACT
UNLABELLED: IFN-gamma a potent antifibrotic activity in interstitial lung diseases (ILD). T cells, both Th1 and Tc1, are considered to be the main local source of IFN-gamma. MATERIAL AND METHODS: BAL fluids of 98 patients with ILD, incl. idiopathic pulmonary fibrosis (IPF/UIP), sarcoidosis, extrinsic allergic alveolitis (EAA), asbestosis and silicosis (n=16, 49, 7, 10, 16 resp.) were tested with ELISA for IFN-gamma levels. Results were compared with BAL cytoimmunology and patients' clinical data. RESULTS: Significantly increased IFN-gamma levels were found in non-treated patients with EAA (7.8 +/- 2.1), IPF (6.1 +/- 1.8), Loefgren's syndrome, LS (11.9 +/- 2.6) and progressive sarcoidosis, PS (6.4 +/- 1.2, p < 0.05 for all), whereas the results in pneumoconioses were comparable to those obtained in controls (2.0 +/- 1.1 pg/ml, median +/- SEM). IFN-gamma results were positively correlated with total number of CD4+ cells (r(s) = +0.38, p < 0.05), CD4+ cells percentage (r(s) = +0.32, p < 0.005) and CD4+/CD8+ ratio (r(s) = +0.38, p = 0.0007), but negatively correlated with CD8+ cell percentage (r(s) = -0.39, p < 0.0005). In IPF patients with CD4/CD8 < or =1 (n=9) IFN-gamma level was lower as compared with the group with CD4/CD8 >1 (n=7), 2.8 +/- 1.3 vs. 7.3 +/- 1.0 pg/ml. In sarcoidosis, IFN-gamma level did not seem to have a prognostic role, since values obtained in PS did not differ remarkably from those in stable sarcoidosis and LS. Moreover, subsequent steroid treatment in 7 patients with progressive sarcoidosis did not change significantly IFN-gamma levels in BAL fluid. CONCLUSIONS: Increased IFN-gamma level was found in non-treated patients with IPF, Loefgren's syndrome and progressive sarcoidosis. CD4+ (Th1), but neither CD8+ (Tc1) nor NK cells seem to be the main local source of IFN-gamma in ILD. Relatively low CD4/CD8 ratio in ILD may indicate the patients with increased risk of lung fibrosis.
