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
INTRODUCTION: In the last years we have used flow cytometry as an auxiliary diagnostic tool in alveolar lymphocyte (i.e. originating from BAL) phenotyping in more than 500 persons suspected for lower airways pathology. MATERIAL AND METHODS: In the study we compared the results of 1) BAL lymphocyte typing by flow cytometry, 2) cytological examination, respectively, in nonsmoking/smoking (NS/S) patients with lung sarcoidosis, n = 56/31, extrinsic allergic alveolitis (EAA), n = 9/5, silicosis, n = 15/18, idiopathic pulmonary fibrosis (IPF), n = 20/7, and pulmonary tuberculosis (TBC), n = 7/6. The results were related to the volume of BAL fluid recovery (higher value reflects the dominance of lower airways content versus bronchial content). RESULTS: In smoking patients, in comparison with respective NS, significantly higher total BAL cell numer (except TBC), higher macrophage percentage, lower lymphocyte percentage and lower CD4/CD8 ratio (except EAA) was found. CD4/CD8 results: 8.26 +/- 0.52 (NS) vs 4.29 +/- 0.65 (S) in sarcoidosis (p < 0.001), 1.18 +/- 0.44 (NS) vs 0.99 +/- 0.43 (S) in IPF (p < 0.05), 1.79 +/- 0.22 (NS) vs 0.73 +/- 0.11 (S) in silicosis (p < 0.001) and 1.64 +/- 0.57 vs 0.88 +/- 0.1 in TBC (p < 0.05). Additionally, cigarette smoking modified BAL pattern: 1. in sarcoidosis and silicosis lower CD4+ cell and higher CD8+ cell percentage; 2. in IPF increase in neutrophil percentage; 3. in TBC higher neutrophil and eosinophil percentage. Both in NS and S, BAL fluid recovery rate is significantly positively correlated with CD4/CD8 ratio and total BAL CD3+ cell number and negatively with BAL CD8+ cell percentage. CONCLUSIONS: Interpreting of BAL material cytoimmunology pattern should take into account data on cigarette smoking and BAL fluid recovery rate. The results obtained in the study may reflect more severe disease course in IPF and TBC.
