Alveolar cell senescence in patients with pulmonary emphysema

RATIONALE AND OBJECTIVES: The prevalence of chronic obstructive pulmonary disease (COPD) is age-dependent, suggesting an intimate relationship between the pathogenesis of COPD and aging. In this study we investigated whether the senescence of alveolar epithelial and endothelial cells is accelerated in emphysematous lungs. METHODS: Samples of lung tissue were obtained from patients with emphysema, asymptomatic smokers, and asymptomatic nonsmokers. Paraffin-embedded lung tissue sections were evaluated for cellular senescence by quantitative fluorescence in situ hybridization to assess telomere shortening, and by immunohistochemistry to assess the expression of senescence-associated cyclin-dependent kinase inhibitors. Tissue sections were also immunostained for proliferating cell nuclear antigen (PCNA), surfactant protein A, and CD31. MAIN RESULTS: The patients with emphysema had significantly higher percentages of type II cells positive for p16INK4a and p21CIP1/WAF1/Sdi1 than the asymptomatic smokers and nonsmokers. They had also significantly higher percentages of endothelial cells positive for p16INK4a than the asymptomatic smokers and nonsmokers, and higher percentages of endothelial cells positive for p21CIP1/WAF1/Sdi1 than the asymptomatic nonsmokers. Telomere length in alveolar type II cells and endothelial cells was significantly shorter in the patients with emphysema than in the asymptomatic nonsmokers. The level of p16INK4a expression was negatively correlated with the level of PCNA expression. The level of alveolar cell senescence was positively correlated with airflow limitation. CONCLUSIONS: These results suggest that the senescence of alveolar epithelial and endothelial cells is accelerated in patients with emphysema. Cellular senescence may explain the abnormal cell turnover that promotes the loss of alveolar cells in emphysematous lungs.     

Cigarette smoking causes accumulation of polymorphonuclear leukocytes in alveolar septum

The polymorphonuclear leukocyte (PMN) may play an important role in the pathogenesis of emphysema. Cigarette smoking is associated with the accumulation of PMN in the lung as determined by bronchoalveolar lavage. We enumerated alveolar wall PMN on histologic sections from lungs of humans and hamsters with and without cigarette smoke exposure. In human nonsmokers, there were 0.9 +/- 0.2 PMN/mm alveolar wall. In cigarette smokers without emphysema, there were 2.1 +/- 0.3 PMN/mm alveolar wall (p less than 0.01), and in cigarette smokers with emphysema, there were 2.4 +/- 0.7 PMN/alveolar wall (p less than 0.05). There were 1.7 +/- 0.3 PMN/mm alveolar wall in the lungs of hamsters unexposed to cigarette smoke compared with 3.1 +/- 0.3 PMN/mm alveolar wall in smoke-exposed hamsters (p less than 0.005). Although cigarette smoking causes PMN to accumulate within alveolar septa, the accumulation does not seem to be closely related to the development of emphysema. This suggests that additional or other factors are important in the pathogenesis of emphysema.     

Differences in the distribution of CD4+ and CD8+ T cells in emphysematous lungs

BACKGROUND: Recent studies have shown that T cells are the predominant cells infiltrating the alveolar wall of patients with emphysema. Since CD4+ and CD8+ cells have dissimilar function, they may have different distributions in emphysematous lungs. OBJECTIVES: The aim of this study was to investigate whether CD4+ and CD8+ are differently distributed in the lungs of patients with emphysema. METHODS: We evaluated CD4+ and CD8+ cell infiltration in lung tissue sections obtained from smokers with pulmonary emphysema who had undergone lung volume reduction surgery (n = 10). Paraffin-embedded lung tissue sections were immunostained using antibodies against CD4+ or CD8+, and distributions of CD4+ and CD8+ cells were evaluated using a morphometric analysis. We determined the degree of emphysema using the area fraction of the alveolar wall (A(A,aw)) and examined its relation to the number of cells per unit area of the alveolar wall (NA). RESULTS: The majority of CD4+ cells were located in the lung parenchyma of severe emphysematous lesions characterized by the lowest alveolar wall fraction (A(A,aw)). In contrast, more CD8+ cells were located in mild emphysematous lesions than in severe lesions. CONCLUSIONS: These results suggest that CD4+ and CD8+ cells are differently distributed according to the severity of emphysema. CD4+ T cells are located preferentially in severe emphysematous lesions whereas CD8+ T cells occur preferentially in mild emphysematous lesions.     

Lymphocyte population and apoptosis in the lungs of smokers and their relation to emphysema.

Previously, it had been shown that T-lymphocytes are the predominant inflammatory cells found in the alveolar wall of smokers and their numbers correlated with the extent of emphysema. However, the phenotype of these cells was not defined. The aim of this study was to describe the different T-cell phenotypes and investigate the possible presence of apoptosis in the lung parenchyma of smokers. Samples from lungs were obtained at surgery from 15 patients who smoked and six who had never smoked. Samples were frozen and prepared for histological and immunocytochemical examination. Slides were stained for CD3+, CD4+, CD8+, gammadelta T-cells, CD56 natural killers ((NK) cells), and elastase (neutrophils). Anti-CD95 monoclonal antibodies and in situ end-labelling techniques were used to detect Fas expression and apoptosis. Positive staining cells were expressed as cells-mm alveolar wall-, percentage of total cells, and Fas/APO and apoptosis index. Emphysema was identified macroscopically, microscopically and reported as present or absent. All subjects had pulmonary function tests before surgery. Neutrophils were the predominant cell in the lung parenchyma of nonsmokers and smokers without emphysema. In smokers with emphysema, the CD3+ and CD8+ were the predominant cells (p<0.05) in the alveolar wall. gammadelta cells were increased in all smokers and no increased numbers of NK cells was found. The T-cell numbers x mm alveolar wall(-1) showed a bilinear relationship with the amount smoked increasing at an inflection point of 30 packs yr(-1) (R2= 0.345; p < 0.01). Apoptosis in smokers showed a bilinear relationship with the amount smoked increasing sharply in smokers with emphysema (R2=0.3613; p < 0.009). It is concluded that the pathogenesis of emphysema might be mediated by T-lymphocytes, mainly CD8+ cytolytic T-cells, and that apoptosis might be one of the mechanisms of lung destruction leading to the development of emphysema. If this is the case, it could be speculated that T-cell inflammation is a response to antigenic stimuli originating in the lung and induced by cigarette smoking.     

Cigarette smoking and lung destruction. Accumulation of neutrophils in the lungs of cigarette smokers

It has been hypothesized that lung destruction in persons with emphysema associated with cigarette smoking is mediated by elastase released by neutrophils that have migrated to the alveolar structures in response to cigarette smoke. To directly evaluate this hypothesis, cell suspensions, isolated from bronchoalveolar lavage fluid and from open lung biopsies of nonsmokers and cigarette smokers with normal lung parenchyma and from open lung biopsies of nonsmokers and cigarette smokers who have sarcoidosis were evaluated for the presence of neutrophils. A significantly increased number of neutrophils was present in the cell suspensions isolated from bronchoalveolar lavage fluid and from open lung biopsies of both normal and sarcoid cigarette smokers compared with that in the nonsmokers (p less than 0.01, each comparison). Evaluation of the alveolar macrophages present in lavage fluid suggested a mechanism by which neutrophils may be attracted to the lungs of cigarette smokers: alveolar macrophages of cigarette smokers release a chemotactic factor for neutrophils, whereas alveolar macrophages of nonsmokers do not. In addition, alveolar macrophages of nonsmokers, after exposure to cigarette smoke, in vitro, are stimulated to release this chemotactic factor. These studies demonstrate that an increased number of neutrophils are present in the lungs of cigarette smokers compared with that in nonsmokers and suggest that cigarette smoke may attract neutrophils to the lung by stimulating alveolar macrophages to release a potent chemotactic factor for neutrophils.     

The destructive index and early lung destruction in smokers

The destructive index (DI), a measure of alveolar septal damage and emphysema, has been proposed as a sensitive index of lung destruction that closely reflects functional abnormalities, especially loss of elastic recoil. To better understand the progression of lung destruction in smokers, we studied the contribution of its principal components: breaks in the alveolar septa (DIb) and the presence of emphysematous spaces (DIe), and compared them to the mean linear intercept (Lm) and DI as originally described. To do this we employed lungs obtained at autopsy from non-smokers and smokers. Lungs were selected by emphysema score (ES) so that all cases were emphysema free (nonsmokers and seven smokers) or had minimal emphysema (nine smokers; ES = 5). Of these indices, only DIb was significantly increased in the lungs of smokers: 17.8 +/- 1.2 versus 12.4 +/- 1.6, p less than 0.05. We also investigated the regional distribution of destruction by comparing results in upper and lower lobes. DIe, but not DIb, was significantly increased in upper lobes of smokers. These data support the notion that increases in DI in the lungs of smokers that occur before increases in Lm or ES reflect the presence of alveolar septal breaks and highlight the importance of alveolar septal destruction as a precursor to the development of airspace enlargement in the lungs of cigarette smokers.     

Airway inflammation and peribronchiolar attachments in the lungs of nonsmokers,current and ex-smokers

to determine the effect of smoking cessation on the number and type of inflammatory cells in the walls of the small airways, we examined the lungs of 13 lifetime nonsmokers, 25 patients who had stopped smoking for at least 6 months, and 49 current smokers. We found that, compared to nonsmokers, both ex-smokers and current smokers had significantly increased numbers of total inflammatory cells and polymorphonuclear leukocytes in the walls of the membranous, but not the respiratory bronchioles. These differences were found even when there was no emphysema present in the gross lung specimen, and current and ex-smokers were matched with the nonsmokers for age. The current and ex-smokers had similar numbers and types of inflammatory cells in the airway wall, and in both current and ex-smokers there was no difference in inflammatory cell number or type when the groups were subdivided based on emphysema score less than or greater than 5. Analysis of peribronchiolar alveolar attachments showed an increase in percentage of alveoli destroyed associated with an increased interalveolar distance in both the current and ex-smokers, which did not change with the presence of emphysema. Pulmonary function was similar in the current and ex-smokers, and the group with emphysema showed greater functional abnormalities compared to the group with little or no emphysema. We conclude that the cigarette smoking habit induces a stereotypical inflammatory response in the small airways. This inflammatory response does not abate after smoking cessation, and in this cross-sectional study, appears to be independent of the presence or absence of emphysema, but related to destruction of the peribronchiolar alveolar attachments.     

Airway inflammation and peribronchiolar attachments in the lungs of nonsmokers, current and ex-smokers

To determine the effect of smoking cessation on the number and type of inflammatory cells in the walls of the small airways, we examined the lungs of 13 lifetime nonsmokers, 25 patients who had stopped smoking for at least 6 months, and 49 current smokers. We found that, compared to nonsmokers, both ex-smokers and current smokers had significantly increased numbers of total inflammatory cells and polymorphonuclear leukocytes in the walls of the membranous, but not the respiratory bronchioles. These differences were found even when there was no emphysema present in the gross lung specimen, and current and ex-smokers were matched with the nonsmokers for age. The current and ex-smokers had similar numbers and types of inflammatory cells in the airway wall, and in both current and ex-smokers there was no difference in inflammatory cell number or type when the groups were subdivided based on emphysema score less than or greater than 5. Analysis of peribronchiolar alveolar attachments showed an increase in percentage of alveoli destroyed associated with an increased interalveolar distance in both the current and ex-smokers, which did not change with the presence of emphysema. Pulmonary function was similar in the current and ex-smokers, and the group with emphysema showed greater functional abnormalities compared to the group with little or no emphysema. We conclude that the cigarette smoking habit induces a stereotypical inflammatory response in the small airways. This inflammatory response does not abate after smoking cessation, and in this cross-sectional study, appears to be independent of the presence or absence of emphysema, but related to destruction of the peribronchiolar alveolar attachments.     

Up-regulated membrane and nuclear leukotriene B4 receptors in COPD

STUDY OBJECTIVES: We investigated the role of two leukotriene B4 (LTB4) receptors, BLT1 and peroxisome proliferator-activated receptor (PPAR)-alpha, in conferring the susceptibility to develop COPD in smokers. Proinflammatory LTB4 activities are mediated by BLT1, while the inactivation of LTB4 is promoted by PPARalpha. PATIENTS AND METHODS: BLT1 and PPARalpha proteins were quantified by immunohistochemistry in specimens obtained during lung surgery from 19 smokers with or without COPD and from 7 nonsmoking subjects. RESULTS: We have shown that the percentages of PPARalpha-positive alveolar macrophages and PPARalpha-positive cells in the alveolar wall were increased in COPD patients compared with control subjects. Moreover, the patients with COPD exhibited a significant increase of BLT1-positive alveolar macrophages compared with nonsmokers and an increased number of BLT1-positive cells in the alveolar walls compared with non-COPD smokers. In contrast, BLT1 and PPARalpha immunoreactivity did not differ significantly between nonsmokers and non-COPD smokers. Most of BLT1-positive cells in the alveolar walls were neutrophils and CD8 cells. While the number of neutrophils infiltrating the lung parenchyma was similar among the three groups, the number of CD8 T cells was increased in COPD patients, but there was no evidence that BLT1 was up-regulated specifically on these cells in COPD patients. CONCLUSION: The results demonstrated that BLT1 and PPARalpha are detectable in alveolar macrophages and CD8 T cells in human lung tissue, and suggest that the dual LTB4 receptor system is up-regulated in the peripheral lungs of smokers who are susceptible to the development of COPD. This system might represent a novel target for therapeutic intervention in COPD patients.     

Cellularity of the alveolar walls in smokers and its relation to alveolar destruction. Functional implications

Inflammatory cells are believed to play an important role in the pathogenesis of emphysema; however, a relationship between presence of cells in the lung parenchyma and its destruction has never been shown. The aim of this study was to quantitate alveolar septal cellularity in smokers' lungs and to investigate its relationship with parenchymal destruction and lung function. The lungs of 23 smokers (SS) undergoing thoracotomy for localized pulmonary lesions were compared with those of eight nonsmokers (NS) and five smokers (AS) who died suddenly of nonrespiratory causes. Pulmonary function tests were performed within 1 wk of surgery in SS. For each subject, we quantitated alveolar wall cells (CELLS), an index of alveolar wall destruction (DI), and the mean linear intercept (Lm). As no significant differences were found between S and AS with regard to these indices, we combined them (Group S) for comparison with NS. Although Lm was not significantly different between S and NS, (0.331 +/- 0.072 versus 0.288 +/- 0.038), CELLS and DI were higher in S than in NS (48 +/- 8 versus 25 +/- 2 cells/mm, p less than 0.001; 47 +/- 20 versus 17 +/- 5, p less than 0.001, respectively). Further, CELLS and DI were significantly correlated (r = 0.799, p less than 0.001). The number of polymorphonuclear cells (PMN) in S can exceed that in NS by as much as 5-fold; however, PMN were inversely correlated with parenchymal destruction (DI) (r = 0.598, p less than 0.01). Thus, smokers' lungs have alveolar septal hypercellularity, possibly inflammatory, and closely related to destruction involving cells other than the PMN.     

Correlation of lung surface area to apoptosis and proliferation in human emphysema.

Pulmonary emphysema is associated with alterations in matrix proteins and protease activity. These alterations may be linked to programmed cell death by apoptosis, potentially influencing lung architecture and lung function. To evaluate apoptosis in emphysema, lung tissue was analysed from 10 emphysema patients and six individuals without emphysema (normal). Morphological analysis revealed alveolar cells in emphysematous lungs with convoluted nuclei characteristic of apoptosis. DNA fragmentation was detected using terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL) and gel electrophoresis. TUNEL revealed higher apoptosis in emphysematous than normal lungs. Markers of apoptosis, including active caspase-3, proteolytic fragment of poly (ADP-ribose) polymerase, Bax and Bad, were detected in emphysematous lungs. Linear regression showed that apoptosis was inversely correlated with surface area. Emphysematous lungs demonstrated lower surface areas and increased cell proliferation. There was no correlation between apoptosis and proliferation, suggesting that, although both events increase during emphysema, they are not in equilibrium, potentially contributing to reduced lung surface area. In summary, cell-based mechanisms associated with emphysematous parenchymal damage include increased apoptosis and cell proliferation. Apoptosis correlated with airspace enlargement, supporting epidemiological evidence of the progressive nature of emphysema. These data extend the understanding of cell dynamics and structural changes within the lung during emphysema pathogenesis.     

Expression of Matrix Metalloproteinase-1 in Alveolar Macrophages,Type II Pneumocytes,and Airways in Smokers: Relationship to Lung Function and Emphysema

Background

An imbalance between proteolytic enzymes and their inhibitors is thought to be involved in the pathogenesis of chronic obstructive pulmonary disease. Matrix metalloproteinase-1, also known as interstitial collagenase, has been implicated as a potentially important proteinase in the genesis of chronic obstructive pulmonary disease and, more specifically, emphysema.

Methods

We performed quantitative immunohistochemical assessment of matrix metalloproteinase-1 expression in the resected lung of 20 smokers/ex-smokers who had varying severity of airflow obstruction and emphysema and compared this with the lungs of 5 nonsmokers. Emphysema was measured using a morphometric measure of the lungs’ surface area/volume ratio and with qualitative and quantitative computed tomography (CT) measures of emphysema.

Results

There were significantly more matrix metalloproteinase-1-expressing alveolar macrophages and type II pneumocytes as well as a greater percentage of small airways that stained positively for matrix metalloproteinase-1 in the lungs of smokers than in those of nonsmokers (p < 0.0001, p < 0.0001, and p = 0.0003, respectively). The extent of staining of type II pneumocytes and airways for matrix metalloproteinase-1 was significantly related to the extent of smoking (p = 0.012 and p = 0.013, respectively). In addition, the extent of matrix metalloproteinase-1 staining of alveolar macrophages was related to the lung surface area/volume ratio and to qualitative estimates of emphysema on CT.

Conclusion

These findings suggest that cigarette smoking increases expression of matrix metalloproteinase-1 in alveolar macrophages as well as in alveolar and small airway epithelial cells. Smokers who develop emphysema have increased alveolar macrophage expression of matrix metalloproteinase-1.     

慢性阻塞性肺疾病患者肺组织中基质金属蛋白酶抑制剂-1及基质金属蛋白酶-9与细胞黏附因子表达的关系

目的 研究COPD患者肺组织中基质金属蛋白酶抑制剂-1(TIMP-1)、基质金属蛋白酶-9(MMP-9)、细胞黏附因子-1(ICAM-1)蛋白和mRNA的分布和表达,探讨其与气流阻塞的关系及吸烟对其影响.方法 取39例因肺癌行肺叶切除的癌旁肺组织标本,其中不吸烟不伴COPD组(A组)9例、吸烟不伴COPD组(B组)11例、吸烟伴COPD组(C组)19例.用免疫组化和逆转录聚合酶链反应方法检测TIMP-1、MMP-9、ICAM-1的蛋白和mRNA表达,并进行相关性分析.结果 MMP-9表达于肺泡上皮细胞、支气管上皮细胞、血管平滑肌细胞、肺泡巨噬细胞、间质细胞,C组MMP-9免疫组化阳性细胞数(54.0±15.0)明显高于A组和B组(1.2±0.7和1.4±0.8);TIMP-1蛋白表达的主要部位为肺泡巨噬细胞、肺泡上皮细胞、毛细血管内皮细胞、血管平滑肌细胞,C组弱表达,A组和B组无表达;ICAM-1主要表达于肺泡上皮细胞,C组ICAM-1免疫组化阳性细胞数(52.1±13.4)明显高于A组和B组(2.1±1.1和4.5±2.4).C组MMP-9、TIMP-1、ICAM-1的mRNA平均吸光度值(0.71±0.16、0.47±0.10、0.62±0.15)明显高于A组(0.17±0.05、0.20±0.06、0.37±0.11)和B组(0.20±0.08、0.26±0.08、0.44±0.12).C组肺组织TIMP-1、MMP-9与ICAM-1的mRNA表达水平呈直线正相关,MMP-9与ICAM-1蛋白表达水平呈显著正相关.MMP-9和ICAM-1的mRNA及蛋白表达水平、TIMP-1的mRNA表达水平与FEV1占预计值%、FEV1/FVC占预计值%均呈显著负相关.结论 TIMP-1、MMP-9和ICAM-1在促进炎性细胞迁移进入细胞外基底膜及气道上皮细胞,导致肺组织破坏和重塑,引起及加重COPD患者的气流阻塞中起着重要作用.     

DNA damage as a molecular link in the pathogenesis of COPD in smokers

In this study, we investigated whether DNA double-strand breaks (DSBs) contribute to the pathogenesis of chronic obstructive pulmonary disease (COPD). We immunofluorescence-stained lung tissue samples obtained from COPD patients, asymptomatic smokers and nonsmokers for markers of DSBs. The numbers of DSB foci (phosphorylated histone 2AX (γH2AX), phosphorylated ATM (ataxia telangiectasia mutated) substrate and phosphorylated p53-binding protein-1 foci) per cell in alveolar type I and II cells and endothelial cells were higher in the COPD patients than in the asymptomatic smokers and nonsmokers. The lung tissue in which type II cells contained higher numbers of γH2AX foci per cell had higher percentages of type II cells that expressed p16(INK4a) (p16), phosphorylated nuclear factor (NF)-κB and interleukin (IL)-6, and of alveolar wall cells that expressed active caspase-3. The type II cells that contained higher numbers of γH2AX foci per cell had higher rates of expression of p16, phosphorylated NF-κB, and IL-6. Half of the alveolar wall cells that expressed active-caspase-3 contained γH2AX foci. Type II cells that stained positive for 8-hydroxy-2-deoxyguanosine contained a higher number of γH2AX foci per cell than the type II cells that stained negative. In conclusion, DSBs, at least in part caused by oxidative stress, appear to contribute to the pathogenesis of COPD by inducing apoptosis, cell senescence and pro-inflammatory responses.     

Destructive index: a measurement of lung parenchymal destruction in smokers

Destruction of alveolar walls is considered by most observers to be the most important part in the definition of emphysema, yet it has never been precisely defined and quantitated. We therefore attempted to devise a reliable microscopic technique to quantitate alveolar destruction that would be both sensitive to disease and easy to perform. Using a point-count system, we obtained an index of parenchymal destruction that represents the percentage of destroyed space as a fraction of the total alveolar and duct space. We have called this measurement the destructive index (DI). In the lungs of 8 nonsmokers and 23 smokers, we quantitated the DI and compared it with the mean linear intercept (Lm) and with pulmonary function in smokers. Although Lm was not significantly different in the 2 groups, significant differences between the DI of smokers and nonsmokers (p less than 0.005) were found. In addition, the DI correlated with FEV1(-0.43, p less than 0.05), MMEF (r = -0.44, p less than 0.05), and recoil pressure at 90% TLC (r = -0.61, p less than 0.05) in smokers. These findings suggest that the destructive component of emphysema can be easily quantitated microscopically, occurs in smokers before dimensional changes are evident (i.e., increased Lm), and influences lung function. Therefore, the quantitation of this destruction (DI) could add greatly to the microscopic definition of emphysema, complementing the information given by the dimensional component of emphysema (Lm).     

Bronchial lavage proteins as correlates of histopathologic airway changes in healthy smokers and patients with pulmonary carcinoma

Cigarette smoking is known to be an important etiologic factor in several lung diseases; however, the number of smokers who develop these diseases represents a small segment of the smoking population. It is possible that evidence of inhalation-induced injury to bronchial epithelial cells of smokers will be reflected in the proteinaceous products of these cells, thereby identifying a high-risk subgroup. We have tested this hypothesis by analysis of 2 proteins, free secretory component (FSC) and the keratins, in lavage fluids obtained from 4 groups of subjects: 30 normal nonsmokers, 15 asymptomatic smokers, 22 symptomatic smokers, and 40 carcinoma patients. Among symptomatic smokers, FSC relative to total protein (FSC/TP) was depressed compared with that in nonsmokers and asymptomatic smokers. The keratins were detected only in symptomatic smokers and correlated with pack/years of smoking history (p = 0.017). Carcinoma patients had depressed FSC/TP and detectable keratin (33 of 38 patients studied). Lung sections from carcinoma patients studied immunohistochemically revealed an apparent inverse relationship between tissue FSC and keratins. This inverse relationship was borne out by analysis of these proteins in the lavage fluid of cancer patients (r = -0.4, p = 0.04). Thus, in cancer patients, immunohistochemical evidence of airway injury correlates with bronchial lavage levels of mucosal epithelial cell proteins. It is possible that smokers with altered levels of these proteins may be the ones at increased risk of smoking-associated lung disease.     

Early Effects of Short-Time Cigarette Smoking on the Human Lung: A Study of Bronchoalveolar Lavage Fluids

We investigated the early effects of cigarette smoking in healthy subjects by means of lung lavage, looking at markers of alveolar permeability, the alveolar cell profile, the immunophenotyping of macrophages and lymphocytes, and the level and profile of surfactant phospholipids. Bronchoal-veolar lavages (BAL) were performed in 33 healthy subjects [20 nonsmokers (nS), 13 moderate and short-time smokers (S)]. In the acellular supernatants we measured the markers of alveolar permeability (i.e., total proteins, albumin, albumin/urea), the alveolar epithelial lining fluid (AELF), the surfactant amounts and profile, and explored the blood lymphocytes by in vitro exposure. The cell pellet established the alveolar formula and a membrane mapping of macrophages (LFA-1 and HLA-DRII expression) and lymphocytes (CD4, CD8, LFA-1, HLA-DRII expression). We found no significant increase of alveolar permeability in our smokers, but an increased alveolar cellularity (more than 3-fold vs nS, P < 0.05) evenly distributed between sub-populations except for an enhanced number of eosinophils in smokers (P < 0.05 vs nS). Smokers’ alveolar macrophages had an overloaded cytoplasm, a decreased percentage of antigen-handling cell expression (HLA DRII: P < 0.05 vs nS) and a low percentage of cell to cell adhesion molecule expression (LFA-1: P < 0.05 vs nS). Smoking history and LFA-1 expression on alveolar macrophages were interrelated. Smokers’ alveolar lymphocyte subsets were more often T suppressor cells (CD8+) and had an increased percentage of antigen-presenting cell expression (HLA DRII: P < 0.05 vs nS). Smokers’ BAL fluid did not show the inhibitory control of phytohemagglutinin-induced lymphocyte proliferation present in nonsmokers’ fluids. Surfactant phospholipid amounts were similar, but phosphatidylethanolamine was raised and the ratio of phosphatidyicholine to sphingomyelin decreased in smokers (P<0.05 vs nS). We observed specific cellular and biochemical alterations in the lung lavage of short-time smokers. Alveolar macrophage and lymphocyte expression of LFA-1 and HLA-DR II molecules was altered. Smokers’ alveolar fluids lost the physiologic regulatory control of T mitogen-induced lymphocyte proliferation. Membrane phospholipids released by cellular damage increased early in tobacco-exposed lung fluids. This profile of alterations may be an early and sensitive marker of smoking-induced lung damage.     

Pulmonary epithelial permeability in ARDS and cardiogenic pulmonary oedema

Clearance of inhaled 99mTechnetium-labelled diethylene triamine pentacetate (99mTc-DTPA) from the lung, an index of pulmonary alveolar epithelial permeability (PAEP), was measured in 13 patients with cardiogenic interstitial pulmonary oedema (CIPO) and in 7 patients with adult respiratory distress syndrome (ARDS). Thirty-five normal subjects (22 nonsmokers and 13 smokers) were evaluated as controls. Half-time clearance (t0.5) values in ARDS patients (mean +/- SD: 15 +/- 2 min) were significantly lower than in CIPO patients (62 +/- 9 min). This PAEP increase in ARDS was impressive, even in comparison to heavy smokers. Loss of the PAEP vertical gradient (apical PAEP greater than base PAEP) was observed in both cardiogenic and ARDS lungs and among smokers.     

CD8+ve cells in the lungs of smokers with chronic obstructive pulmonary disease.

Previous studies have shown an increased number of inflammatory cells and, in particular, CD8+ve cells in the airways of smokers with chronic obstructive pulmonary disease (COPD). In this study we investigated whether a similar inflammatory process is also present in the lungs, and particularly in lung parenchyma and pulmonary arteries. We examined surgical specimens from three groups of subjects undergoing lung resection for localized pulmonary lesions: nonsmokers (n = 8), asymptomatic smokers with normal lung function (n = 6), and smokers with COPD (n = 10). Alveolar walls and pulmonary arteries were examined with immunohistochemical methods to identify neutrophils, eosinophils, mast cells, macrophages, and CD4+ve and CD8+ve cells. Smokers with COPD had an increased number of CD8+ve cells in both lung parenchyma (p < 0.05) and pulmonary arteries (p < 0.001) as compared with nonsmokers. CD8+ve cells were also increased in pulmonary arteries of smokers with COPD as compared with smokers with normal lung function (p < 0.01). Other inflammatory cells were no different among the three groups. The number of CD8+ve cells in both lung parenchyma and pulmonary arteries was significantly correlated with the degree of airflow limitation in smokers. These results show that an inflammatory process similar to that present in the conducting airways is also present in lung parenchyma and pulmonary arteries of smokers with COPD.