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Turkish Respiratory Journal
August 2004, Volume 5, Number 2, Page(s) 097-101
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Bronchoalveolar Lavage Findings in Patients With Mild to Moderate COPD Who do not Currently Smoke
Duygu Ozol, MD1; Tulin Aysan, MD2; Zeynep A. Solak, MD2; Ali Veral, MD3; Filiz Sebik, MD4
1Department of Pulmonology, Fatih University School of Medicine, Ankara, Turkey
2Department of Pulmonology, Ege University, School of Medicine, İzmir, Turkey
3Department of Pathology, Ege University, School of Medicine, İzmir, Turkey
4Department of Immunology, Ege University, School of Medicine, İzmir, Turkey
Keywords: COPD, smoking, bronchoalveolar lavage, lung functions
Summary
In this study, we aimed to examine the effect of smoking cessation on bronchoalveolar lavage (BAL) findings in COPD patients who were heavy or less heavy smokers. Twenty five ambulatory patients with stable mild to moderate COPD according to the GOLD classification were included the study. Sixteen patients had a history of more than 40 pack-years cigarette (Group 1) and 9 had a history of less than 40 pack-years cigarette (Group 2) were compared for the relationship of smoking history and spirometric tests with BAL cell count and interleukin-8 (IL-8) levels.

Mean forced expiratory volume in one second (FEV1) value in Group 1 was significantly lower as compared to Group 2 (1412±106.8 ml and 1488±239.4 ml respectively) (p=0.009). In the total group of patients, a positive correlation was found between intensity of smoking history and increased neutrophils. (p=0.002, r=0.583) and a negative correlation between neutrophils and FEV1 (p=0.003, r=- 0.435). No association was observed between intensity of cigarette smoking history and IL-8, FEV1.

Patients with COPD who had a history of heavy smoking had higher neutrophil counts in the airways as compared to less heavy smokers and increased neutrophil counts were associated with altered lung functions. Despite quitting smoking, neutrophilic inflammation was found to continue in patients with COPD.
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  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
    • Introduction
    Chronic obstructive pulmonary disease (COPD) is characterized by a chronic inflammatory process in the large and small airways, as well as in the lung parenchyma. COPD is the only leading cause of death that is increasing in prevalence and it is expected to be the fourth leading cause of death worldwide in 2020 [1]. The pathogenesis of COPD is multifactorial, involving airway inflammation, protease-antiprotease imbalance, oxidative stress and recurrent infection [2]. As a result of these factors, a persistent airflow obstruction with reduction in the maximum expiratory flow develops in COPD.

    Techniques allowing precise cell phenotyping and determination of cytokine production have allowed for a much better description of the inflammatory profiles in the lungs of smokers, ex-smokers and patients with COPD. There are different techniques to investigate airway inflammation like spontaneous or induced sputum examination, bronchial biopsy and bronchial or bronchoal- veolar lavage (BAL). While induced sputum seems to be a combination of resident mucus and mostly samples the large airways, BAL samples mainly the alveolar compartment [3]. In COPD patients, the inflammatory process which contributes to narrowing in the small airways, leads to an increased number of CD8 lymphocytes, neutrophils, increased connective tissue deposition and epithelial metaplasia [4]. Interleukin-8 (IL-8) that is produced by epithelial cells, macrophages and neutrophils is a cytokine with potent neutrophil chemotactic and activation properties [5]. In normal persons who do not smoke, BAL cellularity consists predominantly of macrophages and neutrophils are usually under 4 percent. Sybill et al showed that, in cigarette smokers, there is an increase in polymorphonuclear neutrophils in peripheral blood as well as in BAL fluid in comparison with nonsmokers [6]. The effect of smoking cessation to the BAL cellularity is controversial. Kulawik reported no significant differences in cell count and macrophage phenotypes between active smokers and ex-smokers in induced sputum in patients with COPD [7]. In contrast, Rutgers showed increased numbers of inflammatory cells in the airways of patients with COPD who were ex-smokers, suggesting that inflammation may persist in the airway once established [8].

    Also in smokers, the relationship between the severity of airflow limitation and the severity of airway inflammation is well documented [9-11], while little is known about the relationship between the severity of airflow limitation and the ongoing inflammation in ex- smokers.

    Survival in COPD directly correlates with the level of forced expiratory volume in one second (FEV1) [12]. As neutrophils and IL-8 may lead to progressive lung damage, the aim of this study was firstly to assess the type of inflammatory process in the BAL of patients with COPD who do not currently smoke and secondly to find out if there is any correlation between smoking history and inflammatory indices in BAL with lung functions, especially with FEV1.
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  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
    • Methods
    A total of 25 ambulatory patients with a medical history and clinical and radiological findings consistent with stable mild to moderate COPD, according to GOLD classification [13], were included in the study. Eight subjects whose postbronchodilator FEV1 value was greater or equal than 80% of the predicted, were considered as mild COPD and 17 subjects whose FEV1 value was in the range between 50-80% of the predicted were considered as moderate (2a) COPD. The patients had all ceased smoking at least one year prior to the enrollment. Cumulative cigarette consumption was measured in pack-years. All subjects had a smoking history of at least 20 pack-years and 16 had a smoking history of more than 40 pack-years.

    Inclusion criteria were: [1] FEV1 value in the range of more than 50% of the predicted value, [2] reversibility with inhaled- beta 2 agonists of less than 15% of predicted FEV1, [3] stable COPD defined as no acute exacerbation within the preceding three months, [4] no history of systemic disease or other pulmonary disease, [5] no therapy with inhaled or systemic corticosteroids within 3 months prior to entry into the study, [6] no history of asthma or atopy.

    All patients were on therapy with inhaled salbutamol and ipratropium bromide. In 9 patients, sustained-released theophyline was also being given.

    Informed consent was obtained from all patients. The study was approved by the Ethics Committee of Ege University. Spirometric tests and BAL analysis with bronchoscopy were ascertained on entry into the study. Pulmonary function tests were performed by the standard method using a dry rollingseal spirometer. Three technically adequate maneuvers were required and the best values for FVC and FEV1 was accepted. Transnasal fiberoptic bronchoscopy was performed using an Olympus flexible fiberoptic bronchoscope, following the guidelines of the National Institutes of Health. Premedication included atropine (0.5 mg-IM) administered 30 minutes before the procedure, with local upper airways anaesthesia with 5 ml of 2% lidocaine. To perform lavage, the bronchoscope was wedged into the segmental bronchus of the middle lobe and 20 ml x 5, a total 100 ml of sterile warmed saline solution was infused. Fluid was gently aspirated immediately after the infusion had been completed and was collected in a sterile container. The fluid was immediately centrifuged at 500xg for 10 minutes. Supernatants were removed and frozen in 1 ml sterile polystyrene tubes at –80°C.

    IL-8 concentrations were determined by two-site sandwich IL-8 specific enzyme-linked immunosorbent assay (ELISAChemikline). The concentration of IL-8 in the samples was calculated by comparison to the curve obtained with different concentrations of standards included in each kit. Tests were done twice for validation.

    For BAL cell counts, the cell pellet was washed with phosphate-buffered saline solution. Cells were resuspended in Hank’s balanced salt solution and counted using a haemochromocytometer chamber. Cytocentrifuges were stained by the May-Grünwald Giemsa method. The differential cell counts of macrophages, lymphocytes, neutrophils and eosinophils were made under light microscopy at x400 magnifications, counting approximately 300 cells. All cell data are expressed as percentages.

    Statistical analysis
    Parametric data are expressed as the mean ±SEM. Parametric data were compared using Student’s t test. Nonparametric data comparisons were made using Mann-Whitney U test between the two groups. The relationship between the proportion of cells and smoking history, expressed as the number of pack years and the results of pulmonary function tests were tested by the Spearman’s correlation test. In each case, a p value of <0.05 was considered significant.
  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
    • Results
    Sixteen patients with a history of more than 40 pack-years cigarette smoking (Group 1) and 9 patients with a history of less than 40 pack-years cigarette (Group 2), with stable COPD were enrolled for the study. Bronchoscopy was performed without any complications. Clinical and demographic findings of the patients are shown in Table 1. Both patient groups were similar with regard to age, sex and mean time period after smoking cessation. Mean FEV1 value and percentage of predicted FEV1 in Group 1 were 1412±106.8 ml (% 60.1±2.2). In Group 2 these values were 1488.8±239.4 ml (% 66.3±1.9) respectively (p=0.009).


    Click to Enlarge    		 Table 1: Clinical and demographic findings

    Analyses of cells recovered in BAL confirmed that bronchial inflammation in COPD patients who do not currently smoke is associated with an increase in polymorphonuclear leukocytes. Since the percentages of eosinophils were very low, these values are not reported. Although there was a tendency of higher mean neutrophil count in Group 1, this was not statistically significant (p=0.52). Other BAL cell findings and IL-8 levels were similar in both groups and there was no significant statistical difference (Table 2, Figure 1).


    Click to Enlarge    		 Table 2: Bronchoalveolar lavage findings


    Click to Enlarge    		 Figure 1: Cell percentage according to smoking history.

    Considering all 25 patients, we found a negative correlation between increased neutrophils and low FEV1 value (Figure 2) and also a positive correlation between increased neutrophils and smoking history (r=0.583, p=0.002) (Figure 3). No association was found between IL-8 and smoking history (r=-0.143, p=0.496).


    Click to Enlarge    		 Figure 2: Relationship between neutrophil percentage in BAL and lung functions.


    Click to Enlarge    		 Figure 3: Relationship between neutrophil percentage and smoking intensity.

  • Top
  • Summary
  • Introduction
  • Methods
  • Results
  • Disscussion
  • References
    • Discussion
    COPD patients who smoked more than 40 pack years had lower FEV1 values and a tendency to have higher neutrophils in their BAL than the patients who smoked less than 40 pack-years. Despite smoking cessation, neutrophils persisted in the airways and increased neutrophils were associated with altered lung functions. We found a weak correlation between a heavy smoking history and low FEV1 and no association was observed between IL-8 and. FEV1.

    Cigarette smoking represents a major risk factor for COPD. The components of cigarette smoke such as nicotine lead to retention of the neutrophils within the pulmonary microvasculature [14]. The first inflammatory cell responding to smoking is most likely the neutrophils. The pathological findings of the airways in COPD patients revealed increased numbers of macrophages and predominantly CD8+ T cells in bronchial biopsy specimens [15], increased mast cells in bronchial glands [16], predominance of neutrophils in BAL and in induced sputum [17, 18]. In our study we focused on neutrophil percentage and IL-8 concentration as a measure of distal airway inflammation and we examined the inflammatory process in BAL which strengths our study as BAL gave a better assessment of small airways and alveolar inflammation. One of the limitations of our study is that we were not able to ascertain total cell counts and T lymphocytes subgroups in the lavage specimens.

    Turato et al examined the number of inflammatory cells, the markers of mononuclear cell activation and the expression of endothelial adhesion molecules and cytokines in the subepithelium by bronchial biopsies and found that there were no differences between current smokers and ex-smokers for any parameter examined [19]. Wright et al found increased numbers of total inflammatory cells and neutrophils in the walls of the membranous bronchioles in current and ex-smokers when compared to nonsmokers. Current and ex-smokers had similar numbers and types of inflammatory cells in the airways, indicating that this inflammatory response does not abate after smoking cessation [20]. COPD patients who were ex-smokers were shown to have increased numbers of inflammatory cells and especially eosinophils in the airways, suggesting the ongoing inflammation once established [8]. We found that despite quitting smoking, neutrophilic inflammation continues in patients with COPD.

    The higher number of neutrophils in the airways could increase the antimicrobial defense but could also impair bronchial cell function. Neutrophils can cause tissue damage through the release of proteases and formation of oxygen radicals. Thompson et al found that in COPD patients with higher bronchial sample neutrophils had significantly more sputum production and lower FEV1, FEV1/FVC and FEF25-75 than did the subjects with lower bronchial sample neutrophils [3]. The superoxide anion release and elastase activity from the neutrophils appears to be correlated with the annual decrease in FEV1 over the course of several years [21]. Correlated with the degree of airflow limitation, there is a causal relationship between the high smoking history and the numbers of neutrophils and lung function decline.

    Interleukin-8, one of the mediators that have a role in the pathogenesis of COPD, along with leucotrien-B4 (LTB4) and tumor necrosing factor-alfa (TNF-α), is a cytokine with potent neutrophil chemotactic and activation properties. Elastase produced by resident neutrophils in airways induces the bronchial epithelium to secrete IL-8, which in turn recruits additional neurophils, which self perpetuates the inflammatory process. Oxidant stress was also found to stimulate IL-8 production in a variety of cell lines [22]. There are other factors affecting neutrophil accumulation in the airways, like oxidant stress, nitric oxide, TNF-α, IL-1 α and IL-1β. Also, the medication which is taken by the patient such as inhaled steroids, may also decrease the percentage of neutrophils in sputum [23] and in BAL fluid [24].

    Compared to current smokers, the ex-smokers had a lower concentration of the chemoattractant IL-8 concentration [25,26]. Another limitation of our study was that we did not have a control group of patients with COPD who currently smoke. By smoking cessation, IL-8 level may have been decreased and that may explain why we could not find a relationship between IL-8 and investigated parameters.

    Longitudinal studies investigating clinical parameters with macrophage activity, neutrophils and CD8+ lymphocytes in the airways before and after smoking cessation is needed for more information about the ongoing inflammation in COPD and this information may contribute to our further understanding of the pathophysiology of COPD.

    Acknowledgment: The authors would like to thank Dr. İpek Akman for her thoughtful review of this manuscript.
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  • Summary
  • Introduction
  • Methods
  • Results
  • Discussion
  • References
    • References

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    2) Cosio MG, Guerassimov A. Chronic obstructive pulmonary disease. Inflammation of small airways and lung parenchyma. Am J Respir Crit Care Med 1999;160:S21-5.

    3) Thompson PB, Daughton D, Robbins GA, Ghafouri MA et al. Intramural airway inflammation in chronic bronchitis: characterization and correlation with clinical parameters. Am Rev Respir Dis 1989;140:1527-37.

    4) Barnes PJ. Mechanisms in COPD, Differences from asthma. Chest 2000;117:10s-4s.

    5) Kanazawa H, Kurihara N, Otsuka T, Fujii T et al. Clinical significance of serum concentration of interleukin-8 in patients with bronchial asthma or chronic pulmonary emphysema. Respiration 1996;63:236-40.

    6) Sibille Y, Marchandise FX. Pulmonary immune cells in health and di- Psease: polymorphonuclear neutrophils. Eur Respir J 1993;6:1529-43.

    7) Kulawik JD, Warzechowska MM, Kraszewska I, Chazan R. The cellular composition and macrophage phenotype in induced sputum in smokers and ex smokers with COPD. Chest 2003;123:1054-9.

    8) Rutgers SR, Postma DS, ten Hacken NH et al. Ongoing airway inflammation in patients with COPD who do not currently smoke. Thorax 2000;55:12-8.

    9) Swan GE, Roby TJ, Hodgin JE, Mitmann C et al. Relationship of cytomorphology to spirometric findings in cigarette smokers. Acta Cytol 1994; 38:547-53.

    10) O’Shaughnessy TC, Ansari TW, Barnes NC. et al. Inflammation in bronchial biopsies of subjects with chronic bronchitis: inverse relationship with CD8+ T lymphocytes with FEV1. Am J Respir Crit Care Med 1997;115:852-7.

    11) Di Stefano A, Capelli A, Lusardi M ET AL. Severity of airflow limitation is associated with severity of airway inflammation in smokers. Am J Respir Crit Care Med 1998;158:1277-85.

    12) Siafakas NM, Vermeire P, Pride NB et al. Optimal assessment and management of chronic obstructive pulmonary disease. Eur Respir J 1995;8:1398-1420.

    13) Gold Workshop Report, Updated 2003. www.goldcopd.com.

    14) Stockley RA. Neutrophils and the pathogenesis of COPD. Chest 2002;121 (Suppl):151S-5S.

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    16) Pesci A,Rossi GA, Bertorelli G et al. Mast cells in the airway lumen and bronchial mucosa of patients with chronic bronchitis. Am J Respir Crit Care Med 1994;149:1311-6.

    17) Yıldız F, Kaur AC, Ilgazlı A, Çelikoglu M, Özkara SK, Paksoy N, Özkarakaş O. Inhaled corticosteroids may reduce neutrophilic inflammation in patients with stable chronic obstructive pulmonary disease. Respiration 2000;67:71-6.

    18) Lacoste JYL, Bousquet J, Chanez P etal. Eosinophilic and neutrophilic inflammation in astma, chronic bronchitis and chronic obstructive pulmonary disease J Allergy Clin Immunol 1993;92:537-8.

    19) Turato G, Di Stefano A, Maestrelli P. Effect of smoking cessation on airway inflammation in chronic bronchitis. Am J Respir Crit Care Med 1995;152:1262-7

    20) Wright JL, Hobson JE, Wiggs B et al. Airway inflammation and peribronchiolar attachment in the lungs of nonsmokers, current and exsmokers. Lung 1988;166:277-86.

    21) Stanescu D, Sanna A, Veriter C, Kostianev S etal. Airways obstruction, chronic expectoration and rapid decline of FEV1 in smokers are associated with increased levels of sputum neutrophils. Thorax 1996;51:267-71.

    22) Martin LD, Rochelle LG, Fischer BM, Krunkosky TM et al. Airway epithelium as an effector of inflammation:molecular regulation of secondary mediators. Eur Respir J 1997;10:2139-46.

    23) Mirici A, Bektas Y, Ozbakis G, Erman Z. Effect of ihaled corticosteroids on respiratory function tests and airway inflammation in stable chronic obstructive pulmonary disease: A randomised, double-blind, placebocontrolled clinical trial. Clin Drug Invest 2001;21:835-42.

    24) Ozol D, Aysan T, Solak ZA, Mogulkoc N, Veral A, Sebik F. The effect of inhaled corticosteroids on bronchoalveolar lavage cells and IL-8 levels in stable COPD patients. Resp Med; 2004 (In Press).

    25) Nagatomo H. Use of expectorated sputum to assess airway inflammation of smoking and its cessation. Nihon Kokyuki Gakkai Zasshi 2001;39:461-5.

    26) Hill AT, Bayley DL, Campbell EJ et al. Airway inflammation in chronic bronchitis: the effects of smoking and alpha1 antitrypsin deficiency. Eur Respir J 2000;15:886-90.
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