Biopsies in Chronic Obstructive
Pulmonary Diseases (A Comparison with Asthma)
Nurdan KÖKTÜRK*, Türkan TATLICIOĞLU*, Leyla MEMİŞ**, Gülten TÜRKKANI**
* Gazi Üniversitesi Tıp Fakültesi Göğüs Hastalıkları Anabilim Dalı,
** Gazi Üniversitesi Tıp Fakültesi Patoloji Anabilim Dalı, ANKARA
SUMMARY
This study was designed to describe the cellular inflammation, thickness of basement membrane (BMT) and epithelial des- quamation (ED) in bronchial biopsies from patients with chronic obstructive pulmonary disease (COPD) compared with asthma and healthy individuals. Thirteen subjects with COPD, 12 asthmatic subjects, and 10 healthy individuals enrolled in the study. Bronchial biopsies obtained by fiberoptic bronchoscopy were stained with hematoxylin and eosin to perform cell counts and descriptive analysis. Neutrophils were found in higher numbers in epithelium and lamina propria in sub- jects with COPD compared with asthma and control groups (p< 0.001, p< 0.05) whereas eosinophils were observed higher in epithelium and lamina propria in asthmatic subjects (p< 0.001, p< 0.001). There were no significant differences in num- bers of neutrophils and eosinophils in submucosa in the three groups (p> 0.05). There were no significant correlations bet- ween the inflammatory cell counts and FEV1or smoking history in either group (p> 0.05). In subjects with COPD only 6 (46.2%) of the subjects showed BMT whilst 2 (15.4%) of the subjects showed ED. In asthma group, 11 (91.7%) of the sub- jects presented increased BMT and ED. In healthy individuals 1 (10%) of the subjects had increased BMT and no one had ED. As a result, we may conclude that the predominant cells of bronchial mucosa in COPD are the neutrophils whereas they are eosinophils in asthma. Thickening of basement membrane and epithelial desquamation are the major features of asthmatics. However in COPD, these features would be focally present and variable.
Key Words:COPD, asthma, pathogenesis.
ÖZET
Kronik Obstrüktif Akciğer Hastalığında Bronş Biyopsi Örneklerinde Hücresel İnflamasyon (Bronş Astımı ile Karşılaştırmalı)
Bu çalışmada kronik obstrüktif akciğer hastalığı (KOAH) hastalarından elde edilen bronş biyopsi örneklerinde hücresel in- filtrasyonun belirlenmesi, yapısal değişikliklere ait patolojik bulguların tanımlanması ve bu bulguların astım hastaları ile karşılaştırılması amaçlanmıştır. Çalışmaya yaş ortalaması 49 ± 15 olan 15’i kadın, 20’si erkek olmak üzere; 13’ü KOAH, 12’si astım ve 10’u sağlıklı kontrol grubunda olan toplam 35 olgu dahil edildi. Alınan bronş biyopsi örneklerinde hematok-
Yazışma Adresi (Address for Correspondence):
Dr. Nurdan KÖKTÜRK, Kızılırmak Sokak No:16/10, 06640, Kocatepe, ANKARA - TÜRKİYE e-mail: [email protected]
Chronic obstructive pulmonary disease (COPD) and asthma are both widely recognized as chro- nic inflammatory diseases. However, the com- ponents of inflammation, structural changes and functional consequences have appeared to be different in these diseases (1).
Studies have demonstrated that T-lymphocytes and eosinophils have major roles in the pathoge- nesis of asthma (1,2). Epithelial desquamation (ED) and thickening of the reticular basement membrane are early and characteristic features of structural changes (airway remodelling). The thickening of bronchial wall contributes to the severity and chronicity of asthma by amplifying airway narrowing.
Unlike asthma, cellular components and struc- tural changes of airways in COPD are not largely described. Although neutrophils, T-lymphocy- tes, macrophages and eosinophils are shown to be involved in inflammation in COPD, data abo- ut predominant cell types has still been variable depending on the methodology (1-5). Additi- onally, airway remodelling in COPD has been described particularly in peripheral airways but the structural changes in large airways have not been largely investigated.
This study was designed to evaluate bronchial biopsies from large airways in patients with COPD for identifying predominant cell types and structural changes such as basement membrane thickness (BMT) and ED and compare them to patients with asthma and healthy individuals.
MATERIALS and METHODS Subjects
The clinical characteristics of the subjects are presented in Table 1.
Thirteen clinically stable COPD subjects (two atopic, 11 nonatopic, mean ages; 59.5 ± 8.3 year), 12 stable mild-moderate asthmatic sub- jects (five atopic, seven nonatopic, mean ages;
44.9 ± 13.2 year), and 10 healthy individuals (two atopic, eight nonatopic, mean ages; 39.2 ± 14.8 year) enrolled in the study. COPD and asth- ma were defined according to the criteria of the American Thoracic Society (6). Asthmatic sub- jects had either reversible airway obstruction or positive methacoline challenge test. The mean FEV1 of subjects with COPD was 61.1 ± 11.8%
of the predicted value whereas it was 82.7 ± 13.5% in the subjects with asthma. All subjects with COPD were smokers (minimum 25 pack/years) and had chronic bronchitis. None of the asthmatic subjects was a current smoker or had smoked during the previous two years. Two of healthy individuals were smokers. None of the healthy individuals had suffered from asthma or chronic bronchitis or received regular medicati- on. They all had normal pulmonary function tests. None of the subjects in three groups was re- ceiving oral or inhaled corticosteroids, long-ac- ting β2 agonists or theophylline during the study and within a month prior to study. All subjects with COPD and asthma were allowed to take short acting β2agonists as needed. Subjects who had experienced symptoms of any bronchial or respiratory tract infection during the month pre- ceding the study were excluded.
The study was approved by Gazi University Et- hical Committee and written informed consent was obtained from the participants.
Study Design
At the initial visit, subjects were evaluated with a full history, pulmonary function tests (FEV1, silen eozin boyası ile elde edilen kesitlerde inflamatuvar hücre sayıları, bazal membran kalınlığı (BMK) ve epitelyal desku- amasyon (ED) değerlendirildi. KOAH grubunda nötrofil düzeyleri diğer gruplardan istatistiksel olarak anlamlı düzeyde yüksek saptanırken, astım grubunda eozinofil düzeyleri yüksek olarak bulundu (p< 0.001, p< 0.05). BMK, KOAH grubun- da 13 olgunun altısında saptanmışken, astımlı olguların biri hariç hepsinde görüldü. Kontrol grubunda yalnızca bir olgu- da BMK saptandı. ED, KOAH grubunda yalnızca iki olguda saptanırken, astım grubunda 12 olgunun 11’inde mevcuttu.
Kontrol grubunda ise hiçbir olguda saptanmadı. Bu verilere dayanarak KOAH’ta bronş mukozasında nötrofillerin, astımda ise eozinofillerin predominant hücreler olduğu, bazal membran kalınlığındaki artış ve epitelyal deskuamasyonun astımda karakteristik olarak saptandığı, bulgulardaki belirgin farklılıklar nedeniyle bronş biyopsi örneklemesinin astım ve KOAH ayırıcı tanısında faydalı bir metod olabileceği ortaya konmuştur.
Anahtar Kelimeler: KOAH, astım, patogenez.
FEV1/FVC, reversibility), methacoline challenge and skin prick tests. After initial evaluation, fi- beroptic bronchoscopy was performed to obtain the bronchial biopsies from large airways. The
number of inflammatory cell counts, BMP, ED were evaluated and compared among the gro- ups.
Table 1. Clinical characteristics of the subjects.
Duration of
symptoms Smoke FEV1 FEV1/ PC20*
Sex Age (Yr) (Pack.Yr-1) Atopy** (%) FVC R*** (mg/mL)
COPD M 56 6 25 N 63 69 11
COPD M 42 10 25 N 66 60 5
COPD M 46 10 28 N 65 70 1
COPD M 61 6 30 N 61 69 7
COPD M 66 6 30 Y 40 46 14
COPD M 70 10 30 N 66 60 10
COPD M 57 15 33 N 71 57 12
COPD M 59 15 40 Y 53 66 2
COPD M 65 6 40 N 53 70 7
COPD M 68 5 40 N 66 78 4
COPD M 62 5 40 N 75 55 11
COPD M 66 30 55 N 39 38 5
COPD M 56 4 60 N 76 65 3
Asthma F 40 2 0 N 72 45 26 -
Asthma F 46 3 0 N 89 77 14 4.44
Asthma F 33 20 0 N 76 83 15 -
Asthma F 50 3 0 Y 85 86 7 2.99
Asthma F 33 3.5 0 Y 84 75 - 0.10
Asthma F 38 4.5 0 Y 73 69 14 0.86
Asthma M 49 25 0 Y 75 52 15 -
Asthma M 34 4 0 N 74 64 4 -
Asthma M 36 2 0 Y 93 83 - 0.84
Asthma F 54 15 0 N 103 90 - 1.19
Asthma M 80 60 0 N 61 65 15 -
Asthma F 46 30 0 N 107 83 - 3.00
Control M 22 0 N 104 89
Control F 35 0 N 113 94
Control F 34 0 N 130 84
Control F 19 0 Y 91 95
Control F 66 0 N 150 83
Control M 32 12 N 81 91
Control F 53 0 Y 101 82
Control M 36 10 N 88 84
Control F 55 0 N 80 87
Control F 40 0 N 93 80
* The concentration of methacoline to produce 20% fall of FEV1.
** Skin prick test (Y: Positive to one or more allergens, N: Negative).
*** Reversibility.
Pulmonary Function Tests, Methacoline Challenge, Skin Prick Tests
Subjects were asked to discontinue inhaled short-acting β2 agonists for a minimum of six hours prior to pulmonary function testing. Me- asurements were performed in sitting position using Sensor Medics Vmax 20 Spirometer. Para- meters were recorded from the best of three for- ced expiration attempts. Reversibility was me- asured after an inhalation of 400 µg of inhaled salbutamol. For methacoline challenge, a stan- dardized bronchial provacation protocol was performed using a nebulized buffered isotonic methacoline solution. Spira Dosimeter (Respira- tory Care Center, Hameenlinna, Finland) was used to perform methacoline challenge. The response of FEV1was expressed as the percen- tage fall from mean baseline value. The con- centration of methacoline (PC20) needed to ca- use a 20% fall in postsaline FEV1 was calcula- ted. Bronchial hyperreactivity was considered when PC20was less than 16 mg/mL. Atopy was considered to be present when skin prick test was positive to at least one of the common aller- gens [dermatofagoides farinea (DF), dermatofa- goides pteronyssinus (DP), mold mix, grass pol- len, tree pollen, aspergillus, cat and dog danders etc].
Fibreoptic Bronchoscopy
Bronchoscopy was performed in accordance with NHLBI guidelines (7).
After premedication with 0.5 mg atropine sulp- hate (s.c), 0.05 mgkg-1midazolam hydrochlori- de (IV) and local anesthesia with topically appli- ed 4% lidocaine spray; a fiberoptic bronhoscope (Olympus BF type 30, Tokyo, Japan) was inser- ted through the nose. Oxygen at 4 L/minutes was administered via nasal cannulae throughout the procedure. All patients had intravenous ac- cess and ECG and oxygen monitorization during the procedure. The biopsies were taken from the subcarina of the right upper lobe by using alliga- tor FB 15C (Olympus, Japan) forceps. Inhalati- on of 200 µg salbutamol was applied after the procedure. All subjects tolerated the procedure well and there were no severe complications.
Pathological Examination of Bronchial Biopsies After fixation with 10% formaldehyde, biopsies were embedded in paraffin blocks and 4 µm sec- tions were obtained. All sections were deparaffi- ned and rehydrated before hematoxylin and eo- sin (HE) staining.
Quantitation
In biopsies stained with HE, inflammatory cell counts, BMT, ED were evaluated by a patholo- gist (Memis) using BH2 light microscope (Olympus) and an eyepiece graticule at a mag- nification of X 400. To avoid observer bias, all slides were coded and read in a blinded fashion.
The number of cells was counted in the whole area of the section including epithelium, lamina propria and submucosa. Cell count was perfor- med on the section that has well-preserved structure. Five of these areas were used to obta- in the total quantitation. The mean number of cells was used for a comparison and expressed as the number per square millimetre. Increased BMT and ED were described as “diffuse”, “ab- sent”, “focal”.
Statistical Analysis
Statistical results were given as means ± SD.
SPSS for “Windows Release 10.01 (1999)” sta- tistical software was used for statistical analysis.
Chi square and Fisher’s exact chi square were performed in order to compare three groups. T- test was applied for a comparison in two groups.
One way ANOVA and Kruskal Wallis ANOVA were used for a comparison in three groups.
When the difference was found between the gro- ups, Tukey’s test was applied to determine the different group.
RESULTS
Mean ages were significantly different in three groups (p< 0.001). As expected by the selection criteria, FEV1(%) and reversibility were signifi- cantly different in the groups (p< 0.001). He- althy individuals had significantly greater FEV1(%) (103.1 ± 22.4) than subjects with asth- ma (82.7 ± 13.5) and subjects with COPD (61.1
± 11.8). Subjects with asthma had significantly higher reversibility (13.8 ± 6.5) than the sub- jects with COPD (7.1 ± 4.2). Atopy status was
not significantly different in the three groups whereas smoking (pack.yr-1) was significantly greater in COPD group (p< 0.001). Results are given in Table 2.
Descriptive Analysis of the Inflammation Although we did not use any specific markers to distinguish the inflammatory cell types, we were able to differentiate them with HE staining beca- use of the distinct shape of the cells. As a result, neutrophils were found in higher numbers in
epithelium and lamina propria in subjects with COPD compared with asthma and control gro- ups (p< 0.001, p< 0.05) whereas eosinophils were observed higher in epithelium and lamina propria in asthmatic subjects (p< 0.001, p<
0.001) (Table 2). There were no significant dif- ferences in numbers of neutrophils and eosinop- hils in submucosa in the three groups (p> 0.05).
The numbers of lymphocytes, plasma cells and monocytes were not significantly different in
Table 2. Clinical characteristics of the subjects and inflammatory cell distribution.
COPD Asthma Controls p value
Number 13 12 10
Age (year) 59.5 ± 8.3 44.9 ± 13.2 39.2 ± 14.8 p< 0.001
FEV1 (%) 61.1 ± 11.8 82.7 ± 13.5 103.1 ± 22.4 p< 0.001
Reversibility 7.1 ± 4.2 13.8 ± 6.5 p< 0.001
Smoke (pack/year) 36.7 ± 10.9 0 2.2 ± 4.7 p< 0.001
Neutrophils
epithelium 6.5 0.08 1.30 p< 0.001
lamina prop 8.08 3.75 3.10 p< 0.05
submucosa 4.25 1.60 3.38 NS
Total neutrophils 18.62 5.17 7.10 p< 0.001
Eosinophils
epithelium 0.62 3.67 0.30 p< 0.001
lamina prop 2.15 6 0.50 p< 0.001
submucosa 0.75 2.40 1.25 NS
Total eosinophils 3.46 11.5 1.80 p< 0.001
Lymphocytes
epithelium 1.38 0.17 0.30 NS
lamina prop 2.38 2.50 1.30
submucosa 1.83 1.20 1.25 NS
Total lymphocytes 5.46 5.33 2.50 NS
Plasma cells
epithelium 1.38 0.17 0.20 NS
lamina prop 5.54 6.67 3.80 NS
submucosa 2.75 0.40 2.25 NS
Total plasma cells 9.46 7.17 5.80 NS
Monocytes
epithelium 0.46 0 0 NS
lamina prop 5.38 2.83 2.40 NS
submucosa 2.50 1.90 2.25 NS
Total monocytes 8.15 4.42 4.20 NS
Results are expressed as means. ± SD. NS: Not significant. Kruskal-Wallis, Fisher’s Exact Chi Square, T test and Tukey’s test were per- formed for statistical analysis.
epithelium, lamina propria and submucosa, in either group (p> 0.05). A comparison of the me- an inflammatory cell counts in different layers has been shown in Figure 1. There were no sig- nificant correlations between the inflammatory cell counts and FEV1 or smoking history in eit- her group (p> 0.05).
HE staining showed striking differences in the structure of bronchi. Thus, in subjects with COPD only 6 (46.2%) of the subjects showed BMT [4 (30.8%) diffuse and 2 (15.4%) focal] and 2 (15.4%) of the subjects showed ED [1 (7.7%) diffuse and 1 (7.7%) focal]. In asthma group, 11 (91.7%) of the subjects presented increased BMT [8 (66.7%) diffuse and 3 (25.0%) focal] and ED [9 (75%) diffuse and 2 (16.7%) focal]. In he- althy individuals 1 (10%) of the subjects had inc- reased BMT and no one had ED.
DISCUSSION
The present study indicates that the inflammati- on in the large airways of subjects with COPD is different from the inflammation present in sub- jects with mild to moderate asthma and that this inflammation differentiates the two diseases.
Furthermore, our findings confirm that the thic- kening of basement membrane and epithelial desquamation are extensively present in asthma but not in COPD. The predominant cell type of bronchial mucosa in COPD is neutrophil where it is eosinophil in asthma.
Increased BMT and ED have been recognised as key features for asthma even in early stages of disease (2,8). Those features have shown to be occurred as consequences of chronic inflamma- tion (9-11). Despite the fact that COPD is also known as a chronic inflammatory disease, structural changes of airways are not well desc- ribed as in asthma. Our findings regarding with structure of the bronchi are in agreement with those of Ollerenshaw et al, and Lacoste et al (12,13). Lacoste et al, described increased BMT and ED extensively present in asthma whereas such changes were lesser extent in the group of COPD. In the other study Ollerenshaw et al, did not find increased BMT in biopsies from nonasth- matic subjects with chronic airflow limitation
whereas biopsies from all asthmatic subjects re- vealed increased BMT.
Thickening of basement membrane and its ca- usative factors have been extensively investiga- ted in asthma. Now, it has been well known that subepithelial fibrosis has occured as a result of impaired tissue repair and excessive fibrosis (14). Such changes have also been described in COPD but the main side of inflammation and structural changes are known to be peripheral airways (15,16). It is more likely that large air- ways do not reflect such changes present in pe- ripheral airways.
ED has been attributed to numerous factors, inc- luding tissue eosinophilia and the release of ma- jor basic protein and superoxides from eosinop- hils (13). As a support of this statement, in our study we have found eosinophils in higher num- bers in the epithelium in the group of asthma. In COPD, epithelial metaplasia is a predominant feature instead of ED.
Eosinophilic inflammation in asthma is well known. Eosinophils release basic proteins that are cytotoxic and lipid mediators that cause air- way obstruction and bronchial epithelial dama- ge (17). This study confirms previous data that shows eosinophilia in bronchial specimen from asthmatics (12,18). On the other hand, in agre- ement with Lacoste et al, we did not find any correlations between the number of eosinophils in bronchial mucosa and airway obstruction.
The present study revealed that neutrophils we- re the predominant cells in the airway mucosa in subjects with COPD.
Interestingly, most of recent studies carried out in subjects with COPD have not found a high number of neutrophils in bronchial biopsies whilst they found neutrophilia in BAL. In an early study, Thompson et al showed high number of neutrophils in BAL of the patients with chronic bronchitis. This was found to be correlated with pack years of cigarettes smoked, lower FEV1 and sputum production (19). However, in more recent studies, Jeffery et al and O’shaughnessy et al have shown CD8+ lymphocytes to be mo- re prominent in COPD. Furthermore they found a negative association between the CD8+
Figure 1. Comparison of the inflammatory cell counts in epithelium, lamina propria and submucosa. Lymp:
Lymphocytes, Plasma: Plasma cells, Mon: Monocytes, Eos: Eosinophils, Neut: Neutrophils.
7
6
5
4
3
2
1
0
Lymp Plasma Mon Eos Neut
Epithelium
Asthma COPD Control
7
6
5
4
3
2
1
0
Lymp Plasma Mon Eos Neut
Lamina Propria
5
4
3
2
1
0 Lymp Plasma Mon Eos Neut
Submucosa
Asthma COPD Control
Control COPD Asthma
lymphocytes and FEV1 (1,20,21). This discre- pancy has led to the hypothesis that the inflam- mation in the lumen may be different from that in the bronchial wall of these patients. The ob- servation of an upregulation of the adhesion mo- lecules on submucosa and airway epithelium of smokers with COPD has supported this hypot- hesis suggesting neutrophil recruitment from the circulation to the lumen through the epithelium (15,22). However, the majority of the studies on bronchial biopsies have been focused on the subepithelial zone, and have not analysed the epithelial and bronchial gland compartments.
Recent studies of surgical specimens and biop- sies showed an increased number of neutrophils in both epithelium and bronchial glands of sub- jects with COPD (23,24). This helps to explain the apparent discrepancy between lumenal and tissue findings in this disease and provide evi- dence for neutrophilia not only in the airway lu- men, but also in specific areas in the airway wall
of these subjects. In agreement with these ob- servations we have found high numbers of neut- rophils in bronchial epithelium and lamina prop- ria whilst no difference was found in submucosa.
Furthermore we did not find any correlations between the cell counts and pulmonary function tests.
In this study we have used visual analysis to co- unt the inflammatory cells. Moreover we did not use any specific markers to identify the cells. In account of those limitations, we may limitedly compare our data to those which was obtained from immunohistochemical and molecular stu- dies. Despite these, we may conclude that the predominant cells of bronchial mucosa in COPD are the neutrophils whereas they are eosinophils in asthma. Thickening of basement membrane and ED are the major features of asthmatics ho- wever in COPD, these features would be focally present and variable.
Figure 2. Biopsy sections, stained with HE from a. A subject with COPD, revealed neutrophilic inflammation in epithelium. In submucosa a mixed cellular inflammation is present (HE x 200). b. A subject with COPD, revealed metaplastic epithelium (HE x 200). c. A healthy individual, revealed intact epithelium. There is no inflammation in submucosa (HE x 100). d. A subject with asthma, revealed epithelial desquamation and basement membrane thickness.
a
c
b
d
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