Archives of Medical Research 40 (2009) 561e564

ORIGINAL ARTICLE

Does Defective Apoptosis Play A Role in Cystic Fibrosis Lung Disease?

Ebru Yalcin, Beril Talim, Ugur Ozcelik, Deniz Dogru, Nazan Cobanoglu, Sevgi Pekcan, and Nural Kiper

Chest Diseases Unit, Department of Pediatrics, Faculty of Medicine, Hacettepe University, Ankara, Turkey

Received for publication November 10, 2008; accepted June 22, 2009 (ARCMED-D-08-00509).

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Background and Aims. Although apoptotic dysfunction has recently been suggested incystic fibrosis (CF), there are few studies reported concerning apoptosis in CF withcontroversial results. The aim of this study was to investigate apoptosis in CF human lungtissues and compare with non-CF bronchiectatic and normal healthy lung tissues. We alsoinvestigated the relation between apoptosis and histopathological features of tissues andmicrobiological factors influencing apoptosis.

Methods. Lung tissue samples from CF (n 5 30), non-CF bronchiectasis (n 5 28, BEgroup) and normal control cases (n 5 24, C group) were included in the study. Histolog-ical examination of H & E-stained archived slides was performed and TUNEL methodwas used to detect DNA fragmentation.

Results. Apoptotic alveolar epithelial cells were significantly increased in the CF groupcompared to BE and C groups ( p 5 0.046). Bronchopneumonia (BP) was present in 15CF cases (50%), whereas none of the cases in C group had BP ( p 5 0.0001). Apoptosiswas significantly increased in cases with BP (n 5 17) compared to cases without BP(n 5 65) ( p 5 0.04).

Conclusions. Apoptotic epithelial cells and BP were significantly increased in the CFgroup and excess level of apoptosis may be the result of enhanced occurrence of BP.Apoptotic cells were alveolar epithelial cells in the great majority of the patients and werenot detected in other locations where CFTR expression is much more prominent thanalveolar cells. We may postulate that increased apoptotic findings in the alveolar epithe-lium were related with the presence of chronic infections rather than CFTRdysfunction. � 2009 IMSS. Published by Elsevier Inc.

Key Words: Apoptosis, Bronchiectasis, Bronchopneumonia, Cystic fibrosis, Lung.

Introduction

Cystic fibrosis (CF) is a single-gene disease and is the mostcommon lethal inherited disease in Caucasians. CF iscaused by mutations in the CF transmembrane regulator(CFTR) gene resulting in absent or deficient expressionand function of CFTR protein (1,2). CFTR protein isexpressed in epithelial cells of the pancreas, intestine, sweatglands, and upper and lower respiratory tracts (3). Althoughit has been demonstrated that CFTR is a Cl� channel, it hasbeen postulated that CFTR may affect other cellular func-tions and participate in the apoptotic process by influencing

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the intracellular pH (1). Apoptosis, programmed cell death,is a physiological process essential for the maintenance ofhomeostasis of epithelial organization and function forclearance of inflammatory cells (2). Although apoptoticdysfunction in CF has recently been suggested, there arefew studies reported concerning apoptosis in CF withcontroversial results (3e5). A high DNA fragmentationand high level of Fas ligand expression have been reportedin both human CF enterocytes and bronchial epithelial cells(1,2). On the other hand, a resistance in the initiation ofapoptosis induced by etoposide was reported in a mutantDF508 epithelial mouse mammary cell line (6).

The aim of this study was to investigate apoptosis in CFhuman lung tissues and compare with non-CF bronchiectaticand normal healthy lung tissues. We also planned to investi-gate the relation between apoptosis and histopathologicalfindings and microbiological factors influencing apoptosis.

ier Inc.

Table 1. Demographic, clinical, histopatological and apoptotic features of patients.

Groups

CF (n 5 30) Bronchiectasis (n 5 28) Control (n 5 24) p

Gender

Female 12 (40%) 15 (54%)

Male 18 (60%) 13 (46%)

Ages 4 dayse15 years 18 monthse15 years 1 monthe6 years

Median age 3 months 9.1 years 16 months

Histopathological findings (n)

BP positive 15 2 0 0.0001

FB positive 0 7 0 0.001

FIB positive 1 19 0 0.0001

Apoptotic cells (n)

Positive 14 (47%) 5 (18%) 6 (25%) 0.046

Negative 16 (53%) 23 (82%) 18 (75%)

Culture positive (n) 16 0 0

DF508 Mutation (%) 30% e e

CF, cystic fibrosis; BP, bronchopneumonia; FB, follicular bronchiectasis; FIB, follicular infective bronchiectasis.

Figure 1. Apoptotic alveolar epithelial cells shown with TUNEL method.

The ratio of TUNEL-positive cells to all epithelial cells was !10% in all

cases. Color version of this figure available online at www.arcmedres.com.

562 Yalcin et al./ Archives of Medical Research 40 (2009) 561e564

Patients and Methods

Between 1979 and 2005, lung tissue samples from CF (25autopsy cases and 5 lobectomy materials; CF group) andnon-CF bronchiectasis (n 5 28, BE group) cases who werebeing followed-up in the Department of Pediatric ChestDiseases and/or had lung tissue samples examined in theDepartment of Pediatric Pathology at the Faculty of Medi-cine of Hacettepe University were included in the study.Normal control lung tissue (n 5 24, C group) was providedfrom autopsy material of non-CF patients.

All tissues (autopsy and lobectomy samples) were fixedin buffered formaldehyde at room temperature. No pre-fixa-tion was applied. Fixation time was similar for all samples(1e2 days).

H & E-stained archived slides were examined and majorpathological findings were classified as bronchopneumonia(BP), follicular bronchiectasis (FB) or follicular infective bron-chiectasis (FIB). The remaining cases showed nonspecificfindings (NS).

To detect DNA fragmentation, we used in situ terminal de-oxynucleotidyl transferase d uridine triphosphate nick endlabelling (TUNEL) method (ApopTag; Qbiogene apoptosisdetection kit, Carlsbad, CA). For TUNEL assay, sectionsprovided within the apoptosis kit (ApopTag) were used as posi-tive control sections and positive staining was obtained withthese. At least 20 fields were examined for each case under20� magnification, and bronchi, bronchioles and submucosalglands were investigated for TUNEL-positive apoptotic cells.Apoptotic cells were alveolar epithelial cells in the greatmajority of the patients. Because the ratio of apoptotic cellsto all epithelial cells was !10% in eachslide, apoptotic activitywas not further graded and reported only as positive or not (7).

Statistical Analysis

The sputum culture positivity, histopathological findings,presence of apoptotic cells and factors which might affect

apoptosis were compared in 3 groups using the c2 test witha statistical software package (SPSS, v.11; SPSS Inc;Chicago, IL). In each situation, a p value of !0.05 wasconsidered significant.

Results

Patient Characteristics

CF group consisted of 30 patients with a median age of3 months (range: 4 days to 15 years). This group included12 girls and 18 boys. There were 28 cases (male/female 5 13/15) in the BE group who had a median ageof 9.1 years (range: 18 months to 15 years). The etiologiesfor bronchiectasis were immune deficiencies (n 5 3),primary ciliary dyskinesia (n 5 3), Kartagener’s syndrome(n 5 2) and unidentified in the remaining 20 cases. C group

50

40

30

20

10

0CF BE C

Pat

ient

s(%

)

Patients with apoptosis detected

Patients who had bronchopneumonia

CF, cystic fibrosis; BE, brochiectasis without CF; C, control

Figure 2. The percentages of bronchopneumonia findings and apoptosis in

the three groups.

563Apoptosis in Cystic Fibrosis

consisted of 24 cases whose ages ranged from 1 month to 6years with a median of 16 months. Before autopsy orbiopsy, sputum cultures were positive in 16 cases withCF, cultures were all negative in the BE and C groups( p 5 0.0001). S. aureus (50%) and P. aeruginosa (40%)were the most common microorganisms identified. Themost common mutation in the CF group was DF508, whichwas identified in nine cases (homozygous in four cases,compound heterozygous in five cases). Demographic andclinical characteristics of the patients are given in Table 1.

Histopathological Findings

BP was present in 15 CF cases (50%) and two BE cases(7%); none of the cases in C group had BP ( p 5 0.0001).FB was not detected in any of the patients in CF or C group,whereas seven cases (25%) in BE group had FB( p 5 0.001). FIB findings were detected in only one patientin CF group (3.3%) compared to 19 cases (67.9%) in BEgroup; none of the cases in C group had FIB( p 5 0.0001). Histopathological characteristics of lungtissues in various groups are given in Table 1.

Apoptotic Findings

Apoptotic cells were detected in 14 cases with CF (47%), fivecases with BE (18%) and six cases in C group (25%).Apoptotic cells were significantly increased in the CF groupcompared to BE and C groups ( p 5 0.046). No significantdifference was detected between the BE and C groups withregard to presence of apoptotic cells ( p 5 0.7). Apoptosiswas detected in a few bronchial epithelial cells of three CF

Table 2. Distribution of all patients according to bronchopneumonia

and apoptotic findings ( p 5 0.04)

BP (þ) BP (�) Total

Apoptosis (þ) 9 16 25

Apoptosis (�) 8 49 57

Total 17 65 82

BP, bronchopneumonia.

cases and two BE cases, whereas none had apoptotic cellsin the glandular epithelium. In all remaining cases, apoptoticcells were found in the alveolar epithelium (Figure 1).Apoptotic findings are displayed in Table 1.

Factors Affecting Apoptosis

In 25 patients, apoptosis was detected in the lung tissues(14 CF, 5 BE, 6 C cases), whereas BP findings were presentin 17 cases (15 CF, 2 BE cases) (Tables 1 and 2). Apoptoticfindings were significantly increased in cases with BP find-ings (n 5 17) compared to cases without (n 5 65)( p 5 0.04). This was regarded as evidence of a positiverelation between the presence of apoptotic cells andBP in the lungs (Table 2 and Figure 2). No significant rela-tion was demonstrated between the bacterial growth rate,presence of FIB or FB and presence of apoptosis.

Discussion

This study demonstrated that apoptotic epithelial cells andBP findings were significantly increased in the CF groupand an excess level of apoptosis may be the result of enhancedoccurrence of BP findings. It is known that extracellularmicroorganisms induce apoptosis on airway epithelial cellsduring pneumonia (8). In our study, S. aureus and P. aerugi-nosa were the most common microorganisms identified inCF patients. Lung of CF patients are often exposed to chronicand persistent pulmonary infections with these two patho-gens. Kahl et al. (9) infected a respiratory epithelial cell linewith S. aureus RN6390, which was derived from a CFpatient. These authors showed that it replicated and inducedapoptosis in pulmonary epithelial cell line. On the other hand,the ability of P. aeruginosa to induce apoptosis in airwayepithelial cells was found to be dependent upon propertiesof host epithelial cell structure and CFTR genotype of the in-fected cells. The 9HTEo� cells that lacked tight junctionintegrity were readily susceptible to apoptosis after exposureto P. aeruginosa. However, cultured cells expressing DF508CFTR showed a delayed apoptotic response to this microor-ganism compared with cells expressing wild-type CFTR(10,11). With prolonged exposure of epithelial cells to P. aer-uginosa and in destroyed epithelial structure in CF lungdisease, P. aeruginosa-induced apoptosis may be expectedin our CF patients (11).

Another important result from our study is that apoptoticcells were detected in alveolar epithelium in all but five casesand none of those had apoptotic cells in the bronchial, bron-chiolar or submucosal glandular epithelium. If increasedapoptosis was associated with CFTR dysfunction in thelungs, we would have expected apoptotic cells in bronchial,bronchiolar and submucosal gland epithelial cells whereCFTR expression is much more prominent than alveolar cells(12). Considering this, we might postulate that the increased

564 Yalcin et al./ Archives of Medical Research 40 (2009) 561e564

apoptotic findings in the alveolar epithelium were relatedwith the presence of chronic infections rather than CFTRdysfunction, which is the basic defect in CF.

The effects of CFTR dysfunction on apoptosis have beenstudied both in vitro and in clinical specimens. Maiuri et al.(1) showed that bronchial (n 5 2) and duodenal (n 5 14)biopsy specimens from CF patients had more apoptoticcells detected by TUNEL method as compared withcontrols, and apoptosis was particularly prominent in areaswith high CFTR expression, such as the submucosal cells.This study showed that inappropriately high DNA fragmen-tation may be a feature of various CF epithelia. However, inthat study the number of cases is small to draw sucha conclusion. On the contrary, a mouse mammary epithelialcell line (C127) transfected with mutant CFTR was foundto be resistant to apoptosis, as compared with cells express-ing the wild-type gene (6). The decrease in the rates ofapoptosis was suggested to be due to consequences ofCl� channel dysfunction and lack of acidification in the cy-toplasma required for caspase activation. Functional CFTRis required for cytoplasmic acidification, which has beenobserved as an early event in the apoptotic cascade due tothe activation of an acid endonuclease required for DNAcleavage (11). Maiuri et al. tried to explain the discrepancybetween these studies by stating that the cells may undergoDNA nicking, as seen by the TUNEL assay. However, finaldegradation by endonucleases does not occur, or otherapoptosis related processes fail to occur. This would indi-cate a situation in which the apoptotic process is initiatedbut then aborted (4,5,11).

Increased susceptibility to apoptosis in epithelial cellsand failed apoptosis in neutrophils would contribute tothe self-perpetuating inflammatory cycle in CF. Indepen-dent of the susceptibility to apoptosis of CF cells, it hasbeen shown that clearance of apoptotic cells is defectiveand that accumulation of such cells may contribute toongoing inflammation in CF patients (5).

The main limitation of our study was based on the TU-NEL method that is sensitive, reproducible, and the preva-lent method to detect apoptosis in tissues. However, it onlydetects DNA fragmentation and is known to be positive alsoin some necrotic cells (its lack of specificity) (1,4,5,13).Used methods may influence TUNEL results. Tateyemaet al. (14) investigated effects of prefixation and fixationtimes on apoptosis detection by in situ end-labeling of frag-mented DNA. They found that prefixation time affects theprecise identification of apoptosis by the TUNEL method.Longer time prefixation intervals cause an incorrect evalu-ation with many more apoptotic cells than expected

(increased false positivity). Prefixation was not applied inour study, so probable false positivity of TUNEL methodcould not be attributed to this. Furthermore, in that study(14), the length of fixation time in formalin seemed to haveno effect on the results obtained by this method.

Because of some pitfalls of the TUNEL methods, largerstudies using more sophisticated techniques to detectapoptosis are required to reveal more precise results. Thus,the exact pathological mechanisms that play a role in theapoptotic processes in CF lung can be elucidated.

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