Neutrophil Elastase Gene Polymorphisms: Modulatorsof Response to Therapy in Childhood Bronchiectasis?

Aleksandra Nikolic • Katarina Milosevic •

Srdjan Boskovic • Branimir Nestorovic

Received: 23 January 2014 / Accepted: 23 April 2014

� Springer Science+Business Media New York 2014

Abstract

Background The aim of this study was to investigate

polymorphisms in the promoter region of the neutrophil

elastase (ELANE) gene as potential modulators of the ther-

apeutic response in children with idiopathic bronchiectasis.

Methods The study included 48 children between 5 and

17 years old who were diagnosed with idiopathic bron-

chiectasis based on high-resolution computed tomography

of the thorax. In all patients therapy included administra-

tion of antibiotics, anti-inflammatory drugs, expectorants,

and postural drainage. Response to therapy was evaluated

by the change in FeNO levels before and after adminis-

tration of therapy. The ELANE promoter region poly-

morphisms were analyzed by PCR-direct DNA sequencing.

Results According to the predicted activity of ELANE

genotypes, subjects were divided into two groups: low/

intermediate activity (n = 18) and high activity (n = 30).

Subjects in the group with high-activity genotype had higher

initial FeNO levels and this difference was statistically

significant (t = 2.906; p = 0.006). The difference between

FeNO levels before and after therapy was also statistically

significantly higher in children with high-activity genotype

(t = 3.329; p = 0.002). Statistically significant correlation

was observed between the change in FeNO levels and

ELANE genotypes (r = 0.350; p = 0.015).

Conclusion Children with high-activity genotype had

higher initial FeNO levels and showed better response to

therapy than children with low/intermediate-activity

genotypes.

Keywords Neutrophil elastase � Bronchiectasis �Gene polymorphism

Introduction

Bronchiectasis is a chronic lung disease in which deregu-

lated inflammatory response and recurrent bacterial infec-

tions result in progressive lung damage and irreversible

dilatation of bronchi. In patients with bronchiectasis, pul-

monary inflammation is dominated by neutrophils and

neutrophil elastase (NE) activity [1]. Neutrophils are

recruited primarily to the airways in response to an initial

infection or other trigger, but the airway epithelium and

neutrophils can become sources of cytokines that lead to

the additional recruitment of neutrophils [2]. The expec-

torated bronchial secretions of patients with bronchiectasis

contain not only NE activity, but also proinflammatory

mediators that can promote the proteolytic action of neu-

trophils leading to damage of the bronchial tissue [3, 4].

Elastase-mediated tissue injury is one of the key compo-

nents in the pathogenesis of bronchiectasis and elastase

activity was shown to be of clinical significance [5].

Human NE is potent in the stimulation of airway

secretion, acceleration of airway inflammation, and

destruction of the airway mucosal tissue in both acute and

chronic pulmonary diseases [6]. The mature 29-kDa

enzyme is produced in granulocyte precursor cells, stored

A. Nikolic (&) � S. Boskovic

Institute of Molecular Genetics and Genetic Engineering,

Vojvode Stepe 444A, PO Box 23, 11010 Belgrade, Serbia

e-mail: aleksni@imgge.bg.ac.rs

K. Milosevic � B. Nestorovic

Department of Pulmonology and Allergology, University

Children’s Hospital, Belgrade, Serbia

B. Nestorovic

Medical Faculty, University of Belgrade, Belgrade, Serbia

123

Lung

DOI 10.1007/s00408-014-9596-3

in azurophilic granules of neutrophils, and released on

surface activation, phagocytosis, or cell death [7, 8]. Its

activity in inflamed pulmonary tissue is counterbalanced

essentially by secretory leukoproteinase inhibitor, a 12-kDa

polypeptide product of the bronchial epithelium and sub-

mucosal glands [9]. The role of the plasma-derived anti-

proteinase a1-antitrypsin, a 52-kDa plasma glycoprotein

synthesized mainly by hepatocytes, becomes important

with increased microvascular permeability [10]. Although

it is unclear how NE activity can remain dominant when

the inflamed bronchial environment is replete with anti-

proteinases, this process is crucial to the pathogenesis of

bronchiectasis [4, 11].

The NE expression levels are known to be affected by

the polymorphisms in the promoter region of the neutrophil

elastase (ELANE) gene. Several polymorphisms were

identified so far in the six repetitive tandem motifs of

the ELANE gene promoter region: -903T/G, -741G/A,

-832G/T, -789C/T, and extra 52 bp between the fourth

and fifth repeats. Polymorphisms -903T/G and -741G/A

were associated with lung cancer risk [12]. Luciferase activity

assays have shown that ELANE gene promoter constructs

carrying -903T/-741G had higher activity compared with

the constructs carrying -903G/-741A. Based on those

results, predicted activity of ELANE genotypes was classified

as low (-903TG), intermediate (-903TT/-741AG and

-903TT/-741AA), or high (-903TT/-741GG) [13].

The treatment of bronchiectasis is aimed at reducing the

inflammatory burden of the lower respiratory tract, mainly

through the use of antibiotics. Considering its role in the

intracellular killing of pathogens, NE significantly contrib-

utes to the immune response of the host and the treatment of

infection [14]. Therefore, the polymorphisms in the ELANE

gene promoter that alter overall NE activity in lungs may be

associated with the therapeutic response in bronchiectasis

patients and be of potential clinical significance.

The aim of this study was to investigate ELANE gene as

potential modulators of the therapeutic response in children

with idiopathic bronchiectasis.

Materials and Methods

Study Subjects

The study included 48 children (19 male and 29 female)

between 5 and 17 years of age who were diagnosed with

idiopathic bronchiectasis at the Department of Allergology

and Pulmonology of the University Children’s Hospital in

Belgrade in the period 2008–2010. Informed consent was

obtained from all patients’ parents and the study was

approved by the Ethics Committee of the Medical Faculty,

University of Belgrade. Bronchiectasis was diagnosed by

high-resolution computed tomography (HRCT) of the

thorax in all patients and was confirmed on pathological

examination of lobectomy material from patients who

underwent surgical removal of lung due to hemorrhage.

Other known causes of bronchiectasis were ruled out in

every patient: cystic fibrosis, immunodeficiency disorders,

ciliary dyskinesia, and tuberculosis. Basic demographic

and clinical data were collected for all study subjects.

Clinical and Laboratory Analyses

The following routine laboratory analyses were performed

in each patient: sedimentation, complete blood count,

C-reactive protein (CRP) in plasma, skin prick tests for

inhalation allergens, pulmonary function tests, cytological

and bacteriological examinations of induced sputum, nitric

oxide in exhaled air (FeNO), chest radiography, HRCT,

and flexible bronchoscopy with alveolar lavage. Cytologi-

cal analysis of sputum was performed by fluorescence

microscopy with a BX41 (Olympus). Serum CRP levels

were measured by turbidimetric analysis on a Dimension

RxL Max Integrated Chemistry System (Siemens). Mea-

surement of FeNO was performed with a NIOX MINO

(Aerocrine). Pulmonary function was evaluated based on

spirometry analysis. Forced expiratory volume in 1 s

(FEV1) and forced vital capacity (FVC) were measured by

spirometer using standard protocols. Lung function was

expressed as percentage of reference values. Response to

therapy was evaluated through change in FeNO levels

before and after administration of therapy.

Therapy

In all patients therapy included administration of antibiotics,

anti-inflammatory therapy, expectorants, and postural

drainage. The choice of antimicrobial therapy was based on

the clinical presentation of each patient and results of

microbiological analysis of BAL or induced sputum

according to the guidelines of the British Thoracic Society

for the treatment of non-cystic fibrosis bronchiectasis [15]. In

patients infected with Streptococcus pneumoniae and

b-lactamase-negative Haemophilus influenzae, amoxicillin

was administered orally for 14 days. In patients with

b-lactamase-positive H. influenzae, amoxicillin with clavu-

lanic acid was administered orally for 14 days. In both cases,

severe infections were treated with intravenous ceftriaxone.

In Pseudomonas aeruginosa infections, ciprofloxacin was

administered orally, while severe infections were treated

with intravenous ceftazidime or intravenous meropenem in

combination with aminoglycosides twice a day via nebulizer.

For Staphylococcus aureus (MSSA), the first line of antibi-

otic treatment was flukloksacilin, followed by clarithromycin

for 14 days. In Moraxella catarrhalis infections, therapy

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consisted of amoxicillin with clavulanic acid for 14 days.

Anti-inflammatory therapy consisted of ibuprofen

(20–30 mg/kg), while N-acetyl cysteine was used as expec-

torant, followed by physical therapy (postural drainage). In

rare cases when pulmonary obstruction was observed, cor-

ticosteroids and b2 agonist salbutamol were used as needed.

The FeNO measurement was chosen as a clinically estab-

lished parameter to measure local inflammation in the air-

ways and hence suitable for follow-up of the response to

therapy, which was expressed as a change between FeNO

levels before and after treatment [16–18]. Patients with

normal flora, improved pulmonary function (increased

FEV1), and decreased amount of sputum after 14 days were

considered as having responded positively to therapy.

Genetic Analysis

Blood samples were taken from all patients and genomic DNA

was extracted using a QIAamp Blood DNA Mini Kit (Qia-

gen). Polymorphisms in the promoter of the ELANE gene were

analyzed by PCR in combination with direct DNA sequenc-

ing. The ELANE promoter region was amplified using the

primers 50-CGCAGTGAGTGCCCGACAC-30 and 50-CTGC

CAAACCTAGACCTGA-30. PCR was conducted in a 50-lL

reaction mixture containing: 1 9 Buffer B (Solis BioDyne),

0.3 mM MgCl2, 0.2 mM each dNTP, 10 pmol of each primer,

2 U of Taq polymerase FIREPol (Solis BioDyne), and

approximately 300 ng of DNA. The amplifications were

performed as follows: initial denaturation for 5 min at 94 �C;

30 cycles consisting of 30 s at 94 �C, 30 s at 58 �C, and 30 s at

72 �C; final extension for 10 min at 72 �C. Before use in

sequencing reactions, PCR products were purified using the

QIAquick PCR Purification Kit (Qiagen). The sequencing

reactions were performed in both forward and reverse direc-

tion, with the same primers used for the amplification, using

the ABI PRISM Big Dye Terminator system (Applied Bio-

systems). Sequences were analyzed using the Sequencing

Analysis software (Applied Biosystems).

Statistical Analysis

The following descriptive statistics methods were applied

for data processing: central tendency measures (arithmeti-

cal mean and median values), variability measures (varia-

tion interval, standard deviation, and interquartile range),

and relative numbers. For statistical analysis methods for

identification of empirical distributions and methods for

assessment of significant differences were used. For sta-

tistically significant differences, Student’s t test and the

rank-sum test were used for numerical variables depending

on the distribution normality, and the v2 test and Fisher’s

test were used for categorical variables. Values of p B 0.05

were considered statistically significant. Statistical analysis

was performed using SPSS for Windows 17.0 (SPSS, Inc.,

Chicago, IL, USA).

Results

Analysis of ELANE gene promoter polymorphisms was

performed for 48 children [19 boys (39.6 %) and 29 girls

(60.4 %)] between 5 and 17 years of age with bronchiec-

tasis. Mean age at diagnosis was 10.6 ± 3.3 years, while

mean age at disease onset was 9.7 ± 3.2 years. Clinical

and laboratory findings of the patients are given in Table 1.

Among the subjects tested in this study, 7 had the low-

activity ELANE genotype (14.6 %), 11 had intermediate-

activity ELANE genotype (22.9 %), and 30 had high-

activity ELANE genotype (62.5 %). According to the pre-

dicted activity of ELANE genotypes, subjects were divided

into two groups: low/intermediate activity (n = 18) and

high activity (n = 30).

The following parameters were compared between the

groups by means of statistical analysis: white blood cell count

in sputum, CRP plasma levels, initial FeNO, and change in

Table 1 Clinical and laboratory findings in the patients’ group

Spirometry (%) (mean ± SD)

FEV1 83.4 ± 16.4

FVC 81.8 ± 14.5

FeNO (ppb) (mean ± SD)

At diagnosis 19.2 ± 10.3

After therapy 13.6 ± 5.5

Difference 5.5 ± 7.2

CRP (mg/L) (mean ± SD) 14.2 ± 11.2

White blood cell count in sputum (%) (mean ± SD)

Neutrophils 57.9 ± 22.0

Macrophages 21.1 ± 17.7

Lymphocytes 9.9 ± 8.5

Eosinophils 9.4 ± 8.9

Monocytes 6.0 ± 3.3

Microorganisms isolated from sputum [n (%)]

Streptococcus pneumoniae 14 (29.2)

Pseudomonas aeruginosa 10 (20.8)

Haemophilus influenzae 10 (20.8)

Moraxella catarrhalis 5 (10.4)

Staphylococcus aureus (MSSA) 4 (8.3)

Acinetobacter 1 (2.1)

Burkholderia cepacia 1 (2.1)

Acinetobacter, Burkholderia cepacia 1 (2.1)

Staphylococcus aureus (coagulase-negative) 1 (2.1)

Normal flora 1 (2.1)

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123

FeNO after therapy. There was no significant difference in

white blood cell count in sputum and CRP plasma levels

between the groups. Subjects in the group with high-activity

genotype had higher initial FeNO levels and the difference

was statistically significant (t = 2.906; p = 0.006). The dif-

ference between FeNO levels before and after therapy was

also statistically significantly higher in children with high-

activity genotype (t = 3.329; p = 0.002). Statistically sig-

nificant correlation was observed between the change in

FeNO levels and ELANE genotypes (r = 0.350; p = 0.015).

The correlation between ELANE genotypes and response to

therapy is shown in Fig. 1. Children with high-activity

genotype had higher initial FeNO levels and showed better

response to therapy than children with low/intermediate-

activity genotypes.

Discussion

This study was conducted in order to test the hypothesis

that ELANE gene act as therapy modulators in bronchiec-

tasis. The study was of a group of pediatric patients diag-

nosed with idiopathic (non-cystic fibrosis) bronchiectasis.

The presence of polymorphisms in the promoter region of

the ELANE gene was determined by sequencing, while

response to therapy was evaluated based on the change in

the FeNO levels before and after therapy. The observed

distribution of ELANE genotypes among the subjects tested

in this study did not differ significantly from previously

published data [12, 13]. The main finding of the study is the

correlation observed between the ELANE genotype and the

response to therapy evaluated based on the change in FeNO

levels before and after therapy administration.

The neutrophilic inflammation of the airways with

increased levels of inflammation markers is characteristic

of bronchiectasis, and although a slight systemic inflam-

mation may be present, the inflammatory response is lar-

gely compartmentalized [19, 20]. There are conflicting

reports in the literature concerning FeNO levels in subjects

with bronchiectasis, with some studies reporting lower

FeNO levels and others reporting levels that do not differ

from those in healthy individuals [21–25]. The most

probable reasons for this inconsistency are differences in

the methodology used and subject selection criteria, most

important of which was the presence of an infection. In our

study, subjects with high predicted ELANE activity had

significantly higher FeNO levels at admission than subjects

with predicted low/intermediate activity. NE may disrupt

the normal functioning of the lung barrier and cause a

release of proinflammatory mediators [26]. Also, NE is

known to reduce the ciliary beat frequency, impairing

mucociliary clearance of pathogens, which is also an

important stimulus of NO production [27]. It is possible

that by these actions NE could cause an increase in NO

production by both resident and inflammatory cells in the

airway.

A significantly greater reduction in FeNO following

therapy was measured among subjects with high predicted

ELANE activity, suggesting that these subjects had a better

response to therapy. Some earlier studies reported no change

in FeNO levels following therapy with either antibiotics or

inhaled corticosteroids [23, 28]. Macrolide antibiotics are

known to inhibit NE, downregulate inflammation, increase

mucus clearance, decrease bacterial virulence, and prevent

biofilm formation [29, 30]. However, the underlying cause

of the observed difference in the reduction of FeNO levels

Fig. 1 Correlation between ELANE genotypes and response to therapy. a FeNO change after therapy in different ELANE genotypes.

b Distribution of therapeutic response according to the ELANE genotypes

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between the two groups with different NE genotypes is not

known. As many factors may influence both antibiotic

treatment and NE activity, the observed phenomenon is

most likely a consequence of a complex interplay of genetic

and nongenetic factors in the lungs.

This was the first study of ELANE gene polymorphisms

in bronchiectasis. Patients with predicted high-activity

ELANE genotypes responded significantly better to therapy

than patients with predicted low/intermediate-activity

genotypes, indicating a potential role of ELANE gene

polymorphisms as therapy modulators in bronchiectasis.

Although based on a relatively small sample, the study

suggests a correlation between the presence of ELANE

gene polymorphisms and response to therapy worth

investigating further. Results obtained in this preliminary

study should be confirmed in a larger group of patients and

several other factors should be taken into consideration,

both genetic and nongenetic. Also, studies of other chronic

lung diseases may provide useful insight into the role

played by NE in response to antimicrobial therapy.

Acknowledgments This study was supported by the project 173008

of the National Ministry of Education, Science and Technological

Development.

Conflict of interest None.

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