Eosinophil-Derived Neurotoxin Versus Immunoglobulin E as Biomarkers for Evaluation of Bronchial Asthma
The aim of this research was to assess the clinical utility of Eosinophil Derived Neurotoxin (EDN) and immunoglobulin E (IgE) as biomarkers for bronchial asthma evaluation as regard type (atopic vs non atopic) and severity. The study included 39 atopic asthmatic patients (group 1), 31 non atopic asthmatic patients (group 2) and 20 age and sex matched controls (group 3) with their age ranged from 7-17 years. Eosinophil count, serum level of immunoglobulin E (IgE), EDN and spirometry were done for all cases. A positive correlation between EDN and eosinophil count was found (r = 0.423 and p<0.01) and between serum EDN and IgE (r = 0.401 and p<0.03). Serum EDN and IgE levels showed statistically significant difference between group 1 and 2 (p<0.001 and 0.01, respectively) and between group 1 and 3 (p<0.001 and 0.003, respectively), but no statistically significant difference was found between group 2 and 3 for both parameters. No correlations were found between EDN or IgE and FEV1 (%) predicted. EDN level showed a statistically significant difference between groups when patients classified into 4 groups based on symptoms and drug use in comparison to IgE which showed no statistically significant difference between the same groups. The study suggests that serum EDN may be superior to IgE as a biomarker for evaluation of asthma regarding its type and severity.
Asthma is a serious public health problem throughout the world, affecting people
of all ages. When uncontrolled, asthma can place severe limits on daily life
and is sometimes fatal (Guill, 2004). Asthma is chronic
inflammatory disorder of the airways in which many cells and cellular elements
play a role, in particular mast cells, eosinophils, t-lymphocytes, neutrophils
and epithelial cells. In susceptible individuals, this inflammation causes recurrent
episodes of wheezing, breathlessness, chest tightness and cough, particularly
at night and in the early morning. These episodes are usually associated with
widespread but variable airflow obstruction that is often reversible either
spontaneously or with treatment. The inflammation also causes an associated
increase in the existing bronchial hyperresponsiveness. The characteristic features
of airway inflammation in asthma are leukocyte infiltration, epithelial sloughing,
basement membrane thickening, edema and hyperplasia of mucous-secreting glands
and hypertrophy of bronchial smooth muscle (Amin el al.,
Atopic predisposition is caused by the interaction between genetic and environmental
factors as well as by the various clinical manifestations that favor a mainly
Thelper type 2 (Th2) response, with interleukins that lead to the formation
of immunoglobulin IgE, pro-inflammatory cytokines and bronchial hyperreactivity.
T cells derived cytokines, IgE and mast cells initiate an early asthmatic reaction
and recruitment and activation of eosinophils. IgE molecules have been found
to play a crucial role in allergic respiratory diseases and possibly cause chronic
airway inflammation in asthma through activation of effector cells via high-affinity
(Fc RI) or low-affinity (Fc RII) IgE receptors (Ozol et
The activated eosinophils release preformed mediators from their granules as
Eosinophil Cationic Protein (ECP), Major Basic Protein (MBP) and Eosinophil
Derived Neurotoxin (EDN) which are highly toxic to epithelial cells in vitro
(Motojima et al., 1989). EDN, also called eosinophil
protein X (EPX) is a glycosylated single-chain protein with a molecular weight
18-21 kD and is a member of ribonuclease A super family along with ECP. Although,
EDN has high sequence homology to ECP, EDN has a 100 fold greater ribonuclease
activity than ECP and neurotoxicity but not cytotoxicity (Silfman
et al., 1989). Few studies have focused on the role of EDN in asthma.
The objective of this study is to evaluate the clinical utility of EDN and IgE as biomarkers for evaluation of bronchial asthma regarding type, atopic vs non atopic and severity.
MATERIALS AND METHODS
Subjects: The study was conducted from May till November 2008 and it
included 70 children and adolescents (age 7-17 years old) with bronchial asthma
and 20 age and sex matched healthy subjects as control group. Cases were recruited
from allergy and outpatient clinics, pediatric and internal medicine departments
Faculty of Medicine, Cairo University. All cases were subjected to proper history
taking and clinical examination with special emphasis on grading of asthma severity
and use of medications, total serum IgE and blood EDN measurements and allergy
skin prick testing to common allergens. Spirometry was performed for all participants
at least six and twelve hours after the use of short-acting or long-acting inhaled
bronchodilator, respectively. The patient had to complete at least three forced
vital capacity maneuvers and the best one was chosen according to the recommendation
of the European Respiratory Society (Miller et al.,
The enrolled asthmatic children and adolescents were defined according to the
criteria of the Global Initiative for Asthma (Global Initiative
for Asthma, 1995). Asthmatic patients with history of systemic steroids,
chest infection in the last month or parasitic infestation and controls with
history of wheeze, atopy, recurrent or chronic diseases, infection in the last
two weeks, parasitic infestation or high IgE were excluded.
Accordingly, the study subjects were classified into three main groups; group
1 included 39 atopic asthmatic patients, group 2 included 31 non-atopic asthmatic
patients and group 3 contained 20 subjects as control group. A positive skin
reaction (mean wheal diameter of at least three mm more than with saline control)
in response to at least one the tested allergens together with positive family
history of other allergic diseases were used to define atopic status. Based
on symptoms frequency and the step of the daily medication regimen that the
patient is currently on, all the asthmatic patients were classified into four
grades of severity according to GINA criteria (Global Initiative
for Asthma, 1995).
EDN and total serum IgE measurements: Venous blood samples were obtained from all subjects and Complete Blood Count (CBC) was performed including the absolute eosinophilic count. Assessment of immunoglobulin E (IgE) was done by rate nephlometry, BN ProSpec, DADE BEHRING. The working range for the assay of total IgE determinations was 0 to 2000 IU mL-1. For this analysis, IgE levels up to 120 IU mL-1 were considered within normal range. For measurement of EDN, blood samples were drawn into vacuum tubes and allowed to clot at room temperature for 60 min, followed by centrifugation at 1300 g for 10 min at 4°C and then samples were stored in fresh plastic tubes at 20°C till time of EDN assay. Serum EDN was measured using ELISA kit (7630, MBL, Nagoya, Japan). A 100 μL of each sample were transferred to a microwell coated with anti-human EDN antibody and incubated at room temperature for 1 h. After rinsing with wash solution, 100 μL of conjugated solution were added to the wells and incubation was continued at room temperature for an additional 1 h. After another washing, TMB chromogen solution (100 μL well-1) was added and allowed to incubate for 5 min at room temperature. Enzyme reaction was stopped with 0.2 M sulfuric acid (100 μL well-1) and the absorbance measured at 450 nm with a microplate ELISA reader, STAT Fax 2100, AWARENESS TECHNOLOGY, INC. within 30 min of stopping. All assays were performed in duplicate for each sample, with the mean values reported here.
Statistical analysis: Statistical analysis was performed using software (SPSS 10.0; Chicago, IL). All results are expressed in Mean±SD. The mean values were compared among study groups using student’s t-test or Mann-Whitney test, according to whether the corresponding values followed a normal distribution or not, as tested by Kolmogorov-Smirnov test. Linear regression analysis was used to investigate the potential relationships between variables. p<0.05 was considered statistically significant.
RESULTS AND DISCUSSION
The study included 70 asthmatic children and adolescents, they were divided into two groups according to results of IgE. Group 1 included 39 atopic asthmatic patients (20 males and 19 females) with mean age 12.3±4.5 years. Group 2 included 31 non-atopic asthmatic patients (15 males and 16 females) with mean age 11.5±5.1 years. The control group (group 3) included 30 age and sex matched healthy children and adolescents (16 males and 14 females) with mean age 12.1±4.3 years. The demographic, clinical and laboratory data are shown in Table 1.
A positive correlation was elicited between serum level of both EDN and IgE with eosinophilic count (r = 0.423, p<0.01 and r = 0.431, p<0.01, respectively). Also there was a positive correlation between serum EDN and IgE (r = 0.401, p<0.03). No correlation was found between both IgE or EDN serum levels and FEV1.
Spirometric and laboratory data of patients categorized according to severity
based on symptoms and medication regimen are shown in Table 2.
The demographic, clinical and laboratory data of study groups
Spirometric and laboratory data of patients categorized according to
severity based on symptoms and medication regimen
Bronchial asthma is a common chronic condition that affects up to 10% of adults
and 35% of children worldwide and it is an increasing public health problem.
Asthma may develop at least in part as a result of genetic or host susceptibility
to allergy or atopy and bronchial hyperresponsiveness (Guill,
2004). In present study, we asses both EDN and IgE for their utility as
biomarkers for evaluation of asthma as regard type and severity.
The study showed both EDN and IgE were positively correlated with the eosinophilic
count. Eosinophils has a crucial role in the pathogenesis and course of asthma,
as most allergic and non-allergic asthmatic patients, including those with mild
asthma, has a bronchial eosinophilia and there is significant association between
eosinophils activation and asthma severity as well as bronchial hyperresponsiveness.
Tissue eosinophilia was found to be significantly greater in fatal asthma (Guill,
2004) than in patients with chronic asthma. Eosinophils are recruited and
found to be activated during segmental allergen challenge. Soluble vascular
cell adhesion molecule-1 (sVCAM-1) levels after segmental antigen challenge
correlate with eosinophil influx, IL-4 and IL-5 production and the late-phase
response (Zangrilli et al., 1995).
The biological properties of eosinophils include the release of toxic granule
proteins, oxygen free radicals, eicosanoids (sulfido-peptide leukotrienes),
Th2-like cytokines and growth factors. Once activated, products
from eosinophils contract human bronchial smooth muscles, increase vascular
permeability and induce airway hyperresponsiveness. Eosinophils are deleterious
in asthma by the release of highly toxic products including Major Basic Protein
(MBP), Eosinophil Cationic Protein (ECP), Eosinophil-Derived Neurotoxin (EDN)
and oxygen free radicals, which induce the shedding of the surface epithelium
in keeping with the hypothesis of eosinophil-induced damage of the bronchi (Busse
and Sedgwick, 1994).
Eosinophils can be important cells of airways remodeling. Eosinophils can release
growth factors, elastase and metalloproteases, involved in the process of tissue
remodeling and fibrosis. Eosinophil products stimulate fibroblasts (Pincus
et al., 1987). Eosinophils appear to be involved in pulmonary fibrosis
or in tropical pulmonary eosinophilia (Schlick, 1993)
and have long been associated with endomyocardial fibrosis (Magnaval
and Bavel, 2005 ) but the involvement of eosinophils in the fibrotic process
is not completely understood. Moreover, in an extensive study, MBP deposition
was found to be present in some, but not all, cases of pulmonary fibrosis at
the site of the fibrotic lesions (Noguchi et al.,
Regarding evaluation of asthma, first the differentiation of atopic and non atopic type, second, the determination of degree of severity and third the diagnosis of asthma per say in pre-school age.
Concerning the differentiation of atopic and non atopic type, (Amin
et al., 2000) reported that in atopic asthmatic subjects, the airway
inflammation is characterized by high numbers of eosinophils, mast
cells and T lymphocytes, whereas non-atopic asthmatic patients mainly display
high numbers of neutrophils and mast cells. There are also distinct structural
alterations in the airway mucosa in patients with atopic asthma that are not
found in non-atopic asthma. Because there is no clear definition of the asthma
phenotype, researchers studying the development of this complex disease turn
to characteristics that can be measured objectively. It has been reported that
atopic patients with asthma have higher concentrations of exhaled Nitric Oxide
(NO) compared with non-atopic patients with asthma who have exhaled NO concentrations
similar to normal values (Lúdvíksdottír
et al., 1999). Also, atopic patients with asthma are more hyperresponsive
to inhaled adenosine-5’-monophosphate (AMP) than non-atopic patients with
asthma (Lúdvíksdottir et al., 2000),
although skin-prick test and IgE are the widely used tools for this purpose.
The study showed that serum EDN level was significantly higher in atopic asthmatic
than non-atopic asthmatic patients and controls. Earlier studies about the relation
of EDN to asthma are deficient. Kim et al. (2007)
reported that EDN and ECP are significantly higher in atopic asthma than non-atopic
asthma and both factors were positively correlated with eosinophilic count.
On the other hand, Nuijsink et al. (2007) found
weak inverse correlation between urinary EDN and FEV1 and borderline
correlation between urinary EDN and PD20 methacholine. No significant
correlation was found between urinary EDN and markers of eosinophilic airway
inflammation including % eosinophils or ECP levels in induced sputum and so
suggested that urinary EDN is not a suitable complementary marker of airway
inflammation in asthma.
The importance of differentiating subtypes of asthma specially in pre-school
age is critically useful as atopic asthma tends to persist in adult life, furthermore,
loss of lung function is more severe and early in atopic asthmatic patients,
that is why the atopic patients are more likely to require daily anti-inflammatory
medications (Chipps et al., 2007).
Second, assessment of severity of asthma was the scope of many studies. As
several eosinophil granular mediators were evaluated for assessment of severity
of asthma. Deposition of MBP has been detected around injured epithelium in
fatal asthma (Fujisawa et al., 1998). Also, MBP
was measured in bronchoalveolar lavage (BAL) and it was found that it alters
bronchial hyper-reactivity via activation of C fibers, bradykinins and interaction
with muscarinic receptors (Gleich, 2000). Its level was
found to be correlated with degree of inflammation, but for ethical purpose,
it is unpractical to do BAL on wide base. On the other hand ECP has been widely
studied to correlate its serum level to asthma symptoms, pulmonary functions,
bronchial hyperresponsiveness and response to therapeutic interventions. Results
showed major debate, as some concluded that ECP is a better marker for monitoring
and predicting clinical course of asthma than pulmonary function tests (Gleich,
2000; Sorkness et al., 2002), while others
concluded that it is a poor indicator of disease activity in chronic asthma
and can not differentiate it from nasal inflammation (Ferguson
et al., 1995). Regarding respiratory function
Bacharier et al. (2004) reported that in children and adolescents,
asthma severity classified by symptoms frequency and medications when categorized
according to the National Asthma Education and Prevention Program Guidelines
does not correlate with forced expiratory volume in 1 sec (FEV1).
Another problem in diagnosing and assessing the severity of asthma is that lung
function testing is often can not be performed in pre-school children.
In this study, it was found that serum EDN level was significantly correlated
with severity of asthma when study groups were categorized based on the frequency
of asthma symptoms and medication use. So, ECP might be a supportive biomarker
for evaluation of asthma severity. On the other hand, inspite of the wide variations
of serum level of IgE in the same groups, they did not mountain to statistically
significant differences between them.
Similar results were obtained by Kim et al. (2007),
as they found that serum EDN is directly correlated with severity of asthma
when study groups are categorized according to both GINA and Joseph-Bowen guidelines
and it was more sensitive than eosinophil cationic protein for severity of asthma.
Also, Ozol et al. (2008) evaluated the relationships
between asthma severity and total IgE in adult female and reported that there
is no statistically significant positive relationships between asthma severity
and total IgE levels and conclude that atopy does not influence asthma severity
in adult female patients.
Lastly, the diagnosis of asthma in children five years and younger presents
a particularly difficult problem. This is because episodic wheezing and cough
are also common in children who do not have asthma, particularly in those under
age of three. Wheezing is usually associated with a viral respiratory illness
predominantly respiratory syncytial virus in children younger than two years
of age and other viruses in older preschool children. This findings point to
the need for a specific biological marker which can diagnose asthma. EDN might
be a feasible biomarker for diagnosis of asthma in this age group. A matter
which necessitate further studies especially that an interaction between atopy
and viral infections appears to be a complex relationship, in which the atopic
state can influence the lower airway response to viral infections, viral infections
can then influence the development of allergic sensitization and interactions
can occur when individuals are exposed simultaneously to both allergens and
viruses (Bacharier et al., 2004).
In conclusion, this study suggest that serum EDN may be superior to IgE as a biomarker for evaluation of asthma and its outcome, as it could help to differentiate atopic from non atopic types in addition to its sensitivity to assess the degree of severity of asthma.
1: Amin, K., D. Ludviksdottir, C. Janson, O. Nettelbladt and E. Bjornsson et al., 2000. Inflammation and structural changes in the airways of patients with atopic and non atopic asthma. Am. J. Respir. Crit. Care Med., 162: 2295-2301.
2: Bacharier, L.B., R.C. Strunk, D. Mauger, D. White, R.F. Lemanske, Jr. and C.A. Sorkness, 2004. Classifying asthma in children, mismatch between symptoms, medication use and lung function. Am. J. Respir. Critical Care Med., 170: 426-432.
CrossRef | Direct Link |
3: Busse, W.W. and J.B. Sedgwick, 1994. Eosinophil eicosanoid relations in allergic inflammation of the airways. Adv. Prostaglandin Thromboxane Leukotriene Res., 22: 241-249.
4: Chipps, B.E., S.J. Szefler, F.E. Simons, T. Haselkorn, D.R. Mink, Y. Deniz and J.H. Lee, 2007. Demographic and clinical characrteristcs of children and adolescents with severe or difficult to treat asthma. J. Allergy Clin. Immunol., 119: 1156-1163.
5: Ferguson, A.C., R. Vaghan, H. Brown and C. Curtic 1995. Evaluation of serum eosinophilic cationic protein as a marker of disease activity in chronic asthma. J. Allergy Clin. Immunol., 95: 23-28.
6: Fujisawa, T., A. Terada, J. Atsuta, K. Iguchi, H. Kamiya and M. Sakurai, 1998. Clinical utility of serum levels of eosinophil cationic protein (ECP) for monitoring and predicting clinical course in childhood asthma. Clin. Exp. Allergy, 28: 19-25.
Direct Link |
7: Gleich, G.J., 2000. Mechanisms of eosinophil-associated inflammation. J. Allergy Clin. Immunol., 105: 651-663.
PubMed | Direct Link |
8: Guill, M.F., 2004. Asthma update: Clinical aspects and management. Pediatr. Rev., 25: 335-344.
PubMed | Direct Link |
9: Kim, K.W., K.E. Lee , E.S. Kim, T.W. Song, M.H. Sohn and K.E. Kim, 2007. Serum Eosinophil-Derived Neurotoxin (EDN) in diagnosis and evaluation of severity and bronchial hyperresponsivenees in childhood asthma. Lung, 185: 97-103.
PubMed | Direct Link |
10: Lúdvíksdottir, D., H. Hedenström, C. Janson, E. Björnsson and G. Strålenheim et al., 2000. Different airway responsiveness profiles in atopic asthma, nonatopic asthma and Sjögren's syndrome. Allergy, 55: 259-265.
11: Lúdvíksdottír, D., C. Janson M. Högman H. Hedenstöm, E. Björnsson and G. Boman, 1999. Exhaled nitric oxide and its relationship to airway responsiveness and atopy in asthma. Respir Med., 93: 552-556.
12: Magnaval, J.F. and A. Berry, 2005. Tropical eosinophilia. Cli. Infect. Dis., 40: 635-636.
PubMed | Direct Link |
13: Motojima, S., E. Frigas, D.A. Loegering and G.J. Gleich, 1989. Toxicicity of eosinophil cationic proteins from guinea pigs tracheal epithelium in vitro. Am. Rev. Resp. Dis., 139: 801-805.
Direct Link |
14: Noguchi, H., G.M. Kephart, T.V. Colby and G.J. Gleich, 1992. Tissue eosinophilia and eosinophil degranulation in syndromes associated with fibrosis. Am. J. Pathol., 140: 521-528.
15: Nuijsink, M., W.C.J. Hop, P.J. Sterk, E.J. Duiverman and P.S. Hiemstra et al., 2007. Urinary eosinophil protein X in children with atopic asthma. Mediators Inflamm., 2007: 49240-49240.
CrossRef | Direct Link |
16: Ozol, D., C. Koca, E. Mete and R. Yigitoğlu, 2008. Influence of atopy on asthma severity in adult female patients. J. Investig. Allergol. Clin. Immunol., 18: 36-40.
17: Pincus, S.H., K.S. Ramesh and D.J. Wyler, 1987. Eosinophils stimulate fibroblast DNA synthesis. Blood, 70: 572-574.
Direct Link |
18: Schlick, W., 1993. Current issues in the assessment of interstitial lung disease. Monaldi Arch. Chest Dis., 48: 237-244.
Direct Link |
19: Silfman, N.R., P. Venge, C.G. Peterson and G.J. Gleich, 1989. Human eosinophil protein X and human eosinophil-derived neurotoxin are likely the same protein. J. Immunol., 143: 2317-2322.
Direct Link |
20: Sorkness ,C., K. McGill and W. Busse, 2002. Evaluation of serum cationic protein as a predictive marker for asthma exacerbation in patients with persistent disease. Clin. Exp. Allergy, 32: 1355-1359.
Direct Link |
21: Zangrilli, J.G., J.R. Shaver, R.A. Cirelli, S.K. Cho and C.G. Garlisi et al., 1995. SVCAM-1 levels after segmental antigen challenge correlate with eosinophil influx, IL-4 and IL-5 production and the late-phase response. Am. J. Respir. Crit. Care Med., 151: 1346-1353.
22: Miller, M.R., J. Hankinson, V. Brusasco, F. Burgos and R. Casaburi et al., 2005. Standardisation of spirometry. Eur. Respir. J., 26: 319-338.
CrossRef | Direct Link |
23: GINA, 1995. Global strategy for asthma management and prevention. NHLBI/WHO Workshop Report, NIH Publication No. 95-3659, U.S. Department of Health and Human Services, Bethesda, MD., USA.