The Predictive Value of Eosinophil Cationic Protein in Asthma - Effect of Different Treatment Regimens.

 

Amina Hamed Ahmad Al Obaidi
Abdul Ghani Mohamed Al Samarai

Departments of Biochemistry and Medicine
Tikrit University College of Medicine
Tikrit
IRAQ

Abstract:


Background: Therapy of asthma may cause decline in eosinophil cationic protein and lung function improvement. During maintenance therapy, serum ECP levels may remain low, but sometimes the values increase with or without a simultaneous drop in lung function; symptoms frequently accompany such ECP increases.
Objective: The purpose of this study was to clarify the effect of different asthmatic therapy on ECP in levels and to evaluate their usage for follow up.
Method: Serum and sputum ECP was determined using enzyme linked immunosorbent assay.
Results: There was a significant reduction (P<0.0001) in serum ECP after treatment with immunotherapy for one year (20.1 µg/l ± 6.5) as compared to the baseline values (54.1 µg/l ± 10.6). The mean concentration of serum ECP was significantly higher during an acute asthma exacerbation (40.3 µg/l , P<0.0001) than when the patients were stable (18.7 µg/l) or when compared to control subjects (7.68 µg/l ). The reduction in serum ECP following treatment was lower for beclomethasone (14.4 µg/l;3 4.6%) as compared to oral prednisolone (21.6 µg/l; 53.5%). Furthermore, the improvement in FEV1 following treatment were lower for beclomethasone (10 ;12.2%) as compared to oral prednisolone (22 ;30.9%). After 4 weeks of treatment with acepifylline, the ECP level of sputum and serum decreased significantly. Systemic salbutamol treatment reduced serum ECP concentration significantly (P<0.001) from 52.1 µg/l at baseline to 34.3 µg/l after treatment In patients group with baseline serum ECP of 58.7 µg/l their post bronchodilator FEV1 less than 75% predicted percent. However, in patients group with baseline serum ECP concentration of 37.6 µg/l show better bronchodilator response (> 75%) following salbutamol treatment
Conclusion: This study provides further evidence that changes in serum ECP may serve as an objective indicator for clinical activity and results of treatment in allergic asthmatic patients. This data suggest that acepifylline exert anti-inflammatory action, but a long course of treatment may be needed to achieve a reduction in ECP lower than or equal to that of healthy control. Thus higher level of serum ECP at acute exacerbation was associated not only with more severe exacerbation as measured by FEV1, but also with a lower degree of bronchodilator responsiveness.
Immunotherapy may be a good adjuvant therapy for allergic bronchial asthma .

 

Introduction:


It is generally agreed that asthma is a chronic inflammatory disease of the respiratory tract. Throughout, just as researchers have done over the past years, asthma will variably be defined by symptoms suggestive of disease, by diagnosed disease and by physiological measures of airway responsiveness, including the bronchial response to histamine, methacholine or exercise, serial measurement of lung function and response to bronchodilators [1].


Although airway inflammation is of crucial pathogenetic importance in asthma, because direct access to the airways is rarely possible, alternative markers of airways inflammation are needed for diagnostic and therapeutic purposes. The allergic immune reaction and the associated inflammation involve many cells which release mediators contributing to the pathogenesis of the disorders [2]. In fact, antibodies and mediators have been used as diagnostic tools for many years. Mediators released from mast cells and eosinophilic granulocytes are becoming increasingly used as markers of the inflammation, and are finding their place in clinical practical work, whereas others, in particular mediators from the immune system such as the cytokines, the interleukins (ILs), and the cell membrane associated markers of differentiation ,(CDs), are still used only in research [2].


Late clinical reactions in asthma are closely associated with the release of ECP in serum. Serum ECP can be used as a measure of allergen provocation causing increased eosinophil activity [3,4]. Serum ECP can be used to assess the exposure to environmental allergens [5], or decreasing presence of allergen in the environment and the need for increase or reduction of therapy [6,7]. Patients with acute asthma exacerbation requiring emergency clinic visits usually have severe ongoing inflammation and increased serum ECP levels in relation to decreased lung function parameters [2]. With rescue medication, serum ECP levels decline rapidly and lung function improves, although in general more slowly. During maintenance therapy, serum ECP levels may remain low, but sometimes the values increase with or without a simultaneous drop in lung function; symptoms frequently accompany such ECP increases [8].
The purpose of this study was to clarify the effect of different asthmatic therapy on ECP in levels and to evaluate their usage for follow up. The study was approved by the ethics committee of our college, and written consent was obtained from all participating subjects.


Materials and Methods:


Patients:
The subjects included in the study were outpatients from the Asthma and Allergy Centre . The diagnosis of asthma was performed by specialist physician and was established according to the National Heart Blood and Lung Institute / World Health Organization (NHLBI/WHO) workshop on the Global Strategy for Asthma [9]. Patients were excluded if they were smokers, if they had respiratory infection within the month preceding the study, a rheumatological illness, malignancy, diabetic, heart failure, history of venous embolisms, coronary heart disease and liver or kidney diseases .
At enrolment, they all underwent full clinical examination, pulmonary function test, and blood sampling. Sputum samples were collected from patients when indicated. Normal volunteers were also enrolled in the study as a healthy control. None of them had any previous history of lung or allergic disease and were not using any medication. They had a normal lung function test (FEV1 > 80%) and negative skin allergy test. General stool examination were performed for all patients and control to exclude parasitic infections.
Asthma severity was classified according to the National Heart Blood and Lung Institute / World Health Organization (NHLBI/WHO) workshop on the Global Strategy for Asthma [9], and patients were classified as intermittent, persistent (mild, moderate or severe ) asthmatics. The severity of asthma was evaluated in a prospective manner, with documented clinical events, lung function values and treatment in the year preceding the study, as previously recommended [10]. Acute asthma exacerbation was defined as dyspnea and wheezing with or without increased coughing [2].


The sampling performed during the period from May 2004 to April 2007. All samples collected at morning following overnight fasting.
ECP measurement as for monitoring of acepifylline ( BP 250 mg tablet, Al Razi Laboratories, Aleppo, Syria) treatment was evaluated in 56 symptomatic asthmatic patients [21 patients defaulted]. All patients had not taken theophylline, corticosteroids, or leukotrines antagonist within 6 weeks before initiation of the study. The asthma symptoms and inhaled B2 agonist ( salbutamol) usage recorded throughout the study period. Before and at the end of the study venous blood and sputum samples were collected for determination of ECP. All patient received acepifylline 250 mg tablet three times daily for 4 weeks.


The effects of beclomethasone dipropionate inhaler (BP 50 ug inhaler, ALDO- UNION, Spain) were compared with those of oral prednisolone (BP 5 mg tablet, SDI, Samara) on marker of eosinophilic inflammation (ECP) in serum of steroid naive asthmatic patients with moderate asthma. A total of 150 patients with symptomatic moderate asthma, recruited from Asthma and Allergy clinic were included. All patients had not received anti inflammatory treatment in the last 3 months. At the time of the study, all patients had active symptoms and were on rescue bronchodilators only. At the screening visit, all patients underwent spirometry to determine FEV1. All patients used B2 agonist bronchodilator ( BP 100 µg inhaler, APOTEX Inc, Canada). There was a 2 week run in period (baseline) to demonstrate the degree of asthma severity. Eligible patients then entered an open label design, parallel group study in which they received either beclomethasone 1000 µg daily in 4 divided doses , or prednisolone 30 mg in three divided doses for 2 weeks. At the end of the study, 60 patients from prednisolone group, and 51 patients from beclomethasone group completed the study. The others , 15 from prednisolone and 24 from beclomethasone groups defaulted.
The relevance of inflammatory marker on the response to short acting B2 agonist (salbutamol) in acute asthmatics was assessed. Thus serum ECP levels were measured in 65 patients with asthma before and after 4 weeks of treatment with inhaled salbutamol 200 µg, four times daily for 4 weeks. After four weeks of treatment the patients divided into two groups: first group include 26 patients with FEV1 of less than 75% and second group include 39 patients with FEV1 of more than 75%. Venous blood samples collected at baseline and 4 weeks after initiation of therapy for determination of serum ECP. The effect of specific immunotherapy for one year on serum ECP was evaluated in 43 asthmatic patients.

Skin Prick Test:
The skin prick tests were performed and evaluated in accordance with European Academy of Allergy and Clinical Immunology subcommittee on allergy standardization and skin tests using standards allergen panel ( Stallergen, France) . The panel included the common inhalant allergens.
Sputum Collection:
Sputum was induced only when it could not be produced spontaneously. Sputum induction was performed as described by Fahy et al [11].
Determination of Serum Eosinophil Cationic Protein :
Serum ECP determined by ELISA kit (MBL MESCACUP ECP TEST) from Medical and Biological Laboratories Co , LTD, Japan. This ELISA detects human ECP with a minimum detection limit of 0.125 ng/ml. The test performed according to the instruction of manufacturer. Briefly, In the wells coated with antihuman ECP monoclonal antibody, 100 µl of diluted serum samples ( 1:5 sample diluent) or standards were added and incubated for 60 minutes at room temperature (20 - 25 0C ). After washing for 4 times, a 100 µl of peroxidase conjugated antihuman ECP polyclonal antibody is added into the wells and incubated for 60 minutes at room temperature. After another 4 times washing, a 100 µl of peroxidase substrate reagent is added to each well and the plate incubated for 10 minutes at room temperature. The add 100 µl of stop solution ( 0.5 mol/l H2SO4) and read the absorbance at 450 nm using a microplate reader. The concentration of ECP is calibrated from a standard curve based on reference standards.

Statistical Analysis
The values are reported as mean ± SD and 95% confidence interval. For statistical analysis between groups paired t test was used . Pearson test was used for correlation analysis. The levels of each marker were compared between the study groups and control group, using SPSS computer package. P values of < 0.05 were considered significant.

Results:


Effect of Specific Immunotherapy on Serum ECP in Asthma:
There was a significant reduction (P<0.0001) in serum ECP after treatment with immunotherapy for one year (20.1 µg/l ± 6.5) as compared to the baseline values (54.1 µg/l ± 10.6). However, the post treatment serum ECP level still significantly higher (P<0.0001) than that of control. Immunotherapy causes a reduction of 63% of serum ECP (Table.1). Furthermore, there was a significant increase in FEV1 (P<0.0001) following immunotherapy and the change was 32% from that of baseline values. In conclusion, immunotherapy may be a good adjuvant therapy for allergic bronchial asthma .
Effect of Prednisolone and Inhaled Corticosteroids (ICS) on Serum ECP in Asthma:
The mean concentration of serum ECP was significantly higher during an acute asthma exacerbation (40.3 µg/l ± 16.2, P<0.0001) than when the patients were stable (18.7 µg/l ± 5.3) or when compared to control subjects (7.68 µg/l ± 5.63). The change in serum ECP following prednisolone therapy was 53.5%. Although, there was a significant reduction in serum ECP after treatment with prednisolone still the level was significantly higher (P<0.001) than that of control (Table.2). FEV1 increased (30.9%) following prednisolone therapy, the change was highly significant (P<0.0001) but still significantly lower (P<0.001) than that in control. Comparison of oral prednisolone (30 mg / day) with inhaled beclomethasone (1000 µg/ day) for 2 weeks treatment course indicated effectiveness of both in improvement of FEV1. Furthermore, beclomethasone causes a significant reduction (P<0.002) in serum ECP from 41.6 µg/l (± 12.7) at baseline to 27.2 µg/l (± 7.6) after treatment for 4 weeks. FEV1 increased from 82 (±9) predicted percent at baseline to 92 (±7) predicted percent following treatment with ICS (Table.2). However, the reduction in serum ECP following treatment was lower for beclomethasone (14.4 µg/l; 34.6%) as compared to oral prednisolone (21.6 µg/l; 53.5%). Furthermore, the improvement in FEV1 following treatment were lower for beclomethasone (10 ;12.2%) as compared to oral prednisolone (22 ;30.9%).
This study provides further evidence that changes in serum ECP may serve as an objective indicator for clinical activity and results of treatment in allergic asthmatic patients.

Effect of Acypifylline on Serum and Sputum ECP in Asthmatic Patients:
The study is performed to investigate the anti-inflammatory effect of acepifylline, a new version of theophylline, in asthma. The level of ECP in sputum of asthmatic subjects at baseline was much higher (703 µg/l ± 256) than that in healthy control ( 136.5 µg/l ± 42.2, P<0.0001). After 4 weeks of treatment with acepifylline, the ECP level of sputum decreased to (231 µg/ l ±191), with high significance (P<0.0001) (Table.3).
The serum ECP of asthmatic patients also was reduced from 49.7 µg/l (±19.5) at baseline to 29.8 µg/l (±13.8) post treatment with highly significant difference (P<0.0001). However both sputum and serum ECP was reduced following treatment with acepifylline, still their post treatment values were significantly higher than that in control (P<0.001, P<0.0001 respectively) (Table.3). This data suggest that acepifylline exert anti-inflammatory action, but a long course of treatment may be needed to achieve a reduction in ECP lower than or equal to that of healthy control.

Effect of Salbutamol on Serum ECP in Asthmatic Patients and its Influence on Bronchodilator Activity:
Systemic salbutamol treatment reduced serum ECP concentration significantly (P<0.02) from 52.1 µg/l (±19.6) at baseline to 34.3 µg/l (± 11.2) after treatment (Table.4). In patients group with baseline serum ECP of 58.7 µg/l their post bronchodilator FEV1 less than 75% predicted percent. However, in patients group with baseline serum ECP concentration of 37.6 µg/l show better bronchodilator response (> 75%) following salbutamol treatment (Table.5). Furthermore, serum ECP concentration after treatment with salbutamol still significantly higher than that in control group (P<0.0001). Thus higher level of serum ECP at acute exacerbation was associated not only with more severe exacerbation as measured by FEV1, but also with a lower degree of bronchodilator responsiveness.

Discussion:


Asthma is a multifactorial disease with many triggers such as allergens, respiratory infections, smoking and psychological stresses [12]. Immunotherapy acts selectively by increasing tolerance to specific antigens to which individuals demonstrate clinical sensitivity [13]. Although, immunotherapy may not replace pharmacotherapy for asthma, there is emerging evidence that immunotherapy can have important effects by modulating the immune system [14]. Indeed, Immunotherapy can alter the production of histamine releasing factors, decrease release of inflammatory mediators by cells following antigen challenge, cause shift in the production of antigen specific and total IgE and IgG and alter T cell subsets [14]. Thus the immune modulation achieved with immunotherapy can potentially provide a long term solution to the bronchial reactivity encountered in asthma.


In fact, the anti – inflammatory potential of immunotherapy measured in the present study is the determination of serum ECP. There was a significant reduction in serum ECP following treatment with immunotherapy for one year as compared to baseline value. The immunotherapy induced a 63.3% reduction in serum ECP. Recently, Kandil et al [15] reported that immunotherapy cause a significant reduction in serum and sputum ECP. Other reported studies [16,17] indicated that immunotherapy for seasonal asthma prevent rises of serum ECP during pollen season and the difference was significant as compared to untreated patients. Thus ECP may be a marker of eosinophilic activity and degranulation that correlates with the severity of bronchial asthma. Hence serum ECP may be a good marker for evaluation of anti inflammatory potential of immunotherapy.
Although there was a significant reduction in serum ECP following immunotherapy, the post treatment values are still significantly higher than those in control. This means that immunotherapy reduced serum ECP following treatment, but it is not so much an effective restitute on the inflammatory responses. So this finding presumed that long course of immunotherapy was needed or adjuvant pharmacotherapy was warranted. To decide which one is effective, clinical trials to evaluate therapeutic effect of long course immunotherapy or combination of pharmacotherapy plus immunotherapy are to be conducted.


An important finding of this study was that immunotherapy for one year course cause significant improvement in FEV1 in association with the reduction of serum ECP. It is now well established that asthma is associated with airway inflammation [18]. ICS are the drug of choice in the treatment of asthma because they provide the best anti – inflammatory treatment available [9]. In this respect, ICS improve asthma symptoms and pulmonary function [1,19], and have been shown to decrease airway inflammation [20,21]. Eosinophils play an important role in the inflammatory events of allergic asthma. Serum ECP is a marker of disease activity and of treatment efficacy in bronchial asthma.


In the present study it was shown that inhaled beclomethasone, at a daily dose recommended by the international guidelines for asthma treatment [9] decreases eosinophil inflammation in blood as measured by serum ECP. However, oral prednisolone (30 mg/day) was with higher significance in reducing serum ECP as that caused by beclomethasone. Also FEV1 improved significantly by both, but the changes in FEV1 was more significant in oral prednisolone treated patients.


Previous studies have reported the effect of ICS or oral corticosteroids on airway inflammation in asthma [22-29]. Bacci and coworkers [30] showed that inhaled beclomethasone at the same dose that is used in this study reduced serum ECP significantly. Fahy and Boushey [31] showed that low steroid doses (168 µg bid) improved clinical and functional data, but had only mild effects on airway inflammation. Jatakenon et al [32] studied a group of mild asthmatic patients treated with very high steroid doses (800 µg bid) and found a significant improvement in both airway function and serum ECP but not in sputum ECP. In another study, the same authors compared the same group of patients with asthmatic patients of a similar degree treated with lower doses of inhaled steroids, and also found that 400 µg budesonide, but not 100 µg significantly improved airway inflammation and lung function [32]. In a study including large number of patients, Meijer et al [27] compared the effects of two different doses of inhaled fluticasone(1000 or 250 µg bid) and oral prednisolone (30 mg daily )on lung function and airway inflammation. They showed that inhaled steroids were at least effective as oral steroids and that the lowest dose of fluticasone had far less systemic effects than the other two treatments. The same finding achieved in the present study concerning inhaled beclomethasone (500 µg bid) and oral prednisolone (30 mg daily). Thus it is now well established that adequate doses of inhaled steroids effectively improve eosinophilic airway inflammation. Beclomethasone inhaled treatment evaluated in this study, causes improve in FEV, but the improvement was lower than that caused by oral prednisolone. However, both inhaled beclomethasone and oral prednisolone decreases serum ECP significantly, still its values were significantly higher than that for control subjects. This means that to achieve a serum ECP level similar or about to be similar to that of control subjects may need longer duration or combined therapy to be suggested.


Oral prednisolone has a greater effect on systemic inflammatory parameters as evaluated by serum ECP. The effect of oral prednisolone on serum ECP was more than that achieved by inhaled beclomethasone. Meijer et al [27] reported that oral prednisolone and inhaled fluticasone at dose of 1000 µg bid caused significant decrease in serum ECP. Tang and Chen [28] and Pizzichini et al [29] reported that oral prednisolone caused significant reduction in serum ECP. The improvement in FEV1 was associated with reduction of serum ECP as our findings indicated and this finding was in consistent with that reported by others [27,30,33].


Inhaled beclomethasone (500 µg bid) reduced significantly serum ECP in asthmatic patients as this study indicated. This might suggest both a direct and indirect effect of beclomethasone on serum ECP. The former consists of a reduction in airway inflammation resulting in lower levels of cytokines, chemokines, and a decreased attraction of blood eosinophils into the lung [34]. Indirect effects of beclomethasone - that is, after systemic absorption from the lung tissue – occur in peripheral blood and bone marrow with suppression of both progenitors of eosinophils in blood and colony forming unit production in the bone marrow, both of which result in reduced numbers of blood eosinophils [35,36]. It is also known that inhalation of high doses of corticosteroids reduces the number of blood eosinophils by increased apoptosis [37]. Our findings are in consistent to statements in international guidelines [9] in which oral prednisolone in a dose of 30 mg / day is regarded as maximum therapy for asthma control at the start of the treatment.


Theophylline is used worldwide for the treatment of asthma and reported studies indicate that theophylline has anti – inflammatory effects [38]. Theophyllin has been shown to inhibit eosinophil degranulation and release of eosinophil basic protein [39], prevent the release and expression of tumor necrosis factor and IL-1B from blood monocytes [40,41], and reduce IL-2 production by T cells and IL-2 dependent T cell proliferation [42]. The use of theophylline, however, has declined owing to the widespread use of inhaled steroids, which remain the most effective treatment for asthma. One of the limitations of theophylline in the past has been the side effects observed in many patients at the traditional bronchodilator doses associated with plasma levels of theophylline between 10 and 20 mg/l. However, anti – inflammatory benefit appear to occur at lower plasma theophylline levels (<10 mg/l), and the incidence of any adverse effects is minimized.


Two studies have demonstrated that low dose of theophylline added inhaled steroids was equally efficacious when compared with increasing the dose of inhaled steroids, in symptomatic patients established on inhaled steroids therapy [43,44]. Previous studies have demonstrated that low dose theophylline reduced the increase in eosinophils in airway biopsies after allergen challenge [45], and withdrawal of theophylline in patients with severe asthma was associated with increase in airway T lymphocytes [38].
Generally, Acepifylline, theophylline salts used in the treatment of asthma. The anti-inflammatory effect of this drug was evaluated in patients with asthma. In this study, the administration of acepifylline (250 mg tds) to 15 asthmatic patients resulted in a significant reduction in serum and sputum ECP. However, the values for both after treatment still significantly higher than that of control. This indicates that the drug therapy for 4 weeks was not enough to reduce serum and sputum ECP within the range of normal control. Horiguchi et al [46] reported that administration of Theophyllin for 8 weeks markedly decreased serum and sputum ECP than that of 4 weeks administration, suggesting that the longer the period of treatment, the more distinct are the anti- inflammatory effects of theophylline.
Basically, our findings indicate that acepifylline had an inhibitory effect on eosinophilic inflammation. This evidence showed that acepifylline was beneficial in the management of asthma by partly acting as an anti – inflammatory drug. A reported studies indicate the anti- inflammatory effect of theophylline [46-51]. In contrast, Basyigit et al [50] reported that theophylline uses in patients with asthma not caused significant reduction in serum and sputum ECP. Surprisingly, they reported that corticosteroids, also not lead to a significant reduction in serum ECP. Aizawa et al [51] reported that 4 weeks treatment with once daily theophylline of subjects with asthma significantly reduced serum ECP. In conclusion, acepifylline in a dose of 250 mg tds is a useful anti – inflammatory agent for chronic inflammation of asthma.
Acute asthma primary therapy was repeated administration of a sympathomimetic bronchodilator to rapidly reverse the airway obstruction [9]. Responsiveness to this therapy, however, varies widely between individuals [1]. In view of the fact that widespread airway obstruction in acute asthma results from bronchial smooth muscle spasm and / or airway inflammation caused by infiltration of inflammatory cells, mucosal oedema and mucus plugging, the extents to which bronchconstriction or airway inflammation contribute to airflow obstruction has been proposed to determine a lower responsiveness to bronchodilator therapy [2]. Eosinophils were the major inflammatory cells infiltrating the mucosa and sub mucosa of patients with bronchial asthma [52]. An increased number of eosinophils are often found in blood and sputum and relationship between this and severity of asthma has been determined [52,53].
Eosinophils are potent effectors cells contributing to inflammation by the release of chemical mediators and cytotoxic granule proteins such as ECP, EPO, and major basic protein [3]. On the other hand T lymphocytes have also received much attention because, once activated, they orchestrate inflammatory reactions by secretion of cytokines that induce the activation and proliferation of other inflammatory cells, inducing eosinophils, basophiles, neutrophils and macrophages [5,54,55]. A direct assessment of inflammation by means of bronchial biopsies or BAL is not feasible in daily clinical practice, especially during an acute exacerbation of asthma, hence, the interest in less invasive markers of inflammation. One of the specific markers that have been most extensively investigated is ECP, the reason obviously being that eosinophil has been given a key role in pathophysiology of asthma. During acute severe asthma, increased numbers of activated blood T cells and a subpopulation of low density eosinophils with increased cytotoxic activity are found [8,56,57]. The relationship between airway inflammation and responsiveness to bronchodilator therapy is insufficiently known. Furthermore, it is not clear whether elevated concentration of serum ECP reduce the response of the patient’s bronchodilator therapy.
This study demonstrated that patients with acute exacerbation of asthma show high serum levels of ECP and more interestingly, that the response to bronchodilator is higher in patients with lower serum ECP levels. This finding was in consistent with that reported by Di Lorenzo [57]. Several studies have demonstrated that serum levels of ECP are elevated in patients with asthma [8,58-66]. Despite having been used for a number of years, the exact role for serum ECP measurements in asthma is still a matter of debate. The degree of airway inflammation in asthma is a dynamic phenomenon. Exposure to factors known to affect the degree of airway inflammation is reflected in changes in serum ECP levels [57].
ECP levels increase during the pollen season or following experimental allergen challenge as this study indicated and that reported before [16,67]. However, a number of studies have failed to find a correlation between ECP and other indices of disease activity in asthma such as symptom score, baseline FEV1 or the degree of bronchial responsiveness [68-70]. Serum ECP levels represent the propensity of eosinophil to release this granule protein during the clotting process. This is considered to reflect the degree of activation of eosinophils, implicating that serum ECP levels might contain additional information, in comparison to mere eosinophil counts [71,72-75]. Furthermore, ECP and eosinophil values might not correlate because a normal or moderately high blood eosinophil count may represent the result of sequestration of eosinophils in the lung during acute exacerbation of asthma [76].
In general, this study demonstrates that levels of serum ECP were higher in patients with a lower percentage of FEV1 predictive value. Besides there was a correlation between serum ECP levels and FEV1 predicted percent. More interesting was the relationship between high initial levels of serum ECP and the degree of airway response to salbutamol. The drug also reduced serum ECP significantly following 4 weeks course, however, the level of serum ECP still significantly higher than control. Therapy for acute exacerbation according to the modified guidelines of the National Institute of Health is based on short acting beta 2 agonist [9]. However, several patients do not show complete crises remission following this therapy. Thus acute asthma exacerbation may be due to both the inflammatory and non inflammatory mechanisms [1,2]. This study results suggest that an inflammation reactivation ( as demonstrated by high levels of serum ECP ) may be responsible for the lack of response to short acting Beta 2 agonist, suggesting that in these patients treatment with steroids that induce eosinophils apoptosis is indicated [77].
Lee et al [66] reported that serum ECP levels during asthma exacerbation inversely correlated with bronchodilator responses of salbutamol therapy. A higher level of serum ECP at acute asthma exacerbation was associated not only with more severe exacerbation but also with a lower degree of bronchodilator responsiveness [66]. These study findings and that of others [57,66] suggest that degree of airway inflammation may be one determinant of degree of responsiveness to initial bronchodilator therapy at acute asthma exacerbation.
In the present study there was an inverse correlation between FEV1 predicted percent and serum ECP levels at baseline for specific immunotherapy, oral prednisolone and acepifylline treated groups. However, there was a significant inverse correlation for all three treatment regimens following therapy, but the level of significance and r values were lower than that at baseline. The explanation for this was that asthma developed in a multi stages with different physiological / pathological processes dominating each stage [78]. In chronic asthmatic the airway hyperreactivity and inflammation quantitatively and qualitatively different from acute stage [78]. Thus the three treatment regimens suppress the inflammation at the beginning of the treatment, but the restore of the airway need a longer time and the correlation in lung function appears to be an adaptive response on the part of the airway tissue, resulting from failure to resolve shortly in consistent with resolving of inflammation.
On the base of our results, the percent of reduction in serum ECP significantly inversely correlated with FEV1 for specific immunotherapy, oral prednisolone and acepifylline. Furthermore, the increase in FEV1 was inversely correlated with the amount of reduction of serum ECP in asthmatic patients following specific immunotherapy, oral prednisolone, and acepifylline. This correlation indicates the anti – inflammatory activity of all the three treatment approach and the role of ECP in the pathogenesis of asthma.


Table:1. Effect of specific immunotherapy [SIT] on serum eosinophil cationic protein (ECP, µg/l ) in asthmatic patients.

Variable

ECP

FEV1

Mean

SD

95%CI

Mean

SD

95%CI

Asthmatic (43)

Baseline

54.10

10.6

50.83- 57.37

76

5

74-78

Post treatment

20.10

6.5

18.10-22.10

100

2

99-101

  

P value    

0.0001

0.0001

Percent           

Reduction

63.3

9.2

60.21-66.39

32.2

7.6

29.65-34.75

Control  (50)

7.68

5.63

6.08-9.68

101

3

100-102

Table.2. The effect of prednisolone and inhaled corticosteroids on serum eosinophil cationic protein (ECP, ug/l).

Variable

 

  Prednisolone 

Mean       SD        95% CI

    

Beclomethason

Mean       SD        95% CI

ECP

  

Baseline

 

 Post- treatment

 

  P value     <

40.3        16.2      36.12-44.28

18.7         5.3       17.3-20.1

0.0001

41.6        12.7    39.6-45.6

27.2        8.8    26.2-28.2

0.002

Control

7.68        5.63       6.08-9.68

7.68        5.63       6.08-9.68

ECP reduction

21.6      11.7     18.6-24.6     

14.4       9.8      11.7-19.8

ECP percent

        Reduction

53.5     16.4        49.3-57.7

34.6       19.1     29.6-39.6

FEV

  Baseline

 Post- treatment

   P value      <

71         11          68-74

93           8          91-95

0.0001

82           9          80 - 84

92           7          90 - 94

0.001

Control

101        3          100-102

101         3          100-102

FEV increase

22         7.6        20 – 24

10           2          129 – 11

FEV percent

Increase

30.9     13.6       26.9-34.9

12.2          2.3        11.2 – 13.2

 

Table:3. Effect of acepifylline on serum and sputum eosinophil cationic protein (ECP, µg/l) in asthmatic patients.

Sample

Eosinophil cationic protein concentration

P value

<

     Before treatment

 Post treatment

Sputum

       Patients

               Mean

                SD

               95%CI

    

       Control

               Mean

               SD

               95% CI

P Value  <

             

              703       

               256

            635 –771

       

136.50

42.2

119.2-153.96

0.001

     

        231  

        191 

205 – 257

0.0001

       Serum

    Patients

         Mean

         SD

         95%CI

       Control

         Mean

         SD

         95%CI

P Value  < 

   

             49.7

             19.5

        44.5-54.9

             

              7.68

              5.63

           6.08-9.68

             0.0001

        

         29.8

         113.8

26.12-33.48

0.0001


Table:4. Effect of salbutamol on serum eosinophil cationic
protein (ECP, µg/l) in asthmatic patients.

          ECP  serum concentrationBefore treatment         After treatment

P value <

Mean

SD

95% CI

52.1                         34.3

19.6                           11.2

    

       49.9-54.68                     31.5- 37.1

0.001

 

Table:5. Influence of serum eosinophil cationic protein
(ECP, µg/l) on salbutamol bronchodilator response.

Post bronchodilator FEV1

No. of patients

Serum ECP

Less than  75

26

58.7

More than  75

39

37.6

P value  <

0.001

  

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First Published May 2007

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