Evaluation of blood eosinophilia and the eosinophil cationic protein (ECP) in the serum of asthmatic children with varying degree of severity
B. Vilá-Indurain, F. Muñoz-López and M. A. Martín-Mateos
Immunology and Paediatric Allergy Department. Hospital Clínico «San Juan de Dios». Faculty of Medicine. University of Barcelona.
Correspondence:
B. Vilá-Indurain
Dpto. de Alergia Pediátrica e Inmunología
Hospital Sant Joan de Déu
Pg. Sant Joan de Déu, 2
08950 Esplugues de Llobregat
Barcelona
SUMMARY
The inflammatory reaction is the main pathogenic mechanism of asthma: as the intensity of inflammation may be related with disease severity, it would be useful to have an easily obtained marker indicating the intensity of inflammation in patients. The best markers are obtained from bronchoalveolar lavage fluid (inflammatory cells and cytochemical mediators), but this technique is not often performed in children. In peripheral blood, eosinophils and eosinophilic enzymes have been related with bronchial inflammatory reactions and their levels have been shown to decline after corticoid treatment by inhalation.
Objective: To determine if blood eosinophil count and eosinophilic cationic protein content (ECP) in serum are related with the intensity of bronchial inflammation and disease severity in asthmatic children.
Material and methods: In a selection of children with bronchial asthma and Dermatophagoides pteronyssinus (D. Pt.) sensitivity, the following groups were made: group 2, 19 children with favorably evolving asthma and normal spirometric values (FEV1 and FMF25-75); group 3, 13 children with below-normal expiratory flow rates that normalized with salbutamol inhalation; group 4, 14 children with below-normal expiratory flow rates who did not recover after the bronchodilation test, suggesting more intense bronchial inflammation. The control group (group 1) contained 11 healthy subjects with negative allergy and respiratory function tests. All participants underwent bronchial challenge with D. Pt. extract. Serum eosinophil count and ECP were evaluated before challenge and 3 hours later. Respiratory function was watched for the next 24 hours.
Results: There were significant differences in the eosinophil count and ECP between the control group and the three patient groups. Values differed before and after challenge in all groups. However, there were no significant differences between the three groups of asthmatic children.
Conclusions: Although the evolution of serum eosinophil count and ECP can be used to differentiate between children with and without asthma, they are not useful for judging the intensity of the bronchial mucosa inflammatory reaction.
Key words: Childhood asthma. Eosinophil count. Eosinophil cationic protein. Bronchial inflammation. Severity of asthma.
OBJECTIVE
Since the above-mentioned severity and persistence of inflammation may be related to the persistence of asthma, it would be of interest and beneficial to have some readily obtainable parameter to indicate the degree of the inflammatory reaction that may be connected with the clinical state of the child with asthma so that we may detect chronicity of the disease early on and thereby adopt suitable therapeutic measures. This was the purpose for which we carried out this study, in which we analyze haematic eosinophil content and eosinophil cationic protein (ECP) in the serum of asthmatic children, who were arranged in three groups according to the degree of severity of the disease. We evaluated these parameters before and after making them inhale an extract of dust mites to which they were sensitized.
MATERIAL AND METHODS
Patients
We included a total of 57 children of both sexes with ages ranging between 8 and 18 years. They were arranged in 4 different groups:
Group 1 (control): 11 healthy children with no clinical suspicion of allergy disease. The IgE levels were normal for their age and the RAST with home dust mites was below 0.35. Skin tests gave negative to common allergens and no bronchial hyperactivity was exhibited as a result of inhalation of methacholine.
Groups 2, 3 and 4: 46 children suffering from extrinsic bronchial asthma with dominant sensitization to Dermatophagoides pteronyssinus. All these children had been receiving care at our department for at least a year and the allergological and respiratory function study, including the methacholine test, had been performed on them at the outset of their attendance. The children were arranged in 3 groups according to the severity of the respiratory function at the outset of this study. The criteria for inclusion in these three groups were: clinical symptoms indicative of bronchial asthma, total serum IgE > 2SD above the mean for their age, skin tests and RAST positive to D. pteronyssinus; almost all the cases were also positive to Dermatophagoides farinae. A few children were also sensitized to other allergens. The distribution of the groups was as follows:
Group 2: 19 children whose basal inter-crisis FEV1 values were >= 80% of the normal values for their height and age. None of them exhibited obstruction of the peripheral airways (small airways) and the FMF25-75 value was equal to 60% of the normality values or higher.
Group 3: 13 children whose FEV1 and FMF25-75 between episodes were lower than 80% and 60%, respectively, of the prediction values for their height and age, with demonstrated obstruction of the major and peripheral airways. The bronchodilator test was performed on then with inhaled salbutamol (200 µg) with spirometric monitoring 15-20 minutes after. The test was considered positive if the basal FEV1 value rose at least 15% above the initial value. This bronchial obstruction reversibility was connected with a possible non-significant degree of inflammation.
Group 4: 14 children with a basal spirometry criterion similar to that of group 3. In these children, however, the increase in FEV1 after salbutamol inhalation was less than 15%, from which we deduced that there was more significant inflammation with greater bronchial rigidity.
We performed the same tests on all the children in the four groups, complying with all the following requirements when conducting them: 1) No manifest symptoms at the time of the study in a normal physical examination. 2) No lower airway infections or asthmatic crises suffered in the previous 6 weeks. 3) Withdrawal of all prior treatment, if any, at least 48 hours before carrying out the study. The signed acceptance of the parents or guardians and of the actual patients themselves if over 12 years old was obtained in all cases.
Bronchial challenge test
This was performed with a Bronchoscreen dosimeter (Jaeger) and the spirometric controls were done with a Pneumoscope (Jaeger). We employed a standardized Dermatophagoides pteronyssinus extract in HEP units (Laboratorio Leti) at an initial concentration of 100 HEP/ml (equivalent to 100,000 Biological Units: BU), obtaining the following concentrations by means of dilution with a physiological saline solution: 0.02, 0.1 and 10 HEP/ml. The test was commenced by carrying out ten inhalations of physiological saline after an initial spirometry, which was repeated 10 minutes after inhalation and then followed up with pulmonary auscultation. If the FEV1 did not drop more than 10%, the test was continued. Mite concentrates were then inhaled, beginning with the highest dilution (0.02 HEP/ml) in volumes of 0.005 ml and continuing with the following concentrations, until the FEV1 dropped 20% (or slightly higher than this figure) (PD20) or a final cumulative dose of 4,428 BU (below the so-called "maximum safety threshold") (1) was reached without a drop in the FEV1 occurring, in which case the test was considered negative. The spirometric controls and auscultation were performed 10 and 30 minutes after each inhalation. The approximate duration time of each test ranged from two to two and a half hours. All the tests were started at around 10 a.m.
Eosinophil Cationic Protein (ECP) Evaluation
This was done in blood serum by means of a radioimmunoassay adhering to the manufacturer''s (Kabbi-Pharmacia) directions, in accordance with a procedure put forward by Peterson (2). The blood serum was separated from the blood sample and frozen at -40° C in a maximum period of 140 minutes post-extraction, in compliance with the maker''s instructions.
Blood eosinophil count
This was done by flow cytometry.
Statistical study
We started off with the descriptive study of every one of the groups, calculating the frequency distributions of the values analyzed and the parameters defining them: means, standard deviation (SD), maximum and minimum values, and frequencies of each group. The analytical study was carried out by relating values to one another in each group and comparing values between the different groups. For making comparisons between the variables we used Student''s t-test or variance analysis, in the case when the variables were adjusted to a standard distribution. The chi-square test (*2) was carried out to study dependence between the qualitative variables. The relationship between the quantitative values was analyzed by means of Pearson''s correlation coefficient.
Study design
1) Patient selection according to the afore-mentioned rules. 2) Spirometry was performed to assure FEV1 stability, while current normality was confirmed by auscultation. 3) Blood extraction by antecubital vein puncture to determine eosinophilia and ECP. 4) Bronchial challenge with D. pt. extract. 5) Three hours after completing the challenge, another blood sample was obtained in order to repeat the same determinations. 6) Clinical and spirometric controls after 3, 24 and 48 hours. Peak- flow evaluation (PEF) every hour for the 12 hours after challenge and every 3 hours thereafter up to 24.
RESULTS
Maximum and minimum values, the mean, the SDs and the degree of significance corresponding to the eosinophils and ECP are shown in figures 1 and 2. In them we may see the following: Eosinophils: in the basal values prior to the challenge no significant differences were observed between the groups of asthmatic children, although there were between these three groups and the control group. After bronchial challenge the asthmatic children underwent a significant drop in the number of eosinophils, which did not take place in the control group. No significant difference was observed, however, between the three asthmatic groups.
Figure 1.--Eosinophils before and after bronchial provocation test.
Figure 2.--ECP Before and after bronchial provocation test.
Cationic protein: there were significant differences in the basal ECP values between the control group and the three groups of asthmatics and, as with the eosinophils, no difference was detected between these three groups. After bronchial challenge a significant drop was recorded in the three groups of asthmatics in relation to the control group, although there was no notable difference between them.
DISCUSSION
Eosinophils are largely responsible for the bronchial mucosa lesion and, therefore, for the inflammatory reaction that takes place as a result of the allergic reaction at that level (3, 4). Various proteolytic enzymes segregated by the eosinophils are the ones that cause the epithelial damage, primarily ECP, PMB, EPX and EPO (5). We know that during the acute phase of the allergic reaction a rise occurs both in the number of eosinophils in peripheral blood and of their enzymes. But the accumulation of cells is higher in the tissues in which the reaction takes place (6, 7). This fact has been confirmed repeatedly in the product obtained by bronchoalveolar lavage both in adults and in children, even though studies at the paediatric age are limited (8, 9).
Some research have been aimed at studying the eosinophil and ECP content in peripheral blood (10) and it has been found that both diminish after treatment with inhaled corticoids, which indicates that their determination could prove useful for verifying the efficacy of medication and favourable evolution of the process, as this fact is related to reduction in bronchial inflammation (11, 12). This means that there may be a certain correlation between blood eosinophilia and serum ECP and the concentration of both in the bronchial mucosa. One study indeed deals with the possibility of evaluating the ECP in other media, such as nasal secretion and urine, for its possible correlation with serum and bronchial content, so that its evaluation may be less distressful in paediatric patients (13).
The result of our study shows that there is a clear difference between eosinophil and ECP content in non-atopic/non-asthmatic and asthmatic children. Although this fact is shown by numerous studies, discrepancies have been found in some trials carried out in adults (14). The main aim of our research was to see whether the haematic content of the parameters studied could be used to distinguish moderate asthma with reversible airway obstruction from severe asthma, considering severity as a function of diminished or no reversibility of the obstruction, which was the criterion that was used to separate patients into the three groups studied. It could be seen that there were no differences in the basal parameter determinations between the three groups. After provocation, in statistical terms there was a decrease both in eosinophils and in ECP, although we found that there were differences between the patients included in each group, since the figures increased in some cases, while in others they remained stable (results not included in the results section). Wohn et al (15) studied the same parameters, before and after challenge with Dermatophagoides pteronyssinus, finding an increase at 30 minutes after provocation, followed by a sharp drop in another 30 minutes, with a subsequent levelling off of the figures. In our study the results obtained 3 hours after provocation present declining figures in this phase. Gruber et al (16) study serum ECP in relation with provocation with cold air and histamine and they reach the same conclusion, namely the lack of correlation between serum ECP content and bronchial reactivity.
In conclusion, it may be contended that, although evaluation of eosinophilia and serum ECP may discriminate between asthmatic and non-asthmatic children, these parameters are not useful for confirming the degree of inflammation and broncholability of paediatric asthma patients.
RESUMEN
La reacción inflamatoria es la principal base patogénica del asma; el grado de la inflamación puede estar relacionado con la gravedad de la enfermedad, por lo que sería conveniente disponer de un marcador fácil de obtener, que pudiese relacionarse con la intensidad de la inflamación en cada paciente. El mejor marcador se obtiene en el producto del lavado broncoalveolar (células y mediadores citoquímicos de la inflamación), pero en la edad infantil, esta molesta técnica no es de uso habitual. En sangre periférica se estudian los eosinófilos y sus enzimas, que se han relacionado con la reacción inflamatoria en los bronquios, habiéndose comprobado la disminución de los niveles tras el tratamiento con corticoides inhalados.
Objetivo: comprobar si el contenido de eosinófilos en sangre y de la proteína catiónica de los eosinófilos (PCE) en suero sanguíneo está en relación con la intensidad de la inflamación bronquial y la gravedad de la enfermedad en los niños asmáticos.
Material y métodos: se han seleccionado niños afectos de asma bronquial, sensibilizados a Dermatophagoides pteronyssinus (D. Pt.), que se han agrupado así: 19 niños con asma de buena evolución, y valores espirométricos (FEV1 y FMF25-75) normales (grupo 2); 13 niños cuyos flujos espiratorios estaban por debajo de los valores normales, pero se normalizaron al inhalar salbutamol (grupo 3); 14 niños, con iguales características de la función respiratoria, pero cuyos valores no se recuperaron tras la prueba broncodilatadora, considerando que la inflamación bronquial era de mayor intensidad (grupo 4). Un grupo control (grupo 1) integrado por 11 sanos, cuyas pruebas alérgicas y funcionales fueron negativas. A todos se les practicó provocación bronquial con extracto de D. Pt., y se valoró la eosinofilia y la PCE antes de la misma y tres horas después, controlándose la función respiratoria durante las 24 horas siguientes.
Resultados: se demostraron diferencias significativas de los eosinófilos y de la PCE entre el grupo control y los tres grupos de pacientes. Hubo diferencia en todos los grupos, en los valores previos a la provocación y los posteriores a la misma. Sin embargo, no se observaron diferencias significativas entre los tres grupos de asmáticos.
Conclusiones: aunque la evaluación de la eosinofilia y de la PCE en suero pueden diferenciar los niños con asma de los que no padecen la enfermedad, sin embargo, no son parámetros útiles para conocer el grado de intensidad de la reacción inflamatoria de la mucosa bronquial.
Palabras clave: Niños asmáticos. Reacción inflamatoria. Edad pediátrica.
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