Serum eosinophil cationic protein (ECP) in chronic asthma. Relationship to spirometry, flow-volume curves, PC20, and exacerbations

Respiratory Medicine (1994) 88, 613-621

Serum eosinophil cationic protein (ECP) in chronic asthma. Relationship to spirometry, flow-volume curves, PC2o , and exacerbations A. M. J. WEVER*w J. WEVER-HESS*, H. E. S. J. HENSGENS'~ AND J. HERMANS~

*Department of Respiratory Medicine and ~fLaboratory of Clinical Chemistry, Red Cross Hospital, The Hague, and ~Department of Medical Statistics, University of Leiden, The Netherlands

Serum ECP was measured in a double-blind study in relation to spirometry, flow-volume curves, and histamine PC2o (30-s tidal breathing method, normal value >32 mg m l - 1) in 20 chronic asthmatic patients (five male), mean age 48.9 yr (sD 11'0), once a month and at additional visits due to acute exacerbations over a 6-month period. All patients were on maintenance inhaled corticosteroid therapy. Serum ECP was considered elevated at >_20pg 1-1. ECP showed significant negative correlations with lung function, especially when ECP was elevated (45/143 observations): r = - 0"48, - 0"48, and - 0.49 for respectively FEV1, PEF, and M M E F (all at baseline and as % predicted, P=0'001), the best correlation being with FEV1/FVC ratio ( r = - 0 . 6 1 , P<0'001). PC2o only had a weak overall correlation with ECP ( r = - 0 ' 2 1 , P=0-015). Eosinophilia at >0.400 x 10 9 cells 1 1 (38/143 observations) did not show any significant correlations with the lung function variables. The patients were subdivided into three groups according to exacerbations. Group A consisted of seven patients with suspected infectious exacerbations (mean 1"4), group B of six patients with suspected inflammatory exacerbations (mean 3-2), and group C of seven patients who did not experience any symptomatic exacerbations. A significant difference in mean ECP was found between group B and A (33-1 vs. 10-2, P=0-003). Group B also differed significantly from group A with respect to lung function and PC2o, but not to atopy, lung function and PC2o being lower and variability higher in group B. The findings of this study suggest that serum ECP is a sensitive marker of inflammatory airflow obstruction in chronic asthma. Elevated ECP seems to denote patients at risk from inflammatory exacerbations, resulting in practical implications for the therapeutic management of chronic asthma.

Introduction

Inflammation is considered the characteristic feature in asthma (1,2), in which the eosinophils play an important role (3,4). The activity of the eosinophils may be measured by the level of eosinophil cationic protein (ECP) in sputum or in serum (4~6). Studies in asthmatic patients have indicated a relation of level of serum ECP to severity and nature of the disease (5,7) especially with respect to late asthmatic response and exercise induced asthma. The inflammatory process appears to diminish with treatment by inhaled corticosteroids (3,8). There are indications that treatment with inhaled corticosteroids reduces serum ECP levels (7,9). Raised ECP levels despite topical steroids may point to an ongoing inflammatory process (10). Therefore, it can be postulated that Received 31 May 1994 and accepted24 June 1994. w whom correspondence should be addressed at: Red Cross Hospital, Sportlaan600, 2566 MJ The Hague, The Netherlands. 0954-6111/94/080613+09 $08.00/0

determination of ECP in serum offers a possibility of monitoring the process of asthmatic inflammation and the efficacy of anti-inflammatory therapy in asthma (11). This study was set up to evaluate monthly measurements of serum ECP over a 6-month period in relation to lung function, bronchial hyperreactivity, and exacerbations in chronic asthmatic patients who were on maintenance inhaled corticosteroid therapy. Methods

PATIENTS Twenty adult asthmatic patients from the outpatients' chest department known to have symptomatic disease were enrolled in the study, which was double-blind with regard to ECP and lung function results. All patients were on maintenance inhaled corticosteroid therapy besides inhaled fl2-agonist therapy. They visited the clinic once a month over a 9 1994W. B. SaundersCompany Ltd

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6-month period in a scheduled way, blood sampling and lung function testing always took place between 08.30 and 10.00h on the same day of the week. A short history was taken and at each visit the patients gave themselves a score expressing their well-being on an analogue scale from 1 (very bad) to 10 (perfect). It was attempted to keep the medication stable during the study period. Some of the patients made additional visits due to an acute exacerbation. Exacerbations were diagnosed when the patient had an increase in complaints and in the use of inhaled fl2-agonists, or was in need of a short course of oral prednisone. On clinical grounds (phlegm production and macroscopic appearance, fever) the patients with exacerbations were divided into two groups: patients with suspected infections exacerbations: group A, and patients with suspected non-infectious i.e. inflammatory exacerbations: group B. The patients who did not experience a symptomatic exacerbation over the study period were grouped into group C. At each visit blood samples were taken for measurement of ECP, total IgE, and cell counts of leucocytes and eosinophils. At the first visit a blood sample was drawn for inhalant allergy testing. Spirometry and flow-volume curves were performed at each visit and the day after the visit a histamine challenge test was done, provided the FEV1 was _> 1.001. Bronchodilator medication was withheld for 12 h in case of short acting fl2-agonists and for 24 h in case of long acting fl2-agonists before lung function testing (spirometry and histamine challenge). LABORATORY TESTS

Blood samples were drawn in Vacutainer SSTtubes and after allowing clotting for 1 h the blood was centrifuged for 3 rain at 3000 r.p.m. The sera were then frozen at - 70~ and kept for analysis at the end of the study period. The following tests were performed: the CAP Phadiatop | F E I A for determination of IgE antibodies against a balanced mixture of relevant inhalant allergens, the Pharmacia CAP total IgE F E I A for determination of total serum IgE, and Pharmacia CAP RAST F E I A for determination of specific IgE antibodies against the following common inhalant allergens: house dust mite, grass pollen, cat dander, dog dander, weed pollen, tree pollen and moulds (D 1, G x l , El, E5, Wx3, Tx9 and Mxl). Phadiatop | and RAST were performed as single tests, total IgE as a double test. The ratio of the Phadiatop | (percent binding of the patient serum to percent binding of a reference serum) was determined and classified as a positive result if the ratio > 1-00 or negative result if _<1"00. The patient was accordingly considered

atopic or non-atopic. The RAST was determined in kUA 1-1 and classified semiquantitatively in classes 0-6. Total IgE concentration was expressed in k U 1 - 1, taking the mean of two determinations. ECP in serum was measured in duplo by the commercially available immunoassay technique Pharmacia CAP ECP FEIA. ECP was considered elevated at levels >20/~g 1 1 (11,12). Total white blood cells count and total number of eosinophils were measured by an automated haematology analyser (Technicon H 1). Elevated values for eosinophils were considered as >0'400 x 109 eosinophils per litre or _>6% of total white blood cells (13). SPIROMETRY AND FORCED EXPIRATORY FLOWS

Forced vital capacity (FVC), inspiratory vital capacity (IVC), and forced expiratory volume in one second (FEV1) were measured by a wet spirometer (Sensormedics, P2450). Expiratory flow-volume curves were obtained from the forced expiration by electronic differentiation of the volume. From the curves the peak expiratory flow (PEF), and the maximal mid-expiratory flow (MMEF) were taken for use in the analysis. Reference values for lung volumes and forced expiratory flows were calculated from the summary equations of the ECSC report on standardized lung function testing (14). Spirometry and flow- volume curves were carried out at baseline and after inhalation of salbutamol 400/lg delivered through the Volumatic spacer device. The reversibility of the bronchial obstruction, measured by FEV 1 and PEF, was calculated as the respective value after bronchodilatation of inhaled salbutamol minus the baseline value divided by the reference value. The variability of FEV 1, PEF, and M M E F over the 6-month period was determined as the respective difference at baseline between the patient's highest value minus the patient's lowest value divided by the highest one. HISTAMINE CHALLENGE TESTS

Standardized inhalation provocation tests with histamine acid phosphate were performed (15), using the 30-s tidal breathing method (normal value of PC2o>32 mg ml l). The aerosols were generated by a DeVilbiss 646 nebulizer, operated by oxygen (output 0.13 ml min 1). During the histamine challenge FEV1 was measured 30 and 90 s after each dose by a wet spirometer (Sensormedics Pulmonet III). The tests were discontinued if FEV 1 decreased by 20% or more from baseline value. The PC20 was calculated by linear interpolation of the last two points of the log dose-response curve and its values were 2log transformed for linearity.

ECP in chronic asthma

615

Table 1 Characteristics of the 20 patients in the study population

Patient

Sex

Age (yr)

1 2 3 4 5 6 7 8~ 9 10 11~ 12 13 14~ 15 16 17 18 19 20

F F F F F F F F M F M F F M F F F M F M

51 34 60 49 44 58 57 45 64 48 34 57 49 22 33 55 46 56 57 59

Smoking habit

Atopy

never yes ex ex ex never ex never never never ex never never yes never never ex ex ex never

+ + + + + + + + + + + + -

RAST*

Inhaled steroids (ug day - 1)

Inhaled fl2_agonists ].

0 1 2 1 3 3 2 0 2 0 4 0 2 4 0 0 0 0 2 0

600-800 400 800 1600 600 1600 400 0-2400 400-800 1600 0-800 400-600 800 300 800 800 400 400-600 100-200 800

s 1 1 1 s s 1 sl s sl s s sl sl 1 s s s sl 1

*Number of RAST-results >_class 2. ]s: short acting, 1: long acting. ~CThese patients did not use inhaled steroids at the first visit. w patient also used sodium cromoglycate.

DATA ANALYSIS The statistical package SPSS/PC + 5.0 was used for the analysis of the data. The d a t a were analysed by linear regression analysis a n d P e a r s o n ' s correlation analysis, Student's t-test on means, a n d in cases w h e n Levene's test for equality o f variance resulted in a Pvalue <0'05 the n o n p a r a m e t r i c M a n n - W h i t n e y U ( M - W ) test was used. ETHICS T h e study was a p p r o v e d by the Medical Ethical C o m m i t t e e of the hospital a n d all patients gave their written consent.

Results T w e n t y chronic a s t h m a t i c patients (5 males, 15 females) t o o k p a r t in the study, the age range being 22-64 years ( m e a n 48.9, SD 11"0). In Table 1 the characteristics o n s m o k i n g habit, atopic status, a n d use of inhaled corticosteroids are given. In total 147 visits were made, o f which seven were additional visits by three patients. In these three patients o n four occasions lung f u n c t i o n testing a n d o n 10 occasions histamine challenge tests could n o t be carried o u t because of the patient's p o o r condition.

RELATIONSHIP OF' ECP TO SPIROMETRY AND PC2o In the regression analysis E C P s h o w e d significant negative correlations with the lung function variables at baseline, especially with E C P levels > 20/~g l - ~, b u t n o t with 21ogPC2o (Table 2). Best results were o b t a i n e d with FEV1/FVC ratio (Fig. 1). A t levels < 2 0 p g 1 - 1 E C P did n o t correlate with these variables. T h e n u m b e r of eosinophils showed similar significant negative correlations with lung f u n c t i o n variables, however w h e n eosinophils were >0"400 • 1 0 9 cells 1 - 1 n o correlations could be established (Table 2). This was also the case when eosinophils > 6% (results n o t shown). W h e n the d a t a were analysed according to atopy, the overall correlations o f E C P a n d o f n u m b e r of eosinophils with lung f u n c t i o n were stronger in the n o n - a t o p i c s t h a n in the atopics. W h e n E C P > 2 0 / ~ g 1 1, however, there were strong correlations in b o t h groups (Table 3), whereas w h e n eosinophils > 0 ' 4 0 0 x 109 cells 1 significant negative correlations were n o t present either in the atopics (23/86 observations) or in the n o n - a t o p i c s (15/57 observations). W h e n the E C P > 20/zg 1 - 1 the m e a n values of the lung function variables were all highly significantly lower t h a n at E C P <20r 1 1: F E V J I V C ratio 59

616

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vs. 71%, F E V J F V C ratio 65 vs. 76%, PEF % predicted 59 vs. 74% (all P<0-001), FEV~ % predicted 72 vs. 83%, M M E F % predicted 39 vs. 62%, and 21ogPC2o 1.7 mg ml - ~ vs. 2-9 mg ml - 1 ( M W test all P_<0-003). Notably the FEVI/FVC ratio was always below the reference value, the M M E F less than 70% of predicted, and 21ogPC20 < 4 mg m l - 1 . Also the reversibility of the bronchial obstruction was significantly greater at ECP ~>20/~g 1- ~ for FEV 1 (mean 17 vs. 13%, P=0.014) and PEF (mean 17 vs. 12%, P=0-017). No significant difference in use of inhaled corticosteroids ~ g d a y - 1 ) was found, the mean for ECP _>20/~g 1-1 being 651/~g day - 1 and for ECP <20#g 1-1 being 851/~g day 1 ( M - W test P=0-59).

RELATIONSHIP OF ECP TO EXACERBATIONS

Over the 6 months study period a total of 29 exacerbations occurred in 13 patients. At the exacerbation visits the patients' scores expressing their well-being were significantly lower than at the visits without exacerbations (4.8 vs. 6'9, P<0-001). However, no significant differences were found in the spirometric values, apart from the reversibility of PEF (9 vs. 14%, M - W test P=0-03), and no significant difference was found in 21ogPC2o either. In seven patients the exacerbations were judged to be of infectious nature (group A: patients 2, 4, 8, 10, 14, 15, and 16) and in six patients of inflammatory nature (group B: patients 1, 3, 7, 18, 19, and 20). Mean number of exacerbations in group A was 1.4 and in group B 3"2. Figure 2 shows the time course of ECP over the study period in group A, B,

and C. Mean ECP at the exacerbation visits vs. mean ECP at the non-exacerbation visits was in group A 9.5/~g 1 ~ vs. 10-4#g 1-1, and in group B 42-5/~g 1 1 vs. 26-4r 1-a (differences not significant). The mean of the individual's mean ECP differed significantly between group B and group A (33'1 vs. 10"2/~g 1- 1, M - W test P=0-003). Group B also differed significantly from group A with respect to most lung function variables and PC2o, lung function and PC2o being lower and reversibility and variability being higher (Table 4). No difference in atopic status was found between A and B as reflected by Phadiatop-result and level of total IgE (Table 4). The mean dose (SD) of the maintenance inhaled corticosteroids was 1047 (622)r d a y - 1 in group A, 563 (257)/~g day - a in group B, and 743 (402)]tg d a y - 1 in group C. The differences did not achieve significance. In group C with a mean ECP of 20.4 r 1- ~ mean values of the lung function variables were: FEV1% predicted 84%, FEVa/IVC ratio 65%, F E V J F V C ratio 70%, PEF % predicted 68%, M M E F % predicted 50%, and 21ogPC2o 1.2 mg ml - 1. In this group the three patients with a mean ECP > 20/~g l - a had a trend towards lower values of lung function and PC2o than the four patients with a mean ECP <20/tg 1-1. If all seven patients with a mean ECP >20/~g 1-1 are considered, the mean lung function values were lower than in the thirteen patients with a mean ECP <20/~g 1-1, reaching significance with respect to F E V J I V C ratio (P=0.007), F E V J F V C ratio (P=0.003), PEF %predicted (P=0.046), and M M E F %predicted (P-~0-031).

618

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Studies on the value of serum ECP in the management of asthma have been mainly related to atopic patients and to special circumstances, such as bronchial challenge situations and the effects of instituting non-steroid and steroid anti-asthma medication (5-7,9,10,16,17). As part of one study ECP was examined in children who were on regular treatment with topical steroids for more than 1 month (10). In this present study serum ECP was evaluated in day-to-day clinical practice in adult chronic atopic and non-atopic asthmatic patients who were on maintenance inhaled corticosteroid therapy during a follow-up once a month over a 6-month period. This study population, consisting of 20 patients, showed a

619

negative relation between ECP and airflow obstruction reflected by FEV 1, PEF, and MMEF, especially when the ECP was elevated, both in atopics and non-atopics. As clustering within patients is a possibility, it is noted that ten patients contributed to the 45 observations when elevated ECP levels were found. When the correlations between ECP and the lung function variables were computed crosssectionally per visit, it appeared that at every visit FEV1/FVC ratio showed a significant negative correlation with ECP, whereas the correlations of the variables expressed as % predicted, although always negative, varied in significance and degree (results not presented). This supports the hypothesis that a lung function result related to the patient's own capacity may be a better measure for current severity of the asthmatic disease than when related to a predicted mean value (10). In several other studies with asthmatic patients a negative correlation between ECP and lung function was established (6,7,10,16), however this was not the case in two allergen challenge studies (20,21), lending support to the hypothesis that ECP levels seem to reflect the chronic rather than the acute phase of asthma (10). At elevated ECP levels, FEV1/VC ratio always proved to be below reference value and the MMEF % predicted to be greatly reduced, although at normal ECP levels there were still some reduced lung function values. The question could be raised that in these cases where the ECP is normal but the lung function is reduced the obstructive component prevails over the inflammatory component, suggesting a need for intensifying bronchodilating rather than antiinflammatory treatment. The suggestion that more severe asthma could have some peripheral bronchial obstruction not always detectable by FEV 1 and/or VC (18) may be supported by the findings in this study of a markedly reduced MMEF, especially at elevated ECP levels. In this study PC2o only had a weak overall correlation with ECP and no correlation with elevated ECP levels. The correlation could have been biased because of missing PC2os, but when the three patients who contributed to the missing data were left out of the analysis similar results were obtained. However, mean PC2o was significantly lower at elevated ECP level than at level <20/~g 1- 1. Also from the data in a study in pollen-allergic patients (19) it can be concluded that the correlation between ECP and PC2o is not a close linear one. It is notable that eosinophilia, both by number and percentage, showed no significant negative correlation with the lung function variables, neither in atopics nor in non-atopics. In this study ECP seems to be a better marker than eosinophils in relation to

620

A. M. J. Wever et al.

Table 4 Mean values of the lung function variables, 21ogPC2o, and indices of atopy in the group of patients with infectious exacerbations (A) and in the group of patients with inflammatory exacerbations (B)

FEVl%pred FEV1/IVC (%) FEVffFVC (%) PEF%pred MMEF%pred % reversibility FEV1 PEF % variability FEV1 PEF MMEF 2logPCzomg ml- 1 l~ kU 1- 1 a~ ratio

A n=7

B n=6

P-value t-test

87 77 81 83 78 7 7 18 26 33 4.1 1.78 0.34

62 58 65 51 35 15 14 35 43 49 2.2 1.94 0.40

0.034 0-003 0.003 0.006 0.010" 0.010 0.062 0-034 0.021 0.052 0.016 0.654 0.879

*Mann-Whitney test. lung function, which is in accordance with other studies in which patients had been treated with (inhaled) corticosteroids (6,7). With respect to exacerbations the patients were divided into group A: infectious exacerbations (seven patients), group B: inflammatory exacerbations (six patients), and group C: no exacerbations (seven patients). The patients in group B proved to have poorer lung function results and higher ECP levels compared to group A. The finding that in group B the mean level of ECP both at exacerbation visits and non-exacerbation visits was elevated suggests an ongoing inflammatory process in these patients. The mean dose of the maintenance inhaled corticosteroids was lowest in group B. They were the most symptomatic group with 19 exacerbations for which 13 short courses of oral prednisone had to be prescribed in contrast to 10 exacerbations with six courses of oral prednisone in group A. As corticosteroids are thought to reduce inflammation and levels of ECP in serum (7,9) it can be postulated that the patients in group B may have been undertreated during the study period, but the study design was to keep the treatment as stable as possible. On clinical grounds three patients in group B had to be given oral corticosteroids in addition to their maintenance treatment of inhaled steroids at the end of the study period, after all these three patients proved to be the ones with the highest mean ECP values. This lends support to the hypothesis that high levels of ECP denotes active disease needing intensifying treatment with anti-inflammatory drugs. The in-between position of group C with regard to the maintenance

inhaled corticosteroid therapy seems to be reflected in the level of ECP and lung function. Of the seven patients without exacerbations three had a mean ECP >20/~g 1 2 As overall the patients with an elevated mean ECP proved to have a poorer lung function and lower PC2o than the patients with a normal mean ECP, and as these three patients likewise suffered from a poor lung function and/or severe bronchial hyperreactivity, it could be argued that the elevated ECP levels reflected an ongoing inflammatory process which was not severe enough to give rise to a symptomatic exacerbation. In this study PC2o seemed to be a poor parameter of the clinical condition of the patient, the individual's PC2o contributing rather to the diagnostic than to the monitoring process of the disease status. As found in other studies (18,22), the patients in this study were still hyperresponsive despite the maintenance corticosteroid treatment, independent of the daily dosage of steroids. It is concluded that in chronic asthmatic patients on inhaled steroids serum ECP seems to be a sensitive marker of inflammatory airflow obstruction. Independent of atopic status, an elevated ECP seems to denote patients at risk from inflammatory exacerbations, and knowledge of the level may be helpful in the therapeutic management of their asthma.

Acknowledgements We wish to thank Pharmacia Nederland B.V. for analysing the patients' sera and Dr M. G. Britton for his critical comments on the manuscript.

E C P in chronic asthma

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