Serum ECP and MPO are increased during exacerbations of chronic bronchitis with airway obstruction

0 2000 Editions

Original

Biomed & Pharmacother 2000 ; 54 : 274-S et medicales Elsevier SAS. All rights reserved

scientifiques

article

Serum ECP and MPO are increased during exacerbations bronchitis with airway obstruction

of chronic

G. Fiorinil, S. Crespil, M. Rinaldi’, E. Oberti2, R. Vigorelli*, G. Palmieril ’ Department

of Internal

Medicine

“Brera”; Niguarda

2 Department of Nuclear Medicine, Ca’ Granda Hospital, Milan, Ital)

Rtrdioinlmllnolo~~

Lahorator?;

Summary - Many studies have demonstrated that, in asthma, serum levels of eosinophil cationic protein (ECP) are related to the activity and severity of the disease and can be used to evaluate the response to steroid treatment. During exacerbations of chronic bronchitis, airway inflammation shows some features of asthmatic inflammatory processes, with recruitment of eosinophils and recovery of significant amounts of ECP in bronchial lavage fluid (BAL). Involvement of neutrophils, with high levels of myeloperoxidase (MPO). is, on the contrary, typical of this latter disease, and not shared with asthma. In spite of the information collected with BAL and bronchial biopsy studies, few data still exist on serum levels of these proteins in chronic bronchitis. The objective of this study was to assess if serum levels of ECP and MPO are specifically increased in exacerbations of chronic bronchitis, as compared to other non-asthmatic acute respiratory disturbances. Serum ECP, MPO and immunoglobulin E (I&E) levels were measured in I7 non-atopic patients with exacerbation of chronic bronchitis with airway obstruction (COPD) and in 1 I control subjects seeking emergency medical treatment for unrelated acute respiratory problems. Spirometry was performed in patients able to give the necessary collaboration. All the subjects of this study were recruited from the emergency department. Both ECP and MPO were significantly increased in serum from patients with exacerbated COPD (22.2 + 4. I vs 9.5 ? 1.4 mcg/L and 853 ? I68 vs 375 k 41 mcg/L) and a strong correlation existed between these tw’o variables (r = 0.782). A further control group was made of 1 I patients with stable COPD. These subjects had levels of both ECP (13.1 r 2.7 mcg/L) and MPO (469 + 7 I) significantly lower than patients with exacerbated disease and higher than those without COPD. We conclude that serum ECP and MPO are increased during the exacerbations of COPD. These observations can give a basis for further studies aimed to evaluate the utility of these two proteins as markers of activity and severity of COPD. 0 2000 Editions scientitiques et medicales Elsevier SAS chronic

obstructive

pulmonary

disease

(COPD)

/ eosinophil

cationic

Chronic bronchitis is a common cause of chronic obstructive pulmonary disease (COPD), a disease etiologically related to cigarette smoking, and clinically characterized by a progressive decline of airflow with intermittent exacerbations [6]. Both in COPD and in asthma, airway inflammation is responsible for airway wall remodelling [2]. Among the inflammatory cells infiltrating the airways of these patients, neutrophils have been demonstrated to prevail in COPD [ 141 and eosinophils in asthma [4, 91. In recent years, the importance of eosinophilic infiltration in the airways of patients with chronic bronchitis has been established, especially during the exacerbations of the disease [ 19. 201.

Correspondence and reprints: Gianfrancesco Fiorini. M.D., Reparto Brera. Ospedale Niguarda Ca’ Granda, Piazza delI’Ospedale Maggiore 3. 20162 Milan, Italy.

protein

(ECP)

I neutrophil

myeloperoxidase

(MPO)

The histologic evidence is paralleled by the recovery of eosinophil cationic protein (ECP), released by activated eosinophils, and other inflammatory markers as neutrophil myeloperoxidase (MPO) [22], in the bronchial lavage [ lo] and induced sputum 17, 171 from patients with COPD. However, while in asthmatics ECP levels in serum are now widely accepted as a marker of activity of the disease and efficacy of treatment [22. 251, in COPD, in spite of the data obtained from lung biopsies and bronchial lavage, there is. to our knowledge, still little information on the possibility to use this protein as a marker of disease activity. This is possibly due to the fact that in basal conditions. serum ECP levels are much lower in patients with COPD than in patients with asthma [26]; therefore. small variations could be less easily detected. On the basis of the similarities with asthma [2], where serum ECP levels are higher during exacerbations [25], it seems likely that increased serum levels of this

Serum ECP and MPO are increased during exacerbations of chronic bronchitis with airway obstruction protein should be high during exacerbations of COPD. On the other hand, the well-established role of neutrophils [ 121 has also to be considered. We therefore thought that an assessment of both ECP and MPO in the serum of patients with exacerbated COPD could help to elucidate the relative contribution of these two cell populations, and could give further indications for the clinical use of these assays. To assess this, we studied the levels of serum ECP and MPO in a group of patients with exacerbated COPD, in a group of patients with acute respiratory failure unrelated to COPD and in a group of subjects with stable COPD.

PATIENTS, MATERIALS

AND METHODS

Patients The group of patients with exacerbated COPD was formed of 17 non-atopic patients with previous documented history of chronic bronchitis and airflow obstruction, fulfilling the criteria of the American Thoracic Society [ 11. They were all seen in the Emergency Department for disease exacerbations requiring admission. To be eligible for the study, either systemic or inhaled steroid treatment should not have been prescribed in the month preceding admission. In addition, no patient was on antibiotic therapy at that time and the standard chest x-ray on admission showed no sign of infectious processes of the lung. Most of them were using oral or spray bronchodilators. The control group was made of 11 subjects seeking emergency medical treatment for respiratory problems unrelated to COPD, asthma and infections (seven cases of pulmonary oedema, three of acute pulmonary thromboembolism and one of large vessel transposition with interventricular communication). All of them were off steroid and antibiotic treatment. Informed consent was obtained from the subjects of both groups. Patients and controls were of comparable age (69.5 f 2 and 70.6 f 4.4 years); both eosinophil count and blood gas analysis showed no significant differences between the two groups. On the contrary, the variables shown in table I were significantly different; these differences will be discussed in the Discussion section. For smokers, the average tobacco consumption was comparable in the two groups (one pack/d). A second group of 11 subjects with stable COPD was also studied. In these subjects, age, sex distribution and smoking habits were not statistically different from those of the patients with exacerbated COPD. In all the subjects of the three groups, skin prick tests for nine common allergens were performed to rule out atopy.

275

Table I. Differences between patients with exacerbated COPD and controls. All the other demographic and routine laboratory data were comparable. Paiients

Sex (M/F) Smokers Total IgE (KU/L)

1314 10 416.6 * 202

Controls

4/l 1 74.2 + 38.8

P < 0.05 < 0.01 NS

Spirometry Twelve of the 17 patients were sufficiently collaborating to undergo spirometry within 48 h from admission. This was performed with a dry wedge spirometer (Hellige, Germany) interfaced with a desktop computer (Olivetti, Italy) that calculated the values in accordance with recommended criteria [16].

Total IgE, ECP and MPO measurement Total IgE, ECP and MPO were measured with commercially available radioimmunoassay kits (Pharmacia Diagnostics, Milan, Italy) in serum obtained after centrifugation of clotted blood samples drawn in Vacutainer SST-tubes. For ECP assays, the blood samples were allowed to clot for exactly 60 minutes. The ECP thus released by activated eosinophils competes with a fixed quantity of radiolabeled ECP for the binding sites of a specific antibody. After separation of free ECP by the addition of a second antibody, the residual radioactivity is measured and it is inversely correlated to the amount of ECPreleased in the sample. ECP standards are calibrated against purified ECP [ 151, and normal values given by the manufacturer are lower than 14.5 mcg/L. The MPO assay is based on comparable principles with normal values less than or equal to 450 mcg/L. The minimum sensitivity of the test is 8 mcg/L [23]. The inter-assay coefficient of variation was < 8% for ECP and < 13% for MPO. Cross-reactivity of these two markers in the same samples was < 0.1%.

Statistical analysis Analysis of variance between independent groups, chisquare test and Spearman’s rank correlation test were used when appropriate. All data are expressed as the mean f standard error of the mean (SEM).

RESULTS Sernm levels of both ECP and MPO were significantly higher in patients with exacerbated COPD than in controls, as shown infigures I and 2, respectively, and their

276

G. Fiorini

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,

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et al

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P< 0.01

P < 0.01

.

2500

. .

l*

2000

. -:.f. - :.: Controls

l* . 2.

ti -;-

;

5 B .

Acute

Stable

Figure 1. Serum ECP levels in patients with exacerbated COPD (acute), stable COPD (stable) andcontrols. The difference between the stable and the control group is also significant (P = 0.03).

mean values (22.2 f 4.1 mcg/L for ECP and 853 f 168 mcg/L for MPO) were well above the upper normal limit given by the manufacturer. In the control group, both ECP and MPO mean values (ECP: 9.5 r 1.4 mcg/L, MPO: 375 k 4 1 mcg/L) fell within the normal range. The same figures show that patients with stable COPD had levels of ECP (13.1 + 2.7 mcg/L) and MPO (469 f 71 mcg/L) significantly lower than their counterpart with exacerbated disease, but higher than those of nonCOPD patients (P = 0.03 for ECP; P = 0.05 for MPO). Patients of the first group had higher IgE levels (table I), but no correlation was found with either ECP or MPO. Since there were significantly more males and smokers in the patients’ group than in the control group, we looked for a possible association of sex and smoke with ECP levels and MPO levels, but none was noted. Moreover, when the patients with exacerbated COPD were subgrouped into smokers and non-smokers, no significant difference was noted in either ECP or MPO levels (ECP: 20.9 + 4 vs 23.9 * 8.5 mcg/L, P = 0.88; MPO: 717.9 1 122.6 vs 1,045.8 + 372.7 mcg/L, P = 0.96). The same occurred when grouping the patients according to sex (ECP: males 25.2 f 5 mcg/L. females 13.6 f 5.4 mcg/L, P = 0.19; MPO: males 8 19.9 * 191, females 960.3 f 395.5 mcg/L, P = 0.78). We also looked for possible correlations between the levels of each of the two proteins and some indicators of lung function (FEV,, PEF, arterial PO,, PCO, and 0,

=i %-il s & c

1.500

i! $ .

loo0 -:-

: 500 -.‘. I

!

:

L

Controls Figure 2. Serum MPO the difference between

Acute

Stable

levels in the three groups. The P-value for the stable and the control group is 0.05.

saturation), but found none. On the contrary, a significant correlation existed between the serum levels of ECP and MPO (figure 3). DISCUSSION ECP can easily be measured in serum, where it is released by activated hypodense blood eosinophils during the clotting process [21]. In asthma its levels are correlated to the severity of disease and response to treatment [25]. MPO also can be measured in serum, where it is released from primary granules of blood neutrophils, but is unaltered in asthma [22].

Serum ECP and MPO

&

are increased

during

exacerbations

of chronic

bronchitis

with airway

obstruction

277

r 0.782 p < 0.001

;

60

z ;

40 20

500

1 .ooo

1.500 SERUM

Figure 3. Correlation

between

serum levels of ECP and MPO

2.000

2.500

MPO @g/L)

in the group of patients

with exacerbated

COPD;

r = Spearman’s

correlation

coefficient.

During exacerbations of chronic bronchitis, both eosinophils and neutrophils infiltrate the bronchial mucosa[ 14, 201,and significant amountsof both ECP and MPO can be recovered in BAL [ 171,possibly with somedifferencesbetweenpatients without chronic airway obstruction and thosewho have already developed COPD [ 131.In spite of this, treatment with glucocorticoids doesnot significantly affect the levelsof ECP and MPO in theselatter patients,andthis is true not only for inhaled steroids, whose delivery to the lung could be hamperedby airflow reduction, but alsofor oral preparations [ 111. In asthmatics,serumECP is higher than in patients with chronic bronchitis either with or without airway obstruction [26] and is a better indicator of disease activity than sputumECP [18]. However, no data are available, to our knowledge, on serumlevelsof this protein and MPO in patients with exacerbationsof COPD. In the presentstudy we demonstratethat both ECP and MPO are increasedin patientswith exacerbatedCOPD. No correlation exists with smoking habits, sex, lung function parameters(FEV, and PEF), serum IgE and blood gasanalysisvalues,as alsofound by other workersboth for ECP andMPO in BAL [171.On the contrary, serumlevels of thesetwo proteinsare closely related. This observationconfirms histological andBAL data demonstratingthe dual nature (eosinophilic and neutrophilic) of acute inflammation in chronic bronchitis. The fact that both thesecell types are simultaneously

activated in exacerbationsof COPD can be explained by a responseto an activating factor acting on both of them, such as granulocyte macrophagecolony stimulating factor (GM-CSF), that is known to be produced by bronchial epithelial cells [24], and interleukin (IL)-8, the levels of which are highly correlated to the levels of both ECP and MPO in induced sputum of patientswith chronic bronchitis [ 171.Alternatively, specific responsesto specific mediators,acting directly on eosinophilsor neutrophilsor indirectly by upregulation of adhesionmolecules[3, 81can be hypothesized. The fact that ECP levels are significantly increased during COPD exacerbationsanddo not return to normal levelsevenduring intercritical periodsgives further support to the possibleoverlapping of COPD and asthma, asproposedby other authors[5]. This is alsosuggested by the high levels of IgE in our patients,in the absence of known allergic diseases;however,the IgE increasein COPD patientscould only be due to cigarette smoking, which is known to affect serumIgE levelsand hasa central role in the aetiology of this disease[6]. Such an explanation is also confirmed by the fact that not only our patientshad IgE levelshigher than controls, but also a much greaterprevalenceof smokers. Our observationscould be the basisfor further follow-up studies aiming to assessif monitoring serum levels of either ECP or MPO or both in chronic bronchitis can have the sameclinical utility of serumECP assayin asthma.

278

G. Fiorini

REFERENCES I ATS. Standards for the diagnosis and care of patients with chronic obstructive pulmonary disease. Am J Respir Crit Care Med 1995 ; 152 : S77-120. 2 Brusasco V. Airway structure to function relationships in asthma and COPD. Monaldi Arch Chest Dis 1997 ; 52 : 597-9. 3 De Stefano A, Maestrelli P, Roggeri G. Turato G, Calabro S, Potena A, et al. Upregulation of adhesion molecules in the bronchial mucosa of subjects with chronic obstructive bronchitis. Am J Respir Crit Care Med 1994 ; 149 : 803-10. 4 Djukanovic R, Wilson JW, Britten KM, Wilson SJ, Walls AF, Roche WR, et al. Quantitation of mast cells and eosinophils in the bronchial mucosa of symptomatic atopic asthmatics and healthy control subjects using immunohistochemistry. Am Re> Respir Dis 1990 : 142 : 863-7 I 5 Dow L. Asthma versus chronic obstructive pulmonary disease-exploring why ‘reversibility versus irreversibility’ is no longer an appropriate approach. Clin Exp Allergy 1999 : 29 : 739-43. 6 Fletcher C, Peto R, Tinker C, Speizer FE. The natural history of chronic bronchitis and emphysema. An eight year study of early COPD in working men in London. Oxford: Oxford University Press; 1976. 7 Gibson PG, Woolley KL. Carty K. Murree-Allen K, Saltos N. Induced sputum eosinophil cationic protein (ECP) measurement in asthma and chronic obstructive airway disease (COAD). Clin Exp Allergy 1998 ; 28 : 1081-8. 8 Gosset P, Lassalle P, Molet S. Tonne] AB. Interactions cellules inflammatoires - endothC1ium dans la rCaction inflammatoire allergique. Rev FrAIlergo 1998 : 38 : 175-81. 9 Humbert M. Cytokines pro-iosinophiles et maladie asthmatique. Rev Fr Allergol 1998 ; 38 : 50-5. 10 Keatings VM, Barnes J. Concentrations of ECP are increased in COPD but these are not modified, by treatment with inhaled corticosteroids [abstract]. Thorax 1995 ; 50 : A33. 11 KeatingsVM, Jatakanon A,Vorsdell MiinY, Barnes PJ. Effects of inhaled and oral glucocorticoids on inflammatory indices in asthma and COPD. Am J Respir Crit Care Med 1997 : 155 : 542-X. I2 Lacoste JY, Bousquet J, Chanez P, Van Vyve T, SimonyLafontaine J, Lequeu N. et al. Eosinophilic and neutrophilic inflammation in asthma, chronic bronchitis, and chronic obstructive pulmonary disease. J Allergy Clin Immunol 1993 ; 92 : 537-48.

et al

I3 Linden M, Rasmussen JB, Piitulainen E, Tunek A, Larson M, Tegner H, et al. Airway inflammation in smokers with nonobstructive and obstructive chronic bronchitis. Am Rev Respir Dis 1993 ; 14X : 1226-32. I4 Martin TR, Raghu G, Maunder RJ. Springmeyer C. The effects of chronic bronchitis and chronic airflow obstruction on lung cell populations recovered by broncho-alveolar lavage. Am Rev Respir Dis 1985 : Ii2 : 254-60. 15 Peterson CGB, Jornvall H. Venge P. Puritication and charactertzation of eosinophil cationic protein from normal human eosinophils. Eur J Haematol 1988 : 40 : 415-23. 16 Quanjer PH. ed. Standardized lung function testing. Bull Eur Physiopathol Respir 1983 : I9 Suppl 5 : l-95. 17 Riise GR, Ahlstedt S. Larsson S, Enander I, Jones J, Larsson P. Andersson B. Bronchial inflammation in chronic bronchitis assessed by measurement of cell products in bronchial lavage fluid. Thorax 1995 ; 50 : 360-5. 18 Ronchi MC. Piragino C. Rosi E. Stendardi L, Tanini A, Galli G, et al. Do sputum eosinophils and ECP relate to the severity of asthma? Eur Renpir J 1997 : IO : 1809-13. 19 Saetta M. Di Stefano A. Maestrelli P. Turato G, Mapp CE. Pieno M, et al. Airway eosinophilia and expression of interleukin-5 protein in asthma and in exacerbations of chronic bronchitis. Clin Exp Allergy 1996 ; 26 : 766-74. 20 Saetta M, Di StefJno A. Maestrelli P, Turato G, Ruggieri MP, Roggeri A. et al. Alrway eosinophilia in chronic bronchitis during exacerbations. Am J Respir Crit Care Med 1994 : 150 : 1646-52. 21 Venge P. Serum measurements of eosinophil cationic protein (ECP) in bronchial asthma. Clin Exp Allergy 1993 ; 23 Suppl2 : 3-7. 22 Venge P. Soluble markers of allergic inflammation. Allergy 1994 ; 49 : l-8. 23 Venge P, Stromberg A, BraconierJH. Roxin LE. Olsson I. Neutrophil and eosinophil granulocytes in bacterial infection. Sequential studies of cellular and rerum levels of granule proteins. Br J Haematol 1978 : 38 : 375-83. 24 Walsh GM. Mechanisms of human cosinophil survival and apoptosis. Clin Exp Allergy 1997 : 27 : 4X2-7. 25 Wever AMJ. Wevcr-Hess J. Herman5 J. The use of serum eosinophil cationic protein (ECP) in the management of steroid therapy in chronic asthma. Clin Exp Allergy 1997 : 27 : 5 19-29. 26 Yoshirawa A, Kamimura M. Sugiyama H, Kudo K, Kabe J. Eosinophil cationic protein in serum from adults with asthma and with chronic obrtructive pulmonary disease. Jpn J Thor Dis 1996 : 34 : 24-9.