- Email: [email protected]
Serum levels of eosinophil cationic protein in allergic diseases and natural allergen exposure
Serum levels of eosinophil cationic protein in allergic diseases and natural allergen exposure Margherita Tomassini, M D , a Laura Magrini, M D , a Guido De Petrillo, MD, a Emilio Adriani, M D , a Stefano Bonini, M D , b Francesco Balsano, M D , a and Sergio Bonini, M D c Rome and Naples, Italy Background: Eosinophil cationic protein (ECP) is a cytotoxic preformed mediator stored in eosinophil granules and released under various in vitro and in vivo conditions. Objective: This study was carried out to evaluate the clinical value of ECP as a marker of allergic inflammation. Methods: ECP was measured by a competitive radioimmunoassay in serum samples from 265 patients and 45 matched control subjects and related to the type of allergic disease (asthma, rhinitis, conjunctivitis) and to the type of allergic sensitization. Results: All the patient groups studied showed significantly higher levels of serum ECP than control groups (p < 0.001). The type of sensitization was shown to be the only variable influencing ECP serum levels. In fact, subjects sensitized to perennial allergens had significantly higher ECP values than subjects with seasonal allergy (p < 0.001), whereas in patients with seasonal allergy ECP levels were significantly increased only during the pollen season. Differences in ECP values between various allergic diseases or age groups were only due to a nonhomogeneous distribution of the type of sensitization or to time of sera collection. Conclusions: Results obtained indicate that persistent natural exposure to a sensitizing allergen is responsible for a measurable increase in serum ECP levels in patients with allergy. (J Allergy Clin ImmunoI 1996;97:1350-5.) Key words: Eosinophil cationic protein, allergic inflammation, eosinophils, perennial and seasonal allergens
It is now well established that the pathophysiology of allergic diseases is characterized by ongoing tissue inflammation, which follows the classical type I immediate hypersensitivity reaction. The activation and recruitment of several effector cells causes morphologic changes, hyperreactivity of target tissues, and, finally, clinical symptoms. 1-3 In From aIstituto di I ° Clinica Medica, Universityof Rome "La Sapienza"; bDepartment of Ophthalmology, University of Rome "Tor Vergata"; and CDepartment of Clinical Immunology and AUergology,2rid Universityof Naples. Margherita Tomassini was supported by the "Istituto PasteurCenci Bolognetti" Foundation, University of Rome "La Sapienza", Italy. Received for publication Jan. 4, 1995; revised July 28, 1995; accepted for publication Aug. 28, 1995. Reprint requests: Sergio Bonini, MD, EAACI, Istituto di I Clinica Medica, Universityof Rome "La Sapienza",Viale del Policlinico, 00161, Rome, Italy. Copyright © 1996 by Mosby-Year Book, Inc. 0091-6749/96 $5.00 + 0 1/1/68857 1350
Abbreviations used ECP: Eosinophil cationic protein HDM: House dust mite PA: Perennial allergen SA: Seasonal allergen
these inflammatory processes eosinophils are considered to play a pivotal role. In fact, the number of eosinophils is increased in blood and tissues in patients with allergic diseases, e, 5 Moreover, eosinophils are massively recruited on specific allergen challenge in several organs and biologic fluids. 3 Finally, in the lungs, nose, skin, and eyes, the release of oxygen radicals, newly generated mediators such as platelet activating factor and leukotriene C4, and granule cationic proteins (major basic protein, eosinophil peroxidase, eosinophil cationic protein [ECP], eosinophil-derived neuro-
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 6
toxin/eosinophil protein X), has been shown to be responsible for basic histopathologic changes of target tissues, such as vasodilation, mucosal edema, epithelial cell damage, and nonspecific hyp erre activity. 1-3,6 A m o n g inflammatory mediators of human eosinophils, granule cationic proteins have been well characterized, 7 and several functions of these proteins have been described. 8 In particular, it has been reported that ECP is a potent toxin for parasites 9 and mammalian cells in vitro. 1° Moreover, it interacts with blood coagulation and fibrinolytic factors n and can activate tissue fibroblasts, promoting their proliferation and collagen deposition. 12 Several reports describe an increase of ECP in serum and biologic fluids in patients with allergic diseases, 13-28 suggesting that the measurement of E C P levels might be a useful clinical test in monitoring disease activity and the efficacy of therapeutic agents. Most of these studies, however, are based on population samples that are small or not well defined in relation to the type of disease, type of sensitization, and other clinical variables. This may account for the great variability of ECP values recorded in nonhomogeneous study groups. In this article, we report data collected from a large population sample, showing that natural allergen exposure is an important factor influencing serum ECP levels in allergic diseases.
METHODS Two-hundred and sixty-five outpatients (58.7% female patients; age range, 6 to 67 years) with bronchial asthma (66 patients with isolated asthma; 28 with asthma and rhinitis; and 30 with asthma, rhinitis, and conjunctivitis), rhinitis (60 patients with rhinitis and 42 with rhinoconjunctivitis), or isolated conjunctivitis (39 patients) were included in this study. The presence of a specific sensitization to common inhalant allergens was documented in 195 patients by clinical history, objective examination, skin prick tests, and the measurement of specific IgE serum levels by RAST (Kabi Pharmacia AB, Uppsala, Sweden). Subjects sensitized to grass (n = 65) and those sensitized to Dermatophagoides pteronyssinus, Parietaria officinalis, or both (n = 130) were considered as separate groups. In this second group, 43 subjects were sensitized to house dust mites (HDMs), 31 to Parietaria species and grass, 37 to HDMs and grass, and 19 to HDMs and Parietaria species. In fact, in our climate, grass pollen is strictly a seasonal allergen (SA), whereas HDM and Parietaria species are present throughout the year. At the time of blood collection, no patients was receiving any therapy, and none had received specific immunotherapy in the 5 years before the study. Forty-five disease-free matched subjects (15 children
T o m a s s i n i et al.
1351
under 12 and 30 adults; 30 male and 15 female) who had no history of allergy, blood eosinophil count in the normal range, and total IgE levels below 250 kU/L were selected as control subjects. The mean age of the control group was 27.5 + 17 years (range, 7 to 63 years); the mean total IgE level was 50.92 _+ 56.82 kU/L (range, 0 to 222 kU/L). Venous peripheral blood (10 ml) was drawn, after informed consent was obtained, into preservative-free tubes. Blood was allowed to clot at constant room temperature (20° to 22 ° C) for 60 _+ 10 minutes, centrifuged at 1250 g for 20 minutes at 4° C, and stored at - 2 0 ° C until assayed. ECP was measured in serum samples by a competitive radioimmunoassay (ECP-RIA kit, Kabi Pharmacia) according to the manufacturer's instructions. All samples were run in duplicate. The range of the standard curve was 2 to 200 p~g/L.With this procedure, according to the manufacturer, normal ranges of serum ECP are 2 to 16 Ixg/L. Because it has been suggested that ECP serum levels can be influenced by the coagulation process and by in vitro handling of the samples, 29-31 all tests were performed in the same run to avoid interassay variations. Moreover, clotting procedures were performed under the same temperature conditions. Parametric statistical analysis of variance was performed to compare patients with allergy and control subjects, and multiple analysis of variance was performed for type of sensitization and type of clinical disease. In fact, the distribution of ECP values, preliminarily evaluated, appeared to be normal. Moreover, our large samples allowed the choice of parametric tests. Total IgE levels are not normally distributed. Accordingly, a nonparametric test (Spearman's correlation) was performed to analyze the relationshi p between serum ECP levels and total IgE. A p value of 0.05 or less was considered statistically significant.
RESULTS Serum E C P levels in the patient group (22.7 _+ 18.2 ~g/L) were significantly higher than those in the control group (6.1 + 4.8 p~g/L) (t --- 6.06, d f = 308, p < 0.001) (Fig. 1). Patients with allergy had significantly higher E C P levels than control subjects, but were not different from those of patients without clinical evidence of sensitization. Analysis of variance performed with the Student-Newmann-Keuls test showed that among variables studied, the type of allergen responsible for sensitization was the only factor influencing the high variability of values observed in the allergic group. In fact, patients with allergy sensitized to perennial allergens (PAs) had significantly higher ECP serum values than patients sensitized to seasonal allergens (n = 130, 35.1 + 33.4 ~g/L vs n =
1352
ECP
Tomassini et al.
J ALLERGYCLIN IMMUNOL JUNE 1996
IJg/I
ECP pg/I
50
80~
22.7--- 18.2
70
35.1 _+33.4
40 60
p<0.001
p<0.001 50
30
t i
40 30 6.1 _+4.8 20
10
15.6_+12.2
10 0
Controls n=45
Patients n=265
FIG. 1. Serum levels !means _+ SD) of ECP were significantly higher in allergic patients than in control subjects (p < 0.001).
65, 15.6 _+ 12.2 p~g/L;p < 0.001) (Fig. 2). Moreover, although E c p levels did not change significantly throughout the year when PAs were responsible for clinical symptoms, mean ECP levels of subjects sensitized to SAs were in the normal range during winter, showed a slight increase at the beginning of the pollen season, and became as high as ECP values observed for subjects sensitized t o PAs during the pollen season (Fig. 3). No age or sex dependence of serum ECP levels was observed in our study group. Apparently, serum ECP levels were significantly higher in patients with asthma and conjunctivitis than in Patients with rhinitis (p < 0.01). However, analysis of variance showed that differences observed were due to differences in the distribution of the type of sensitization to the various allergens studied in the different disease groups. In fact, in our patients rhinitis was more often caused by SAs (grass, Olea europea), whereas asthma and conjunctivitis were mainly caused by PAs (Dermatophagoides spp., P. officinalis, cat and dog danders). Neither severity nor duration of clinical symptoms, nor levels of specific IgE (assessed by RAST class) could account for the differences found. No significant difference in ECP serum levels was found between subjects with positive RAST results and those with negative RAST results for various diseases considered. The relationship between serum ECP levels and total eosinophil count was analyzed in 39 patients with or without peripheral eosinophilia. The number of circulating eosinophils was found to be related to ECP serum levels (r = 0.477, t = 3.299, df = 37, p < 0.005).
0 PA n--130
$A n=65
FIG. 2. Serum levels (means _+ SD) of ECP were significantly higher in patients sensitized to perennial allergens (PA) than in patients sensitized to seasonal allergens (SA) (p < 0.001).
The relationship between serum ECP levels and total IgE was evaluated in 85 patients, independently of the clinical picture, by Spearman's correlation test. The correlation between these two parameters was significant, in spite of the low value of rs (rs = 0.293, df = 83, p < 0.01) (Fig. 4). DISCUSSION
Our results show that serum ECP levels are significantly increased in patients with allergic disease compared with normal control subjects, even when the number of circulating eosinophils is in the normal range. It has been reported that ECP measured in the blood is mainly released in vitro during the blood coagulation process. 29 Accordingly, plasma and serum levels may be different, and results also depend on procedures used in handling blood samples. 3°,31 We have measured ECP in sera with a standard procedure for all samples. This should avoid pitfalls in ECP determination. Moreover, if the above-reported hypothesis is true, our results imply that allergic diseases, independently of the clinical picture, (i.e., asthma, rhinitis, o r conjunctivitis) are "systemic" diseases, because primed eosinophils are present in the circulation and not only in the target tissue. Several reports describe an increase of ECP in serum and biologic fluids in patients with allergic diseases, 13-z8 suggesting that the measurement of ECP levels might be a useful clinical test for monitoring disease activity and the effects of therapeutic agents. Most of these studies, however, are based on population samples that are small or not
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 6
Tomassini
e t al.
1353
ECP/~g/I 120
100
80
60 m |
.
: : •
:
•
.
m
•
:.
40 i
20
,
n
•
:1 : .... ,
'
!
m
PA SA Oct-Jan p<0.001
:::::I
'
•
PA SA Feb-Mar n.s.
'
PA SA Apr-Jun n.s.
FIG. 3. ECP serum levels at various periods of collection of sera in patients with hypersensitivity to perennial allergens (PA) and seasonal allergens (SA). Total IgE kU/L 1,200
1,000 '
:
=I
800 S
:i: ~:ii
600 p<0,O1
A
400
200
m
'b
m
mm m
i
#m
• •
m
•
•
•
m l
0 0
10
20
30
40
50
60
70
80
90
100 110 120 ECP/~g/L
FIG. 4, Serum ECP and total IgE levels were significantly correlated in patients with allergy (rs = 0.293, p < 0.01).
well defined in relation to the type of disease, type of sensitization, and other clinical variables. This might account for the great variability of ECP values in nonhomogeneouS study groups.
In this article, we report data collected from a large population sample, showing that natural allergen exposure is an important factor influencing serum ECP levels in patients with allergic diseases.
1354
T o m a s s i n i et al.
J ALLERGY CLIN IMMUNOL JUNE 1996
In fact, when analyzing the relationship between specific sensitization and serum E C P levels, we f o u n d that, independently of the clinical picture, serum E C P levels are higher in subjects sensitized to PAs than in subjects sensitized to SAs. Moreover, in patients allergic to grass, serum E C P levels reach high values only during the pollen season, This implies that, independently of the biochemical features of the specific allergen responsible for clinical symptoms, long-term allergen exposure is a crucial factor for the continuous activation of the allergic reaction in h u m a n beings, as it is in rodents. 32 It is well known that long-term stimuli can induce both an increase in total I g E levels and peripheral eosinophilia. In fact, total I g E levels were significantly correlated to E C P serum levels (p < 0.01), although the low correlation coefficient (0.293) indicates that a "private" regulatory m o d ulation is present in addition to c o m m o n regulatory factors. In reference to these factors, it must be noted that both of these parameters are highly influenced by the degree of T-cell activation. In particular, a T H 2 cell subset has been invoked to m o d u l a t e the allergic response t h r o u g h its peculiar cytokine profile. 33 A clonal expansion of TH2 cells in vivo has b e e n recently d e m o n s t r a t e d in a case of hypereosinophilia. 34 In fact, IL-3 can influence mast cell and basophil activation and releasability; IL-4 and IL-13 total l g E and I g G 4 production; IL-5 maturation, activation, and recruitment; and survival of eosinophilsY -37 Interestingly enough, all of these cytokines and granulocyte-macrophage colony-stimulating factor are u n d e r the control of genes closely located on h u m a n c h r o m o s o m e 5. 38 O n the other hand, specific I g E response is also influenced by other genetic (major histocompatibility complex genes) 39 and environmental factors. O n the basis of the above-reported data, we might suggest that phenotypic markers of the overexpression 4° of the 5q cytokine-gene cluster (including total I g E and E C P ) can represent a c o m m o n hallmark of all allergic diseases, whereas specific I g E antibodies are detectable only in some patients with allergy. In fact, E C P serum levels were significantly increased in both I g E - m e d i a t e d and nonI g E - m e d i a t e d forms of diseases studied. The clinical counterpart o f this point of view is that I g E tests, so widely p e r f o r m e d in patients with allergy, should be c o m p l e m e n t e d by other, new allergy tests. Only m o r e extensive and prospective studies can validate the usefulness of E C P determination in both I g E - m e d i a t e d and n o n - I g E - m e diated clinical allergies.
We thank Professor Francesco Vecchi (Department of Human Biology, University of Rome "La Sapienza") for assistance with statistical analysis.
REFERENCES
1. Bousquet J, Chanez P, Campbell AM, et al. Inflammatory processes in asthma. Int Arch Allergy Appl Immunol 1991;94:227-32. 2. Weller PF. The immunobiology of eosinophils. N Engl J Med 1991;324:1110-8. 3. Hallgren R, Venge P. The eosinophil in inflammation. In: Matsson P, Ahlstedt S, Venge P, Thorell J, eds. Clinical impact of the monitoring of allergic inflammation. London: Academic Press, 1991:119-40. 4. Fukuda T, Gleich GJ. Heterogeneity of human eosinophils. J Allergy Clin Immunol 1989;83:369-73. 5. Capron M, Kusnierz JP, Prin L, et al. Cytophilic IgE on human blood and tissue eosinophils: detection by flow microfluorometry. J Immunol 1985;134:3013-8. 6. Bousquet J, Chanez P, Lacoste JY, et al. Eosinophilic inflammation in asthma. Int Arch Allergy Appl Immunol 1991;94:227-32. 7. Dvorak AM, Ackerman SJ, Weller PF. Subcellular morphology and biochemistry of eosinophils. In: Harris JR, ed. Blood cell biochemistry. Megakaryocytes, platelets, macrophages and eosinophils. London: Plenum Publishing 1990; 2:237-44. 8. Gleich GJ, Adolphson CR. The eosinophilic leukocyte: structure and function. Adv Immunol 1986;39:177-253. 9. Mc Laren DJ, Mc Kean JR, Olsson I, Venge P, Kay AB. Morphological studies on the killing of schistosomula of Schistosoma mansoni by human eosinophils and neutrophil cationic proteins in vitro. Parasite Immunol 1981;3:359-73. 10. Young DE, Peterson CGB, Venge P, Cobn ZA. Mechanism of membrane damage mediated by human eosinophil cationic protein. Nature 1986;321:613-8. 11. Dahl R, Venge P. Activation of blood coagulation during inhalation challenge tests. Allergy 1981;36:129-33. 12. Venge P. The eosinophil. In: Godard Ph, Bousquet J, and Michel FB, eds. Advances in allergology and clinical immunology. Cornforth, U.K.: The Parthenon PuNishing Group Ltd, 1992:175-85. 13. Carlson M, Hakansson L, Peterson C, Stalenheim G, Venge P. Secretion of granule proteins from eosinophiis and neutrophils is increased in asthma. J Allergy Clin Immunol 1991;87:27-33. 14. Dahl R, Venge P. Are blood eosinophil number and activity important for the development of the late asthmatic reaction after allergen challenge? Eur Respir J 1989;2:430s434s. 15. Bisgaard H, Grenborg H, Mygind N, et al. Allergeninduced increase of eosinophil cationic protein in nasal lavage fluid: effect of the glucocorticoid budesonide. J Allergy Clin Immunol 1990;85:891-5. 16. Klementsson H, Andersson M, Pipkorn U. Allergen-induced increase in non specific nasal reactivity is blocked by antihistamines without a clear-cut relationship to eosinophil influx. J Allergy Clin Immunol 1990;86:466-72. 17. Svensson C, Andersson M, Persson CGA, et al. Albumin, bradykinins and eosinophil cationic protein on the nasal mucosal surface in patients with hay fever during natural allergen exposure. J Allergy Clin Immunol 1990;85:828-33. 18. Venge P, Hakansson L, Peterson CGB. Eosinophil activa-
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 6
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
tion in allergic diseases. Int Arch Allergy Appl Immunol 1987;82:333-7. Rak S, Lowhagen O, Venge P. The effect of immunotherapy on bronchial hyperresponsiveness and eosinophil cationic protein in pollen-allergic patients. J Allergy Clin Immunol 1988;82:470-80. Bousquet J, Chanez P, Lacoste JY, et al. Indirect evidence of bronchial inflammation assessed by titration of inflammatory mediators in BAL fluid of patients with asthma. J Allergy Clin Immunol 1991;88:649-60. Klementsson H, Svensson C, Andersson M, et al. Eosinophils, secretory responsiveness and glucocorticoid-induced effects on the nasal mucosa during a weak pollen season. Clin Exp Allergy 1991;21:705-10. Durham SR, Loegering DA, Dunnette S, Gleich GJ, Kay AB. Blood eosinophils and eosinophil-derived proteins in allergic asthma. J Allergy Clin Immunol 1989;84:931-6. Andersson M, Andersson P, Venge P, Pipkorn U. Eosinophils and eosinophil cationic protein in nasal lavages in allergen-induced hyperresponsiveness: effects of topical glucocorticosteroid treatment. Allergy 1989;44:342-8. Dahl R, Venge P, Olsson I. Variations of blood eosinophils and eosinophil cationic protein in serum in patients with bronchial asthma. Allergy 1978;33:211-5. Pipkorn U, Karlsson G, Enerback L. The cellular response of the human allergic mucosa to natural allergen exposure. J Allergy Clin Immunol 1988;82:1046-54. Winqvist I, Olsson I, Werner S, Stenstam M. Variations of cationic proteins from eosinophil leukocytes in food intolerance and allergic rhinitis. Allergy 1981;56:419-23. Sugai T, Sakiyama Y, Matumoto S. Eosinophil cationic protein in peripheral blood of pediatric patients with allergic diseases. Clin Exp Allergy 1992;22:275-81. Venge P, Zetterstrom O, Dahl R, Roxin LE, Olsson J. Low levels of eosinophil cationic protein in patients with asthma. Lancet 1977;2:373-5. Venge P. Serum measurements of eosinophil cationic pro-
T o m a s s i n i et al.
30.
31.
32. 33.
34.
35.
36. 37.
38.
39.
40.
1355
tein (ECP) in bronchial asthma. Clin Exp Allergy 1993; 23(suppl 2):3-7. Reimert CM, Venge P, Kharazni A, Bendtzen K. Detection of eosinophil cationic protein (ECP) by an enzyme-linked immunosorbent assay. J Immunol Methods 1991;138:285-90. Peterson CGB, Enander I, Nystrand J, et al. Radioimmunoassay of human eosinophil cationic protein (ECP) by an improved method. Establishment of normal levels in serum and turnover in vivo. Clin Exp Allergy 1991;21:561-7. Gray D, Matzinger P. T-cell memory is short-lived in the absence of antigen. J Exp Med 1991;174:969-74. Mosmann TR, Coffman RL. Thl and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1989;7:145-73. Cogan E, Schandeye A, Crusiaux P, et al. Clonal proliferation of type 2 helper T cells in a man with the hypereosinophilic syndrome. N Engl J Med 1994;330:535-8. Arai K, Yokota T, Watanabe S, Arai N, Miyajima A. The regulation of allergic response by cyytokine receptor networks. ACI News 1992;4:113-20. Romagnani S. Regulation and deregulation of IgE synthesis. Immunol Today 1990;11:316-21. Lopez AF, Sanderson CJ, Gamble JR, et al. Recombinant human IL-5 is a selective activator of human eosinophil function. J Exp Med 1988;167:219-24. Van Leeuwen BH, Martinson ME, Webb GGC, Young IG. Molecular organization of the cytokine gene cluster involving the human IL-3, IL-4, IL-5 and GM-CSF genes on human chromosome 5. Blood 1989;73:1142-8. Bonini S, Magrini L, Rotiroti G, Ronchetti MP, Onorati P. Genetic and environmental factors in the changing incidence of allergy. Allergy 1994;49:6-14. Kay AB, Ying S, Varney V, et al. Messenger RNA expression of the cytokine gene cluster, interleukin-3 (IL-3), IL-4, IL-5 and GGM-CSF, in allergen-induced late-phase cutaneous reactions in atopic subjects. J Exp Med 1991;173: 775-8.
It is now well established that the pathophysiology of allergic diseases is characterized by ongoing tissue inflammation, which follows the classical type I immediate hypersensitivity reaction. The activation and recruitment of several effector cells causes morphologic changes, hyperreactivity of target tissues, and, finally, clinical symptoms. 1-3 In From aIstituto di I ° Clinica Medica, Universityof Rome "La Sapienza"; bDepartment of Ophthalmology, University of Rome "Tor Vergata"; and CDepartment of Clinical Immunology and AUergology,2rid Universityof Naples. Margherita Tomassini was supported by the "Istituto PasteurCenci Bolognetti" Foundation, University of Rome "La Sapienza", Italy. Received for publication Jan. 4, 1995; revised July 28, 1995; accepted for publication Aug. 28, 1995. Reprint requests: Sergio Bonini, MD, EAACI, Istituto di I Clinica Medica, Universityof Rome "La Sapienza",Viale del Policlinico, 00161, Rome, Italy. Copyright © 1996 by Mosby-Year Book, Inc. 0091-6749/96 $5.00 + 0 1/1/68857 1350
Abbreviations used ECP: Eosinophil cationic protein HDM: House dust mite PA: Perennial allergen SA: Seasonal allergen
these inflammatory processes eosinophils are considered to play a pivotal role. In fact, the number of eosinophils is increased in blood and tissues in patients with allergic diseases, e, 5 Moreover, eosinophils are massively recruited on specific allergen challenge in several organs and biologic fluids. 3 Finally, in the lungs, nose, skin, and eyes, the release of oxygen radicals, newly generated mediators such as platelet activating factor and leukotriene C4, and granule cationic proteins (major basic protein, eosinophil peroxidase, eosinophil cationic protein [ECP], eosinophil-derived neuro-
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 6
toxin/eosinophil protein X), has been shown to be responsible for basic histopathologic changes of target tissues, such as vasodilation, mucosal edema, epithelial cell damage, and nonspecific hyp erre activity. 1-3,6 A m o n g inflammatory mediators of human eosinophils, granule cationic proteins have been well characterized, 7 and several functions of these proteins have been described. 8 In particular, it has been reported that ECP is a potent toxin for parasites 9 and mammalian cells in vitro. 1° Moreover, it interacts with blood coagulation and fibrinolytic factors n and can activate tissue fibroblasts, promoting their proliferation and collagen deposition. 12 Several reports describe an increase of ECP in serum and biologic fluids in patients with allergic diseases, 13-28 suggesting that the measurement of E C P levels might be a useful clinical test in monitoring disease activity and the efficacy of therapeutic agents. Most of these studies, however, are based on population samples that are small or not well defined in relation to the type of disease, type of sensitization, and other clinical variables. This may account for the great variability of ECP values recorded in nonhomogeneous study groups. In this article, we report data collected from a large population sample, showing that natural allergen exposure is an important factor influencing serum ECP levels in allergic diseases.
METHODS Two-hundred and sixty-five outpatients (58.7% female patients; age range, 6 to 67 years) with bronchial asthma (66 patients with isolated asthma; 28 with asthma and rhinitis; and 30 with asthma, rhinitis, and conjunctivitis), rhinitis (60 patients with rhinitis and 42 with rhinoconjunctivitis), or isolated conjunctivitis (39 patients) were included in this study. The presence of a specific sensitization to common inhalant allergens was documented in 195 patients by clinical history, objective examination, skin prick tests, and the measurement of specific IgE serum levels by RAST (Kabi Pharmacia AB, Uppsala, Sweden). Subjects sensitized to grass (n = 65) and those sensitized to Dermatophagoides pteronyssinus, Parietaria officinalis, or both (n = 130) were considered as separate groups. In this second group, 43 subjects were sensitized to house dust mites (HDMs), 31 to Parietaria species and grass, 37 to HDMs and grass, and 19 to HDMs and Parietaria species. In fact, in our climate, grass pollen is strictly a seasonal allergen (SA), whereas HDM and Parietaria species are present throughout the year. At the time of blood collection, no patients was receiving any therapy, and none had received specific immunotherapy in the 5 years before the study. Forty-five disease-free matched subjects (15 children
T o m a s s i n i et al.
1351
under 12 and 30 adults; 30 male and 15 female) who had no history of allergy, blood eosinophil count in the normal range, and total IgE levels below 250 kU/L were selected as control subjects. The mean age of the control group was 27.5 + 17 years (range, 7 to 63 years); the mean total IgE level was 50.92 _+ 56.82 kU/L (range, 0 to 222 kU/L). Venous peripheral blood (10 ml) was drawn, after informed consent was obtained, into preservative-free tubes. Blood was allowed to clot at constant room temperature (20° to 22 ° C) for 60 _+ 10 minutes, centrifuged at 1250 g for 20 minutes at 4° C, and stored at - 2 0 ° C until assayed. ECP was measured in serum samples by a competitive radioimmunoassay (ECP-RIA kit, Kabi Pharmacia) according to the manufacturer's instructions. All samples were run in duplicate. The range of the standard curve was 2 to 200 p~g/L.With this procedure, according to the manufacturer, normal ranges of serum ECP are 2 to 16 Ixg/L. Because it has been suggested that ECP serum levels can be influenced by the coagulation process and by in vitro handling of the samples, 29-31 all tests were performed in the same run to avoid interassay variations. Moreover, clotting procedures were performed under the same temperature conditions. Parametric statistical analysis of variance was performed to compare patients with allergy and control subjects, and multiple analysis of variance was performed for type of sensitization and type of clinical disease. In fact, the distribution of ECP values, preliminarily evaluated, appeared to be normal. Moreover, our large samples allowed the choice of parametric tests. Total IgE levels are not normally distributed. Accordingly, a nonparametric test (Spearman's correlation) was performed to analyze the relationshi p between serum ECP levels and total IgE. A p value of 0.05 or less was considered statistically significant.
RESULTS Serum E C P levels in the patient group (22.7 _+ 18.2 ~g/L) were significantly higher than those in the control group (6.1 + 4.8 p~g/L) (t --- 6.06, d f = 308, p < 0.001) (Fig. 1). Patients with allergy had significantly higher E C P levels than control subjects, but were not different from those of patients without clinical evidence of sensitization. Analysis of variance performed with the Student-Newmann-Keuls test showed that among variables studied, the type of allergen responsible for sensitization was the only factor influencing the high variability of values observed in the allergic group. In fact, patients with allergy sensitized to perennial allergens (PAs) had significantly higher ECP serum values than patients sensitized to seasonal allergens (n = 130, 35.1 + 33.4 ~g/L vs n =
1352
ECP
Tomassini et al.
J ALLERGYCLIN IMMUNOL JUNE 1996
IJg/I
ECP pg/I
50
80~
22.7--- 18.2
70
35.1 _+33.4
40 60
p<0.001
p<0.001 50
30
t i
40 30 6.1 _+4.8 20
10
15.6_+12.2
10 0
Controls n=45
Patients n=265
FIG. 1. Serum levels !means _+ SD) of ECP were significantly higher in allergic patients than in control subjects (p < 0.001).
65, 15.6 _+ 12.2 p~g/L;p < 0.001) (Fig. 2). Moreover, although E c p levels did not change significantly throughout the year when PAs were responsible for clinical symptoms, mean ECP levels of subjects sensitized to SAs were in the normal range during winter, showed a slight increase at the beginning of the pollen season, and became as high as ECP values observed for subjects sensitized t o PAs during the pollen season (Fig. 3). No age or sex dependence of serum ECP levels was observed in our study group. Apparently, serum ECP levels were significantly higher in patients with asthma and conjunctivitis than in Patients with rhinitis (p < 0.01). However, analysis of variance showed that differences observed were due to differences in the distribution of the type of sensitization to the various allergens studied in the different disease groups. In fact, in our patients rhinitis was more often caused by SAs (grass, Olea europea), whereas asthma and conjunctivitis were mainly caused by PAs (Dermatophagoides spp., P. officinalis, cat and dog danders). Neither severity nor duration of clinical symptoms, nor levels of specific IgE (assessed by RAST class) could account for the differences found. No significant difference in ECP serum levels was found between subjects with positive RAST results and those with negative RAST results for various diseases considered. The relationship between serum ECP levels and total eosinophil count was analyzed in 39 patients with or without peripheral eosinophilia. The number of circulating eosinophils was found to be related to ECP serum levels (r = 0.477, t = 3.299, df = 37, p < 0.005).
0 PA n--130
$A n=65
FIG. 2. Serum levels (means _+ SD) of ECP were significantly higher in patients sensitized to perennial allergens (PA) than in patients sensitized to seasonal allergens (SA) (p < 0.001).
The relationship between serum ECP levels and total IgE was evaluated in 85 patients, independently of the clinical picture, by Spearman's correlation test. The correlation between these two parameters was significant, in spite of the low value of rs (rs = 0.293, df = 83, p < 0.01) (Fig. 4). DISCUSSION
Our results show that serum ECP levels are significantly increased in patients with allergic disease compared with normal control subjects, even when the number of circulating eosinophils is in the normal range. It has been reported that ECP measured in the blood is mainly released in vitro during the blood coagulation process. 29 Accordingly, plasma and serum levels may be different, and results also depend on procedures used in handling blood samples. 3°,31 We have measured ECP in sera with a standard procedure for all samples. This should avoid pitfalls in ECP determination. Moreover, if the above-reported hypothesis is true, our results imply that allergic diseases, independently of the clinical picture, (i.e., asthma, rhinitis, o r conjunctivitis) are "systemic" diseases, because primed eosinophils are present in the circulation and not only in the target tissue. Several reports describe an increase of ECP in serum and biologic fluids in patients with allergic diseases, 13-z8 suggesting that the measurement of ECP levels might be a useful clinical test for monitoring disease activity and the effects of therapeutic agents. Most of these studies, however, are based on population samples that are small or not
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 6
Tomassini
e t al.
1353
ECP/~g/I 120
100
80
60 m |
.
: : •
:
•
.
m
•
:.
40 i
20
,
n
•
:1 : .... ,
'
!
m
PA SA Oct-Jan p<0.001
:::::I
'
•
PA SA Feb-Mar n.s.
'
PA SA Apr-Jun n.s.
FIG. 3. ECP serum levels at various periods of collection of sera in patients with hypersensitivity to perennial allergens (PA) and seasonal allergens (SA). Total IgE kU/L 1,200
1,000 '
:
=I
800 S
:i: ~:ii
600 p<0,O1
A
400
200
m
'b
m
mm m
i
#m
• •
m
•
•
•
m l
0 0
10
20
30
40
50
60
70
80
90
100 110 120 ECP/~g/L
FIG. 4, Serum ECP and total IgE levels were significantly correlated in patients with allergy (rs = 0.293, p < 0.01).
well defined in relation to the type of disease, type of sensitization, and other clinical variables. This might account for the great variability of ECP values in nonhomogeneouS study groups.
In this article, we report data collected from a large population sample, showing that natural allergen exposure is an important factor influencing serum ECP levels in patients with allergic diseases.
1354
T o m a s s i n i et al.
J ALLERGY CLIN IMMUNOL JUNE 1996
In fact, when analyzing the relationship between specific sensitization and serum E C P levels, we f o u n d that, independently of the clinical picture, serum E C P levels are higher in subjects sensitized to PAs than in subjects sensitized to SAs. Moreover, in patients allergic to grass, serum E C P levels reach high values only during the pollen season, This implies that, independently of the biochemical features of the specific allergen responsible for clinical symptoms, long-term allergen exposure is a crucial factor for the continuous activation of the allergic reaction in h u m a n beings, as it is in rodents. 32 It is well known that long-term stimuli can induce both an increase in total I g E levels and peripheral eosinophilia. In fact, total I g E levels were significantly correlated to E C P serum levels (p < 0.01), although the low correlation coefficient (0.293) indicates that a "private" regulatory m o d ulation is present in addition to c o m m o n regulatory factors. In reference to these factors, it must be noted that both of these parameters are highly influenced by the degree of T-cell activation. In particular, a T H 2 cell subset has been invoked to m o d u l a t e the allergic response t h r o u g h its peculiar cytokine profile. 33 A clonal expansion of TH2 cells in vivo has b e e n recently d e m o n s t r a t e d in a case of hypereosinophilia. 34 In fact, IL-3 can influence mast cell and basophil activation and releasability; IL-4 and IL-13 total l g E and I g G 4 production; IL-5 maturation, activation, and recruitment; and survival of eosinophilsY -37 Interestingly enough, all of these cytokines and granulocyte-macrophage colony-stimulating factor are u n d e r the control of genes closely located on h u m a n c h r o m o s o m e 5. 38 O n the other hand, specific I g E response is also influenced by other genetic (major histocompatibility complex genes) 39 and environmental factors. O n the basis of the above-reported data, we might suggest that phenotypic markers of the overexpression 4° of the 5q cytokine-gene cluster (including total I g E and E C P ) can represent a c o m m o n hallmark of all allergic diseases, whereas specific I g E antibodies are detectable only in some patients with allergy. In fact, E C P serum levels were significantly increased in both I g E - m e d i a t e d and nonI g E - m e d i a t e d forms of diseases studied. The clinical counterpart o f this point of view is that I g E tests, so widely p e r f o r m e d in patients with allergy, should be c o m p l e m e n t e d by other, new allergy tests. Only m o r e extensive and prospective studies can validate the usefulness of E C P determination in both I g E - m e d i a t e d and n o n - I g E - m e diated clinical allergies.
We thank Professor Francesco Vecchi (Department of Human Biology, University of Rome "La Sapienza") for assistance with statistical analysis.
REFERENCES
1. Bousquet J, Chanez P, Campbell AM, et al. Inflammatory processes in asthma. Int Arch Allergy Appl Immunol 1991;94:227-32. 2. Weller PF. The immunobiology of eosinophils. N Engl J Med 1991;324:1110-8. 3. Hallgren R, Venge P. The eosinophil in inflammation. In: Matsson P, Ahlstedt S, Venge P, Thorell J, eds. Clinical impact of the monitoring of allergic inflammation. London: Academic Press, 1991:119-40. 4. Fukuda T, Gleich GJ. Heterogeneity of human eosinophils. J Allergy Clin Immunol 1989;83:369-73. 5. Capron M, Kusnierz JP, Prin L, et al. Cytophilic IgE on human blood and tissue eosinophils: detection by flow microfluorometry. J Immunol 1985;134:3013-8. 6. Bousquet J, Chanez P, Lacoste JY, et al. Eosinophilic inflammation in asthma. Int Arch Allergy Appl Immunol 1991;94:227-32. 7. Dvorak AM, Ackerman SJ, Weller PF. Subcellular morphology and biochemistry of eosinophils. In: Harris JR, ed. Blood cell biochemistry. Megakaryocytes, platelets, macrophages and eosinophils. London: Plenum Publishing 1990; 2:237-44. 8. Gleich GJ, Adolphson CR. The eosinophilic leukocyte: structure and function. Adv Immunol 1986;39:177-253. 9. Mc Laren DJ, Mc Kean JR, Olsson I, Venge P, Kay AB. Morphological studies on the killing of schistosomula of Schistosoma mansoni by human eosinophils and neutrophil cationic proteins in vitro. Parasite Immunol 1981;3:359-73. 10. Young DE, Peterson CGB, Venge P, Cobn ZA. Mechanism of membrane damage mediated by human eosinophil cationic protein. Nature 1986;321:613-8. 11. Dahl R, Venge P. Activation of blood coagulation during inhalation challenge tests. Allergy 1981;36:129-33. 12. Venge P. The eosinophil. In: Godard Ph, Bousquet J, and Michel FB, eds. Advances in allergology and clinical immunology. Cornforth, U.K.: The Parthenon PuNishing Group Ltd, 1992:175-85. 13. Carlson M, Hakansson L, Peterson C, Stalenheim G, Venge P. Secretion of granule proteins from eosinophiis and neutrophils is increased in asthma. J Allergy Clin Immunol 1991;87:27-33. 14. Dahl R, Venge P. Are blood eosinophil number and activity important for the development of the late asthmatic reaction after allergen challenge? Eur Respir J 1989;2:430s434s. 15. Bisgaard H, Grenborg H, Mygind N, et al. Allergeninduced increase of eosinophil cationic protein in nasal lavage fluid: effect of the glucocorticoid budesonide. J Allergy Clin Immunol 1990;85:891-5. 16. Klementsson H, Andersson M, Pipkorn U. Allergen-induced increase in non specific nasal reactivity is blocked by antihistamines without a clear-cut relationship to eosinophil influx. J Allergy Clin Immunol 1990;86:466-72. 17. Svensson C, Andersson M, Persson CGA, et al. Albumin, bradykinins and eosinophil cationic protein on the nasal mucosal surface in patients with hay fever during natural allergen exposure. J Allergy Clin Immunol 1990;85:828-33. 18. Venge P, Hakansson L, Peterson CGB. Eosinophil activa-
J ALLERGY CLIN IMMUNOL VOLUME 97, NUMBER 6
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
tion in allergic diseases. Int Arch Allergy Appl Immunol 1987;82:333-7. Rak S, Lowhagen O, Venge P. The effect of immunotherapy on bronchial hyperresponsiveness and eosinophil cationic protein in pollen-allergic patients. J Allergy Clin Immunol 1988;82:470-80. Bousquet J, Chanez P, Lacoste JY, et al. Indirect evidence of bronchial inflammation assessed by titration of inflammatory mediators in BAL fluid of patients with asthma. J Allergy Clin Immunol 1991;88:649-60. Klementsson H, Svensson C, Andersson M, et al. Eosinophils, secretory responsiveness and glucocorticoid-induced effects on the nasal mucosa during a weak pollen season. Clin Exp Allergy 1991;21:705-10. Durham SR, Loegering DA, Dunnette S, Gleich GJ, Kay AB. Blood eosinophils and eosinophil-derived proteins in allergic asthma. J Allergy Clin Immunol 1989;84:931-6. Andersson M, Andersson P, Venge P, Pipkorn U. Eosinophils and eosinophil cationic protein in nasal lavages in allergen-induced hyperresponsiveness: effects of topical glucocorticosteroid treatment. Allergy 1989;44:342-8. Dahl R, Venge P, Olsson I. Variations of blood eosinophils and eosinophil cationic protein in serum in patients with bronchial asthma. Allergy 1978;33:211-5. Pipkorn U, Karlsson G, Enerback L. The cellular response of the human allergic mucosa to natural allergen exposure. J Allergy Clin Immunol 1988;82:1046-54. Winqvist I, Olsson I, Werner S, Stenstam M. Variations of cationic proteins from eosinophil leukocytes in food intolerance and allergic rhinitis. Allergy 1981;56:419-23. Sugai T, Sakiyama Y, Matumoto S. Eosinophil cationic protein in peripheral blood of pediatric patients with allergic diseases. Clin Exp Allergy 1992;22:275-81. Venge P, Zetterstrom O, Dahl R, Roxin LE, Olsson J. Low levels of eosinophil cationic protein in patients with asthma. Lancet 1977;2:373-5. Venge P. Serum measurements of eosinophil cationic pro-
T o m a s s i n i et al.
30.
31.
32. 33.
34.
35.
36. 37.
38.
39.
40.
1355
tein (ECP) in bronchial asthma. Clin Exp Allergy 1993; 23(suppl 2):3-7. Reimert CM, Venge P, Kharazni A, Bendtzen K. Detection of eosinophil cationic protein (ECP) by an enzyme-linked immunosorbent assay. J Immunol Methods 1991;138:285-90. Peterson CGB, Enander I, Nystrand J, et al. Radioimmunoassay of human eosinophil cationic protein (ECP) by an improved method. Establishment of normal levels in serum and turnover in vivo. Clin Exp Allergy 1991;21:561-7. Gray D, Matzinger P. T-cell memory is short-lived in the absence of antigen. J Exp Med 1991;174:969-74. Mosmann TR, Coffman RL. Thl and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 1989;7:145-73. Cogan E, Schandeye A, Crusiaux P, et al. Clonal proliferation of type 2 helper T cells in a man with the hypereosinophilic syndrome. N Engl J Med 1994;330:535-8. Arai K, Yokota T, Watanabe S, Arai N, Miyajima A. The regulation of allergic response by cyytokine receptor networks. ACI News 1992;4:113-20. Romagnani S. Regulation and deregulation of IgE synthesis. Immunol Today 1990;11:316-21. Lopez AF, Sanderson CJ, Gamble JR, et al. Recombinant human IL-5 is a selective activator of human eosinophil function. J Exp Med 1988;167:219-24. Van Leeuwen BH, Martinson ME, Webb GGC, Young IG. Molecular organization of the cytokine gene cluster involving the human IL-3, IL-4, IL-5 and GM-CSF genes on human chromosome 5. Blood 1989;73:1142-8. Bonini S, Magrini L, Rotiroti G, Ronchetti MP, Onorati P. Genetic and environmental factors in the changing incidence of allergy. Allergy 1994;49:6-14. Kay AB, Ying S, Varney V, et al. Messenger RNA expression of the cytokine gene cluster, interleukin-3 (IL-3), IL-4, IL-5 and GGM-CSF, in allergen-induced late-phase cutaneous reactions in atopic subjects. J Exp Med 1991;173: 775-8.