Annals of Clinical & Laboratory Science 36:262-266 (2006)
© 2006 Association of Clinical Scientists
Plasma Eosinophil Cationic Protein, Interleukin-5, and ECP/Eo Count Ratio in Patients with Various Eosinophilic Diseases
Yeon-Joon Park1,
Eun-Jee Oh1,
Jong-Won Park2,
Myungshin Kim1 and
Kyungja Han1
1 Departments of Laboratory Medicine and 2 Internal Medicine, College of Medicine, Catholic University of Korea, Seoul, Korea
Address correspondence to Eun-Jee Oh, M.D., Department of Laboratory Medicine, College of Medicine, Catholic University of Korea, 505 Banpo-dong Seocho-ku, Seoul, 137-040, Korea (South); tel 82 2 590 2221; fax 82 2 536 1594; e-mail ejoh{at}catholic.ac.kr
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Abstract
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Hypereosinophilia is associated with clonal disorders, reactive conditions, and rarely with idiopathic hypereosinophilic syndrome (IHES). We investigated whether measurement of eosinophilic activity using the plasma eosinophil cationic protein (ECP) level, interleukin-5 (IL-5) level, and the ratio of eosinophilic cationic protein/eosinophil count (ECP/Eo) could improve the early differentiation among various eosinophilic diseases: IHES (n = 9), clonal disorder (n = 35), reactive eosinophilia with malignancy (n = 30), and reactive eosinophilia with inflammation (n = 46). The 120 eosinophilic patients had higher plasma ECP and IL-5 levels than the non-eosinophilic control group (p <0.05). The 9 patients with IHES had significantly higher plasma ECP and IL-5 levels than patients with other eosinophilic diseases (p <0.05). The plasma levels of ECP and the ECP/Eo ratio were higher in patients with non-haematologic malignancy than in those with other reactive eosinophilias (p <0.05). This study shows that levels of plasma ECP, IL-5, and ECP/Eo ratio may assist in the clinical differentiation of various eosinophilic diseases.
Keywords: eosinophils, eosinophil cationic protein (ECP), ECP/Eo ratio, interleukin-5, hypereosinophilic syndrome
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Introduction
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Hypereosinophilia is a common finding in clinical practice and is associated with clonal disorders and reactive conditions [1–3]. Patients with clonal eosinophilia display either cytogenetic evidence or bone marrow histological evidence of an otherwise classified hematologic malignancy such as acute leukemia or chronic myeloid disorder. The causes of reactive eosinophilia include tissue-invasive parasitosis, allergic or inflammatory conditions, and malignancy.
In malignancy, eosinophils are not considered to be part of the neoplastic process and eosinophilia is thought to be the result of eosinophilopoietic cytokines, including interleukin-5 (IL-5) and other cytokines or chemokines that are elaborated by tumor cells [4]. However, eosinophilia may precede the clinical diagnosis of malignancy and may require a prolonged period of observation. Therefore, when dealing with a patient presenting hypereosinophilia, the clinician’s major task is to assess the underlying disease [2], and the method of treatment will vary depending on whether a clonal disorder of bone marrow or a malignancy has been identified as the cause of the eosinophilia. The current evaluation of patients with eosinophilia includes a thorough patient history, basic laboratory tests, and bone marrow examination with cytogenetics [3]. Thus, a non-invasive specific laboratory test would be helpful for differential diagnosis of eosinophilia. When thorough evaluation fails to reveal any clonal abnormality or reactive cause, the term idiopathic hypereosinophilic syndrome (IHES) is used. IHES is a rare disease and is defined as a persistent marked hypereosinophilia of unknown origin, generally complicated by end-organ damage [1]. For patients with IHES, the clinical appearance and tissue damage are the results of accumulation of eosinophils along with their toxic granules, and the clinical treatment consists of controlling the eosinophil levels and the eosinophilic activity.
IL-5 is a major eosinophilopoietic cytokine and is produced by lymphocytes, malignant cells, and the eosinophil itself [5,6]. The overproduction of eosinophilopoietic cytokine is sufficient to induce blood and tissue eosinophilia by stimulating bone marrow generation and inhibiting peripheral destruction [7]. Eosinophil cationic protein (ECP) is a cytotoxic protein that originates from eosinophil granules, and the plasma levels of ECP may reflect the degree of eosinophilic activation [8]. While previous reports have shown higher levels of ECP in patients with IHES or some solid tumors than in healthy controls [9,10], the eosinophil activity has not been compared between patients with clonal eosinophilia and patients with reactive eosinophilia. Moreover, the clinical usefulness of plasma ECP is still uncertain. Some recent studies in patients with asthma have shown that the ratio of eosinophil cationic protein/eosinophilic count (ECP/Eo) may be a marker that is better than using the serum ECP and eosinophilic blood count separately [11,12]. So, we also used this parameter to compare the different eosinophilic diseases.
We hypothesized that we could improve the early identification of clinical subtypes of eosinophilia by measuring the eosinophilic activity via plasma ECP, ECP/Eo ratio, and IL-5 levels. The aims of the present study were to determine whether the eosinophil activities differ among patients having clonal eosinophilia, reactive eosinophilia, and IHES, and whether the plasma ECP, ECP/Eo ratio, and IL-5 levels are helpful for phenotypic differentiation of eosinophilia.
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Methods
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Patients and their clinical characteristics.
This study consisted of 25 non-eosinophilic controls and 120 eosinophilic patients who underwent a bone marrow (BM) examination before receiving treatment for their specific disease; 9 patients had IHES, 35 patients had clonal disorder, 30 patients had reactive eosinophilia with malignancy, and 46 patients had reactive eosinophilia with inflammation. The eosinophilic patients included those who had blood eosinophilia (absolute eosinophil count [AEC] 
600 cells/µl) or BM eosinophilia (8% eosinophils and eosinophilic precursors). Based on the clinical investigations, a diagnosis of IHES was made (AEC 1500 cells/µl for at least 6 mo and target organ damage), and this was confirmed upon review of the BM slides. Acute myeloid leukemia, chronic myeloid leukemia, and myelo-proliferative disorder were categorized as clonal disorders. Lymphoma and non-hematologic cancer including lung cancer and metastatic cancer were categorized as reactive eosinophilia with malignancy. Allergies such as asthma or atopic dermatitis, lung diseases, gastrointestinal diseases, and idiopathic/autoimmune inflammatory conditions were considered as allergic inflammation.
Eosinophil, plasma ECP, ECP/Eo ratio, and IL-5 assays.
The AEC in peripheral blood was measured with a SE 9000 electronic counter (Sysmex Co.), and expressed as the number of cells/µl. The % of eosinophils in BM was calculated from the differential count of 500 nucleated cells by two of the authors, and the BM eosinophil index was used to obtain the absolute count in the BM with the following formula: BM eosinophil index = (% eosinophils in BM x cellularity)/100. The plasma concentration of ECP released per peripheral blood eosinophil for each patient was expressed as the ratio of the plasma ECP concentration (µg/L) to the eosinophilic count (cells/µl) using the following formula: ECP/Eo ratio (pg/cell) = ECP (µg/L)/number of eosinophils (cells/µl).
Plasma ECP and IL-5 levels were measured with a commercial ELISA kit for ECP (Medical & Biological Laboratories, Japan) and a Quantikine human IL-5 kit (R&D Systems, Minneapolis, MN, USA) according to the manufacturers’ instructions. The plasma specimen was obtained at diagnosis prior to any treatment, and the aliquots of plasma were stored at –80°C before being analyzed. Because cells in the EDTA blood are inactivated and do not release any granules ex vivo [8], EDTA plasma was used in this study. Spearman’s test was used to investigate the correlations between the ECP, AEC, and the bone marrow eosinophil count. Statistical tests were performed with Windows SPSS (version 10.1) and differences between groups were evaluated by the Mann-Whitney U-test.
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Results
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The plasma levels of ECP and IL-5 are listed in Table 1
according to the underlying disease and the AEC. The 120 eosinophilic patients had higher plasma ECP and IL-5 levels than the 25 non-eosinophilic control subjects (p <0.05), but there was no difference of ECP/Eo ratio. The ECP level showed no correlation with the BM eosinophil index (r = 0.16) and weak correlation with the AEC (r = 0.49) (Fig. 1). Among the eosinophilic patients, the 9 patients with IHES had higher levels of ECP (mean ± SD, 243.0 ± 64.8 µg/L) and IL-5 (14.2 ± 3.1 pg/ml) than patients with other eosinophilic diseases (ECP: 90.6 ± 98.4 µg/L, p <0.05, IL-5: 9.7 ± 4.0 pg/ml, p <0.05). The ECP/Eo ratio in patients with IHES was similar to those in other eosinophilic groups.
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Fig. 1. A weak positive correlation was noted between the absolute eosinophilic counts and the plasma eosinophilic cationic protein (ECP) levels (asterisks = diopathic hypereosinophilic syndrome; solid circles = clonal disorder; open circles = reactive eosinophilia with malignancy; open triangles = reactive eosinophilia with inflammation).
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In comparisons of eosinophilic activities among the 3 eosinophilic populations (clonal disorder, reactive eosinophilia with malignancy, and reactive eosinophilia with inflammation), although there was no statistical significance, the patients with clonal disease (101.7 ± 99.0 µg/L) and the patients with reactive eosinophilia with malignancy (103.8 ± 104.3 µg/L) had higher plasma ECP levels than the patients with reactive eosinophilia with inflammation (75.0 ± 92.3 µg/L) (p = 0.11 and p = 0.12, respectively) (Fig. 2). There was no difference of plasma IL-5 levels between patients with clonal disorders and those with reactive eosinophilia (p >0.05).
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Fig. 2. The levels of plasma eosinophil cationic protein (ECP) (a) and IL-5 (b) in non-eosinophilic controls (n = 25) and 120 eosinophilic patients who were categorized as patients with idiopathic hypereosinophilic syndrome (IHES) (n = 9), clonal disorder (n = 35), reactive eosinophilia with malignancy (n = 30), and reactive eosinophilia with inflammation (n = 46). The IHES patients had higher levels of plasma ECP and IL-5 than the other eosinophilic patients (*p <0.05).
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Among the patients with reactive eosinophilia, those with non-hematologic malignancy had significantly higher plasma ECP levels (158.2 ± 75.1 µg/L) and ECP/Eo ratios (0.26 ± 0.16) than the other patients (p <0.05, Table 1
).
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Discussion
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Our findings indicate that the plasma ECP level showed no correlation with the BM eosinophil index and weak correlation with the AEC. These results are consistent with a previous study, and support the finding that the ECP measurement provides different and more useful information about the eosinophilic activity compared to mere enumeration of the eosinophil count [8]. The reason for this finding is that the eosinophil count in blood reflects a balance between the BM production of eosinophils, tissue egress, eosinophil apoptosis, and cytolytic degradation [13,14], while most eosinophils are normally found in tissues.
The patients with IHES showed higher levels of plasma ECP and IL-5 than the patients with other eosinophilic diseases. These findings support the findings that more aggressive tissue damage and clinical manifestations in patients with IHES are a direct result of the accumulation of eosinophils and increased eosinophilopoietic activity. Because distinguishing the rare, but more serious, IHES disease from the other eosinophilic disorders is a difficult task and prolonged diagnostic procedures are sometimes required, the measurement of plasma ECP and IL-5 seems a useful way to differentiate IHES from the other hypereosinophilic diseases. However, in a recent analysis of patients with eosinophilia, the discovery of the cryptic FIP1L1-PDGFRA gene fusion in cytogenetically normal patients with eosinophilia has redefined these diseases as clonal eosinophilia [15]. As the clinical presentation and course of FIP1L1-PDGFRA positive cases is presently unknown, and this study used phenotypically classified IHES, further studies should be performed focused on the molecularly characterized genotypes of clonal eosinophilia in association with plasma ECP and IL-5 levels.
Because previous studies showed high levels of ECP in patients with Hodgkin’s lymphoma and renal cell carcinoma [10,16], we compared the eosinophilic activities among the 3 eosinophilic populations (clonal disorder, reactive eosinophilia with malignancy, and reactive eosinophilia with inflammation). Although there was no statistical significance, our results suggested that patients with clonal disease or malignancy had higher plasma ECP levels than patients with inflammation. When we compared the ECP levels in patients with reactive eosinophilia, patients with non-hematologic malignancy had significantly higher plasma ECP levels than patients with reactive eosinophilia. For patients with malignant disease, eosinophils are known to be cytotoxic against tumor cells [17], and eosinophilia in the blood can be induced by the chemotactic factors released from malignant cells [18–20]. Thus, our results suggest that the propensity of eosinophils to secrete their granule proteins may reflect the killing effect of eosinophils.
In this study, we also evaluated ECP/Eo ratio as a marker of eosinophilic activation and degranulation. The patients with eosinophilia had various results for their ECP/Eo ratios and showed no correlation between the plasma ECP level and the ECP/Eo ratio. However, patients suffering with non-hematologic malignancies had higher ECP/Eo ratios, in addition to their plasma ECP levels, than the other patients with reactive eosinophilia. Thus, our study suggests that the plasma ECP level and the ECP/Eo ratio may serve as tools for the early differentiation of reactive eosinophilia. Further studies, including patients with tissue invasive parasitosis, are required to reach firm conclusions as to the diagnostic value of the plasma levels of ECP and the ECP/Eo ratio.
IL-5 dramatically increases the life span of eosinophils by inhibiting their apoptotic cell death in vitro [14]. A similar effect of IL-5 in the peripheral blood would explain the persistent and extreme eosinophilia seen in IHES patients [21]. However, measurement of plasma IL-5 alone could not distinguish the clonal disorders from reactive eosinophilia, and this is probably due to the fact that the eosinophils themselves secrete IL-5 [6]. More precise methods such as IL-5 measurements in isolated cell populations or the detection of the IL-5 mRNA in purified eosinophils will be needed for further clarification.
In conclusion, although the usefulness of these markers as a diagnostic tool must be confirmed in larger studies, the levels of plasma ECP and IL-5 may discriminate IHES from other eosinophilic conditions, and the plasma ECP level and ECP/Eo ratio may differentiate eosinophilic patients with non-hematologic malignancy from those with other forms of reactive eosinophilia.
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Acknowledgements
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This work was supported by the Catholic Medical Center Research Foundation in program year 2004, and by the Post-Doctoral Fellowship Program of the Korea Science and Engineering Foundation.
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Abstract
Introduction
