INTRODUCTION

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Chronic airway inflammation is thought to play a fundamental role in the pathogenesis of asthma. The inflammatory response in asthma involves an increased number of eosinophils, basophils, mast cells, and, most importantly, type-2 helper T-lymphocytes (Th2), which can be isolated from the lungs of patients with asthma (1, 2). Immunopathological studies of biopsy specimens and bronchoalveolar lavage (BAL) fluid from patients with asthma have demonstrated that Th2-dominant chronic airway inflammation is associated with high concentrations of Th2 cytokines, such as interleukin-4 (IL-4), IL-5, and IL-13 (3). The importance of Th2 cytokines in asthma has been demonstrated in animal models. Either disruption of the genes encoding these proteins or their antibody-mediated neutralization prevents eosinophilia and attenuates various pathological changes, such as airway hyperreactivity, associated with experimental asthma (7). Th2 cells are essential in the initiation and prolongation of an asthmatic response because they regulate the growth, differentiation, and recruitment of mast cells, basophils, and eosinophils (3, 11). Therefore, inappropriate adaptive Th2-polarized immune responses to environmental antigens play a critical role in the development of asthma (2, 12).

Three distinct types of ST2 gene products, a soluble secreted form (ST2), a transmembrane receptor form (ST2L), and a variant form (ST2V), have been cloned (13). The ST2 gene, also known as T1, Fit-1, and DER4, was originally identified as a gene induced by serum stimulation of fibroblasts (13, 16), and has recently been demonstrated to be overexpressed preferentially on Th2 effector cells, but not on Th1 cells (19, 20). Furthermore, in a murine model of Th2-dependent allergic airway inflammation, administration of a neutralizing antibody against ST2 partially inhibited the development of Th2 effector functions in vivo (21, 22). Therefore, ST2 seems to play an essential role in the development of Th2 responses in patients with asthma.

These findings suggesting that ST2 expression is critical both for Th2 responses and for effector functions of Th2 in murine models of asthma led us to evaluate the clinical functions of ST2 in patients with bronchial asthma. We have recently generated an enzyme-linked immunosorbent assay (ELISA) system to quantify the soluble form of human ST2 (hST2) protein (23). In the present study, using this system, we measured serum levels of hST2 protein in patients with atopic asthma with or without acute exacerbation, and evaluated the clinical roles of this protein in bronchial asthma.

	METHODS

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Subjects

The study protocol was approved by our institutional review board for human studies, and informed consent was obtained from all subjects. The subjects consisted of 56 patients with bronchial asthma and 200 healthy volunteers (Table 1). Of the 56 patients with asthma, 30 exhibited an acute exacerbation during the follow-up. Upon enrollment, all patients underwent a full clinical examination, pulmonary function test, chest radiograph, full blood count, total serum immunoglobulin E (IgE), and radioallergosorbent test (RAST). The patients with asthma had a current history of asthma including documented reversible airway obstruction. Atopy was defined as a positive prick test or RAST to one or more common aeroallergens, and all patients with atopic asthma in this study were reactive to either house dust mites or mixed grass pollen. None of the patients with asthma smoked, had complications of other lung diseases, or had a history suggesting intolerance to nonsteroidal antiinflammatory drugs. The cause of acute exacerbation in 30 patients seemed to be allergen exposure of house dust mites and mixed grass pollen, as they had no symptom of respiratory infection prior to asthma attack. Two hundred healthy volunteers were studied as a control. They had no history of atopic factors or allergic diseases, and no evidence of any lung disease.

Classification According to Severity of Asthma

The classification proposed by the NHLBI/WHO Workshop on the Global Strategy for Asthma (GINA guidelines) was used to classify disease severity of asthma (Tables 1 and 2) and severity of acute exacerbation (Table 3) (24). Acute exacerbation was defined as a state in which both respiratory wheeze in the bilateral lung fields and a > 20% fall in percentage of predicted peak expiratory flow (%PEF) were observed.

Measurement of hST2 Protein and IL-5 Levels in Sera

The level of soluble hST2 protein in serum was measured by a sandwich ELISA that we developed as previously described (23, 25). The concentration of IL-5 in serum was measured by a sandwich ELISA kit (BioSource International Inc., Camarillo, CA). All samples were assayed in duplicate.

Immunoprecipitation, Glycosidase Digestion, and Immunoblotting

The supernatant from COS7 cells transfected with pEF-BOS containing human ST2 cDNA (23, 26) or serum samples from patients with asthma and healthy controls were immunoprecipitated with anti-hST2 monoclonal antibody (2A5)-coated TOYOPEARL beads (TOSOH, Tokyo, Japan) (23, 26). Some of these samples were digested with peptide-N-glycosidase F (26). The reaction mixture was processed for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequent immunoblotting. The immunoblotting was carried out with the mixture of HRP-labeled anti-hST2 monoclonal antibodies, G7 and HB12 as described (23).

Statistical Analysis

Data are expressed as median values with ranges. Differences between two groups were compared by the Mann-Whitney U test. Comparisons of clinical parameters and hST2 levels among all the groups were made by the Kruskal-Wallis test. Differences in hST2 levels of patients with asthma between those with nonattack and those with attack were analyzed by Wilcoxon signed-ranks test. Associations between serum ST2 levels and various parameters were analyzed by Spearman's correlation coefficient. p < 0.05 was considered significant.

	RESULTS

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Detection of hST2 Protein in Serum of Patients with Atopic Asthma

We analyzed expression of serum hST2 protein in patients with asthma by immunoblotting. Using monoclonal antibodies against hST2 protein, a band of about 60 kD could be detected in the serum of patients with atopic asthma, but not in that of the healthy control volunteer (Figure 1, lanes 2 and 3). The mobility of the protein in the serum of the patient with asthma was slightly different from that of the recombinant ST2 protein released from COS7 cells (Figure 1, lanes 1 and 2). However, after treatment with peptide-N-glycosidase F, the apparent molecular weight of both bands shifted to 37 kD (Figure 1, lanes 4 and 5), corresponding to the molecular weight of deglycosylated ST2 protein (26). Therefore, a protein band of about 60 kD detected in the serum of the patient was confirmed to be serum hST2 protein (Figure 1, lane 2).

View larger version (33K): [in this window] [in a new window]   Figure 1.   Detection of human ST2 protein in the serum of a patient with atopic asthma. The supernatant from COS7 cells transfected with pEF-BOS containing human ST2 cDNA (lanes 1 and 4, positive control) or serum samples of patients with asthma (lanes 2 and 5) and healthy control (lanes 3 and 6) were immunoprecipitated with anti-hST2 monoclonal antibody, 2A5. The aliquots of each sample were incubated overnight with peptide-N-glycosidase F (PNGase F) (lanes 4, 5, and 6) or without enzyme (lanes 1, 2, and 3), and the reaction mixture was processed by immunoblotting using the mixture of HRP-labeled anti-hST2 monoclonal antibodies, G7 and HB12. The arrow and arrowhead indicate the positions of the glycosylated ST2 protein and deglycosylated ST2 protein, respectively.

Serum hST2 Levels in Patients with Bronchial Asthma

Although the serum hST2 levels in the 56 patients with asthma having no attacks were as low as those in the 200 healthy volunteers (median values of nonattack in asthma versus healthy control, 0.493 ng/ml versus 0.415 ng/ml, respectively), there was a significant difference in serum ST2 levels between the control subjects and all the subgroups of patients with asthma (p = 0.04, Table 2). However, no significant difference was found among the subgroups of patients with asthma (p = 0.18), and there was no correlation between the serum hST2 level and the severity of asthma (data not shown). Furthermore, multiple comparisons showed no significant differences in blood eosinophils, total serum IgE, or serum hST2 among subgroups of patients with asthma regardless of the severity of the asthma (Table 2).

Of the 56 patients with atopic asthma with nonattack, 30 exhibited an acute exacerbation, and the serum hST2 levels were compared between nonattack and attack groups. Serum ST2 levels during attack were significantly elevated in comparison with those during nonattack (p < 0.0001, Figure 2). Patients with asthma with acute exacerbation were classified into two groups according to the severity of exacerbation (mild to moderate, and severe), and laboratory data as well as serum hST2 levels were compared between the two groups (Table 3). The serum hST2 levels in patients with severe attacks were significantly higher than those in patients with mild or moderate attacks (p = 0.0047), while blood eosinophils and total serum IgE did not differ significantly between the groups. The serum IL-5 levels in patients with asthma with severe exacerbation were significantly higher than those in patients with asthma with mild to moderate exacerbation (p = 0.048).

View larger version (27K): [in this window] [in a new window]   Figure 2.   Increase in serum hST2 levels in patients with asthma with an acute exacerbation (n = 30). *p < 0.0001 between groups with nonattack and attack.

On the basis of %PEF or Pa_CO2, patients with asthma with acute exacerbation were divided into two groups, and their serum hST2 levels were compared. Serum hST2 levels in the group of %PEF < 60% were significantly higher than those in the group of %PEF  60% (p < 0.05, Figure 3A), and serum hST2 levels in the group of Pa_CO2  45 mm Hg were significantly higher than those in the group of Pa_CO2 < 45 mm Hg (p < 0.005, Figure 3B).

View larger version (24K): [in this window] [in a new window]   Figure 3.   Comparison of serum hST2 levels in patients with asthma with an acute exacerbation according to %PEF (A) and PaCO2 (B). Values are presented as upper and lower quartiles and range (box), median value (horizontal line), and the middle 90% distribution (whisker line). *p < 0.05 versus patients with asthma with %PEF of more than 60%; p < 0.005 versus patients with asthma with PaCO2 of less than 45 mm Hg. %PEF, percentage of predicted peak expiratory flow.

Correlation between Serum hST2 Levels and Various Clinical Parameters

Factors associated with the serum ST2 levels in patients with atopic asthma having attacks were analyzed using Spearman's rank correlation test (Table 4). In patients during asthmatic attack, the serum hST2 level was inversely correlated with %PEF (r = 0.634, p = 0.004), and was positively correlated with Pa_CO2 (r = 0.516, p = 0.003). There was no significant correlation between the serum IL-5 concentration and hST2 levels, but the p value was 0.052, which is close to the p < 0.05 significance level. Other factors, such as the blood eosinophil count, total serum IgE, and Pa_O2, did not correlate with the serum ST2 level.

	DISCUSSION

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Little is known so far about the role and function of human ST2 in pathological conditions. The objective of this study was to determine the clinical role of ST2 in patients with bronchial asthma. To our knowledge, this is the first report implicating ST2 in human disease. The results of this study demonstrate that serum ST2 levels in patients with stable asthma are marginally higher than those in healthy control subjects, and that the serum ST2 levels of patients with asthma rise in correlation with the severity of asthma exacerbation.

We first succeeded in detecting the soluble form of ST2 protein by immunoprecipitation and immunoblotting analysis (Figure 1). Although we used the supernatant from ST2 cDNA-transfected COS7 cells as a positive control of ST2 protein in this assay, the electrophoretic mobility was different from that of patients with asthma. This can be explained by the difference in the extent or pattern of glycosylation of ST2 protein between a natural human cell system and an artificial simian cell system, because treatment of both samples with peptide- N-glycosidase F resulted in a mobility shift from around 60 kD to 37 kD, which corresponds to naked ST2 protein. And, this 37-kD protein has been confirmed to be really the ST2 gene product in our previous study (26).

There have recently been several reports on the function of ST2 protein in Th2-mediated inflammation in murine models. In vivo depletion of ST2 by treatment with anti-ST2 antibody abrogated both the accumulation of eosinophils in the airway and the secretion of Th2 cytokines in a murine model of Th2-dependent allergic airway inflammation (21, 22). These findings were further supported by the results of a study using ST2-deficient mice in a pulmonary granuloma model induced by Schistosoma mansoni eggs (27). This study showed that Th2 cytokine production is impaired after immunization in the absence of ST2 expression. In contrast, other investigations have showed no difference in Th2 response in wild-type versus ST2-deficient mice after parasitic infection with the helminth Nippostrongylus braziliensis or after allergen-induced airway inflammation (28). There are no obvious explanations for these discrepancies, however, because such discrepancies may be attributed to differences in experimental design, including antigen challenge, route of administration, and antigen-specific restimulation, the possibility that ST2 plays an important role in the development and function of Th2 cells cannot be excluded.

Furthermore, some studies have reported that administration of recombinant ST2 fusion protein attenuates eosinophilic inflammation of the airway and suppresses IL-4 and IL-5 production in murine asthma models (21, 22). These data suggest that soluble forms of ST2 protein can inhibit the interaction of membrane-bound ST2 with its putative ligand, resulting in downregulation of Th2 effector function. Since human helper T cells cannot be as clearly divided into two subsets, Th1 and Th2, as murine helper T cells (29), it would seem unwise to extrapolate these results of murine experiments to directly explain the function of hST2 in asthma in all its clinical settings. However, recent studies have shown that activated CD4 T lymphocytes in the peripheral blood or airway tissue of patients with asthma exacerbation express protein or mRNA of cytokines in the context of a Th2-type pattern (30, 31). Del Prete and coworkers reported that remarkable proportions of CD4 T cell clones derived from the airway mucosa after experimental bronchial challenge with positive allergens in patients with atopic asthma exhibited allergen-specific activated T cells with a Th2 profile of cytokine production (31). Therefore, it is possible to speculate that hST2 during acute exacerbation may also downregulate the Th2-mediated allergic inflammation by competition with the putative ligand in the ligation with receptor-type ST2, named ST2L (14). The significant evidence of the present study, that the serum hST2 level in patients with atopic asthma correlates well with severity of asthma exacerbation, suggests that ST2 protein production may be required to increase for suppression of allergic inflammation, in proportion to the severity of airway inflammation contributing to acute exacerbation.

Although there are reports indicating that airway inflammation is prominent at the time of symptomatic exacerbation (30, 32), the degree of airway inflammation cannot be readily quantified by direct methods such as BAL or bronchial biopsy, especially upon acute exacerbation. The results of our study indicate that serum hST2 levels during exacerbation of asthma inversely correlate with the %PEF, and positively correlate with Pa_CO2, suggesting that serum hST2 levels may correlate well with airway inflammation inducing airflow obstruction. Furthermore, there was a weak, though not significant, correlation (p = 0.052) of serum hST2 levels with IL-5, which is one of Th2-derived cytokines and plays an important role in eosinophilic airway inflammation (1, 2, 30, 31). Taken together, these data suggest that serum ST2 is a promising candidate for a marker to monitor the degree of airway inflammation in asthma exacerbation.

Asthma is characteristic of Th2-mediated eosinophilic airway inflammation (1, 2), and ST2 has been reported to be expressed predominantly on Th2 cells, but not on Th1 cells (19, 20). Such evidence suggests that the serum hST2 protein detected in this study may be derived from circulating Th2 cells. However, the recent demonstration of mRNA encoding ST2 in murine mast cells, in monoblastic cell lines, and in megakaryoblastic cell lines raises the possibility that ST2 may originate from cells other than Th2 cells (19, 28). We also observed ST2 mRNA expression in human bronchial epithelial cells and lung fibroblasts (data not shown). Airway inflammation in asthma involves eosinophils, mast cells, neutrophils, and Th2 cells (33). Further analysis using flow cytometry, immunohistochemical staining, or in situ hybridization should be required to determine from which cells hST2 protein originates in asthma.

In summary, our results showed that the serum-soluble hST2 protein level was significantly elevated in patients with atopic asthma, and that this level correlated with the severity of acute asthma exacerbation. These findings suggest that the serum ST2 level may reflect the severity of Th2-dominant inflammation. Further elucidation of the roles and functions of hST2 in bronchial asthma will require longitudinal studies and detailed molecular research comparing the immunopathological characteristics of asthma in diverse clinical settings by using samples of induced sputum, BAL fluid, or bronchial biopsy specimens. At present, a ligand for ST2 has not yet been identified. Identification of such a ligand is expected to improve our understanding of the biological and pathological functions of ST2, particularly in Th2-mediated allergic disease.