INTRODUCTION

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Immunocytochemical studies of bronchial biopsies have shown that asthma is a chronic inflammatory disease in which the airway mucosa is infiltrated by activated T cells and eosinophils (1, 2). Numerous studies have demonstrated that CD4+ T lymphocytes regulate the events that initiate and maintain the inflammatory response observed in asthma (3, 4).

For the recruitment of effector T cells to airway, close interaction between T cells and antigen-presenting cells (APC) is essential (5). Naive T cells stimulated by APC differentiate into memory/effector T cells that are classified into T helper cell, type 1 (Th1) and type 2 (Th2) subsets based on their profiles of cytokine production; Th1 cells secrete cytokines such as interferon (IFN)- to promote cellular immune responses, whereas Th2 cells release interleukin (IL)-4 and IL-5 to promote allergic responses (6). Adhesion molecules and chemokines selective for Th1 and Th2 cells are involved in the differential recruitment of these two subsets (7, 8). In this context, differential expressions of certain chemokine receptors in Th1 and Th2 cells have been identified. CXC chemokine receptor (CXCR) 3 has been found to be preferentially expressed on Th1 cells and CC chemokine receptor (CCR) 4 is selectively expressed on Th2 cells (9, 10). We have recently developed a method to measure Th1 and Th2 helper T cells by the selective expression of chemokine receptors using flow cytometer (11).

In the treatment of patients with asthma, the value of corticosteroids is well documented, and the use of inhaled corticosteroids in asthma control is widely accepted (12, 13). Corticosteroids reduce airway inflammation as reflected in a reduction of the number of eosinophils and local T cells expressing the activation markers human leukocyte-associated antigen-DR (HLA-DR) and CD25 (14). Corticosteroids have also been shown to inhibit the production of cytokines, including IL-4 and IL-5 in vitro and in vivo (15, 16). However, whether these inhibitory effects of corticosteroids involve the changes of blood Th1/Th2 balance has not been established.

In this study we semiquantitatively measured CXCR3- expressing and CCR4-expressing CD4+ T cells by flow cytometer in patients with asthma and in normal control subjects. To examine the effects of corticosteroid on blood CXCR3+ and CCR4+ cells, we performed 2 wk of oral prednisolone therapy in a randomized, double-blind, placebo-controlled parallel group study.

	METHODS

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Patients

Twenty-eight patients with atopic asthma (15 male, 13 female, age 16 to 68 yr) with baseline FEV₁ values of 55.7 to 112.0% (mean ± SEM, 84.3 ± 3.1%) of predicted values were studied. Male and female patients were eligible for the study if they were at least 15 yr of age and satisfied the American Thoracic Society (ATS) definition of asthma, with symptoms of episodic wheezing, cough, and shortness of breath responding to bronchodilators and reversible airflow obstruction (more than 15% reversibility in terms of FEV₁) documented on at least one pulmonary function study (17). All patients had a positive serum IgE to house dust mite. Thirteen patients had a positive serum IgE to pollens. None of the patients had a history of excessive mucus production, and thin-slice chest computed tomography (CT) showed no low attenuation area in any of the patients. None of the patients had taken theophylline, antihistamines, sodium cromoglycate, or oral corticosteroids for at least 2 mo before the study, and none experienced an upper respiratory tract infection in the preceding month or during the study. Permitted medication, which remained unchanged during the study, included inhaled 2-agonists and inhaled beclomethasone less than 400 µg in 19 patients with mild persistent asthma. This study was carried out while the patient's symptoms were mild and stable.

Normal Control Subjects

Baseline values of CXCR3-expressing and CCR4-expressing memory CD4+ T cells were measured in 81 normal control subjects. Fifty-two nonatopic normal control subjects who had no history of allergic disease and never had symptoms such as wheezing, chest tightness, and dyspnea were screened by questionnaire. Twenty-nine atopic normal control subjects had history of allergic disease such as allergic rhinitis and atopic dermatitis, but never had symptoms such as wheezing, chest tightness, and dyspnea according to questionnaire. Among the control subjects, 13 individuals volunteered to enter the trial (6 nonatopic and 7 atopic control subjects; 5 with history of allergic rhinitis and 2 with history of atopic dermatitis). All patients with asthma and normal control subjects gave informed consent before entry into the study. This study was approved by the ethics committee of our hospital.

Study Design

The study was performed in a randomized, double-blind, placebo-controlled, parallel group fashion. Placebo or prednisolone in a dose of 20 mg was given orally once a day, 30 min after breakfast, for 2 wk. At the time of entry day and at the last day of the study, pulmonary functions and blood CXCR3-expressing and CCR4-expressing memory CD4+ T lymphocytes were measured. Patients with asthma were asked to record their clinical symptoms on a standard chart consisting of checklists such as dyspnea, wheezing, chest tightness, cough, sputum, and peak flow in the morning and at 5 P.M. to assess if there was any major asthmatic attack during the study. The clinical effect of treatment was only evaluated by the changes of FVC and FEV₁. In normal control subjects, blood CXCR3-expressing and CCR4-expressing memory CD4+ T lymphocytes were measured at the time of entry day and at the last day of the study.

CXCR3+ and CCR4+ Lymphocytes in Blood Memory T Cells

The percentages of CXCR3+ and CCR4+ lymphocytes in peripheral memory T cells were measured with FACStar (Becton Dickinson, Mountain View, CA) by 3-color staining for CD4/CD45RO/CXCR3 or CD4/CD45RO/CCR4, respectively (9). We used anti-CD45RO monoclonal antibodies (mAbs) to distinguish between naive and memory T cells in CD4-positive cells. First, peripheral blood mononuclear cells (PBMC) were obtained from patients using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden). PBMC (5 × 10⁵) were centrifuged at 15,000 rpm for 5 min at 4° C, and supernatant was aspirated. For blocking, 2 µl of normal murine serum (DAKO Japan, Tokyo) was added to the pellet and incubated on ice for 5 min. Cells were then incubated with antibodies (1 µl of CXCR3-fluorescein isothiocyanate [FITC] or CCR4-FITC/1 µl of CD45RO-PE/1 µl of CD4-Cy chrome) on ice for 20 min. After incubation, cells were washed with 3% fetal bovine serum-phosphate-buffered saline (FBS-PBS), resuspended with 250 µl of 3% FBS-PBS, and analyzed with FACStar. The following murine antibodies were used: anti-CD4-Cy chrome (DAKO Japan), anti-CD45RO-PE (PharMingen, San Diego, CA), anti-CXCR3-FITC (DAKO Japan, Tokyo), and anti-CCR4-FITC (18).

Statistics

Demographic data for the patients are presented as mean ± SEM. Values for CXCR3+ T cells, CCR4+ T cells, CXCR3+/CCR4+ ratio, and CD45RO+ T cells are expressed as mean with horizontal bar. Statistical differences between before and after treatments were determined by Student's paired t test. Statistical differences among control subjects and asthmatics were determined by Scheffé analysis. Spearman rank tests were used for correlations. Levels of significance were set at the 95% cutoff point.

	RESULTS

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Randomization resulted in 15 patients with asthma receiving prednisolone and 13 patients receiving placebo treatment. In normal control subjects, six subjects received prednisolone and seven subjects received placebo. All subjects completed the study, and none reported side effects during the treatment.

In patients with asthma, there were no significant differences between steroid and placebo group in age, FVC, FEV₁, and serum IgE before treatment (Table 1). There was considerable heterogeneity in the percentages of CXCR3+ T cells (from 33.8 to 63.7% in memory T cells), CCR4+ T cells (from 15.6 to 44.0% in memory T cells), and memory T cells (from 31.9 to 65.6% in CD4+ cells); however, there were no significant differences between steroid and placebo groups in these parameters at baseline.

After treatment, both steroid and placebo groups showed no significant differences in CXCR3+ T cells (Figure 1; mean ± SEM; before steroid therapy, 50.6 ± 1.9%; after steroid therapy, 51.9 ± 1.8%, not significant [NS]; before placebo, 47.5 ± 2.3%; after placebo, 48.7 ± 2.3%; NS). On the other hand, there was a significant decrease in CCR4+ T cells after 2 wk of corticosteroid therapy compared with baseline values (Figure 2; mean ± SEM; before steroid therapy, 29.3 ± 1.8%; after steroid therapy, 20.3 ± 1.5%, p < 0.0001), whereas there was no significant difference in the placebo group (Figure 2; mean ± SEM; before placebo, 27.1 ± 1.7%; after placebo, 27.7 ± 1.4%, NS). As a result, the ratio of CXCR3+ cells and CCR4+ cells significantly increased in the steroid-treated group (Figure 3; mean ± SEM; before the steroid-therapy, 1.86 ± 0.19; after steroid therapy, 2.89 ± 0.32, p = 0.0047), whereas there was no significant difference in the placebo group (Figure 3; mean ± SEM; before placebo, 1.87 ± 0.18; after placebo, 1.83 ± 0.15, NS). As shown in Figure 4, baseline values of CCR4+ T cells from patients with asthma were significantly higher than those from normal control subjects (mean ± SEM; 21.6 ± 0.7%, nonatopic control subjects, n = 52; 23.0 ± 0.7%, atopic control subjects, n = 29), whereas there was no difference in CXCR3+ T cells (mean ± SEM; 52.8 ± 1.1%, nonatopic control subjects, n = 52; 50.1 ± 0.8%, atopic control subjects, n = 29). In contrast to patients with atopic asthma, there was no significant change in CXCR3+ or CCR4+ T cells in control subjects after treatments of prednisolone (n = 6) or placebo (n = 7) (Table 2).

View larger version (20K): [in this window] [in a new window]   Figure 1.   Percentages of blood CXCR3+ cells in effector/memory T cells (CD4+/CD45RO+ cells) before and after treatment with prednisolone or placebo in patients with asthma. Horizontal line represents the mean.

View larger version (20K): [in this window] [in a new window]   Figure 2.   Percentages of blood CCR4+ cells in effector/memory T cells (CD4+/CD45RO+ cells) before and after treatment with prednisolone or placebo in patients with asthma. Horizontal line represents the mean.

View larger version (17K): [in this window] [in a new window]   Figure 3.   Ratio of CXCR3+ and CCR4+ cells in blood memory T cells before and after treatment with prednisolone or placebo in patients with asthma. Horizontal line represents the mean.

View larger version (21K): [in this window] [in a new window]   Figure 4.   Box-plot summary of CXCR3+ and CCR4+ memory T cells in patients with asthma and in normal subjects. Horizontal line represents the median, the box encompasses the 25th to 75th percentile, and the error bars encompass the 10th to 90th percentile.

During the 2-wk period, both steroid and placebo groups in patients with asthma showed no significant differences in CD45RO+ memory T cells in CD4+ cells (Figure 5; mean ± SEM; before steroid therapy, 47.3 ± 3.6%; after steroid therapy, 46.4 ± 4.1%, NS; before placebo, 46.6 ± 2.5%; after placebo, 46.4 ± 2.5%; NS). There was no significant change in CD45RO+ memory T cells in normal subjects either after treatments of prednisolone (n = 6) or placebo (n = 7) (data not shown).

View larger version (18K): [in this window] [in a new window]   Figure 5.   Percentages of blood effector/memory T cells (CD4+/ CD45RO+ cells) in helper T cells (CD4+ cells) before and after treatment with prednisolone or placebo in patients with asthma. Horizontal line represents the mean.

Steroid-treated patients showed significant increase in FVC (7.5 ± 2.5%, p < 0.05) and FEV₁ (10.3 ± 3.5%, p < 0.05) whereas the placebo-treated group showed no significant increase in FVC (1.0 ± 1.2%, NS) or FEV₁ (1.1 ± 1.4%, NS). There was no significant relationship between the observed changes in FEV₁ and changes in the percentages of Th2 cells in the steroid-treated group (data not shown).

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this double-blind, placebo-controlled, parallel group study, we investigated the effect of oral prednisolone on the balance of blood CXCR3+ and CCR4+ T cells in patients with stable mild asthma. The results showed that 2 wk of oral prednisolone 20 mg/d reduced the percentage of blood CCR4-expressing CD4+ T cells, resulting in the shift to CXCR3-expressing CD4+ T cells. Baseline values of CCR4+ T cells were higher in patients with atopic asthma than those of atopic control or nonatopic control subjects. Moreover, the effect of oral steroid on CCR4+ T cells appears to be specific to patients with atopic asthma because there was no effect of oral steroids on CCR4+ T cells in normal control subjects.

Previous methods for the identification of Th1 and Th2 cells by flow cytometer were based on the intracellular cytokine production after stimulation of the cells (19, 20). Recently, the preferential expression of some cytokine receptors on the in vitro polarized Th1 and Th2 cells or cell lines has been reported. Bonecchi and coworkers demonstrated that Th1 cell lines preferentially expressed CCR5 and CXCR3 messenger RNA (mRNA), whereas Th2 cell lines preferentially expressed CCR4 and, to a lesser extent, CCR3 mRNA (10). Other groups reported that CCR5 is another marker for Th1 cells (21), but Sallusto and coworkers showed that CCR5 expressions on Th1 cells were not consistent whereas CXCR3 expressed at high levels on Th1 (9). Yamamoto and coworkers recently showed that the cells capable of producing Th2 cytokines such as IL-4, IL-5, and IL-13, were restricted to the CCR4-expressing population within memory CD4+ T cells, whereas for Th1 cytokine production, IFN--producing cells resided exclusively in CXCR3- expressing memory CD4+ T cells (11).

Corticosteroids are effective in reducing airway inflammation through multiple mechanisms, including the modulation of cytokine and chemokine production in lymphocytes and endothelial cells. Corticosteroids bind to specific receptors in the cytosol, are translocated to the nucleus, and bind to specific regulatory sequences of target genes (22). Corticosteroids have been shown to act directly on T cells, suppressing mRNA expression for IL-2 and IFN- (25). By regulating cytokine gene expression (25), corticosteroids may critically modulate effector functions of T cells. The results of this study showed the differential effects of corticosteroids on CXCR3+ T cells and CCR4+ T cells. The mechanisms and kinetics of the reduction of CCR4+ T cells after steroid treatment in patients with asthma are unclear, but our preliminary data of five patients in the steroid-treated group showed that CCR4+ T cells decreased after 1-wk treatment of oral prednisolone, but it did not reach statistical significance (data not shown). It has been reported that several cytokines and drugs differently modify Th1 and Th2 functions, and in rare case switch to the opposite phenotype. Human Th2 clones can transiently express IFN- after IL-12 treatment (28). Th1 cells are reported to be irreversible, although mouse Th1 cells can produce IL-4 when stimulated in the presence of IL-4 (29). Corticosteroids have been observed to inhibit IL-4 production in human lymphocytes (24), but other investigators have demonstrated that IL-4 synthesis in T cells is increased by in vivo and in vitro treatment with steroids (30, 31). Blotta and coworkers demonstrated that corticosteroids reduced the production of IL-12 in macrophages, resulting in a decreased ability to induce IFN- and an increased ability to induce IL-4 in T cells (32). However, a recent clinical study demonstrated that prednisolone treatment of asthmatic patients resulted in a specific reduction in mRNA for IL-4 and IL-5, and an increase in mRNA for IFN- in bronchoalveolar lavage (BAL) lymphocytes (16), although cytokine mRNA was determined semiquantitatively, and the results cannot exclude Th0 cells or CD8+ Tc cells which can also produce IL-4 and IL-5, and IFN- (33). Our data express the percentage in memory T cells, and do not show the direct numbers of CXCR3+ and CCR4+ T cells in the blood; however, our data support the notion that corticosteroids suppress allergic reaction in part by modulating the balance of Th1 and Th2.

In this study, one patient out of 15 in the steroid-treated group showed increased CCR4+ cells (from 15.0 to 18.1%) and decreased CXCR3+/CCR4+ ratio (from 3.54 to 2.37) after treatment. We have no data to explain the difference in this patient; however, it is noteworthy that the baseline value of CCR4+ cells of this patient was lowest in the steroid-treated group. Leung and coworkers reported that, after prednisolone treatment, steroid-sensitive asthmatics had significant decrease in the numbers of BAL cells expressing mRNA for IL-4 and IL-5, whereas steroid-resistant asthmatics had no significant change in the number of BAL cells expressing mRNA for IL-4 or IL-5 (36). However, all steroid-treated patients in our study were steroid-sensitive in a sense that all showed decrease or disappearance of wheezing and cough after steroid treatment. The subject who showed increased CCR4+ cells after steroid therapy also showed a 7.7% increase in FEV₁. Moreover, the changes of CCR4+ cells were not associated with the changes of FEV₁ (data not shown). The discrepancies of these observations suggest that further studies would be needed to elucidate the role of blood CCR4-expressing CD4+ memory T cells in bronchial asthma.

We assessed the percentages of CD45RO+ memory T cells in total CD4+ cells as the balance of naive and memory T cells. It has been recognized that CD45RO+ and CD45RO subsets in human CD4+ T cells have different sensitivity to corticosteroids (37). Corticosteroids reduce clonal expansion of CD4+ CD45RO "naive" T cells, but CD4+CD45RO+ "memory" T cells are 100-fold less sensitive. Two weeks of oral corticosteroids did not alter this balance, but it would be of importance for the control of asthma whether longer use of corticosteroids could reduce memory T cells which can react to airborne antigens.

In summary, we have demonstrated that the balance of CCR4+ T cells to CXCR3+ T cells were downregulated by 2 wk of 20 mg/d prednisolone therapy. It may explain in part the beneficial effects of corticosteroids in the treatment of atopic asthma.