INTRODUCTION

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Allergic asthma is characterized by mucosal infiltration of the airways by eosinophils and lymphocytes. Elevated numbers of activated eosinophils and CD4⁺ T lymphocytes, both in bronchial mucosa and in bronchoalveolar lavage (BAL) fluid, are constant features of asthma (1, 2). Increases in these cells have been associated with enhanced bronchial hyperreactivity (BHR) and disease severity (1, 3). Data on asthma suggest that the onset of the asthmatic response is controlled by CD4⁺ cells which produce a characteristic T helper cell type 2 (Th2) pattern of cytokine production, such as interleukin-4 (IL-4), IL-5, and so on (4).

In the previous studies, we have provided direct evidence that IL-5 is capable of inducing eosinophil infiltration into the asthmatic airways, as well as the activation of infiltrating eosinophils, and thus promotes the development of BHR (5, 6). The hypothesis that IL-4 is responsible for airway eosinophilia and BHR in humans is based on the findings of a significant correlation between elevated levels of IL-4 and airway eosinophilia or BHR in asthmatics (7, 8). To our knowledge, the present study was designed for the first time to investigate the effects of the administration of recombinant human (rh) IL-4 by inhalation on airway responsiveness to methacholine as well as the number of eosinophils in induced sputum.

	METHODS

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Subjects and Study Design

The study protocol was approved by the ethics committee of Guangxi Medical University, People's Republic of China, and all subjects provided written consent. Eight nonsmoking patients (3 males, 5 females; 18 to 48 yr of age), who met the criteria for a diagnosis of asthma as defined by the American Thoracic Society (9) were enrolled in this study. All patients had mild atopic asthma, with baseline forced expiratory volume in one second (FEV₁) greater than 70% of predicted value (mean ± standard error of mean [SEM] = 96 ± 3% of predicted), requiring only intermittent use of inhaled ₂-agonists. All patients had a provocative concentration of methacholine producing a 20% fall in FEV₁ (PC₂₀ Mch) < 8 mg/ml. Each patient had one or more documented positive skin prick test responses to aeroallergens, but none were receiving immunotherapy including the use of inhaled corticosteroid therapy.

A randomized double-blind, placebo-controlled study design was employed in which each subject acted as his or her own control.

Assessment of Bronchial Responses

Methacholine inhalation tests were carried out as previously described (6). Briefly, methacholine chloride (Sigma Chemical Company, St. Louis, MO) was dissolved in normal saline to make solutions of doubling concentrations from 0.04 to 20 mg/ml. Normal saline and methacholine solutions were inhaled from a nebulizer (Liuzhou Medical Instrument Ltd., Guangxi, China) operated by compressed air at 5 L/min. Normal saline was inhaled first for 2 min, and FEV₁ was measured using dry wedge spirometry (Autospiro AS-600; Minato Ltd., Osaka, Japan). If the change in FEV₁ from the baseline value after inhalation of normal saline was 10% or less, inhalation of methacholine was started. When inhaled normal saline caused a greater change in FEV₁, the test was stopped or postponed. Methacholine was inhaled for 2 min by mouth tidal breathing wearing a noseclip, and this was followed immediately by measurements of FEV₁. Increasing concentrations of methacholine were inhaled until a fall of 20% or more in FEV₁ was obtained. The measured values were plotted on semilogarithmic graph paper, and PC₂₀ Mch was determined. FEV₁ was measured three times, and the best FEV₁ value of three attempts was recorded each time. Following this, subjects underwent sputum induction.

Administration of rhlL-4

The following afternoon (24 h after initial methacholine challenge), 20 µg of rhIL-4 (Genzyme Co., Boston, MA) in vehicle (0.1% bovine serum albumin in 0.9% saline) or vehicle only was inhaled as a 0.5-ml nebulized solution, the chamber was refilled twice with 0.5 ml vehicle, and the nebulization was repeated to scavenge any remaining rhIL-4. The dose of rhIL-4 was based upon a preliminary study involving two asthmatic patients.

In addition, rhIL-4 used for inhalation in this study was obtained commercially, which was contaminated with endotoxin (0.02 ng/µg, indicated by manufacturers), thus the total dose of endotoxin inhaled by each subject was 0.4 ng. To exclude the possibility that the observed effects caused by IL-4 were endotoxin related, 0.4 ng lipopolysaccharide (LPS, Escherichia coli serotype 026:B6 [Sigma]) was added to vehicle only when control experiments involving vehicle inhalation were carried out.

Methacholine challenge and sputum induction were repeated 2, 24, and 48 h after the inhalation of rhIL-4 or vehicle. Each patient experienced both inhalations of rhIL-4 and vehicle, at least 4 wk were allowed to elapse between the two inhalations, and the order of inhalation of rhIL-4 or vehicle was randomized.

Sputum Induction and Examination

Sputum induction was performed as previously described (6). Briefly, subjects inhaled nebulized 3.5% saline and gargled orally with water prior to voluntary coughing every 2.5 min until 20 min had elapsed or until 5 ml of sputum had been expectorated. The induced sputum was added with an equal volume of 1 mmol/L dithiothreitol (Sigma) in Hanks' buffered salt solution and then mixed gently by vortex mixer and incubated at 37° C for 15 min to ensure complete homogenization. After incubation, aliquots of homogenized samples were removed for total cell counts by hemocytometer. The cell pellets were prepared for differential cell counts by Diff-Quik staining of cytocentrifuge preparations. A total of 400 cells not including squamous cells were enumerated for differential cell counts. The absolute number of eosinophils was calculated.

Statistical Analysis

Results are expressed as arithmetic mean ± SEM for eosinophil numbers. PC₂₀ Mch values were log10-transformed for analysis and reported as geometric mean and geometric standard error of mean (GSEM). GSEM is the antilog of SEM of the log PC₂₀ Mch values and is a factor multiplication. Statistical analysis was done by repeated measure analysis of variance (ANOVA) for data conforming to a normal distribution, and by Friedman's test for those data with a nonparametric distribution (confirmed by the Shapiro-Wilk W test). Paired t test was used to compare the data obtained at the same time points between rhIL-4 and the control inhalations. p Values < 0.05 were considered significant.

	RESULTS

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Effects of IL-4 on Methacholine Responsiveness

All subjects tolerated the procedures without complications such as fever, increased cough, or any other increased symptoms during or after the 48-h follow-up period.

Baseline measurements of PC₂₀ Mch before both challenges showed no significant difference (0.49 ± 1.76 mg/ml with vehicle, and 0.43 ± 1.81 mg/ml with rhIL-4, respectively) (Figure 1). PC₂₀ Mch within vehicle group did not appear to change from baseline value at any time throughout the study. Two hours after rhIL-4 inhalation, PC₂₀ Mch (0.33 ± 1.87 mg/ ml) was not different from baseline value (p > 0.05). PC₂₀ Mch fell from baseline to 0.22 ± 1.73 mg/ml (p < 0.01) at 24 h, and to 0.21 ± 1.74 mg/ml (p < 0.01) at 48 h after rhIL-4 inhalation.

View larger version (13K): [in this window] [in a new window]   Figure 1.   Changes in log PC20 Mch (mean ± SEM) after inhalation of vehicle control and recombinant human interleukin-4 in patients with allergic bronchial asthma. n = 8 for each group. *p < 0.01 compared with baseline measurement. The scale along the x-axis is nonlinear.

In addition, when comparison was made between rhIL-4 and control inhalations, no significant changes in the airway reactivity could be seen at 2 h (p > 0.05) after inhalations. However, PC₂₀ Mch values at 24 and 48 h after rhIL-4 inhalation were significantly lower than those after vehicle inhalation, respectively (both p < 0.01).

Effects of IL-4 on Eosinophil Numbers in Sputum

The absolute numbers of sputum eosinophils are presented in Figure 2. After allergic asthmatics were challenged with vehicle only, we did not observe increases of eosinophil counts in induced sputum obtained at three time points when compared with baseline measurement. Compared with baseline value (2.6 ± 1.2 × 10⁵/ml), no significant increase in the number of eosinophils could be found at 2 h (3.9 ± 1.9 × 10⁵/ml, p > 0.05) after rhIL-4 inhalation. The number of eosinophils increased with time, reaching a significantly higher level at 24 h (5.0 ± 2.0 × 10⁵/ml, p < 0.05); this significant sputum eosinophilia lasted at least 48 h (5.2 ± 1.4 × 10⁵/ml, p < 0.05).

View larger version (36K): [in this window] [in a new window]   Figure 2.   Comparison of eosinophil numbers (mean ± SEM) in induced sputum from allergic asthmatics challenged with vehicle and recombinant human IL-4. n = 8 for each group. *p < 0.05 compared with baseline measurement.

	DISCUSSION

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It has been reported that the production of IL-4 in peripheral blood mononuclear cell cultures from atopic children was significantly higher than those from both nonatopic ones with asthma and nonatopic control subjects who did not have asthma (10). An elevation of serum IL-4 concentration has also been found in asthmatic children (11). Sun and coworkers have revealed that there were significant increases in percentages of IL-4 mRNA⁺ cells in both BAL fluids and bronchial biopsies from atopic asthmatics when compared with nonasthmatic subjects (12, 13). Furthermore, decreased bronchial responsiveness in asthma after prednisonlone treatment was accompanied by a reduction in numbers of BAL cells expressing mRNA for IL-4 (14). The importance of IL-4 is also demonstrated by animal studies showing that in vivo treatment with anti-IL-4 antibodies inhibits the infiltration of eosinophils into the mouse airways (15). These data suggest an IL-4-dependent mechanism for the induction of eosinophil recruitment and the development of BHR.

The most important findings in this study were that rhIL-4 inhalation, not vehicle inhalation, of allergic asthmatics produced a marked progressive influx of eosinophils into the airways when studied by differentials of cells in induced sputum. The changes were time-course-related, in that the response was most marked at 24 h. Our present study also showed increased airway responsiveness to methacholine after rhIL-4 inhalation in patients with allergic bronchial asthma. The changes were also time-course-related and coincided with an eosinophil recruitment. These data indicated directly that IL-4 is capable of inducing BHR and airway eosinophilia in asthmatics.

The exact mechanism by which IL-4 contributes to BHR is still unknown. Because IL-4 has an ability to attract eosinophil recruitment into the asthmatic airways, it is possible that IL-4 induces BHR by means of a role for eosinophils in the asthmatic process. Eosinophils are known to secrete a number of basic proteins, including major basic protein, eosinophil cationic protein, eosinophil-derived neurotoxin, and eosinophil peroxidase that have profound effects on airway cells (16, 17). Release of these products is associated with increases in vascular permeability, bronchoconstriction, and destruction of airway epithelial cells (17). In particular, major basic protein has been shown to cause BHR in animal models of asthma (18, 19). In addition, eosinophil activation also results in the release of a number of important lipid mediators, including leukotriene C₄ and platelet-activating factor, which can contract airway smooth muscle as well as increase bronchial responsiveness (20). Thus, eosinophils possess properties that can directly or indirectly cause airway obstruction and promote BHR.

We have shown that 10 µg of IL-5 increased sputum eosinophils 6-fold and decreased PC₂₀ Mch by 64% in asthmatics at 24 h after inhalation (6). We noted in this study that 20 µg of IL-4 increased sputum eosinophils by 48% and decreased PC₂₀ Mch by 49% at the same time point after inhalation. Based on this comparison, it might be concluded that the responses of airway eosinophils and airway reactivity to IL-4 are more modest than those to IL-5. In addition, IL-5 selectively recruited eosinophils, but not any other type of cells, into the asthmatic airways (6). However, IL-4 recruited not only eosinophils but also neutrophils into the airways (data not shown). The airway neutrophilia induced by IL-4 might also make a contribution to the observed BHR (21, 22).

In summary, we have shown that rhIL-4 is functionally important in causing BHR to methacholine. This IL-4-induced BHR was accompanied by a significant airway eosinophilia. These data suggest that drugs that interfere with IL-4 synthesis or IL-4 receptor antagonists could be beneficial in the treatment of asthma.