INTRODUCTION

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It is well established that the underlying immunological pathomechanism of allergic asthma is a chronic inflammation characterized by activated mast cells, eosinophils, and T cells (1). While mast cells and eosinophils play a major role as potent effector cells responsible for tissue damage by the release of mediators, T cells are able to orchestrate the inflammatory process through the release of cytokines. Increasing evidence has been accumulated that in atopic asthma the inflammation is associated with a T-helper-2 (Th2) profile, namely interleukin 4 (IL-4) and interleukin-5 (IL-5) (2). Because IL-4 promotes the switching of B-cell isotypes to IgE production and IL-5 activates selectively human eosinophils, these cytokines may be of particular clinical importance. However, limited data exist, which definitively show the expression of IL-4 and IL-5 protein in individual T cells from asthmatics. Furthermore, it remains unclear whether or not a simultaneous production of both cytokines, which one might expect when produced by Th2 cells, is present in asthma, as appropriate single cell assays to study this question have not been employed. We have previously shown that after in vitro stimulation of freshly isolated peripheral blood T cells from healthy donors coexpression of IL-4 and IL-5 was rarely detectable, indicating an independent regulation of these Th2 cytokines (7).

Although most data on T-cell cytokine production in asthma have been obtained from adults, allergic asthma is thought to represent the same disease in children as in adults. It is possible, however, that the underlying mechanism might change with duration or late onset of the disease. Therefore, the aim of our study was first to determine the frequencies of IL-4 and IL-5 positive T cells in the peripheral blood of patients with atopic asthma of different age groups and to correlate it to the number of eosinophils, the degree of hyperresponsiveness, and the concentration of total IgE. The hypothesis was proposed that in allergic asthma there is enhanced expression of IL-4 and IL-5 protein in circulating T cells of both children and adults. Second, we determined the frequency of T cells that stained positively for both cytokines and hypothezised that coexpression of IL-4 and IL-5 is present in T cells in asthma. We have employed a flow cytometric assay (8), which allows the simultaneous detection of T cells and two intracellular cytokines in unseparated blood samples after short-term in vitro activation.

	METHODS

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Subjects

Nine atopic asthmatic children, nine atopic asthmatic adults, and for each age group nine age-matched atopic control subjects as well as nine nonatopic control subjects participated in the study (clinical and demographic characteristics are shown in Table 1). FEV₁ and MEF₂₅ were recorded in all subjects from the maximum flow expiratory volume curve and expressed as percent of the expected value. Increasing doubling concentrations of histamine were administered as previously described (9) and the concentration of histamine producing a decrease of 50% from baseline in the specific conductance [PC₅₀SGaw] was calculated by linear interpolation between the last two points of the log concentration-response curve. Nonspecific bronchial hyperresponsiveness was defined as a PC₅₀SGaw < 8 mg/ml.

All asthmatics had mild allergic asthma as defined in the International Consensus Report (10). They had a history of intermittent wheeze, chest tightness, cough, and sputum production either spontaneously, after allergen exposure, or after exercise together with repeated and reversible airflow obstruction. All asthmatics had an increased airway hyperresponsiveness to histamine. Atopy of asthmatic and atopic control subjects was defined by positive skin prick tests (wheal diameter  4 mm; Bencard, Neuss, Germany) and elevated specific IgE (Phadebas CAP System; Pharmacia Diagnostics, Uppsala, Sweden) for at least one of the following aeroallergens: Dermatophagoides pteronyssinus, grass pollen, tree pollen, cat fur, dog hair, Alternaria tenuis, Cladosporium spp. Total serum IgE (Phadezym PRIST, Pharmacia Diagnostics) was determined according to the manufacturer's directions. All asthmatic children were being treated with sodium cromoglycate and three children and two adults with inhaled steroids. ₂-agonists were used when required for relief of symptoms. None received oral steroids or theophylline. The atopic control subjects had a history of allergic rhinitis and/or conjunctivitis but had never suffered from asthmatic symptoms. Lung function tests were normal, but mild bronchial hyperreactivity was present in children (see Table 1). They had no history of other diseases and did not take any medications. The normal control subjects had no history of allergic or other diseases, negative skin prick tests, normal total IgE ( 100 IU/ml), normal lung function tests, and no bronchial hyperresponsiveness (PC₅₀SGaw > 8 mg/ml).

All study subjects were nonsmokers, no subject had an acute bronchitis 3 wk prior to the investigations, and all studies were done outside the relevant allergy season. All subjects were volunteers and, after fully informed about the purpose and the nature of the study, a written consent was obtained from the patient, or if a minor, from his or her parents. The study was approved by the ethics committee of the Ruhr-Universität Bochum.

Anti-cytokine Monoclonal Antibodies

Anti-IL-4 (mouse IgG₁, 4D9) (11) and anti-IL-5 (mouse IgG₁, 445-25) (7) were generated at Novartis Pharma AG, Basel. Whereas the anti-IL-4 antibody has been well characterized and shown to be highly specific and suitable for intracellular cytokine detection previously (8, 11), binding specificity for intracellular staining of the monoclonal antibody (mAb) 445-25 has been reconfirmed using Chinese hamster ovary (CHO) cells transfected with complementary DNA (cDNA) for human IL-5 or IL-4: CHO cells were fixed with paraformaldehyde, permeabilized with saponin (as described subsequently) and fluorescein isothiocyanate (FITC)-labeled mAb 445-25 (0.5 µg/100 µl) was added to the cells. Positive staining was detected by flow cytometry, which could be inhibited when mAb 445-25 was preincubated with 50 µg/100 µl rhIL-5 (Figure 1). No binding was detected with IL-4-transfected or untransfected CHO cells. The same staining pattern was seen using other well-characterized anti-IL-5 mAbs (TRFK-5 [12] and mAb7 [13], data not shown). Furthermore, human Th2 cell clones were used as a positive control for coproduction of IL-4 and IL-5 (7). In nonpermeabilized cells no staining was detectable, demonstrating the intracellular origin of the signal (data not shown).

View larger version (16K): [in this window] [in a new window]   Figure 1.   Specificity of intracellular staining for IL-5. IL-5-transfected CHO cells were fixed, permeabilized, and incubated with FITC-labeled anti-IL-5 (1 µg/100 µl). Intracellular staining was detected by flow cytometry which could be inhibited by preincubation of anti-IL-5 with rhIL-5 (50 µg/100 µl).

Anti-IL-5 was labeled with FITC using a FITC-labeling kit (Calbiochem, La Jolla, CA) and anti-IL-4 was biotinylated using a biotin labeling kit (Molecular Probes, Eugene, OR) according to the manufacturer's instructions.

Processing of Cells and Intracellular Staining Procedure

Heparin anticoagulated blood was collected from all subjects at the time of lung function tests under sterile conditions. The number of eosinophils was determined by staining with Dunger solution and positively staining cells were counted on a Fuchs-Rosenthal hemocytometer. For intracellular T-cell cytokine detection we employed a flow cytometric method, which has been described elsewhere (8, 14). Briefly, 20 µl whole blood was resuspended in 180 µl culture medium containing 10% fetal calf serum (Biochrom, Berlin, Germany). Cells were cultured in 96-well flat-bottom plates (Nunc, Wiesbaden, Germany) and 20 wells were used for each experiment. Cells were stimulated with 12-myristate 13-acetate (PMA, 10 ng/ml) and ionomycin (1 µM) in the presence of monensin (2.5 µM). After incubation for 5 h at 37° C in a humidified atmosphere of 8% CO₂ in air, cells were washed in phosphate-buffered saline (PBS) and fixed in 1 ml 4% ice cold paraformaldehyde (Riedel de Haen, Seelze, Germany) for 10 min. After a further wash in PBS, cells were resuspended in 100 µl saponin buffer (PBS containing 0.1% saponin [Riedel de Haen] and 0.01 M HEPES buffer [Serva, Heidelberg, Germany]). Lysis of erythrocytes occurred during the incubation with saponin. Anti-IL-5-FITC and biotinylated anti-IL-4 (each 1 µg/ml) together with 10 µl of anti-CD3-Tricolor (Medac, Hamburg, Germany; dilution 1:20 in saponin buffer) were added to the cell suspension and incubated for 20 min at 4° C in the dark. After a further wash in saponin buffer, cells were incubated with 1 µl streptavidin-PE (Serva) for 20 min. After a final wash in saponin buffer, cells were resuspended in PBS and kept in the dark at 4° C until flow cytometric evaluation (same day). As controls FITC-labeled and biotinylated nonspecific mouse IgG₁ antibodies were used (Becton Dickinson, Heidelberg, Germany and Medac) followed by incubation with streptavidin-PE. If not indicated differently, reagents were purchased from Sigma (St. Louis, MO).

Flow Cytometric Analysis

A FACScan flow cytometer (Becton Dickinson) equipped with a 15-mW argon ion laser and filter settings for FITC (530 nm) (FI-1), PE (585 nm) (FI-2), and Tricolor (tandem dye of PE linked to Cy5; Medac) emitting in the deep red (> 650 nm) (FI-3) was used. Ten thousand cells were computed in list mode and analyzed using Lysis II software (Becton Dickinson). An electronic gate was set on the lymphocytes on the forward and side scatter plot, and CD3⁺ stained cells falling within the gated area were then identified by detection of FI-3. The cytokines were then analyzed by detection of FI-1 and FI-2 using double gates on FI-3-positive cells and lymphocytes. Cells staining positively for cytokines were expressed as a percentage of the CD3⁺ cells (Figure 2). The flow cytometric analysis was done by one investigator blinded to the source of the samples.

View larger version (18K): [in this window] [in a new window]   Figure 2.   Typical flow cytometric detection of intracellular IL-4 and IL-5 in T cells from whole blood samples in an asthmatic child.

Statistical Analysis

When multiple comparisons were made between groups, significant between-group variability was first established using the Kruskal-Wallis test. The Mann-Whitney U test was then used for intergroup comparison. Correlation coefficients were obtained by Spearman's rank-order method. Probability values of p < 0.05 were accepted as significant.

	RESULTS

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Figure 3 shows the percentage of CD3⁺ T cells staining positively for the cytokines IL-4 and IL-5 after 5 h of stimulation with PMA and ionomycin in the presence of monensin. No cytokines were detectable without stimulation, and appropriate control antibodies showed no positive staining. In asthmatic children there was an increased percentage of T cells producing IL-5 (median 4.9% [range 1.1 to 8.9%]) compared with atopic control subjects (0.3% [0.2 to 0.9%]; p = 0.003) and nonatopic control subjects (0.4% [0.1 to 3.8%]; p = 0.001). The frequency of IL-4-producing T cells was increased in atopic asthmatics (1.2% [0.2 to 2.6%]; p = 0.011) and atopic control subjects (0.8% [0.4 to 3.7%]; p = 0.007) compared with nonatopic controls (0.4% [0.2 to 0.9%]). The percentage of T cells staining positively for both cytokines was in the range of the detection limit of the method (0.1 [0.0% to 0.2%]) in all three groups without significant differences. In children there was a significant correlation between the percentage of IL-5-producing T cells and the number of eosinophils in peripheral blood (n = 27; R = 0.60, p = 0.006) and the level of hyperresponsiveness assessed as the log PC₅₀SGaw (n = 18, nonatopic control subjects were not included because PC₅₀SGaw was determined as > 8 only; R = 0.71, p = 0.003) (Figure 4). Furthermore, there was a good correlation between the percentage of IL-4-producing T cells and the concentration of total IgE (n = 27; R = 0.59, p = 0.002). A significant correlation was found between the frequency of positive skin prick tests and the percentage of IL-4-producing T cells (n = 27; R = 0.57, p = 0.003) or IL-5-producing T cells (n = 27, R = 0.40, p = 0.004). No significant correlations were found between cytokine expression and FEV₁ or MEF₂₅. The number of eosinophils correlated with FEV₁ (n = 27; R = 0.54, p = 0.01) and PC₅₀SGaw (n = 18; R = 0.83, p = 0.004) but not with MEF₂₅. In contrast, when asthmatic adults were compared with their age-matched control groups there was no difference in the percentage of T cells staining positively for IL-5 (AA: 0.7% [0.2 to 2.6%]; AN: 0.5% [0.3 to 1.9%]; NN: 1.0% [0.5 to 3.8%]); and IL-4 (AA: 0.9% [0.3 to 9.0%]; AN: 1.6% [1.0 to 2.7%]; NN: 1.1% [0.6 to 1.7%]). As in children the percentage of T cells coexpressing IL-5 and IL-4 was hardly detectable (0.1% [0.0 to 0.3%] in all three groups). No significant correlations were found between cytokine expression and lung function, eosinophilia, total IgE, or the frequency of skin prick tests. The number of eosinophils correlated with the level of hyperresponsiveness assessed as the log PC₅₀SGaw (n = 9, atopic and nonatopic control subjects were not included because PC₅₀SGaw was determined as > 8 only; R = 0.63, p = 0.05) but not with FEV₁ and MEF₂₅.

View larger version (12K): [in this window] [in a new window]   Figure 3.   Percentage of CD3+ lymphocytes with positive intracellular staining for IL-4 and IL-5 in peripheral blood from normal control, atopic nonasthmatic, and atopic asthmatic children (top, n = 9) and adults (bottom, n = 9). Bars indicate median values, and statistical comparison was performed by the Mann-Whitney U test.

View larger version (8K): [in this window] [in a new window]   Figure 4.   Correlations between the percentage of IL-5-positive T cells and the number of peripheral eosinophils; the percentage of IL-5-positive T cells and the bronchial hyperresponsiveness; and between the percentage of IL-4-positive T cells and IgE concentrations in children.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

In this study we have evaluated the ability of peripheral blood T cells from mild asthmatics to produce IL-4 and IL-5 after short-term in vitro stimulation at the single cell level. In asthmatic children compared with atopic and nonatopic control subjects there was an increased percentage of T cells producing IL-5 related to eosinophilia and airway hyperresponsiveness, whereas the expression of IL-4 was elevated in both atopic groups related to IgE levels. These data are in accordance with the proposed hypothesis, that in children there is an increased activity of Th2-type cytokines in asthma and that T cells are an important source of these cytokines. Furthermore our data support the hypothesis that IL-4-producing T cells are rather associated with IgE synthesis and atopic status than with asthma and that IL-5-producing T cells regulate airway hyperresponsiveness through eosinophil recruitment. A limited number of other studies is available which address the role of cytokines in children with atopic asthma: Two studies demonstrated increased concentrations of IL-4 in serum (15, 16) and others have found increased production of IL-4 of PHA-stimulated peripheral blood mononuclear cells (PBMCs) from atopic asthmatics (17). Furthermore an increased expression of IL-4 and IL-5 mRNA in PBMCs (18, 19) and T cells (20, 21) has been demonstrated in atopic asthmatic children.

When atopic adult asthmatics were compared with age-matched atopic and nonatopic control groups we surprisingly did not find differences in the percentage of T cells staining positively for IL-4 and IL-5. Although other investigators have shown an increased release of IL-4 and IL-5 from peripheral T cells (3), measured by ELISA, and an increased expression of mRNA encoding for IL-4 and/or IL-5 in peripheral CD4⁺ lymphocytes (5) and PBMCs (22) from adult asthmatics, our data do not support the hypothesis that in adults there is enhanced IL-4 and IL-5 protein expression in circulating T cells. Because different techniques have been applied to detect cytokine gene or protein expression, the results from these studies are not directly comparable: Gene expression does not necessarily correlate with protein production and the number of individual T cells, positive for a specific cytokine, is not necessarily related to the amount of cytokine produced by the whole cell population (as measured by ELISA). The single cell assay of intracellular cytokine detection used in this study has the important advantage of simultaneously identifying the phenotype of the cytokine-producing cells, but one possible limitation of the method might be the lower sensitivity of cytokine detection compared with others. A further possible explanation for the divergent results might be differences in study populations. In this regard asthma was more severe in two of the cited studies compared with our study population (5, 22) and in one study no comparison was possible owing to the lack of detailed clinical data (3).

Allergic asthma in children and adults is thought to represent the same disease with similar underlying mechanisms, but very limited data exist comparing cytokine expression in atopics between both age groups with identical methodology: Yabuhara and colleagues (18) investigated cytokine expression of house dust mite-stimulated PBMCs and showed increased IL-4/IL-5 mRNA expression in 2- and 5-yr-old atopics as well as in adults with highest values at the age of 5 yr. Together with these results the differences in cytokine production found in our study suggest that IL-4- and IL-5-producing T cells seem to play a more important role in children. Two major differences between our study groups were the duration of asthma (5 ± 3 yr versus 11 ± 9 yr) and especially the age of onset of asthma (4 ± 3 yr versus 25 ± 8 yr). Therefore, one could hypothesize that with late onset and with duration of asthma the clear-cut initial Th2-cell-driven regulatory mechanism becomes less important in the self-perpetuating chronic disease. Other cells or cytokines might become more relevant in adult asthmatics. However, differences in the severity of asthma could have influenced the cytokine profile in our study: While FEV₁, MEF₂₅, IgE, and the frequency of positive skin prick tests did not differ between children and adults, bronchial hyperresponsiveness was slightly higher in children and a trend for higher eosinophil counts was found in children. All children (9 × sodium cromoglycate, 3 × inhaled steroids) but only two adults (2 × inhaled steroids) have been receiving anti-inflammatory drugs at the time of our study. Therefore, it is possible that in a group of more severe adult asthmatics the number of IL-4- and IL-5-producing T cells would have been more elevated than in our study. However, because there was not even a trend for an increased population of IL-4- and IL-5-producing T cells in adults, we do not believe that only differences in asthma severity account for the different T-cell cytokine production. In addition to T cells, mast cells (23, 24) and basophils (25, 26) are able to produce IL-4 and IL-5 and one must not forget that these cells might significantly contribute to Th2-type cytokine production in allergic disease. However, data on age-dependent cytokine production of these cells are not available.

We have shown that in patients, where T-cell production of IL-4 and IL-5 was enhanced, these cytokines are not produced by the same T cell when activated in vitro. Therefore, we are unable to support the hypothesis that coexpression of IL-4 and IL-5 is present in circulating T cells in asthma. This segregation of cytokine production has also been observed in human CD4⁺ and CD8⁺ memory T cells from healthy volunteers, patients with hyper-IgE syndrome (7) and in CD4⁺ cells in mice (27) using single-cell assays. Furthermore, in Th2 clones a substantial proportion of statistically overlapping IL-4/IL-5 double-positive coexpression and a proportion of single-positive or even completely negative cells were found (7). These findings suggest that close to the in vivo situation the expression of each cytokine is under independent control but that under clonal culture conditions tendencies for concordant cytokine expression may be achieved after prolonged stimulation and repeated restimulation.

Future studies, using single-cell assays, should focus on T cells from the airways obtained by bronchoalveolar lavage rather than on circulating T cells to get a closer insight into the local cytokine expression at the site of the allergic reaction. Furthermore, the exploration of the cytokine coexpression in different T-cell subsets could significantly contribute to our understanding of the regulation of the T-cell-derived cytokines in asthma.