INTRODUCTION

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Keywords: interleukin-4; splice variant; asthma; atopy

Atopy is an immunoglobulin E (IgE)-associated disorder manifest as hypersensitivity to environmental antigens and it clinically prediposes to asthma, allergic rhinitis, and eczema. Asthma and atopy are biologically linked by type-2 cytokine-driven inflammatory processes. Interleukin-4 (IL-4), in particular, is central to the maturation of T helper type-2 (Th2) cells and IgE class switching (1). Activated IL-4-secreting T cells are found in peripheral blood (2), mucosal biopsies (3), and bronchoalveolar lavage (BAL) T cells (4) during acute episodes of asthma, and the levels of IL-4 gene expression correlate with clinical measures of asthma severity (4).

Given the importance of IL-4 in the immunopathogenesis of asthma, the relative role of IL-4 and its splice variant (IL-42) in the disease process needs to be understood. IL-42 mRNA has been found in human peripheral T cells (5), lymphoid tissue (6), and nasal and endobronchial biopsy specimens (7). IL-4 and IL-42 mRNA are coexpressed in healthy people but vary in relative ratio from individual to individual, and also between different tissue sites in the same individual (5). Some normal people have higher levels of expression of IL-42 than of IL-4 mRNA (5).

Work with recombinant IL-42 protein suggests that it antagonizes the effects of IL-4 on T cells, B cells, and macrophages (8, 9). One aim of asthma therapy is to reverse the effects of IL-4 and of other type-2 cytokines (10), so IL-42 may provide a novel treatment strategy. Moreover, an inappropriately low level of expression of IL-42 relative to IL-4 could be an important but hitherto unexplored reason for the pathology in patients with severe asthma.

It is not currently possible to measure IL-42 protein levels in biological samples using antibody-based assays, because epitopes unique to IL-42 and absent from IL-4 have yet to be identified. However, mutually exclusive primers can be designed for reverse-transcription polymerase chain reaction (RT-PCR) to distinguish the two cytokines. Previous studies that have used sequences flanking exon 2 as primers for PCR or probes for hybridization have not distinguished IL-4 from IL-42.

Therefore in this study we have used appropriate RT-PCR protocols to measure separately mRNA copy numbers of IL-4 and IL-42 in a group of patients with chronic asthma, atopy, and high serum IgE titers against one or more aeroallergens. As a control pulmonary disease we have included patients with tuberculosis (TB), because although type-1 cytokines form the dominant response in TB, these patients also have a significant increase in type-2 cytokine expression (11, 12).

	METHODS

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Subjects and Clinical Criteria for Inclusion

Informed consent was obtained from all subjects involved and the protocols were approved by the respective institutional review boards.

Patients with atopic asthma. The nine patients studied (age range was 30-55, male:female ratio 1:2) were part of the Oxford Asthma Research project (13) conducted at the Churchill Hospital, Oxford, UK, and had both chronic asthma and atopy. Asthma was diagnosed by a specialist physician and all had recurrent breathlessness and chest tightness requiring on-going treatment, physician-documented wheeze, and documented labile airflow obstruction with variability in serial peak expiratory flow rates > 30%. The asthma had been chronic since diagnosis, with no period when medication was not required. In addition, all had experienced at least one of the following: allergic rhinitis, eczema, and anaphylactoid reactions to one or more food substances. They showed positive skin-prick test of > 5 mm against any one of the antigens tested or a RAST score of more than 2. Atopy was defined by a high concentration of total serum IgE and/or a positive specific IgE titer against one or more aeroallergens. The specific IgE against house dust mite and a grass mix, and total serum IgE were measured by the CAP immunoassay system (Pharmacia, Uppsala, Sweden).

Patients with TB. The 18 patients (age range 18-64, male:female ratio 2:1) with their first episode of culture-proven pulmonary TB were attending the Middlesex Hospital (London, UK) (11). Chest X-rays (CXR) at presentation were consistent with pulmonary TB. Blood samples were taken just before or within the first month of initiating treatment. Patients with multiple lung pathologies, a history of asthma of any duration, any experience of symptoms of atopy, long-term steroid therapy, or other chronic illness requiring long-term medication were excluded

Healthy subjects. There were 18 healthy subjects (age range 23-67, male:female ratio 2:1). Exclusion criteria were a recent history of acute respiratory illness, receipt of any medication within the past 4 wk, previous long-term steroid therapy, a history of asthma of any duration, any experience of symptoms of atopy, history of tuberculosis, or any chronic illness requiring long-term medication. The CXR, total white cell counts and differential counts, serum C-reactive protein levels, and erythrocyte sedimentation rates of all these subjects were tested and confirmed by a physician to be within normal limits.

RNA Extraction and RT-PCR

RNA was extracted from peripheral blood mononuclear cells (PBMCs) using the Quickprep Micro mRNA purification kit (Amersham Pharmacia Biotech, Little Chalfont, Bucks, UK). The first-strand cDNA synthesis kit (Amersham Pharmacia Biotech) was used for reverse transcription. Quantitative nested RT-PCR for IL-4 and IL-42 was performed as previously described (14), by one operator using the same batch of RNA standards throughout. The cytokine mRNA copy numbers were expressed relative to -actin mRNA copy numbers.

Statistical Analysis

The nonparametric Mann-Whitney U test and Spearman rank correlation were used respectively for statistical comparisons between two groups of subjects and for studying correlations between two variables.

	RESULTS

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IL-4 and IL-42 expression were closely correlated in each of the three groups (patients with asthma: p = 0.00088; patients with tuberculosis: p = 0.0016; control subjects: p = 0.044) (Figure 1). The median IL-4 mRNA copy number in the cells from the patients with asthma was 2.8 logs higher than in the cells from patients with tuberculosis (p = 0.00047) and 4.5 logs higher (p = 0.00039) than in the cells from healthy control subjects (Figure 2A). Possibly due to the small size of the asthma group, the bimodal distribution of IL-42 was not as distinct as in the other two groups (Figure 2B), and we did not perform statistical analysis on the two clusters separately. The IL-42 mRNA levels of the asthma group were neither higher than those of the patients with tuberculosis (p = 0.92) nor the control subjects (p = 0.090).

View larger version (21K): [in this window] [in a new window]   Figure 1.   Relationship between IL-4 and IL-42 mRNA copy numbers (squares, asthma, triangles, tuberculosis, circles, control subjects). The regression line is shown for the asthma group. The statistics by Spearman rank correlation for each group are as follows: asthma: r = 0.97, p = 0.000876; tuberculosis: r = 0.61, p = 0.0016; control subjects: r = 0.31, p = 0.044.

View larger version (12K): [in this window] [in a new window]   Figure 2.   IL-4 (A) and IL-42 (B) mRNA copy numbers in each group of subjects; each data point represents the median result of duplicate experiments performed using cells from one subject. The median value in each group is indicated by a horizontal bar. TB, tuberculosis.

The ratio of IL-4 to IL-42 mRNA copy number was calculated for each subject and this was very much higher (median 501) in the group with asthma than in the tuberculosis group (median 1.17; p = 0.000031) and healthy control group (median 0.693, p = 0.00021) (Figure 3). The IL-4:IL-42 ratio did not correlate with age, sex, total serum IgE levels, or the incidence of eczema, rhinitis, or anaphylaxis in the group with asthma (p > 0.05).

View larger version (11K): [in this window] [in a new window]   Figure 3.   The ratio of IL-4 to IL-42 copy numbers was computed for each subject and the median ± 2 SD (error bars) for each group is represented in this figure. Where the error bars are not seen, the error falls within the symbol (asthma n = 9, tuberculosis [TB] n = 18, control subjects n = 18).

	DISCUSSION

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This study is the first to characterize IL-42 gene expression in patients with significant clinical and laboratory-documented atopy and asthma that has been persistent since diagnosis. Atopy has been particularly associated with high levels of IL-4 produced by mitogen- or antigen-stimulated cells from blood and BAL in previous studies (reviewed in [1]). Our study extends these observations to fresh unstimulated peripheral blood mononuclear cells (PBMCs) directly ex vivo, which we believe more accurately reflect the physiological situation. We have been able to measure IL-4 mRNA in unstimulated cells from healthy control subjects for comparison with the atopic subjects, due to the use of a sensitive method that has been validated for quantifying mRNA even for very low copy number cytokines (14).

The most significant finding is that there was a 500-fold difference between the group with asthma and the other groups in the relative expression of IL-4 and IL-42 (Figure 3). It is currently believed that IL-42 may be a functional IL-4 antagonist that competes for receptor binding (8). Hence, a low level of IL-42 expression, relative to IL-4 expression, could be a factor in the pathobiology of asthma.

It is interesting to note the results of a recent study on endobronchial biopsy specimens from patients whose asthma was stable and unchanged for 4 wk on inhaled corticosteroids or without medication. In these patients with well-controlled disease IL-4 mRNA levels were unremarkable, but their IL-42 expression levels were significantly elevated, when compared with healthy subjects (7). This is consistent with the hypothesis that the ratio of IL-4 to IL-42 may be important.

Subepithelial basement membrane collagen deposition occurs in the airway remodeling process in patients with chronic asthma (1). The role of IL-42 in inducing collagen 2(I) mRNA production in several fibroblast cell lines has been reported (15), and in this role it appears to synergize with IL-4. The authors suggest that whether IL-42 acts as an IL-4 agonist or antagonist may depend on their relative binding to different IL-4 receptors on different cell types. All human lung fibroblasts have functional but different IL-4 and IL-13 receptors. When activated by IL-4 and IL-13, different subsets of lung fibroblasts may act as effector cells in lung remodeling processes, and may also differentially contribute to trigger and maintain the recruitment, homing, and activation of inflammatory cells (16). However, the activity of IL-4 has not been distinguished from that of IL-42 because validated protein assays that specifically detect IL-42 (as distinct from IL-4) have yet to be developed. Interestingly, BAL cells from some patients with systemic sclerosis and severe pulmonary fibrosis expressed IL-42 with no detectable mRNA for IL-4 itself (15). Hence, IL-42 is potentially responsible for some of the profibrotic activity previously ascribed to IL-4, and the observation that in patients with tuberculosis there is a simultaneous and parallel rise in mRNA for both IL-4 and IL-42 (Figure 3, [11, 14]) is compatible with the view that in TB the Th2-like component is driving fibrosis, which is a major feature of the disease, but very little IgE (though IgE antibody to M. tuberculosis is present in TB) (17). Our data therefore indicate that the IL-4 response in TB is radically different from that seen in allergic asthma.

There is an apparent paradox that type-2 cytokines, which inhibit proinflammatory cytokines such as tumor necrosis factor-alpha and interferon-gamma (IFN-) in many other disease models (18), are implicated in the airway inflammation of patients with atopic asthma (1). However, this study highlights the diversity of IL-4-expressing T cells. In other diseases the cells may in reality be expressing IL-42, or they may be IL-4-expressing regulatory cells rather than Th2 effector cells (19).

In conclusion, the relative roles of IL-4 and IL-42 in the pathogenesis of atopic and fibrotic aspects of pulmonary disease deserve further investigation. Disproportionate levels of IL-4 relative to IL-42 may contribute to the pathogenesis of atopic asthma, but the potential association of IL-42 with pulmonary fibrosis, possibly manifested in tuberculosis and systemic sclerosis, may limit its utility in therapeutics.