Introduction

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Allergic rhinitis is an IgE-mediated inflammatory disorder with high morbidity. The allergen-induced late nasal response (LNR) has provided a clinical model for allergic rhinitis. This reaction occurs from 6 to 24 h after local allergen challenge of subjects with atopic rhinitis. The LNR is associated with cellular infiltration of the nasal mucosa with activated CD4⁺ T-lymphocytes and eosinophils (1). Recent studies have implicated cytokines as important mediators in this inflammatory reaction (2).

We previously showed that the LNR is associated with increases in cells expressing Th2-type cytokines, in particular interleukin-4 (IL-4) and IL-5, in vivo in the nasal mucosa of allergic rhinitic subjects 24 h after local allergen challenge (3, 4). IL-4 is believed to be important in the LNR through the recruitment of eosinophils to the nasal mucosa by upregulating the endothelial expression of vascular cell adhesion molecule-1 (VCAM-1) (5), and possibly in the propagation of IgE synthesis through the induction of immunoglobulin isotype switching to IgE (6). A central role for IL-4 in the LNR is supported by recent investigations showing that inhibition of this response with topical glucocorticoid treatment is accompanied by a local attenuation of IL-4, but not IL-5, messenger RNA (mRNA) expression (7).

Given the apparent significance of IL-4 in regulating LNR-associated inflammation, the investigation of IL-13 may be of considerable importance. IL-13 has functional similarities to IL-4 (8, 9). These include the capacity to induce immunoglobulin isotype switching to IgE in B cells (10), as well as VCAM-1 expression on endothelial cells (11). Functional heterogeneity between IL-4 and IL-13 has been observed on activated T-cell clones, with IL-4, but not IL-13, having a proliferative effect (9). Recent reports have suggested that IL-4 and IL-13 may be under differential regulation (12), supporting the notion that these cytokines may hold distinct functions in some physiologic circumstances.

We recently showed increased expression of IL-13 mRNA in skin lesions of patients with atopic dermatitis (16). The present study was undertaken to investigate IL-13 transcript expression and immunoreactivity in the LNR and, in the context of a double-blind trial, to examine the influence of topical corticosteroids on IL-13 expression. These experiments were performed on sections from nasal biopsies of subjects with seasonal allergic rhinitis obtained at baseline (out of season), as well as from nonrhinitic control subjects. We also compared the expression of IL-13 mRNA with that of IL-4 mRNA under the same conditions.

	Materials and Methods

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Subjects and Study Design

The study was approved by the Ethics Committees of the Sahlgren Hospital, Gothenburg, and The Royal Brompton National Heart and Lung Hospital, London. Ten normal volunteers (three men and seven women, mean age: 31 yr) and 20 subjects with a clinical history of seasonal hay fever (eight men and 12 women, mean age: 30 yr) were recruited, and informed written consent was obtained from all. Inclusion criteria for the subjects with allergic rhinitis included: (1) history of summer hay fever for at least 2 yr; (2) a positive skin-prick test (> 5 mm) to Timothy grass pollen extract (Soluprick, Phleum pratense; ALK, Horsholm, Denmark). Patients were excluded if they had received topical or oral medication in the previous 6 mo or immunotherapy in the previous 5 yr. Baseline nasal biopsies were obtained from all subjects, using a Gerritsma forceps, after providing local anesthesia with 3% cocaine and 0.025% adrenaline. All subjects were studied outside the grass-pollen season, at a time when they were asymptomatic. Patients were randomized to receive either a 6-wk treatment with topical fluticasone propionate 200 µg twice daily or matched placebo nasal spray in a double-blind fashion. Following treatment, local nasal allergen provocation was performed by applying a 4-mm filter-paper disk containing normal saline-diluted grass-pollen extract to the undersurface of the nasal inferior turbinate for 10 min, as previously described (1). Nasal biopsies were taken 24 h after the allergen challenge, as described.

Tissue Preparation

Nasal biopsies were prepared for immunocytochemistry and in situ hybridization procedures. Tissues were immediately fixed in 4% paraformaldehyde for 2 h, washed twice in 15% sucrose in 0.1 M phosphate-buffered saline (PBS) (pH 7.4), embedded in ornithyl carbamyl transferase (OCT) compound, and snap-frozen in liquid nitrogen-cooled isopentane. Cryostat sections (8 µm thick) were cut and mounted on 0.1% poly-L-lysine-coated slides, and air-dried overnight at 37°C.

Immunocytochemistry

Immunocytochemistry was performed with antihuman IL-13 (PeproTech Inc., Rocky Hill, NJ) antibody. We used the alkaline phosphatase antialkaline phosphatase (APAAP) technique as previously described (1). The reaction was visualized with fast red TR (Sigma Chemical Co., St. Louis, MO). Sections were then counterstained with hematoxylin.

Preparation of Riboprobes

Riboprobes were prepared from complementary DNA (cDNA) for IL-13 (a gift from Dr. Adrian Minty, Sanofi Recherche, Labège Cedex, France) and IL-4 (a gift from the Glaxo Institute for Molecular Biology, Geneva, Switzerland) as previously described (16, 17). cDNA were inserted into pGEM vectors and linearized with the appropriate enzymes prior to in vitro transcription. Transcription was performed in the presence of [³⁵S]uridine triphosphate ([³⁵S]UTP) and RNA polymerases to generate antisense (complementary to mRNA) and sense (identical to mRNA) probes.

In Situ Hybridization

In situ hybridization was performed as previously described (16, 17). Briefly, cryostat sections were first permeabilized by immersion in 0.3% Triton X-100 in PBS for 10 min, followed by exposure to proteinase K solution (1 mg/ ml in 20 mM Tris-HCl and 1 mM ethylenediamine tetraacetic acid [EDTA], pH 7.2) for 30 min at 37°C. The slides were then prehybridized with 50% formamide in 2 × standard saline citrate (SSC) for 15 min at 37°C. Hybridization was performed with [³⁵S]UTP-labeled riboprobes (either antisense or sense) for 16 h at 42°C. Posthybridization washings were done with SSC solutions (4 × SSC and 0.1 × SSC) followed by ribonuclease (RNase) treatment to remove unhybridized single-stranded RNA. The preparations were dehydrated, immersed in emulsion, and then subjected to autoradiography for 14 days. The autoradiograms were developed and subsequently counterstained with hematoxylin. As a negative control, sections were hybridized with the sense probe or were pretreated with RNase prior to hybridization with the antisense probe.

Combined Immunocytochemistry-In Situ Hybridization

To identify the percentage of IL-13 mRNA positive cells coexpressing the T-cell marker CD3 or the mast cell marker tryptase, colocalization studies were done on sections prepared from nasal biopsies obtained 24 h after allergen challenge from five allergic rhinitic subjects given placebo. This technique has been described elsewhere (3, 4). The specimens were first immunostained with the APAAP technique to phenotype cells, using a monoclonal antibody directed against human T-cell receptor (anti-CD3; Becton Dickinson, Mississauga, ON, Canada) or a monoclonal antibody directed against human mast-cell tryptase (antitryptase; Chemicon International Inc., Temecula, CA). In order to avoid RNase contamination, all solutions used were made up in 0.1% diethylpyrocarbonate (DEPC)- treated distilled water. After immunochemical staining of biopsy sections, the specimens were processed for in situ hybridization, using a digoxigenin-labeled IL-13 antisense riboprobe, as previously described (3, 4). Cell counts were expressed as the percentage of IL-13 mRNA-positive cells that coexpressed immunoreactivity for CD3 or tryptase.

Double In Situ Hybridization

To colocalize IL-13 and IL-4 mRNA, double in situ hybridization was performed. The IL-13 antisense riboprobe was labeled with [³⁵S]UTP and the IL-4 probe was labeled with biotin-11-UTP (Enzo Diagnostic, New York, NY), as previously described (18). Biopsy sections following allergen challenge from six randomly selected allergic rhinitic subjects given placebo were analyzed. Sections were treated similarly for single in situ hybridization, but were hybridized simultaneously with radiolabeled IL-13 and nonradiolabeled IL-4. Biotin-labeled cRNA-mRNA hybrids were visualized with an avidin-biotin complex and diaminobenzidine (brown stain). Sections were then dried and covered with emulsion to develop the radiolabeled cRNA-mRNA hybrid. Double hybridization signals were demonstrated by the presence of silver grains in brown-staining cells.

Counting and Data Analysis

Nasal biopsy sections were coded and counting was accomplished in a blinded fashion with an Olympus microscope with an eyepiece graticule at ×100 magnification. Cells that were mRNA-positive or immunoreactive for IL-13 and IL-4 were counted and results were expressed as mean counts ± SD per field (3, 4). Cells that were positive for IL-13 mRNA and CD3 or tryptase were counted and data were presented as the percentage of IL-13 mRNA-positive cells ± SD that coexpressed CD3 or coexpressed tryptase. For double in situ hybridization studies, results were expressed as the percentage (± SD) of IL-13 mRNA-positive cells coexpressing IL-4 mRNA and as the percentage (± SD) of IL-4 mRNA-positive cells coexpressing IL-13 mRNA.

The expression of each cytokine, IL-13 and IL-4, in nasal biopsies obtained from subjects with allergic rhinitis and normal controls was compared through the use of Student's t test. Comparison of the mean counts of cells expressing each cytokine at baseline and after allergen challenge was done with a paired t test in each study group (placebo- and steroid-treated groups). Changes in the mean counts of IL-13 or IL-4 mRNA-positive cells observed after allergen challenge as compared with baseline values were expressed as delta values, and were compared through the use of Student's t test. Correlations were performed through linear regression analysis and the Spearman's rank method. Significance was accepted at the 5% level of confidence. Statistical tests were done with a standard computer package (Systat version 5.03; Systat Inc., Evanston, IL).

	Results

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Baseline Expression of IL-13 mRNA and Immunoreactivity in Nasal Mucosa

At baseline, the number of IL-13 mRNA-positive cells was small and there was no significant difference between normal (0.96 ± 0.56 mRNA-positive cells/field) and allergic rhinitic (0.82 ± 0.67 mRNA-positive cells/field, P = NS) subjects in mean counts for IL-13 mRNA-positive cells (Figure 1A).

View larger version (11K): [in this window] [in a new window]   Figure 1.   IL-13 expression in nasal biopsy specimens from normal subjects and allergic rhinitic subjects studied out of season as assessed by: (A) in situ hybridization for IL-13 mRNA; and (B) immunocytochemistry for IL-13. Results are expressed as the number of positive cells per field. Statistical analysis was done with an unpaired t test. There were no statistical differences between allergic rhinitic subjects and normal controls in the numbers of IL-13 mRNA-positive cells and IL-13-immunoreactive cells.

IL-13 immunoreactivity was observed in nine of 10 normal controls and in all rhinitic subjects. There was no difference between normal controls (2.01 ± 1.61 positive cells/field) and allergic rhinitic subjects (3.02 ± 1.50 positive cells/field, P = NS) in the number of IL-13-immunoreactive cells (Figure 1B).

IL-13 mRNA and Immunoreactivity Expression Following Antigen Challenge

When biopsies from fluticasone- and placebo-treated subjects were examined at baseline, no difference was found between the two groups for either IL-13 mRNA expression or IL-13 immunoreactivity. In the subjects given placebo, a significant increase in IL-13 mRNA-positive cells was observed 24 h after the allergen challenge (before allergen: 1.28 ± 2.05 mRNA-positive cells/field; after allergen: 8.74 ± 3.44 mRNA-positive cells/field, P < 0.001) (Figures 2 and 3A). In contrast, in the fluticasone-treated group, the number of IL-13 mRNA-positive cells did not increase significantly after allergen challenge (before allergen: 0.86 ± 0.65 mRNA-positive cells/field; after allergen: 1.22 ± 0.93 mRNA-positive cells/field, P = NS) (Figure 3A). Similarly, a significant increase in the number of IL-13-immunoreactive cells was observed 24 h after allergen challenge in the subjects given placebo but not in the fluticasone-treated group (Figure 3B).

View larger version (60K): [in this window] [in a new window]   Figure 2.   (A) Representative autoradiograph (darkfield), at 24 h after antigen challenge, of in situ hybridization of nasal biopsies from allergic rhinitic subjects given placebo. Arrows indicate some of the IL-13 mRNA-positive cells. (B) A representative example, at 24 h after antigen challenge, of immunostaining for IL-13 in a nasal biopsy specimen from an allergic rhinitic subject given placebo. IL-13 immunoreactivity was observed in small mononuclear cells (large arrows) and some larger mononuclear cells (small arrow). (C) Nasal biopsy section after allergen challenge of an allergic rhinitic subject given placebo, that was double-stained for IL-13 and IL-4 mRNA by double in situ hybridization. Double hybridization signals (black arrow) were demonstrated by the presence of silver grains (IL-13 mRNA-positive) in brown-staining cells (IL-4 mRNA-positive). The white arrow indicates a single staining cell expressing IL-13 but not IL-4 mRNA.

View larger version (17K): [in this window] [in a new window]   Figure 3.   Effects of allergen challenge on (A) IL-13 mRNA and (B) immunoreactivity expression in nasal biopsy specimens from allergic rhinitic subjects given placebo and those treated with steroids. Biopsies were taken at baseline (before) and 24 h after allergen challenge (after). Results are expressed as the number of positive cells per field. Statistical analyses were done with a paired t test. There were statistically significant increases in the numbers of IL-13 mRNA-positive cells and IL-13-immunoreactive cells at 24 h after allergen challenge in the subjects given placebo. *P < 0.001.

The morphology of most of the IL-13-immunoreactive cells was consistent with lymphocytes. However, some IL-13-positive cells were large mononuclear cells (Figure 2). Colocalization studies conducted in five rhinitic subjects given placebo after allergen challenge showed that 76.9 ± 5.5% of these subjects' IL-13 mRNA-positive cells were positive for the T-lymphocyte cell surface antigen CD3. In contrast, 11.2 ± 2.7% of the IL-13 mRNA-positive cells coexpressed immunoreactivity for the mast-cell marker tryptase (Table 1).

In contrast to IL-13 mRNA-positive cells, significantly more IL-4 mRNA-positive cells were found in allergic rhinitic subjects at baseline (1.90 ± 1.58 mRNA positive cells/ field) than in the nonatopic controls (0.14 ± 0.19 mRNA-positive cells/field, P < 0.001) (Table 2). In subjects given placebo, a significant increase in IL-4 mRNA-positive cells was observed 24 h after the allergen challenge (P < 0.001). In contrast, in the fluticasone-treated group, the number of IL-4 mRNA-positive cells did not increase significantly after allergen challenge. In the subjects given placebo, the increase in the numbers of cells expressing IL-4 mRNA (delta value: 5.4 ± 2.8) following allergen challenge was significantly smaller than the increase in the numbers of cells expressing IL-13 mRNA (delta IL-13: 8.9 ± 3.1, P = 0.001). As shown in Figure 4, there was no correlation between the numbers of IL-13 mRNA- and IL-4 mRNA-positive cells (r = 0.305, P = 0.392).

View larger version (11K): [in this window] [in a new window]   Figure 4.   Linear regression analysis indicated no correlation between numbers of IL-4 mRNA- and IL-13 mRNA-positive cells per field in rhinitic subjects given placebo after allergen challenge. r = 0.305, P = 0.392.

Double in situ hybridization analyses were performed on biopsy sections following allergen challenge from six allergic rhinitic subjects given placebo, to determine whether IL-13 and IL-4 transcripts were coexpressed by the same cells in the nasal mucosa (Figure 2). The percentage of IL-13 mRNA-positive cells coexpressing IL-4 mRNA ranged from 52.6% to 81.3%, and the mean was 66.6 ± 10.5%. In contrast, 100% of the IL-4 mRNA-positive cells expressed IL-13 transcripts in sections from each of the six subjects.

	Discussion

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We and others have previously shown that the allergen- induced LNR is associated with an increase in the numbers of cells expressing mRNA for Th2-type cytokines, particularly IL-4 and IL-5, but not IL-2 or interferon- (IFN-) (2). This suggests a selective recruitment and/or local activation of Th2-type cytokine-producing cells in the atopic nasal mucosa following antigen provocation. We investigated the expression of IL-13, a recently described cytokine with functional similarities to IL-4. The results of the present study demonstrate no significant difference in baseline IL-13 expression in allergic rhinitic subjects as compared with normal controls, indicating that the presence of IL-13 in the nasal mucosa may not be associated with atopy. However, a pronounced increase in the numbers of IL-13 mRNA-positive cells was observed 24 h after local allergen challenge of rhinitic subjects given placebo, suggesting that IL-13 is involved in allergen-induced LNR. To determine if this increase in cytokine mRNA also occurs at the protein level, we examined the effects of allergen challenge on IL-13 immunoreactivity. Immunocytochemical analyses confirmed a corresponding upregulation of translated IL-13 protein.

In vitro experiments have shown that IL-13 is produced by mast cells, basophils, and T cells of both the CD4⁺ and CD8⁺ phenotypes (19, 20). In the present study, we identified IL-13 transcripts and immunoreactivity in at least two different cell types based on morphologic criteria. IL-13-positive cells appeared as both small and large mononuclear cells, the former resembling lymphocytes. The morphology of the larger cells is consistent with that of mast cells or basophils. Colocalization studies performed on biopsies obtained from five allergic rhinitic subjects following allergen provocation demonstrated that 76.9 ± 5.5% of the IL-13 mRNA-positive cells were CD3⁺, indicating that T cells are the major source of IL-13 in the nasal mucosa. These results are in agreement with our previous observations that IL-13 mRNA is predominantly expressed in CD3⁺ T cells in the bronchial mucosa of atopic asthmatic individuals (21). In earlier work, nasal IL-13 production was colocalized to F_cRI⁺ cells that morphologically resembled mast cells (22). In the nasal mucosa of five allergic rhinitic subjects given placebo, we found that 11.2 ± 2.7% of IL-13 mRNA-positive cells were tryptase-positive mast cells at 24 h after allergen provocation. Granulocytes do not appear to be a cellular source of IL-13 at sites of allergen challenge in patients with asthma (23).

Previous studies have shown that topical corticosteroid treatment results in ablation of the LNR (24), possibly in relation to the selective inhibition of cytokine expression. We have previously shown that attenuation of the LNR following corticosteroid therapy is associated with cytokine inhibition for IL-4, but not IL-5, transcripts (7). These results reveal some selectivity in the repressing effect of topical glucocorticoids, as well as a potential hierarchy in cytokine function in the nasal mucosal microenvironment. In the present study, we examined the effect of 6-wk topical glucocorticocoid treatment on allergen-induced IL-13 upregulation in the nasal mucosa. A marked reduction in the number of IL-13-immunoreactive cells was identified after allergen challenge in the corticosteroid-treated group as compared with matched controls given placebo. A corresponding decline in the number of IL-13 mRNA-positive cells was observed, verifying that like other targets of steroid action, gene transcription is a likely site of IL-13 repression.

The suppression of IL-13 expression in the nasal mucosa by topical corticosteroids suggests a putative mechanism by which these drugs inhibit the LNR. Support for the notion that glucocorticoids may alleviate symptoms of the LNR partly by attenuating IL-13 is derived from investigations of the biologic activity of this cytokine. In vitro studies have shown that IL-13 augments the endothelial expression of VCAM-1 (11), an adhesion molecule thought to be crucial in the recruitment of eosinophils and CD4⁺ T lymphocytes into inflammatory sites. We have previously reported a decrease in the numbers of these cell types in the nasal mucosa following allergen challenge in corticosteroid-treated allergic rhinitic subjects (7). We postulate that the suppression of cellular infiltration after allergen challenge in subjects undergoing glucocorticoid therapy occurs partly through the inhibition of IL-13, effectively blocking the VCAM-1/very late antigen-4 (VLA-4) adhesion pathway.

In addition to upregulating endothelial VCAM-1, IL-13 shares many other biologic properties with IL-4, including the stimulation of immunoglobulin isotype switching to IgE (10). IL-13, like IL-4, downregulates IL-12 and IFN- production (8), thus favoring a Th2-type response. Functional redundancy between these cytokines may be partly explained by their sharing of a receptor subunit (25). IL-4 and IL-13 also have distinct activities. IL-4 stimulates the proliferation of activated T lymphocytes, whereas IL-13 does not have this potential (9). In addition, it was recently reported that IL-13, but not IL-4, promotes the chemotaxis and prolongs the survival of eosinophils in vitro (26).

To clarify the relative roles and importance of IL-13 and IL-4 in the LNR, we examined the expression of IL-4 mRNA in the same allergic rhinitic subjects before and after local allergen challenge and also in response to topical glucocorticoid treatment. Concording with previous studies (7), our findings confirm that IL-4 is upregulated 24 h after allergen challenge and, as with IL-13, this effect is inhibited by topical corticosteroid therapy. We used double in situ hybridization to determine whether IL-4 and IL-13 transcripts were expressed by the same cells in the nasal mucosa of six rhinitic subjects given placebo after allergen challenge. Whereas 100% of the IL-4 mRNA-positive cells expressed IL-13, 66.6 ± 10.5% of cells expressing IL-13 transcripts also expressed IL-4. This suggests that there may be some level of IL-4 and IL-13 transcriptional coregulation; however, this coregulation appears to be incomplete. Another possibility is that IL-13 mRNA has a longer half-life than IL-4 transcripts. We recently obtained similar results in the bronchial mucosa of atopic asthmatic individuals in whom we showed that 60% of the IL-13 mRNA-positive cells were expressing IL-4 mRNA and 100% of the IL-4 mRNA-positive cells were coexpressing IL-13 transcripts (21).

Several lines of evidence derived from our data support the idea that IL-4 and IL-13 hold distinct roles in development of the LNR. First, whereas the baseline expression of IL-13 in allergic rhinitic subjects showed no significant difference from that of normal subjects, IL-4 mRNA-positive cells were present in much larger numbers in the atopic rhinitic subjects than in normal controls. Second, a comparison between IL-4 and IL-13 mRNA expression in rhinitic subjects at baseline revealed that in the absence of allergen, IL-4 mRNA was produced by a greater number of cells than was IL-13 mRNA. In contrast, following allergen challenge, the increase in IL-13 mRNA was much more pronounced than the increase in IL-4 mRNA (delta values). Moreover, IL-4 and IL-13 mRNA expression showed no statistical correlation at baseline or 24 h after allergen challenge. At the latter time point, approximately 30% of the IL-13 mRNA-positive cells did not express IL-4 transcripts.

Taken together, these findings suggest a model in which IL-4 and IL-13 are under some level of independent regulation. Since mRNA was examined, differential regulation would seem to occur at the level of transcription. Furthermore, because IL-4 transcripts are found in a significantly greater number of cells at baseline in rhinitic subjects than in normal controls, and show a much lower allergen-induced upregulation than IL-13 mRNA, it can be postulated that the presence of IL-4 in the cytokine milieu of the nasal mucosa acts as a predisposing factor for allergic rhinitis. On the other hand, since IL-13 levels at baseline in rhinitic subjects do not differ from those of normal subjects, yet allergen provocation of the rhinitic individuals results in substantial augmentation of IL-13-positive cells, the upregulation of this cytokine could in part account for the clinical manifestations of the LNR.

In conclusion, these experiments show that IL-13 expression is a prominent feature of LNR. In addition, the results of our study suggest that inhibition of the LNR following topical glucocorticoid therapy may in part be attributable to inhibition of IL-13 expression.