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Background: Allergen challenge in allergic rhinitis patients leads to local eosinophilia and Th2-type cytokine expression. Natural exposure to grass pollen is additionally characterized by epithelial mast-cell infiltration. We hypothesized that perennial allergic rhinitis is also associated with T-cell and eosinophil infiltration of the nasal mucosa, local Th2-type cytokine expression, and increased numbers of nasal epithelial mast cells.
Methods: Nasal biopsies from perennial allergic rhinitis patients and controls were analysed by immunocytochemistry for different cell populations and in situ hybridization for cytokine mRNA-expressing cells.
Results: Perennial allergic rhinitis was associated with increased numbers of submucosal CD3+ T cells (P=0.05), EG2+ activated eosinophils (P=0.01), and CD68+ macrophages (P=0.01) compared to controls. Epithelial, but not submucosal, tryptase-positive mast cells were also elevated in rhinitics compared to controls (P=0.01). The numbers of cells expressing interleukin (IL)-5 were higher (P=0.01) and the numbers of cells expressing IL-2 were lower (P=0.04) in rhinitic patients than controls. There were no significant differences for either IL-4 or interferon-gamma between the groups.
Conclusions: Perennial allergic rhinitis is characterized by mast-cell migration into the epithelium; submucosal infiltration by T cells, eosinophils, and macrophages; and an imbalance in local T-cell cytokine production in favour of enhanced IL-5 and reduced IL-2 expression.
In patients with allergic rhinitis, allergen provocation results in early (0–1 h)- and late (2–24 h)-phase clinical responses, characterized by nasal discharge, sneeze, and/or blockage. While mast cells are presumed to play a role in the early nasal response and eosinophil recruitment and activation, release of lipid mediators, such as the cysteinyl leukotrienes, is thought to make a significant contribution to the late response.
Late-phase nasal responses induced by allergen challenge are accompanied by infiltration of the nasal mucosa by activated T cells, eosinophils, and neutrophils ( 1), and local expression of the “Th2-type” cytokines interleukin (IL)-4 and IL-5 ( 2). IL-4 is required for IgE synthesis by B cells and may play a role in IgE heavy-chain switching within the nasal mucosa ( 3) and upregulation of VCAM-1 expression on vascular endothelial cells ( 4). On the other hand, IL-5 has various specific effects on eosinophils, promoting maturation, endothelial adhesion, activation, survival (5–7), release from bone marrow ( 8), and responsiveness to chemoattractants such as RANTES ( 9). We have also previously shown that seasonal allergic rhinitis is characterized by nasal eosinophilia, increases in the numbers of epithelial mast cells ( 10), and increases in IL-4 ( 11) and IL-5 mRNA-expressing cells ( 12) in the nasal mucosa during the pollen season. Although cytokine immunoreactivity has been studied within the nasal mucosa in perennial allergic rhinitis (13, 14), the patterns of cytokine mRNA expression have not yet been investigated.
In the present study, we have examined both cytokine mRNA expression and epithelial and submucosal cell counts in perennial allergic rhinitic patients and controls. We set out to test the following hypotheses: first, that the symptoms of perennial allergic rhinitis are associated with infiltration of the nasal mucosa by T cells and eosinophils, but not neutrophils; second, that macrophages are recruited to the nasal mucosa in perennial allergic rhinitis, thus potentially increasing local antigen presentation to T cells; and, third, that the numbers of mast cells are selectively increased in the nasal epithelium of patients with perennial allergic rhinitis, as we previously showed to be the case in seasonal allergic rhinitis. Finally, we hypothesized that the symptoms of perennial allergic rhinitis arise in part through expression of Th2-type cytokines in the nasal mucosa, with IL-4 and IL-5 driving IgE synthesis and tissue eosinophilia, respectively. Nasal biopsies were collected from patients with symptomatic perennial allergic rhinitis and a group of normal control subjects. Cell populations in the mucosa were quantified by immunocytochemistry, and the numbers of IL-5, IL-4, interferon-gamma (IFN-γ), and IL-2 mRNA-expressing cells were determined by in situ hybridization with specific riboprobes.
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- Material and methods
This study represents the first systematic comparison of both cell populations and cytokine mRNA expression in perennial allergic rhinitis patients and normal controls. We report that perennial allergic rhinitis is associated with significantly increased numbers of submucosal CD3+ T cells, CD68+ macrophages, and EG2+ activated eosinophils compared to normal controls. Recruitment of T cells and activated eosinophils was restricted to the submucosa, since cell numbers within the epithelium were equivalent in rhinitic and control groups. For tryptase-positive mast cells, the opposite pattern was observed; i.e., higher numbers of epithelial, but not submucosal, mast cells were evident in rhinitics than normal controls. Finally, we report that perennial allergic rhinitis is associated with increased numbers of nasal mucosal cells expressing mRNA encoding the proeosinophilic cytokine IL-5, although, surprisingly, we did not find evidence of increased IL-4 expression in the same patients. Although the numbers of IFN-γ-expressing cells were also statistically equivalent in both groups, the numbers of IL-2 mRNA-expressing cells were lower in the rhinitic group.
Evidence of T-cell recruitment to the nasal mucosa in allergic rhinitis is controversial. In nasal biopsies collected from seasonal rhinitis patients before and during the grass-pollen season, natural allergen exposure was not accompanied by a significant increase in numbers of either CD3+ or CD4+ T cells ( 10). Similarly, no increases were observed in numbers of submucosal CD3+ T cells after allergen challenge, although CD4+ numbers did increase ( 1). Nevertheless, our present data agree with those of Calderón et al. ( 16), who also found increased numbers of submucosal CD3+ T cells in perennial allergic rhinitis. These findings suggest that the extent of CD3+ T-cell infiltration in allergic rhinitis might depend on the chronicity of allergen exposure as well as the severity of disease.
Our demonstration of increased numbers of submucosal CD68+ macrophages in perennial allergic rhinitis is novel and is supported by studies performed on nasal biopsies ( 17) and nasal scrapings ( 18) from patients with seasonal allergic rhinitis. However, the exact role of macrophages in the allergic response is unknown. The recent demonstration of high-affinity IgE receptor alpha subunit (FcεRIα) expression by these cells ( 19) suggests that IgE-mediated antigen processing by nasal mucosal macrophages may play a role in driving local T-cell activation and cytokine production in response to inhaled allergen.
Local tissue eosinophilia and associated Th2-type cytokine expression are well established features of cutaneous ( 20) and nasal late-phase responses to allergen ( 2). Like the mast cell, the eosinophil is a source of proinflammatory lipid mediators and cytokines, and is considered to be an effector cell in allergic inflammation. The demonstration of eosinophil cationic protein in nasal lavage fluid after local allergen provocation is consistent with eosinophil activation and degranulation occurring within the nasal mucosa of rhinitics (21, 22). We have extended these observations to show that perennial allergic rhinitis caused by natural exposure to allergen, is also, like seasonal allergic rhinitis ( 10), characterized by significant recruitment of activated eosinophils into the nasal submucosa. However, activated eosinophil cell numbers in the nasal epithelium were not significantly different between perennial allergic rhinitics and normal controls. Although we also observed a trend for increases in total eosinophil numbers in the nasal submucosa of perennial allergic rhinitics, it only approached statistical significance (P=0.06). In contrast to eosinophils, mast cells were selectively recruited to the nasal epithelium in perennial allergic rhinitis patients. These data are consistent with previous studies comparing normal controls and patients with seasonal ( 10) or perennial allergic rhinitis (13, 23) induced by natural allergen exposure, and suggest that during chronic allergen exposure, mucosal-type mast cells are induced either to migrate from the submucosa into the epithelium, or undergo proliferation ( 24) or maturation from precursors in situ. These findings are also consistent with a recent study suggesting that nasal mast cells undergo functional and phenotypic changes suggestive of activation in perennial allergic rhinitis ( 25).
The numbers of nasal mucosal cells expressing mRNA for the Th2-type cytokine IL-5 were significantly elevated in the perennial allergic rhinitics compared to normal controls. These data are consistent with previous reports of the upregulation of IL-5 mRNA expression in rhinitis induced by nasal allergen provocation ( 2) or seasonal exposure to grass pollen ( 12). Although we did not colocalize cytokine mRNA to different cell types, our previous studies in the nose (12, 26) and lung ( 27) suggest that most IL-5 mRNA-expressing cells in allergic tissue reactions are CD3+ T cells, with lesser contributions from mast cells and eosinophils. Previous studies have also demonstrated increased IL-4 mRNA expression after allergen challenge ( 2) or during the pollen season ( 11), although we were unable to demonstrate increased numbers of IL-4 mRNA-expressing cells in the nasal mucosa in perennial allergic rhinitis. One explanation for this may be that we selected patients with relatively mild disease, as reflected by a modest medium nasal symptom score of 4.75 out of the maximum score of 12. It is also possible that the pattern of cytokine expression in perennial allergic rhinitis differs from that observed after more acute exposure to allergen (such as an artificial challenge or seasonal exposure) with marked upregulation of IL-5, but not IL-4, mRNA expression. Thus, IL-4 mRNA expression could transiently increase after allergen exposure and play a role in inducing short-term symptoms.
Bradding et al. examined IL-4, IL-5, and IL-6 immunoreactivity in nasal biopsies and reported a significant increase in IL-4 immunostaining in perennial allergic rhinitis patients ( 13). Increases in IL-4, IL-5, and IL-6 immunoreactivity in nasal biopsies of perennial allergic rhinitics have also been reported by Saito et al. ( 14). However, immunostaining biopsy sections with monoclonal antibodies does have the disadvantage of preferentially identifying preformed and stored cytokine protein within granular cells (28, 29).
In contrast, in situ hybridization identifies all cells actively expressing cytokine mRNA, including T cells, which secrete but do not store cytokines and are therefore not detectable by immunostaining techniques.
Cell numbers expressing the archetypal Th1-cytokines IFN-γ and IL-2 were lower in the perennial allergic rhinitis group than in normal controls. For IL-2 mRNA expression, the difference between the two groups was significantly different. In vitro studies in human subjects previously showed reduced secretion of IL-2 by Th2-type allergen-specific ( 30) T-cell clones, and in vivo studies demonstrated preferential expression of IL-2 mRNA in tuberculin-driven delayed-type hypersensitivity, but not allergen-driven late-phase reactions ( 31). Therefore, these data support the conclusion that in perennial allergic rhinitis there is an imbalance in T-cell cytokine production in the nasal mucosa, in favour of a Th2-type response.
In summary, we have shown that perennial allergic rhinitis is associated with submucosal infiltration by T cells, activated eosinophils, and macrophages, and infiltration of the epithelium by mast cells. Mast cells represent an alternative source of preformed cytokines, including IL-4 and IL-5, which may be released immediately after allergen challenge. Production of Th2-type cytokines by T helper cells within the nasal mucosa is likely to represent a more sustained source of chronic inflammatory changes in perennial allergic rhinitis.