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Background IL-9 is an important stimulus for tissue infiltration by mast cells, a feature requiring concomitant activation of c-Kit. Objectives We assessed IL-9 expression and c-Kit + mast cells in the nasal mucosa of patients with allergic rhinitis during seasonal pollen exposure and observed the effects of allergen immunotherapy. Methods We studied 44 patients with seasonal rhinitis and asthma before and 2 years after a double-blind trial of grass pollen immunotherapy. Nasal mucosal IL-9 + cells and c-Kit + mast cells were assessed by means of immunochemistry. Cell types expressing IL-9 protein were determined by means of dual immunofluorescence. IL-9 mRNA-positive cells were assessed by means of in situ hybridization, and their phenotype was determined by using sequential immunohistochemistry and in situ hybridization. Results Nasal mucosal c-Kit + mast cells were increased during the pollen season ( P = .0001). IL-9 mRNA-positive cells also tended to increase ( P = .1) and correlated with nasal EG2 + eosinophils ( r = 0.47, P = .05) and IL-5 mRNA-positive cells ( r = 0.54, P = .02). The cell sources of IL-9 included T cells, eosinophils, neutrophils, and mast cells. When compared with placebo, successful pollen immunotherapy markedly inhibited seasonal increases in nasal mucosal c-Kit + mast cells ( P = .001) and the seasonal expression of IL-9 mRNA-positive cells ( P = .06). Immunotherapy also inhibited IL-9 protein expression from nonendothelial cell sources ( P = .0007). Conclusion IL-9 is upregulated in the nasal mucosa during the pollen season and correlates with tissue infiltration by eosinophils. Successful pollen immunotherapy is associated with inhibition of seasonal increases in both nasal c-Kit + mast cells and eosinophils. This effect might be explained, at least in part, by the reduced local expression of IL-9.

Specific allergen injection immunotherapy is highly effective in IgE-mediated diseases, such as allergic rhinitis and venom anaphylaxis. Immunotherapy inhibits both early and late responses to allergen exposure. Immunotherapy is accompanied by increases in allergen-specific IgG, particularly the IgG4 isotype, which blocks not only IgE-dependent histamine release from basophils but also IgE-mediated antigen presentation to T cells. Immunotherapy acts on T cells to modify peripheral and mucosal T(H)2 responses to allergen in favor of T(H)1 responses. Recent studies have identified increased IL-10 production in peripheral blood and mucosal surfaces after immunotherapy. IL-10 has numerous potential antiallergic properties, including suppression of mast cell, eosinophil, and T-cell responses, as well as acting on B cells to favor heavy chain class switching to IgG4. These IL-10-producing cells might be so-called regulatory T cells and appear to be identified by the CD4(+)CD25(+) phenotype. Studies in mice suggest that dendritic cells play a vital role in induction of regulatory T cells. Novel approaches to immunotherapy currently being explored include the use of adjuvants, such as monophosphoryl lipid A or nucleotide immunostimulatory sequences derived from bacteria that potentiate T(H)1 responses. Alternative strategies include the use of allergen-derived peptides or modified recombinant allergen vaccines that act on T cells while minimizing the IgE-dependent mast cell activation that is dependent on the native allergen conformation.

We have previously shown increased expression of the CD4(+) cell chemoattractant IL-16 in bronchial mucosa of patients with asthma. We investigated the effects of allergen challenge on airway IL-16 expression.We investigated the expression of IL-16 immunoreactivity in bronchial biopsy samples obtained from atopic asthmatic subjects (n = 19) and normal subjects (n = 6) 24 hours after segmental allergen challenge. Control biopsy samples were obtained either at baseline or after diluent challenge. IL-16 expression was correlated to numbers of CD4(+) cells, CD25(+) cells, and activated eosinophils. IL-16 bioactivity was assessed in bronchoalveolar fluid obtained from patients with asthma.IL-16 expression was higher in control biopsy specimens obtained from subjects with asthma compared with normal subjects (P.05). In patients with asthma, numbers of IL-16 immunoreactive cells were significantly higher in biopsy specimens obtained after allergen challenge compared with control biopsy specimens (P.001). Allergen provocation was associated with release of IL-16 in bronchoalveolar fluid in patients with asthma. In normal subjects, there was no difference in the number of IL-16-immunoreactive cells in biopsy specimens obtained after allergen challenge compared with biopsy specimens obtained after diluent challenge. Allergen challenge was associated with an increase in the numbers of EG2(+) eosinophils in patients with asthma but not in normal subjects. IL-16 expression correlated with the numbers of CD4(+) cells and CD25(+) cells after allergen challenge in asthmatic subjects with a provocative concentration required to decrease the FEV(1) by 20% of its baseline value (PC(20)FEV(1))4 mg/mL. IL-16-immunoreactive cells were identified mainly as T cells and eosinophils in asthmatic subjects after allergen challenge.Endobronchial allergen provocation in atopic asthmatic patients resulted in increased airway expression of IL-16 and release of bioactive IL-16 in airways. IL-16 may contribute to the immunoregulation of the inflammatory infiltrate in the airways in response to antigen.

Background Regulatory T (Treg) cells play an important role in controlling allergic inflammation. The transcription factor Foxp3 regulates the development and function of natural and adaptive CD4 + CD25 + Treg cells. Objectives We sought to examine the effect of grass pollen injection immunotherapy on the numbers of Foxp3 + CD4 + and Foxp3 + CD25 + T cells in and out of season and their expression of IL-10 in the nasal mucosa of patients with hay fever. Methods Nasal biopsy specimens were obtained from untreated patients with hay fever, participants with grass pollen allergy who had received 2 years of immunotherapy, and healthy control subjects. Dual-immunofluorescence microscopy was used to enumerate and colocalize Foxp3 expression to CD4 + and CD25 + T cells in the nasal mucosa. Triple staining was performed to colocalize Foxp3 + cells to CD3 + CD25 + and CD3 + IL-10–expressing cells. Results At peak season, numbers of Foxp3 + CD25 + ( P = .02) and Foxp3 + CD4 + ( P = .03) cells were significantly increased in the nasal mucosa of immunotherapy-treated patients compared with numbers before treatment. Foxp3 + CD25 + ( P = .03) and Foxp3 + CD4 + ( P = .04) cells were also greater in immunotherapy-treated patients out of season compared with those in untreated patients with hay fever. Within the immunotherapy-treated group, 20% of CD3 + CD25 + cells expressed Foxp3, and 18% of Foxp3 + CD3 + cells were IL-10 positive. Conclusion The presence of local Foxp3 + CD25 + CD3 + cells in the nasal mucosa, their increased numbers after immunotherapy, and their association with clinical efficacy and suppression of seasonal allergic inflammation support a putative role for Treg cells in the induction of allergen-specific tolerance in human subjects.

Rationale In murine studies IL-9 is involved in mast cell infiltration of tissues in a c-Kit dependent fashion and stimulates eosinophil development via upregulation of IL-5 receptor. IL-9 is increased in the bronchial mucosa in asthma (Shimbara et al, JACI 1999; 105:108). Grass pollen immunotherapy was effective in hayfever and inhibited seasonal increases in eosinophils in the nasal mucosa (Wilson et al Clin Exp Allergy; 2001: 1705). In the same biopsies we measured IL-9 expression before/after immunotherapy and related IL-9 to seasonal changes in local mast cells and eosinophils. Methods In a randomized blinded trial in 44 adults with seasonal rhinitis, nasal biopsies were taken from the inferior turbinate before treatment and during the pollen season after 2 years of treatment. Biopsies were processed by in situ hybridization to detect IL-9 mRNA+ cells and immunohistochemistry to detect IL-9 protein+ cells, eosinophils and mast cells. Results Seasonal increases in nasal mucosal IL-9 mRNA+ cells and IL-9 protein+ cells were inhibited after immunotherapy (between group differences p=0.06 and p=0.04, respectively). Seasonal increases in nasal c-Kit+ mast cells and EG2+ eosinophils were similarly inhibited (p=0.001 and p=0.03). In the placebo-treated group, the number of IL-9 mRNA+ cells correlated with IL-9 protein+ cells (r=0.47, p=0.06). There were also significant correlations (both groups combined) between IL-9 mRNA + cells and EG2 + cells (r=0.46, p=0.004) and IL-5 mRNA+ cells (r=0.41, p=0.01). Conclusions Local expression of IL-9 in the nasal mucosa during natural seasonal pollen exposure may contribute to local eosinophil (and possibly mast cell) accumulation and is inhibited following successful immunotherapy.

Rationale Grass pollen injection immunotherapy (IT) is associated with a suppressive response mediated by IL-10 and TGF-β, inducing respectively IgG4 and IgA production. We assessed the IgA response in patients with severe seasonal rhinitis during a double-blind, placebo-controlled trial of grass pollen IT, and related the findings to TGF-β expression in the nasal mucosa. Methods Serum concentrations of IgA1, IgA2 and joining chain-containing IgA antibodies to grass pollen (GP extract or Phl p 5 ) were measured by ELISA using specific monoclonal antibodies (and expressed as arbitrary units) before treatment and after 2 years of IT. TGF-β mRNA was assessed by in situ hybridization in nasal biopsies taken before and after 2 year IT. Results Serum IgA2 antibodies to GP were significantly increased after 2 years of IT as compared with placebo (median: 35.65, IQ range: 29.4-49.9 versus 8.15, 1-17.78, p U -test), as also observed for IgA2 to Phl p 5 . In contrast, IgA1 antibodies to GP were increased to a much lower extent and only at the peak pollen season. Mucosal-derived, joining chain-containing IgA to Phl p 5 was significantly increased after 2 years of IT as compared with placebo (108, 94.11-151.33 versus 80.22, 54.39-116.47, p=0.02). Moreover, levels of serum IgA2 antibodies to Phl p 5 significantly correlated with TGF-β expression in the nasal mucosa (p=0.01, r=0.60). Conclusions The IgA response to grass pollen immunotherapy is mostly of the IgA2 subclass, and grass pollen-specific serum IgA2 antibodies could be, at least partly, produced within the nasal mucosa and associated with local TGF-β expression.