FcgammaRIIb inhibits allergic lung inflammation in a murine model of allergic asthma

Abstract

Allergic asthma is characterized by airway eosinophilia, increased mucin production and allergen-specific IgE. Fc gamma receptor IIb (FcgammaRIIb), an inhibitory IgG receptor, has recently emerged as a negative regulator of allergic diseases like anaphylaxis and allergic rhinitis. However, no studies to date have evaluated its role in allergic asthma. Our main objective was to study the role of FcgammaRIIb in allergic lung inflammation. We used a murine model of allergic airway inflammation. Inflammation was quantified by BAL inflammatory cells and airway mucin production. FcgammaRIIb expression was measured by qPCR and flow cytometry and the cytokines were quantified by ELISA. Compared to wild type animals, FcgammaRIIb deficient mice mount a vigorous allergic lung inflammation characterized by increased bronchoalveolar lavage fluid cellularity, eosinophilia and mucin content upon ragweed extract (RWE) challenge. RWE challenge in sensitized mice upregulated FcgammaRIIb in the lungs. Disruption of IFN-gamma gene abrogated this upregulation. Treatment of naïve mice with the Th1-inducing agent CpG DNA increased FcgammaRIIb expression in the lungs. Furthermore, treatment of sensitized mice with CpG DNA prior to RWE challenge induced greater upregulation of FcgammaRIIb than RWE challenge alone. These observations indicated that RWE challenge upregulated FcgammaRIIb in the lungs by IFN-gamma- and Th1-dependent mechanisms. RWE challenge upregulated FcgammaRIIb on pulmonary CD14+/MHC II+ mononuclear cells and CD11c+ cells. FcgammaRIIb deficient mice also exhibited an exaggerated RWE-specific IgE response upon sensitization when compared to wild type mice. We propose that FcgammaRIIb physiologically regulates allergic airway inflammation by two mechanisms: 1) allergen challenge mediates upregulation of FcgammaRIIb on pulmonary CD14+/MHC II+ mononuclear cells and CD11c+ cells by an IFN-gamma dependent mechanism; and 2) by attenuating the allergen specific IgE response during sensitization. Thus, stimulating FcgammaRIIb may be a therapeutic strategy in allergic airway disorders.

Figure 1. Role of FcγRIIb in allergic airway inflammation.

(A, B, C and D) Total inflammatory cells (A), eosinophils (B), macrophages (C) and lymphocytes (D) were quantified in BAL of C57Bl6 RWE-sensitized WT and FcγRIIb KO mice challenged with either PBS (WT PBS and KO PBS) or RWE (WT RWE and KO RWE). *, p<0.05.

Figure 2. Role of FcγRIIb in allergic airway inflammation.

(A, B and C) Total inflammatory cells (A), eosinophils (B) and lymphocytes (C) were quantified in BAL of Balb/c RWE-sensitized WT and FcγRIIb KO mice challenged with either PBS (WT PBS) or RWE (WT RWE and KO RWE). (D, E and F) Lung sections were obtained from RWE-sensitized WT and FcγRIIb KO mice challenged with either PBS (WT PBS) or RWE (WT RWE & KO RWE). These sections were stained with PAS to identify mucin containing cells. (G) Mucin containing cells in the lung sections were analyzed by morphometric analyses of PAS staining area. (H) Mucin was quantified in BAL samples by ELISA using biotinylated mucin binding lectin. (I) WT and FcγRIIb KO mice were sensitized with RWE and challenged with either PBS (WT PBS) or RWE (WT & KO RWE). PENH was measured by Buxco whole body plethysmography. *, p<0.05.

Figure 3. Expression of FcγRIIb in the lungs after RWE challenge.

(A) Balb/c mice sensitized with RWE and challenged with either RWE (filled squares) or PBS (open diamond). Mice were sacrificed 1, 4, 24, 72 and 240 h after challenge, lungs were collected and RNA was extracted. Quantitative PCR (qPCR) analysis for FcγRIIb was performed on these RNA samples using SYBR green Real time PCR kit (Applied biosystems). (B) Wild-type and INF-γ deficient BALB/c mice were sensitized with RWE, and challenged with PBS or RWE. 4 h later, the lungs were collected and qPCR for FcγRIIb was performed. (C) Naïve wild-type mice were challenged with PBS, CpG DNA or GpC DNA. 4 hours post-challenge lungs were collected and FcγRIIb expression was quantified by qPCR. (D) Wild-type BALB/c mice were sensitized with RWE. The mice were pre-treated with PBS (PBS challenge or RWE challenge group) or 35 µg CpG oligonucleotide intranasally (CpG → RWE) 48 h prior to RWE challenge. 4 h post-challenge, lungs were collected and qPCR for FcγRIIb was performed. * = p<0.05.

Figure 4. Identification of cells in the lungs that upregulate FcγRIIb after RWE challenge.

Single cell lung and spleen suspensions were prepared from RWE-sensitized BALB/c mice that were challenged with PBS or RWE. A multi-color FACS analysis for FcγRIIb and cell specific markers (CD14/MHC II for macrophages, CD11c for dendritic cells and B220 for B cells) was performed on these cells. FcγRIIb expression is shown for PBS challenged (grey histogram) and RWE challenged (black histogram) mice. FcγRIIb expression is increased on CD14+/MHC II+ and CD11c+ gated cells. Data from one representative animal in each group. MFI, Mean fluorescence intensity.

Figure 5. Role of FcγRIIb on serum IgE levels and antigen-induced Th2 cytokine production.

(A) RWE-specific IgE levels in serum were quantified in sensitized WT and FcγRIIb KO mice. (B, C and D) Splenocytes from sensitized wild-type and FcγRIIb KO mice were cultured with PBS or RWE for four days, and the cell supernatants were analyzed for IL-4, IL-5 and IL-13 levels by ELISA. *, P<0.05; NS, not significant.