Role of FcγRIIb in allergic airway inflammation.

<p>(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.</p

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

<p>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.</p

Primers used for quantitative PCR analyses.

<p>Primers used for quantitative PCR analyses.</p

Expression of FcγRIIb in the lungs after RWE challenge.

<p>(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.</p

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

<p>(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.</p

Role of FcγRIIb in allergic airway inflammation.

<p>(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.</p