Figure 2. Complete depletion of surface IgE does not occur with desensitization.
(A) IgE-sensitized mice were desensitized (DS), challenged with antigen (Ag), or injected with PBS (IgE). At the end of desensitization, mice were sacrificed for peritoneal lavage. Lavage cells were incubated with A647-labeled TNP-OVA for 1 hour on ice, then washed 5 times with PBS before fixation and assay via FACS. Data were expressed as fold median fluorescence intensity (MFI) over IgE alone. Data are combined from 2 experiments (n = 10). (B) 5 × 105 IgE-sensitized BMMCs per sample in quintuplicate were desensitized (blue), Ag challenged (red), or untreated (green), then labeled with A647–TNP-OVA as in A. BMMCs without IgE were also incubated with labeled TNP-OVA as control (gray). A histogram representative of at least 3 experiments is shown. (C) 5 × 105 BMMCs per sample of IgE-alone (IgE), Ag-challenged (Ag), or desensitized (DS) cells were washed 3 times in PBS before resuspension in 500 μl RPMI without IgE. Zero to 72 hours later, cells were collected and challenged with 10 ng/ml Ag. Degranulation was measured using a β-hexosaminidase assay. (D) Control (IgE), Ag-challenged (Ag), or desensitized (DS) cells were incubated for 0 or 48 hours before being assayed for surface staining of IgE via FACS. Data are representative of at least 3 independent experiments. Data were analyzed via 1-way (A) or 2-way (C and D) ANOVA. Error bars indicate SEM. *P < 0.05, **P < 0.01, ***P < 0.001, #P < 0.001 compared with all other groups.