Figure 2. CAR4 protein expression on lung eosinophils during allergic inflammation.

A, Eosinophils (~10⁶) are present in the airway in the mold allergen (Aspergillus fumigatus)–induced experimental asthma murine model. A FACS serial gating strategy identifies bronchoalveolar (BALF) and lung tissue eosinophils (EOS) as SSC^highSiglec-F⁺CD11b⁺GR-1⁺CD11c⁻ events with > 99% purity confirmed by sorting. B, Following allergen challenge, CAR4 surface expression (αCAR4) on Car4^+/+ (red histogram) and Car4^−/− (blue histogram) blood and BALF eosinophils are illustrated after serial gating (solid grey, IgG control). Representative FACS histograms of at least 5 independent experiments are shown. C, Following allergen challenge, similar CAR4 expression pattern is shown on lung tissue eosinophils (gated) acquired after tissue digestion and anti-CAR4 staining (αCAR4, solid grey, wild-type [WT]; red, CD2-IL5 transgenic mice [IL5 Tg]). D, With experimental asthma induced in CC10-IL13 transgenic mice and driven by 4 weeks of doxycycline exposure, CAR4 surface expression on BALF eosinophils was assessed (red) in reference to IgG staining (solid grey). E, Flow-imaging (Imagestreams) micrographs of the surface expression of CAR4 together with eosinophil markers. F, With Aspergillus challenges, BALF cells were FACS sorted for eosinophils by the aforementioned gating strategy. CAR4⁺ (red histogram) and CAR4⁻ (blue histogram) eosinophils were back-run by FACS to confirm sorting purity. Car4 quantitative PCR was performed with RNA extracted from sorted CAR4⁺ and CAR4⁻ eosinophils, which were also used to prepare a cytospin slide for morphological evaluation under 400× microscopy (25-μm bar superimposed). (***, p < 0.001, two-tailed student t-test, mean ± SEM)