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. 2012 Sep 4;109(38):15413–15418. doi: 10.1073/pnas.1204525109

Fig. 5.

Fig. 5.

TIPE2 controls the strength of phagocytosis through Rac. (A) BMDMs from WT and Tipe2−/− mice were incubated with or without apoptotic GFP thymocytes (GFP-Thy) for 30 min at a ratio of 1:5. One group of BMDMs was also pretreated with 5 μM Cytochalasin B (Cyto B) for 5 min as indicated. After incubation, cells were fixed, stained with an antibody to the macrophage marker F4/80, and analyzed by flow cytometry. Only cells in the macrophage gates set based on the FSC/SSC values are shown. The number in each contour plot represents the percentage of GFP+F4/80+ cells (macrophages with ingested thymocytes) in the gated area. Solid bar, WT BMDMs; open bar, Tipe2−/− BMDMs. Phagocytic index = the percentage of gated fluorescent macrophages × mean fluorescence intensity of gated macrophages. (B and C) WT (solid bars) and Tipe2−/− BMDMs (open bars) were fed with 2-μm fluorescent beads at the indicated ratio with or without Cyto B (B) or CFSE-labeled live or heat-killed L. monocytogenes (Lm) at a ratio of 1:10 (C) and incubated for 20 min. Phagocytic indexes were determined as in A. (D) WT and Tipe2−/− BMDMs were infected with retroviruses that carried either nerve growth factor receptor (NGFR) or NGFP plus TIPE2 cDNAs (TIPE2) as described in Methods. Phagocytic indexes were determined as in A. Only data from NGFR-positive macrophages are shown. (E) WT (solid bars) and Tipe2−/− BMDMs (open bars) were infected with retroviruses that carried NGFR or NGFP plus wild-type Rac1 (Rac1-WT), constitutively active Rac1 (Rac1-61L), or dominant-negative Rac1 (Rac1-17N) cDNAs. Phagocytic indexes were determined as in A. Only data from NGFR-positive macrophages are shown. (F) WT (solid bars) and Tipe2−/− BMDMs (open bars) were pretreated with Rac1 inhibitor NSC24766 at the indicated concentrations for 24 h and incubated with GFP-Thy for 30 min at a ratio of 1:5. Phagocytosis index was determined as in A. (G) WT (solid bars) and Tipe2−/− BMDMs (open bars) were treated with LPS (100 ng/mL) for the indicated times. After the treatment, cells were subjected to the phagocytosis assay as in A. The fold increase in the phagocytic index is shown, with the value of the untreated cells set as 1. Results shown are means ± SEM. Statistics were performed on pooled data from three independent experiments. **P < 0.01.