CR3 and Dectin-1 Collaborate in Macrophage Cytokine Response through Association on Lipid Rafts and Activation of Syk-JNK-AP-1 Pathway
Fig 2
Clustering of CR3 and Dectin-1 on lipid rafts is required for their collaboration in cytokine production and signaling activation.
(A and C) Macrophages were stimulated with or without (0 min) HK H. capsulatum for 15 or 30 min. Cells were fixed and stained for CR3 (red) and Dectin-1 (green) (A), or for p-Syk (green) (C). Cells were viewed under confocal microscope. Lipid raft was identified by staining with cholera toxin B (CTB) (violet). Nuclear compartment was stained by DAPI (blue). Arrowheads in the DIC/DAPI fields point to H. capsulatum yeasts. The intensity of different fluorochromes along the white arrow in the merged images is shown in the histogram on the right. (B) Macrophages from WT mice were stimulated with or without (control) HK H. capsulatum for 30 min. Cell lysates were subjected to sucrose gradient ultracentrifugation. The fractions were collected and subjected to Western blotting and probed with anti-CD11b, anti-Dectin-1 and anti-flotillin-1 antibodies. (D and E) Macrophages were untreated or treated with methyl-β-cyclodextrin (MβCD, 10 mM) for 30 min. To reconstitute cholesterol, MβCD-treated cells were cultured in medium containing water-soluble cholesterol (MβCD-CHO, 400 μg/ml) for 1 h. After washing, macrophages were stimulated with HK H. capsulatum for 6 h (D), or 30 min (E). The concentrations of TNF and IL-6 in culture supernatants were analyzed by ELISA. Mean ± SD are shown (n = 10 from 4 independent experiments) (D). Cell lysates were analyzed by Western blotting by using antibodies against p-Syk, Syk, and β-actin. Data are representative of 2 (A and C) and 3 (B and E) independent experiments with similar results. ** p ≦ 0.01, *** p ≦ 0.001. NS, not significant [one-way ANOVA with Tukey post-hoc analysis (D)].