Fig. 1. Cholesterol depletion inhibits the ability of mouse macrophages to phagocytose P. gingivalis.

(A) J774A.1 macrophages were pretreated for 30 min with 10 mM MCD to deplete cholesterol, or were pretreated for 30 min with 10 mM MCD followed by addition of 1 mM cholesterol for an additional 30 min. The MCD- and MCD/cholesterol-pretreated macrophages, as well as cells pretreated with medium only, were subsequently incubated for 30 min with FITC-labeled P. gingivalis (MOI = 10:1). Association (i.e., representing both adherence and phagocytosis) or phagocytic indices were determined by flow cytometry, as outlined in Experimental Procedures, using the formula (% positive cells × MFI)/100. (B) The adherence index was calculated as above with the exception that the cells were additionally treated with cytochalasin D (5 µg/ml) to prevent phagocytosis and allow for monitoring of adherence only. (C) Levels of cellular cholesterol in untreated and MCD-treated cells were quantified using the Amplex Red cholesterol assay kit. (D) Visualization of lipid rafts in untreated, MCD-treated, and cholesterol reconstituted MCD-treated cells by confocal microscopy using staining with Alexa Fluor 594-CTB followed by anti-CTB antibody to crosslink lipid rafts into distinct patches on the cell membrane. Numerical data are presented as means ± SDs (n = 3), from one of three independent sets of experiments that yielded similar results. Asterisks indicate statistically significant (p < 0.05) inhibition of indicated activity (A,B) or significant (p < 0.05) reduction of cholesterol content (C) in MCD-treated compared to untreated cells.