Fig 6.

Arg mediates amastigote engulfment. (A) Imatinib is not toxic to amastigotes. Shown is a representative experiment of two experiments following the number of amastigotes per ml in control, DMSO-treated, or imatinib-treated medium over 6 days. (B) Imatinib decreases amastigote uptake. Mϕs were pretreated with 3.3 μM imatinib or DMSO. Mouse anti-P8 IgG1 or freshly isolated mouse serum was used to opsonize amastigote surfaces with IgG1 or C3bi. Amastigotes were incubated with Mϕs for 90 min (unopsonized) or 20 min (opsonized). Two-color IF with anti-P8 distinguished internalized from external amastigotes. The graph shows the mean PI ± SE for imatinib-treated Mϕs, normalized to DMSO-treated Mϕs, of opsonized (+ C3bi, + IgG) and unopsonized (Amastigotes) L. amazonensis parasites. P = 0.0024 (**) for DMSO- versus imatinib-treated Mϕs for unopsonized amastigotes, P = 0.013 (*) for C3bi-opsonized amastigotes, and P = 0.0063 (**) for IgG-opsonized amastigotes by one-sample t test. (C) IgG1-opsonized amastigote uptake decreases in Mϕs lacking Abl and Arg. WTLM versus dKO Mϕs were incubated with IgG1-opsonized amastigotes for 20 min. The graph shows the mean PI ± SE by dKO Mϕs, normalized to WTLM Mϕs (labeled WT). *, P = 0.038 for WTLM versus dKO Mϕs by one-sample t test. (D) Arg mediates IgG-opsonized amastigote uptake. Bars show the mean PI ± SE of amastigotes by abl^−/− and arg^−/− Mϕs, normalized to WTLM (labeled “WT”). *, P = 0.034 for WTLM versus arg^−/− Mϕs by one-sample t test. (E) An FcRγIII-blocking antibody (F16/32) decreases amastigote uptake. RAW 264.7 cells were preincubated with F16/32 or M1/70 prior to incubation with IgG1-opsonized amastigotes. The graph shows the mean PI + SE for untreated, M1/70-treated, or F16/32-treated RAW 264.7 cells. **, P < 0.01 for F16/32-treated cells by one-way ANOVA (n = 3 experiments).