Fig 5.

Abl mediates promastigote engulfment. (A) Time course for cultured promastigote uptake. Promastigotes were incubated with primary Mϕs for the time indicated, and the PI was calculated. Shown is the absolute PI for a representative experiment of two experiments. (B) The effect of C3bi opsonization on uptake decreases over time. Shown is the PI ± SE of cultured and opsonized promastigotes over time, normalized to the PI of cultured promastigotes at 20 min, from a representative experiment of 2. (C) Imatinib decreases uptake of L. amazonensis promastigotes. Mϕs were treated with 3.3 μM imatinib or DMSO. Promastigotes were grown in culture (Promastigotes) or preincubated with mouse serum (+C3bi) or mouse IgG1 anti-gp46 (+IgG) and incubated with Mϕs for 20 min (+C3bi, +IgG) or 90 min (Promastigotes). An antibody to gp46 differentiated internalized (green) from external (orange) promastigotes. The graph shows the average PI ± SE for imatinib-treated Mϕs for cultured promastigotes, C3bi-opsonized promastigotes, and IgG-opsonized promastigotes, each normalized to their respective DMSO-treated Mϕs for 3 experiments. *, P = 0.024 for uptake by DMSO- versus imatinib-treated Mϕs for cultured promastigotes, P = 0.011 for C3bi-opsonized promastigotes, and P = 0.032 for IgG-opsonized promastigotes by one-sample t test. (D) Imatinib continues to decrease parasite uptake even after 3 h. The graph shows the average PI ± SE for imatinib-treated Mϕs, normalized to their respective DMSO-treated Mϕs. *, P < 0.05 by one-tailed t test (n = 3 experiments). (E) Imatinib is not toxic to promastigotes. Shown is a representative experiment of two experiments following promastigotes per ml of triplicate culture in control, DMSO-treated, or 3.3 μM imatinib-treated media over 3 days. (F) C3bi-opsonized promastigote uptake is decreased in Mϕs lacking Abl and Arg. WTLM versus dKO Mϕs were incubated with C3bi-opsonized promastigotes for 20 min. The graph shows the mean PI ± SE by dKO Mϕs, normalized to WTLM Mϕs (labeled WT). **, P = 0.0068 for WTLM versus dKO Mϕs by one-sample t test. (G) Abl mediates C3bi-opsonized promastigote uptake. The graph shows the mean PI ± SE by abl^−/− and arg^−/− Mϕs, normalized to WTLM Mϕs (labeled WT). **, P = 0.0046 for WTLM versus abl^−/− Mϕs by one-sample t test (n = 3 experiments). (H) A CR3-blocking antibody (M1/70) decreases promastigote uptake. RAW 264.7 cells were preincubated with M1/70 or F16/32 (an FcR blocking antibody; both rat IgG2) prior to incubation with C3bi-opsonized promastigotes. The graph shows the mean PI + SE for untreated, M1/70-treated, or F16/32-treated RAW 264.7 cells. **, P < 0.01 for M1/70-treated cells by one-way ANOVA (n = 3 experiments).