Effect of JNK inhibitor on release of KC, JNK activity and intramacrophagic parasite growth.

<p>(A) Resident macrophages were infected or not with <i>L. major</i> in the presence of solvent or the indicated MAPK inhibitors. The levels of KC were determined by ELISA after 20 h of infection. (B) Macrophages were infected or not in the presence of solvent or JNK inhibitor SP600125. After 4 h, the levels of JNK and p-JNK were determined by western blotting. (C) Macrophages were infected overnight and cultured for additional 3 d in the presence of solvent or MAPK inhibitors. Intracellular load of parasites was evaluated. Results are mean and SE of the number of extracellular promastigotes produced. *<i>P</i><0.05, compared to treatment with solvent.</p

Infection with <i>L. major</i> increases FasL expression, but does not induce cell death.

<p>(A) Resident B6 macrophages, either wild-type (wt) or FasL-deficient <i>gld</i>, were infected or not for 20 h. Monolayers were detached and stained for FCM. Gated populations comprise F4/80⁺ CD11b⁺7AAD⁻ viable macrophages. Results indicate contour plots of Annexin V versus FasL staining. Numbers indicate percentages of cells in each quadrant. (B) Levels of soluble FasL in the supernatants of either control or infected resident macrophages 28 h after infection. (C) Resident macrophages were infected with <i>L. major</i> and cultured for 48 h. Supernatants were assayed for lyzozyme activity. As a control, macrophages were treated with paraformaldehyde (PFA) before collecting the supernatant. Results are mean and SE of triplicate cultures. **<i>P</i><0.01.</p

Induction of cytokine and chemokine release by <i>L. major</i> infection.

<p>(A) Resident B6 macrophages were infected (closed bars) or not (open bars) for 20 h with <i>L. major</i>, and supernatants were probed with a mouse cytokine array. The intensity of the labeling for each cytokine/chemokine/mediator was quantitated and normalized as percentage of a positive control provided in the kit. Data indicate mean and SD of two independent arrays. Infected versus uninfected values were compared using non-parametric Mann-Whitney U-test. Cytokines showing a significant (P<0.05) increase following infection are indicated by an asterisk. (B) Supernatants were also probed by ELISA for IL-1β, KC, IL-6 and TNF-α, as indicated. **<i>P</i><0.01.</p

Effects of antioxidants on ROS generation, JNK activation, KC release, and intramacrophagic parasite growth.

<p>(A, B) Macrophages were loaded with DCFH-DA, washed and infected or not with <i>L. major</i> for 4 h in the presence of medium, antioxidants DFO (A), or NAC (B). Results indicate arbitrary units of fluorescence and are mean and SE of triplicates. (C, D) Macrophages were infected or not in the presence of medium, DFO (C), or NAC (D). After 4 h, the levels of JNK and p-JNK were determined by western blotting. (E, F) Macrophages were infected or not in the presence of medium, DFO (E), or NAC (F). The levels of KC were determined by ELISA after 20 h of infection. (G) Macrophages were infected overnight and cultured for additional 3 d in the presence of medium, DFO or NAC. Intracellular load of parasites was evaluated. Results are mean and SE of extracellular promastigotes produced. *<i>P</i><0.05, **<i>P</i><0.01.</p