Figure 2. Ara-LAM mediated effector function depends on the reciprocal activation of p38MAPK and ERK–1/2.

Peritoneal macrophages (2×10⁶cells/mL) were treated with SB203580 (SB) or PD098059 (PD) for 2 h, followed by Ara-LAM treatment for 3 hr. The cells were then infected with Leishmania parasite for 24 h and assayed for the levels of IL-12 (A) and IL-10 (C) in the culture supernatant by ELISA as described in Methods. ELISA data are expressed as means standard deviations of values from triplicate experiments that yielded similar observations. Macrophages cultured in a 24-well plate (1×10⁶ cells/mL) were pretreated and infected as described above and assayed for the levels of extracellular NO as described in the Materials and Methods (E). Asterisks indicate statistically significant induction of nitrite generation, compared with Ara-LAM–pretreated infected macrophages. *P<.001. From a separate set of cells (2×10⁶ cells/mL) RNA was isolated and levels of mRNA expression for IL-12p40 (B), IL-10 (D), inducible nitric oxide synthase 2 (iNOS2) (F) were determined by quantitative RT-PCR. Results are presented as changes (nfold) relative to uninfected control cells. The data represent the mean values± standard deviation of results from 3 independent experiments that all yielded similar results. In a separate experiment, the macrophages were cultured in coverglasses treated with SB or PD for 2 h, subsequently followed by 3 hr of Ara-LAM treatment and 4 h of Leishmania infection. After indicated time of incubation intracellular parasite number were assessed as described in methods. Pretreatment with SB significantly inhibited Ara-LAM –mediated parasite killing compared with levels in corresponding Ara-LAM pretreated infected controls (G). *P<.001 for SB.