Figure 4

TgCtwh6 induced high levels of NO whereas Wh3-Mφ had enhanced arginase activity. (A) On day 3 postinfection with TgCtwh3 or TgCtWh6, mice were euthanized, and adherent peritoneal macrophages from harvested PECs were cultured for 12 h, then supernatants were collected and assayed for NO. (B) Arginase activity was measured in Wh3-Mφ or Wh6-Mφ lysates by the conversion of L-arginine to urea. Bars represent means ± SD from five independent experiments (^#p>0.05, ^*p<0.05, ^**p<0.01,^***p<0.001, Wh3-Mφ or Wh6-Mφ vs uninfected Mφ). (C) As in (B), infected BMMφs or RAW264.7 cells (2:1 ratio of tachyzoites to cells) were lyzed at 24 h postinfection, and arginase activity was measured (^#p>0.05, ^***p<0.001, TgCtwh3 or TgCtwh6 infected cells vs uninfected cells). (D) RNA was extracted from either TgCtwh3- or TgCtwh6-infected or uninfected BMMφs at 24 h postinfection, and mRNA was analyzed by real time-PCR. Housekeeping gene GAPDH allowed normalization of the expression of the interested genes. Relative expressions of iNOS, Ym1 and Arginase-1 were displayed. Bars represent means ± SD from three independent experiments (^#p>0.05, ^**p<0.01, ^***p<0.001, TgCtwh3- or TgCtwh6- infected cells vs uninfected cells). (E) Cell lysates collected from BMMφs infected with RH, PRU, TgCtwh3, TgCtwh6 or left uninfected at 24 h postinfection were subjected to Western blotting using antibodies to iNOS, Ym1 and Arginase-1. (F) A quantitative analysis of the Western blotting using densitometry (normalized to β-actin) is shown (^#p>0.05, ^*p<0.05, ^**p<0.01, ^***p<0.001, either RH- or PRU- or TgCtwh3- or TgCtwh6-infected cells vs uninfected cells). This experiment was repeated three times with similar results.