Nuclear translocation of NFκB and PPAR-γ activation in BMDMs primed with GM-CSF.

<p>NFκBp65, IκBα expression and NFκB nuclear translocation were investigated in BMDMs from WT mice. Graphic (<b>A</b>) percentage of relative expression of total NFκBp65, phosphorylated NFκBp65, (<b>B</b>) total IκBα and phosphorylated IκBα compared with non-stimulated control cell lysate (dotted line). Cells were pre-incubated for 24 h with GM-CSF, IFN-γ or GM-CSF plus IFN-γ and then stimulated with LM (5000 ng/mL) for 2 h <i>in vitro.</i> Total cellular proteins were collected to assay the relative expression by ELISA (PathScan Sandwich ELISA kit). The percentage of positive cells for NFkB nuclear translocation is shown for macrophages primed with GM-CSF for 24 h and then stimulated with different TLR2 agonists or LPS for 2 h, as analyzed by confocal microscopy. The <b>panel</b> depicts the expression of nuclear NFκB in GM-CSF primed BMDMs stimulated with medium (<b>vii</b>), AraLAM (<b>viii</b>), LM (<b>ix</b>), Malp2 (<b>x</b>), Pam₃-CSK₄ (<b>xi</b>) or LPS (<b>xii</b>) and the contrast DIC (i<b>, ii, iii, iv, v</b> and <b>vi</b>) on immunofluorescence slides. To evaluate the role of PPAR-γ in pro-inflammatory mediator release, WT primary macrophages were treated with 1 µM of PPAR-γ antagonist (GW 9662) 1 h before stimulus. TNF-α (<b>C</b>) and PGE₂ production (<b>D</b>) after 24 h of incubation were measured directly in the supernatant by ELISA. The graphic results are the average ± S.E.M for cells (<i>n</i> = 6 per group) from two independent experiments; *, p<0.05 compared with unstimulated cells. The images from confocal microscopy were representatives from two independent experiments.