Bone-marrow derived macrophages at the end of 6-day culture were detached, washed, and resuspended in Nucleofector solution of 2x10^6/100 µL containing nonspecific siRNA (0.5 µg), SHIP-1-specific siRNA (0.5 µg), WT-Akt (1 µg) or dominant-negative Akt (2 µg). Transfections were performed using program Y-001. Following the resting period, transfected cells were either pelleted, and lysates were prepared for Western blot analysis or LPS stimulation.
Transfection efficiency was determined using pmaxGFP vector and yielded in consistently effencies of >85 % using flow cytometric analysis 16 h post transfection.
The immunomodulatory effects of glucocorticoids (GCs) have been described as bimodal, with high levels of GCs exerting immunosuppressive effects and low doses of GCs being immunopermissive. While the mechanisms used by GCs to achieve immunosuppression have been investigated intensely, the molecular mechanisms underlying the permissive effects of GCs remain uncharacterized. Herein, we demonstrate that GC conditioning during the differentiation of myeloid progenitors into macrophages (Mphis) results in their enhanced LPS responsiveness, demonstrated by an overexpression of the inflammatory cytokines TNF-alpha, IL-6, and IL-12. Inflammatory cytokine overexpression resulted from an increased activation of NF-kappaB and the MAPK signaling cascade and a reduced activation of the PI3K-Akt pathway following LPS stimulation. GC conditioning during Mphi differentiation induced an increase in the expression of SHIP1, a phosphatase that negatively regulates the PI3K signaling pathway. Small interfering RNA-mediated knockdown of SHIP1 expression increased PI3K-dependent Akt activation and subsequently decreased inflammatory cytokine expression, suggesting GC-mediated up-regulation of SHIP1 expression is responsible for the augmentation in inflammatory cytokine production following LPS stimulation. We also show that splenic Mphis purified from normal mice that were implanted with timed-release GC pellets exhibited an enhanced LPS responsiveness and increased SHIP1 expression, indicating that GCs can regulate SHIP1 expression in vivo. Our results suggest that minor fluctuations in physiological levels of endogenous GCs can program endotoxin-responsive hemopoietic cells during their differentiation by regulating their sensitivity to stimulation.