GM-CSF Stimulates IL-8 Expression in Human Bone Marrow Stromal Cells Via Activation of NADPH Oxidases.

The self-renewal, proliferation, and mobilization of hematopoietic progenitor cells (HPC) depend on complex interactions involving cytokines and bone marrow (BM) stromal microenvironment. Little is known about the effect of mobilizing agents such as granulocyte-macrophage colony-stimulating factor (GM-CSF) on BM stromal cells and their contribution to the mobilization of peripheral blood stem cells. In the present study we show that, 3 hours after addition, GM-CSF distinctly upregulated IL-8 mRNA expression and protein secretion in cultured human BM stromal cells. Furthermore, GM-CSF stimulated the generation of reactive oxygen species (ROS) in a dose- and time-dependent manner. Intracellular levels of ROS correlated with the extent of IL-8 production and IL-8 promotor activity as determined by luciferase reporter assay. Using western blot analysis, we showed that BM stromal cells expressed both phagocytic and non-phagocytic NAD(P)H oxidase components such as gp91phox, NOX1, p22phox, p47phox, p67phox, and NOXO1, suggesting that the upregulation of IL-8 expression induced by GM-CSF may be associated with the activation of stromal cell NADPH oxidase activity. GM-CSF directly stimulated the phosphorylation of p47phox, a key event in NAD(P)H oxidase activation. Inhibitors of the ERK1/2 and AKT pathways abrogated GM-CSF-induced phosphorylation of p47phox, indicating that GM-CSF-induced phosphorylation of p47phox is mediated by these pathways in BM stromal cells. Accordingly, the transactivation of IL-8 promotor activity induced by GM-CSF was suppressed by these inhibitors and the antioxidants diphenylene iodonium (DPI) and N-acetyl-l-cysteine (NAC). In conclusion, these findings show that GM-CSF elicits the activation of the redox-sensitive signaling via ROS, resulting in stromal cell function such as cytokine production and therefore possibly impacts on hematopoiesis.