Abstract 11738: Novel Crosstalk of Toll-Like Receptor 2/6 and the NADPH Oxidase in Endothelial Cells: Implication for the Regulation of the Growth Factor GM-CSF
Background: Stimulation of Toll-like receptors (TLRs) by pathogen-associated molecular patterns leads to a direct activation of the NADPH oxidase (NOX) in immune cells in order to generate reactive oxygen species (ROS) for oxidative burst during pathogen defense. In contrast, NOX-dependent ROS-formation in vascular cells is crucial for intracellular signaling. However, little is known about an interaction of TLRs and NOX in endothelial cells and was therefore the aim of this study.
Methods and Results: Human endothelial cells (HUVEC, HUAEC, HMVEC) were stimulated with the TLR2/6 agonist MALP-2, a bacterial lipopeptid. We observed a fast ROS-induction (DCF-fluorescence, ESR-spectroscopy) and a strong gene induction and protein secretion of the granulocyte macrophage colony-stimulating factor (GM-CSF) (DNA/Protein-array, qRT-PCR, ELISA). As is known, GM-CSF is important for progenitor cell mobilization and differentiation into granulocyte and macrophage lineages. Accordingly, MALP-2 injection (i.p.) increased GM-CSF plasma level (ELISA) and subsequently progenitor cells in blood and spleen (CFU, FACS) and granulocytes in blood and peritoneum (FACS). ROS scavenger (Tempol, Tiron) as well as NOX inhibitors (DPI, apocynin, VAS2870) completely blocked GM-CSF release from cultured endothelial cells following MALP-2 stimulation (ELISA). Furthermore, explanted aortic rings from mice deficient for the NOX subunit p47phox showed significantly reduced GM-CSF release in response to MALP-2. Contrary, inhibition of other ROS-producing systems in endothelial cells (NO-synthase, xanthine oxidase, cyclooxygenase, mitochondria) did not show any effect on MALP-2-induced GM-CSF release underlining the importance of NOX in this context. Neither NOX activators (PMA, angiotensin II, thrombin) nor supply of intracellular radicals (Menadione, DMNQ, H2O2, SNAP) alone were able to induce GM-CSF. However, additional superoxide supply (Menadione, DMNQ) during MALP-2 stimulation had additive effects on GM-CSF secretion from endothelial cells.
Conclusion: Our findings identify a novel interaction of TLR2/6 and the NADPH oxidase for the induction of the growth factor GM-CSF in endothelial cells with potential implications for the immune response.
- Stem/progenitor cells
- Growth factors
- Reactive oxygen intermediates
- Free radicals/Free-radical scavengers
- © 2011 by American Heart Association, Inc.