Zhao, G; Thomas, S; Kowalsky, G; Christman, JW; Qian, F; Levitan, I; Deng, J; Xiao, L
RATIONALE: Endotoxin LPS is recognized by Toll-like receptor 4 (TLR4) and is a potent activator of the mammalian immune system. It was reported that LPS stimulation induces generation of reactive oxygen species (ROS) in macrophages, and the ROS generated from NOX play an important role in host defense. However, the mechanism between the activations of TLR4 and NOX has not been clearly defined. Our aim was to investigate the role of LPS in NOX activation in BMDM.
METHODS AND RESULTS: Mouse bone marrow cells were cultured for 7 days in L929 cell-conditioned medium and then exposed to LPS (1 and 5 µg/ml) for up to 16 hr. Using real-time quantitative RT-PCR, we found that the mRNA expression of all major NOX subunits was significantly up-regulated after 16 hr of LPS treatment. The mRNA expression of gp91^phox, p67^phox, p47^phox, p40^phox and p22^phox was approximately increased by four-, two-, three-, two-, and two-fold, respectively. This increased NOX expression in BMDM was also confirmed by Western blot analysis, at 2, 6 and 16 hr after LPS stimulation. The protein expression of the above NOX subunits was markedly increased in a time-dependent manner. In contrast, LPS stimulation didn’t affect the expression of several other non-inducible proteins in BMDM. In addition, we found that LPS-induced up-regulation of NOX was prevented by the NOX inhibitor diphenyleneiodonium. Similarly, up-regulation of NOX expression was abolished in BMDM from the p47^phox knockout mice, suggesting that LPS-induced up-regulation of NOX subunits requires a functional NOX enzyme. We also measured ROS production using dihydroethidium staining or chemiluminescence methods with or without the ROS scavenger MnTMPyP. There was no significant difference in ROS generation in BMDM between the control and the LPS directly treated (1 hr to 16 hr) groups without LPS priming, which was also not affected by 50 µM MnTMPyP pretreatment, suggesting that the LPS-induced up-regulation of NOX expression is not ROS-dependent. In contrast, dramatically increased ROS generation was observed when using zymosan as a second stimulation after BMDM being first primed with LPS for 16h, suggesting that NOX activation requires additional stimuli such as zymosan.
CONCLUSION: Taken together, our data suggest that LPS treatment up-regulates the protein and mRNA expressions of all major subunits of NOX in BMDM, but may not directly trigger the generation of ROS; whereas LPS-primed BMDM may express higher level of NOX and thus are prone to ROS generation from NOX upon additional stimulations.
