Redox-active metals in commercial preparations of lipopolysaccharide: implications for studies of cellular responses to bacterial products.
The mechanisms by which lipopolysaccharide (LPS) activates cells have been the subject of intense investigation for many years. Whereas much information on this process has been collected for mammalian species, little is known about the signalling path-ways operative in other animals. One general mode of cellular activation that has been recently pro-posed for pathways independent of the primary mammalian LPS receptor, CD14, involves reactive oxygen species (ROS) as intermediates in LPS-induced signalling pathways. Therefore, we used 2',7'-dichlorodihydrofluorescein, a fluorogenic probe of redox activity, to examine LPS-induced oxidative responses of a macrophage-like cell line from the rainbow trout, RTS11. Lipopolysaccharide dose-dependently increased oxidation of this probe by RTS11 cells, and a variety of other cell lines. This process was inhibited by catalase, superoxide dismutase and NG-methylarginine citrate, an inhibitor of nitric oxide synthases, suggesting the involvement of a diverse assortment of cellular ROS. More careful dissection of this phenomenon led us to conclude that the increase in oxidation was, in fact, due almost entirely to metals, particularly copper, in some LPS preparations, which is something to consider when experimenting with LPS.
Brubacher
John L
JL
Department of Biology, University of Waterloo, 200 University Ave W., Waterloo, ON, N2L 3G1, Canada.
DeWitte-Orr
Stephanie J
SJ
Zorzitto
Joanna R
JR
Playle
Richard C
RC
Bols
Niels C
NC
D000818:Animals; D001419:Bacteria / Q000378:metabolism*; D002374:Catalase / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System; D002614:Chelating Agents / Q000378:metabolism; D002951:Citrates / Q000378:metabolism; D003300:Copper / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000378:metabolism; D005452:Fluoresceins / Q000378:metabolism; D016166:Free Radical Scavengers / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008670:Metals / Q000378:metabolism*; D017686:Oncorhynchus mykiss; D016877:Oxidants / Q000378:metabolism; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D000077559:Sodium Citrate; D013482:Superoxide Dismutase / Q000378:metabolism
D002614:Chelating Agents; D002951:Citrates; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D016166:Free Radical Scavengers; D008070:Lipopolysaccharides; D008670:Metals; D016877:Oxidants; D010618:Phenanthrolines; D017382:Reactive Oxygen Species; C065013:2',7'-dichlorodihydrofluorescein; D000077559:Sodium Citrate; C028559:bathocuproine sulfonate; D003300:Copper; D006861:Hydrogen Peroxide; D002374:Catalase; D013482:Superoxide Dismutase