ABSTRACT
Previous studies have shown that apolipoprotein E (apoE) plays a role in immune function by modulating tissue redox balance. Using a mouse macrophage cell line (RAW 264.7), we have examined the mechanism by which apoE regulates nitric oxide (NO) production in macrophages. ApoE potentiates NO production in immune activated RAW cells in combination with lipopolysaccharide or polyinosinic:polycytidylic acid (PIC), agents known to induce expression of inducible nitric oxide synthase mRNA and protein. The effect is not observed with apolipoprotein B or heat-inactivated apoE. The combination of PIC plus apoE produced more NO than the level expected from an additive effect of PIC and apoE alone. Furthermore, this increase was observed at submaximal extracellular arginine concentrations, suggesting that apoE altered arginine (substrate) availability. Examination of [(3)H]arginine uptake across the cell membrane demonstrated that arginine uptake was increased by PIC but further increased by PIC plus apoE. Treatment of RAW cells with apoE was associated with an increased apparent V(max) and decreased affinity for arginine as well as a switch in the induction of mRNA for subtypes of cationic amino acid transporters (CAT). Treatment of RAW cells with PIC plus apoE resulted in the loss of detectable CAT1 mRNA and expression of CAT2 mRNA. Regulation of arginine availability is a novel action of apoE on the regulation of macrophage function and the immune response.
Subject(s)
Apolipoproteins E/pharmacology , Arginine/metabolism , Macrophages/drug effects , Nitric Oxide/biosynthesis , Animals , Apolipoproteins B/pharmacology , Arginine/pharmacology , Biological Transport/drug effects , Cell Line , Dose-Response Relationship, Drug , Drug Synergism , Interferon-gamma , Lipopolysaccharides , Macrophages/immunology , Macrophages/metabolism , Mice , Nitric Oxide Synthase/biosynthesis , Nitric Oxide Synthase Type II , Poly I-C , RNA, Messenger/biosynthesis , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Induction of oxidative stress has been implicated as a causative factor in fetal alcohol syndrome although the source of reactive oxygen species is not clear. One potential source is the microglia, the CNS macrophage, which generate superoxide anion as part of their normal immune function. Our data indicate that chronic exposure to ethanol alters the function of cultured neonatal hamster microglia by inducing superoxide anion production in resting (nonstimulated) cells. An increase in superoxide anion was seen at 24 or 48 hr of ethanol treatment but was not seen during acute exposures of up to 3 hr. This effect was dose dependent and was maximal at 20 mM ethanol. Treatment with ethanol did not directly activate the respiratory burst seen in microglia and did not act as a priming agent to enhance phorbol-ester-stimulated superoxide anion production. Lipopolysaccharide-mediated priming of microglial superoxide anion production was also not affected by exposure to 20 mM ethanol for 24 hr. Ethanol treatment, however, did depress nitric oxide (NO) levels in hamster microglia which had been stimulated to produce NO by polyinosinic:polycytidylic acid (poly I:C). Uptake of latex beads was increased by 24 hr of exposure to ethanol. The overall action of ethanol on neonatal hamster microglia is to shift the balance between the production of superoxide anion and NO. Because NO is protective to mammalian cells, these changes predict that oxidative stress in the CNS would be enhanced.
