Nramp transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on oxidative burst and nitric oxide pathways.
BACKGROUND
The Ity/Lsh/Bcg gene on mouse chromosome 1 regulates priming/activation of macrophages for antimicrobial and tumouricidal activity. A candidate gene expressed in macrophages has been identified by positional cloning and full-length sequence analysis, and encodes the Natural resistance-associated macrophage protein (Nramp). In this study, we have tested the hypothesis that the Nramp gene corresponds to Ity/Lsh/Bcg.


METHODS
In vitro transfection was used to introduce the resistant allele into the macrophage cell line RAW 264.7 derived from the recessive susceptible BALB/c mouse strain. Expression of the transgene was monitored on the background of the endogenous susceptible allele by allele-specific oligonucleotide hybridization.


RESULTS
Expression of the transgene correlated with three Lshr-associated lipopolysaccharide/interferon-gamma-regulated macrophage activation phenotypes: respiratory burst, nitrite release, and uptake of L-arginine. Endogenous and stimulated L-arginine fluxes were inhibitable with the radical scavengers nordihydroguaiaretic acid and butylated hydroxyanisole. The mitochondrial electron transport inhibitors, rotenone and thenoyltrifluoroacetone, inhibited respiratory burst, and rotenone suppressed L-arginine flux, implying that mitochondrial-derived oxygen radicals are important mediators in Nramp-regulated signal transduction pathways.


CONCLUSIONS
These data provide the first direct evidence that Nramp is the product of the Ity/Lsh/Bcg gene, and are consistent with the hypothesis that the many pleiotropic effects of this gene on macrophage activation may all derive from the requirement for mitochondrial generation of oxygen radicals for intracellular signaling.
University of Cambridge Clinical School, Department of Medicine, Addenbrooke's Hospital, United Kingdom.
C H
CH
Barton
S H
SH
Whitehead
J M
JM
Blackwell
D000818:Animals; D001120:Arginine / Q000378:metabolism; D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics* / Q000502:physiology; D027682:Cation Transport Proteins; D002460:Cell Line; D004579:Electron Transport / Q000187:drug effects; D016166:Free Radical Scavengers / Q000494:pharmacology; D007371:Interferon-gamma / Q000494:pharmacology; D033862:Iron-Binding Proteins; D008070:Lipopolysaccharides / Q000494:pharmacology; D008262:Macrophage Activation; D008264:Macrophages / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics* / Q000502:physiology; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009569:Nitric Oxide / Q000378:metabolism*; D009573:Nitrites / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D017382:Reactive Oxygen Species / Q000378:metabolism / Q000494:pharmacology; D016897:Respiratory Burst; D014162:Transfection; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2
D001120:Arginine; D002352:Carrier Proteins; D027682:Cation Transport Proteins; D016166:Free Radical Scavengers; D007371:Interferon-gamma; D033862:Iron-Binding Proteins; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D009569:Nitric Oxide; D009573:Nitrites; D017382:Reactive Oxygen Species; D000099173:Natural Resistance-associated Macrophage Protein 1; D000099236:Solute Carrier Family 11, Member 2