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 primi ng/activation of macrophages for antimicrobial and tumouricidal activi ty. A candidate gene expressed in macrophages has been identified by p ositional cloning and full-length sequence analysis, and encodes the N atural resistance-associated macrophage protein (nramp). In this study , we have tested the hypothesis that the Nramp gene corresponds to Ity /Lsh/Bcg. Materials and Methods: In vitro transfection was used to int roduce the resistant allele into the macrophage cell line RAW 264.7 de rived from the recessive susceptible BALB/c mouse strain. Expression o f the transgene was monitored on the background of the endogenous susc eptible allele by allele-specific oligonucleotide hybridization. Resul ts: Expression of the transgene correlated with three Lsh(r)-associate d lipopolysaccharide/interferon-gamma-regulated macrophage activation phenotypes: respiratory burst, nitrite release, and uptake of L-argini ne. Endogenous and stimulated L-arginine fluxes were inhibitable with the radical scavengers nordihydroguaiaretic acid and butylated hydroxy anisole. The mitochondrial electron transport inhibitors, rotenone and thenoyltrifluoroacetone, inhibited respiratory burst, and rotenone su ppressed L-arginine flux, implying that mitochondrial-derived oxygen r adicals are important mediators in Nramp-regulated signal transduction pathways. Conclusions: These data provide the first direct evidence t hat Nramp is the product of the Ity/Lsh/Bcg gene, and are consistent w ith the hypothesis that the many pleiotropic effects of this gene on m acrophage activation may all derive from the requirement for mitochond rial generation of oxygen radicals for intracellular signaling.