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Nramp transfection transfers Ity/Lsh/Bcg-related pleiotropic effects on macrophage activation: influence on oxidative
C H Barton1, S H Whitehead, J M Blackwell
1University of Cambridge Clinical School, Department of Medicine, Addenbrooke's Hospital, United Kingdom.
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.
Materials And 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.
Insights
The Natural resistance-associated macrophage protein (Nramp) gene is confirmed as the Ity/Lsh/Bcg gene. This finding links macrophage antimicrobial and tumoricidal activity to mitochondrial oxygen radical signaling pathways.
Area of Science:
- Immunology
- Genetics
- Cell Biology
Background:
- The Ity/Lsh/Bcg gene on mouse chromosome 1 influences macrophage antimicrobial and tumoricidal activity.
- A candidate gene, Natural resistance-associated macrophage protein (Nramp), has been identified in macrophages.
Purpose of the Study:
- To test the hypothesis that the Nramp gene is equivalent to the Ity/Lsh/Bcg gene.
- To investigate the role of Nramp in macrophage activation.
Main Methods:
- In vitro transfection of the Nramp resistant allele into RAW 264.7 macrophage cell line.
- Monitoring transgene expression using allele-specific oligonucleotide hybridization.
- Assessing macrophage activation phenotypes: respiratory burst, nitrite release, and L-arginine uptake.
Main Results:
- Transgene expression correlated with lipopolysaccharide/interferon-gamma-regulated macrophage activation.
- Mitochondrial electron transport inhibitors affected respiratory burst and L-arginine flux.
- Mitochondrial-derived oxygen radicals are implicated in Nramp-regulated signaling.
Conclusions:
- Direct evidence confirms Nramp as the product of the Ity/Lsh/Bcg gene.
- Macrophage activation effects of Ity/Lsh/Bcg may stem from mitochondrial oxygen radical generation for intracellular signaling.