Nramp1 Functionality Increases Inducible Nitric Oxide Synthase Transcription Via Stimulation of IFN Regulatory Factor 1 Expression

Abstract

Natural-resistance associated macrophage protein 1 (Nramp1) encodes a transmembrane phagolysosomal protein exerting resistance toward infections with intracellular pathogens by a mechanism not fully elucidated so far. We used the murine macrophage cell line RAW264.7, stably transfected with functional (RAW-37) or nonfunctional (RAW-21) Nramp1, to study for differences in the expression of NO, a central antimicrobial effector molecule of macrophages. Following stimulation with IFN-γ and LPS, Nramp1-expressing cells exhibit higher enzymatic activity of inducible NO synthase (iNOS) and increased cytoplasmic iNOS mRNA levels than RAW-21 cells. Time-course experiments showed that iNOS-mRNA levels remain increased in RAW-37 cells after prolonged cytokine stimulation while they decrease in RAW-21 cells. Reporter gene assays with iNOS-promoter luciferase constructs demonstrated an increased and prolonged promoter activity in Nramp1-resistant vs susceptible cells. This was paralleled by increased IFN regulatory factor 1 (IRF-1) expression and binding affinity to the iNOS promoter in RAW-37 cells, which may be related to enhanced STAT-1 binding affinity in these cells. A point mutation within the IRF-1 binding site of the iNOS promoter abolished the differences in iNOS transcription between RAW-21 and RAW-37 cells. Cells carrying functional Nramp1 express increased amounts of NO, which may be related to STAT-1-mediated stimulation of IRF-1 expression with subsequent prolonged activation of iNOS transcription. Enhanced NO expression may partly underlie the protection against infection with intracellular pathogens by Nramp1 functionality.

Coexpression of NRAMP1, iNOS, and Nitrotyrosine in Bovine Tuberculosis

“…2 In this regard, it is interesting to mention a recent study describing upregulation of iNOS by NRAMP1 that is associated with increased expression of interferon regulatory factor 1 and augmented binding affinity to the iNOS promoter. 18 In conclusion, our current observations suggest that M. bovis infection promotes the expression of NRAMP1 and iNOS by bovine MØs. Evidence that this expression is particularly strong in the granuloma suggests that these molecules are involved in the host defense against mycobacteria in cattle TB.…”

“…3,4,35 In a recent study we demonstrated high expression of NRAMP1 in granulomas of M. bovis-infected cattle; 15 since granulomas are protective inflammatory reactions, 17,20,21 it was thought that NRAMP1 plays a defensive role against mycobacteria locally. This finding, in addition to recent studies showing a regulatory role for NRAMP1 in iNOS production, 2,18 prompted this study, which aims to analyze the expression of NRAMP1 and iNOS in M. bovis-infected cattle. In addition, we analyzed nitrotyrosine (NT) expression as an indication of iNOS enzymatic activity and NO production.…”

The kinase inhibitor imatinib mesylate inhibits TNF-α production in vitro and prevents TNF-dependent acute hepatic inflammation

“…The following oligonucleotides were used: NF-B sense, 5Ј-AGCTTCA-GAGGGGACTTTCCGAGAGG-3Ј and NF-B, antisense 5Ј-TCGACCTCTCGGAAAGTCCCCTCTGA-3Ј. Gel mobilityshift assay and supershift assays were performed as described (13). For supershift assays, 1 g of the appropriate anti-NF-B subunit antibody (p50, p65; Santa Cruz Biotechnology) was added.…”

Slc11a1 (formerly NRAMP1) gene modulates both acute inflammatory reactions and pristane-induced arthritis in mice

“…Slc11a1 is pleiotropic, interfering with macrophage activation, 17 oxidative and nitrosamine bursts, 18 TNFa, IFNg, and IL-1 production, 19 and the expression of MHC class II molecules. 20,21 The mutation corresponding to the Slc11a1 S allele determines a gly169asp substitution resulting in a nonfunctional protein 22 that promotes an accumulation of ions inside the phagosome that favors pathogen replication.…”