Regulation of Iron Metabolism in Murine J774 Macrophages: Role of Nitric Oxide–Dependent and –Independent Pathways Following Activation With Gamma Interferon and Lipopolysaccharide

Abstract

To elucidate the pathways by which nitric oxide (NO) influences macrophage iron metabolism, the uptake, release, and intracellular distribution of iron in the murine macrophage cell line J774 has been investigated, together with transferrin receptor (TfR) expression and iron-regulatory protein (IRP1 and IRP2) activity. Stimulation of macrophages with interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS) decreased Fe uptake from transferrin (Tf), and there was a concomitant downregulation of TfR expression. These effects were mediated by NO-dependent and NO-independent mechanisms. Addition of the NO synthase (NOS) inhibitor N-monomethyl arginine (NMMA) partially restored Fe uptake but either had no effect on or downregulated TfR expression, which suggests that NO by itself is able to affect iron availability. Analysis of the intracellular distribution of incorporated iron revealed that in IFN-gamma/LPS-activated macrophages there was a decreased amount and proportion of ferritin-bound iron and a compensatory increase in insoluble iron, which probably consists mainly of iron bound to intracellular organelles. Finally, although NO released by IFN-gamma/LPS-activated macrophages increased the iron-responsive element (IRE)-binding activity of both IRP1 and IRP2, IFN-gamma treatment decreased IRP2 activity in an NO-independent manner. This study demonstrates that the effect of IFN-gamma and/or LPS on macrophage iron metabolism is complex, and is not entirely due to either NO-or to IRP-mediated mechanisms. The overall effect is to decrease iron uptake, but not its utilization.