Method for Measuring Macrophage Iron Efflux in Vitro and in Vivo Using Magnetic Resonance Imaging

Anemia of inflammation (AI) is the disturbance of iron homeostasis manifest by a decrease in absorption of dietary iron from the gastrointestinal tract and by iron sequestration by reticuloendothelial macrophages. It is widely recognized that elevated levels of proinflammatory cytokines and other mediators from activated immune cells are the primary causes of AI. More specifically, it is the abnormally decreased rate of iron efflux from macrophages and enterocytes that appear to be the crucial pathophysiology. Increasingly, the central role of the liver-derived factor hepcidin is recognized in mediating the disturbances of iron absorption, sequestration and utilization that characterize AI. Hepcidin decreases iron absorption from the duodenal enterocytes and promotes the sequestration of iron in the reticuloendothelial macrophages by down regulating the transmembrane iron export protein, ferroportin. Thus the regulation of hepatic hepcidin expression becomes of paramount interest in the study of AI. We have shown that bone morphogenetic protein is critical for the regulation of hepcidin expression. The aim of our study is to develop a model to study iron efflux both in vitro and in vivo using magnetic resonance imaging (MRI). This was done by experimentally measuring spin-spin relaxation times (T2 measured in sec-1) through multi-echo, spin-echo technique (16 echoes) on a 4.7 T Bruker pharmacan experimental MRI imager. Iron loaded cells exhibit lower T2 values than normal control cells. In vitro, hepatomas Hep3B and macrophages J774 and Raw 264.7 were loaded with magnetic iron oxide nanoparticles (MION) overnight. The next morning (baseline), the intracellular and extracellular iron content was serially measured for up to 24 hours using MRI. As expected, both J774 and Raw 264.7 cells lines displayed lower intracellular T2 values in comparison to baselines. In addition, both ferroportin and the iron storage protein ferritin were modulated following overnight MION treatment and 24 hours post baseline. In contrast, Hep3B cells did not take up any significant MION following overnight treatment. In vivo, normal mice on a normal diet were given an intravenous MION injection dosed at 10mg/kg iron. MRI was performed at baseline, 24, 48, 72 and 96 hrs post injection. 24 hours post injection, T2 increased in both liver and spleen (liver > spleen). Following the 24 hour spike, the T2 values in the mice decreased for up to 72 hours after injection with a rate in the liver mildly increased relative to the rate of decrease in the spleen.