Erythrophagocytosis by Liver Macrophages (Kupffer Cells) Promotes Oxidative Stress, Inflammation, and Fibrosis in a Rabbit Model of Steatohepatitis

Abstract

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. Here, we report a molecular aspect of this disease elucidated using rabbits fed a cholesterol-rich high-fat diet and exhibiting insulin resistance. The liver in this model showed steatohepatitis with fibrosis and high mRNA expression for some cytokines, heme oxygenase-1, transforming growth factor-beta1, and collagen alpha1(I). Erythrocytes isolated from the model showed marked fragility and the externalization of phosphatidylserine (PS) on the outer leaflet of the membrane and were frequently engulfed by Kupffer cells/macrophages in the hepatic sinusoids. Expression of milk fat globule-epidermal growth factor (EGF)-factor 8, a PS-binding protein, was augmented in the liver. In culture, RAW 264.7 cells engulfed erythrocytes oxidized by tert-butyl hydroperoxide, a process that was inhibited by anti-milk fat globule-EGF-factor 8 antibody. In addition, PS-positive erythrocytes appeared entrapped in the model liver in ex vivo perfusion experiments. Finally, in specimens from NASH patients, the aggregation of erythrocytes in inflammatory hepatic sinusoids was notable. These results indicate that the engulfment of PS-externalized, apoptotic signal-positive, erythrocytes by hepatic macrophages may lead to the deposition of iron derived from hemoglobin in the liver and be involved in the pathogenesis of steatohepatitis.

Mechanisms Linking Glucose Homeostasis and Iron Metabolism Toward the Onset and Progression of Type 2 Diabetes

“…It is interesting that aggregates of erythrocytes were documented in microscopic areas of inflammation from liver biopsies of patients with NAFLD (34). Excess iron and erythrocytes engulfed by Kupffer cells were also reported in a rabbit model of familial hypercholesterolemia that exhibits steatohepatitis and fibrosis (34).…”

Dysregulation of iron and copper homeostasis in nonalcoholic fatty liver

“…This was documented in rabbits on a highfat diet and the phagocytosis of fragile erythrocytes was observed in vitro. Accumulation of erythrocytes was microscopically detected in inflamed regions in human NAFLD [127] suggesting that uptake of hemeiron via erythrophagocytosis may contribute to NAFLD iron accumulation, then promoting oxidative stress and inflammation. Although cellular iron uptake via TfR1 is the most important route of iron uptake under physiological circumstances TfR1 appears not to be involved in excess iron uptake in NAFLD [128,129] .…”

Reversibility of fibrosis, inflammation, and endoplasmic reticulum stress in the liver of rats fed a methionine–choline-deficient diet

“…For semi-quantitative analysis, the number of TUNEL-positive cells was counted in five randomly selected fields by viewing each slide at a magnification of  400, and the average number in each group was calculated, as described previously. 27 BrdU Assay for Hepatocyte Proliferation For the detection of hepatocyte proliferation, rats were i.p. injected with BrdU (100 mg/kg) 2 h before being killed.…”

“…29 The expression of mRNA was measured using TaqMan One-Step RT-PCR Master Mix Reagents (Applied Biosystems, Foster City, CA, USA) or using the One-Step SYBR RT-PCR Kit (Perfect Real Time; Takara Bio, Ohtsu, Japan), and Applied Biosystems Prism 7700 (Applied Biosystems) according to a previously reported procedure. 27 Primers and oligonucleotide probes were designed using Primer Express (Sigma Chemical), and are listed in Table 1. Each PCR amplification was performed on five rats in both experimental and control groups.…”