Dysfunction of the heme recycling system in heme oxygenase 1–deficient mice: effects on macrophage viability and tissue iron distribution

Abstract

To better understand the tissue iron overload and anemia previously reported in a human patient and mice that lack heme oxygenase-1 (HO-1), we studied iron distribution and pathology in HO-1(Hmox1) ؊/؊ mice. We found that resident splenic and liver macrophages were mostly absent in HO-1 ؊/؊ mice. Erythrophagocytosis caused the death of HO-1 ؊/؊ macrophages in in vitro experiments, supporting the hypothesis that HO-1 ؊/؊ macrophages died of exposure to heme released on erythrophagocytosis. Rupture of HO-1 ؊/؊ macrophages in vivo and release of nonmetabolized heme probably caused tissue inflammation. In the spleen, initial splenic enlargement progressed to red pulp fibrosis, atrophy, and functional hyposplenism in older mice, recapitulating the asplenia of an HO-1-deficient patient. We postulate that the failure of tissue macrophages to remove senescent erythrocytes led to intravascular hemolysis and increased expression of the heme and hemoglobin scavenger proteins, hemopexin and haptoglobin. Lack of macrophages expressing the haptoglobin receptor, CD163, diminished the ability of haptoglobin to neutralize circulating hemoglobin, and iron overload occurred in kidney proximal tubules, which were able to catabolize heme with HO-2. Thus, in HO-1 ؊/؊ mammals, the reduced function and viability of erythrophagocytosing macrophages are the main causes of tissue damage and iron redistribution. IntroductionHumans and mice contain 2 well-characterized heme oxygenase (HO) enzymes: HO-1, which is inducible, and HO-2, which is constitutively expressed in most tissues. 1,2 HO metabolizes heme and releases free iron, carbon monoxide, and biliverdin, which quickly undergoes conversion to bilirubin. Red blood cells (RBCs) contain very high concentrations of hemoglobin (Hb), 3,4 but HO allows efficient recycling of the iron that is bound to Hb molecules in RBCs. On phagocytosis of senescent RBCs, macrophages increase their expression of HO-1 to efficiently degrade heme, and iron returns to the circulation through the iron exporter ferroportin. 5 Excess free heme is highly toxic in the circulation, 4 and protective systems exist that enable animals to avoid toxicity caused by free heme and free Hb. Hemopexin (Hpx) is a heme-binding serum protein that scavenges free heme in the circulation, 6,7 and haptoglobin (Hp) binds free Hb,8,9 whereupon the Hb-Hp complex is endocytosed through the CD163 receptor 10 and is metabolized by macrophages.Previous work in mouse models has shown that the lack of both HOs is embryonically lethal, whereas work on an HO-1 Ϫ/Ϫ mouse model 11,12 and a single HO-1-deficient human patient revealed that both the HO-1 Ϫ/Ϫ mouse and the human patient were anemic. 13 However, splenomegaly was described in the mouse model, 11,12 whereas hyposplenia was present in the human patient. 14 Hepatic and renal iron overload was observed in the patient and in mouse models, but the mechanism for iron redistribution was not clear. HO-1 deficiency was discovered in a single patient with hemolytic anemia when his physicians ...

The Mononuclear Phagocyte System in Homeostasis and Disease: A Role for Heme Oxygenase-1

“…Hmox1 -/ -mice first exhibit splenomegaly, but as they age the relative spleen size diminishes and the red pulp is progressively replaced by fibrotic tissue resulting from progressive depletion of splenic macrophages. Therefore, the asplenia observed in the HO-1-deficient human could have been secondary to heme-toxicity mediated by ROS in the splenic microenvironment and depletion of intra-splenic macrophages that recycle heme after phagocytosis of senescent red blood cells (90). Collectively, these findings suggest that both the innate and adaptive immunity are strongly affected by HO-1 deficiency.…”

“…MP infiltrating sites of tissue injury robustly express HO-1 (48,97). Further, HO-1 deficiency in vivo leads to chronic inflammation, perturbed distribution and function of the MPS, and exaggerated inflammatory responses in disease models (65,83,90,131).…”

“…Investigations of MP biology in the context of heme recycling and HO-1 activity provide compelling evidence that protection from oxidative damage is only one of the modes by which HO-1 exerts functional effects on the immune response (20,24,90,174). The literature on the role of HO-1 in immune signaling pathways is complex because of the variety of experimental systems used, the ability of nearly all cells to make HO-1 under certain conditions, the dependence of certain observations on substrate availability, and because pharmacologic modulators of HO-1 expression are not entirely specific (105).…”

“…These results suggest that HO-1 deficiency potentiates the pro-inflammatory activation of stimulated MP. In both humans (187) and mice (90), the cytoarchitecture of the secondary lymphoid tissues is markedly abnormal. The two published reports of human HO-1 deficiency indicated that the patients were essentially asplenic (61,187).…”

Iron homeostasis: An anthropocentric perspective

“…To date, mammalian models lacking HRG-1 have not been reported, but suppression of HRG-1 in developing zebrafish results in developmental and erythropoietic defects (26). Heme degradation is catalyzed by the enzyme heme oxygenase 1 (HO-1), and the loss of HO-1 function causes macrophage death attributed to heme accumulation during erythrophagocytosis (27). An alternative mechanism for iron export from the phagolysosome may utilize DMT1 and its paralog Nramp1 transporting ferrous iron from the phagolysosome to the cytoplasm (28).…”