Haptoglobin genotype-dependent differences in macrophage lysosomal oxidative injury.
The major function of the Haptoglobin (Hp) protein is to control trafficking of extracorpuscular hemoglobin (Hb) thru the macrophage CD163 receptor with degradation of the Hb in the lysosome. There is a common copy number polymorphism in the Hp gene (Hp 2 allele) that has been associated with a severalfold increased incidence of atherothrombosis in multiple longitudinal studies. Increased plaque oxidation and apoptotic markers have been observed in Hp 2-2 atherosclerotic plaques, but the mechanism responsible for this finding has not been determined. We proposed that the increased oxidative injury in Hp 2-2 plaques is due to an impaired processing of Hp 2-2-Hb complexes within macrophage lysosomes, thereby resulting in redox active iron accumulation, lysosomal membrane oxidative injury, and macrophage apoptosis. We sought to test this hypothesis in vitro using purified Hp-Hb complex and cells genetically manipulated to express CD163. CD163-mediated endocytosis and lysosomal degradation of Hp-Hb were decreased for Hp 2-2-Hb complexes. Confocal microscopy using lysotropic pH indicator dyes demonstrated that uptake of Hp 2-2-Hb complexes disrupted the lysosomal pH gradient. Cellular fractionation studies of lysosomes isolated from macrophages incubated with Hp 2-2-Hb complexes demonstrated increased lysosomal membrane oxidation and a loss of lysosomal membrane integrity leading to lysosomal enzyme leakage into the cytoplasm. Additionally, markers of apoptosis, DNA fragmentation, and active caspase 3 were increased in macrophages that had endocytosed Hp 2-2-Hb complexes. These data provide novel mechanistic insights into how the Hp genotype regulates lysosomal oxidative stress within macrophages after receptor-mediated endocytosis of Hb.
From the Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel and the Department of Cardiology and Coronary Care Unit, Rambam Health Care Center, Haifa 31096, Israel rasleh@tx.technion.ac.il.
Rabea
R
Asleh
From the Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel and.
John
J
Ward
From the Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel and.
Nina S
NS
Levy
From the Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel and.
Shady
S
Safuri
the Department of Cardiology and Coronary Care Unit, Rambam Health Care Center, Haifa 31096, Israel.
Doron
D
Aronson
From the Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa 31096, Israel and.
Andrew P
AP
Levy
D000818:Animals; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D005838:Genotype; D006242:Haptoglobins / Q000235:genetics*; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D018384:Oxidative Stress
D006242:Haptoglobins
Apoptosis; Atherosclerosis; Genetic Polymorphism; Hemoglobin; Lysosome; Macrophage; Oxidative Stress; α-Tocopherol