Abstract
Diseases that cause hemolysis or myonecrosis lead to the leakage of large amounts of heme proteins. Free heme has proinflammatory and cytotoxic effects. Heme induces TLR4-dependent production of tumor necrosis factor (TNF), whereas heme cytotoxicity has been attributed to its ability to intercalate into cell membranes and cause oxidative stress. We show that heme caused early macrophage death characterized by the loss of plasma membrane integrity and morphologic features resembling necrosis. Heme-induced cell death required TNFR1 and TLR4/MyD88-dependent TNF production. Addition of TNF to Tlr4(-/-) or to Myd88(-/-) macrophages restored heme-induced cell death. The use of necrostatin-1, a selective inhibitor of receptor-interacting protein 1 (RIP1, also known as RIPK1), or cells deficient in Rip1 or Rip3 revealed a critical role for RIP proteins in heme-induced cell death. Serum, antioxidants, iron chelation, or inhibition of c-Jun N-terminal kinase (JNK) ameliorated heme-induced oxidative burst and blocked macrophage cell death. Macrophages from heme oxygenase-1 deficient mice (Hmox1(-/-)) had increased oxidative stress and were more sensitive to heme. Taken together, these results revealed that heme induces macrophage necrosis through 2 synergistic mechanisms: TLR4/Myd88-dependent expression of TNF and TLR4-independent generation of ROS.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Blotting, Western
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Cell Survival / drug effects
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Cells, Cultured
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Dose-Response Relationship, Drug
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Heme / pharmacology*
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Heme Oxygenase-1 / genetics
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Heme Oxygenase-1 / metabolism
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Imidazoles / pharmacology
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Indoles / pharmacology
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JNK Mitogen-Activated Protein Kinases / metabolism
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Macrophages / cytology
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Macrophages / drug effects*
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Macrophages / metabolism
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Mice
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Mice, Inbred C57BL
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Mice, Knockout
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Myeloid Differentiation Factor 88 / genetics
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Myeloid Differentiation Factor 88 / metabolism
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NIH 3T3 Cells
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Necrosis
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Reactive Oxygen Species / metabolism*
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Receptor-Interacting Protein Serine-Threonine Kinases / antagonists & inhibitors
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Receptor-Interacting Protein Serine-Threonine Kinases / genetics
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Receptor-Interacting Protein Serine-Threonine Kinases / metabolism
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Receptors, Tumor Necrosis Factor, Type I / genetics
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Receptors, Tumor Necrosis Factor, Type I / metabolism
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Toll-Like Receptor 4 / genetics
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Toll-Like Receptor 4 / metabolism
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Tumor Necrosis Factors / metabolism*
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Tumor Necrosis Factors / pharmacology
Substances
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Imidazoles
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Indoles
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Membrane Proteins
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Myd88 protein, mouse
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Myeloid Differentiation Factor 88
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Reactive Oxygen Species
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Receptors, Tumor Necrosis Factor, Type I
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Tlr4 protein, mouse
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Toll-Like Receptor 4
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Tumor Necrosis Factors
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necrostatin-1
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Heme
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Heme Oxygenase-1
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Hmox1 protein, mouse
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Receptor-Interacting Protein Serine-Threonine Kinases
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Ripk3 protein, mouse
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JNK Mitogen-Activated Protein Kinases