RESUMEN

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.

Asunto(s)

Hemo/farmacología , Macrófagos/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Factores de Necrosis Tumoral/metabolismo , Animales , Western Blotting , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Hemo-Oxigenasa 1/genética , Hemo-Oxigenasa 1/metabolismo , Imidazoles/farmacología , Indoles/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Células 3T3 NIH , Necrosis , Proteína Serina-Treonina Quinasas de Interacción con Receptores/antagonistas & inhibidores , Proteína Serina-Treonina Quinasas de Interacción con Receptores/genética , Proteína Serina-Treonina Quinasas de Interacción con Receptores/metabolismo , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Factores de Necrosis Tumoral/farmacología