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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 hemeinduced 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. (Blood. 2012;119(10): 2368-2375) IntroductionThe term programmed cell death was used for many years as a synonym of apoptosis, whereas necrosis in the opposite extreme was considered an abrupt and uncontrolled type of cell death. However, recent evidence clearly shows that several nonapoptotic cell death modes including autophagy, pyroptosis, and necrosis also involve elaborate molecular circuitry. 1,2 This scenario was originally revealed in a study showing that depending on the cell type, tumor necrosis factor (TNF) could trigger different cellular fates including survival, apoptosis, and necrosis. 3 On blockage of protein synthesis or NF-B, activation of death cytokine receptors of the TNF superfamily triggers caspase-dependent apoptosis, whereas simultaneous inhibition of caspase reorients the cell death to necrosis. [4][5][6][7] Receptor-interacting protein 1 (RIP1, also known as RIPK1) regulates survival and cell death fates. Mice deficient in Rip1 present extensive apoptosis, dying early after birth. The increased sensitivity to TNF-mediated cell death in Rip1 Ϫ/Ϫ cells correlates with a failure to activate NF-B. 8 Recent work shows that necrotic cell death is highly regulated by the RIP1 and RIP3 kinases (also known as RIPK3). 6,7,9-11 Programmed necrosis can be initiated by several stimuli including DNA damage, oxidative stress, infection, and activation of pattern recognition receptors. 1,2,[12][13][14][15][16][17] Intra or extra vascular hemolysis, rhabdomyolysis, and extensive cell damage cause the release of large quantities of hemeproteins. The oxidation of some hemeproteins including hemoglobin and myoglobin can release the heme moiety promoting further oxidation an...

Infectious diseases that cause hemolysis are among the most threatening human diseases, because of severity and/or global distribution. In these conditions, hemeproteins and heme are released, but whether heme affects the inflammatory response to microorganism molecules remains to be characterized. Here, we show that heme increased the lethality and cytokine secretion induced by LPS in vivo and enhanced the secretion of cytokines by macrophages stimulated with various agonists of innate immune receptors. Activation of nuclear factor B (NF-B) and MAPKs and the generation of reactive oxygen species were essential to the increase in cytokine production induced by heme plus LPS. This synergistic effect of heme and LPS was blocked by a selective inhibitor of spleen tyrosine kinase (Syk) and was abrogated in dendritic cells deficient in Syk. Moreover, inhibition of Syk and the downstream molecules PKC and PI3K reduced the reactive oxygen species generation by heme. Our results highlight a mechanism by which heme amplifies the secretion of cytokines triggered by microbial molecule activation and indicates possible pathways for therapeutic intervention during hemolytic infectious diseases.A general consequence of infectious diseases that cause hemolysis, internal hemorrhage, or extensive cell damage is the release of hemeproteins. Upon oxidation, hemeproteins release heme, a potentially harmful molecule (1). Heme-binding plasma proteins, such as hemopexin or albumin, remove the intravascular free heme, subsequently degraded by heme oxygenase-1 (HO-1), generating equimolar amounts of biliverdin, carbon monoxide, and free iron (2, 3). HO-1-deficient mice (Hmox Ϫ/Ϫ ) have high plasma concentrations of heme and show increased susceptibility to LPS-induced lethality, associated with inflammation and oxidative damage (4). Accumulation of large amounts of heme might overwhelm the capacity of heme scavengers and degrading system, thus causing oxidative stress and inflammation (5, 6). In fact, recent studies suggest that heme, in combination with ROS 3 and inflammatory mediators, increase blood brain barrier leakage and hepatocyte necrosis in models of malarial infection (7,8).Hemolysis or hemoglobinemia are associated with increased mortality in septic patients (9, 10). Hemoglobin increases the secretion of TNF triggered by LPS, whereas globin has an inhibitory effect (11), suggesting that heme is responsible for the cytokine amplification. Heme has several pro-inflammatory activities, including leukocyte activation and migration, upregulation of adhesion molecules, ROS production, and induction of cytokine expression (12-14). Recently, we have shown that heme is able to activate Toll-like receptor 4 (TLR4) inducing TNF on macrophages and dendritic cells (DC) (15).Mammalian pattern recognition receptors (PRRs) recognize conserved microbial molecules from all classes of microorganisms (16,17). The activation of these receptors elicits selective intracellular signaling cascades that result in the production of cytokines, chemokin...