Synergistic Inflammation Is Induced by Blood Degradation Products with Microbial Toll‐Like Receptor Agonists and Is Blocked by Hemopexin

Abstract

Detection of microbial components by immune cells via Toll-like receptors (TLRs) with subsequent induction of inflammation is essential for host defense. However, an overactive immune response can cause tissue damage and sepsis. The endogenous molecule hemoglobin and its derivative heme are often released into tissue compartments where there is infection in the presence of degrading blood. We found that hemoglobin synergizes with multiple TLR agonists to induce high levels of tumor necrosis factor and interleukin-6 from macrophages and that this synergy is independent of TLR4 and MyD88. In contrast, heme synergized with some but not all TLR agonists studied. Furthermore, the synergy of both hemoglobin and heme with lipo-polysaccharide was suppressed by hemopexin, a plasma heme-binding protein. These studies suggest that hemoglobin and heme may substantially contribute to microbe-induced inflammation when bacterial or viral infection coexists with blood degradation and that hemopexin may play a role in controlling inflammation in such settings.

Hemolysis and free hemoglobin revisited: exploring hemoglobin and hemin scavengers as a novel class of therapeutic proteins

“…78 Within these complex disease processes, hemin could act as an activator of innate immunity via pattern recognition receptor activation (eg, TLR-4), via synergistic effects with microbial products or endogenous pro-inflammatory ligands (eg, HMGB1), or via peroxidative tissue damage. 24,79,80 Studies in mice suggested that the net effect of free hemin in polymicrobial sepsis could be detrimental and that neutralization of hemin by Hpx administration ameliorates this effect. 78 The protective action of Hpx in sepsis may involve the control of excessive hemin peroxidative activity, inhibition of innate immunity receptor interactions, and redirection of free hemin away from susceptible tissues toward clearance in the liver.…”

Purified and Recombinant Hemopexin: Protease Activity and Effect on Neutrophil Chemotaxis

“…Macrophage Culture and Cytokine Assays Bone marrow-derived macrophages (BMDMs) were prepared from mice as we described previously (12). BMDMs were washed three times in serum-free medium, followed by incubation overnight with LPS with or without hemin or hemoglobin in the presence or absence of Fc-hHx or Fc or hHx-B in the culture as noted.…”

“…Hemin chloride was purchased from Frontier Scientific Inc. Hemin solutions were made immediately before use in the dark as described previously (12). Chemoattractants N-formyl-l-methionyll-leucyl-l-phenylalanine (fMLF) and leukotriene B4 (LTB4) were purchased from Sigma-Aldrich and Cayman Chemical, respectively.…”

“…This binding affinity is so high that Hx "steals" heme from hemoglobin (21). Heme and hemoglobin are synergistic with toll-like receptor (TLR) agonists a more conservative transfusion strategy (1)(2)(3)(4), studies suggesting that older stored blood is associated with higher morbidity than fresh blood (5)(6)(7)(8)(9), studies that older blood has higher levels of cell-free hemoglobin (10,11), in vitro studies in which hemoglobin and heme potentiate release of cytokines from macrophages (12)(13)(14), inTRODUCTiOn There has been increasing appreciation that cell-free plasma hemoglobin and secondarily released free heme may be toxic in critical care settings. Evidence for this concept comes from studies in which liberal transfusion is associated with equivalent or worse outcome than 2 Frank E Riley, perform amidolytic assay: H-D-IIe-ProArg-pNA⋅2HCl (S2288) that is sensitive to a broad spectrum of serine protease, and H-D-Phe-Pip-Arg-pNa⋅2HCl (S2238) that is sensitive to thrombin.…”

Cell-Free Hemoglobin and Its Scavenger Proteins: New Disease Models Leading the Way to Targeted Therapies

“…Blood transfusion leading to stimulation of the heme breakdown pathway and accumulation of heme catabolites was associated with anti-inflammatory and immunosuppressive effects in multiple animal models, and such effects could contribute to the immunecompromised state of severely ill and massively transfused patients (Yazdanbakhsh et al 2011). On the other hand, proinflammatory effects may be triggered by free heme through the stimulation of toll-like receptor 4 (TLR-4), by reaction intermediates emanating from Hb/ heme peroxidative reactions with phospholipids, or by Hb/heme breakdown products (D'Agnillo and Alayash 2001; Jeney et al 2002;Jia et al 2007;Lin et al 2010;Yazdanbakhsh et al 2011). In a mouse blood transfusion model, older stored RBCs induced an acute phase cytokine release response that accompanied the clearance of transfused RBCs in the spleen (Hod et al 2010).…”