Revisiting the putative role of heme as a trigger of inflammation

Activation of the innate immune system by free heme has been proposed as one of the principal consequences of cell‐free hemoglobin (Hb) exposure. Nonetheless, in the absence of infection, heme exposures within a hematoma, during hemolysis, or upon systemic administration of Hb (eg, as a Hb‐based oxygen carrier) are typically not accompanied by uncontrolled inflammation, challenging the assumption that heme is a major proinflammatory mediator in vivo. Because of its hydrophobic nature, heme liberated from oxidized hemoglobin is rapidly transferred to alternative protein‐binding sites (eg, albumin) or to hydrophobic lipid compartments minimizing protein‐free heme under in vivo equilibrium conditions. We demonstrate that the capacity of heme to activate human neutrophil granulocytes strictly depends on the availability of non protein‐associated heme. In human endothelial cells as well as in mouse macrophage cell cultures and in mouse models of local and systemic heme exposure, protein‐associated heme or Hb do not induce inflammatory gene expression over a broad range of exposure conditions. Only experiments in protein‐free culture medium demonstrated a weak capacity of heme‐solutions to induce toll‐like receptor‐(TLR4) dependent TNF‐alpha expression in macrophages. Our data suggests that the equilibrium‐state of free and protein‐associated heme critically determines the proinflammatory capacity of the metallo‐porphyrin. Based on these data it appears unlikely that inflammation‐promoting equilibrium conditions could ever occur in vivo.