Identification of hemopexin as an anti-inflammatory factor that inhibits hemoglobin synergy with HMGB1 in sterile and infectious inflammation (172.11)

Abstract In many clinical settings such as trauma, infections and other hemorrhagic diseases, multiple endogenous substances are released into the extracellular space, including hemoglobin (Hb) from degrading RBC and HMGB1, an intracellular DNA-binding protein that has been shown to be pro-inflammatory, from injured or immune cells. We studied the effects of Hb and HMGB1 on the activation of macrophages. In the supernatant of cultured bone marrow-derived macrophages from C57BL/6 or C3H/HeN mice, significantly increased levels of pro-inflammatory cytokines (TNF and IL-6) were detected when Hb was added with HMGB1 compared to the culture with HMGB1 alone ( P < 0.01), while Hb itself did not induce detectable TNF and IL-6. This synergistic effect was also present in the culture of TLR2 knockout and TLR4 deficient (C3H/HeJ) macrophages. Addition of hemopexin (Hx), an endogenous heme-binding plasma protein, significantly decreased TNF and IL-6 induced by Hb and HMGB1 (P < 0.01). Co-incubation of microbial ligands LPS or Pam3Cys with Hb and HMGB1 in the culture resulted in further dramatic increases of TNF and IL-6 that were also significantly decreased by Hx (P < 0.01). These findings suggest that Hb may play an important role in amplifying sterile as well as infectious inflammation, and that endogenous Hx may play a role in controlling it. Administration of Hx could be a beneficial strategy in clinical settings where extracellular Hb and HMGB1 are both present.