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.
Hemoglobin is released from lysed RBCs in numerous clinical settings. High mobility group box 1 (HMGB1) is a nuclear and cytosolic DNA-binding protein released from injured cells that has been shown to play an important role in inducing inflammation. Because both of these endogenous molecules are frequently present in sites of necrosis and inflammation, we studied their interaction on the activation of macrophages. We report in this article that hemoglobin and HMGB1 synergize to activate mouse macrophages to release significantly increased proinflammatory cytokines. Addition of microbial ligands that activate through TLR2 or TLR4 resulted in further significant increases, in a “three-way” synergy between endogenous and microbial ligands. The synergy was strongly suppressed by hemopexin (Hx), an endogenous heme-binding plasma protein. The findings suggest that hemoglobin may play an important role in sterile and infectious inflammation, and that endogenous Hx can modulate this response. Administration of Hx may be beneficial in clinical settings characterized by elevated extracellular hemoglobin and HMGB1.
Background
Transfusion of human blood stored for over 2 weeks is associated with increased mortality and morbidity. During storage, packed erythrocytes progressively release hemoglobin, which avidly binds nitric oxide. We hypothesized that the nitric oxide mediated hyperemic response following ischemia would be reduced after transfusion of packed erythrocytes stored for 40 days.
Methods and results
We conducted a cross-over randomized interventional study, enrolling 10 healthy adults. Nine volunteers completed the study. Each volunteer received one unit of 40-day and one of 3-day stored autologous leukoreduced packed erythrocytes, on different study days according to a randomization scheme. Blood withdrawal and reactive hyperemia index measurements were performed before and 10 min, 1 h, 2 h, and 4 h after transfusion. The reactive hyperemia index during the first 4 h after transfusion of 40-day as compared to 3-day stored packed erythrocytes was unchanged. Plasma hemoglobin and bilirubin levels were higher after transfusion of 40-day than after 3-day stored packed erythrocytes (p = 0.02, [95% CI difference 10-114mg/l] and 0.001, [95% CI difference 0.6-1.5mg/dl], respectively). Plasma levels of potassium, Lactate dehydrogenase, haptoglobin, cytokines, as well as blood pressure, did not differ between the two transfusions and remained within the normal range. Plasma nitrite concentrations increased after transfusion of 40-day stored packed erythrocytes, but not after transfusion of 3-day stored packed erythrocytes (p = 0.01, [95% CI difference 0.446 - 0.66 μM]).
Conclusions
Transfusion of autologous packed erythrocytes stored for 40 days is associated with increased hemolysis, an unchanged reactive hyperemia index, and increased levels of plasma nitrite.
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.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.