Hemolysis Induced Activation of Monocytes Is Followed By Inactivation Via Heme-Induced HO-1 Expression

Introduction: Sickle cell disease (SCD) is characterized by profound erythrocyte hemolysis. Hemolytic events, both chronic and acute, lead to elevated levels of both hemoglobin and heme in patients. Both hemoglobin and heme are toxic in the vasculature, but the relative contribution of each to the overall state of health in patients is unknown. Nonetheless, hemolysis is emerging as a clear contributor to inflammation and coagulation activation. The mechanism through which hemolysis may contribute to either inflammation or coagulation activation is not well defined. The monocyte, however, is the only white blood cell in the bloodstream that is uniquely positioned to influence both coagulation activation and inflammation. Monocytes can express tissue factor (TF) on their surface and, as such, activate the extrinsic pathway of thrombin generation. Furthermore, monocytes are activated in SCD, and likely contribute to the overall inflammatory state of the disease. Thus, we reasoned that monocyte activation is a likely mechanism through which hemolysis, either via free hemoglobin or free heme, could influence both coagulation and inflammation. Another aspect of hemolysis is the persistent induction of heme-oxygenase-1 (HO-1) in the presence of elevated levels of plasma heme. We also reasoned that any interrogation of the effects of hemolysis on monocyte activation would induce the significant expression of this heme-degrading enzyme potentially mitigating long term hemolytic stress.

Objectives: Determine the effects of free hemoglobin, heme, and heme-induced HO-1 expression on monocyte inflammation and pro-coagulant function.

Methods: Monocyte activation was measured in response to either purified hemoglobins A or S, or heme, and was measured in THP-1 monocytes. Simultaneous analysis of 84 heme-induced inflammatory genes was conducted via qRT-PCR using the Qiagen RT Profiler PCR array. HO-1 was induced by pre-conditioning monocytes overnight in heme (5uM). Monocyte inactivation was measured using this pre-conditioning, followed by an additional treatment with 10uM heme. Monocyte TF expression was determined via flow cytometry analysis. TF specific pro-coagulant activity was determined in a one stage clotting assay. The expression of HO-1 protein in THP-1 cells was analyzed using ELISA.

Results: We found, in terms of monocyte function, purified hemoglobins A or S were essentially inert. Free heme at physiological levels in patients with SCD, however, was a powerful monocyte activator. We found that, in naïve cells, heme induced profound TF expression at the cell surface, increased monocyte TF mRNA, and significantly increased the pro-coagulant potential of monocytes in a TF-dependent manner. Furthermore, we noted an acute increase in heme-modulated inflammatory gene expression in monocytes using a global gene expression analysis. Of note, there was a profound induction of inflammatory genes in the TGF-β family by heme, including BMP7. Members of the growth and differentiation family were heme-inducible as well - including GDF1,2,& 3. Expression of interleukins 3, 6, 8, 9, & 25 was all significantly increased in response to an acute heme exposure. All genes responded in a dose dependent manner to heme. There was no heme-dependent induction of TNFα suggesting a non-classical monocytic response to heme stimulation. However, sustained exposure to heme inactivated the monocyte inflammatory gene response. Furthermore, monocyte pre-conditioning in heme rendered the cells resistant to subsequent heme-induced inflammation or pro-coagulant function. This heme induced inactivation of monocyte was coincident with and linked to a robust monocyte expression of HO-1.

Conclusions: We report a novel heme-induced activation of monocytes that can be directly inhibited by heme-induced HO-1 expression. Our data therefore provide evidence for the first time of a link between hemolysis and monocyte inflammatory and pro-coagulant function. These data potentially suggest that, in patients, higher levels of baseline hemolysis might be protective against the sudden, acute hemolytic event in SCD. These events often precede acute pain crisis and acute chest syndrome. Further study of monocyte activation and levels of HO-1 in patients during these events is merited.