In contrast, less ATP depletion was observed during exposure to heme-albumin, and no effect could be detected when either cell type was exposed to metHb or oxyHb at concentrations of up to 500 lmolL1, even in the absence of serum.
These experiments confirm a number of earlier reports and support the idea that certain solutions derived from purified/crystalline heme have the potential to induce weak TLR4-mediated inflammatory responses in macrophages in the complete absence of plasma derived proteins.
By modeling the equilibrium concentration in the experiments performed with heme-albumin we could show that the free heme is the active neutrophil-stimulating component, which is inactivated by protein association (Figure S2).
Purified heme was found to be an activator of TLR4 8,11,27 in some studies and of the inflammasome 9 in others, and these activities were considered to be the molecular mechanism behind the coexistence of inflammation and hemolysis.
Only under protein-free conditions did we observe a limited heme-induced TNF-alpha response in cultured macrophages, which was triggered via signaling of the classical TLR4–MyD88–TRIF pathway of NF-kB activation.28
In subsequent reactions, metHb or metHb-derived heme participates in redox chain reactions that lead to the accumulation of modified lipids and proteins, as well as to heme degradation and to the release of free iron.19,25
Exposure of washed human peripheral blood leukocytes to protein free crystalline heme that has been dissolved in NaOH (100 mmolL1) with subsequent adjustment to pH 7.8 resulted in a drastic reduction in cell surface CD62L expression on CD14 low CD15 high neutrophil granulocytes after 30 minutes of exposure (Figure 1A).
These observations suggest that in any physiological scenario in which heme might be present in extracellular spaces as a component of a natural hemoprotein, the concentration of free or quasi-free heme can be expected to be very low, possibly below the minimum range required to trigger granulocyte activation and other innate immunity responses.
In serum-free conditions, NaOH-dissolved heme (final pH 7.8) reduced cellular ATP in both cell types in a dose-dependent manner after 8 hours of exposure (Figure 2A)
This experiment demonstrated that under serum-free conditions, 4 hours of exposure to NaOH-dissolved heme induced complete deterioration of mitochondrial respiration at concentrations as low as 10 lmolL1, while exposure to albumin-associated heme in the presence of 2% FCS stimulated respiratory capacity to levels above baseline.
However, a weak TNF-alpha signal appeared in macrophages that were treated with the highest concentration of NaOH-dissolved heme (500 lmolL1).
The second biochemical process relates to the ability of Hb to facilitate oxidative reactions outside of the reducing environment of the RBC, leading to the accumulation of ferric metHb(Fe3+) in tissue.24
However, similar to Hb, heme-albumin did not induce an inflammatory response in macrophages or endothelial cells in the presence of low concentrations of serum.
The first process links the nitric oxide (NO) reactivity of oxyHb to a cascade of cell-free Hb extravasation, hemolysis-associated NO-depletion, and vasoconstriction.21–23
In endothelial cells, we found that, in the absence of serum, heme-albumin triggered a dose-dependent HMOX1 protein expression signal in an exposure range of 0–175 lmolL1.
heme-albumin is also a robust inducer of dose-dependent HMOX1 expression in BMDMs (Figure 2D).
heme-albumin, in contrast, induced HMOX1 in a dose-dependent manner.
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If you find BEL Commons useful in your work, please consider citing: Hoyt, C. T., Domingo-Fernández, D., & Hofmann-Apitius, M. (2018). BEL Commons: an environment for exploration and analysis of networks encoded in Biological Expression Language. Database, 2018(3), 1–11.