(538) The Effect of Free Heme Combined With Different Serum Proteins on Immune Cells

Introduction: Red blood cell transfusion is common in critical care settings, with 60% of all patients receiving at least one unit of packed red blood cells (PRBC), with a mean of 5 transfused units. Hemolysis in PRBCs increases with storage duration and releases heme. Free heme, released either by in vivo hemolysis or introduced via transfusion of aged PRBCs, is known to have pro-inflammatory effects and to decrease survival in sepsis models. Heme binds to serum proteins, however its unknown how this functionally affects immune cells.

Methods: Peripheral blood mononuclear cells (PBMCs) from healthy donors were incubated with free heme in a concentration of either 25 or 80 μM, to which equimolar amounts of albumin, Low-Density-Lipoprotein (LDL), High-Density-Lipoprotein (HDL), haptoglobin or hemopexin were added. After 12 hours the PBMCs were collected and used for flow cytometry (n=7) to differentiate between monocytes, CD4+ and CD8+ T-Cells and regulatory T-Cells and asses their proliferation rate as well as the rate of apoptosis. We also conducted Enzyme Linked Immunospot assays (n=4) after 24 hours of incubation to assess functional production of tumor necrosis factor α (TNF)and interferon-γ (IFNy)

Results: TNF production was increased by incubation with heme, this effect was reduced by coincubation with haptoglobin and vanished with hemopexin, IFNy production was not significantly affected. Heme led to an increased rate of apoptosis in CD4+ and CD8+ T-Cells but did not change rates in monocytes. Coincubation of heme with LDL resulted in severely increased apoptosis rates in all cell populations and reduced proliferation in CD4+ T-Cells. The mean fluorescence intensity (MFI) of Monocyte CD163 and HLA-DR was also decreased after incubation with heme and LDL or HDL, while haptoglobin increased the MFI of CD163.

Conclusions: Free heme is negatively impacting T Cell and monocyte survival, with the effect being heavily amplified by coincubation with LDL. This result may be due to heme binding to the LDL which could result in a cytotoxic compound, partially explaining the negative effect heme has on survival in murine and canine sepsis models. However further research is needed to assess whether heme is actually bound to LDL and if the resulting compound leads to reduced functionality.