D. Petrov1, C. Lin2, M. Mazer2,3, M. Moorman5, K.E. Remy2,6, J.T. Ross2,5 1Northeast Ohio Medical University, School Of Medicine, Rootstown, OH, USA 2Case Western Reserve University School Of Medicine, The Blood, Heart, Lung, And Immunology Research Center, Cleveland, OH, USA 3University Hospital Cleveland, Division Of Pediatric Critical Care Medicine, Cleveland, OH, USA 4University of California Davis, Department Of Surgery, Sacramento, CA, USA 5University Hospitals Cleveland, Department Of Surgery, Cleveland, OH, USA 6University Hospitals Cleveland, Division Of Pulmonary Critical Care Medicine, Cleveland, OH, USA
Introduction: Cell-free hemoglobin (CFH) and free heme mediate endotheliopathy and organ injury in sepsis, but their role in trauma is unclear. Traumatic injury produces early endogenous hemolysis, which temporarily overwhelms scavenging mechanisms. Elevated CFH and heme, detectable at ED arrival after trauma, are associated with longer hospital stays, septic complications and AKI. We hypothesize that CFH and heme mediate septic complications in trauma through a pro-inflammatory effect on circulating leukocytes.
Methods: Venous blood was drawn from six healthy volunteers. Fresh whole blood was incubated at 37°C with phosphate-buffered saline (PBS, negative control), lipopolysaccharide (LPS 100 ng/ml, positive control), 40 μM cell-free hemoglobin (CFH), 20 μM heme, or a combination of CFH + LPS or Heme + LPS. TNFα production was quantified by enzyme-linked immunosorbent spot (ELIspot) assay after 4- and 18-hour incubations. ELIspot allows visualization of TNFα production from individual cells, which each generate distinct spots. Exposures were conducted in triplicate and results are expressed as mean spots per 1,000 cells. This study was approved by our institutional IRB.
Results: Six healthy volunteers were enrolled (median age 28, 83% male; to represent a trauma cohort). We found that both 4 and 18 hour exposures to CFH stimulated a greater than 5-fold increase in TNFα release compared to PBS alone (p = 0.002), Figure 1. Notably, CFH exposure produced a nearly 2-fold greater increase in TNFα production compared to LPS (p=0.002), but the combination of CFH and LPS did not significantly alter TNFα production compared to CFH alone. In contrast, Heme exposure did not stimulate significant TNFα release alone and the combination of Heme and LPS did not significantly alter TNFα production compared to LPS alone.
Conclusions: Our study demonstrates that clinically relevant concentrations of cell-free hemoglobin stimulate a robust pro-inflammatory response in whole blood. While ex-vivo stimulation of whole blood ignores the important role of the vascular endothelium, our findings suggest that circulating leukocytes play a role in mediating the inflammatory response to hemolysis. This is an intriguing avenue for research, as multiple existing therapies block components of the hemolysis pathway. We believe that it is possible to leverage those treatments to moderate the burden of acute and chronic inflammation resulting from traumatic injury and associated intravascular hemolysis.