Free hemoglobin enhances tumor necrosis factor-α production in isolated human monocytes

Background: A systemic inflammatory response (SIR) is seen in approximately 75% of patients with complex blunt liver injuries treated nonoperatively. Many feel this response Is caused by blood, bile, and necrotic tissue accumulation in the peritoneal cavity. Our current treatment for these patients is a delayed laparoscopic washout of the peritoneal cavity, resulting in a dramatic resolution of the SIR. Spectrophotometric analysis of the intraperitoneal fluid has confirmed the presence of high concentrations of free hemoglobin (Hb). We hypothesize that free Hb enhances the local peritoneal response by increasing tumor necrosis factor-alpha (TNF-alpha) production by monocytes, contributing to the local inflammatory response and SIR. Methods: Monocytes from five healthy volunteers were isolated and cultured in RPMI-1640 for 24 hours. Treatment groups included saline controls, lipopolysaccharide ([LPS], 10 ng/mL, from Escherichia coli), human Hb (25 mug/mL), and Hb + LPS. Supernatants were analyzed by enzyme-linked immunosorbent assay. Student's t test with Mann-Whitney posttest was used for statistical analysis with p less than or equal to 0.05 considered significant. Results: Free Hb significantly increased TNF-alpha production 915 +/- 223 pg/mL versus saline (p = 0.02). LPS and Hb + LPS further increased TNF-alpha production (2294 pg/mL and 2501 pg/mL, respectively, p < 0.001) compared with saline controls. Conclusion: These data confirm that free Hb is a proinflammatory mediator resulting in the production of significant amounts of TNF-alpha These in vitro findings support our clinical data in which timely removal of intraperitoneal free hemoglobin helps prevent its deleterious local and systemic inflammatory effects in patients with complex liver injuries managed nonoperatively.