Hemoglobin participates in enhancing tumor necrosis factor (TNF) production by stimulated macrophages

TNF, a cytokine mediator of septic shock and tissue injury, is overproduced in endotoxemic hypophysectomized animals. We recently identified and isolated a serum protein from hypophysectomized animals that significantly increased TNF production In LPS-stimulated macrophages. Amino acid sequence analysis identified the protein as hemoglobin A. Here we investigate the direct activity of hemoglobin as an enhancer of TNF production. Murine macrophage-like RAW 264.7 cells were plated at 2×10⁶ cells/ml in 100 λ of serum-free RPMI medium (+pen/strep & L-glutamine) per well in a 96-well plate and incubated at 37°C for 3 hrs. In a second 96-well plate, IPS (10 ng/ml, 0111:B4, Sigma) and hemoglobin (Hb A, Sigma) were preincubated in the concentrations shown at 37°C for 30 min. At t=0 the media was aspirated from the cultures and replaced with the preincubated experimental media. After a 4 hr incubation (37°C), the conditioned supernatants were collected and assayed for TNF by ELISA. TNF production (pg/ml) in the cultures is shown In the Table (mean ±s.e., n=6/condition) [Hemoglobin] (μ/ml) Addition 0 12.5 125 500 HbA Alone 24±12 10±5 94±21 255±42 HbA + LPS 3662±450*5086±213*6311± 503*14472±689**P<0.05 vs HbA-alone by ANOVA It is now plausible to investigate the result of inhibiting HbA enhancement of TNF effect in diseases complicated by hemolysis and hemoglobinemia, such as trauma, cardiopulmonary bypass, and critical illness.