Mechanism of the adjuvant effect of hemoglobin in experimental peritonitis: VIII. A leukotoxin is produced by Escherichia coli metabolism in hemoglobin

Hemoglobin, but not albumin, has long been recognized as an infection potentiating factor in experimental Escherichia coli peritonitis, but the mechanism has defied definition. We have shown previously that stroma-free hemoglobin is not toxic to polymorphonuclear neutrophils. To test the hypothesis that hemoglobin provides a nutritional boost to the growth of E. coli in vivo, we inoculated E. coli into dialysis bags containing equivalent amounts of stroma-free hemoglobin or albumin. These bags were implanted into the peritoneal cavity of rats and at intervals the fluid was removed and the bacteria enumerated. This technique allows for intraperitoneal bacterial growth but eliminates the variables of lymphatic clearance and phagocytic ingestion. The growth rate of E. coli was the same irrespective of the nutritional supplement in the bag. Thus there is no experimental support for the notion that hemoglobin directly accelerates E. coli proliferation under in vivo conditions. To test the hypothesis that a leukocyte toxin may result from E. coli growth in hemoglobin, we exposed normal human neutrophils to the sterilized contents of the peritoneal dialysis bags. In vitro function of the neutrophils (viability, random migration, chemotaxis, phagocytosis, bacterial killing, and chemiluminescence) was significantly depressed by prior exposure to hemoglobin supernatants that had supported E. coli proliferation in vivo. Stroma-free hemoglobin had minimal adverse effects. Albumin supernatants that had supported E. coli proliferation in vivo had significantly less effect on neutrophil function even though the endotoxing levels were identical to the hemoglobin E. coli solutions. We must conclude that leukotoxins result from E. coli growth in solutions of pure hemoglobin. The data support the idea that the infection potentiating effect of hemoglobin in vivo is due to such leukotoxins.