Inactivation of Endotoxin by a Humoral Component. Vi. Two Separate Systems Required for Viable and Killed Salmonella Typhosa

Abstract

The blood of mammalian species contains a component which inactivates endotoxins in vitro (1). This endotoxin-detoxifying component (EDC) is identified by attributes which distinguish it from other substances in blood of known biologic activity (2, 3). Following appropriate interaction in vitro with this component, endotoxins from gram-negative bacteria are so altered that they no longer elicit such characteristic host reactions as the production of antibody (4), tumor damage, local skin reactivity and fatal shock. Bacterial endotoxins elicit the same host reactions which are evoked by the intact gram-negative bacilli from which they are isolated (5). It was therefore expected that the endotoxin in its native state on the bacterial surface would also be susceptible to inactivation by an agent with the distinctive properties of EDC.This communication records the results of experiments which show that, by appropriate interaction in vitro with serum, it is possible to eliminate characteristic host-reactive properties of a typical gram-negative species. In the course of these studies with Salmonella typhosa, EDC was found to inactivate the endotoxin of bacilli which had been killed by heat or by chemical means, but not that in viable organisms; however, in the latter the endotoxin was inactivated by the serum bactericidal system consisting of antibody and complement. It is thus evident that two humoral mechanisms can function to inactivate the endotoxic properties of intact gram-negative bacilli. MATERIALS AND METHODSCollection of serum. Pooled serum specimens were prepared from the blood (10 or more individual donors) of mice, guinea pigs and rats. In some experiments human serum, pooled from 100 or more individuals, was employed. In other instances, animal and human serum samples were individual specimens. All serum samples * Present address: Children's Hospital, Boston, Mass.were stored at -20°C in sealed glass ampoules. Plasma was obtained from blood collected in heparin, sodium citrate or ACD solution,' and by passage of blood through a Fenwal ion exchange pack; the latter is referred to as "resin-treated" plasma.Preparation of complemnent reagentts. The reagents were prepared by standard methods (6) and each was checked for absence of hemolytic and/or bactericidal activity. Human serum was made deficient in the first component of complement (C'1) by dialysis against acetate buffer (pH 5.5, u = 0.02) for 48 hours at 40 C. The serum was then centrifuged, and the supernatant fluid was adjusted to the desired pH and ionic strength with NaOH and NaCl. This reagent, deficient in C'1, is referred to as RI.The precipitate obtained in this way (by dialysis and centrifugation) was washed twice with the dialyzing buffer and taken up in saline to original volume. This reagent is deficient in the second component of complement (C'2) and is referred to as R2.Human serum was made deficient in the third component of complement (C'3) by incubation for 1 hour at 370 C with zymosan (Fleischmann; 2 mg per ml serum).This reagent ...

Bactericidal Method for the Measurement in Normal Serum of Antibody to Gram-Negative Bacteria

“…The bactericidal action of fresh normal serum on gram-negative bacteria is attributable to a system comprised of specific antibody, the recognized components of complement, and divalent cation. The essential findings of a previous studv (Landy, Trapani, and Rosen, 1960), which detailed these requirements for S. t!lphosa 0901, are summarized in removal, inactivation, or complexing of any of the components of this system results in loss of bactericidal activity. When all other components were present in excess, as little as 0.002 iAg of antibody N was sufficient to fully restore bactericidal activity to the system.…”

“…Investigations which reaffirmed the specificity of the antibody requirement have, in addition, shown that the quantity of this component which suffices to restore bactericidal activity to absorbed serum is exceedingly small (Muschel and Treffers, 1956;Landy, Trapani, and Rosen, 1960). It was this minimal antibody requirement which suggested to us that the bactericidal system could be utilized for the detection and measurement of the small amounts of antibody present in normal serum.…”