The interaction of Escherichia coli endotoxin with leukocytes

Abstract

Rabbit polymorphonuclear leukocytes obtained from acute peritoneal exudates were used to study the interaction of endotoxin and leukocytes. Saline-washed cells were incubated with 32P-labeled endotoxin prepared from Escherichia coli, and the amount of bound endotoxin was measured in cells separated from mixtures by filtration. Maximum removal of endotoxin from the soluble phase of mixtures was obtained in 10 min. The amount of endotoxin bound was proportional to the amount of endotoxin added over a wide range of endotoxin concentration. The binding process was independent of temperature and was not inhibited by such reagents as iodoacetate, N-ethylmaleimide and NaCN. It was inhibited by rabbit serum, sodium oxalate, and ethylenediaminetetraacetic acid. The results of the binding study are best rationalized in terms of the interaction of two charged species mediated by cell-bound cations. After binding to leukocytes endotoxin was, in part, degraded. Gel filtration of the supernatant fluid from an incubated 14C-labeled endotoxin leukocyte mixture showed radioactivity in a low molecular weight fraction associated with protein. Examination of the nature of the radiolabeled products by extraction and separation of lipids showed free fatty acids as well as neutral—and phospholipids in both the supernatant fluid and cells. N-Ethylmaleimide, EDTA, and protamine sulfate inhibited formation of the lipids.