Molecular interactions between bacterial endotoxins and mammalian cells. A spin-label study

Endotoxin molecules from E. coli 0111:B4, J5 and lipid A have been spin-labelled with 2,2,6,6-tetramethylpiperidin-N-oxyl(TEMPO) free radicals in their sugar residues. Measurement of the rotational correlation times indicates that these saccharide residues do not bind to cell membrane surface structures. A lipid-labelled derivative of the lipid A component of LPS has also been synthesized with the spin-label group positioned on the myristic acid chain. When this is incubated with macrophage cells, an EPR signal characteristic of a spin-label motionally restricted in a lipid bilayer is obtained. This indicates that this LPS derivative binds to the cell membrane through its lipid component. The majority of this 'binding' (66%) is shown to be due to non-specific insertion in the bilayer. The membrane-intercalated LPS appears to aggregate into patches of high endotoxin concentration, which are stabilised by their strong interaction with membrane phospholipids (phosphatidylethanolamine). Uptake of these spin-labelled derivatives into cells is found to be dependent on a chemical energy source (ATP) and an intact cytoskeleton. We speculatively suggest that the aminophospholipid translocase enzyme present in cell membranes might be responsible for cellular uptake of endotoxin.