Synthesis of [H-3]-labeled bioactive lipid A analogs and their use for detection of lipid A-binding proteins on murine macrophages

Both endotoxic and antagonistic [H-3]-labeled 2-(phosphonooxy)ethyl (PE) an alogs of lipid A were synthesized with high purity and high specific radioa ctivity. Lipid A-binding proteins were detected by using the endotoxic anal og of hexaacyl Escherichia coli-type designated [H-3] PE-506. The plasma me mbrane fractions from peritoneal macrophages derived from LPS-responder C3H /HeN mice and LPS-hyporesponder C3H/HeJ mice were separated by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were then incubat ed with the [H-3] PE-506. Several [H-3] PE506 binding proteins were detecte d in both C3H/HeN and C3H/HeJ macrophages. Unlabeled hexaacyl lipid A inhib ited the interaction between [H-3] PE-506 and these proteins. The result su ggests that there exist multiple binding sites for lipid A on macrophages. LPS-induced change in the profile of the cell surface lipid A binding prote ins was observed in C3H/HeN macrophages, but not in C3H/HeJ macrophages, by preincubation of macrophages with LPS.