Binding of rough lipopolysaccharides (LPS) to human leukocytes. Inhibition by anti-LPS monoclonal antibody

The binding of rough LPS (ReLPS from Salmonella minnesota R595) to human peripheral blood polymorphonuclear leukocytes (PMN), monocytes, and lymphocytes was examined by using fluorescein-labeled LPS and flow cytometry. At 4°C, FITC-ReLPS bound rapidly in a concentration- and time-dependent way to PMN, monocytes, and lymphocytes. Because mononuclear cells showed both binding and nonbinding cell populations, FITC-ReLPS was used in conjunction with specific phycoerythrin-labeled mAb to identify these cell subpopulations. In contrast to T lymphocytes and NK cells, all monocytes and B lymphocytes efficiently bound FITC-ReLPS. PMN and monocytes showed two to three times more cell-associated FITC-ReLPS when cells were incubated at 37°C compared with incubation at 4°C. Binding of FITC-ReLPS to lymphocytes was similar for both 4°C and 37°C incubation conditions. In contrast to 4°C, at 37°C cell-associated LPS reflects surface-bound as well as internalized LPS, as demonstrated with fluorescence quenching of extracellular FITC-ReLPS by trypan blue. At 4°C, binding of FITC-ReLPS was inhibited by polymyxin B. In addition, purified IgM mAb directed against hydrophobic acyl residues of ReLPS showed more than 95% inhibition of ReLPS binding to leukocytes, indicating the ability of specific mAb to prevent LPS-cell interactions necessary to exert biologic effects. The use of mAb, directed against different parts of the LPS molecule, provides an alternative method for LPS binding-inhibition studies.