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 degrees 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 degrees C compared with incubation at 4 degrees C. Binding of FITC-ReLPS to lymphocytes was similar for both 4 degrees C and 37 degrees C incubation conditions. In contrast to 4 degrees C, at 37 degrees 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 degrees 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.
Weersink
A J
AJ
Eijkman-Winkler Laboratory of Medical Microbiology, University of Utrecht, Medical School, The Netherlands.
Van Kessel
K P
KP
Torensma
R
R
Van Strijp
J A
JA
Verhoef
J
J
D000911:Antibodies, Monoclonal / Q000378:metabolism; D001667:Binding, Competitive; D005434:Flow Cytometry; D006801:Humans; D066298:In Vitro Techniques; D007962:Leukocytes / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D009000:Monocytes / Q000378:metabolism; D009504:Neutrophils / Q000378:metabolism; D011112:Polymyxin B / Q000494:pharmacology; D012475:Salmonella; D013481:Superoxides / Q000378:metabolism; D013696:Temperature
D000911:Antibodies, Monoclonal; D008070:Lipopolysaccharides; D013481:Superoxides; D011112:Polymyxin B