LIPOPOLYSACCHARIDE COMPLEXED WITH SOLUBLE CD14 BINDS TO NORMAL HUMAN MONOCYTES

Using flow cytometry we have compared the binding of Neisseria meningi tidis lipopolysaccharide labeled with fluorescein isothiocyanate (FITC -LPS) to normal human monocytes in whole blood with the binding to chi nese hamster ovary (CHO) cells transfected with human CD14 gene (hCD14 -CHO cells). Binding of FITC-LPS to cells was dose dependent, saturabl e and enhanced in the presence of increasing concentrations of serum. Blockade of membrane CD14 with saturating concentrations of anti-CD14 monoclonal antibody (mAb) My4 inhibited 50% of the binding of FITC-LPS to monocytes and 100% to hCD14-CHO cells. Similarly, removal of membr ane CD14 by phosphatidylinositol phospholipase C (PI-PLC) treatment of the cells partially decreased the binding of FITC-LPS to monocytes bu t totally inhibited the binding to hCD14-CHO-transfected cells. These results suggest that binding of FITC-LPS to monocytes is not only medi ated by membrane CD14. Using two-color flow cytometry, we observed tha t FITC-LPS binds to My4-saturated monocytes in association with solubl e (s)CD14 present in serum as revealed by staining with rhodamine-labe led My4 mAb. The binding of FITC-LPS/sCD14 complexes to monocytes trea ted with saturating amounts of unlabeled My4 prior to addition of the complexes was completely inhibited by anti-CD14 mAb 10G33. When cells were first saturated with a mixture of My4 and 10G33 mAb, washed and f urther incubated with FITC-LPS/sCD14, inhibition of the binding of LPS was similar to that observed with cells saturated with My4 alone, sho wing that the binding of FITC-LPS is not mediated by the 10G33 epitope present on mCD14. These results suggest that either the 10G33 epitope on sCD14 is involved in the binding of LPS/sCD14 complexes to the cel ls, or that 10G33 mAb inhibits the binding of FITC-LPS to sCD14. Taken together, these data indicate that sCD14 which is present in normal s erum, in addition to membrane CD14, enables LPS to bind monocytes thro ugh an as yet unidentified molecule and that sCD14 does not simply ser ve as a shuttle for transfer of LPS to membrane CD14.