IDENTIFICATION OF A DOMAIN IN SOLUBLE CD14 ESSENTIAL FOR LIPOPOLYSACCHARIDE (LPS) SIGNALING BUT NOT LPS BINDING

CD14 is a 55-kDa glycoprotein that binds lipopolysaccharide (LPS) and enables LPS dependent responses in a variety of cells. Monoclonal anti bodies of CD14 such as 3C10 and MEM-18 are known to neutralize biologi cal activity of CD14. Recently, it has been demonstrated that MEM-18 r ecognizes the LPS-binding site of CD14, between amino acids 57 and 64. It has also been shown that 3C10 recognizes a distinct epitope from t hat of MEM-18, indicating that 3C10 may yet define another functional domain of CD14. In order to identify the epitope for 3C10, we construc ted a series of alanine substitution mutants of soluble CD14 (sCD14). BIAcore analyses showed that regions between amino acids 7 and 10 and between amino acids 11 and 14 are required for 3C10 binding. To assess the effect of altering the 3C10 epitope in CD14, we generated a stabl e cell line expressing a mutant sCD14 containing alanine substitutions in the region between amino acids 7 and 10, sCD14((7-10)A), and purif ied this protein to homogeneity. sCD14((7-10)A) has impaired ability t o mediate LPS-dependent IL-6 up-regulation in U373 cells, integrin act ivation in neutrophils, and NF-kappa B activation in U373 cells. Purif ied sCD14((7-10)A) was, however, capable of forming a stable complex w ith LPS in an LPS binding protein-facilitated and LPS binding protein- independent fashion. The ability of sCD14((7-10)A) to bind LPS was als o demonstrated in assays in which excess sCD14((7-10)A) inhibited LPS- mediated tumor necrosis factor-alpha production in whole blood and adh esion of polymorphonuclear leukocytes to fibrinogen. These data strong ly suggest that a region recognized by neutralizing monoclonal antibod y 3C10 contains a domain required for cellular signaling but not for L PS binding.