SummaryCD14 is a 55-kD protein found both as a glycosylphosphatidyl inositol-linked protein on the surface of mononudear phagocytes and as a soluble protein in the blood. CD14 on the call membrane (mCD14) has been shown to serve as a receptor for complexes of lipopolysaccharide (LPS) with LPS binding protein, but a function for soluble CD14 (sCD14) has not been described. Here we show that sCD14 enables responses to LPS by cells that do not express CD14. We have examined induction of endothdial-leukocyte adhesion molecule 1 expression by human umbilical vein endothdial cells, interleukin 6 secretion by U373 astrocytoma cells, and cytotoxicity of bovine endothelial ceUs. None of these cell types express mCD14, yet all respond to LPS in a serumdependent fashion, and all responses are completely blocked by anti-CD14 antibodies. Immunodepletion of sCD14 from serum prevents responses to LPS, and the responses are restored by addition of sCD14. These studies suggest that a surface anchor is not needed for the function of CD14 and further imply that sCD14 must bind to additional proteins on the cell surface to associate with the cell and transduce a signal. They also indicate that sCD14 may have an important role in potentiating responses to LPS in cells lacking mCD14.

This study demonstrates that lipopolysaccharide (LPS) mediates induction of transcription factor NFB and activation of the cytomegalovirus (CMV) promoterenhancer in the SW480 cell line. These cells do not express a functional membrane CD14. The LPS response in SW480 cells was weaker and markedly slower than the tumor necrosis factor (TNF) response. Pretreatment with TNF for 72 h inhibited both TNF, tumor necrosis factor receptor (TNFR) p55, TNFR p75, and LPS-mediated activation of nuclear factor -B (NFB), whereas pretreatment with LPS only inhibited the LPS response. TNFR p55 antibody pretreatment resulted in marked inhibition of the LPS response, while pretreatment with TNFR p75 antiserum only had a weak inhibitory effect. Flowcytometric analysis showed that LPS binding as well as expression of TNFR p55 and TNFR p75 were not affected by LPS or TNF pretreatment, indicating that the observed inhibition is not due to reduction of specific binding sites at the cell surface. The results suggest that LPS signaling in SW480 cells involves intracellular components which may be depleted or inactivated via TNFR p55, indicating that the LPS and TNFR p55 pathways overlap. We propose that TNFR p55 can mediate activation of NFB and cytomegalovirus promoter-enhancer in SW480 cells via two distinct mechanisms, one which is activated only via TNFR p55 and leads to rapid activation of NFB, and another which is overlapping with the LPS pathway. Lipopolysaccharide (LPS),1 a major membrane component of Gram-negative bacteria, plays an important role in the pathogenesis of Gram-negative sepsis leading to septic shock (1). LPS is a potent stimulator of monocytes and macrophages which respond by production of tumor necrosis factor (TNF), interleukin (IL)-1, IL-6, IL-8, eicosanoids (2), and nitric oxide (3). LPS activates monocytes and macrophages via CD14, a glycosyl-phosphatidylinositol-anchored surface protein (4). However, LPS receptors other than CD14 may also contribute to LPS signaling (5-7).A wide variety of other cell types are also affected by LPS, and some of these cells do not express membrane CD14. Thus, LPS has been reported to stimulate arachidonate metabolism and surface expression of adhesion molecules in endothelial cells, and it can induce aggregation of platelets, stimulate cytokine release from mast cells and fibroblasts, and lead to generation of chemotactic factors in epithelial cells (8). Although CD14 is not present on the plasma membrane of these cells, soluble (s)CD14 present in serum is essential for their stimulation by LPS (9, 10).Transcription factors activated by LPS include NFB (10, 11) and NF-IL6 (12, 13). TNF is also known as a potent inducer of NFB as well as of other transcription factors including AP-1, NF-IL6, IRF-1, and NF-GMa (14). NFB belongs to the rel family of transcription factors which form a number of different hetero-and homodimers participating in the regulation of a large number of genes involved in the immune response (15). NFB proteins are constitutively expressed in the cytoplasm, b...

In this study we examined the involvement of human serum, recombinant lipopolysaccharide binding protein (rLBP), recombinant (r)CD14, CD14 antibodies and recombinant bactericidal permeability-increasing factor (rBPI) in the induction of TNF by Salmonella minnesota LPS of different polysaccharide chain lengths. Soluble rCD14 and rLBP markedly enhanced LPS 6261 TNF production and to a lesser degree also enhanced TNF production from Re 595 LPS and lipid A DP. Addition of anti-CD14 antibodies resulted in nearly complete inhibition of LPS 6261-induced TNF production and partial inhibition of Re 595 LPS and lipid A DP-induced TNF release. The ability of lipid A MP to induce TNF production increased with addition of rCD14. Addition of rLBP or anti-CD14 antibodies had no detectable effect on lipid A MP-induced TNF production. The effect of rBPI was also tested and the results showed that only the TNF-inducing ability from smooth LPS was completely inhibited by rBPI. Recombinant BPI was considerably less effective in inhibiting Re 595 LPS-induced TNF production, and lipid A DP was not affected by rBPI. Our data suggest that the ability of rLBP, rCD14, CD14 antibodies and rBPI to modulate LPS induced TNF production is strongly dependent on the LPS polysaccharide chain length.

Well-defined polysaccharides, such as ␤1-4-linked D-mannuronic acid (poly[M]) derived from Pseudomonas aeruginosa, induce monocytes to produce tumor necrosis factor (TNF) through a pathway involving membrane CD14. In this study we have investigated the effects of soluble CD14 (sCD14), lipopolysaccharide-binding protein (LBP), and bactericidal/permeability-increasing factor (BPI) on poly(M) binding to monocytes and induction of TNF production. We show that LBP increased the TNF production from monocytes stimulated with poly(M). Addition of sCD14 alone had only minor effects, but when it was added together with LBP, a rise in TNF production was seen. BPI was found to inhibit TNF production from monocytes stimulated with poly(M) in the presence of LBP, LBP-sCD14, or 10% human serum. Binding studies showed that poly(M) bound to LBP-and BPI-coated immunowells, while no significant binding of poly(M) to sCD14-coated wells in the absence of serum was observed. Binding of poly(M) to monocytes was also examined by flow cytometry, and it was shown that the addition of LBP or 10% human serum clearly increased the binding of poly(M) to monocytes. BPI inhibited the binding of poly(M) to monocytes in the presence of LBP, LBP-sCD14, or 10% human serum. Our data demonstrate a role for LBP, LBP-sCD14, and BPI in modulating TNF responses of defined polysaccharides.