Binding of the lipid A portion of bacterial lipopolysaccharide (LPS) to leukocyte CD14 activates phagocytes and initiates the septic shock syndrome. Two lipid A analogs, lipid IVA and Rhodobacter sphaeroides lipid A (RSLA), have been described as LPS-receptor antagonists when tested with human phagocytes. In contrast, lipid IVA activated murine phagocytes, whereas RSLA was an LPS antagonist. Thus, these compounds displayed a species-specific pharmacology. To determine whether the species specificity of these LPS antagonists occurred as a result of interactions with CD14, the effects of lipid IVA and RSLA were examined by using human, mouse, and hamster cell lines transfected with murine or human CD14 cDNA expression vectors. Several precursors and analogs of the toxic lipid A moiety from Escherichia coli LPS have been shown to inhibit LPS activation of lymphocytes, neutrophils, monocytes, and macrophages. By increasing the concentration of LPS relative to the concentration of antagonist, inhibition by these agents was overcome, suggesting that they competed with LPS for binding to a specific component of the LPS-signaling system. For example, Rhodobacter sphaeroides lipid A (RSLA) exhibited LPS antagonist properties in both murine (15-17) and human LPS-responsive cells (15,18). In contrast to RSLA, the tetraacyldisaccharide lipid A precursor, designated lipid IVA, inhibited LPS-induced activation of human cells (15,18,19) but acted as an LPS mimetic in murine cells (15,18), demonstrating a species-specific effect of these LPS-receptor antagonists.Kitchens et al. (20,21) reported that lipid IVA, when used in nanomolar concentrations under physiologic conditions, effectively blocked LPS-induced activation of human monocytes, whereas micromolar concentrations of lipid IVA were required to block specific binding of LPS to surface CD14. The difference between the concentration of LPS antagonists required to inhibit signal transduction compared to concentrations required to block specific binding of LPS to CD14 suggested that CD14 was not the cellular target for antagonists such as lipid IVA. Collectively, these findings are consistent with a model of signal transduction in which LPS-bound CD14 interacts with an as-yet-unidentified protein(s) present in limiting quantities on endotoxin-responsive cells that then induces a signal-transduction event across the plasma membrane. However, a definitive interpretation of these cellbinding studies (20,21) is complicated by the problems inherent with lipophilic and amphipathic ligands such as LPS. For Abbreviations: LPS, lipopolysaccharide; FBS, fetal bovine serum; NF-KB, nuclear factor KB; IFN-y, interferon -y; RSLA, Rhodobacter sphaeroides lipid A; GPI, glycosyl-phosphatidylinositol; EMSA, electrophoretic mobility-shift assay.

Phagocytic leukocytes respond to a variety of bacterial products including Gram-negative bacterial LPS and mycobacterial lipoarabinomannan (LAM). Anti-CD14 mAbs have been shown to block LPS and LAM activation of myeloid cells, suggesting that CD14 is required for cellular recognition of both ligands. Activation of undifferentiated promonomyelocytic THP-1 cells with LAM or LPS under serum-free conditions was enhanced in the presence of recombinant soluble CD14 (rsCD14). LPS binding protein (LBP), which is present in normal serum, further enhanced the sensitivity of undifferentiated THP-1 cells to both ligands even in the absence of rsCD14. Although CD14-transfected Chinese hamster ovary and human HT1080 fibrosarcoma cell lines can be activated by LPS, neither cell line was activated by LAM. Furthermore, U373 astrocytoma cells, which respond to LPS using sCD14 and LBP, failed to be activated by LAM in the presence of rsCD14 and rLBP. We then tested the effects of lipid IVA and Rhodobacter sphaeroides lipid A, compounds that function as endotoxin inhibitors in human cells by interacting with a molecule thought to be a CD14-dependent LPS signal transducer. Both lipid IVA and R. sphaeroides lipid A inhibited the effects of LPS and LAM in THP-1 cells. Thus, the LPS and LAM receptors share CD14, LBP, and a putative endotoxin antagonist-inhibitable signal transducing component. However, the LAM signaling system appears to require an additional receptor component whose expression is restricted to cells of hemopoietic origin.