Decay-accelerating factor (DAF/CD55) is a functional active element of the LPS receptor complex.

Previously, we identified an 80 kDa membrane protein (LMP80) that is capable of binding to LPS and lipid A in the presence of LBP and sCD14. LMP80 could also be detected after immuno-coprecipitation of cell membranes with LPS and lipid A, indicating a physical contact of LMP80 and LPS/lipid A. Further analysis and peptide sequencing revealed that LMP80 is identical to CD55 (decay accelerating factor, DAF), a regulatory molecule of the complement cascade. Transfection of LPS-hyporesponsive Chinese hamster ovary (CHO) cells with human CD55 resulted in the translocation of NF-B upon stimulation with LPS or lipid A. Our results demonstrate a new functional role of CD55 as a molecule able to mediate LPS-induced activation of cells that may be part of a multimeric LPS receptor complex.

The involvement of CD14 in the activation of human monocytes by peptidoglycan monomers.

BACKGROUND: Cell-wall components of Gram-positive and Gram-negative bacteria induce the production of cytokines in human peripheral blood mononuclear cells. These cytokines are the main mediators of local or systemic inflammatory reaction that can contribute to the development of innate immunity. AIMS: This study was performed to analyze the involvement of CD14 molecule in the activation of human monocytes by peptidoglycan monomer (PGM) obtained by biosynthesis from culture fluid of penicillin-treated Brevibacterium divaricatum NRLL-2311. METHODS: Cytokine release of interleukin (IL)-1, IL-6 and tumor necrosis factor-alpha from human monocytes via soluble CD14 (sCD14) or membrane-associated (mCD14) receptor using anti-CD14 monoclonal antibody (MEM-18) or lipid A structure (compound 406) was measured in bioassays. RESULTS: The results demonstrated that PGM in the presence of human serum might induce the monokine release in a dose-dependent manner. The addition of sCD14 at physiologic concentrations enhanced the PGM-induced monokine release, while the monokine inducing capacity of PGM in the presence of sCD14 was inhibited by MEM-18. Effects of PGM were also blocked by glycolipid, compound 406, suggesting the involvement of binding structures similar to those for lipopolysaccharide. CONCLUSION: Activation of human monocytes by PGM involves both forms of CD14 molecule, sCD14 and mCD14.

The internalization time course of a given lipopolysaccharide chemotype does not correspond to its activation kinetics in monocytes.

The prerequisites for the initiation of pathophysiological effects of endotoxin (lipopolysaccharide [LPS]) include binding to and possibly internalization by target cells. Monocytes/macrophages are prominent target cells which are activated by LPS to release various pro- and anti-inflammatory mediators. The aim of the present study was to establish a new method to determine the binding and internalization rate of different LPS chemotypes by human monocytes and to correlate these phenomena with biological activity. It was found that membrane-bound LPS disappears within hours from the surface being internalized into the cell. Further, a correlation between the kinetics of internalization and the length of the sugar chain as well as an inverse correlation between the time course of internalization and LPS hydrophobicity was revealed. Comparison of the internalization kinetics of different LPS chemotypes with kinetics of tumor necrosis factor alpha release and kinetics of oxidative burst did not reveal any correlation of these parameters. These findings suggest that cellular internalization of and activation by LPS are mechanisms which are independently regulated.

Identification of the 80-kDa LPS-binding protein (LMP80) as decay-accelerating factor (DAF, CD55).

The activation of immunocompetent cells by lipopolysaccharide (LPS) during severe Gram-negative infections is responsible for the pathophysiological reactions, possibly resulting in the clinical picture of sepsis. Monocytes recognize LPS mainly through the LPS receptor CD14, however, other cellular binding structures have been assumed to exist. In previous studies, we have described an 80-kDa LPS-binding membrane protein (LMP80), which is present on human monocytes as well as endothelial cells. Here we demonstrate that LMP80 is widely distributed and that it forms complexes together with LPS and sCD14. Furthermore, we report on the biochemical purification of LMP80 and its identification as decay-accelerating factor, CD55, by amino acid sequencing and cloning techniques. Our results imply a new feature of CD55 as a molecule which interacts with LPS/sCD14 complexes. However, the involvement of CD55 in LPS-induced signaling remains to be elucidated.

Components of gut bacteria as immunomodulators.

In 1885 Louis Pasteur was the first to propose that the human immune system may be influenced by microorganisms. A large body of data has since been accumulated proving this assumption to be correct. Bacteria constitute the main constituents of the microbial flora of the human digestive tract and compounds of the bacterial cell wall have been shown to play an important role in the interaction of microbes with higher organisms. These components include peptidoglycan (PG) and lipopolysaccharide (LPS) of gram-negative bacteria. Both types of molecules are potent activators of the human immune system and exert their activity through the induction of endogenous mediators which are endowed with biological activity. This review focuses on the structure and activity of LPS and PG and illustrates how these bacterial factors stimulate the immune cells resulting in desired physiological or dramatic pathophysiological responses of the host organism.

Lipopolysaccharide and peptidoglycan: CD14-dependent bacterial inducers of inflammation.

Surface structures of bacteria contribute to the microbial pathogenic potential and are capable of causing local and generalized inflammatory reactions. Among these factors, endotoxin and peptidoglycan are of particular medical importance. Both toxic bacterial polymers are now recognized to interact with the same cellular receptor, the CD14 molecule, which is expressed on different types of immune cells, in particular, monocytes/macrophages. The interaction between these bacterial activators and CD14 leads to the production of endogenous mediators such as tumor necrosis factor alpha, interleukin 1 (IL-1), and IL-6, which are ultimately responsible for phlogistic responses. The fact that CD14 recognizes not only endotoxin and peptidoglycan but also other glycosyl-based microbial polymers suggests that this cellular surface molecule represents a lectin.

Detection of lipopolysaccharide (LPS)-binding membrane proteins by immuno-coprecipitation with LPS and anti-LPS antibodies.

In this study we describe a general method for the detection and characterization of endotoxin-(lipopolysaccharide, LPS)-binding membrane proteins. In the past, experimental procedures to detect LPS-binding sites on cells were generally performed with chemically modified LPS derivates. Since any modification of a ligand may lead to a modification of its binding characteristics, the results of those studies are controversial. In our assay, cell membrane preparations are treated with free lipid A, the endotoxic center of LPS, in the presence of normal human serum. After binding of lipid A, membrane proteins are solubilized by mild detergent treatment without disruption of the lipid A-protein complexes. Addition of anti-(lipid A) mAbs and subsequent adding of protein A agarose lead to the precipitation of complexes of lipid A and its binding proteins. By SDS/PAGE and western blot, these precipitates can be screened for the presence of LPS/lipid A-binding proteins. We describe the use of this method for the immuno-coprecipitation of lipid A (or LPS) with an 80-kDa LPS-binding membrane protein (LMP80), which we have previously identified on several human cells. In addition, CD14, the well-known functional LPS receptor on monocytes and macrophages, can be detected. By means of this immuno-coprecipitation approach we could demonstrate binding of either purified LPS preparations or synthetic lipid A to these LPS/lipid A-binding membrane proteins at physiological pH under conditions in which the proteins are in their natural membranous environment.