As a key receptor for lipopolysaccharide (LPS) on the surface of monocytes and macrophages, the CD14 molecule is primarily involved in non-specific host defense mechanisms against gram-negative bacteria. To delineate the structural basis of LPS binding, 23 mutants in the N-terminal 252 amino acids of human CD14 were generated and stably transfected into CHO cells. In each mutant, a block of five amino acids was substituted by alanine. Reactivity of the mutants with anti-CD14 mAbs, and their ability to interact with LPS and Escherichia coli were tested. serum, which is directly secreted or derived from protease-dependent shedding of the membrane-bound molecule [l 1 -141. As a soluble LPS-receptor, sCD14 competes with mCDt4 for LPS binding and is able to neutralize LPS-induced responses in vitro and in vivo [7,[15][16][17]. In addition to this, however, sCD14 mediates the LPS-induced activation of non-CD14-expressing endothelial, epithelial and smooth-muscle cells 118-221.Because the LPS molecule and its receptor and carrier proteins are primary targets for therapeutic-intervention strategies, the knowledge of ligand-receptor interactions on a molecular level may provide a rationale for the development of specific drugs that interfere with LPS binding or with LPS signaling. Therefore, different approaches have been used to identify the LPS-binding domain of human CDI 4. Viriyakosol and Kirkland 1231 constructed a series of small deletion mutants of the hydrophilic regions within the first 65 residues of the mature protein. They could not demonstrate serum-dependent binding of 'H-labeled LPS to any of their mutant proteins, though only CHO cells transfected with a deletion mutant spanning amino acids 35-39 failed to respond to LPS by translocation of NFKB. By sequential deletion of the C-terminal part of the molecule, Juan et al. [24] showed that CD14-(1-152)-peptide is able to recognize LPS and to mediate cellular responses induced by LPS. In a further study from the same group, an epitope of CD14 was defined, which is protected by LPS from cleavage by endoproteinase Asp-N 1251. They showed that a deletion mutant covering amino acids 57-64 does not interact with LPS In this study we have performed an alanine scan of amino acids 1 to 152 of human CD14. 23 alanine-substitution mutants have been constructed and stably expressed in CHO cells. We analyzed whether mutant proteins expressed on the surface of 1261.
Legionella pneumophila, a gram-negative bacterium causing Legionnaires’ disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in theLimulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.
The CD14 molecule expressed on monocytes and macrophages is a high-affinity receptor for bacterial lipopolysaccharide (LPS) and hence an important component of the innate immune system. LPS binding protein (LBP) is required to facilitate the binding of LPS to CD14 in vitro and is necessary for the induction of an inflammatory response to LPS in vivo. Here we show that CD14 and LBP can also bind to lipoteichoic acid from the gram-positive bacterium Bacillus subtilis. Although CD14 does not interact with intact B. subtilisorganisms, a brief exposure of the bacteria to serum converts them into a form which can bind to CD14 in an LBP-dependent reaction. When serum-pretreated B. subtilis organisms are incubated with the myelomonocytic cell line U937, which expresses CD14, the bacteria are rapidly phagocytosed. The phagocytosis is strictly dependent both on LBP and on CD14. These in vitro results suggest that LBP plays a role in the innate response not only to gram-negative but also to gram-positive infections.
Legionella pneumophila, a gram-negative bacterium causing Legionnaires' disease and Pontiac fever, was shown to be highly reactive in in vitro gelation of Limulus lysate but not able to induce fever and the local Shwartzman reaction in rabbits and mice. We analyzed the capacity of purified L. pneumophila lipopolysaccharide (LPS-Lp) to induce activation of the human monocytic cell line Mono Mac 6, as revealed by secretion of proinflammatory cytokines and desensitization to subsequent LPS stimulation. We showed that despite normal reactivity of LPS-Lp in the Limulus amoebocyte lysate assay, induction of cytokine secretion in Mono Mac 6 cells and desensitization to an endotoxin challenge required LPS-Lp concentrations 1,000 times higher than for LPS of Salmonella enterica serovar Minnesota. Therefore, we examined the interaction of LPS-Lp with the LPS receptor CD14. We demonstrated that LPS-Lp did not bind to membrane-bound CD14 expressed on transfected CHO cells, nor did it react with soluble CD14. Our results suggest that the low endotoxic potential of LPS-Lp is due to a failure of interaction with the LPS receptor CD14.
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