Membrane-anchored forms of lipopolysaccharide (LPS)-binding protein do not mediate cellular responses to LPS independently of CD14.
Inflammatory responses of myeloid cells to LPS are mediated through CD14, a glycosylphosphatidylinositol-anchored receptor that binds LPS. Since CD14 does not traverse the plasma membrane and alternatively anchored forms of CD14 still enable LPS-induced cellular activation, the precise role of CD14 in mediating these responses remains unknown. To address this, we created a transmembrane and a glycosylphosphatidylinositol-anchored form of LPS-binding protein (LBP), a component of serum that binds and transfers LPS to other molecules. Stably transfected Chinese hamster ovary (CHO) fibroblast and U373 astrocytoma cell lines expressing membrane-anchored LBP (mLBP), as well as separate CHO and U373 cell lines expressing membrane CD14 (mCD14), were subsequently generated. Under serum-free conditions, CHO and U373 cells expressing mCD14 responded to as little as 0.1 ng/ml of LPS, as measured by NF-kappaB activation as well as ICAM and IL-6 production. Conversely, the vector control and mLBP-expressing cell lines did not respond under serum-free conditions even in the presence of more than 100 ng/ml of LPS. All the cell lines exhibited responses to less than 1 ng/ml of LPS in the presence of the soluble form of CD14, demonstrating that they are still capable of LPS-induced activation. Taken together, these results demonstrate that mLBP, a protein that brings LPS to the cell surface, does not mediate cellular responses to LPS independently of CD14. These findings suggest that CD14 performs a more specific role in mediating responses to LPS than that of simply bringing LPS to the cell surface.
Tapping
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Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Orr
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SL
Lawson
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EM
Soldau
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Tobias
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PS
D000209:Acute-Phase Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001254:Astrocytoma; D001483:Base Sequence; D016466:CHO Cells; D002352:Carrier Proteins / Q000096:biosynthesis / Q000378:metabolism / Q000502:physiology*; D002462:Cell Membrane / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D006224:Cricetinae; D004306:Dose-Response Relationship, Immunologic; D006801:Humans; D007111:Immunity, Cellular; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis; D015850:Interleukin-6 / Q000096:biosynthesis; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008070:Lipopolysaccharides / Q000276:immunology* / Q000378:metabolism* / Q000494:pharmacology; D008562:Membrane Glycoproteins; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism; D011994:Recombinant Proteins / Q000276:immunology; D014407:Tumor Cells, Cultured; D000099231:Lipopolysaccharide-Binding Protein
D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D018799:Intercellular Adhesion Molecule-1; D015850:Interleukin-6; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D016328:NF-kappa B; D011994:Recombinant Proteins; D000099231:Lipopolysaccharide-Binding Protein