title: Lipopolysaccharide-binding protein is bound and internalized by host cells and colocalizes with LPS in the cytoplasm: Implications for a role of LBP in intracellular LPS-signaling.
abstract: The lipopolysaccharide-binding protein (LBP) is critically involved in innate immune responses to Gram-negative infections. We show here that human peripheral blood-derived monocytes, but not lymphocytes, stain positive for endogenous LBP on the cell surface. Studies on human macrophages demonstrate LBP binding at normal serum concentrations of 1-10 μg/ml. Binding was increased in a concentration-dependent manner by lipopolysaccharide (LPS). Fluorescence quenching experiments and confocal microscopy revealed constitutive and LPS-induced internalization of LBP by macrophages. Experiments with macrophages and HEK293 cell lines showed that binding and uptake of LBP do not depend on the LPS receptors CD14 and TLR4/MD-2. Fractionation of Triton X-100 solubilized cytoplasmic membranes revealed that LBP was primarily localized in non-raft domains under resting conditions. Cellular LPS stimulation elevated LBP levels and induced enrichment in fractions marking the transition between non-raft and raft domains. LBP was found to colocalize with LPS at the cytoplasmic membrane and in intracellular compartments of macrophages. In macrophages stimulated with LPS and ATP for inflammasome activation, LBP was observed in close vicinity to activated caspases. Furthermore, LBP conferred IL-1β production by LPS in the absence of ATP. These data establish that LBP serves not only as an extracellular LPS shuttle but in addition facilitates intracellular transport of LPS. This observation adds a new function to this central immune regulator of LPS biology and raises the possibility for a role of LBP in the delivery of LPS to TLR4-independent intracellular receptors.
authors.affiliation: Division of Immunobiophysics, Priority Area Infections, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, 23845 Borstel, Germany.
authors.forename: Franziska
authors.initials: F
authors.lastname: Kopp
authors.affiliation: Division of Immunobiophysics, Priority Area Infections, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, 23845 Borstel, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Kupsch
authors.affiliation: Division of Immunobiophysics, Priority Area Infections, Research Center Borstel, Leibniz-Center for Medicine and Biosciences, 23845 Borstel, Germany. Electronic address: aschromm@fz-borstel.de.
authors.forename: Andra B
authors.initials: AB
authors.lastname: Schromm
mesh_terms: D000209:Acute-Phase Proteins / Q000378:metabolism*; D001692:Biological Transport; D002352:Carrier Proteins / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D004705:Endocytosis; D057809:HEK293 Cells; D006801:Humans; D007963:Leukocytes, Mononuclear / Q000378:metabolism; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D015398:Signal Transduction / Q000502:physiology; D014018:Tissue Distribution; D051197:Toll-Like Receptor 4 / Q000378:metabolism; D000099231:Lipopolysaccharide-Binding Protein
chemical_list: D000209:Acute-Phase Proteins; D002352:Carrier Proteins; D008070:Lipopolysaccharides; D008562:Membrane Glycoproteins; D051197:Toll-Like Receptor 4; D000099231:Lipopolysaccharide-Binding Protein; C495345:TLR4 protein, human
keywords: Lipopolysaccharide; Lipopolysaccharide-binding protein; Macrophage; TLR4