Bacterial lipopolysaccharide can enter monocytes via two CD14-dependent pathways.
Host recognition and disposal of LPS, an important Gram-negative bacterial signal molecule, may involve intracellular processes. We have therefore analyzed the initial pathways by which LPS, a natural ligand of glycosylphosphatidylinositol (GPI)-anchored CD14 (CD14-GPI), enters CD14-expressing THP-1 cells and normal human monocytes. Exposure of the cells to hypertonic medium obliterated coated pits and blocked 125I-labeled transferrin internalization, but failed to inhibit CD14-mediated internalization of [3H]LPS monomers or aggregates. Immunogold electron microscope analysis found that CD14-bound LPS moved principally into noncoated structures (mostly tubular invaginations, intracellular tubules, and vacuoles), whereas relatively little moved into coated pits and vesicles. When studied using two-color laser confocal microscopy, internalized Texas Red-LPS and BODIPY-transferrin were found in different locations and failed to overlap completely even after extended incubation. In contrast, in THP-1 cells that expressed CD14 fused to the transmembrane and cytosolic domains of the low-density lipoprotein receptor, a much larger fraction of the cell-associated LPS moved into coated pits and colocalized with intracellular transferrin. These results suggest that CD14 (GPI)-dependent internalization of LPS occurs predominantly via noncoated plasma membrane invaginations that direct LPS into vesicles that are distinct from transferrin-containing early endosomes. A smaller fraction of the LPS enters via coated pits. Aggregation, which greatly increases LPS internalization, accelerates its entry into the nonclathrin-mediated pathway.
Kitchens
R L
RL
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235, USA. RKITCH@mednet.swmed.edu
Wang
P
P
Munford
R S
RS
D000215:Acylation; D000595:Amino Acid Sequence; D002460:Cell Line; D002746:Chlorpromazine / Q000494:pharmacology; D002966:Clathrin / Q000502:physiology; D003034:Coated Pits, Cell-Membrane / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D004705:Endocytosis / Q000187:drug effects / Q000235:genetics / Q000276:immunology*; D019286:Endopeptidase K / Q000378:metabolism; D005110:Extracellular Space / Q000201:enzymology / Q000378:metabolism; D017261:Glycosylphosphatidylinositols / Q000378:metabolism; D006801:Humans; D006982:Hypertonic Solutions; D018950:Lipopolysaccharide Receptors / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D008070:Lipopolysaccharides / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009000:Monocytes / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D010587:Phagocytosis / Q000187:drug effects; D010873:Pinocytosis / Q000187:drug effects; D011973:Receptors, LDL / Q000235:genetics / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D015398:Signal Transduction / Q000276:immunology; D013395:Sucrose / Q000494:pharmacology; D014168:Transferrin / Q000378:metabolism
D002966:Clathrin; D017261:Glycosylphosphatidylinositols; D006982:Hypertonic Solutions; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D011973:Receptors, LDL; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D013395:Sucrose; D019286:Endopeptidase K; D002746:Chlorpromazine