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Related Experiment Videos

CD14-dependent endotoxin internalization via a macropinocytic pathway

C Poussin1, M Foti, J L Carpentier

  • 1Division of Medical Intensive Care, Department of Medicine, University Hospital of Geneva, 24 r. Micheli-du-Crest, 1211 Geneva 14, Switzerland.

The Journal of Biological Chemistry
|August 1, 1998
PubMed
Summary

Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) internalization and CD14 glycoprotein trafficking occur via macropinocytosis, a pathway distinct from classical endocytosis. This process is independent of LPS-induced cell activation.

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Area of Science:

  • Immunology
  • Cell Biology
  • Microbiology

Background:

  • Gram-negative bacterial endotoxin (lipopolysaccharide, LPS) binds to CD14, a myeloid glycoprotein, initiating cell activation and LPS clearance.
  • The precise mechanisms of LPS and CD14 internalization and the role of CD14's GPI anchor in this process remain incompletely understood.

Purpose of the Study:

  • To investigate the internalization pathways of LPS and CD14.
  • To determine the influence of the CD14 GPI anchor on its endocytic route.
  • To explore the relationship between LPS/CD14 internalization and LPS-induced cell activation.

Main Methods:

  • Utilized THP-1 cells expressing different CD14 forms (GPI-anchored vs. chimeric integral).
  • Employed sucrose density gradients to analyze membrane subdomains.

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  • Conducted electron microscopy and confocal microscopy for internalization pathway analysis.
  • Investigated the effect of cytochalasin D (macropinocytosis inhibitor) on LPS/CD14 uptake and cell activation.

    • Main Results:

    • Both GPI-anchored and chimeric CD14 localized to microvilli/ruffles but sorted to distinct membrane subdomains.
    • CD14 internalization occurred via macropinocytosis, forming large vesicles and subsequently phago-lysosomes.
    • LPS internalization paralleled CD14 uptake, with co-localization observed at the cell surface and in endosomes.
    • Cytochalasin D inhibited LPS and CD14 internalization but did not affect LPS-dependent cell activation.

    Conclusions:

    • CD14 and LPS are internalized through macropinocytosis, a pathway separate from clathrin-coated pits and caveolae.
    • CD14's GPI anchor influences its plasma membrane localization but not its macropinocytosis-mediated internalization.
    • LPS internalization and LPS-induced cell activation are distinct, dissociable processes.