CD14-dependent endotoxin internalization via a macropinocytic pathway

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.
• 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.