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An interplay between scavenger receptor A and CD14 during activation of J774 cells by high concentrations of LPS

Maciej Czerkies1, Kinga Borzęcka, Mykola I Zdioruk

  • 1Department of Cell Biology, Nencki Institute of Experimental Biology, 02-093 Warsaw, Poland.

Immunobiology
|May 15, 2013
PubMed

Insights

High concentrations of lipopolysaccharide (LPS) are internalized by macrophages via scavenger receptor A (SR-A), independent of TLR4. SR-A is crucial for maximal TNF-α production, highlighting its role in inflammatory responses.

Area of Science:

  • Immunology
  • Cell Biology
  • Molecular Biology

Background:

  • Lipopolysaccharide (LPS) activates macrophages through TLR4/MD-2, initiating pro-inflammatory pathways via MyD88 and TRIF.
  • High-dose LPS can be internalized for detoxification, a process influenced by endocytic pathways.

Purpose of the Study:

  • To investigate the mechanisms of high-concentration LPS uptake and its impact on macrophage inflammatory mediator production.
  • To elucidate the role of scavenger receptor A (SR-A) and its interaction with CD14 and TLR4 in LPS-induced responses.

Main Methods:

  • Utilized macrophage-like J774 cells treated with high-concentration LPS (1000ng/ml).
  • Investigated the effects of inhibiting clathrin- and dynamin-mediated endocytosis on LPS uptake and mediator production.
  • Examined the role of SR-A, CD14, and TLR4 using specific antibodies, dextran sulfate, and gene silencing (siRNA).
  • Quantified the production of tumor necrosis factor-alpha (TNF-α) and RANTES.

Main Results:

  • Inhibition of clathrin/dynamin-mediated endocytosis increased LPS uptake but reduced TNF-α and RANTES production.
  • LPS binding and uptake at high concentrations were mediated by SR-A and CD14, independent of TLR4.
  • SR-A occupation enhanced LPS-induced TNF-α and RANTES production, with CD14 acting as a limiting factor.
  • Dextran sulfate increased cell surface TLR4 and CD14 levels, potentially contributing to enhanced pro-inflammatory responses.
  • Silencing SR-A inhibited LPS-induced TNF-α production, while RANTES release remained unaffected.

Conclusions:

  • Scavenger receptor A (SR-A) plays a critical role in the maximal production of TNF-α following LPS stimulation in macrophages.
  • SR-A may modulate inflammatory responses by influencing the cell surface expression of TLR4 and CD14.
  • These findings reveal a novel pathway for LPS interaction with macrophages involving SR-A, distinct from the canonical TLR4 pathway for high-dose uptake.
Keywords:
CD14CTX-FITCEndocytosisHEPES-buffered salineLBPLPS conjugated with Alexa Fluor 488LPS-AF488LPS-binding proteinLipopolysaccharidePD bufferSR-AScavenger receptorTLRpoly(I:C)polyinosinic–polycytidylic acidscavenger receptor Asubunit B of cholera toxin conjugated with FITCtransferrin conjugated with Alexa Fluor 647transferrin-AF647

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