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Cross-linking of lipopolysaccharide (lps) to cd14 on thp-1 cells mediated by lps-binding protein

Academic Article
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Overview

authors

  • Tobias, Peter
  • Soldau, K.
  • Kline, Lawrence
  • Lee, Jiing-Dwan
  • Kato, K.
  • Martin, T. P.
  • Ulevitch, Richard

publication date

  • 1993

journal

  • Journal of Immunology  Journal

abstract

  • Recent work has established that bacterial endotoxin (LPS) binds to the plasma protein LPS-binding protein (LBP) forming high affinity complexes (LPS-LBP), that LBP is an opsonin for LPS-bearing particles, and that LPS-LBP complexes are potent agonists for monocytic cells (MO). mAb to the MO plasma membrane protein, CD14, inhibit LBP-dependent binding of LPS to MO, and LPS-LBP-dependent stimulation of cytokine release from MO. These data suggest that CD14 functions as a membrane receptor for LPS but do not demonstrate a direct association of LPS with CD14. Calcitriol was used to induce a high level of CD14 expression in the human monocyte-like cell line THP-1, resulting in enhanced responses of these cells to LPS-LBP complexes manifested by enhanced binding of LPS and a decrease in the amount of LPS needed to induce IL-8 release. An Re595 LPS derivative containing a radioiodinated, photoreactive, phenyl azide (125I-ASD-LPS) was used in cross-linking experiments to identify membrane proteins in calcitriol-treated THP-1 cells that interact with LPS. 125I-ASD-LPS was added to calcitriol-induced THP-1 cells in the presence or absence of LBP, the mixture photolyzed, and the resultant radioiodinated proteins analyzed by SDS-PAGE and autoradiography. We observed strong cross-linking of 125I-ASD-LPS to a 55-kDa membrane protein when LBP was present, but failed to observe radiolabeling of any other proteins with apparent molecular masses distinct from CD14. The cross-linked product was identified as CD14 by immunoprecipitation with anti-human CD14 mAb. Studies with human CD14 expressing transfectants of the murine B cell line 70Z/3 also revealed LBP-dependent cross-linking of 125I-ASD-LPS to CD14. These data document binding of LPS to a specific membrane protein that serves as an LPS receptor.

subject areas

  • Acute-Phase Proteins
  • Animals
  • Antigens, CD
  • Antigens, CD14
  • Antigens, CD18
  • Antigens, Differentiation, Myelomonocytic
  • B-Lymphocytes
  • Calcitriol
  • Carrier Proteins
  • Cell Line
  • Cross-Linking Reagents
  • DNA
  • Humans
  • Lipopolysaccharides
  • Membrane Glycoproteins
  • Mice
  • Monocytes
  • Transfection
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Identity

International Standard Serial Number (ISSN)

  • 0022-1767

PubMed ID

  • 7681085
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Additional Document Info

start page

  • 3011

end page

  • 3021

volume

  • 150

issue

  • 7

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