title: The Role of Lipopolysaccharide Structure in Monocyte Activation and Cytokine Secretion.
abstract: BACKGROUND
  The lipopolysaccharide (LPS) molecule is composed of a hydrophobic lipid region (Lipid A), an oligosaccharide core, and an O-Antigen chain. Lipid A has been described as the molecular region responsible for inducing activation of immune cells. We hypothesize that the O-Antigen plays a critical role in the activation and responsiveness of mononuclear cell immune function.
  
  
  METHODS
  Peripheral blood mononuclear cells (PBMCs) from healthy volunteers were stimulated with LPS, LPS with attenuated O-Antigen (RF5), or Lipid A (DPL), which lacks an O-Antigen. Selected cells were pretreated with a blocking antibody to CD14. Western blots were performed to determine activation of mitogen-activated protein kinases (MAPK) p38, ERK, and JNK at selected time-points. RNA was extracted for RT-PCR quantification of TNF-α and IL-10 gene transcription. Supernatants were harvested and analyzed by ELISA for tumor necrosis factor alpha (TNF-α) and interleukin 10 (IL-10).
  
  
  RESULTS
  LPS elicited maximal response, including phosphorylation of p38, ERK, and JNK, synthesis of TNF-α and IL-10 mRNA, and secretion of TNF-α and IL-10. Stimulation with RF5 activated the same pathways to a lesser degree. DPL led to increased phosphorylation of p38 and ERK and increased secretion of IL-10. CD14 blockade was associated with a significant decrease in cytokine secretion by LPS, and abolished cytokine secretion in cells stimulated with RF5 or DPL.
  
  
  CONCLUSIONS
  Structural variants of LPS activate monocytes differentially. The complete O-Antigen is important for maximal activation of MAPK, cytokine synthesis, and cytokine secretion. LPS with attenuated O-Antigen and Lipid A activate only certain components of these pathways. LPS with a complete O-Antigen stimulates cytokine secretion that is partially independent of CD14, but shortening or removal of the O-Antigen inhibits this secretion.
authors.affiliation: Department of Surgery, Harborview Medical Center, University of Washington, Seattle, Washington.
authors.forename: Rebecca E
authors.initials: RE
authors.lastname: Plevin
authors.forename: Megan
authors.initials: M
authors.lastname: Knoll
authors.forename: Meghan
authors.initials: M
authors.lastname: McKay
authors.forename: Saman
authors.initials: S
authors.lastname: Arbabi
authors.forename: Joseph
authors.initials: J
authors.lastname: Cuschieri
mesh_terms: D002478:Cells, Cultured; D016207:Cytokines / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D016753:Interleukin-10 / Q000096:biosynthesis / Q000235:genetics; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D008050:Lipid A / Q000276:immunology; D018950:Lipopolysaccharide Receptors / Q000276:immunology; D008070:Lipopolysaccharides / Q000737:chemistry / Q000276:immunology*; D008297:Male; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism; D019081:O Antigens / Q000276:immunology; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D014409:Tumor Necrosis Factor-alpha / Q000096:biosynthesis / Q000235:genetics
chemical_list: D016207:Cytokines; C508609:IL10 protein, human; D008050:Lipid A; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D019081:O Antigens; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D016753:Interleukin-10; D020929:Mitogen-Activated Protein Kinase Kinases