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.
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.
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