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 pre-treated 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 which is partially independent of CD14, but shortening or removal of the O-Antigen inhibits this secretion.

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“…In particular, the level of IL-33 and the magnitude of 15,18 Previous studies implicated that LPS signal transduction lead to activation of MAP kinases and cytokine productions in monocytes. 19,20 Our results showed that treatment with LPS activated all p38, JNK and ERK kinases in monocytes. In addition, the pretreatment of IL-33 in monocytes synergically promoted the LPS-stimulated cytokine secretion through ERK1/2, but not p38 and JNK, suggesting the molecular mechanism involved in IL-33 signalling in response to the overwhelming systemic inflammation.…”

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