Meconium-induced release of cytokines is mediated by the TRL4/MD-2 complex in a CD14-dependent manner

Mol Immunol. 2010 Mar;47(6):1226-34. doi: 10.1016/j.molimm.2009.12.015. Epub 2010 Jan 22.

Abstract

Objective: Meconium, the first intestinal discharge of the newborn, contains material accumulated during fetal life. Meconium activates complement and CD14 and may induce a systemic inflammatory response. Toll-like receptors are classical pattern-recognition receptors recognizing both exogenous and host-derived ligands. The cyanobacterial product CyP is a potent LPS antagonist binding to the TLR4/MD-2 complex. The aim of the present study was to investigate the role of the CD14/TLR4/MD-2 complex in meconium-induced inflammation.

Methods: Whole blood from six donors was preincubated with anti-CD14 or CyP. Meconium was added and the samples were incubated for 4h. Twenty-seven inflammatory mediators were measured in a Bioplex Array Reader. Human embryonic kidney cells transfected with plasmids containing NF-kappaB dependent luciferase reporter, human MD-2, TLR4, TLR2 and/or CD14, were incubated with meconium or LPS for 18 h. Luciferase activity in cytoplasmic extracts was measured using a Luciferase Assay System kit.

Results: Meconium induced formation of a broad panel of inflammatory mediators. CyP and anti-CD14 significantly (p<0.001) inhibited meconium-induced formation of (a) proinflammatory cytokines (TNF-alpha, IL-1beta, IL-6, IFN-gamma) by 60-80% and 72-94%, respectively, (b) anti-inflammatory cytokines (IL-10, IL-1Ra) by 58-59% and 50-65%, respectively, (c) chemokines (IL-8, MCP-1, MIP-1alpha, MIP-1beta, eotaxin, IP-10) by 43-77% and 57-87%, respectively, and (d) growth factors (G-CSF, GM-CSF, basic FGF, PDGFbb) by 53-71% and 40-78%, respectively, with no statistical significant difference between Cyp and anti-CD14. The inflammatory response could only partly be explained by LPS, suggesting that endogenous components of meconium contribute to the inflammatory response. Meconium activated NF-kappaB dose-dependently in cells expressing TLR4/MD-2 together with CD14, while no effect was seen in cells expressing TLR4/MD-2 alone or in TLR2/CD14 transfected cells.

Conclusions: The results indicate that the CD14-dependent meconium-induced inflammatory reaction is mediated through the TLR4/MD2 complex. These data may have implications for future therapeutic strategies for meconium aspiration syndrome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adult
  • Anti-Inflammatory Agents / metabolism
  • Cell Line
  • Chemokines / biosynthesis
  • Cyanobacteria / chemistry
  • Cytokines / metabolism*
  • Humans
  • Infant, Newborn
  • Inflammation / pathology
  • Inflammation Mediators / metabolism
  • Lipopolysaccharide Receptors / metabolism*
  • Lymphocyte Antigen 96 / metabolism*
  • Meconium / drug effects
  • Meconium / metabolism*
  • NF-kappa B / metabolism
  • Polysaccharides, Bacterial / pharmacology
  • Toll-Like Receptor 4 / metabolism*
  • Transfection

Substances

  • Anti-Inflammatory Agents
  • Chemokines
  • Cytokines
  • Inflammation Mediators
  • LY96 protein, human
  • Lipopolysaccharide Receptors
  • Lymphocyte Antigen 96
  • NF-kappa B
  • Polysaccharides, Bacterial
  • TLR4 protein, human
  • Toll-Like Receptor 4