Meconium-induced release of cytokines is mediated by the TRL4/MD-2 complex in a CD14-dependent manner.
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.
Salvesen
Bodil
B
Institute of Immunology, University of Oslo and Rikshospitalet University Hospital, Oslo, Norway. bodil.salvesen@rr-research.no
Stenvik
Jørgen
J
Rossetti
Carlo
C
Saugstad
Ola D
OD
Espevik
Terje
T
Mollnes
Tom E
TE
D000328:Adult; D000893:Anti-Inflammatory Agents / Q000378:metabolism; D002460:Cell Line; D018925:Chemokines / Q000096:biosynthesis; D000458:Cyanobacteria / Q000737:chemistry; D016207:Cytokines / Q000378:metabolism*; D006801:Humans; D007231:Infant, Newborn; D007249:Inflammation / Q000473:pathology; D018836:Inflammation Mediators / Q000378:metabolism; D018950:Lipopolysaccharide Receptors / Q000378:metabolism*; D051218:Lymphocyte Antigen 96 / Q000378:metabolism*; D008470:Meconium / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D011135:Polysaccharides, Bacterial / Q000494:pharmacology; D051197:Toll-Like Receptor 4 / Q000378:metabolism*; D014162:Transfection
D000893:Anti-Inflammatory Agents; D018925:Chemokines; D016207:Cytokines; D018836:Inflammation Mediators; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D051218:Lymphocyte Antigen 96; D016328:NF-kappa B; D011135:Polysaccharides, Bacterial; C495345:TLR4 protein, human; D051197:Toll-Like Receptor 4