A differential role for ceramide kinase in antigen/FcɛRI-mediated mast cell activation and function.
BACKGROUND
Mast cells are specialized secretory cells releasing multiple inflammatory mediators when activated. Activation requires antigen/IgE cross-linking of FcɛRI receptors, initiating a complex intracellular signalling cascade. Ceramide kinase (CERK) is a novel lipid kinase implicated in several inflammatory cellular signalling processes.


OBJECTIVE
We sought to investigate a role for CERK in FCɛRI/IgE-mediated mast-cell activation.


METHODS
The rat and human mast cell-lines RBL-2H3 and LAD-2, respectively, were stimulated via FcɛRI or with the active product of CERK [ceramide-1-phosphate (C-1P)]. Multiple end-points were measured by enzyme-linked immunosorbent assay; histamine (pre-formed early-phase mediator), prostaglandin D₂ (PGD₂ - rapidly metabolized early-phase mediator) and interleukin (IL) -13 (de novo transcribed late-phase mediator).


RESULTS
We demonstrated that C-1P alone induced release of histamine and PGD₂ and was additive to antigen-mediated activation. C-1P did not stimulate IL-13 by a statistically significant amount. Using a specific inhibitor of CERK, antigen-mediated release of histamine and PGD₂ was significantly inhibited. Finally, we identified that, for histamine, CERK was downstream of spleen tyrosine kinase, phosphoinositol-3 kinases and phospholipase C, but upstream of c-jun N-terminal kinase (JNK); while for PGD₂ CERK was positioned upstream of JNK, mitogen-activated protein kinase kinase and cyclooxygense.


CONCLUSIONS
We have identified a differential role for CERK in mast-cell activation and begun to elucidate its position in the mast cell-signalling cascade, thereby suggesting a model by which CERK may be mediating its effects. This type of study is essential for complete understanding of activation pathways that may eventually be used to identify new targets for drug discovery in inflammatory diseases.
Research Enabling Group, Pfizer Global Research and Development, Sandwich, Kent CT13 9NJ, UK. Chris.Hewson@pfizer.com
C A
CA
Hewson
J R
JR
Watson
W L
WL
Liu
M D
MD
Fidock
D000818:Animals; D015153:Blotting, Western; D015550:Cell Degranulation / Q000276:immunology; D002460:Cell Line; D004797:Enzyme-Linked Immunosorbent Assay; D006636:Histamine Release / Q000276:immunology; D006801:Humans; D008407:Mast Cells / Q000276:immunology* / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000276:immunology* / Q000378:metabolism; D015230:Prostaglandin D2 / Q000378:metabolism; D051381:Rats; D017455:Receptors, IgE / Q000276:immunology*; D015398:Signal Transduction / Q000276:immunology*
D017455:Receptors, IgE; D017853:Phosphotransferases (Alcohol Group Acceptor); C066324:ceramide kinase; D015230:Prostaglandin D2