Specific signaling pathways in the regulation of TNF-alpha mRNA synthesis and TNF-alpha secretion in RBL-2H3 mast cells stimulated through the high affinity IgE receptor.
OBJECTIVE
In the present study, we investigated signal transduction pathways involved in TNF-alpha gene expression and TNF-alpha secretion by mast cells stimulated through the high affinity IgE receptor (FcepsilonRI).


METHODS
TNF-alpha mRNA steady state levels and TNF-alpha secretion in the presence of specific pharmacological agents were monitored using rat basophilic leukemia cells (RBL-2H3) stimulated through FcepsilonRI. Relative amounts of TNF-alpha mRNA versus beta-actin levels were quantified by RNase protection and RT-PCR assays. TNF-alpha secretion was measured by a current ELISA test.


RESULTS
We show that EGTA (5 mM) prevented TNF-alpha mRNA expression and TNF-alpha secretion in antigen-stimulated cells. The protein kinase C (PKC) inhibitor bisindolylmaleimide I substantially blocked TNF-alpha secretion at 2 microM but had only a marginal effect on TNF-alpha mRNA expression. The results were similar when PKC isoforms were depleted by long-term exposure to 100 nM phorbol ester (PMA). The PI 3-kinase inhibitor wortmannin blocked TNF-alpha secretion at low doses (EC50= 13 nM), but only partially affected mRNA expression.


CONCLUSIONS
Our results show that in FcepsilonRI-stimulated RBL-2H3 cells calcium mobilization, activation of PKC and PI 3-kinase are necessary for TNF-alpha secretion while for the increased TNF-alpha mRNA expression PKC activity is dispensable and PI 3-kinase activity only partially required.
Pelletier
C
C
Institut Pasteur, Unité d'Immuno-Allergie, Paris, France.
Guérin-Marchand
C
C
Iannascoli
B
B
Marchand
F
F
David
B
B
Weyer
A
A
Blank
U
U
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D004533:Egtazic Acid / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D007211:Indoles / Q000494:pharmacology; D015471:Leukemia, Basophilic, Acute; D008301:Maleimides / Q000494:pharmacology; D008407:Mast Cells / Q000276:immunology / Q000502:physiology*; D000081082:Phosphoinositide-3 Kinase Inhibitors; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D012333:RNA, Messenger / Q000096:biosynthesis; D051381:Rats; D017455:Receptors, IgE / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012260:Ribonucleases; D015398:Signal Transduction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics* / Q000378:metabolism*; D000077191:Wortmannin; D001619:beta-N-Acetylhexosaminidases / Q000378:metabolism
D000730:Androstadienes; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D000081082:Phosphoinositide-3 Kinase Inhibitors; D012333:RNA, Messenger; D017455:Receptors, IgE; D014409:Tumor Necrosis Factor-alpha; D004533:Egtazic Acid; D011493:Protein Kinase C; D012260:Ribonucleases; D001619:beta-N-Acetylhexosaminidases; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D000077191:Wortmannin