Thymoquinone suppressses in vitro production of IL-5 and IL-13 by mast cells in response to lipopolysaccharide stimulation.
OBJECTIVE
Activated mast cells produce Th2 cytokines that regulate allergic inflammation. We have previously shown that thymoquinone (TQ) attenuated airway inflammation in a mouse model of allergic airway inflammation. The present study investigated whether TQ affects Th2 cytokine response in vitro in lipopolysaccharide (LPS)-activated rat mast cells, RBL-2H3.


METHODS
RBL-2H3 cells were stimulated for 12 h with 0.1 microg/ml LPS in the presence or absence of 10 microM TQ. Th2 cytokine production was measured in the culture supernatants by ELISA. The mRNA expression of IL-5, IL- 13 and GATA transcription factors was determined by RT-PCR. The expression of the transcription proteins c-Fos, c- Jun and phospho-c-Jun were determined by western blotting. The in vivo binding of GATA, AP-1 and NF-AT transcription factors to IL-5 promoter was assessed by chromatin immunoprecipitation analysis.


RESULTS
TQ significantly (p <0.05) inhibited LPS-induced IL-5 and IL-13 mRNA expression and protein production. However, TQ did not affect IL-10 production. GATA transcription factors are involved in the transcription of IL-5 and IL-13. TQ had no effect on the expression of AP-1 protein subunits, c-Jun and c-Fos, but markedly reduced the transcription of GATA-1 and -2 genes. Chromatin immunoprecipitation revealed that GATA, AP-1 and NF-AT binding to IL-5 promoter was induced by LPS stimulation and that TQ inhibited GATA binding at the IL-5 promoter but did not affect AP-1 and NF-AT binding.


CONCLUSIONS
These results suggest that TQ inhibits LPS-induced proinflammatory cytokine production in RBL-2H3 cells by blocking GATA transcription factor expression and promoter binding which demonstrates the anti-inflammatory effect of TQ.
Department of Medicine, University of Colorado Health Sciences Center, Denver, CO 80262, USA. melgazza@wfubmc.edu
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El Gazzar
D000818:Animals; D000893:Anti-Inflammatory Agents / Q000494:pharmacology; D016227:Benzoquinones / Q000494:pharmacology*; D002478:Cells, Cultured; D050982:GATA1 Transcription Factor / Q000235:genetics; D050989:GATA2 Transcription Factor / Q000235:genetics; D018793:Interleukin-13 / Q000096:biosynthesis*; D015848:Interleukin-5 / Q000096:biosynthesis* / Q000235:genetics; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008407:Mast Cells / Q000187:drug effects / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism; D011401:Promoter Regions, Genetic; D051381:Rats; D018808:Transcription Factor AP-1 / Q000378:metabolism
D000893:Anti-Inflammatory Agents; D016227:Benzoquinones; D050982:GATA1 Transcription Factor; D050989:GATA2 Transcription Factor; C494690:Gata1 protein, rat; C494713:Gata2 protein, rat; D018793:Interleukin-13; D015848:Interleukin-5; D008070:Lipopolysaccharides; D016328:NF-kappa B; D018808:Transcription Factor AP-1; C003466:thymoquinone