Pyrrolidine dithiocarbamate inhibits the production of interleukin-6, interleukin-8, and granulocyte-macrophage colony-stimulating factor by human endothelial cells in response to inflammatory mediators: modulation of NF-kappa B and AP-1 transcription factors activity.
Endothelial cells (EC) play a key role in the inflammatory response, both by the production of proinflammatory cytokines and by their interaction with leukocytes. Molecular genetic analysis has demonstrated that functional NF-kappa B sites are involved in the transcription of interleukin-6 (IL-6), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes in response to inflammatory mediators. Thus, we have explored the effect of two inhibitors of the NF-kappa B activation, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), on the production of these cytokines by EC. Both PDTC and NAC inhibited, in a dose-dependent manner, the synthesis of IL-6, IL-8, and GM-CSF induced by tumor necrosis factor (TNF)-alpha or bacterial lipopolysaccharides (LPS) in human umbilical vein endothelial cells (HUVEC). PDTC appeared to prevent IL-6, IL-8, and GM-CSF gene transcription, as it blocked the induction of specific mRNA by TNF-alpha or LPS. The TNF-alpha mediated transcriptional activation of a chloramphenicol acetyltransferase (CAT) plasmid containing three copies of the -72 kappa B binding site from the IL-6 promoter was abrogated by PDTC. According to transfection experiments, electrophoretic mobility shift assays (EMSA) demonstrated that the antioxidant prevented the induction of NF-kappa B DNA-binding activity by TNF-alpha. Under the same conditions, PDTC by itself or in combination with TNF-alpha, enhanced the DNA-binding activity of AP-1, as well as c-fos and c-jun mRNA levels. Altogether, these results indicate that the antioxidant PDTC specifically inhibits the transcription of IL-6, IL-8, and GM-CSF genes through the inhibition of the NF-kappa B activation, while increasing the expression of AP-1. Our data make evident the antiinflammatory and immunoregulatory potential of the pharmacological inhibition of the NF-kappa B activation. In addition, PDTC and related molecules may be a useful tool to explore the expression of genes involved in the inflammatory response.
Muñoz
C
C
Servicio de Inmunología, Hospital de la Princesa, Universidad Autónoma de Madrid, Spain.
Pascual-Salcedo
D
D
Castellanos
M C
MC
Alfranca
A
A
Aragonés
J
J
Vara
A
A
Redondo
J M
JM
de Landázuri
M O
MO
D000111:Acetylcysteine / Q000494:pharmacology; D000975:Antioxidants / Q000494:pharmacology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000378:metabolism*; D016166:Free Radical Scavengers / Q000494:pharmacology; D005786:Gene Expression Regulation; D016179:Granulocyte Colony-Stimulating Factor / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D015850:Interleukin-6 / Q000096:biosynthesis* / Q000235:genetics; D016209:Interleukin-8 / Q000096:biosynthesis* / Q000235:genetics; D016328:NF-kappa B / Q000235:genetics; D011759:Pyrrolidines / Q000494:pharmacology*; D013859:Thiocarbamates / Q000494:pharmacology*; D018808:Transcription Factor AP-1 / Q000235:genetics*
D000975:Antioxidants; D016166:Free Radical Scavengers; D015850:Interleukin-6; D016209:Interleukin-8; D016328:NF-kappa B; D011759:Pyrrolidines; D013859:Thiocarbamates; D018808:Transcription Factor AP-1; D016179:Granulocyte Colony-Stimulating Factor; C020972:pyrrolidine dithiocarbamic acid; D000111:Acetylcysteine