NF-kappaB activation is required for C5a-induced interleukin-8 gene expression in mononuclear cells.
C5a, a potent peptide chemoattractant, stimulates interleukin-8 (IL-8) secretion from peripheral blood mononuclear cells (PBMC). Experiments were conducted to understand the mechanisms for C5a-induced IL-8 production, which was 14-fold greater than that in unstimulated cells by 2 hours. IL-8 secretion was accompanied by accumulation of IL-8 mRNA in the cytosol and by nuclear expression of a kappaB DNA binding activity within 30 minutes. AP-1 but not NF-IL-6 DNA binding activity was also detected in C5a-stimulated PBMC; however, its delayed expression (maximal at 4 hours) suggested a less important role in the rapid production of IL-8. The correlation between C5a-induced kappaB binding activity and IL-8 gene expression was examined in the RAW264.7 macrophage cells using reporter genes directed by the kappaB sequence from IkappaBalpha and IL-8 promoter regions. C5a-induced reporter gene expression was abolished by introducing mutations into the kappaB sites and by coexpression of a dominant negative IkappaBalpha construct resistant to agonist-induced phosphorylation. Pertussis toxin, which ADP-ribosylates the Gi proteins known to couple to the C5a receptor, produced minimal inhibition of C5a-induced IL-8 expression and had little effect on C5a-induced calcium mobilization in RAW264.7 cells. These results suggest that NF-kappaB activation is required for C5a-induced IL-8 gene expression and that this response is mediated primarily through a pertussis toxin-insensitive pathway.
Hsu
M H
MH
Department of Immunology, The Scripps Research Institute, La Jolla, CA, USA.
Wang
M
M
Browning
D D
DD
Mukaida
N
N
Ye
R D
RD
D000818:Animals; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015936:Complement C5a / Q000276:immunology / Q000494:pharmacology*; D003600:Cytosol / Q000276:immunology; D005786:Gene Expression Regulation / Q000276:immunology*; D017930:Genes, Reporter; D006801:Humans; D016209:Interleukin-8 / Q000096:biosynthesis / Q000235:genetics*; D007700:Kinetics; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000494:pharmacology; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D016209:Interleukin-8; D016328:NF-kappa B; D011994:Recombinant Proteins; D018808:Transcription Factor AP-1; D015936:Complement C5a