ABSTRACT
NF-kappaB is a dimeric transcription factor which regulates transcription of a number of different genes including IL-8 and p53. In resting cells NF-kappaB is usually retained in an inactive state in the cytoplasm through binding to a member of the inhibitory kappaB (IkappaB) protein family. The purpose of this study was to determine the effect of 1alpha,25(OH)(2)D(3) on NF-kappaB activation in both unstimulated and stimulated (IL-1alpha) cultured normal human keratinocytes. NF-kappaB DNA binding activity was determined by EMSA using two different oligonucleotides containing the kappaB sequence from either the IL-8 or the p53 promoter. IkappaBalpha and p53 expression was determined by Western blotting and IL-8 expression by ELISA. In unstimulated keratinocytes no NF-kappaB binding to the IL-8 kappaB binding sequence was detectable, whereas stimulation with IL-1alpha (10 ng/ml) led to a significant ( P<0.05) induction of NF-kappaB binding. In contrast NF-kappaB binding to the p53 kappaB binding sequence was detectable in unstimulated cells, although it was significantly increased after IL-1alpha (10 ng/ml) stimulation. Incubation with 1alpha,25(OH)(2)D(3) (10(-8)-10(-7) M) was shown to significantly ( P<0.05) stimulate the expression of IkappaBalpha and in parallel experiments with normal human keratinocytes stimulated with IL-1alpha (10 ng/ml) a significant ( P<0.05) time and dose-dependent decrease in NF-kappaB binding to the IL-8 kappaB binding sequence and in IL-8 expression were seen. A less-pronounced decrease in NF-kappaB binding to the p53 kappaB response element was seen after preincubation with 1alpha,25(OH)(2)D(3) and IL-1alpha stimulation, and it did not result in any change in p53 expression. These results demonstrate that 1alpha,25(OH)(2)D(3) inhibits NF-kappaB binding to the IL-8 kappaB binding sequence more potently than binding to the p53 kappaB binding sequence. We propose that this selectivity may be mediated through an increased expression of IkappaBalpha which leads to an inhibition of specific NF-kappaB subunits resulting in a selective regulation of NF-kappaB-induced gene transcription.
Subject(s)
DNA/metabolism , I-kappa B Proteins/metabolism , Keratinocytes/metabolism , NF-kappa B/genetics , Vitamin D/analogs & derivatives , Vitamin D/pharmacology , Adult , Blotting, Western , Cells, Cultured , Electrophoresis , Enzyme-Linked Immunosorbent Assay , Humans , Interleukin-1/pharmacology , Interleukin-8/metabolism , Tumor Suppressor Protein p53/metabolismABSTRACT
Tumor cells treated with IL-10 were shown to have decreased, but peptide-inducible expression of MHC class I, decreased sensitivity to MHC class I-restricted CTL, and increased NK sensitivity. These findings could be explained, at least partially, by a down-regulation of TAP1/TAP2 expression. In this study, IT9302, a nanomeric peptide (AYMTMKIRN), homologous to the C-terminal of the human IL-10 sequence, was demonstrated to mimic these previously described IL-10 effects on MHC class I-related molecules and functions. We observed a dose-dependent down-regulation of MHC class I at the cell surface of melanoma cells after 24-h treatment with IT9302. The IL-10 homologue peptide also caused a dose-dependent inhibition of the IFN-gamma-mediated surface induction of MHC class I in a melanoma cell line. We demonstrated, using Western blot and flow cytometry, that IT9302 inhibits the expression of TAP1 and TAP2 proteins, but not MHC class I H chain or low molecular protein molecules. Finally, peptide-treated melanoma cells were shown to be more sensitive to lysis by NK cells in a dose-dependent way. Taken together, these results demonstrate that a small synthetic peptide derived from IL-10 can mimic the Ag presentation-related effects mediated by this cytokine in human melanomas and increase tumor sensitivity to NK cells, which can be relevant in the designing of future strategies for cancer immune therapy.
Subject(s)
ATP-Binding Cassette Transporters/biosynthesis , Eye Neoplasms/metabolism , Gene Expression Regulation, Neoplastic/drug effects , Histocompatibility Antigens Class I/biosynthesis , Interleukin-10/agonists , Melanoma/metabolism , Neoplasm Proteins/biosynthesis , Oligopeptides/pharmacology , ATP Binding Cassette Transporter, Subfamily B, Member 2 , ATP Binding Cassette Transporter, Subfamily B, Member 3 , ATP-Binding Cassette Transporters/genetics , Cell Line, Tumor/drug effects , Cell Line, Tumor/metabolism , Cysteine Endopeptidases/biosynthesis , Cysteine Endopeptidases/genetics , Cytotoxicity, Immunologic , Dose-Response Relationship, Drug , Eye Neoplasms/pathology , Genes, MHC Class I , Humans , Interferon-gamma/antagonists & inhibitors , Interferon-gamma/pharmacology , Interleukin-10/chemistry , Killer Cells, Lymphokine-Activated/immunology , Melanoma/pathology , Neoplasm Proteins/genetics , Protein Structure, Tertiary , Recombinant ProteinsABSTRACT
In a recent study we have demonstrated that interleukin 8 (IL-8) and tumour necrosis factor alpha (TNF-alpha) serum levels correlate positively with the severity of acute pancreatitis (AP), induced by bile acid injected into the pancreatic duct of rabbits. In this article we describe the effect of an IL-10 analogue IT9302 and a monoclonal anti-IL-8 (mon. IL-8) antibody on the content of several pro-inflammatory cytokines in the serum of rabbits, after induction of AP. We found that the serum content of inflammatory cytokines IL-8, IL-1beta, TNF-alpha and monocyte chemoattractant protein 1 (MCP-1) are increased during AP. Injection of IT9302 or mon. IL-8 antibody, diminish the concentration of these cytokines in the serum, with the exception that mon. IL-8 antibody actually increased the circulating level of MCP-1. In addition, intravenous administration of IT9302 increased the serum levels of IRAP, an IL-1beta receptor antagonistic cytokine. Furthermore, intravenous injection of mon. IL-8 antibody increased serum levels of IL-4. It can be concluded that both the human IL-10 analogue IT9302 and mon. IL-8 antibody are able to alter the pro-inflammatory cytokine levels in rabbits suffering from experimentally induced AP.
