ABSTRACT
BACKGROUND: Although reactive oxygen species (ROS) have been produced in both mouse bone marrow-derived dendritic cells (DCs) and XS-106 DCs by contact sensitizers and irritants in previous studies, the generation of ROS in human monocyte-derived DCs (MoDCs) and their role in contact hypersensitivity (CHS) has yet to be elucidated. OBJECTIVE: The purpose of this study was to determine whether contact allergens and irritants induce ROS in MoDCs and, if so, to evaluate the role of contact allergen and irritant induced-ROS in MoDCs in CHS. METHODS: Production of ROS was measured by 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCFDA) assay. Surface CD86 and HLA-DR molecules were detected by flow cytometry. Protein carbonylation was detected by Western blotting. RESULTS: ROS were produced by contact allergens such as dinitrochlorobenzene (DNCB) and thimerosal and the irritant benzalkonium chloride (BKC). DNCB-induced, but not BKC-induced, ROS increased surface CD86 and HLA-DR molecules on MoDCs and induced protein carbonylation. These changes were reduced in the presence of antioxidant N-acetyl cysteine. CONCLUSION: Our results suggest that DNCB-induced ROS may be different from those induced by irritant BKC. The DNCB-induced ROS may be associated with the CHS response, because they activate surface molecules on DCs that are important for generating immune reactions.
Subject(s)
Animals , Humans , Mice , Allergens , Benzalkonium Compounds , Blotting, Western , Cysteine , Dendritic Cells , Dermatitis, Contact , Dinitrochlorobenzene , Flow Cytometry , HLA-DR Antigens , Irritants , Protein Carbonylation , Reactive Oxygen Species , ThimerosalABSTRACT
PURPOSE: Echinacea, a traditional plant medicine has been used as immune-stimulant. Recent studies have revealed that extract of Echinacea has immunostimulatory effects on human blood mononuclear cells. This study was designed for the purpose of screening the genes associated with immunologic effects of Echinacea on monocytes and dendritic cells using a cDNA microarray chip. METHODS: CD14+monocyte cells were cultured for one day with Echinacea extract (final concentration: 50 microgram/mL) in experiment 1, but were cultured without Echinacea in experiment 2. The gene expression of these cultured monocytes was analyzed using the cDNA microarray chip. Dendritic cells produced from CD14+monocyte were cultured for five days with GM-CSF and IL-4, and then cultured for one day with Echinacea in experiment 3, but were done without Echinacea in experiment 4. RESULTS: In experiments 1 and 2, there were 17 significantly expressed genes with average expression ratios above 2.5, including interferon gamma-inducible protein 30 (IFI 30), CDC (cell-division-cylcle)-like kinase 2 (CLK 2), syndecan binding protein (syntenin), superoxide dismutase 2, etc. In experiments 3 and 4, there were 24 gene, with significantly expressed genes were 24 genes, which were insulin-like growth factor 2 (somatomedin A), methyl-CpG binding domain protein 3, IFI 30, small inducible cytokine subfamily A, member 22, etc. The genes encoding CD44, IFI 30, mannose receptor C type 1 (MRC 1), chemokine receptor 7 (CCR 7), CLK 2, syntenin and cytochrome C oxidase subunit VIII were significantly expressed in both monocytes and dendritic cells cultured with Echinacea. CONCLUSION: This study employed a cDNA microarray chip to elicit the immune-associated gene profile; the expression was enhanced by Echinacea in CD14+monocytes and dendritic cells. Thus we laid the basis for the quantitative and functional analysis of genes induced by Echinacea in monocytes and monocyte-derived dendritic cells.
Subject(s)
Humans , Carrier Proteins , Dendritic Cells , Echinacea , Electron Transport Complex IV , Gene Expression , Granulocyte-Macrophage Colony-Stimulating Factor , Interferons , Interleukin-4 , Mannose , Mass Screening , Monocytes , Oligonucleotide Array Sequence Analysis , Phosphotransferases , Plants , Superoxide Dismutase , Syndecans , SynteninsABSTRACT
Cutaneous dendritic cells (DCs), Langerhans cells (LCs) and dermal dendritic cells (DDCs), are present in an immature state. The maturation of DCs is crucial for initiating an immune response. Since HLA-DM has an important role for antigen presentation, an increase in HLA-DM expression according to the maturation of blood monocyte-derived dendritic cells (MoDCs), which have similar characteristics with DDCs, is expected. Therefore, the aim of this study was to determine whether or not HLA-DM expression in MoDCs is related to maturation at each culture day (from day 0 to day 13) by flow cytometry. This was compared with the functional changes related to the maturation of MoDCs. MoDCs were generated by culturing human peripheral blood monocytes in the presence of GM-CSF and IL-4 for 7 days, which were followed by subsequent treatment with a cytokine cocktail (GM-CSF, IL-4, IL-1beta, TNF-alpha, IL-6 and PGE2) for the maturation of MoDCs. The intracellular HLA-DM was expressed in the immature MoDC. A sudden 3 to 8 fold increase in the intracellular HLA-DM expression was observed after treatment with a cytokine cocktail. HLA-DM was weakly expressed on the surface of the immature MoDC, but it seemed to be decreased with maturation. This study indicated that the intracellular HLA-DM expression increased, but not on the MoDC surface during maturation. This was despite the fact that HLA-DM expression was noted not only on the surface but also in the intracellular in the MoDC.