| Eczema is a heterogeneous disease and the pathophysiological mechanism should be clarified in each disease. Allergic contact dermatitis and atopic dermatitis are representative diseases showing eczematous reaction. A major player in both allergic contact dermatitis and atopic dermatitis is skin-specific T lymphocytes. Activation and skin-selective homing of T cells and effector functions in the skin represent sequential events in the pathogenesis of eczematous dermatitis. The evidence showing that infiltrating T cells participate in the formation of eczematous reaction through Fas-induced apoptosis is important in our understanding of eczematous reactions and also for treatment. It is generally accepted that the Fas-Fas ligand (FasL) pathway is involved in T cell–mediated keratinocyte apoptosis. In eczematous skin lesions, T cells upregulate the expression of Fas antigen on keratinocytes through IFN-γ and induce apoptosis through Fas Ligand, which are released by the activated T cells. The apoptosis is initiated by pretreatment with a neutralizing anti-Fas antibody and inhibited by pretreatment with Fas-Fc fragment (Fas-Fc). Previous studies suggested that damage to epidermis, which serves as a barrier, can lead to spongiosis, and subsequently can amplify the inflammatory process. Thus, in the process of chronic eczematous dermatitis, the apoptosis of keratinocytes is not only a turning point, but also a new target of drug screening. In this study, Fas-induced keratinocyte apoptosis in a keratinocyte cell line, colo-16, cocultured with IFN-γ and anti-Fas monoclonal antibody (AntiFasmab) served as an in vitro model of eczematous dermatitis. A colorimetric test was investigated using sulforhodamine B (SRB), which stains protein synthesized by cells, as an end-point marker. We assumed that the decreases of cell proliferation in Fas-induced keratinocyte apoptosis obtained by SRB assay correlated well with results obtained by flow cytometry. Therefore, the SRB assay permitted rapid and reliable assessment of keratinocyte apoptosis in our system and screened favourably with therapeutic agents of anti- eczematous dermatitis. Finally, we tested, in this model, whether ginsenosides Rb1 and Re were able to inhibit apoptosis of keratinocytes.Methods: The cultured keratinocyte colo-16 was exposed to IFN-γ and AntiFasmab at various time and in various concentrations, respectively or together in order to select the optimum condition of keratinocyte apoptosis. Fas-Fc and ginsenosides were tested at the optimum. The induction of cell apoptosis was validated by flow cytometry to compare with inhibition of cell proliferation measured by SRB assay. Morphologic changes for keratinocyte apoptosis were observed by inverted microscope and by acridine orange stained fluorescent microscopic analysis.Results: (1) Exposed to IFN-γ and AntiFasmab, typical morphologic changes were presented as cell rounding, shrinkage and cytoplamic blebbing, observed by inverted microscope, and as reduction of cell quantities, chromatin condensation and yellow staining by fluorescent microscope in colo-16 cells,. (2) Up-regulated the expression of Fas antigen on colo-16 was remarkably showed in addition of IFN-γ alone by flow cytometry. In addition of IFN-γ at 0ng/ml,1ng/ml,10ng/ml,25ng/ml and 50ng/ml, the quantity of Fas antigen expressed on colo-16 was 49.57%7.05, 62.63%6.31, 71.33%6.14, 77.49%6.06 and 79.22%4.94, respectively. (3) Cell proliferation of colo-16 was not inhibited when the cell was exposed alone to IFN-γ at concentration from 0ng/ml to 50ng/ml by SRB assay. (4) When AntiFasmab was at the concentration of 1μg/ml, apoptotic rate of colo-16 cell, after exposed to the above five concentrations of IFN-γ, was 5.76%1.33, 14.33%3.87, 27.95%3.92, 26.19%6.10 and 53.14%8.48, respectively. This result demonstrated that the apoptosis was directly correlated with the expression of Fas antigen, which induced by IFN-γ, and initiated in combination of AntiFasmab. (5) In the combined treatment with IFN-γ and AntiFa... |
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