| Chronic relapsing inflammatory skin disorders, such as psoriasis and allergic conFK-506ct dermatitis, have major impacts on patients’quality of life. They are characterized by an intense infiltrate of activated T cells, which release T cell lymphokines influencing the immune functions of resident keratinocytes. At the same time, skin provides a complicated micro-environment for the initiation and maintenance of T cell-mediated immunoresponses. The interaction between keratinocytes and infiltrated T cells plays a key role in the inflammation. In the Thl dominant skin inflammation, also named IFN-y-dependent inflammation, IFN-y is one of the most potent proinflammatory cytokines secreted by infiltrated Thl lymphocytes. It transforms keratinocytes in strong producers of inflammatory mediators, such as interleukin 8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1). To understand the mechanisms by which IFN-y signaling in keratinocytes is switched off could be helpful to direct rational treatment for Thl dominant skin inflammation, and could be helpful for the discovery of new anti-inflammatory drug targets.Tacrolimus (FK-506) is an immunosuppressant and anti-inflammtory drug by systemic or topical application. Tripterygium wilfordii exerts its immunosuppressive and anti-inflammtory function by oral administration. Clobetasol propionate is one of topical corticosteriods.13-hexyl-berberine hydrochloride (HB-13) and 13-hexyl-palmatine hydrochloride (HP-13) are novel topical compounds with anti-inflammatory potent. The aim of the current study is to investigate the effects of those drugs on IFN-y signal transduction in a spontaneously transformed human epidermal cell line HaCaT. The paper consists of five chapters.Chapter I Effects of drugs on the secretion of IL-8 and the expression of ICAM-1 induced by IFN-y in human keratinocytesObjective To investigate the effects of drugs on the secretion of IL-8 and the expression of ICAM-1 induced by IFN-y in HaCaT keratinocytes. Methods The secretion of IL-8 was detected by ELISA and the expression of ICAM-1 determined by cell-ELISA.Results HaCaT keratinocytes were found to secrete IL-8 constitutively. The secretion was induced by 500-2000U/mL rhIFN-y stimulation for 48 hours. Pretreatment with FK-506, HB-13 or HP-13 for 2 hours dose-dependently inhibited IL-8 secretion induced by 500U/mL rhIFN-y and the IC50 values were 0.0076μg/mL,0.269μg/mL and 0.365μg/mL repectively. Results of immunoperoxidase-cell-ELISA indicated that unstimulated HaCaT cells constitutively expressed a low level of ICAM-1. Stimulation with 500U/ml rhIFN-y for 24 hours dramatically enhanced the expression of ICAM-1. Pretreatment with FK-506, triptolide, clobetasol propionate, HB-13 or HP-13 for 2 hours dose-dependently inhibited ICAM-1 expression induced by 500U/ml IFN-y and the IC50 values were 0.86μg/mL,2.10×10-4μg/mL,0.024μg/mL,0.279μg/mL and 0.268μg/mL respectively. Conclusion The above-metioned five drugs can inhibit the expression of inflammatory cytokines and adhesion molecules induced by IFN-y in keratinocytes.ChapterⅡEffects of drugs on IFN-yRa expression induced by rhIFN-y in human keratinocytesObjective To investigate the effects of drugs on IFN-yRa expression induced by rhIFN-y in HaCaT keratinocytes. Methods The expression of IFN-yRa was examined by Western blotting. Results IFN-yRa was found to express constitutively in HaCaT keratinocytes. The amount of expression increased 5 minutes after stimulation with 500U/mL rhIFN-y, and maintained at a high level at least for 2 hours. IFN-yRa expression could be induced by 250～1000U/mL rhIFN-y and there were no significant differences among 250,500 and 1000U/ml groups. Pretreatment with FK-506, triptolide, HB-13, HP-13 or clobetasol propionate for 2 hours dose- dependently inhibited the expression of IFN-yRa induced by 500U/mL rhIFN-y for 5 minutes and the IC50 values were about 0.43μg/mL, 4.94×10-3μg/mL,0.351μg/mL,2.190μg/mL and 0.061μg/mL respectively. Conclusion The above-metioned five drugs can inhibit IFN-y signal transduction by inhibiting the expression of IFN-yRa in HaCaT keratinocytes.Chapter III Effects of drugs on pJAK2 expression induced by rhIFN-y in HaCaT keratinocytesObjective To investigate the effects of drugs on pJAK2 expression induced by rhIFN-y in HaCaT keratinocytes. Methods The expression of pJAK2 was detected by Western blotting. Results The results suggested that pJAK2 expression could almost not be detected in normal HaCaT cells. It was immediately induced (5 minutes) after rhIFN-y stimulation and the expression disappeared 15 minutes later. The expression of pJAK2 could be induced by 250～1000U/ml rhIFN-y and there were no significant differences among 250,500 and 1000U/ml groups. Pretreatment with FK-506, triptolide, HB-13, HP-13 or clobetasol propionate for 2 hours dose-dependently inhibited the expression of pJAK2 induced by 5 minutes of incubation with 500U/mL rhIFN-y and the IC50 values were about 0.224μg/mL,1.02×10-3μg/mL,0.386μg/mL,2.049μg/mL and 0.095μg/mL respectively. Conclusion The above-metioned five drugs can inhibit IFN-y signal transduction by inhibiting the phosphorylation of JAK2 in HaCaT keratinocytes.ChapterⅣEffects of drugs on pSTAT-1 expression induced by rhIFN-y in HaCaT keratinocytesObjective To investigate the effects of drugs on pSTAT-1 expression induced by rhIFN-γin HaCaT keratinocytes. Methods The expression of pSTAT-1 was detected by Western blotting. Results The results showed that pSTAT-1 was not detecFK-506ble in nonactivated HaCaT cells. Strong expression was found 5 minutes after IFN-y stimulation and a high level of expression lasted at least 2 hours. The expression of pSTAT-1 could be induced by 250～1000μ/mL rhIFN-y and there were no significant differences among 250,500 and 1000U/ml groups. Pretreatment with FK-506, triptolide, HB-13, HP-13 or clobetasol propionate for 2 hours dose-dependently inhibited the expression of pSTAT-1 induced by 30 minutes of stimulation with 500U/mL rhIFN-y and the IC50 values were about 0.95μg/mL,4.64×10-4μg/mL, 0.303μg/mL,2.102μg/mL and 0.609μg/mL respectively. Conclusion The above-metioned five drugs can inhibit IFN-y signal transduction by inhibiting the phosphorylation of STAT-1 in HaCaT keratinocytes.Chapter V Effects of drugs on SOCS-1 expression induced by rhIFN-y in HaCaT keratinocytesObjective To investigate the effects of drugs on the expression of SOCS-1 induced by rhIFN-γin HaCaT keratinocytes. Methods The expression of SOCS-1 was analysed by Western blotting. Results Results revealed that SOCS-1 was constitutively expressed in HaCaT keratinocytes at a low level. The expression of SOCS-1 was induced after 30 minutes of stimulation by 500U/mL rhIFN-y and the high level of expression maintained at least 4 hours. SOCS-1 expression could be induced by 250～1000U/mL rhIFN-y and there were no significant differences among 250,500 and 1000U/mL groups. Pretreatment with FK-506, triptolide, HB-13, HP-13 or clobetasol propionate for 2 hours dose-dependently up-regulated the expression of SOCS-1 induced by an hour of stimulation with 500U/mL rhIFN-γand the ED50 values were about 0.015μg/mL, 1.10×10-5μg/mL,0.051μg/mL,1.777μg/mL and 0.29μg/mL respectively. Conclusion The above-metioned five drugs can inhibit IFN-γsignal transduction by upregulating the expression of SOCS-1 in HaCaT keratinocytes. |
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