Chemokine IP-10 expression in cultured human keratinocytes
Chemokine IP-10 expression in cultured human keratinocytes
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DOI：
10.1007/s004030050314
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发表时间：
1998-06-01
影响因子：
3
通讯作者：
Stoof, TJ
中科院分区：
文献类型：
--
作者：
Boorsma, DM;Flier, J;Stoof, TJ
IP-10是CXC趋化因子家族的成员，被认为通过其T细胞趋化性和促进粘附特性在炎症中发挥重要作用。银屑病、迟发型超敏反应、皮肤T细胞淋巴瘤和固定药疹的表皮中IP-10水平升高促使我们研究其在角质形成细胞中的表达。使用敏感的RT-PCR方法可以检测到IF-10 mRNA，但在来自未刺激的正常培养角质形成细胞的RNA制剂中不能通过北方印迹法检测到，表明IF-10 mRNA的稳态水平较低。在用IFN-γ刺激后，发现IF-10 mRNA以时间和剂量依赖性方式大量积累。在IFN-γ和TNF-α或IL-1的组合下发现超表达，尽管这些后者的细胞因子本身不诱导IP-10 mRNA的积累。进行核连续实验以研究IP-10 mRNA表达的调节，在未受刺激的角质形成细胞中显示出非常高的IP-10基因的组成型转录活性，其不受IFN-γ、TNF-α或IFN-γ和TNF-α的组合的刺激的影响。蛋白激酶C（PKC）被证明参与IP-10 mRNA的表达，因为PKC抑制剂H7减少IP-10 mRNA的积累。使用HPLC技术从刺激的角质形成细胞的培养上清液中分离蛋白质，并且通过序列分析发现其与IP-10相同。通过ELISA监测的IP-10蛋白质的分泌动力学显示与mRNA表达平行。
IP-10, a member of the CXC family of chemokines, is considered to play an important role in inflammation via its T-cell chemotactic and adhesion-promoting properties. Elevated IP-10 levels in the epidermis of psoriasis, delayed-type hypersensitivity reactions, cutaneous T-cell lymphoma and fixed drug eruptions prompted us to study its expression in keratinocytes, IF-10 mRNA could be detected using the sensitive RT-PCR method, but not by Northern blotting in RNA preparations from unstimulated normal cultured keratinocytes, indicating a low steady-state level of IF-10 mRNA. Upon stimulation with IFN-gamma, IF-10 mRNA was found to accumulate in high amounts in a time- and dose-dependent manner, Superexpression was found with the combination of IFN-gamma and TNF-alpha or IL-1, although these latter cytokines by themselves did not induce accumulation of IP-10 mRNA, Nuclear run-on experiments performed tio investigate the regulation of IP-10 mRNA expression, showed a very high constitutive transcriptional activity of the IP-10 gene in unstimulated keratinocytes, which was not affected by stimulation with IFN-gamma, TNF-alpha, or a combination of IFN-gamma and TNF-alpha. Protein kinase C (PKC) was shown to be involved in IP-10 mRNA expression since the PKC inhibitor H7 decreased IP-10 mRNA accumulation. A protein was isolated from culture supernatants of stimulated keratinocytes using HPLC techniques and, by sequence analysis, was found to be identical to IP-10, The dynamics of secretion of IP-10 protein as monitored by ELISA was shown to parallel the mRNA expression.