Boorsma D M, Flier J, Sampat S, Ottevanger C, de Haan P, Hooft L, Willemze R, Tensen C P, Stoof T J
Department of Dermatology, University Hospital Vrije Universiteit, Amsterdam, The Netherlands.
Arch Dermatol Res. 1998 Jun;290(6):335-41. doi: 10.1007/s004030050314.
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. IP-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 IP-10 mRNA. Upon stimulation with IFN-gamma, IP-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 to 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.
IP-10是CXC趋化因子家族的成员之一，因其具有T细胞趋化和促进黏附的特性，被认为在炎症反应中发挥重要作用。银屑病表皮、迟发型超敏反应、皮肤T细胞淋巴瘤和固定性药疹中IP-10水平升高，促使我们研究其在角质形成细胞中的表达。使用灵敏的RT-PCR方法可检测到IP-10 mRNA，但在未刺激的正常培养角质形成细胞的RNA制剂中，通过Northern印迹法却检测不到，这表明IP-10 mRNA的稳态水平较低。用γ干扰素刺激后，发现IP-10 mRNA会以时间和剂量依赖性方式大量积累。虽然γ干扰素与肿瘤坏死因子-α或白细胞介素-1单独使用时不会诱导IP-10 mRNA的积累，但发现它们联合使用时会使IP-10超表达。为研究IP-10 mRNA表达的调控而进行的核转录实验表明，在未刺激的角质形成细胞中，IP-10基因具有非常高的组成型转录活性，γ干扰素、肿瘤坏死因子-α或γ干扰素与肿瘤坏死因子-α联合刺激均不会影响该活性。蛋白激酶C（PKC）被证明参与了IP-10 mRNA的表达，因为PKC抑制剂H7可减少IP-10 mRNA的积累。使用高效液相色谱技术从刺激的角质形成细胞培养上清液中分离出一种蛋白质，经序列分析发现其与IP-10相同。通过酶联免疫吸附测定法监测发现，IP-10蛋白的分泌动态与mRNA表达平行。
