Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Medical University of Vienna, Allgemeines Krankenhaus, Währinger Gürtel 18-20, Vienna, Austria.
J Invest Dermatol. 2010 Jun;130(6):1611-23. doi: 10.1038/jid.2010.31. Epub 2010 Feb 25.
Skin inflammation can induce local expression of CCL21, which is subsequently drained to lymph nodes (LNs) influencing their cellular composition. To determine whether the same can be achieved by dermal administration of a plasmid DNA (pDNA) encoding CCL21, we generated a pDNA-based gene construct allowing high-level expression of CCL21. Expression and secretion of biologically active CCL21 were confirmed in vitro by immunohistochemistry, western blot analysis, ELISA, and transwell chemotactic assays. In vivo experiments showed cellular expression of transgenic CCL21 after particle-mediated gene gun delivery of pDNA into skin. CCL21 was expressed in the epidermis, consequently secreted into the upper dermis, and transported into the draining LNs, which resulted in increased CCL21 concentration, total cell number, and frequencies of CD11c(+) DCs and CD4(+)/CD62L(+) naïve, CD4(+)/CD62L(-), and CD8(+)/CD62L(-) effector memory T-cells (expressing CCL21 receptors CCR7 or CXCR3), as well as retention of adoptively transferred T-lymphocytes, in the draining LNs of plt/plt mice (lacking endogenous expression of CCL21). Our studies show that biologically active CCL21 can be overexpressed by genetic means in vitro and in vivo. This strategy allows reconstitution of a genetic defect and colocalization of different cell types in the secondary lymphoid organs, an important prerequisite for targeted cell migration.
皮肤炎症可以诱导 CCL21 的局部表达，随后这些 CCL21 被引流到淋巴结（LNs），影响它们的细胞组成。为了确定通过皮内给予编码 CCL21 的质粒 DNA（pDNA）是否可以达到同样的效果，我们构建了一个基于 pDNA 的基因构建体，允许高水平表达 CCL21。通过免疫组织化学、Western blot 分析、ELISA 和 Transwell 趋化实验，在体外证实了具有生物活性的 CCL21 的表达和分泌。体内实验表明，在皮肤经粒子介导的基因枪递送 pDNA 后，转基因 CCL21 可在细胞内表达。CCL21 在表皮中表达，随后分泌到真皮上层，并被运送到引流的 LNs，导致 CCL21 浓度、总细胞数以及 CD11c(+) DC 和 CD4(+)/CD62L(+) naive、CD4(+)/CD62L(-)和 CD8(+)/CD62L(-)效应记忆 T 细胞（表达 CCL21 受体 CCR7 或 CXCR3）的频率增加，同时在 plt/plt 小鼠（缺乏内源性 CCL21 表达）的引流 LNs 中保留了过继转移的 T 淋巴细胞。我们的研究表明，生物活性 CCL21 可以通过遗传手段在体外和体内进行过表达。这种策略允许重建遗传缺陷，并使不同类型的细胞在次级淋巴器官中重新聚集，这是靶向细胞迁移的重要前提。
