Matsue H, Cruz P D, Bergstresser P R, Takashima A
Department of Dermatology, University of Texas Southwestern Medical Center, Dallas 75235.
J Invest Dermatol. 1992 Nov;99(5):537-41. doi: 10.1111/1523-1747.ep12667296.
Immunocompetent cells of the epidermis can interact by the elaboration and recognition of cytokines. Although much new information has been reported concerning the cytokines secreted by keratinocytes, little is known about cytokines derived from Langerhans cells (LC). To address this deficiency, we examined cytokine mRNA profiles in different epidermal preparations from BALB/c mice, taking advantage of the sensitive technique of polymerase chain reaction (PCR), after reverse transcription of mRNA. In assays of epidermal sheets separated from dermis by ammonium thiocyanate, mRNA for IL-1 alpha, IL-1 beta, IL-6, IL-7, tumor necrosis factor alpha (TNF alpha), TNF beta, granulocyte macrophage/colony-stimulating factor (GM-CSF), and macrophage inflammatory protein-1 alpha (MIP-1 alpha) were unequivocally present. By contrast, faint bands were detected for IL-4, IL-5, and interferon gamma (IFN gamma), and no PCR signal was detected for IL-2. Importantly, assays of epidermal cells (EC) dissociated with trypsin revealed similar mRNA profiles. To determine the effects of cell isolation, fluorescence-activated cell sorter (FACS)-purified Ia- EC were first analyzed; all of the previously cited cytokine mRNA were present except for IL-1 beta and MIP-1 alpha. EC depleted of LC by a second technique, lysis using anti-Ia monoclonal antibody and complement, revealed similar profiles, with substantially reduced PCR signals for IL-1 beta and MIP-1 alpha. Finally, FACS-purified LC (Ia+ EC) clearly expressed IL-1 beta and MIP-1 alpha mRNA, a finding that was verified by Southern blotting using internal oligo probes. We conclude that these cell-isolation procedures did not produce substantial alterations in basal mRNA profiles and that LC are the principal source of mRNA for IL-1 beta and MIP-1 alpha among unstimulated EC in mice.
表皮的免疫活性细胞可通过细胞因子的产生和识别来相互作用。尽管关于角质形成细胞分泌的细胞因子已有许多新信息报道，但对于源自朗格汉斯细胞（LC）的细胞因子却知之甚少。为了弥补这一不足，我们利用聚合酶链反应（PCR）的灵敏技术，在mRNA逆转录后，检测了BALB/c小鼠不同表皮制剂中的细胞因子mRNA谱。在用硫氰酸铵从真皮分离的表皮片检测中，明确检测到了白细胞介素-1α（IL-1α）、白细胞介素-1β（IL-1β）、白细胞介素-6、白细胞介素-7、肿瘤坏死因子α（TNFα）、肿瘤坏死因子β（TNFβ）、粒细胞巨噬细胞集落刺激因子（GM-CSF）和巨噬细胞炎性蛋白-1α（MIP-1α）的mRNA。相比之下，白细胞介素-4、白细胞介素-5和干扰素γ（IFNγ）检测到的条带较淡，而白细胞介素-2未检测到PCR信号。重要的是，用胰蛋白酶解离的表皮细胞（EC）检测显示出相似的mRNA谱。为了确定细胞分离的影响，首先分析了荧光激活细胞分选仪（FACS）纯化的Ia + EC；除了IL-1β和MIP-1α外，所有上述细胞因子mRNA均存在。通过第二种技术（使用抗Ia单克隆抗体和补体裂解）去除LC的EC显示出相似的谱，IL-1β和MIP-1α的PCR信号大幅降低。最后，FACS纯化的LC（Ia + EC）明显表达IL-1β和MIP-1α mRNA，这一发现通过使用内部寡核苷酸探针的Southern印迹得到了验证。我们得出结论，这些细胞分离程序在基础mRNA谱中未产生实质性改变，并且在未受刺激的小鼠EC中，LC是IL-1β和MIP-1α mRNA的主要来源。
