In vivo imaging demonstrates ATP release from murine keratinocytes and its involvement in cutaneous inflammation after tape stripping.
In vivo imaging demonstrates ATP release from murine keratinocytes and its involvement in cutaneous inflammation after tape stripping.
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DOI：
10.1038/jid.2013.163
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发表时间：
2013-10
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通讯作者：
Toshiya Takahashi;Y. Kimura;K. Niwa;Y. Ohmiya;T. Fujimura;K. Yamasaki;S. Aiba
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文献类型：
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作者：
Toshiya Takahashi;Y. Kimura;K. Niwa;Y. Ohmiya;T. Fujimura;K. Yamasaki;S. Aiba
在各种体外应激模型中，已经观察到角质形成细胞释放5 ' -三磷酸腺苷（ATP），但研究表明，体内表皮ATP释放有限。为了在体内可视化细胞外ATP (eATP)，我们开发了固定化在琼脂糖珠上的增强型绿色荧光素酶（lucu -琼脂糖）。在腹腔注射荧光素后，在BALB/c小鼠背侧皮肤皮下注射e -琼脂糖和ATP，使用anin体内成像系统产生可检测和可测量的生物发光。我们在体内证明，反复剥去小鼠皮肤的胶带后1 ~ 20分钟可观察到明亮的生物发光。这种生物发光被局部给药apyrase抑制。仅在经皮失水（TEWL）在100 ~ 140 g m2h−1之间的胶带剥离皮肤中观察到双糖琼脂糖生物发光，表明过度的胶带剥离（TEWL>140 g m−2h−1）导致了生物发光的损失。组织学上，eATP可检测的条带剥离皮肤有存活的表皮和表皮下中性粒细胞浸润，给药apyrase可减少炎症浸润。在过度剥离胶带后，既未观察到存活的表皮，也未观察到上真皮中性粒细胞浸润。这些结果表明，胶带剥离促进活的角质形成细胞释放ATP，从而促进炎症细胞迁移。在小鼠皮肤病模型中，琼脂糖可能在体内检测eATP是有用的。
Adenosine 5′-triphosphate (ATP) release from keratinocytes has been observed in various stress modelsin vitro, but studies demonstrating epidermal ATP releasein vivoare limited. To visualize extracellular ATP (eATP)in vivo, we developed enhanced green-emitting luciferase immobilized on agarose beads (Eluc-agarose). Subcutaneous injection of Eluc-agarose together with ATP into the dorsal skin of BALB/c mice following intraperitoneal luciferin injection produced detectable and measurable bioluminescence using anin vivoimaging system. Using Eluc-agarose, we demonstratedin vivothat bright bioluminescence was observed from 1 to 20 minutes after repeated tape stripping of murine skin. This bioluminescence was suppressed by the local administration of apyrase. Eluc-agarose bioluminescence was observed only in tape-stripped skin with transepidermal water loss (TEWL) between 100 and 140 g m2h−1, indicating a loss of bioluminescence with excessive tape stripping (TEWL>140 g m−2h−1). Histologically, tape-stripped skin with detectable eATP had a viable epidermis and a subepidermal neutrophil infiltrate, and administration of apyrase reduced the inflammatory infiltrate. Neither a viable epidermis nor an upper dermal neutrophil infiltrate was observed after excessive tape stripping. These results suggest that tape stripping prompts ATP release from viable keratinocytes, which facilitates inflammatory cell migration. Eluc-agarose may be useful in thein vivodetection of eATP in murine models of skin diseases.