Dias Felipe F, Amaral Kátia B, Carmo Lívia A S, Shamri Revital, Dvorak Ann M, Weller Peter F, Melo Rossana C N
Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil (FFD,KBA,LASC,RCNM)Department of Pathology (AMD)Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts (RS,PFW,RCNM).
Laboratory of Cellular Biology, Department of Biology, Federal University of Juiz de Fora, UFJF, Juiz de Fora, MG, Brazil (FFD,KBA,LASC,RCNM)Department of Pathology (AMD)Department of Medicine, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts (RS,PFW,RCNM)
J Histochem Cytochem. 2014 Jun;62(6):450-459. doi: 10.1369/0022155414531437. Epub 2014 Mar 26.
Protein disulfide isomerase (PDI) has fundamental roles in the oxidative folding of proteins in the endoplasmic reticulum (ER) of eukaryotic cells. The study of this molecule has been attracting considerable attention due to its association with other cell functions and human diseases. In leukocytes, such as neutrophils, PDI is involved with cell adhesion, signaling and inflammation. However, the expression of PDI in other leukocytes, such as eosinophils, important cells in inflammatory, allergic and immunomodulatory responses, remains to be defined. Here we used different approaches to investigate PDI expression within human eosinophils. Western blotting and flow cytometry demonstrated high PDI expression in both unstimulated and CCL11/eotaxin-1-stimulated eosinophils, with similar levels in both conditions. By using an immunogold electron microscopy technique that combines better epitope preservation and secondary Fab-fragments of antibodies linked to 1.4-nm gold particles for optimal access to microdomains, we identified different intracellular sites for PDI. In addition to predictable strong PDI labeling at the nuclear envelope, other unanticipated sites, such as secretory granules, lipid bodies and vesicles, including large transport vesicles (eosinophil sombrero vesicles), were also labeled. Thus, we provide the first identification of PDI in human eosinophils, suggesting that this molecule may have additional/specific functions in these leukocytes.
蛋白质二硫键异构酶（PDI）在真核细胞内质网（ER）中蛋白质的氧化折叠过程中发挥着重要作用。由于该分子与其他细胞功能及人类疾病相关，对其研究一直备受关注。在白细胞中，如中性粒细胞，PDI参与细胞黏附、信号传导和炎症反应。然而，PDI在其他白细胞中的表达情况，如嗜酸性粒细胞（炎症、过敏和免疫调节反应中的重要细胞），仍有待确定。在此，我们采用不同方法研究人嗜酸性粒细胞内的PDI表达。蛋白质印迹法和流式细胞术表明，在未受刺激和CCL11/嗜酸性粒细胞趋化因子-1刺激的嗜酸性粒细胞中，PDI均有高表达，且两种情况下表达水平相似。通过使用一种免疫金电子显微镜技术，该技术结合了更好的表位保存以及与1.4纳米金颗粒相连的抗体二级Fab片段，以便最佳地进入微结构域，我们确定了PDI在细胞内的不同定位。除了在核膜处可预测的强烈PDI标记外，其他意外的定位，如分泌颗粒、脂质体和囊泡，包括大型转运囊泡（嗜酸性粒细胞草帽样囊泡），也有标记。因此，我们首次在人嗜酸性粒细胞中鉴定出PDI，表明该分子可能在这些白细胞中具有额外/特定功能。
