Human Eosinophil Leukocytes Express Protein Disulfide Isomerase in Secretory Granules and Vesicles

Abstract

SummaryProtein 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. (J Histochem Cytochem 62:450-459, 2014)

Myeloid-derived growth factor is a resident endoplasmic reticulum protein

“…MYDGF co-localized most extensively with P4HB (ER marker) around the periphery of the nucleus and in the centralized patch between nuclear lobes, with overlapping immuno-staining accounting for over 90% of staining relative to either antibody alone in unactivated or IL5-activated eosinophils ( Table 1). The immuno-fluorescence pattern of P4HB is consistent with previous immuno-gold electron microscopic studies of eosinophils that localized P4HB to the nuclear envelope (11). MYDGF in the patch co-localized with RCAS1, a Golgi marker (Fig.…”

“…When we realized that C19orf10 had been characterized functionally as MYDGF (9), we asked whether eosinophils represent a second myeloid source of releasable MYDGF. As described herein, however, upon immuno-staining of eosinophils we found that MYDGF did not localize to eosinophil granules but rather to the Golgi and endoplasmic reticulum (ER), which in eosinophils is restricted to the nuclear envelope (11).…”

Pre-embedding immunogold labeling to optimize protein localization at subcellular compartments and membrane microdomains of leukocytes

“…5a), MBP 17 (Fig. 6a) and PDI 27 (Fig. 6b), within human leukocytes isolated from the peripheral blood.…”

“…17,24,26) and protein disulfide isomerase (PDI) 27 in leukocytes. We demonstrated that CD9 is abundant on the surface of eosinophil leukocytes, presenting the first EM data of the ultrastructural immunolocalization of CD9 in human eosinophils, which represents a novel insight into the organization of the antigen presentation complex of these cells 24 .…”

“…Our methodology, a combination of different strategies for optimal labeling and morphology preservation, has been used for almost 10 years (refs. 17,20–24,27). Figure 2 presents a summary of all the stages of the protocol.…”