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

Abstract

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.

Keywords: cell activation; endoplasmic reticulum; human eosinophils; immunonanogold electron microscopy; lipid bodies; protein disulfide isomerase; secretory granules; transmission electron microscopy; vesicular traffic.

Figure 1.

Protein disulfide isomerase (PDI) expression in human eosinophils. (A) The expression of PDI in fresh isolated human eosinophils (lane 1) or after 1 hr incubation (Inc.) at 37C in the absence (lane 2) or presence (3) of CCL11 was detected by western blotting, as detailed in the Materials & Methods. (B) The intracellular content of PDI in freshly isolated human eosinophils (Bi), or after 1 hr incubation at 37C in the absence, or presence of CCL11 (Bii) was measured by flow cytometry, as detailed in the Materials & Methods. IC, irrelevant antibody control. Representative blot and histograms are shown.

Figure 2.

Immunonanogold electron microscopy of protein disulfide isomerase (PDI) in human eosinophils. (A, B) PDI is densely labeled at the nuclear envelope (highlighted in yellow in A). Gold particles are indicated by arrowheads in (B). Arrows point to PDI-positive vesicles. Eosinophils were isolated from the blood of healthy donors, stimulated with CCL11 and processed for immunonanogold electron microscopy as before (Melo et al. 2005a). N, nucleus, Gr, secretory granule. Scale bars: (A) 2.0 μm; (B) 0.8 μm.

Figure 3.

Immunonanogold electron microscopy demonstrates protein disulfide isomerase (PDI) labeling within vesicles and lipid bodies (LBs). (Ai-Aii) Large vesicles (circles in Aii) around and in association with LBs are densely positive for PDI. PDI labeling is also seen within LBs (arrowheads in Aii). Note in (Ai) the consistent PDI labeling (arrowheads) around the nuclear envelope. (Ai) and (Aii) are boxed areas of (A) seen in high magnification. N, nucleus, Gr, secretory granule; LB, lipid body. Scale bars: (A) 1.1 μm; (Ai) 0.9 μm; (Aii) 0.5 μm.

Figure 4.

Different subcellular compartments are labeled for protein disulfide isomerase (PDI) within human eosinophils. (A, Ai) A typical Eosinophil Sombrero Vesicle (EoSV, Ai) is observed in the cytoplasm of a human eosinophil by transmission electron microscopy after immunonanogold labeling for PDI. Note that PDI is localized within the vesicle lumen and that the vesicle is close to a secretory granule (Gr). (Ai) is the boxed area of (A) seen in high magnification. Arrowheads indicate PDI labeling at the nuclear envelope. (B and C) Quantitation of PDI immunogold labeling in eosinophil subcellular compartments. Data shown represent the mean and SEM of gold particle counts for each compartment. The total number of organelles/structures evaluated was as follows: 386 secretory granules, 24 lipid bodies and 314 vesicular compartments. The entire extension of the nuclear envelope of 30 cell sections was measured and the number of gold particles counted. Eosinophils were isolated from the blood of healthy donors, stimulated with CCL11 and processed for immunonanogold electron microscopy as before (Melo et al. 2005a). Scale bars: (A) 1.9 μm; (Ai) 70 nm. N, nucleus; LB, lipid body.

Figure 5.

Eosinophil secretory granules (Gr) are positive for protein disulfide isomerase (PDI). (A) Gold particles (arrows) are seen inside granules and close to a lipid body (LB). In (B), a representative control cell, for which the primary antibody was replaced by an irrelevant antibody, was negative for PDI. N, nucleus. Scale bars: (A, B) 400 nm.