Person:

Melo, Rossana

Precise immunolocalization of proteins within a cell is central to understanding cell processes and functions such as intracellular trafficking and secretion of molecules during immune responses. Here we describe a protocol for ultrastructural detection of proteins in leukocytes. The method uses a pre-embedding approach (immunolabeling before standard processing for transmission electron microscopy (TEM)). This protocol combines several strategies for ultrastructure and antigen preservation, robust blocking of nonspecific binding sites, as well as superior antibody penetration for detecting molecules at subcellular compartments and membrane microdomains. A further advantage of this technique is that electron microscopy (EM) processing is quick. This method has been used to study leukocyte biology, and it has helped demonstrate how activated leukocytes deliver specific cargos. It may also potentially be applied to a variety of different cell types. Excluding the initial time required for sample preparation (15 h) and the final resin polymerization step (16 h), the protocol (immunolabeling and EM procedures) can be completed in 8 h.

Major basic protein (MBP), the predominant cationic protein of human eosinophil specific granules, is stored within crystalloid cores of these granules. Secretion of MBP contributes to the immunopathogenesis of varied diseases. Prior electron microscopy (EM) of eosinophils in sites of inflammation noted losses of granule cores in the absence of granule exocytosis and suggested that eosinophil granule proteins might be released through piecemeal degranulation (PMD), a secretory process mediated by transport vesicles. Because release of eosinophil granule-derived MBP through PMD has not been studied, we evaluated secretion of this cationic protein by human eosinophils. Intracellular localizations of MBP were studied within nonstimulated and eotaxin-stimulated human eosinophils by both immunofluorescence and a pre-embedding immunonanogold EM method that enables optimal epitope preservation and antigen access to membrane microdomains. In parallel, quantification of transport vesicles was assessed in eosinophils from a patient with hypereosinophilic syndrome (HES). Our data demonstrate vesicular trafficking of MBP within eotaxin-stimulated eosinophils. Vesicular compartments, previously implicated in transport from granules to the plasma membrane, including large vesiculotubular carriers termed eosinophil sombrero vesicles (EoSVs), were found to contain MBP. These secretory compartments were significantly increased in numbers within HES eosinophils. Moreover, in addition to granule-stored MBP, even unstimulated eosinophils contained appreciable amounts of MBP within secretory vesicles, as evidenced by immunonanogold EM and immunofluorescent colocalizations of MBP and CD63. These data suggest that eosinophil MBP, with its multiple extracellular activities, can be mobilized from granules by PMD into secretory vesicles and both granule- and secretory vesicle-stored pools of MBP are available for agonist-elicited secretion of MBP from human eosinophils. The recognition of PMD as a secretory process to release MBP is important to understand the pathological basis of allergic and other eosinophil-associated inflammatory diseases.

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.