Autophagy Protects against Eosinophil Cytolysis and Release of DNA.

The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Eosinophils deposit their damaging products in airway tissue, likely by degranulation and cytolysis. We previously showed that priming blood eosinophils with IL3 strongly increased their cytolysis on aggregated IgG. Conversely, IL5 priming did not result in significant eosinophil cytolysis in the same condition. Therefore, to identify critical events protecting eosinophils from cell cytolysis, we examined the differential intracellular events between IL5- and IL3-primed eosinophils interacting with IgG. We showed that both IL3 and IL5 priming increased the eosinophil adhesion to IgG, phosphorylation of p38, and production of reactive oxygen species (ROS), and decreased the phosphorylation of cofilin. However, autophagic flux as measured by the quantification of SQSTM1-p62 and lipidated-MAP1L3CB over time on IgG, with or without bafilomycin-A1, was higher in IL5-primed compared to IL3-primed eosinophils. In addition, treatment with bafilomycin-A1, an inhibitor of granule acidification and autophagolysosome formation, enhanced eosinophil cytolysis and DNA trap formation in IL5-primed eosinophils. Therefore, this study suggests that increased autophagy in eosinophils protects from cytolysis and the release of DNA, and thus limits the discharge of damaging intracellular eosinophilic contents.

Observation and Quantification of Eosinophil Motility.

Eosinophils are important for tissue homeostasis and host responses to pathogens and allergens. The impact of eosinophils within tissues depends in part on whether cytotoxic proteins in crystalloid granules are released. Determinants of eosinophil motility and loss of granule contents are incompletely understood. The goal of this chapter is to present methods to study the effects of potential mediators on purified human blood eosinophils interacting with adhesive proteins found in extracellular matrix. We show that differential interference contrast video-enhanced microscopy and a bead-clearing assay provide complementary information about how different mediator-adhesive protein combinations direct eosinophil motility and granule fate. The former method is rich in information about cell shape, pattern of movement, and state of granules whereas the latter method lends itself to quantification and interrogation of multiple conditions in replicate.

Plasma P-Selectin Is Inversely Associated with Lung Function and Corticosteroid Responsiveness in Asthma.

BACKGROUND: Severe asthma has multiple phenotypes for which biomarkers are still being defined. Plasma P-selectin reports endothelial and/or platelet activation. OBJECTIVE: To determine if P-selectin is associated with features of asthma in a longitudinal study. METHODS: Plasmas from 70 adult patients enrolled in the Severe Asthma Research Program (SARP) III at the University of Wisconsin-Madison were analyzed for concentration of P-selectin at several points over the course of 3 years, namely, at baseline (BPS), after intramuscular triamcinolone acetonide (TA) injection, and at 36 months after baseline. Thirty-four participants also came in during acute exacerbation and 6 weeks after exacerbation. RESULTS: BPS correlated inversely with forced expiratory volume in 1 s (FEV1) and with residual volume/total lung capacity, an indicator of air trapping. BPS was inversely associated with FEV1 change after TA, by regression analysis. FEV1 did not change significantly after TA if BPS was above the median, whereas patients with BPS below the median had significantly increased FEV1 after TA. BPS was higher in and predicted assignment to SARP phenotype cluster 5 ("severe fixed-airflow asthma"). P-selectin was modestly but significantly increased at exacerbation but returned to baseline within 3 years. CONCLUSIONS: High BPS is associated with airway obstruction, air trapping, the "severe fixed-airflow" cluster, and lack of FEV1 improvement in response to TA injection. P-selectin concentration, which is a stable trait with only modest elevation during exacerbation, may be a useful biomarker for a severe asthma pheno- or endotype characterized by low pulmonary function and lack of corticosteroid responsiveness.

Eosinophil cytolysis on Immunoglobulin G is associated with microtubule formation and suppression of rho-associated protein kinase signalling.

BACKGROUND: The presence of eosinophils in the airway is associated with asthma severity and risk of exacerbations. Cell-free eosinophil granules are found in tissues in eosinophilic diseases, including asthma. This suggests that eosinophils have lysed and released cellular content, likely harming tissues. OBJECTIVE: The present study explores the mechanism of CD32- and αMß2 integrin-dependent eosinophil cytolysis of IL3-primed blood eosinophils seeded on heat-aggregated immunoglobulin G (HA-IgG). METHODS: Cytoskeletal events and signalling pathways potentially involved in cytolysis were assessed using inhibitors. The level of activation of the identified events and pathways involved in cytolysis was measured. In addition, the links between these identified pathways and changes in degranulation (exocytosis) and adhesion were analysed. RESULTS: Cytolysis of IL3-primed eosinophils was dependent on the production of reactive oxygen species (ROS) and downstream phosphorylation of p-38 MAPK. In addition, formation of microtubule (MT) arrays was necessary for cytolysis and was accompanied by changes in MT dynamics as measured by phosphorylation status of stathmin and microtubule-associated protein 4 (MAP4), the latter of which was regulated by ROS production. Reduced ROCK signalling preceded cytolysis, which was associated with eosinophil adhesion and reduced migration. CONCLUSION AND CLINICAL RELEVANCE: In this CD32- and αMß2 integrin-dependent adhesion model, lysing eosinophils exhibit reduced migration and ROCK signalling, as well as both MT dynamic changes and p-38 phosphorylation downstream of ROS production. We propose that interfering with these pathways would modulate eosinophil cytolysis and subsequent eosinophil-driven tissue damage.

Control of cytokine-driven eosinophil migratory behavior by TGF-beta-induced protein (TGFBI) and periostin.

Periostin, which is induced by interleukin (IL)-13, is an extracellular matrix (ECM) protein that supports αMß2 integrin-mediated adhesion and migration of IL-5-stimulated eosinophils. Transforming growth factor (TGF)-ß-induced protein (TGFBI) is a widely expressed periostin paralog known to support monocyte adhesion. Our objective was to compare eosinophil adhesion and migration on TGFBI and periostin in the presence of IL-5-family cytokines. Eosinophil adhesion after 1 h and random motility over 20 h in the presence of various concentrations of IL-5, IL-3, or granulocyte macrophage-colony stimulating factor (GM-CSF) were quantified in wells coated with various concentrations of TGFBI or periostin. Results were compared to video microscopy of eosinophils. Cytokine-stimulated eosinophils adhered equivalently well to TGFBI or periostin in a coating concentration-dependent manner. Adhesion was blocked by anti-αMß2 and stimulated at the lowest concentration by GM-CSF. In the motility assay, periostin was more potent than TGFBI, the coating-concentration effect was bimodal, and IL-3 was the most potent cytokine. Video microscopy revealed that under the optimal coating condition of 5 µg/ml periostin, most eosinophils migrated persistently and were polarized and acorn-shaped with a ruffling forward edge and granules gathered together, in front of the nucleus. On 10 µg/ml periostin or TGFBI, more eosinophils adopted a flattened pancake morphology with dispersed granules and nuclear lobes, and slower migration. Conversion between acorn and pancake morphologies were observed. We conclude that TGFBI or periostin supports two modes of migration by IL-5 family cytokine-activated eosinophils. The rapid mode is favored by intermediate protein coatings and the slower by higher coating concentrations. We speculate that eosinophils move by haptotaxis up a gradient of adhesive ECM protein and then slow down to surveil the tissue.

Myeloid-derived growth factor is a resident endoplasmic reticulum protein.

Human myeloid-derived growth factor (MYDGF; also known as C19orf10) is named based on its identification as a secreted monocyte/macrophage-derived mediator of cardiac repair following myocardial infarction in mice. Homologs of MYDGF, however, are present in organisms throughout and outside of the animal kingdom, some of which lack hematopoietic and circulatory systems. Moreover, the UPF0556 protein domain, which defines these homologs, lacks a known structure. As a result, the functions and properties of MYDGF are unclear. Our current work was initiated to test whether MYDGF is present in secretory vesicles of eosinophils as it was recently reported to be abundant in these cells. However, we could not demonstrate secretion and unexpectedly discovered that MYDGF colocalizes with P4HB in the nuclear envelope, which comprises the bulk of endoplasmic reticulum (ER) in eosinophils, and with P4HB and RCAS1 in Golgi. We noted a ubiquitous C-terminal sequence, BXEL (B, basic; X, variable residue; E, Glu; L, Leu), that has the potential to retain human MYDGF and its homologs in the ER. To test the functionality of this sequence, we expressed full-length human MYDGF or MYDGF lacking the C-terminal Glu-Leu residues in monolayers of human embryonic kidney 293 (HEK293) cells. Full-length MYDGF accumulated in cells, whereas truncated MYDGF appeared in the medium. These observations reveal that MYDGF resides in the ER and Golgi and provide a new framework for investigating and understanding this intriguing protein.