IL-5 Induces Suspended Eosinophils to Undergo Unique Global Reorganization Associated with Priming

Abstract

The experiments described herein define a unique program of polarization of suspended human eosinophils stimulated with IL-5 family cytokines. We found that eosinophil granules and the nucleus move in opposite directions to form, respectively, a granular compartment and the nucleopod, a specialized uropod occupied by the nucleus and covered with adhesion receptors, including Pselectin glycoprotein ligand-1, CD44, and activated a M b 2 integrin. Ligated IL-5 family receptors localize specifically at the tip of the nucleopod in proximity to downstream signaling partners Janus tyrosine kinase 2, signal transducer and activator of transcription-1 and -5, and extracellular signal-regulated kinase. Microscopy and effects of cytochalasin B and nocodazole indicate that remodeling of filamentous actin and reorientation of the microtubule network are required for eosinophil polarization and nucleopod formation. IL-5 induces persistent polarization and extracellular signal-regulated kinase redistribution that are associated with eosinophil priming, a robust response on subsequent stimulation with N-formylmethionyl-leucyl-phenylalanine. Global reorganization of cytoskeleton, organelles, adhesion receptors, and signaling molecules likely facilitates vascular arrest, extravasation, migration, granule release, and survival of eosinophils entering inflamed tissues from the bloodstream.

Myeloid-derived growth factor is a resident endoplasmic reticulum protein

“…1B). We had hypothesized that MYDGF would be packaged for secretion in the large phase-dense granules that fill the cytoplasm of unactivated eosinophils and move to the granular pole when eosinophils are induced to polarize with IL5 (13). We detected no signal in granules above the auto-fluorescence (due to a high content of FAD (14)) found in the specificity control.…”

“…Human blood eosinophils were purified to >98% purity by Percoll centrifugation and negative selection for neutrophils and monocytes as described previously (13). The cells were received without identifying information in accord with a protocol approved (#2013-1570) by the University of WisconsinMadison Center for Health Sciences Institutional Review Board.…”

The Peripheral Blood Eosinophil Proteome

“…To assess the impact of platelet contamination, we purified an additional sample of eosinophils from a single donor in which negative selection was accomplished with the antibody cocktail described above or with the cocktail plus an antibody to CD61, the β 3 subunit of the major platelet integrin. Purified eosinophils, received on ice in 2% calf serum, were resuspended in RPMI at 2.5 × 10 6 cells/ml, incubated at 37 °C for 1 h, 14 and then centrifuged at 1200 rpm (260 g in a Sorvall Technospin R centrifuge, Du Pont, Wilmington, DE) for 10 min at 4 °C. The supernatant was aspirated, and the cell pellet was immediately frozen by immersing the tube in N 2 ( l ).…”

Anti-CD69 monoclonal antibody treatment inhibits airway inflammation in a mouse model of asthma

“…IL-5 has been shown to activate eosinophils to produce more IL-5 (Spencer et al, 2009), thus forming a positive feedback loop to expand the eosinophil reservoir in peripheral blood and bone marrow reacting to the allergen challenge. IL-5 also regulates the expression of genes involved in growth, survival, and effector function of eosinophils (Takatsu and Nakajima, 2008;Han and Mosher, 2014). Therefore, decreasing IL-5 levels in the local microenvironment may dramatically shorten the life span of T cells and eosinophils.…”