IL‐5‐stimulated eosinophils adherent to periostin undergo stereotypic morphological changes and ADAM8‐dependent migration

Abstract

Summary Background IL-5 causes suspended eosinophils to polarize with filamentous (F)-actin and granules at one pole and the nucleus in a specialized uropod, the “nucleopod”, which is capped with P-selectin glycoprotein ligand-1 (PSGL-1). IL-5 enhances eosinophil adhesion and migration on periostin, an extracellular matrix protein upregulated in asthma by type 2 immunity mediators. Objective Determine how the polarized morphology evolves to foster migration of IL-5-stimulated eosinophils on a surface coated with periostin. Methods Blood eosinophils adhering to adsorbed periostin were imaged at different time points by fluorescent microscopy, and migration of eosinophils on periostin was assayed. Results After 10 min in the presence of IL-5, adherent eosinophils were polarized with PSGL-1 at the nucleopod tip and F-actin distributed diffusely at the opposite end. After 30–60 min, the nucleopod had dissipated such that PSGL-1 was localized in a crescent or ring away from the cell periphery, and F-actin was found in podosome-like structures. The periostin layer, detected with monoclonal antibody Stiny-1, shown here to recognize the FAS1 4 module, was cleared in wide areas around adherent eosinophils. Clearance was attenuated by metalloproteinase inhibitors or antibodies to disintegrin metalloproteinase 8 (ADAM8), a major eosinophil metalloproteinase, previously implicated in asthma pathogenesis. ADAM8 was not found in podosome-like structures, which are associated with proteolytic activity in other cell types. Instead, immunoblotting demonstrated proteoforms of ADAM8 that lack the cytoplasmic tail in the supernatant. Anti-ADAM8 inhibited migration of IL-5-stimulated eosinophils on periostin. Conclusions and Clinical Relevance Migrating IL-5-activated eosinophils on periostin exhibit loss of nucleopodal features and appearance of prominent podosomes along with clearance of the Stiny-1 periostin epitope. Migration and epitope clearance are both attenuated by inhibitors of ADAM8. We propose, therefore, that eosinophils remodel and migrate on periostin-rich extracellular matrix in the asthmatic airway in an ADAM8-dependent manner, making ADAM8 a possible therapeutic target.

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

“…Therefore, we proposed that migration arrest and lack of motion might be an important event preceding or concomitant with cytolysis. To quantify the hypothesized lack of eosinophil migration on IgG, we used a previously described bead‐based cell motility assay . Figure shows that IL3‐primed eosinophils in the absence of IgG (IL3) displayed some migration, whereas in the presence of IgG (IL3IgG) lack of migration or only small circular traces but no migration tracks were observed.…”

“…Purified blood eosinophils suspended in 1 × 10 6 /mL in complete medium were cultured with IL3 (2 ng/mL) for 20 h. Cell motility was assessed in a bead clearing assay as previously described with the following modifications. Wells had been coated overnight at 37°C with HA‐IgG (10 µg/mL, 50 µL per well), or rh periostin (R&D Systems, Minneapolis, MN, 5 µg/mL in Tris‐buffered saline, 100 µL per well), or were not coated.…”

Possible Mechanisms of Eosinophil Accumulation in Eosinophilic Pneumonia

“…Furthermore, IL-5-activated eosinophils migrate on periostin in an ADAM8-dependent manner [77], suggesting that periostin has a role in the eosinophil accumulation in EP. Actually, the concentration of periostin is elevated in the BALF of EP patients [19,78], and it correlates with the eosinophil and lymphocyte counts and the concentrations of IL-5, IL-13, and transforming growth factor β1 [19].…”

Comorbidity of Neurally Mediated Syncope and Allergic Disease in Children

“…In the pathogenesis of AS, exposure to allergens induces helper T cell type 2 (Th2) to produce a series of inflammatory mediators, such as interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-13 (IL-13). IL-5 promotes the aggregation of eosinophils ( 43 ), and IL-4 and IL-13 trigger downstream signaling and upregulate the synthesis of inducible nitric oxide synthase (iNOS) mRNA, leading to a significant increase in the levels of fractional exhaled nitric oxide (FeNO) ( 44 ). iNOS can be detected in the airway epithelial cells of patients with AS ( 45 ), and the levels of FeNO in AS are significantly increased ( 42 ).…”