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

Abstract

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 minutes 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 minutes, 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.

Keywords: ADAM8; FAS1 modules; IL-5; Stiny-1; eosinophils; migration; periostin; podosomes.

Fig. 1

Alterations in the morphology of eosinophils upon adhesion to periostin. (A-H) Localization of PSGL-1 (green), F-actin (red), and nucleus (blue) in eosinophils adherent to periostin in the presence (A-D, H) or absence (E-G) of IL-5. Cells were allowed to adhere for 10 (A, B, and E), 30 (C and F), or 60 (D, G, and H) min before fixation and staining. (H) Confocal image, 0.5 μm slice + xz and yz planes. Cells were analyzed by immunofluorescent staining using mAb to PSGL-1 and FITC- or Alexa Fluor 488-conjugated secondary antibody. F-actin was stained with rhodamine-phalloidin and nuclei with DAPI. Arrows, punctate F-actin-containing structures (podosomes). Bar, 10 μm. (I-L) Quantitation of changes in morphology and F-actin and PSGL-1 localization in eosinophils adherent to periostin in the presence of IL-5 for 10, 30 or 60 min, using the Fiji version of ImageJ (http://fiji.sc/Fiji): (I) cell circumference, (J) cell area, (K) peripheral F-actin staining as percentage of circumference, and (L) peripheral PSGL-1 staining as percentage of circumference (mean ± SEM, n = 30 cells at each time point, *P ≤ 0.05, ***P ≤ 0.001 versus 10 min).

Fig. 2

Gelsolin and Arp-3 colocalize with F-actin in podosomes in IL-5-stimulated eosinophils adherent to periostin, whereas cortactin does not. Localization of gelsolin (green, A and C), Arp-3 (green, D and F), cortactin (green, G and H), F-actin (red, B, C, E, F, and H), and nucleus (blue, C and G) in eosinophils adherent to periostin for 1 h in the presence of IL-5. (G and H) Confocal images, 0.3 μm slices close to the substrate level. Cells were analyzed by immunofluorescent staining using mAb to gelsolin or cortactin, or polyclonal antibody to Arp-3, and Alexa Fluor 488-conjugated secondary antibody. F-actin was stained with rhodamine-phalloidin and nuclei with DAPI. Arrows, podosomes. Bars, 10 μm.

Fig. 3

The Stiny-1 periostin epitope is cleared around adherent eosinophils in a manner attenuated by the metalloproteinase inhibitor BB94. (A-C) Clearance of the periostin substrate layer by eosinophils adherent for 10 (A) or 60 (B, C) min in the presence of IL-5. (C) Confocal image, volume view. (D, E) Clearance at 60 min by adherent eosinophils not treated with IL-5 and incubated with DMSO vehicle (1:200) (D) or DMSO and 50 μM BB94 (E). The periostin layer was visualized with mAb Stiny-1 and Alexa Fluor 488-conjugated secondary antibody (green). F-actin was stained with rhodamine-phalloidin (red, A-C) and nuclei with DAPI (blue). Arrowheads, areas of periostin clearance. Arrows, podosomes. Bar, 10 μm. (F) Quantification of cells associated with clearance as described [36] (mean ± standard error of the mean [SEM], n = 3 donors, *P ≤ 0.05 versus control without BB94).

Fig. 4

Stiny-1 mAb binds to the FAS1 4 module of periostin. (A) SDS-PAGE Gelcode Blue staining of PN0 and periostin FAS1 modules (reduced, 1 μg/well). (B) Western blot of PN0 and periostin FAS1 modules (4 pmol/well) using Stiny-1 mAb. PN0, shortest C-terminus splice variant of periostin lacking exons 17, 18, 19, and 21 (80 kDa); 1–2, periostin FAS1 1 to FAS1 2 (32 kDa); 2, periostin FAS1 2 (16 kDa); 2–3, periostin FAS1 2 to FAS1 3 (30 kDa); 3–4, periostin FAS1 3 to FAS1 4 (31 kDa); 3-C, periostin FAS1 3 to the C-terminus (42 kDa). Molecular size markers (kDa) on the left in (A) and (B).

Fig. 5

Stiny-1 periostin epitope clearance is attenuated by the ADAM inhibitor TAPI-1 or by anti-ADAM8 ectodomain antibodies. Clearance from the periostin substrate layer by IL-5-treated eosinophils adherent to periostin for 1 h in presence of DMSO vehicle (A), DMSO and 25 μM TAPI-1 (B), non-immune control (ctrl) goat IgG (C), or 10 μg/ml anti-ADAM8 ectodomain polyclonal antibodies (D). The periostin layer was visualized with mAb Stiny-1 to periostin and Alexa Fluor 488-conjugated secondary antibody (green). Nuclei were stained with DAPI (blue). Arrowheads, areas of periostin clearance. Bar, 10 μm. (E) Quantitation of cells associated with clearance as described [36] (mean ± SEM, n = 3 donors, *P ≤ 0.05 versus DMSO or IgG control). (F) Quantification of clearance as percentage of field area using Fiji (mean ± SEM, n = 4 fields for each treatment, *P ≤ 0.05 versus IgG control).

Fig. 6

Immunofluorescence microscopy and immunoblotting indicate that ADAM8 in adherent eosinophils does not localize to podosomes and that ADAM8 is shed to the medium. (A) Detection of regions recognized by anti-ectodomain and anti-cytoplasmic domain polyclonal antibodies on a schematic modified from [54] of the canonical form or isoform 1 of ADAM8 (UniProt identifier No. P78325–1). Black box, signal peptide; PRO, prodomain; CATA, catalytic (metalloproteinase) domain; DIS, disintegrin domain; CR, cysteine-rich domain; EGF, epidermal growth factor-like domain; TM, transmembrane segment; IC, intracellular or cytoplasmic domain. (B) Localization of ADAM8 (green) and F-actin (red) in eosinophils adherent to periostin for 1 h in the presence of IL-5. Confocal images, 0.3 μm slices close to the substrate level. (C) Localization of ADAM8 (green) and F-actin (red) in eosinophils adherent to periostin for 1 h in the absence of IL-5. Confocal images, 0.3 μm slices. Cells in (B) and (C) were analyzed by immunofluorescent staining using polyclonal anti-ectodomain or cytoplasmic domain antibodies, or control goat or rabbit IgG and Alexa Fluor 488-conjugated secondary antibody. F-actin was stained with rhodamine-phalloidin and nuclei with DAPI (blue). Bar, 10 μm. (D) Recombinant human ADAM8 encompassing amino acids No. 158–497 (predicted molecular mass 38 kDa, 3 pmol/well) immunoblotted with anti-ADAM8 ectodomain or cytoplasmic domain antibody under non-reducing conditions. (E) Cell lysates (lanes labeled C) and supernatants (lanes labeled S) of eosinophils incubated in suspension in the presence of IL-5 for 1 h, immunoblotted with anti-ADAM8 ectodomain or cytoplasmic domain antibody under non-reducing conditions. Arrowheads point to ~60–65 kDa bands in the supernatant reactive with anti-ectodomain but not with anti-cytoplasmic domain antibodies. A similar pattern was found when IL-5 was omitted during the one-hour incubation (not shown, figure shows the lanes from a single blot with the samples from IL-5-stimulated cells from a representative donor). Arrows, molecular size (kDa) markers.

Fig. 7

Eosinophil motility on periostin is inhibited by anti-ADAM8. Paths of eosinophils treated with IL-5 migrating in the presence of non-immune control (ctrl) goat IgG (A) or 10 μg/ml anti-ADAM8 ectodomain polyclonal antibody (B) were revealed by perturbation of a monolayer of 1-μm diameter latex beads. Wells were photographed after 20 h. Bar, 100 μm. (c) Morphometric analysis of track areas using Fiji (mean ± SEM, n = 3 donors, **P ≤ 0.01 versus goat IgG control).