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Eosinophils Adhere to Vascular Cell Adhesion Molecule-1 via Podosomes

Departments of Medicine and Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin

Address correspondence to: Mats W. Johansson, Ph.D., Department of Medicine, University of Wisconsin, 4285A, Medical Sciences Center, 1300 University Avenue, Madison, WI 53706-1532. E-mail: mwj{at}medicine.wisc.edu

Vascular cell adhesion molecule (VCAM)-1 supports specific eosinophil adhesion via ₄ß₁ integrin. We tested the hypothesis that adhesive contacts formed by eosinophils on VCAM-1 are different from focal adhesions formed by adherent fibroblasts. Eosinophils adherent on VCAM-1 formed punctate adhesions that fit the criteria for podosomes, highly dynamic structures found in adherent transformed fibroblasts, osteoclasts, and macrophages. The structures contained ß₁ integrin subunit, phosphotyrosine-containing proteins, punctate filamentous actin, and gelsolin, a podosome marker. In contrast, nontransformed fibroblasts on VCAM-1 formed peripheral focal adhesions that were positive for ₄, ß₁, phosphotyrosine, vinculin, talin, and paxillin; negative for gelsolin; and associated with microfilaments. Phorbol myristate acetate or tumor necrosis factor- and interleukin-5 stimulated podosome formation in adherent eosinophils. Because podosomes in tumor cells are associated with extracellular matrix degradation, we analyzed the VCAM-1 layer. VCAM-1 was lost under adherent eosinophils but not under adherent fibroblasts. This loss was inhibited by the metalloproteinase inhibitor ortho-phenanthroline and correlated with expression and podosome localization of a membrane-tethered metalloproteinase, a disintegrin and metalloproteinase domain 8. Podosome-mediated VCAM-1 clearance may be a mechanism to regulate eosinophil arrest and extravasation in allergic conditions such as asthma.

Abbreviations: a disintegrin and metalloprotease, ADAM • bovine serum albumin, BSA • cluster of differentiation, CD • Chinese hamster ovary, CHO • fluorescence-activated cell sorting, FACS • filamentous actin, F-actin • fetal bovine serum, FBS • fluorescein isothiocyanate, FITC • formyl-methionylleucylphenylalanine, FMLP • fibronectin, FN • granulocyte-macrophage–colony stimulating factor, GM-CSF • 12(S)-hydroxy-(5Z,8Z,10E,14Z)-eicosatetraenoic acid, 12(S)-HETE • intercellular cell adhesion molecule, ICAM • immunoglobulin, Ig • interleukin, IL • monoclonal antibody, mAb • matrix metalloproteinase, MMP • ortho-phenanthroline, OP • platelet activating factor, PAF • pAcGP67.coco plasmid, pCOCO • phosphate-buffered saline, PBS • phorbol myristate acetate, PMA • phenylmethylsulfonyl fluoride, PMSF • transforming protein of Rous sarcoma virus, Src • Tris-buffered saline, TBS • tumor necrosis factor, TNF • vascular cell adhesion molecule, VCAM

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