Expression of a functional laminin receptor (alpha 6 beta 1, very late activation antigen-6) on human eosinophils

Abstract

The mechanisms responsible for the accumulation of eosinophils at sites of allergic and other inflammatory reactions are unknown, but recent studies have implicated both eosinophil and endothelial adhesion molecules in this process. However, less well studied have been the adhesive interactions between eosinophils and the subendothelial basement membrane and interstitial connective tissues. To test the hypothesis that eosinophils might interact with extracellular matrix proteins, we analyzed purified human eosinophils for the expression and function of various beta 1 integrins. Using indirect immunofluorescence and flow cytometry, purified eosinophils from mildly allergic donors were found to consistently express the integrin subunits beta 1 (CD29), alpha 4 (CD49d, very late activation antigen [VLA]-4 alpha), and alpha 6 (CD49f, VLA-6 alpha). No significant expression of the alpha 1, alpha 2, alpha 3, alpha 5, or beta 4 subunits was detected. Platelet contamination of the eosinophil preparations was excluded by light microscopy and by the inability to detect expression of platelet glycoproteins alpha v, CD41b, and CD42b. Immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of eosinophils confirmed the expression of cell-surface beta 1, alpha 4, and alpha 6. Furthermore, eosinophils purified from allergic donors were shown to adhere to plate-bound laminin, but not to type 1 or type 4 collagen. Adhesion to laminin was concentration-dependent, required divalent cations, and was completely and specifically inhibited by the anti-alpha 6 monoclonal antibody (MoAb) GoH3 and by the anti-beta 1 MoAb 33B6. Interestingly, the anti-beta 1 MoAb 18D3 (which like 33B6 blocks eosinophil binding to VCAM-1) did not inhibit eosinophil adhesion to laminin, suggesting that there are functionally distinct epitopes on the beta 1 subunit. Eosinophils purified from 4 healthy, nonallergic donors also showed alpha 6-dependent adhesion to laminin, although these cells adhered less well. These studies establish the expression of alpha 6 beta 1 on human eosinophils and document its function as a laminin receptor. Interaction of eosinophil alpha 6 beta 1 with laminin, eg, in basement membranes, may contribute to the localization of these cells at inflammatory sites in vivo.

3D Timelapse Analysis of Muscle Satellite Cell Motility

“…Neutralizing sterile monoclonal antibodies to integrin chains were purchased from BioLegend (San Diego, CA, http://www.biolegend.com ) (anti-α2, clone HMα2 [ 19 ]; anti-α4, clone 9C10(MFR4.B) [ 20 ]; anti-α4, clone R1-2 [ 21 ]; anti-α5, clone 5H10-27(MFR5) [ 20 ]; anti-α5, clone HMα5-1 [ 22 ]; anti-α6, clone GoH3 [ 23 ]; anti-αV, clone RMV-7 [ 24 ]; anti-β1, clone HMβ1-1 [ 25 ]; and anti-β2, clone M18/2 [ 26 ]) or MBL International (Nagoya, Japan, http://www.mblintl.com ) (anti-α7, clone 6A11, [ 27 ]); all antibodies were tested for adhesion and blocking of substrate adhesion to confirm bioactivity. Collagen gel fiber preparations were treated with 25 μg/ml concentrations of individual antibodies except for anti-β1 which was at 50 μg/mL; when two antibodies were available to the same integrin chain, they were both added to achieve maximal blocking.…”

Eosinophils: Biological Properties and Role in Health and Disease

“…The firm adhesion of the eosinophil, and transmigration across the vascular epithelium into tissues is regulated by coordinated interaction between networks involving chemokine and cytokine signaling, eosinophil adhesion molecules (e.g., selectins and integrins), and integrin receptors such as vascular cell adhesion molecule (VCAM)-1, mucosal addressin cell adhesion molecule (MAdCAM)-1 and intercellular adhesion molecule (ICAM)-1 expressed on vascular endothelial cells (Kunkel & Butcher 2002;Hogan et al 2004). Integrins are heterodimeric surface molecules consisting of an a and b chain and eosinophils express members of the b1 (a4b1 and a6b1), b2 (aLb2, aMb2, aXb2, and aDb2) and a7 (a4b7) integrin families (Georas et al 1993;Grayson et al 1998;Tachimoto & Bochner 2000;Bochner & Schleimer 2001;Tachimoto et al 2002). These various integrin molecules selectively interact with adhesion receptors (VCAM-1, MAdCAM-1, ICAM-1, -2 and -3, and fibrinogen) expressed on the vascular endothelium.…”

The laminin β1-competing peptide YIGSR induces a hypercontractile, hypoproliferative airway smooth muscle phenotype in an animal model of allergic asthma

“…Eosinophils express a number of integrins, of which the α6β1 mediates adhesion to laminin, but not to collagen type I or type IV [ 45 , 46 ]. Eosinophils isolated from allergic donors show higher adhesion to laminin than those isolated from healthy subjects [ 46 ]. Migration of eosinophils through matrigel, a basement membrane extract containing laminin-111, also requires interaction with β1-integrins [ 46 ].…”

“…Eosinophils isolated from allergic donors show higher adhesion to laminin than those isolated from healthy subjects [ 46 ]. Migration of eosinophils through matrigel, a basement membrane extract containing laminin-111, also requires interaction with β1-integrins [ 46 ]. These findings suggest that laminin-competing peptides could affect allergen-induced airway infiltration of inflammatory cells.…”