Abstract
On circulating leukocytes, including monocytes and lymphocytes, α4-integrins are expressed in a low-affinity conformation. Low-affinity interactions with its ligand, vascular cell adhesion molecule-1 (VCAM-1), result in leukocyte tethering and rolling under flow (1,2), whereas high-affinity interactions mediate leukocyte arrest (3). Rapid triggering of integrin-mediated arrest occurs upon leukocyte stimulation with chemoattractants and chemokines (4,5). In monocytes, α4-integrin affinity is rapidly upregulated and mediates arrest (6). Changes in affinity may be monitored by binding of a soluble ligand because high-affinity α4-integrins form stable interactions with a soluble ligand, unlike low-affinity α4-integrins (7). In this protocol, binding of recombinant chimeric human VCAM-1 is used to identify high-affinity α4-integrins by flow cytometry. In addition, a recently reported method for monitoring α4-integrin affinity in real time using a ligand-mimetic peptide, which contains the leucine, aspartic acid, valine (LDV) consensus binding sequence for α4-integrin, is described (8). The cell line U937, transfected with the human formyl peptide receptor (FPR), and stimulated with fMLP or SDF-1α is used as a model. These methods allow for analysis of α4-integrin activity without the complications of postligand-binding events inherent in cell adhesion-based assays.
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References
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© 2004 Humana Press Inc., Totowa, NJ
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Chan, J.R., Cybulsky, M.I. (2004). Detection of High-Affinity α4-Integrin Upon Leukocyte Stimulation by Chemoattractants or Chemokines. In: D’Ambrosio, D., Sinigaglia, F. (eds) Cell Migration in Inflammation and Immunity. Methods in Molecular Biology™, vol 239. Humana Press. https://doi.org/10.1385/1-59259-435-2:261
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DOI: https://doi.org/10.1385/1-59259-435-2:261
Publisher Name: Humana Press
Print ISBN: 978-1-58829-102-8
Online ISBN: 978-1-59259-435-1
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