Detection of High-Affinity 4-Integrin Upon Leukocyte Stimulation by Chemoattractants or Chemokines

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.