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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Alon, R., Kassner, P. D., Carr, M. W., Finger, E. B., Hemler, M. E., and Springer, T. A. (1995) The integrin VLA-4 supports tethering and rolling in flow on VCAM-1. J. Cell. Biol. 128, 1243–1253.
Berlin, C., Bargatze, R. F., Campbell, J. J., et al. (1995) alpha 4 integrins mediate lymphocyte attachment and rolling under physiologic flow. Cell 80, 413–422.
Chen, C., Mobley, J. L., Dwir, O., et al. (1999) High affinity very late antigen-4 subsets expressed on T cells are mandatory for spontaneous adhesion strengthening but not for rolling on VCAM-1 in shear flow. J. Immunol. 162, 1084–1095.
Campbell, J. J., Qin, S., Bacon, K. B., Mackay, C. R., and Butcher, E. C. (1996) Biology of chemokine and classical chemoattractant receptors: differential requirements for adhesion-triggering versus chemotactic responses in lymphoid cells. J. Cell. Biol. 134, 255–266.
Campbell, J. J., Hedrick, J., Zlotnik, A., Siani, M. A., Thompson, D. A., and Butcher, E. C. (1998) Chemokines and the arrest of lymphocytes rolling under flow conditions. Science 279, 381–384.
Chan, J. R., Hyduk, S. J., and Cybulsky, M. I. (2001) Chemoattractants induce a rapid and transient upregulation of monocyte alpha4 integrin affinity for vascular cell adhesion molecule 1 which mediates arrest: an early step in the process of emigration. J. Exp. Med. 193, 1149–1158.
Jakubowski, A., Rosa, M. D., Bixler, S., Lobb, R., and Burkly, L. C. (1995) Vascular cell adhesion molecule (VCAM)-Ig fusion protein defines distinct affinity states of the very late antigen-4 (VLA-4) receptor. Cell Adhes. Commun. 3, 131–142.
Chigaev, A., Blenc, A. M., Braaten, J. V., et al. (2001) Real time analysis of the affinity regulation of alpha 4-integrin. The physiologically activated receptor is intermediate in affinity between resting and Mn(2+) or antibody activation. J. Biol. Chem. 276, 48,670–48,678.
Takagi, J., Petre, B., Walz, T., and Springer, T. (2002) Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 110, 599–611.
Constantin, G., Majeed, M., Giagulli, C., et al. (2000) Chemokines trigger immediate beta2 integrin affinity and mobility changes: differential regulation and roles in lymphocyte arrest under flow. Immunity 13, 759–769.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2004 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
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
Download citation
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
eBook Packages: Springer Protocols