Real-time Analysis of Very Late Antigen-4 Affinity Modulation by Shear

Abstract

Shear promotes endothelial recruitment of leukocytes, cell activation, and transmigration. Mechanical stress on cells caused by shear can induce a rapid integrin conformational change and activation, followed by an increase in binding to the extracellular matrix. The molecular mechanism of increased avidity is unknown. We have shown previously that the affinity of the ␣ 4 ␤ 1 integrin, very late antigen-4 (VLA-4), measured with an LDV-containing small molecule, varies with cellular avidity, measured from cell disaggregation rates. In this study, we measured in real time affinity changes of VLA-4 in response to shear. The resulting affinity was comparable with the state mediated by receptor signaling and corresponded in time with intracellular Ca 2؉ responses. Ca Leukocytes are recruited to endothelial cells in a multistep process using selectin and integrin adhesion molecules (1, 2). These molecules allow a cell to tether, roll, adhere, and transmigrate along and across an endothelial layer. Selectin and some integrin molecules and their associated ligands mediate tethering and rolling interactions. Firm adhesion is mediated by vascular ligands of the immunoglobulin superfamily such as vascular cell adhesion molecule 1 (VCAM-1) 1 and their associated integrins (1, 2). The adhesive strength or avidity (3) of cells expressing integrins can be rapidly modulated by chemokines and chemoattractants, which also regulate leukocyte recruitment and migration across vascular endothelium. The rapid changes in avidity have been attributed to changes in the number of interacting molecules or valency due to molecular redistribution or clustering and to changes in the affinity of the individual receptor-ligand bonds (3-10).Physiological shear can also regulate leukocyte traffic by stimulating mechanosensors on neutrophils, monocytes, lymphocytes, erythrocytes, and platelets (see Ref. 11 and references therein). Shear arises from bifurcating blood vessels or rapid changes in blood vessel diameters. Shear acting on leukocytes, bound to endothelial cells, produces mechanical stress on the cells or their receptors, regulating cell growth and proliferation, protein synthesis, gene expression, and blood cell recruitment (12, 13). Integrins (such as ␣ v ␤ 3 , ␣ 5 ␤ 1 , ␣ 4 ␤ 1 , and ␣ 2 ␤ 1 ) on endothelial cells can act as mechanosensors to changes in blood flow (13,14) and trigger an intracellular signaling pathway involving focal adhesion kinase and mitogen-activated protein kinase cascades. How shear specifically induces blood cell adhesiveness or recruitment through mechanosensors is unknown. Indirect evidence shows that increased integrin binding to the extracellular matrix occurs when shear acts on cells or their mechanosensors to induce intracellular signaling. For example, intracellular signaling leads to conformational changes and activation of ␣ v ␤ 3 on endothelial cells and ␣ 4 ␤ 1 and ␣ 5 ␤ 1 integrins on monocytic cells (15, 16). Shear acting on endothelial cells affects the GTPase Rho signaling pathway and in monocyti...

Immobilized Stromal Cell-Derived Factor-1α Triggers Rapid VLA-4 Affinity Increases to Stabilize Lymphocyte Tethers on VCAM-1 and Subsequently Initiate Firm Adhesion

“…The small molecule has several technical advantages over mAbs and large soluble proteins; primarily being that its size is less likely to perturb cell function and secondarily, that its affinity for ligand is much more easily measurable than is the case with the large molecules. Indeed, Scatchard analysis has shown the small molecule blocker to have monospecific and saturable binding to VLA-4, with distinct affinities for resting and activated VLA-4 (18,20). In contrast to peptides that are unable to discriminate between high-and low-affinity VLA-4, the LDV-based small molecule has a 40-fold higher affinity for the Mn 2ϩ -induced state of VLA-4 than for resting VLA-4, thereby allowing functional discrimination between activated and nonactivated VLA-4.…”

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