A miniature Couette to generate shear for flow cytometry: studying real-time modulation of intracellular calcium in monocytic cells.

Extracellular hydrodynamic forces may be transmitted to the interior of cells through the alteration of integrin conformation and affinity. Integrin activation regulates leukocyte recruitment, cell activation, and transmigration. The cellular and molecular mechanisms for integrin activation are not precisely known, although intracellular calcium signaling is involved. Flow cytometry offers a versatile way to study intracellular calcium signaling in real-time. We report a novel method to generate defined shear by using a miniature Couette. Testing involved measuring shear-induced intracellular calcium signals of human monoblastoid U937 cells in suspension. The Couette was connected externally to a flow cytometer and pressurized at 6 PSI (4.1 N/m(2) ). Cells were subjected to a well-defined shear between 0 and 1,000 s(-1) and delivered continuously within 10 s to a FACScan at 1 µl/s. Intracellular calcium levels and the percentage of cells activated increased as shear increased in duration and intensity.

Regulation of cell adhesion by affinity and conformational unbending of alpha4beta1 integrin.

Rapid activation of integrins in response to chemokine-induced signaling serves as a basis for leukocyte arrest on inflamed endothelium. Current models of integrin activation include increased affinity for ligand, molecular extension, and others. In this study, using real-time fluorescence resonance energy transfer to assess alpha(4)beta(1) integrin conformational unbending and fluorescent ligand binding to assess affinity, we report at least four receptor states with independent regulation of affinity and unbending. Moreover, kinetic analysis of chemokine-induced integrin conformational unbending and ligand-binding affinity revealed conditions under which the affinity change was transient whereas the unbending was sustained. In a VLA-4/VCAM-1-specific myeloid cell adhesion model system, changes in the affinity of the VLA-4-binding pocket were reflected in rapid cell aggregation and disaggregation. However, the initial rate of cell aggregation increased 9-fold upon activation, of which only 2.5-fold was attributable to the increased affinity of the binding pocket. These data show that independent regulation of affinity and conformational unbending represents a novel and fundamental mechanism for regulation of integrin-dependent adhesion in which the increased affinity appears to account primarily for the increasing lifetime of the alpha(4)beta(1) integrin/VCAM-1 bond, whereas the unbending accounts for the increased capture efficiency.

Real-time analysis of very late antigen-4 affinity modulation by shear.

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 alpha(4)beta(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(2+) ionophores and N,N'-[1,2-ethanediyl-bis(oxy-2,1-phenylene)]bis[N-[2-[(acetyloxy)methoxy]-2-oxoethyl]]-, bis[(acetyloxy)methyl]ester demonstrate that the affinity regulation of VLA-4 in the presence of shear was related to Ca(2+) signaling. Pertussis toxin treatment implicates G(i) in an unknown pathway that connects shear, Ca(2+) elevation, VLA-4 affinity, and cell avidity.

Conformational regulation of alpha 4 beta 1-integrin affinity by reducing agents. "Inside-out" signaling is independent of and additive to reduction-regulated integrin activation.

The alpha(4)beta(1)-integrin (very late antigen-4 (VLA-4), CD49d/CD29) is an adhesion receptor involved in the interaction of lymphocytes, dendritic cells, and stem cells with the extracellular matrix and endothelial cells. This and other integrins have the ability to regulate their affinity for ligands through a process termed "inside-out" signaling that affects cell adhesion avidity. Several mechanisms are known to regulate integrin affinity and conformation: conformational changes induced by separation of the C-terminal tails, divalent ions, and reducing agents. Recently, we described a fluorescent LDV-containing small molecule that was used to monitor VLA-4 affinity changes in live cells (Chigaev, A., Blenc, A. M., Braaten, J. V., Kumaraswamy, N., Kepley, C. L., Andrews, R. P., Oliver, J. M., Edwards, B. S., Prossnitz, E. R., Larson, R. S., and Sklar, L. A. (2001) J. Biol. Chem. 276, 48670-48678). Using the same molecule, we also developed a fluorescence resonance energy transfer-based assay to probe the "switchblade-like" opening of VLA-4 upon activation. Here, we investigated the effect of reducing agents on the affinity and conformational state of the VLA-4 integrin simultaneously with cell activation initiated by inside-out signaling through G protein-coupled receptors or Mn(2+) in live cells in real time. We found that reducing agents (dithiothreitol and 2,3-dimercapto-1-propanesulfonic acid) induced multiple states of high affinity of VLA-4, where the affinity change was accompanied by an extension of the integrin molecule. Bacitracin, an inhibitor of the reductive function of the plasma membrane, diminished the effect of dithiothreitol, but had no effect on inside-out signaling. Based on this result and differences in the kinetics of integrin activation, we conclude that conformational activation of VLA-4 by inside-out signaling is independent of and additive to reduction-regulated integrin activation.