Outside-in signal transmission by conformational changes in integrin Mac-1.

Intracellular signals associated with or triggered by integrin ligation can control cell survival, differentiation, proliferation, and migration. Despite accumulating evidence that conformational changes regulate integrin affinity to its ligands, how integrin structure regulates signal transmission from the outside to the inside of the cell remains elusive. Using fluorescence resonance energy transfer, we addressed whether conformational changes in integrin Mac-1 are sufficient to transmit outside-in signals in human neutrophils. Mac-1 conformational activation induced by ligand occupancy or activating Ab binding, but not integrin clustering, triggered similar patterns of intracellular protein tyrosine phosphorylation, including Akt phosphorylation, and inhibited spontaneous neutrophil apoptosis, indicating that global conformational changes are critical for Mac-1-dependent outside-in signal transduction. In neutrophils and myeloid K562 cells, ligand ICAM-1 or activating Ab binding promoted switchblade-like extension of the Mac-1 extracellular domain and separation of the alpha(M) and beta(2) subunit cytoplasmic tails, two structural hallmarks of integrin activation. These data suggest the primacy of global conformational changes in the generation of Mac-1 outside-in signals.

Activation of human neutrophil Mac-1 by anion substitution.

Substituting the medium chloride with glucuronate or glutamate causes a rapid, 10 to 30-fold, increase in the binding of the monoclonal antibody, CBRM1/5, which recognizes the high-affinity conformation of the Mac-1 integrin. This change is reflected in functional adhesion assays that show increased adhesion to ICAM-1 coated beads. Blocking antibodies indicate that the increased adhesion is almost entirely due to Mac-1. The inhibitor NPPB (100 microM) reduces Cl(-) efflux into low Cl(-) medium by 75%, and blocks increased CBRM1/5 binding after stimulation with fMLP or TNF-alpha, but has no effect on the anion substitution induced increase in CBRM1/5 binding or adhesion to immobilized ICAM-1. Thus, changes in external anion composition, not internal chloride or increases in Cl(-) efflux, are responsible for Mac-1 activation. This effect is substantial. The percentage of Mac-1 in the high affinity state approaches 100% in glutamate and 50% in glucuronate, a far greater response than what is observed after stimulation with fMLP.

Mechanical shedding of L-selectin from the neutrophil surface during rolling on sialyl Lewis x under flow.

The interaction of L-selectin expressed on leukocytes with endothelial cells leads to capture and rolling and is critical for the recruitment of leukocytes into sites of inflammation. It is known that leukocyte activation by chemoattractants, the change of osmotic pressure in cell media, or cross-linking of L-selectin all result in rapid shedding of L-selectin. Here we present a novel mechanism for surface cleavage of L-selectin on neutrophils during rolling on a sialyl Lewis x-coated surface that involves mechanical force. Flow cytometry and rolling of neutrophils labeled with Qdot(R)-L-selectin antibodies in an in vitro flow chamber showed that the mechanical shedding of L-selectin occurs during rolling and depends on the amount of shear applied. In addition, the mechanical L-selectin shedding causes an increase in cell rolling velocity with rolling duration, suggesting a gradual loss of L-selectin and is mediated by p38 mitogen-activated protein kinase activation. Thus, these data show that mechanical force induces the cleavage of L-selectin from the neutrophil surface during rolling and therefore decreases the adhesion of cells to a ligand-presenting surface in flow.

Volume-sensitive chloride channels do not mediate activation-induced chloride efflux in human neutrophils.

Many agents that activate neutrophils, enabling them to adhere to venular walls at sites of inflammation, cause a rapid Cl(-) efflux. This Cl(-) efflux and the increase in the number and affinity of beta(2) integrin surface adhesion molecules (up-regulation) are all inhibited by ethacrynic acid and certain aminomethyl phenols. The effectiveness of the latter compounds correlates with their inhibition of swelling-activated Cl(-) channels (I(Clvol)), suggesting that I(Clvol) mediates the activator-induced Cl(-) efflux. To test this hypothesis, we used whole-cell patch clamp in hypotonic media to examine the effects of inhibitors of up-regulation on I(Clvol) in neutrophils and promyelocytic leukemic HL-60 cells. Both the channel blocker 5-nitro-2-(3-phenylpropylamino)benzoic acid and [3-methyl-1-p-sulfophenyl-5-pyrazolone-(4)]-[1,3-dibutylbarbituric acid]-pentamethine oxonol (WW781), a nonpenetrating oxonol, inhibited I(Clvol) at concentrations similar to those that inhibit beta(2) integrin up-regulation. However, ethacrynic acid, at the same concentration that inhibits activator-induced Cl(-) efflux and up-regulation, had no effect on I(Clvol) and swelling-activated Cl(-) efflux, providing evidence against the involvement of I(Clvol) in the activator-induced Cl(-) efflux.