(645b) Manganese and Gadolinum Regulate Neutrophil Cd18 Integrin Affinity Via Distinct Mechanisms

Affinity regulation of leukocyte integrins plays an important role in controlling the progress of normal immune response and inflammatory ailments. Such modulation of receptor-ligand binding kinetics is a key step during leukocyte-endothelial cell and leukocyte-leukocyte binding in the human vasculature. The divalent cation Manganese (Mn2+) has been traditionally used to alter the affinity state of integrins and to study the effect of resulting changes on molecular function. In the current paper, we introduce another cation, the trivalent lanthanide Gadolinum (Gd3+), as another tool that can be used in studies of integrin biology. In homotypic neutrophil aggregation experiments, we observe that while neutrophil-neutrophil adhesion is reversible following stimulation with a range of chemokines (fMLP, PMA, IL-8), cell adhesion is irreversibly sustained upon addition of either μM concentrations of Gd3+ or mM quantities of Mn2+. Both Gd3+ and Mn2+ mediate homotypic cell aggregation via common mechanisms involving L-selectin and two members of the CD18 integrin family, LFA-1 and Mac-1. The effects of these cations on integrin function are however distinct since Gd3+ augments both LFA-1 and Mac-1 affinity, while Mn2+ only upregulates Mac-1 function. The effect of Gd3+ on LFA-1 affinity was confirmed in cell-free studies where the binding of dimeric ICAM-1 to beads bearing immobilized LFA-1 was augmented in the presence of the cation. In further support of the proposition that Gd3+ and Mn2+ bind integrins, both antibodies were observed to enhance the binding of 327C (an antibody that specifically recognizes the active CD18 integrin) to isolated human neutrophils. In these studies, 327C binding was enhanced by cations both in the presence and absence of exogenous IL-8 stimulation. Mn2+ caused upto ~8-fold increase in 327C binding to neutrophils while Gd3+ only increased antibody binding by 1.5-2 fold. Since 327C binding was induced by Mn2+ even at low concentrations which did not support irreversible neutrophil aggregation, we conclude that changes in Mn2+ induced integrin conformation do not correlate tightly with integrin adhesive function. In other studies that examined potential mechanisms by which cations can affect leukocyte function, we observed that Mn2+ but not Gd3+ altered the rates of calcium release from neutrophil granules. Overall, while both Gd3+ and Mn2+ enhanced integrin affinity and reduced the rates of active integrin turnover, Gd3+ appeared to do this more directly by affecting integrin conformation. Mn2+ plays other roles including altering cellular calcium levels.