Differential regulation of neutrophil CD18 integrin function by di- and tri-valent cations: Manganese vs. Gadolinium

Affinity regulation of integrin function plays an important role during both leukocyte-endothelial and leukocyte-leukocyte interactions. We compared the roles of Mn²⁺ (Manganese) and Gd³⁺ (Gadolinium) in regulating leukocyte CD18-integrin function. We observed that: (i) Both cations prolonged neutrophil homotypic aggregation following chemoattractant IL-8 stimulation, with Gd³⁺ being effective at doses two orders of magnitude (10 μM range) lower that Mn²⁺. (ii) While both Gd ³⁺ and Mn²⁺ mediate homotypic cell aggregation via l-selectin and CD18 integrins, their effects on the integrin subunits, LFA-1 (CD11a/CD18) and Mac-1 (CD11b/CD18), was different. Gd³⁺ altered both LFA-1 and Mac-1 function, while the dominant effect of Mn²⁺ was on Mac-1. This effect of Gd³⁺ on LFA-1 function was confirmed in cell-free studies that measured the binding of recombinant ICAM-1 to LFA-1 immobilized on beads. (iii) Both ions augmented the binding of 327C, an antibody that recognizes active CD18 on human neutrophils, both in the presence and absence of exogenous IL-8. The effects of Mn²⁺ was more pronounced since it caused 3-4-fold increase in mAb 327C binding to neutrophils compared to Gd³⁺ which increased antibody binding by only ̃80%. 327C binding was partially reduced by Ca²⁺. Further, 327C binding induced by Mn²⁺ did not correlate tightly with cell adhesion function. (iv) In studies that monitored intracellular Ca²⁺ ([Ca²⁺] _i), the addition of Mn²⁺ but not Gd³⁺ to neutrophils altered [Ca²⁺]_i levels. Overall, while both Gd³⁺ and Mn²⁺ stabilize high affinity CD18 mediated cell adhesion, Gd³⁺ affects integrin conformation while Mn²⁺ may also trigger other effects.