Substrate‐induced eosinophil adhesion requires intracellular Ca 2+ mobilization and PI‐3 kinase activation.

Abstract

Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added signaling molecules. Adhesion initially involves formation of a tethered attachment, followed by a rapid increase in intracellular Ca2+. In the present study we developed a method for characterizing adhesion in real time by measuring changes in impedance as eosinophils adhere to the surface of gold-film electrodes placed on the surface of a tissue culture dish. Impedance measurements were made in serum-free, HCO3 buffered HybriCare or DMEM media maintained in a humidified 5% CO2 incubator at 37 °C. Impedance increased by more than 1 k within minutes after eosinophils (5 x 105 cells/well) made contact with the electrode and reached a peak within approximately 15 minutes. Blocking the increase in intracellular Ca2+ that precedes adhesion with BAPTA-AM (10 μM) completely inhibited the increase in impedance along with changes in cell shape typically observed in adherent cells. Moreover, pretreatment with anti-CD18 antibody to block substrate interactions with β2 integrins or jasplakinolide (2 μM), to induce actin polymerization, also abolished the increase in impedance and adherent morphology of the cells. Pretreatment of eosinophils with the PI-3 kinase inhibitors Wortmannin (5 μM) or LY294002 (5 μM) significantly reduced the increase in impedance, but only a relatively small decrease in impedance was detected when cells were treated with the pan-specific PKC inhibitor GF-109203X (2 μM). These results demonstrate a novel method for measuring eosinophil adhesion and show that Ca2+ mobilization and PI-3 kinase activation are essential for substrate-induced adhesion to occur.