Beta2-integrin-mediated adhesion and intracellular Ca2+ release in human eosinophils.
Human eosinophils spontaneously adhere to various substrates in the absence of exogenously added activators. In the present study a method was developed for characterizing eosinophil adhesion by measuring changes in impedance. Impedance measurements were performed in HCO(3)-buffered HybriCare medium maintained in a humidified 5% CO(2) incubator at 37 degrees C. Impedance increased by more than 1 kOmega within minutes after eosinophils made contact with the substrate, reaching a peak within 20 min. Blocking mobilization of intracellular [Ca(2+)] that precedes adhesion with BAPTA-AM (10 microM) completely inhibited the rise in impedance as well as the changes in cell shape typically observed in adherent cells. However, lowering the extracellular [Ca(2+)] with 2.5 mM EGTA did not inhibit the increase in impedance. Pretreatment with anti-CD18 antibody to block substrate interactions with beta(2)-integrins, or jasplakinolide (2 microM) to block actin reorganization, abolished the increase in impedance and adherent morphology of the cells. Exposure of eosinophils to the phosphatidylinositol 3 kinase inhibitor LY294002 (5 microM) or treatment with protein kinase C zeta pseudosubstrate to competitively inhibit activity of the enzyme significantly reduced the increase in impedance and inhibited the cell spreading associated with adhesion. These results demonstrate a novel method for measuring eosinophil adhesion and showed that, following formation of a tethered attachment, a rapid increase in intracellular [Ca(2+)] precedes the cytoskeletal rearrangements required for cell shape changes and plasma membrane-substrate interactions associated with adhesion.
Bankers-Fulbright
Jennifer L
JL
Department of Biology, Augsburg College, Minneapolis, MN 55454, USA.
Bartemes
Kathleen R
KR
Kephart
Gail M
GM
Kita
Hirohito
H
O'Grady
Scott M
SM
D018821:CD18 Antigens / Q000378:metabolism / Q000502:physiology*; D002118:Calcium / Q000378:metabolism*; D002448:Cell Adhesion / Q000502:physiology*; D002478:Cells, Cultured; D004804:Eosinophils / Q000166:cytology* / Q000378:metabolism*; D006801:Humans; D007150:Immunohistochemistry; D018613:Microscopy, Confocal
D018821:CD18 Antigens; D002118:Calcium