| Budget Amount *help |
¥3,200,000 (Direct Cost: ¥3,200,000)
Fiscal Year 2001: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 2000: ¥1,700,000 (Direct Cost: ¥1,700,000)
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| Research Abstract |
β_2-integrins are essential for transmigration of neutrophils through the endothelium^1; however, the mechanisms underlying pseudopod formation and force generation in this process are still unknown. This study shows that, in suspended human neutrophils, local ligation of CD18 morecules, brought by an anti-CD18 coated polystyrene bead captured in an optical trap, induced pseudopod formation at the cell membrane presentation site. By contrast, local presentation of mouse Ig G_1 coated bead did not induce pseudopod, indicating that pseudopod formation by an anti-CD18 coated bead presentation was not caused by mechanical stimuli or nonspecific If presentation. Prevention of myosin light chain phosphorylation with myosin light-chain kinase inhibitor ML-9 or ML-7,and inhibition of myosin ATPase butanedione monoxime (BDM), dose-dependently inhibited the pseudopod formation induced by CD18 ligation. The actin polymerization inhibitor cytochalasin D inhibited the pseudopod formation, whereas the microtubule polymerization inhibitor nocodazole had no effect on it. These results suggest that, local activation of β_2-integrins at the cell membrane induces pseudopod formation at that site, and that site, and that these events occur via an actomyosin dependent but microtubule independent pathway. The former finding implies that neutrophils may extend pseudopod toward the local surface of endothelial cells where more ICAM-1 molecules are expressed and thus may initiate transendothelial migration, and the latter finding implies that the force generation for this protrusion may be driven by hydraulic pressure.
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