| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 1998: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1997: ¥2,100,000 (Direct Cost: ¥2,100,000)
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| Research Abstract |
We developed a monoclonal antibody (mAb), termed anti-4C8, that blocks transmigration but not adhesion in a culture system in which high CD26 expressing (CD26^<hi>) T cells preferentially migrate through human umbilical vein endothelial cell (HUVEC) monolayers cultured on collagen gels. This mAb also appears to inhibit TNF-alphat production by the interaction between T cells and synoviocytes from patients with rheumatoid arthritis. Anti-4C8 reacted with all CD3+ T cells and monocytes, but not neutrophils or HUVECs. The structure defined by this antibody was an 80 kDa molecule. The mAb at 1 mug/ml inhibited 80-90% of migration of CD3+ T cells through unstimulated and IFN-gamma-stimulated HUVEC monolayers without interfering with adhesion and cell motility. When added to the cultures after the adhesion, anti-4C8 completely blocked subsequent transmigration of adherent T cells. Phase-contrast and electron microscopy revealed that T cells are arrested at the intercellular junctions of HUVE
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Cs in the presence of anti-4C8. Anti-4C8 exhibited agonistic effects on resting T cells without other stimuli under culture conditions in which anti-4C8 can stimulate T cells. First, in the checkerboard assay using collagen gels, the antibody promoted chemokinetic migration of the cells in a dose-dependent manner from 0.1 to 10 mug/ml. The predominant population of T cells that migrated into collagen gels with impregnated anti-4C8 were CD26^<hi>. Second, solid-phase immobilized anti-4C8 induced adhesion of T cells to the substrate, often with polarizations in cell shape and large pseudopods rich in filamentous (F-) actin. Third, soluble anti-4C8 augmented F-actin content preferentially in CD26^<hi> T cells when added to T cells at a high dose of 10 gg/ml. These findings suggest that stimulation via the 4C8 antigen increases cell motility of CD26^<hi> cells with profound cytoskeletal changes through signaling pathways including G proteins. The 4C8 antigen may be involved in preferential transmigration of CD26^<hi> cells adherent to HUVEC.The cloning of the gene coding this antigen is proceeding now. Less
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