Engagement of the T-cell antigen receptor by anti-CD3 monoclonal antibody causes a rapid increase in lymphocyte F-actin.
Activation of protein kinase C (PKC) causes a rapid and sustained increase in the F-actin of T lymphocytes. Because the phosphatidylinositol pathway and the cytoskeleton play a role in lymphocyte activation, we examined the relationship between signal transduction and the F-actin increase in human blood T cells. Anti-CD3 monoclonal antibodies (mAbs) initiate signals which result in activation of T lymphocytes through the T-cell receptor (TCR), involving the phosphatidylinositol pathway, activation of PKC, and increasing intracellular calcium (Cai2+). The fluorescent probe NBD-phallacidin was used to examine the conformational state of actin following stimulation of T lymphocytes with anti-CD3 mAb. Each of three different murine anti-CD3 mAbs caused rapid increases in lymphocytic F-actin content, which was enhanced by cross-linking with a goat anti-mouse IgG. A maximally effective dose of the mAb Leu 4 caused a rise in cellular F-actin of 1.8-fold at 2 minutes and a three-fold increase in Cai2+. Ionomycin, 100 nM, caused a Cai2+ rise similar in magnitude to that caused by anti-CD3 mAb but had no effect on F-actin content. Inhibitors of PKC, 1(5-isoquinolinylsulfonyl)-2-methylpiperazine (H7), sphingosine, and sphinganine lowered the resting cellular F-actin and partially blocked the increase in F-actin caused by either anti-CD3 mAb or ionomycin; however, they had no effect on the rise in Cai2+. Cells leached of Ca2+ with EGTA and ionomycin exhibited no Cai2+ increase in response to anti-CD3 mAb or ionomycin; such cells retained the F-actin increase caused by anti-CD3 mAb. We conclude that stimulation of human T lymphocytes via the TCR causes an early rapid increase in F-actin content. Activation of PKC may play a role but the concomitant Cai2+ increase is neither sufficient nor necessary for the F-actin increase.
Phatak
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PD
Department of Medicine, University of Rochester School of Medicine and Dentistry, New York 14642.
Packman
C H
CH
D000199:Actins / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000276:immunology*; D002118:Calcium / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D015759:Ionomycin / Q000494:pharmacology; D008214:Lymphocytes / Q000378:metabolism*; D047428:Protein Kinase Inhibitors; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*
D000199:Actins; D000911:Antibodies, Monoclonal; D047428:Protein Kinase Inhibitors; D011948:Receptors, Antigen, T-Cell; D015759:Ionomycin; D002118:Calcium