A Role for Phosphatidylinositol 3-Kinase in the Regulation of 131 Integrin Activity by the CD2 Antigen

The rapid and reversible upregulation of the functional activity of integrin receptors on T lymphocytes is a vital step in the adhesive interactions that occur during successful T cell recognition of foreign antigen and transendothelial migration. Although the ligation of several different cell surface receptors, including the antigen-specific CD3/T cell receptor complex, the CD2, CD7, and CD28 antigens, as well as several chemokine receptors, has been shown to rapidly upregulate integrin function, the intracellular signaling events that initiate this increase in adhesion remain poorly defined. In this study, we have used DNA-mediated gene transfer to explore the role of phosphatidylinositol 3-kinase (PI 3-K) in the upregulation of 131 integrin functional activity mediated by the CD2 antigen. CD2 was expressed in the myelomonocytic cell line HL60, which expresses 131 integrins that mediate adhesion to fibronectin and VCAM-1 in an activationdependent manner. Antibody stimulation of CD2 expressed on HL60 transfectants resulted within minutes in increased 131-mediated adhesion to fibronectin and VCAM-1 at levels comparable to that obtained upon stimulation with the phorbol ester PMA. A role for PI 3-K in CD2-mediated increases in 131 integrin function is suggested by: (a) the ability of the PI 3-K inhibitor wortmannin to completely inhibit CD2-induced increases in 131 integrin activity; (b) the association of PI 3-K with CD2; and (c) induced PI 3-K activity upon CD2 stimulation. The mode of association of PI 3-K with CD2 is not mediated by tyrosine phosphorylationdependent binding of PI 3-K via SH2 domains, since: (a) PI 3-K is associated with CD2 in unstimulated cells; (b) CD2 stimulation fails to increase the amount of associated PI 3-K; and (c) the CD2 cytoplasmic domain lacks tyrosine residues. A role for both protein kinase C and cytoskeletal rearrangements in CD2 regulation of integrin activity is also suggested, since a PKC inhibitor partially inhibits CD2-induced increases in 131 integrin function, and CD2 stimulation increases F-actin content in a wortmannin-sensitive manner. Analysis of human peripheral T cells indicated that CD2 stimulation also results in PI 3-K-dependent upregulation of 131 integrin activity. Thus, these results demonstrate that CD2 can function as an adhesion regulator in the absence of expression of the CD3/T cell receptor complex; and directly implicate PI 3-K as a critical intracellular mediator involved in the regulation of 131 integrin functional activity by the CD2 antigen. T HE precise regulation of the interaction of immune cells, such as T lymphocytes, with other cells and with components of the extracellular matrix (ECM) 1 is vital to the successful response of the immune system to a foreign challenge. Integrin adhesion receptors are a major group of adhesion molecules used by circulating leukoAddress all correspondence to Yoji Shimizu, Dept. of Laboratory Medicine and and Pathology, University of Minnesota Medical School, Box 609 UMHC, 420 Delaware St. SE, Minneapolis, MN 55455. Tel.: (612) 6266849. FAX: (612) 625-2199. 1. Abbreviat ions used in this paper: ECL, enhanced chemiluminescence; ECM, extracellular matrix; FN, fibronectin; HGF, hepatocyte growth factor; PDGF, platelet-derived growth factor; PI 3-K, phosphatidylinositol 3-kinase; PKC, protein kinase C; SH2, src-homology 2. cytes to interact with and respond to the extraceUular environment (32, 67). Since the primary function of a T cell involves recirculation in an ongoing search for foreign antigen, it is not surprising that T cells use several mechanisms of regulating integrin receptor functional activity (41). One major mode of regulating integrin function that is used by T cells as well as other circulating leukocytes involves a rapid increase in integrin functional activity after activation of the cell (17, 63, 78). Thus, while human T cells bind poorly to most integrin ligands and counterreceptors, such as the LFA-1 counter-receptor ICAM-1, the et4131 counter-receptor VCAM-1, and the ct4131 and t~5~l ECM ligand fibronectin (FN), activation quickly increases integrin-mediated T cell function (17, 60, 62, 63, 70, 78, 82). Such activation-dependent upregulation of in© The Rockefeller University Press, 0021-9525/95/12/1867/14 $2.00 The Journal of Cell Biology, Volume 131, Number 6, Part 2, December 1995 1867-188