Rafts CD147 Through Novel Modulation of Lipid Selective
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The Journal of Immunology Selective Inhibition of T Cell Activation Via CD147 Through Novel Modulation of Lipid Rafts1 Gu¨nther Staffler,2* Andreas Szekeres,* Gerhard J. Schu¨tz,† Marcus D. Sa¨emann,* Elisabeth Prager,* Maximilian Zeyda,‡ Karel Drbal,§ Gerhard J. Zlabinger,* Thomas M. Stulnig,‡ and Hannes Stockinger3*§ The plasma membrane is compartmentalized into microdomains and the association/dissociation of receptors and signaling mol- ecules with/from these membrane domains is a major principle for regulation of signal transduction. By following the reorgani- zation of microdomains on living cells and performing biochemical studies, we show that Ab targeting of the T cell activation- associated Ag CD147 prevents TCR stimulation-dependent reorganization and clustering of microdomains. Triggering CD147 induces a displacement of the GPI-anchored coreceptors CD48 and CD59 from microdomains in human T lymphocytes. This perturbation of microdomains is accompanied by a selective inhibition of TCR-mediated T cell proliferation. The CD147-inhibited cells secret normal levels of IL-2 but acquire reduced amounts of the IL-2 receptor ␣-chain CD25. These results indicate that negative regulating signals can modulate microdomains and suggest a general mechanism for inhibition of receptor signaling. The Journal of Immunology, 2003, 171: 1707–1714. here is increasing evidence that the plasma membrane is and Ca2ϩ flux in response to stimulation using CD3 mAb OKT3 organized in microdomains. This compartmentalization of (2). In addition, it was shown that these antifungal agents also T the plasma membrane appears, on the one hand, to be inhibited mAb-induced internalization of the GPI-anchored protein important to separate reactive components (i.e., receptors and as- CD59 (9). Furthermore, it was recently shown that depletion of sociated signal-transducing molecules) in unprimed resting cells, cholesterol using methyl--cyclodextrin induced uncontrolled T and, on the other hand, to cluster and concentrate them when de- cell activation by transient tyrosine phosphorylation of multiple termined for execution of signal transduction and function in ac- proteins, including -associated protein 70 (ZAP-70),4 linker for tivated cells (1, 2). These membrane compartments are relatively activation of T cells (LAT), and phospholipase C␥1 (10). How- resistant to solubilization by many nonionic detergents. They con- ever, not only a change of membrane cholesterol alters signaling tain GPI-anchored proteins, glycosphingolipids, sphingomyelins, and receptor function, but also modification of the saturation index cholesterol, Src family protein tyrosine-kinases and G proteins (3, of the fatty acids in the membrane. Shifting the index toward un- 4) and are, therefore, also called GPI microdomains (1), glyco- saturation by feeding cells with polyunsaturated fatty acids re- sphingolipid-cholesterol rafts, detergent-insoluble glycolipid-en- sulted in an inhibition of signal transduction, which is accompa- riched domains (5) or glycosphingolipid-enriched membrane do- nied by a specific displacement of intracellular signaling molecules mains (6). from microdomains (11). The primary components responsible for the integrity of GPI With the exception of fractions of some transmembrane proteins microdomains have been shown to be cholesterol and saturated including integrins (12), CD4 and CD8 (13, 14), most transmem- fatty acids mainly associated with sphingolipids, GPI proteins and brane proteins are excluded in the resting state of the cell from GPI Src kinases (7). Consequently, also these lipids play a major role microdomains (1). Upon receptor ligation/cell activation, however, to enable signaling. Reduction of the cholesterol content inhibited several recent studies show association of the TCR, Fc⑀R1, or signaling via GPI-anchored proteins (8). Disruption of cholesterol- ␣ rich membrane domains in T cells by polyene antifungal agents Fc R with GPI microdomains and subsequent signal transduction decreased tyrosine phosphorylation of CD3-, phospholipase C␥1, (2, 15–17). The mechanisms ruling over association/dissociation of transmembrane receptors with microdomains are not clear yet. However, CD28 engagement induces redistribution and polariza- *Institute of Immunology, University of Vienna, Vienna, Austria; †Institute for Bio- tion of GPI microdomains into caps at the site of TCR engagement. physics, University of Linz, Linz, Austria; and ‡Department of Internal Medicine III, Thus, CD28 appears to promote association of TCR with microdo- University of Vienna, Vienna, Austria; and §Competence Center for Biomolecular Therapeutics, Vienna, Austria. mains (18). The existence of regulators, which amplify association Received for publication November 6, 2002. Accepted for publication June 17, 2003. of molecules with GPI microdomains, implies that there might also exist receptors that trigger dissociation of molecules from mi- The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance crodomains, resulting in inhibition/deactivation of signal transduc- with 18 U.S.C. Section 1734 solely to indicate this fact. tion. Searching for molecules that might execute negative regulat- 1 This work was supported by the Competence Center for Biomolecular Therapeutics, ing signals via modification of GPI microdomains, we found by the GEN-AV program of the Austrian Federal Ministry of Education, Science and Culture, and by grants SFB00503, P15025-B08, and P13507-B01 from the Austrian CD147 to be a potential candidate. Science Fund. 2 Current address: Intercell, Campus Vienna Biocenter 6, A-1030 Vienna, Austria. 3 Address correspondence and reprint requests to Dr. Hannes Stockinger, Institute of Immunology, University of Vienna, Brunner Strasse 59, A-1235 Vienna, Austria. 4 Abbreviations used in this paper: ZAP-70, -associated protein 70; LAT, linker for E-mail address: [email protected] activation of T cells; PR, phenol red. Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 1708 CD147-INDUCED MODULATION OF T CELL ACTIVATION CD147, also known as M6 Ag (19), extracellular matrix metal- the distribution of the fluorescence intensity on the plasma membranes of loproteinase inducer (20), basigin or neurothelin (21, 22), is a 50- MEM-102-Cy5-stained cells using SPOT Advanced Software (Diagnostics to 60-kDa type I transmembrane glycoprotein belonging to the Ig Instruments, Sterling Heights, MI). Cells displaying a 3-fold increase of fluorescence intensity on one site of the plasma membrane were counted as superfamily (19). CD147 is widely expressed on hemopoietic and cells with caps. Three hundred cells per sample were evaluated and the nonhemopoietic cells. It is strongly up-regulated on T cells upon experiment was repeated once. activation, indicating a function in T cell biology (19, 23). Indi- Analysis of protein tyrosine phosphorylation cation of a potential negative regulatory function of CD147 in T cell regulation was shown by enhanced mixed lymphocyte re- Purified human T cells were rested overnight in complete RPMI 1640 sponses of lymphocytes from CD147 knockout mice (24) and by a medium. Before stimulation, T cells were incubated in RPMI 1640 medium supplemented with 1% FCS for4hat37°C. Cells (3 ϫ 106) were stim- mAb that inhibited T cell proliferation (23). In this study, we show ulated for 5 min at 37°C using CD3 mAb OKT3 (10 g/ml) along with that triggering the CD147 molecule on T cells by this inhibitory CD28 mAb Leu-28 (2 g/ml) in the presence or absence of CD147 mAbs, mAb results in a characteristic modulation of microdomains, or they were left unstimulated. The reaction was stopped by addition of which is associated with impaired signaling for expression of the ice-cold washing buffer (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 5 ϫ IL-2R ␣-chain CD25. mM EDTA). After centrifugation (2 min, 850 g,4°C), cells were im- mediately lysed for 30 min in ice-cold TBS (20 mM Tris-HCl (pH 7.5) and 150 mM NaCl) containing 1% Nonidet P-40 (Pierce, Rockford, IL), 1 mM Materials and Methods sodium orthovanadate, 20 mM NaF, 5 mM EDTA, and protease inhibitors Cell preparation (5 mM aprotinin, 5 mM leupeptin, 1 mM PMSF, and 1 M pepstatin; all from Sigma-Aldrich). After centrifugation (5 min, 14,000 ϫ g,4°C), ly- Peripheral blood was taken from healthy donors and PBMCs were obtained sates were analyzed by nonreducing SDS-PAGE (12% gel) and immuno- by density gradient centrifugation using Ficoll-Hypaque (Pharmacia, Upp- blotting using HRP-labeled anti-phosphotyrosine Ab 4G10 (1/4000; Up- sala, Sweden). T cells were enriched by removing adherent cells on nylon state Biotechnology, Lake Placid, NY). wool (Robbins Scientific, Sunnyvale, CA) and purified by negative deple- tion of CD14-, CD16-, CD20-, and CD56-positive cells using the respec- Multiprobe RNase protection assay tive specific mAbs (CD14 mAb MEM-18, CD16 mAb MEM-154, CD20 Purified human T cells were rested overnight in RPMI 1640 medium sup- mAb MEM97, CD56 mAb MEM-188, all of which were produced by Dr. plemented with 10% FCS. Cells (4 ϫ 107) were stimulated by plate-bound I. Hilgert and Dr. V. Horejsi, Academy of Sciences of the Czech Republic, OKT3 (immobilized at 0.1 g/ml) along with soluble Leu-28 (0.5 g/ml) Prague, Czech Republic) and the MACS technique of Miltenyi Biotec alone or in the presence of soluble CD147 mAbs (1 g/ml). After a 12-h (Bergisch Gladbach, Germany).