Int. J, lmmunopharmac., Vol. 14, No. 4, pp. 583-593, 1992.

0192-0561/92 $5.00 + .00 Pergamon Press Ltd. International Society for Immunopharmacology.

Printed in Great Britain.

T U N I C A M Y C I N INHIBITS F U N C T I O N A N D E X P R E S S I O N OF THE H I G H - A F F I N I T Y IL-2 R E C E P T O R IN A M U R I N E I L - 2 - D E P E N D E N T CELL LINE* O L I V E R J . SEMMES, t M A R C E L O S. SZTEIN, *§ J . M A R T Y N BAILEY a n d W I L L I A M D . M E R R I T T II

Departments of Biochemistry and Molecular Biology and of *Medicine, The George Washington University School of Medicine and Health Sciences, Washington, DC. 20037, U.S.A. (Received 27 August 1991 and in final form 14 November 1991)

-Murine interleukin-2-dependent T-lymphocytes (CT6) were treated with tunicamycin, an inhibitor of both glycoprotein and ganglioside synthesis, to study the involvement of glycosylation in the IL-2 proliferative response. Tunicamycin inhibited proliferation in a dose-dependent manner at concentrations which did not inhibit protein synthesis (10-50 ng/ml). Swainsonine, a glycoprotein processing inhibitor, had no effect on proliferation. Inhibition of proliferation by tunicamycin was accompanied by an inhibition of binding of ~25I-IL-2to its high-affinity receptor. Scatchard analysis showed that receptor number was decreased by tunicamycin treatment. On the other hand, tunicamycin did not affect either the binding of the monoclonal antibody 7D4, specific for the 55 kDa low-affinity protein subunit of the IL-2 receptor, or the recycling of the IL-2 receptor. To determine the specific effects of tunicamycin on the biosynthesis of particular CT6 glycoconjugates, cells were radiolabeled with 3Hglucosamine and incorporation into ganglioside, neutral glycolipid and glycoprotein fractions was measured. Low doses of tunicamycin inhibited ganglioside synthesis and glycoprotein glycosylation to the same extent, whereas no effect on neutral glycolipid synthesis was observed. These results suggest that glycosylation of glycoprotein and/or gangliosides might play an important role in the formation of a functional high-affinity IL-2 receptor complex in CT6 cells. Abstract

The proliferative response o f T-lymphocytes to antigenic stimuli is directly dependent on the interaction o f interleukin-2 (IL-2) with its receptor (Cantrell & Smith, 1984). Initial characterization of this receptor in both human (Leonard, Depper, Uchiyama, Smith, W a l d m a n n & Greene, 1982) and murine (Malek, R o b b & Shevach, 1983) T-cells revealed a 55,000 kDa glycoprotein (p55). No evidence exists as yet regarding the function of the p55 low-affinity IL-2 binding protein. F o r m a t i o n of a functional high-affinity IL-2 receptor requires the association of at least two protein chains, p55 and a second, intermediate-affinity IL-2 binding protein, p75 (Tsudo, Kozak, G o l d m a n & Waldmann, 1986;

Robb, Rusk, Yodoi & Greene, 1987; Dukovich et al., 1987). Cross-linking studies of the murine t h y m o m a high-affinity IL-2 receptor with a monoclonal rat anti-murine IL-2 receptor antibody (7D4) demonstrate that the high-affinity receptor exists as a complex of p55 and p75, and that several other high molecular weight proteins appear to be in association with this heterodimer (Saragovi & Malek, 1988). Studies of the biosynthesis of the human and murine p55 IL2 receptor protein show that this protein is derived from a p32 kDa polypeptide precursor which becomes heavily glycosylated with both N-linked and O-linked carbohydrate residues through posttranslational modifications (Leonard, Depper, Robb,

*This work was supported by Grant No. CA35978 (to W.D.M.) from the National Institutes of Health. tCurrent address: Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892, U.S.A. ~Current address: Center for Vaccine Development, Dept. of Pediatrics, Univ. of Maryland at Baltimore, Baltimore, MD 21201, U.S.A. I"o whom correspondence should be addressed at: Center for Cancer and Transplantation Biology, Children's Research Institute, 111 Michigan Avenue, NW, Washington, DC. 20010, U.S.A. 583

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Waldmann & Greene, 1983; Leonard, Depper, Kr6nke, Robb, Waidmann & Greene, 1985; Malek & Korty, 1986). These modifications of the human p55 IL2 receptor are not required for IL-2 binding, since p33, p35 and p37 precursor polypeptides can bind IL-2 (Leonard et al., 1985). The role of glycosylation of this subunit as well as of other putative molecules in the formation of a functional high-affinity IL2 receptor complex is still unclear. The function of carbohydrates in cellular processes has been studied with the aid of antibiotics that block glycosylation. One such antibiotic, tunicamycin (TM), inhibits glycosylation of asparaginelinked glycoproteins by blocking formation of the first intermediate, glucosamine-diphosphodolicol (Tkacz & Lampen, 1975; Elbein, 1987). This agent was used, for instance, to show that N-linked carbohydrates are required for human natural killer cell function (Kornbluth, 1985). Tunicamycin also inhibits the synthesis of gangliosides and neutral glycolipids in neural cells at concentrations which inhibit glycoprotein synthesis, and therefore can be considered a general inhibitor of glycosylation (Guarnaccia, Shaper & Schnaar, 1983). Swainsonine is an antibiotic which blocks glycoprotein processing by inhibiting mannosidase II, leading to an accumulation of mannose-rich N-linked glycoproteins (Elbein, 1987; Tulsiani, Harris & Touster, 1982). No effects on glycolipids have been described for this antibiotic. We have utilized both of these agents to assess the role of glycoconjugates on the binding of IL-2 to the high-affinity IL-2 receptor in intact murine CT6 cells and the effects on the IL-2induced proliferation of this IL-2-dependent cell line.

EXPERIMENTAL PROCEDURES

Culture o f CT6 cells, proliferation assay, and reagents The murine cytotoxic T-cell line CT6 was cultured in growth media consisting of RPMI 1640 (Mediatech, Herndon, VA), 10% fetal bovine serum (Gibco, Grand Island, NY), I0 mM HEPES buffer, 50 U/ml penicillin/streptomycin solution, 100 U/ml kanamycin sulfate, 5 × 10 5 M 2-mercaptoethanol, and 30% IL-2-containing supernatants from concanavalin A-stimulated rat splenocytes. To

measure proliferation, cells were washed three times in IL-2-free media, and then seeded in 96-well microtiter plates at an initial concentration of 5 × 104 cells/ml in growth media (200/al total volume). Cells were cultured for 24 or 48 h, and incorporation of 3H-methyl-thymidine into cellular DNA was measured as described previously (Merritt, Bailey & Pluznik, 1984). Tunicamycin and swainsonine were obtained from Sigma Chemical Co. and were dissolved in RPMI media immediately prior to use.

Assay o f glycosylation and protein synthesis To determine the relative inhibitory effects of tunicamycin on protein synthesis and glycosylation, CT6 cells were incubated in growth media in the presence or absence of tunicamycin for 48 h with 2 ~Ci/ml L-3H-leucine (55 Ci/mmol, New England Nuclear) and 0.5/aCi/ml D-L4C-glucosamine-HC1 (285 mCi/mmol, New England Nuclear). Ceils were harvested onto glass fiber filter discs (Whatmann GFB), and the filters were washed alternately with 7070 trichloroacetic acid and 95070 ethanol three times. The filters were dried at room temperature, and radioactivity was determined by scintillation counting utilizing a LKB Rackbeta scintillation counter with a dual isotope counting program which corrects for channel spill.

IL-2 receptor binding assay High-affinity IL-2 receptor binding was measured as described by Robb et al. (Robb, Mayer & Garlick, 1985). CT6 cells (5 × 105) were incubated in microcentrifuge tubes in 100/al RPMI 1640 media containing 1°70 bovine serum albumin (RPMI - BSA) and ~zsI-IL-2 (I00 pM final concentration, New England Nuclear). Tubes were incubated at 37°C for 15 min with constant shaking. These conditions were adopted after we established in preliminary experiments the optimal incubation conditions for CT6 cell binding of IL-2. Non-specific binding was determined by incubation in parallel tubes with a 100 times excess unlabeled human rlL-2 (a gift from Cetus Corporation, Emeryville, CA). Binding was halted by the addition of 1 ml ice-cold RPMI - BSA and centrifugation at 9000g for 30 s in a microcentrifuge. The supernatant was removed and saved for determination of unbound IL-2. To remove residual radioactivity, the pellet was redissolved in 100/A RPMI-BSA, layered over a 200/al mixture of 84°70 silicone oil (Sigma Chemical

Tunicamycin Inhibition of the IL-2 Receptor Co.) and 16o70 paraffin oil (Fischer Scientific Co.), and the tubes were centrifuged at 9000 g for 90 s. The supernatants were discarded, and the tips were counted with a Beckman Gamma 5000 counter. For determining saturation binding of IL-2 to CT6 cells, ~25I-IL-2 was serially diluted (12.5-400 pM, final concentration) and incubated in duplicate, inclusive of a parallel tube containing excess unlabeled IL-2, with CT6 cells as described above. The data were analyzed by the method of Scatchard with the assistance of computerized data analysis and curve fitting (Munson & Rodbard, 1980; LIGAND, version 2.3.11, P.J. Munson, NIH, Bethesda, MD).

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goat FITC-conjugated anti-rat IgM were used as control stains in these studies. Fluorescence was determined using a FACS IV equipped with a 2020 series Spectra Physics argon laser (Becton-Dickinson, Oxnard, CA). The excitation wavelength was 488 nm at 500 mW. FITC-fluorescence was collected using a 5 1 0 - 5 4 0 band-pass filter and the data were stored and analyzed with a PDP 11/73 digital computer. The percentage of positive cells was estimated against a background of cells stained with normal rat IgM. Antigen density between the different populations was estimated by comparing the logarithm of the mean channel fluorescence intensity of the positive cell populations.

Determination of IL-2 receptor recycling Recycling of the IL-2 receptor was determined as described (Kumar, Moreau, Gilbert & Th6ze, 1987). Briefly, cells were incubated in microcentrifuge tubes at 4°C for 1 h in the presence of 300 pM ~25I-IL-2. Internalization of bound IL-2 was determined by subsequent incubation at 37°C for varying lengths of time. Internalization was halted by adding 1 ml icecold R P M I - BSA containing 0.1% sodium azide, and centrifugation at 9000 g for 60 s at 4°C. The cells were washed once more with RPMI - BSA, and the pellet was resuspended in 1 ml of 0.2 M acetic acid, 0.5 M NaCI, pH 2.5 at 4°C for 5 min. The hydrolysis was halted with 0.7 ml of 0.1 M NaOH, and centrifugation as above. The supernatant was counted to determine acid-sensitive, surface-bound IL-2. The microfuge tips containing cell pellets were cut and counted to determine internalized IL-2.

Indirect immunofluorescence with antibody 7D4 and flow cytometry

monoclonal

The rat IgM monoclonal antibody 7D4, specific for the 55 kDa component of the murine IL-2 receptor (Malek et al., 1983) was obtained from Dr E. Shevach, NIAID, National Institutes of Health. Cells were suspended in phosphate-buffered saline containing 1070 bovine serum albumin ( P B S - BSA) at 2 × 1 0 6 cells/ml and pelleted. The pellet was resuspended in 100/al PBS - BSA containing 7D4 at 1 : 3 0 dilution and incubated at 4°C for 30 min. After washing, the pellet was incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-rat IgM (Cappel Products, West Chester, PA) for 30 min at 4°C in the dark. The cells were washed twice in P B S - B S A plus 0.1070 sodium azide and then in fixative solution containing 2°7o formaldehyde, P B S - BSA, and 0.1 °7o sodium azide. Normal rat IgM (Accurate Chemical, Westbury, NY) and

Assay of ganglioside, glycoprotein biosynthesis

neutral

glycolipid

and

CT6 cells were incubated for 12 h in growth media with 2 taCi/ml 1,6-3H-glucosamine-HC1 (39.9 Ci/mmol, New England Nuclear), and in the presence or absence of tunicamycin. Cells ( 1 - 2 × 107) were harvested, washed twice with PBS, and processed for incorporation into gangliosides, neutral gtycolipids, and glycoproteins utilizing a modification of the method of Fishman et al. (Fishman, Moss & Manganiello, 1977). Specifically, cell pellets were vortexed vigorously in 0.5 ml distilled water, and lipids were extracted with 12 ml of chloroform : methanol, 1 : 1 (v/v) for 4 h with constant rotation. Extracts were pelleted at 400 g for 15 min, the pellet was washed three times with the above solvent, and the supernatants were combined for further analysis. The pellets were redissolved in 1 ml distilled water and 50 tal were removed for determination of radiolabel incorporation into glycoproteins. The supernatants were evaporated to dryness under nitrogen gas, redissolved in chloroform : methanol, 2 : 1 (v/v). The total lipid fraction was partitioned by adding 0.2 vol. of distilled water, vortexing, and centrifugation at 250 g for 15 min. The upper phase was removed to a 50 ml tube, the lower phase was washed five times with methanol : water, 1 : 1 (v/v), and the upper phases from these washes were combined with the original upper phase. The lower organic phase, consisting of neutral lipids, was evaporated to dryness, resuspended to a known volume of chloroform : methanol and counted for determination of radiolabel incorporation into neutral glycosphingolipids. The combined aqueous phases were partially dried to remove methanol, and then lyophilized. The residue was dissolved in 1 ml

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Tunicamycin (ng/ml)

Fig. 1. Influence of tunicamycin on glycosylation and protein synthesis in CT6 cells. Cells were incubated for 48 h with radiolabel in the presence and absence of tunicamycin. Values shown are representative of two separate experiments, and are averages of triplicate determinations; standard errors were less than 10%.

1 10 0.01 0.1 Concentration (fig/ml)

100

Fig. 2. Influence of tunicamycin and swainsonine on proliferation of CT6 cells. Cells were incubated with agents for 48 h and assayed for DNA synthesis with a terminal 7 h pulse of ‘H-thymidine. The results shown are representative of five separate experiments, and values are averages triplicate determinations. The standard errors were less than 10%.

chloroform : methanol : water, 60 : 30 : 4.5, and passed through a 1 g Sephadex G-25 Fine column. Polar lipids including radiolabeled gangliosides were eluted in the void volume with 5 ml of the above solvent, and free unreacted radiolabel remained on the column. The ganglioside-rich fraction was evaporated to dryness and analyzed for radioactivity

by scintiliation

counting.

RESULTS

Inhibition of glycosylation and protein synthesis by tunicamycin in CT6 cells Since tunicamycin has been reported to inhibit both glycosylation of proteins and de novo protein synthesis (Elbein, 1987), we first established the differential effects of TM on glycosylation and protein synthesis in CT6 cells. As shown in Fig. 1, TM inhibited incorporation of “C-glucosamine into TCA-precipitable molecules at low ng concentrations concentrations of whereas, (Wo = 20 ng), TM>100 ng/ml were required to inhibit protein synthesis (EDGE= 175 ng). A concentration of 50 ng/ml TM was chosen as a dose which significantly inhibited glycosylation but had no effect on protein synthesis in CT6 cells.

0

10

20 30 Time (hours)

40

50

Fig. 3. Influence of tunicamycin on CT6 cell proliferation as a function of time of exposure. Cells cultured in the presence and absence of TM were sequentially pulsed with ‘H-thymidine and harvested at 8 h intervals. Values shown are representative of three separate experiments, and are averages of triplicate determinations. The standard errors were less than 5%.

IL-2-dependent Effects of tunicamycin on proliferation of CT6 ceils CT6 cells were cultured in the presence of varying concentrations of TM and effects on

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Fig. 4. Recovery of CT6 cell proliferation after removal of tunicamycin. Cells were treated for either ]2 h (A), 24 h (B), or 48 h (C) with ] 0 - 500 ng/m] TM. Cells were washed and then replated in TM-free medium, and wells were sequentially

pulsed with 3H-thymidine and harvested in 12 h intervals. Results shown are representative from two separate experiments and are averages of triplicate determinations. The standard errors were less than 5%.

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Fig. 5. ~25I-IL-2 bound to CT6 cells as a function of time and temperature. Cells (10 6) were incubated with t25I-IL-2at 4°C (11), 20°C (©) and 37°C (O) and specific binding was assessed. Also shown are values associated with nonspecific binding at 37°C (ZI). Values are means of triplicate determinations with the standard error less than 10%. proliferation were determined by measuring 3H-thymidine incorporation. As shown in Fig. 2, T M inhibited proliferation of CT6 cells in a dose-dependent fashion (EDs0 = 4 0 - - 5 0 ng). Proliferation was inhibited at concentrations which inhibited glycosylation but not protein synthesis ( 1 0 - 5 0 n g / m i , Fig. 1). Higher doses o f T M completely blocked proliferation. On the other hand, the glycoprotein processing inhibitor swainsonine did not inhibit growth o f CT6 cells at all doses tested.

A kinetic analysis of growth inhibition by tunicamycin was conducted to determine the m i n i m u m time of exposure required to observe inhibition. Proliferation was determined at 8 h intervals in the presence of various concentrations of T M (Fig. 3). High doses of T M showed immediate inhibitory effects on proliferation, whereas doses of T M