CCA (disodium 4-chloro-2,2′-iminodibenzoate) inhibits progression of human T cell proliferation triggered by PHA

CLINICAL

IMMUNOLOGY

AND

56, 384-392

IMMUNOPATHOLOGY

(l%o)

CCA (Disodium 4-Chloro-2,2’-iminodibenzoate) Inhibits Progression of Human T Cell Proliferation Triggered by PHA KOKO

KATAGIRI, YOICHI

**’ TOSHIAKI ICHIKAWA,$

NAKANO,? YUTAKA SuGAw,m&t AND TAKESHI YOSHIDA*

*Tokyo Institute for Immunopharmacology, Inc., 41-8, Takada 3 chome, Toshimaku, Tokyo 171; fResearch Laboratories, Chugai Pharmaceutical, Inc., 41-8, Takada 3 chome, Toshimaku, Tokyo 171; and SDepartment of Internal Medicine, Keio University, 35. Shinanomachi, Shinjukuku, Tokyo 160. Japan The effect of the anti-rheumatic drug CCA (disodium 4-chloro-2,2’-iminodibenzoate) on the proliferation of T cells activated by PHA was examined. Cell cycle analysis showed that CCA blocked the transition of the cells from G, to S (progression), but had little effect on the G, -+ G, transition (initiation). CCA had no significant effect on IL-2 receptor expression, an early G, event, but did inhibit transfertin receptor expression, a late Gi event. CCA did not inhibit IL-2 production by PHA-activated T cells, but did block IFN--y production at 72 hr after the stimulation. CCA failed to inhibit c-myc mRNA induction, but did delay the decrease in c-myc mRNA levels that normally occurs with the onset of DNA synthesis. These results indicate that CCA inhibits the progression, but not initiation, of human T cell proliferation. o IWO Academic press, hc.

INTRODUCTION

Lobenzarit disodium (CCA; disodium 4-chloro-2,2’-iminodibenzoate) is a newly synthesized anti-rheumatic drug which has been shown to be clinically effective. CCA has immunosuppressive effects; however, it has no anti-in0ammator-y effects (l-3). CCA inhibits the proliferation of and the production of antibodies by human B cells that have been stimulated with mitogens or lymphokines and also inhibits the proliferation of human T cells stimulated with mitogens (4; Takeda er al., manuscript in preparation). These effects of CCA may explain the important therapeutic action of the drug, since it has been well established that hyperactivation of T and B cells plays a major role in the pathophysiology of rheumatoid arthritis. However, the precise mechanism for the inhibitory effects of CCA remains unknown. To date, the molecular events required for mitogen-stimulated T cells to progress through the cell cycle have been partly elucidated. After stimulation of resting (GO) peripheral blood mononuclear cells (PBMC) with mitogens, the level of c-myc mRNA is enhanced (5, 6). T cells then enter into the G, phase of the cell cycle where they produce interleukin 2 (IL-2) and express its receptor (7, 8). The interaction of IL-2 with the IL-2 receptor (IL-2R) on activated T cells promotes progression through the G, to the S phase. In this way, a critical threshold of intracellular signals is eventually attained; sequential activation of many genes including the gene for transfer-r-in receptor (9) occurs, permitting the cells to enter ’ To whom correspondence should be addressed. 384 0090-1229/90

$1.50

Copyright 0 I!390 by Academic Press, Inc. AJI rights of reproduction in any form reserved

CCA

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the DNA replication phase. The binding of transferrin to its receptor during the late G, of the cell cycle is believed to be necessary for T cells to change from the G, to the S phase (9). In the present study, we examined the effects of CCA on IL-2R and TfR expression and IL-2 and interferon-y (IFN-y) production of PBMC stimulated with a T-cell mitogen, phytohemagglutinin (PHA). We also assessedits effect on the accumulation and reduction of c-myc mRNA in cells activated with PHA. These data provide evidence indicating that CCA may suppress cell proliferation by affecting a specific site in the cell cycle which differs from that affected by other immunosupressive agents such as cyclosporin A or dexamethasone. MATERIALS

AND METHODS

Cell cultures. Human PBMC isolated by Ficoll-Hypaque discontinuous gradient centrifugation were cultured at 1 x lo6 cells/ml in RPM1 1640 medium supplemented with 5% heat-inactivated (56”C, 30 min) fetal bovine serum (FBS) and 100U/ml penicillin/100 ~&ml streptomycin at 37°C in a humidified atmosphere of 5% CO, in air. The cells were stimulated with PHA at 1 pg/ml (Wellcome Reagents Ltd., Beckenham, UK). Cultures were performed in the absence or presence of various concentrations of CCA. Proliferation studies. After 24,48, and 72 hr in culture, equal volumes (200 pl) of each sample were plated in triplicate into %-well plates. Cells were pulsed with 1 t&i of [3H]thymidine (13HlTdR; ICN, Irvine, CA; sp act 10 Ci/mM) for 8 hr. They were harvested onto glass fiber filters and their radioactivities were measured with a liquid scintillation counter. Antibodies. Fluorescein isothiocyanate (FITC)-conjugated monoclonal antibodies directed against the IL-2R and TfR were purchased from Becton Dickinson, (Oxnard, CA). Fluorescence staining and flow microjluorometry analysis. Direct immunofluorescence staining was used in all instances. Hanks’ balanced salt solution (HBSS, pH 7.2) containing 3% FBS, 0.1% sodium azide, and 10 mM HEPES buffer was used as the staining medium. Samples of 0.5 to 1 x lo6 cells were incubated in 100 ~1 of staining medium for 30 min at 4°C with a FITC-conjugated monoclonal antibody. After washing three times, the cells were analyzed by flow cytometry (FACS440, Becton Dickinson). Cell cycle analysis. The effects of CCA on T lymphocyte activation and proliferation were analyzed by using the conventional method which exploits the differential staining of cellular DNA and RNA by the metachromatic fluorescent dye acridine orange (AO) (10). In brief, lymphocytes were removed from culture, washed, and then resuspended at 2.5 x lo6 cells/ml. Aliquots of the cells (0.4 ml) were added to 1 ml of solution containing 0.1% Triton X-100 (Sigma), 0.2 M sucrose, low4 M EDTA, and 2 X lo-’ M citrate phosphate buffer (pH 3). The cells were stained 1min later by the addition of 2 ml of solution containing 0.002% A0 (Polysciences, Inc., Warrington, PA), 0.1 M NaCl, and lo-’ M citrate phosphate buffer at pH 3.8. After 5 min of equilibration at room temperature, the fluorescent of individual cells was measured using FACS440. Assay of IL-2 and ZFN-y production. Supematants were harvested and frozen

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at -20°C before the assays. The level of IFN-y was analyzed by a solid-phase radioimmunoassay (RIA) (Centocor Corp., Malvern, PA) that utilized two murine monoclonal antibodies to different epitopes of biologically active IFN-y. Antibody coupled to polystyrene beads was incubated with culture supematant and subsequently washed, and a second ‘251-labeled antibody was added to different non-cross-reacting epitopes of IFN-y. After washing, binding of the second antibody to beads was quantified by using a gamma-counter. Levels of IFN-y were then expressed as reference units on the basis of values obtained with a standard. The levels of IL-2 were analyzed by a competitive RIA (Amersham Co., Arlington Heights, IL) that utilized a high specific activity ‘25I (human, recombinant) tracer together with a highly specific and sensitive antiserum. Antiserum was incubated with culture supernatant for 4 hr and subsequently ‘251-IL-2 was added and incubated 16-24 hr at 4°C. An Amerlex-M second antibody preparation (donkey antirabbit serum coated onto magnetizable polymer particles) was added. The antibody-bound fraction was separated from free fraction using centrifugation and was quantified by a gamma-counter. Levels of IL-2 were then expressed as reference units on the basis of values obtained with a standard. RNA preparation. Total cellular RNA from the cultured cells was prepared by guanidinium thiocyanate-CsCI gradient centrifugation. The washed cells were resuspended in solution A (4 M guanidinium isothiocyanate/25 mM sodium citrate/ 0.1 M 2-ME/0.5% sarcosyl, pH 7.0) and layered onto solution B (5.7 M CsCV25 mM sodium acetate, pH 5.0). RNA was collected after centrifugation at 36,000 rpm for 16 hr, dissolved in diethylpyrocarbonate-treated water, and precipitated with a mixture of 3 M sodium acetate and 95% ethanol. The amount of RNA was determined spectrophotometrically, with the concentration adjusted to 1 mg/ml, and stored at - 80°C. DNA probe. The c-myc probe was provided by the Japanese Cancer Research Resources Bank. Whole probes were radiolabeled with [a-32P]dCTP (3000 Ci/ mmol, Amersham Co.) by nick-translation. Northern blot analysis. RNA samples (10 l&lane) were denatured in 1 M deionized glyoxaYSO% (v/v) DMSO/lO mit4 sodium phosphate buffer, pH 7.0, at 50°C for 1 hr and size-fractionated by electrophoresis in 1% agarose gels (0.1 M sodium phosphate buffer, pH 7.0). They were transferred to a nitrocellulose filter for blotting. Hybridization of RNA. The Northern blot filters were prehybridized and then hybridized to 32P-radiolabeled DNA probes as previously described (11). Hybridization with “P-labeled cDNA (lo6 cpm/ml) was performed at 42°C for 24 hr in Denhardt’s solution/5 x SSC(1 x SSC is 0.15 M sodium chloride, 0.015 M sodium citrate)/40% (v/v) deionized formamide/lOO l&ml sonicated denatured salmon sperm DNA/IO% (w/v) dextran sulfate. The nitrocellulose filters were washed with 2 x SSC-O. 1 x SSC-O. 1% SDS at 42°C dried, and exposed to X-ray film for l-2 days. RESULTS Effects of CCA on T Lymphocyte Mitogenesis: Cell Cycle Analysis

CCA has been shown to inhibit

PHA-stimulated

proliferation

of PBMC

in a

CCA

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dose-dependent fashion (4). As shown in Fig. 1, we also observed statistically significant inhibition (25%) of T cell growth at the same concentration of CCA (50 ~,ig/ml)as that detected in the set-aof patients with continuous administration of 240 mg/day of CCA. To determine the stage of T cell growth where CCA acts, PBMC were incubated with PHA in the absence or presence of CCA (50 *g/ml) for 65 hr and then analyzed by A0 staining. As shown in Fig. 2, when cells were treated with PHA and CCA, the cell number increased in the G, compartment (65% in PHA/CCA treated, 54% in PHA alone treated) with a corresponding decrease of that in the S phase (7% in PHA/CCA treated, 20% in PHA alone treated) as compared to when the cells were incubated with PHA alone. CCA had little inhibitory effect on G, + G, transition (Fig. 2). Effects

of CCA on IL-2 and Transferrin

Receptor

Expression

Next, the effects of CCA on the expression of the growth-associated receptors (IL-2R and TfR) on the T cells were investigated by flow cytometry. CCA treatment had little or no effect on the expression of IL-2R at 42 hr after the stimulation of T cells with PHA (Table 1). In contrast, CCA showed statistically significant inhibition (33%) of the expression of TfR in PHA-stimulated T cells (Table 1). Effect of CCA on IL-2 and ZFNy Production

IL-2 is well known to be essential for the proliferation of lectin-stimulated T 3H-TdR cm

uptake

2

1

0 Time

after

PHA,

day

FIG. 1. Inhibition of [‘H]TdR incorporation by PHA-stimulated PBMC by CCA. PBMC were cultured with PHA in the presence (@) or absence (0) of CCA or in medium alone (A). After 2 days, [‘H]TdR uptake of the cells was examined and is expressed as the mean f SE value of three experiments from a different donor.

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ET AL.

p (65hr after PI-IA) ’

7

0 RNA

d

RNA

C

s 1.“...*I I 100

10’

102

103

104

RNA

FIG. 2. A0 cell cycle analysis. Human PBMC (106/m1) were stimulated with PHA in the absence (B) or presence of CCA (50 pg/ml) (C) for 65 hr, washed, stained with AO, and simultaneously assessed for cegular DNA and RNA content of lo4 cells by cytofluorography as described under Materials and Methods. Regions were set by using freshly prepared PBMC (A) for the G, (Region l), and the two compartments of G,, G,, (Region 2) and Gm (Region 3) were determined on the basis of RNA content of PHA-stimulated control PBMC, with G,, representing an increase in the amount of cellular RNA content that would enable DNA synthesis. Region 4 shows S phase. The results are representative of one of three. similar experiments.

lymphocytes (7). IFN--r is produced by activated T cells in response to mitogen or antigen and has a broad range of immunoregulatory activities, including macrophage activation (12). We examined the effects of CCA on the production of IL-2 and IFN-v by PBMC. The production of IL-2 by PHA-stimulated lymphocytes was not inhibited by CCA at the concentration of 100 ~&nil (Fig. 3). In contrast, CCA blocked IFN-y production in a concentration-dependent fashion 3 days after stimulation (maximum inhibition: 30%) (Fig. 3).

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TABLE 1 EFFECT OF CCA ON IL-2R AND TfR EXPWSION OF HUMAN PBMC STIMULATED WITH PHA Positive (%) Treatment

IL-2R

TfR

None PHA’ PHA + CCA (50 &nl)

4.3 It 0.3” 85.3 f 1.8 84.3 2 2.9

3.0 + 0.6 36.0 + 3.2b 24.3 +- 2.3’

b These responses were significantly different (P < 0.05). c Human PBMC were stimulated with 1 &rl of PHA for 42 hr as described under Materials and Methods.

Eflect of CCA on c-myc mRNA

Level

Previous studies of human peripheral T lymphocytes have shown that the c-myc mRNA level reaches a plateau within several hours after mitogen stimulation, and returns to baseline as the cells begin DNA synthesis (13, 14). As shown in Fig. 4, PHA induced a marked increase in the levels of c-myc transcripts at 3 to 14 hr after stimulation; those levels decreased at 38 hr (Fig. 4). CCA did not inhibit the IFN y

IL2

CCA

btgfmt)

CCA

hzfmt)

6

2

0

24

Time after

40 PHA. hr

0

24 T,me after

40

12

PHA. hr

FIG. 3. Effects of IL-2 and IF&I-~ production by CCA. PBMC were cultured (2 x 106 cells/ml) with PHA for various times in the absence (0) or presence (0) of various concentrations of CCA and in medium alone (0). Supematants were then assayed for IL-2 and IFN-7 by RIA. The results indicate the mean value of triplicate assays and the bars show the SE value of each assay.

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Exp.1 3hr after

=l 2

a 2

ET AL.

Exp.ll 14hr after

PHA

PHA l

.

I

+c-myc

* c-myc

38hr

after

PHA

FIG. 4. Effect of CCA on levels of c-myc mRNA. Total cellular RNA was isolated from PBMC after culture for 3 hr (Expt l), 14 hr, 38 hr (Expt 2) with PHA in the presence or absence of CCA (50 &ml). Total cellular RNA was analyzed by Northern blot for c-myc mRNA. The results are representative of one of three similar experiments

induction of c-myc mRNA in PHA-stimulated cells at 3 and 14 hr, but did cause the three to fivefold increase in relative levels of c-myc transcripts at 38 hr (Fig. 4). Thus, CCA appears to delay the decrease in c-myc mRNA levels of activated T cells that normally occurs with the onset of DNA synthesis. DISCUSSION

By analyzing the cell cycle of PHA-stimulated human T cells, we found that CCA inhibited the progression of the cells from the G, to S phase, while the early stage of T cell activation (G, to G, transition) was unaffected. The fact that CCA does not affect the transition of T cells from GO to G, was further demonstrated by its inability to modulate IL-2 receptor (Tat antigen) induction and IL-2 production which are characteristic of early T cell activation (7, 8). In contrast, CCA significantly inhibited the expression of the transferrin receptor. Previous works have shown that induction of the transferrin receptor follows IL-2R expression, occurring in the late G, or S phase (9). The different effects of CCA on TfR and IL-2R expression further support the notion that CCA acts specifically on the Gi + S transition phase in T cell growth. IFN-y production by PBMC was inhibited by CCA 3 days after PHA stimulation. IL-2 has been reported to up-regulate the IFN-7 synthesis by T cells, and agents that prevent IL-2 production have been shown to inhibit the production of IFN--y (15, 16). Because CCA did not inhibit IL-2 production, CCA seems to directly inhibit the production of IFN-y synthesis. The mechanisms of inhibition of IFNy production by CCA are under investigation. The increase of c-myc mRNA level has been shown to occur upon exposure of cells to mitogens and to be correlated with DNA replication (17). CCA did not inhibit the induction of c-myc mRNA in the PHA-stimulated cells, but delayed the

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decrease in c-myc mRNA levels that normally occurs with the onset of DNA synthesis. Thus, CCA may prevent cells from reaching a point in the cell cycle where an unknown signal that turns off c-myc gene expression is generated. The normal decline of c-myc mRNA levels might be necessary for the proliferation and differentiation of the cells. The recent findings showed that the continuous expression of c-myc inhibits the maturation of HL-60 cells to monocyte/ macrophage (18, 19). The delayed decline of c-myc mRNA levels may inhibit the functional development of T cells. Thus, the effect of CCA on c-myc mRNA levels observed here might be associated with the inhibition of T cell function. Reed et al. (20) showed that the level of c-myc mRNA was regulated in normal human T lymphocytes by various modulators of cell proliferation at several points in the cell cycle. Cyclosporin A and dexamethasone, which interfere with early events of T cell activation, diminished the PHA-induced accumulation of c-myc mRNA. In contrast, hydroxyurea, which impairs the C, -j S transition in cycling cells, failed to inhibit c-myc expression and instead delayed the decrease in c-myc mRNA levels. The results in this paper show that CCA shares the site of action in T cell activation with hydroxyurea, but differs from cyclosporin A and dexamethasone. The data indicate that the site of CCA action in human T cell proliferation resides at the late G, phase in the cell cycle and CCA may suppress phasespecifically the excessive proliferation of lymphocytes characteristic of autoimmune diseases including rheumatoid arthritis. This provides new information concerning the mechanisms of the therapeutic effect of CCA on rheumatoid arthritis and may lead to better approaches in the treatment of patients. ACKNOWLEDGMENTS We are grateful to Dr. Y. Takeda and Dr. T. Katagiri for their encouragement and support

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9. Leonard, M. N., and Cossman, J., Transferrin receptor inhibition in mitogen-stimulated human T lymphocytes is required for DNA synthesis and all division and is regulated by interleukin 2. hoc. Natl. Acad. Sci. USA 80, 3494-3498, 1983. IO. William, F. C., Noelle, R. J., Krause, K., and Fanger, M. W., The effects of 1,25dihydroxyvitamin D on human T lymphocyte activation and proliferation: A cell cycle analysis. J. Immunol. 135, 227%2286, 1985. II. Tomas, P. S., Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Natl. Acad. Sci. USA 77, 5201-5205, 1980. 12. Friedman, R. M., and Vogel, S. N., Interferons with special emphasis on the immune system. Adv. Immunol. 34, 97-140, 1983. 13. Kelly, K., Cochran, B. H., Stile, S. D., and Leder, P., Cell-specific regulation of the c-myc gene by lymphocyte mitogens and platelet-derived growth factor. Cell 35, 603-610, 1983. 14. Reed, J. C., Alpers, J. D., Nowell, P. C., and Hoover, R. G., Sequential expression of protooncogenes during lectin-stimulated mitogenesis of normal human lymphocytes. Proc. Natl. Acad. Sci. USA 83, 3982-3985, 1986. 15. Reem, G. H., Cook, L. A., and Vileck, J., Gamma interferon synthesis by human thymocytes and T lymphocytes inhibited by cyclosporin A. Science 221, 63-65, 1983. 16. Kelso, A., and Munck, A., Glucocorticoid inhibition of lymphokine secretion by alloreactive T lymphocyte clones. J. Immunol. 133, 784-791, 1984. 17. Persson, H., Hennighausen, L., Taub, R., Dugrade, W., and Leder, P., Antibodies to human c-myc oncogene product: Evidence of an evolutionarily conserved protein induced during cell proliferation. Science 225, 687-693, 1984. 18. Holt, J. T., Render, R. L., and Nienhuis, A. W., An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation. Mol. Cell. Biol. 8, %3-973, 1988. 19. Anfossi, G., Gewirtz, A. M., and Calabretta, B., An ohgomer complementary to c-myc-encoded mRNA inhibits proliferation of human myeloid leukemia cell lines. hoc. Natl. Acad. Sci. USA 86, 3379-3383, 1989. 20. Reed, J. C., Nowell, P. C., and Hoover, R. G., Regulation of c-myc mRNA levels in normal human lymphocytes by modulators of cell proliferation. Proc. Natl. Acad. Sci. USA 82, 42214224, 1985. Received January 1I, 1990; accepted with revision May 9, 1990