Downregulation of Interleukin-2 and α-Chain Interleukin-2 Receptor Biosynthesis by Cisplatin in Human Peripheral Lymphocytes

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Vol. 79, No. 1, April, pp. 43–49, 1996 Article No. 0049

Downregulation of Interleukin-2 and a-Chain Interleukin-2 Receptor Biosynthesis by Cisplatin in Human Peripheral Lymphocytes P. P. SFIKAKIS,* V. L. SOULIOTIS,* N. KATSILAMBROS,* K. MARKAKIS,* G. VAIOPOULOS,* G. C. TSOKOS,† P. PANAYIOTIDIS*

AND

*Research Immunology Laboratory, First Department of Propedeutic Medicine, Athens University Medical School, Athens, Greece; Laboratory of Chemical Carcinogenesis, National Hellenic Research Foundation, Athens, Greece; and †Department of Clinical Physiology, Walter Reed Army Institute of Research, Washington, DC 20307-5100

potential, especially against solid tumors such as bladder, ovarian, and testicular carcinomas. Although the mechanism of action of cisplatin is not definetely known, its effect on cancer cells has been attributed to direct damage to the DNA template with subsequent cell death (1, 2). However, accumulated data on the effects of cisplatin on various in vitro and in vivo immune functions indicate that administration of this agent also results in modification of naturally occurring biological response (3–9). In addition, the efficacy of cisplatin on suppression of graft rejection (10), on adjuvant-induced arthritis in rats (11, 12), as well as on refractory rheumatoid arthritis (RA) in a patient treated for a concomittant adenocarcinoma (13), has been described. The mechanism(s) underlying the immunomodulatory effects of cisplatin has not been elucidated. It has, however, been demonstrated that cisplatin complexes inhibit B-cell surface membrane immunoglobulin capping (6), suppress chemotaxis of monocytes (7), and inhibit the proliferation of peripheral lymphocytes in response to mitogens or allogeneic cells, as well as the growth of T and B cells in culture (8). T cells that are stimulated either by mitogens or by antigens in the presence of an antigen-presenting cellderived costimulatory signal produce interleukin-2 (IL2), and express activation markers including IL-2 receptors (IL-2R) (reviewed in 14). Recent studies have shown that the molecular basis of effective antigeninduced activation of T cells for IL-2 production involves specifically the costimulatory molecules of the B7 family, CD80 (or B7-1 or B7/BB1) and CD86 (or B7-2), on the antigen-presenting cells that interact with the CD28 and CTLA4 molecules on T cells (reviewed in 15, 16). In addition to IL-2 secretion and IL-2R surface expression, activated T cells secrete a form of the a-chain (or p55 or CD25 or Tac antigen) of IL-2R, known as soluble IL-2Ra (17). Binding of IL-2 on its specific, high-affinity, surface receptor (a complex of a, b, and g chains of IL-2R) results in vigorous lymphocyte proliferation, which is a pivotal step in the generation and regulation of immune responses (14, 15).

To further investigate the mechanisms by which the antineoplastic agent cisplatin interferes with immune function, we studied its effect on the biosynthesis of interleukin-2 (IL-2) and its a-chain receptor (IL-2Ra). Normal human peripheral blood lymphocytes (PBL) were activated in vitro with phytohemagglutinin (PHA), and anti-CD3 antibody in the presence of various concentrations of cisplatin. Purified T cells were also cultured with anti-CD3 antibody and costimulated by CD80 (B7-1, B7/BB1)-transfected P815 mastocytoma cells in the presence of cisplatin. Tritiated thymidine incorporation assays, an enzyme-linked immunosorbent assay for soluble IL-2Ra determination, and RNA dot-blot analysis and hybridization with IL-2and IL-2Ra-specific probes were used. PHA-induced and anti-CD3 antibody-induced proliferation of PBL were significantly inhibited by cisplatin at concentrations attainable in vivo. This inhibition was not due to direct cell death as shown by the absence of trypan blue uptake in the presence of high concentrations of cisplatin. Therapeutic concentrations of cisplatin (1 mg/ml) also inhibited the IL-2-dependent proliferation of purified T cells, mediated via the CD28–CD80 costimulatory pathway. In addition, the amount of soluble IL-2Ra released in the T cell culture supernatants was decreased by cisplatin in a dose-dependent fashion, suggesting that inhibition of cell proliferation was associated with a parallel decrease in IL-2Ra production. These effects correlated with a specific cisplatin-induced downregulation of both IL-2 and IL2Ra messenger RNA accumulation in PHA-stimulated PBL that was dependent on the concentration of the drug. These findings suggest that the immunomodulatory effects of cisplatin may result in part from its capacity to directly downregulate the IL-2/IL-2R system in activated lymphocytes. © 1996 Academic Press, Inc.

INTRODUCTION

cis-Diamminodichloroplatinum(II) (cis-DDP or cisplatin) is an antineoplastic agent with vast therapeutic 43

0090-1229/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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To further investigate the mechanisms by which cisplatin interferes with immune function, and in view of the biological significance of the IL-2/IL-2R system, we studied the in vitro effects of cisplatin on IL-2mediated proliferative responses of lymphocytes, as well as on IL-2 and IL-2Ra biosynthesis, at concentrations that are attainable in vivo during chemotherapy. Our findings suggest that therapeutic cisplatin may inhibit lymphocyte activation and subsequent proliferation and modulate immune function in vivo by directly suppressing the expression of IL-2 and IL-2Ra genes.

for 18 hr before being harvested in glass fiber filter using a semiautomatic cell harvester (Skatron, Lier, Norway) and counted in a Packard liquid scintillation analyzer (Downers Grove, IL). Results were expressed as mean counts/minute (cpm) from triplicate cultures. The percentage of inhibition of cell proliferation was calculated using the formula

MATERIALS AND METHODS

where experimental and control represent cpm in the presence of absence, respectively, of cisplatin, and background represents cpm in CM alone, in the absence of cells. Evaluation of control T cell proliferative responses to CD3-mediated and B7/BB1-induced costimulation was determined by subtracting the cpm in the presence of irradiated P815–B7/BB (−) cells from the cpm in the presence of irradiated P815–B7/BB1 (+), as described (18). The ratio of 1 SD to the mean was always <10%.

Cell cultures. Peripheral blood lymphocytes (PBL) from healthy concenting volunteers were isolated by gradient centrifugation over Ficoll–Hypaque (Flow Laboratories, Irvine, Scotland) of heparinized venous blood or lymphapheresis preparations. To obtain purified T cells, PBL (5 × 106/ml) were immediately suspended in warm complete culture medium (CM). CM consisted of RPMI 1640 (Flow) supplemented with 20 mM Hepes buffer, 2 mM L-glutamine, and penicillin/ streptomycin (Gibco, Grand Island, NY), 10% heatinactivated fetal calf serum (FCS; Sera-Lab, Sussex, England). Monocytes were depleted by 2 hr incubation of PBL and the nonadherent cells were rosetted twice with sheep red blood cells (SRBC) as described (18). The percentage of nonrosetting cells in the final preparations was always <2%. SRBC in the T cell fraction were lysed with a KHCO3-buffered ammonium chloride solution. Viability always exceeded 95%, as determined by the trypan blue exclusion test. PBL (0.5 × 106/ml) were cultured in CM alone or in the presence of 1 mg/ml PHA (HA-16; Wellcome, Beckenham, England) or optimal concentrations of soluble OKT3 (lgG2a mitogenic anti-CD3 antibody; Ortho, Raritan, NJ) at a final volume of 0.2 ml for 4 days. OKT3-stimulated T cells were also cultured in the presence of optimal quantities of functional CD80 (B7/ BB1) antigen. For the B7/BB1-induced costimulation the murine mastocytoma cell line P815–B7/BB1(+) (19) (a kind gift from Dr. L.L. Lanier, DNAX, Palo Alto, CA) was used. Transfectant cells were maintained in CM containing 1 mg/ml G418 (Gibco). Parental P815 cells that were negative for surface expression of B7/BB1, referred to as P815–B7/BB1(−) were used as control cells. Microcultures were performed as described in detail (18). Cisplatin (Mr 300.1, Platinol, Bristol Myers Co., U.S.A.), at the indicated concentrations, was always added at the initiation of cultures. Dexamethasone (Mr 392.5, Sigma, St. Louis, MO) was also used in control experiments. Tritiated thymidine ([3H]Tdr) incorporation. The microcultures were incubated at 37°C in a humidified 5% CO2 atmosphere and treated with [3H]thymidine

% inhibition 4

F

11

G

(experimental − background) × 100, (control − background)

Determination of soluble IL-2Ra in culture supernatants. The concentration of IL-2Ra in soluble form was measured in supernatants from 4-day cultures that were set up under the same experimental conditions as those for the assessment of the T cell proliferative response to CD3-mediated and B7/BB1induced costimulation, as described (18). Levels of soluble IL-2Ra were determined with a sandwich enzyme-linked immunosorbent assay (T cell Sciences, Cambridge, MA) employing two different mAs against the a-chain of the IL-2R. Undiluted supernatants were tested in duplicate; soluble IL-2Ra concentration values are expressed in units/ml, relative to a set of standards supplied by the manufacturer. RNA dot-blot analysis and hybridization. Total cellular RNA was prepared according to the method described by Berger (20) from PBL that had been cultured with PHA for 8, 15, 24, and 35 hr RNA (2 mg) was then blotted onto Hybond N+ membrane (Amersham) according to the procedure of Thomas (21). Hybridization of 32P-labeled oligonucleotide probes for IL-2 (OSF/IL-2, P2-1; Oncogene Science, Uniondale, NY), for IL-2Ra (OS1/IL-2R, P2-3; Oncogene), and for b-actin (complementary DNA probe; kindly provided by Dr. N. Tac, NCl, Bethesda, MD) to filter-bound RNA was performed as follows: Prehybridization was carried out in 1 M NaCl, 50 mM Tris– HCl, pH 7.5, 10% dextran sulfate, 1% sodium dodecyl sulfate (SDS), and 100 ml/ mg denatured nonhomologous DNA, for at least 1 hr at 65°C. Hybridization was performed in the same solution containing approximately 2.5 × 106 cpm of 59 endlabeled probe, for 16 hr at 65°C.

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Following hybridization, filters were washed for two 10-min periods in 2X SSC (1X SSC40.15 M NaCl and 0.015 M sodium citrate), 0.1% SDS at room temperature, for 30 min in 2X SSC, 0.1% SDS at 65°C, for 5 min in 2X SSC, 0.1% SDS at room temperature, and then briefly in 2X SSC at room temperature. Filters were blotted dry and exposed to X-ray film (Eastman– Kodak, Rochester, NY). The relative intensity of spots/ bands on autoradiograms was quantified with a spectrophotometer (Cary 210; Varian, Palo Alto, CA) at 600 nm. In order to rehybridize the filters, the probe was removed using standard methods. RESULTS

Cisplatin-Induced Inhibition of PHA- and Anti-CD3-Induced Proliferation of PBL Is Not Caused by Direct Cytotoxicity We first investigated the in vitro effect of 0.1–10 mg/ ml of cisplatin on mitogenic lectin-induced PBL proliferation as well as on anti-CD3 antibody-induced proliferation, which represent a polyclonal and a T cell receptor (TCR)-mediated pathway of lymphocyte activation, respectively. The range of concentrations of cisplatin (0.1–10 mg/ml) used in these experiments include the levels of cisplatin that are attained in vivo during chemotherapy (22–25). Cisplatin inhibited the PHA-induced and anti-CD3 antibody-induced [3H]TdR uptake by proliferating normal PBL (Figs. 1A and 1B respectively). To examine whether cisplatin exerted any direct cytotoxic effect, cell viability was assessed using staining with the vital dye trypan blue, after 22 hr of incubation, in the presence of 0–100 mg/ml of cisplatin. As shown in Table 1, the viability of PBL was

45

TABLE 1 Effect of Cisplatin on PBL Viabilitya Cisplatin concentration 0 0.1 mg/ml 1 mg/ml 10 mg/ml 100 mg/ml

Experiment 1 1.76 1.60 1.61 1.76 1.81

× × × × ×

6

10 106 106 106 106

Experiment 2 1.70 1.74 1.76 1.86 1.66

× × × × ×

106 106 106 106 106

a 2 × 106 PBL were cultured in triplicates in 2 ml of culture medium with or without the indicated concentrations of cisplatin for 22 hr. The number of cells that excluded trypan blue at the end of culture was counted using an hemocytometer. Results of two independent experiments are shown.

not decreased even in the presence of high concentrations of cisplatin. Cisplatin Inhibits the T Cell Proliferative Response Mediated by TCR/CD3 in the Presence of B7/BB1 Costimulation The importance of the CD28–CD80 pathway in IL-2 production by T cells stimulated via the TCR/CD3 complex has been documented in several studies (reviewed in 15, 16). Therefore, purified T cells as described above were tested for their ability to proliferate in response to a soluble anti-CD3 mAb and to the B7/Bb1 costimulatory molecule, in the presence or absence of cisplatin. As determined in previous experiments, the optimal T cell proliferative response in this culture system occurs at 4 days of culture, while blocking assays using antiCD28 and anti-CD80 mAbs have shown that the TCR/ CD3-mediated T cell proliferative response in the pres-

FIG. 1. Cisplatin inhibits the PHA-induced (A) and anti-CD3-induced (B) proliferation of human peripheral blood lymphocytes, and the proliferation of purified T cells mediated by TCR/CD3 in the presence of B7/BB1 costimulation (C). Cultures were performed as described under Materials and Methods. Proliferation was assessed by tritiated thymidine incorporation assays. Values are the mean of eight experiments. Vertical lines represent SEM.

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ence of irradiated P815–B7/BB1(+) cells is entirely dependent upon the CD28–B7/BB1 interaction (18). Cisplatin had no effect either on spontaneous proliferation of nonirradiated or on the viability of irradiated P815 cells. Purified T cells could not proliferate in the presence of soluble OKT3 alone (not shown), but a vigorous proliferative response was observed in the presence of B7/BB1 costimulation. As depicted in Fig. 1C, the optimal TCR/CD3 complex-mediated and B7/BB1costimulated proliferation of normal T cells was inhibited by cisplatin in a dose-dependent fashion. The calculated percentage of inhibition of B7/BB1-induced T cell proliferation was always higher than the inhibition that was observed in PHA-stimulated and anti-CD3stimulated PBL in the presence of the same concentrations of cisplatin. Production of Soluble IL-2Ra by Activated T Cells Is Suppressed by Cisplatin Subsequently, we studied the effects of cisplatin on the release of the soluble form of IL-2Ra in supernatants of purified T cells cultured with soluble OKT3 mAb and the B7/BB1 costimulatory molecule, in the presence or absence of cisplatin. Cisplatin concentrations of 5, 1, and 0.1 mg/ml decreased the amount of soluble IL-2Ra by approximately five-, three-, and twofold, respectively (mean of three experiments). A representative experiment showing the cisplatin-induced, dose-dependent suppression of soluble IL-2Ra production by activated T cells is shown in Fig. 2. There was no release of IL-2Ra in supernatants of purified T cells cultured with soluble OKT3 antibody alone.

Cisplatin Inhibits the Lymphocyte Activation-Induced IL-2 and IL-2Ra mRNA Accumulation in a Dose-Dependent Fashion It has been previously reported that PHA-induced proliferation of normal PBL is regulated by IL-2 and inhibition of IL-2 production, e.g., with dexamethasone, arrests these cells in the G1a phase (26). Therefore, PHA-stimulated PBL represent an appropriate in vitro system to study the effect of cisplatin on the accumulation and kinetics of IL-2 and IL-2Ra mRNA. Because levels of ribosomal RNA increase in PBL after stimulation with PHA, equal amounts of total cellular RNA rather than RNA from equal numbers of cells were compared. Figure 3 shows that no effect on the mRNA accumulation of the “housekeeping” gene b-actin was observed in the presence of either cisplatin or dexamethasone. In contrast, incubation of normal PBL with cisplatin at concentrations of 5, 1, and 0.1 mg/ml resulted in dose-dependent decrease of IL-2 mRNA accumulation, by 91, 50, and 10%, respectively, at the peak time point of IL-2 mRNA induction (8 hr) in this experiment (Fig. 3). At this same time point, dexamethasone (5 mM), a known inhibitor of IL-2 (27), caused an 84% decrease in IL Il-2 mRNA. IL-2Ra mRNA accumulation was reduced by dexamethasone to a lesser extent than IL-2 (by 63% in the experiment presented in Fig. 3), as expected (27). The concentrations of 5, 1, and 0.1 mg/ml of cisplatin decreased IL2Ra mRNA accumulation by 85, 25, and 10%, respectively. These inhibitory effects were observed at 15 hr (the peak time IL-2Ra mRNA accumulation; Fig. 3) and were less prominent compared to the effect of the same cisplatin concentrations on IL-2 mRNA accumu-

FIG. 2. Cisplatin-induced inhibition of the release of soluble a-chain interleukin-2 receptors (IL-2Ra) in supernatants of T cells cultured in the presence of soluble OKT3 antibody, with or without B7/BB1 costimulation. Cultures were performed as described under Materials and Methods. Soluble IL-2Ra were measured by ELISA. Representative of three independent experiments.

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FIG. 3. Effects of cisplatin on interleukin-2 (IL-2), a-chain interleukin IL-2 receptor (IL-2Ra), and b-actin messenger RNA accumulation and kinetics in PHA-stimulated human peripheral lymphocytes. Cells were cultured in the presence of medium alone (lane a), PHA (lane b), PHA + dexamethasone, 5 mM (lane c), PHA + cisplatin, 5 mg/ml (lane d), PHA + cisplatin, 1 mg/ml (lane e), and PHA + cisplatin, 0.1 mg/ml (lane f) and harvested at the indicated times. Total RNA was extracted, blotted onto nylon membranes, and hybridized with 32P-labeled oligonucleotide probes as described under Materials and Methods. Cisplatin induced a dose-dependent inhibition of IL-2 and IL-2Ra mRNA accumulation and had no effect on the accumulation of b-actin mRNA. Representative of three independent experiments.

lation. We did not observe any accumulation of mRNA for IL-2/IL-2Ra at any time interval in nonstimulated cultures, in either the presence or absence of cisplatin. DISCUSSION

Our findings clearly show that cisplatin inhibited the IL-2-dependent proliferation of T cells stimulated by TCR/CD3 complex in the presence of the costimulatory signal mediated via the CD28-CD80 pathway. Although to a lesser extent, the mitogen-induced and anti-CD3-induced proliferation of PBL was also inhibited by cisplatin in a dose-dependent fashion. Inhibition of the lymphocyte proliferative responses was correlated with downregulation of both IL-2 and IL-2Ra mRNA in short-term cultures of PHA-stimulated PBL. Because induction of IL-2Ra gene occurs several hours prior to the IL-2Ra (CD25) appearance on the cell membrane, immunofluorescence analysis of IL-2Ra expression was not included in the short-term cultures performed for the mRNA studies. The T cell activationdependent release of soluble IL-2Ra, which is in proportion to its surface expression (17), was indeed decreased in the presence of cisplatin. No previous study to our knowledge has investigated the in vitro effects of cisplatin on the IL-2/IL-2R system. This system is piv-

otal in the generation and regulation of physiologic and autoimmune responses (14, 15). Downregulation of IL-2 and IL-2Ra by cisplatin was comparable to the effect of dexamethasone, which is a known inhibitor of both genes expression (27). In contrast, the mRNA accumulation of the housekeeping gene b-actin was not affected by either cisplatin or dexamethasone, suggesting a specific effect of cisplatin on IL-2/IL-2Ra gene expression. The dose-dependent downregulation of IL-2 and IL2Ra biosynthesis was achieved by concentrations of cisplatin that are attainable in the sera of patients receiving chemotherapy (22–25). After intravenous administration, cisplatin undergoes rapidly a ligandexchange reaction producing a mixture of proteinbound platinum (fixed metabolite), ultrafiltrable platinum complexed with several nucleophilic functional groups (mobile metabolites), and unchanged cisplatin. Patients infused with a conventional therapeutic dose of 80 mg cisplatin/m2 over 2 hr, followed by hydration for 4 hr, sustain a plasma concentration of 1.2 mg/ml of unchanged cisplatin 8 hr after the infusion (25). Also, the plasma concentration of filterable platinum species following bollus injection of 100 cisplatin/ m2 in cancer patients is approximately 0.8 mg/ml 1 hr later (24). Because the culture medium employed in

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our experiments contains fetal calf serum as well as several supplemental amino acids, and because the half-life of cisplatin bond in this type of culture medium is less than 2 hr, it seems reasonable to speculate that an initial cisplatin concentration of 1 mg/ml of culture medium mimics the in vivo milieu in cancer patients receiving the drug. It is well known that successful antineoplastic treatment does not depend solely on the direct action of a drug on tumor cells, but also on a combination of this direct action with the complex condition of the organism. Among other factors, immune defense against tumor antigens plays a leading role. The ability of therapeutic levels of cisplatin to downregulate IL-2 may be important in this setting, since IL-2 exerts its in vivo effects not only directly on IL-2R-bearing cells but also by inducing a set of other immunimodulator cytokines that are probably indispensable in perpetuating a host response against tumor cells. Along this line, our observations offer a possible explanation for the beneficial results of the combination of IL-2 and high-dose cisplatin in patients with metastatic melanoma, in whom the addition of IL-2 was more effective in achieving complete response than cisplatin therapy alone (28). Therefore, studies to investigate whether therapeutic cisplatin-induced downregulation of endogenous IL-2 relates to specific cases of patients with melanoma or other cancers who are refractory to cisplatin treatment are warranted. Downregulation of the IL-2/IL-2Ra system by cisplatin may interpret its ability to delay the development of adjuvant-induced arthritis in rats (11, 12), as well as the suppression of graft rejection (10, 12). Our findings also support the hypothesis that the desirable immunomodulatory effects of cisplatin treatment in a patient with refractory RA (13) may result, at least in part, from downregulation of the IL-2/IL-2Ra system in T cells. Indeed, several studies have demonstrated abnormal IL-2 production and upregulation of IL-2R on T lymphocytes in the peripheral blood, synovial tissue, and synovial fluid of patients with RA (reviewed in 29). Additional in vivo evidence supporting this hypothesis is provided by the successful treatment of two RA patients with a specific antibody against IL-2Ra (30), as well as by the observation that chronic inflammatory arthritis may develop in cancer patients treated with therapeutic high-dose IL-2 (31). Similarly to cisplatin, the gold compounds that are used successfully in the treatment of patients with RA are also potent inhibitors of IL-2 gene expression (32). Since gold compounds and cisplatin are both complexes of the related transition-state metals (Au, Pt) and display a significant number of molecular, chemical, and biological similarities (33), the use of low, periodic doses of cisplatin may prove of therapeutic importance in the treatment of selected patients with refractory RA and/ or other chronic rheumatic diseases.

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Received August 28, 1995; accepted with revision November 22, 1995

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