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The Journal of Immunology, 1999, 163: 4182-4191.
Copyright © 1999 by The American Association of Immunologists

Nitric Oxide Suppresses Human T Lymphocyte Proliferation Through IFN-{gamma}-Dependent and IFN-{gamma}-Independent Induction of Apoptosis1

Alessandra Allione, Paola Bernabei, Marita Bosticardo, Silvia Ariotti, Guido Forni and Francesco Novelli2

Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human normal and malignant T cells cease to proliferate, down-modulate Bcl-2 expression, and undergo apoptosis when cultured in the presence of NO-donor compounds (sodium nitroprusside and NOC12) for 48 h. At 72 h, cells that evade apoptosis start to proliferate again, overexpress both chains of the IFN-{gamma}R, and thus become susceptible to apoptosis in the presence of IFN-{gamma}. By contrast, in the presence of IFN-{gamma}, no apoptosis, but an increase of proliferation was displayed by control cultures of T cells not exposed to NO and not overexpressing IFN-{gamma}R chains. The NO-induced cell surface overexpression of IFN-{gamma}R chains did not affect the transduction of IFN-{gamma}-mediated signals, as shown by the expression of the transcription factor IFN regulatory factor 1 (IRF-1). However, transduction of these signals was quantitatively modified, because IFN-{gamma} induces enhanced levels of caspase-1 effector death in NO-treated cells. These findings identify NO as one of the environmental factors that critically govern the response of T cells to IFN-{gamma}. By inducing the overexpression of IFN-{gamma}R chains, NO decides whether IFN-{gamma} promotes cell proliferation or the induction of apoptosis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Interferon-{gamma} induces a variety of biological responses, e.g., antiviral, antiproliferative, and immunomodulatory activities in sensitive cells (1). The first event in the induction of these responses is the specific binding of IFN-{gamma} to its cell surface receptor (IFN-{gamma}R), which is composed of at least two chains, IFN-{gamma}R1 (2) and IFN-{gamma}R2, the latter cloned as accessory factor-1 (3). Although IFN-{gamma}R1 alone binds IFN-{gamma} with high affinity, its interaction with the other chain is required for IFN-{gamma}-mediated signaling (4).

We have previously shown that IFN-{gamma} plays a key role in regulating both the proliferation and the apoptosis of normal and malignant T cells, and that this double effect is correlated with differences in the expression of its receptor chains (5, 6, 7). When T cells express high IFN-{gamma}R1 and low IFN-{gamma}R2 levels (6, 7), IFN-{gamma} promotes their proliferation, whereas it induces their apoptosis when high levels of both chains are expressed (5, 6, 7). In vitro, several treatments cause a quick and dramatic increase in the surface expression of IFN-{gamma}R chains by T cells and render them susceptible to IFN-{gamma}-mediated apoptosis. They include TCR engagement (6), exposure to dexamethasone (5), chemotherapeutic drugs (8), x-rays (5), negative growth factors such as ß-galactoside-binding protein (9), and deletion of growth factors such as IL-2 (6, 7) and serum (5) from the culture medium. Thus, the regulation of IFN-{gamma}R chains is a critical event that influences the cell’s fate and responsiveness to IFN-{gamma}, and may itself be regulated by a series of specific, receptor-mediated signals or by environmental factors acting as nonspecific tissue mediators.

One of these mediators is NO, a short-lived messenger molecule involved in neurotransmission, regulation of blood pressure, and cytotoxicity (10), and generated during the oxidation of L-arginine to L-citrulline by at least three isoforms of the enzyme NO synthase (NOS).3 Two isoforms, neuronal and endothelial, are constitutively expressed, whereas the third (iNOS) is a transcriptionally inducible isoform (11). Maximal expression of iNOS mRNA in murine macrophages is achieved by stimulation with IFN-{gamma} plus bacterial LPS (12). Human monocytes/macrophages express all three isoforms upon stimulation (13, 14), and the expression of endothelial NOS in human B and T cells has also been demonstrated (15). Moreover, NO has also been shown to regulate the expression of molecules involved in apoptosis, such as Fas (16) and Fas ligand (FasL) (17).

We (5, 6, 7) and others (18, 19) have shown that the outcome of the signal delivered by IFN-{gamma} on T lymphocytes critically depends on the presence or absence of costimulatory signals provided by accessory cells. In the absence of accessory cells or costimulatory signals, IFN-{gamma} mediates the apoptosis of T lymphocytes (5, 18). By contrast, when accessory cells are present and costimulatory signals are provided, IFN-{gamma} promotes the progression of T lymphocyte activation (5, 6, 7). NO produced by monocytes/macrophages following interaction with IFN-{gamma} or bacterial products (12, 13), or following a decrease of oxygen partial pressure (20, 21), may interact with IFN-{gamma}-producing T cells. There is a body of evidence that different processes that induce NO production by macrophages also induce immune suppression (22, 23, 24, 25, 26). Antitumor therapy with IL-12 (25) or trypanosome infection (26) induces IFN-{gamma}-mediated NO production by macrophages, which suppress T cell proliferative responses. Thus, the interplay between NO and IFN-{gamma} may be critical in deciding both the proliferative and the apoptotic response of an activated T lymphocyte. To determine whether NO influences the T lymphocyte response to IFN-{gamma}, we evaluated its effect in regulation of IFN-{gamma}R chain expression.

This study investigates the role of NO on the apoptosis and IFN-{gamma}R expression of three malignant lines corresponding to distinct stages of T lymphocyte differentiation. Exposure to NO from a brief delivery NO donor transiently inhibited the proliferation of all three lines. This was due to NO triggering of an IFN-{gamma}-independent apoptosis, because these lines did not produce IFN-{gamma} constitutively, nor after the exposure. The surviving cells started to proliferate again, but were tagged by high expression of both chains that made them susceptible to IFN-{gamma}-mediated apoptosis. In effect, the addition of IFN-{gamma} completely abolished their growth and induced their apoptosis through expression of caspase-1 effector death.

In conclusion, these data indicate that NO induces an IFN-{gamma}-independent apoptosis of human T cells. They also identify NO as one of the environmental factors that, by inducing the recruitment of IFN-{gamma}R chains from granule stores, increase their surface expression and convert the signal delivered by IFN-{gamma} from growth promoting into apoptotic.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials

RPMI 1640 was from BioWhittaker (Walkersville, MD); FCS, L-glutamine, penicillin, streptomycin, gentamicin, and trypan blue were from Life Technologies (Grand Island, NY); EDTA, Triton X-100, Tween 20, PBS, BSA, HEPES, MgCl2, KCl, PMSF, DTT, pepstatin A, aprotinin, leupeptin, benzamidine, glycerol, NaCl, sodium azide (NaN3), bromophenol blue, Tris-HCl, sodium nitroprusside (SNP), propidium iodide (PI), and paraformaldehyde were from Sigma (St. Louis, MO); FITC-conjugated mouse anti-Bcl-2, FITC-conjugated mouse IgG1 negative control, FITC-conjugated mouse anti-CD3, FITC-conjugated mouse anti-CD25, mouse IgG1 and IgG2a negative control, biotin-conjugated rabbit anti-mouse, and PE-streptavidin were from Dako (Glostrup, Denmark); anti-IRF-1, anti-caspase-1 rabbit polyclonal Abs, and HRP-conjugated goat anti-rabbit IgG were from Santa Cruz Biotechnology (Santa Cruz, CA); NOC12 was from Dojindo Laboratory (Kumamoto, Japan); and 6-anilino-5,8-quinolinedione (LY83583) was from ICN Pharmaceuticals (Costa Mesa, CA).

Malignant T lymphocyte cultures

ST4 cells (CD1+, CD2-, CD3-, CD4-, CD8+, CD25-) display large irregular nuclei with deep indentations typical of childhood, convoluted-type T cell lymphoma; PF382 is a human T acute lymphoblastic leukemia (CD1+, CD2-, CD3-, CD4-, CD8+, CD25-) stabilized both in vitro and in nu/nu mice starting from biopsy material (27, 28). Jurkat is a human T acute lymphoblastic (CD1+, CD2+, CD3+, CD4+, CD8-, CD25-) line. All three lines were routinely cultured in RPMI 1640 medium containing penicillin, streptomycin, and gentamicin, and supplemented with 10% FCS (complete medium).

Normal T lymphocyte cultures

Human PBL from heparinized venous blood from healthy donors were isolated by Lymphoprep gradient (Ficoll-Type 400; Pharmacia, Uppsala, Sweden) centrifugation, stimulated (1 x 106/ml) with 2.5 µg/ml PHA (Sigma), and cultured in complete medium. The cells from 5 days of culture (T lymphoblasts, 94–98% CD3+, 93–95% CD25+) were treated with SNP (1 mM) or NOC12 (0.1 mM) added once at the start and 60 U/ml rIL-2 (EuroCetus, Milan, Italy). Cells were examined every 24 h.

Cell proliferation assay

ST4, PF382, and Jurkat cells were cultured (0.2 x 106/ml) in complete medium in the absence or presence of scalar (from 0.01 to 1 mM) doses of SNP or NOC12 added once at the start. T lymphoblasts were cultured as described above, but in the presence of 60 U/ml rIL-2. Parallel cultures of malignant T cells were set up with NOC12 (0.1 mM) added at the start and replaced every 24 h. In a few wells of normal and malignant T cells cultured in the presence of 1 mM SNP or 0.1 mM NOC12, scalar doses (from 1 to 0.1 mM) of soluble guanylate cyclase inhibitor, LY-83583, were added. A small aliquot of the cell suspension was removed every 24 h: 50 µl were mixed with 10 µl of trypan blue dye and viable cells were counted. The results are expressed as the arithmetic mean ± SD of viable cells from triplicate cultures. Representative results of at least three experiments are shown.

Determination of NO production

To evaluate the kinetics of NO released by scalar concentration of NOC12 or by an IFN-{gamma}- and LPS-stimulated macrophage murine cell line J774 (29), supernatants were harvested and the nitrite levels were determined by the Griess reaction (30). Briefly, a 50-µl aliquot of cell culture medium was mixed with 50 µl of Griess reagent (1 vol of 0.2% naphthylethylenediamine dihydrochloride in distilled water plus 1 vol of 2% p-aminobenzene-sulfonamide in 5% of phosphoric acid). The mixture was incubated in 96-well plates for 10 min at room temperature. A scalar dilution of NaNO2 was used as standard. OD were measured at 550 nm.

Human rIFN-{gamma} and mAb to IFN-{gamma}R

IFN-{gamma} {Delta}10 (specific antiviral activity 108 U/mg) was kindly provided by Dr. G. Garotta, Hoffman-LaRoche (Basel, Switzerland). It shows the NH2-terminal MQDP and lacks the COOH-terminal 10-aa residues encoded by IFN-{gamma} (31). Mouse mAb {gamma}R99 is an IgG1 that specifically interacts with the extracellular domain of human IFN-{gamma}R1 and inhibits the binding of IFN-{gamma} (32); mouse mAb C.11 is an IgG2a that specifically interacts with the extracellular domain of the human IFN-{gamma}R2 (6, 7).

Flow cytometry

Malignant T cells and T lymphoblasts recovered at the times indicated were washed twice in PBS supplemented with 0.2% BSA and 0.1% NaN3 and incubated with {gamma}R99 or C.11 mAb 30 min at 4°C. As negative control, cells were incubated with mouse IgG1 or IgG2a, respectively. As secondary Ab we used rabbit anti-mouse Ig biotin conjugated, followed by staining with streptavidin PE. An FITC-conjugated anti-CD25 mAb was used to follow the kinetics of IL-2R{alpha}-chain expression on T lymphoblasts (6). Intracellular staining of Bcl-2 was performed following the procedure by Schmidt et al. (33). Briefly, cells were resuspended in 0.875 ml of cold PBS. Next, 0.125 ml of cold 2% paraformaldehyde solution was added, and the samples were incubated in ice for 1 h. The fixed cells were washed and gently resuspended in 1 ml of 0.2% Tween 20 in PBS and incubated for 20 min at 37°C. One milliliter of PBS supplemented with 2% FCS and 0.1% NaN3 was added, and the suspension was spun for 5 min at 1300 rpm before staining with FITC-conjugated anti-Bcl-2 mAb for 30 min at 4°C. Membrane and internal Ag expression were analyzed with a FACScan flow cytometer (Becton Dickinson, Milan, Italy). Each analysis represents the results from 10,000 events.

Western blot analysis

ST4 cells were cultured in the absence or presence of SNP or NOC12 NO-donor compounds. After 48 h, each culture was divided and cultured in the absence or presence of 1000 U/ml IFN-{gamma} for an additional 24 h. Treated cells (5 x 106) were washed twice in cold PBS and then collected by centrifugation. For the extraction of total protein, the pellet was resuspended in four packed cell volumes of 20 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, pH 7.9), 50 mM NaCl, 10 mM EDTA, 2 mM EGTA, 0.5% Nonidet P-40, 10 mM sodium molybdate, 10 mM sodium orthovanadate, 100 mM NaF, 0.5 mM PMSF, and 10 µg/ml leupeptin. The suspension was centrifuged at 10,000 rpm for 10 min in an Eppendorf centrifuge, and the supernatants were stored at -80°C. For the extraction of nuclear proteins, the pellet was resuspended in 400 µl of 10 mM HEPES, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM PMSF. The cells were allowed to swell on ice for 15 min, after which 25 µl of a 10% solution of Nonidet P-40 was added. The suspension was centrifuged at 14,000 rpm for 30 s in an Eppendorf centrifuge. The nuclear pellet was resuspended in 50 µl ice-cold buffer containing 20 mM HEPES, 0.4 M NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, and 1 mM PMSF, and the tube was vigorously rocked at 4°C for 15 min on a shaking platform. The nuclear extract was centrifuged at 14,000 rpm for 5 min in an Eppendorf centrifuge, and the supernatants were stored at -80°C until use. Extracts (25 or 30 µg of protein) were separated on SDS-PAGE at 140 V on 8% miniprotein gels. Gels were electroblotted onto a polyvinylidene fluoride membrane (Bio-Rad, Richmond, CA) at 100 V for 1 h, and the equality of the amount of protein analyzed was checked by nonspecific staining with Ponceau S. The membranes were blocked with TTBS (20 mM Tris-HCl, pH 7.5, 500 mM NaCl, and 0.05% Tween 20) and 5% nonfat dry milk overnight and then incubated with a dilution of 1/1000 of anti-IRF-1, or anti-caspase-1 rabbit polyclonal Abs. After washing with TTBS, blots were reacted with 1:2000 HRP-conjugated goat anti-rabbit IgG Ab. Ab reactions were visualized by enhanced chemoluminescence reagents according to the manufacturer’s instructions (ECL plus; Amersham International, Bucks, U.K.).

RT-PCR

ST4 and PF382 cells (1 x 106/ml) were cultured: 1) in the absence, 2) in the presence of SNP (1 mM), or 3) in the presence of NOC12 (0.1 mM). After 24 h, IFN-{gamma}R1 and IFN-{gamma}R2 mRNA expression was evaluated by RT-PCR on the recovered cells, as previously described (7). Total cellular RNA was extracted by using the Ultraspec RNA solution (Biotecx, Houston, TX). All reagents for cDNA synthesis and PCR were from Promega (Madison, WI). Specific ß2-microglobulin primer pairs were obtained from Clontech (Palo Alto, CA). Specific primers for IFN-{gamma}R1 and IFN-{gamma}R2 were designed on the basis of published sequences (2, 3): IFN-{gamma}R1, 5'-GTCCTCAGTGCCTACACCAACTAA and 3'-CCACACATGTAAGACTCCTCCTGC (amplified fragment of 594 bp); IFN-{gamma}R2, 5'-GCAAGATTCGCCTGTACAACGCA and 3'-GTCACCTCAATCTTTTCTGGAGGC (amplified fragment of 339 bp). Primer pairs were used in the following conditions: IFN-{gamma}R1 and IFN-{gamma}R2, 94°C, 1 min; 65°C, 1 min; and 72°C, 1 min for 30 cycles. Fifteen microliters of PCR product were electrophoresed in a 2% agarose gel in Tris/boric acid/EDTA buffer. Gels were stained with ethidium bromide (Sigma) and photographed.

Apoptosis TUNEL analysis

Apoptosis was evaluated by the fluorochrome labeling of DNA strand breaks by terminal deoxynucleotidyl transferase assay (34) using the Apo-Direct kit from PharMingen (San Diego, CA). Briefly, 1 x 106 cells for each sample were suspended in 0.5 ml of PBS, 5 ml of 1% paraformaldehyde in PBS was added, and the suspension was placed on ice for 15 min. Cells were then washed twice in 5 ml of PBS; 5 ml of ice-cold 70% ethanol was added and the samples were stored at -20°C until use. Staining was performed according to the manufacturer’s instructions. Each sample was incubated for 60 min at 37°C with terminal deoxynucleotidyl transferase enzyme and FITC-dUTP in a reaction buffer. The cells were washed and resuspended in 1 ml of PI and RNase solution and then incubated for 30 min at room temperature. Samples were analyzed by flow cytometry within 3 h of staining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of NO on T cell proliferation: time course and reversibility

The effects of NO on the proliferation of malignant T cells were investigated by using SNP and NOC12, two donor compounds that release NO for no longer than 100 min (35, 36). Malignant T cells (ST4, Jurkat, and PF382) were cultured in the absence or presence of SNP (1 mM) or NOC12 (0.1 mM) added once at the start, and their proliferation was evaluated every 24 h by trypan blue dye exclusion. Parallel cultures of malignant T cells were set up in the presence of NOC12 (0.1 mM) added daily to evaluate the effects of continuous exposure to NO.

The presence of the two NO donors almost completely abrogated proliferation in all three malignant T cell lines for 48–72 h (Fig. 1Go). The cells then began to proliferate again.



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  		FIGURE 1. Kinetics of proliferation of malignant T cells cultured with NO donors. ST4, PF382, and Jurkat cells were cultured in complete medium, SNP (1 mM) or NOC12 (0.1 mM) added once at the start. Parallel cultures were set up in the presence of NOC12 (0.1 mM), added at the beginning of the culture, and replaced every 24 h (NOC12 daily). The number of viable cells was evaluated by trypan blue dye exclusion every 24 h for 120 h. Results are expressed as the arithmetic mean ± SD of cell numbers from triplicate cultures. Data from one of three experiments are shown.

 
SNP only produced CN- in amounts that are equimolar to the amounts of NO generated (37). However, because the reprise of proliferation was equally observed in the presence of both NO donors, the toxic effect of CN- can be ruled out. However, this reprise did not occur when NOC12 was added daily.

Similar results were observed when the kinetics of malignant T cell proliferation in the presence of SNP or NOC12 was measured by means of the (3) [H]TdR uptake assay (data not shown).

Effect of NO on T cell proliferation: dose-response and physiologic relevance

A linear relationship between the concentration of NOC12 and the proliferative inhibition of malignant T cell lines was observed after 72 h (Fig. 2GoA).



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  		FIGURE 2. Dose response and physiologic relevance of NO donor concentration on T cell proliferation. A, ST4, Jurkat, and PF382 cells (0.25 x 106/ml) were cultured in the absence or presence of scalar doses of NOC12. After 72 h, cells were recovered and counted by using the trypan blue dye technique. Results are indicated as percentages of inhibition of proliferation and were calculated by comparing the number of viable cells recovered from the cultures set up in the presence and absence of NOC12. B, Kinetics of nitrite released by complete medium alone, or by medium containing SNP (1 mM) or NOC12 (0.1 mM), or by medium from a culture of J774 cells stimulated with LPS and IFN-{gamma}. At the indicated time, an aliquot of medium was harvested and the nitrite concentration was determined by the Griess reaction technique. Results are representative of five independent experiments.

 
In effect, donors release NO for a short time (35, 36), and this could contrast with physiologic conditions under which T lymphocytes are likely to be exposed to lower concentration of NO for longer periods (38).

To better understand the physiologic relevance of the NO concentration able to inhibit T cell proliferation, we compared the kinetics of nitrite levels released in the culture medium by 0.1 mM of NOC12 or 1 mM of SNP and by 5 x 105/ml J774 murine macrophage cells stimulated with 1 µg/ml LPS plus 100 U/ml murine IFN-{gamma}. Murine J774 cell line releases NO in response to IFN-{gamma} and LPS (29) and provides a somewhat realistic model to compare the physiological production of NO (detected as nitrite accumulation) with that released by two NO donors.

As shown in Fig. 2GoB, no nitrite accumulation was detected in the culture medium alone. A progressive accumulation of nitrite was observed in the medium from J774 cells stimulated with LPS and IFN-{gamma}. Nitrite accumulation in the medium containing NOC12 had already reached after 24 h similar levels to that released by J774 after 96 h, whereas in the medium containing SNP, it was lower and peaked at 96 h.

These results indicated that 0.1 mM of NOC12 and 1 mM SNP release NO in amounts comparable with or even lower than that released physiologically by stimulated J774 cells.

Although IFN-{gamma} plus LPS-stimulated J774 produced nitrite amounts comparable with that released by NOC12 and SNP (see Fig. 2GoB), the growth of normal and malignant T cells was significantly inhibited by coculture with J774 cells, irrespective of their activation with IFN-{gamma} and LPS (data not shown). Coculture is thus an inefficient way of investigating the role of endogenously NO produced in human system, as other unknown factors produced by murine macrophages may inhibit human T cell proliferation and mask the effect of NO.

Direct effect of NO on T cell apoptosis and Bcl-2 expression

Because the aim of this study was to determine whether NO modulates the proliferation of T cells and their responsiveness to IFN-{gamma}, we evaluated the response of malignant T cells to exposure to NO for a short period only, because continuous exposure irreversibly inhibited their proliferation (see Fig. 1Go). So the next set of experiments was set up in the presence of SNP and NOC12 added once at the beginning of culture only.

To determine whether the NO-induced inhibition was merely due to suppression of the cell’s proliferative program or to apoptosis, malignant T cells were cultured in the presence of SNP (1 mM) or NOC12 (0.1 mM) and cell apoptosis was evaluated by TUNEL analysis after staining DNA strand breaks with dUTP-FITC and DNA content with PI (Fig. 3Go, left panels). After 48 h, the percentage of apoptotic ST4 cells was almost nil in the cultures in medium only. It was higher when the medium was supplemented with SNP, and even higher in the presence of NOC12 (Fig. 3Go, left panels). Similar data were obtained with PF382 and Jurkat cells. NO donor-induced ST4 and Jurkat cell apoptosis was also confirmed by electrophoretic analysis of DNA fragmentation (data not shown).



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  		FIGURE 3. Analysis of apoptosis induced by NO donors on malignant T cells. ST4 were cultured in complete medium alone, or in the presence of SNP (1 mM) or NOC12 (0.1 mM) added once at the start. After 48 h, cells were recovered and apoptosis (left panels) and Bcl-2 expression (right panels) were evaluated. Percentage of apoptotic cells obtained by the TUNEL technique is indicated in the two regions. Percentage of Bcl-2-positive cells and relative means of fluorescence (MFI) were calculated by subtracting the values of control, as indicated by the marker (M1). Data from one of three experiments are shown.

 
Flow cytometry showed that ST4 apoptosis is accompanied by a dramatic down-modulation of expression of Bcl-2, a mitochondrial protein that protects from apoptosis (39) (Fig. 3Go, right panels). Although the percentage of Bcl-2-positive cells was slightly reduced only in ST4 exposed to NOC12 (control culture, 99%; SNP, 98%; NOC12, 85%), the mean fluorescence intensity (MFI) values indicated that Bcl-2 intensity was reduced to about one-third in the presence of both SNP and NOC12. Bcl-2 MFI was 144 in the control culture, 43 in cultures with SNP, and 38 in those with NOC12. This dramatic reduction in Bcl-2 expression indicated that both NO donors equally triggered the apoptotic program in ST4 cells. Furthermore, the percentage of apoptotic cells evaluated by dUTP-FITC and PI at the same time point was higher with NOC12 than with SNP.

Effect of NO on T cell apoptosis: time course and reversibility

Because SNP and NOC12 added once release NO for only a short period, it was arguable that they were unable to induce the apoptosis of all seeded malignant T cells (see Fig. 1Go). Therefore, we evaluated the kinetics of the apoptosis of ST4 and Jurkat cells cultured in the absence and presence of SNP or NOC12 added once at the start. In their absence, very low levels of apoptosis were detected (Fig. 4Go), whereas in their presence the percentage of apoptosis of both lines increased after 24 h, was high between 48 and 72 h, and then decreased to become undetectable after 96–120 h (Fig. 4Go).



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  		FIGURE 4. Kinetics of apoptosis induced by NO on malignant T cells. ST4 and Jurkat cells were cultured in complete medium, in the absence or presence of SNP (1 mM) or NOC12 (0.1 mM) added once at the start. Percentage of apoptotic cells was evaluated every 24 h by TUNEL analysis. Results are expressed as arithmetic mean ± SD of the percentage of apoptotic cells from three independent experiments.

 
cGMP is not involved in NO-mediated T cell apoptosis

NO increases cellular cyclic GMP (cGMP) by activating soluble guanylate cyclase (40). Because it has been reported that cGMP promotes apoptosis of nerve cells (41), we investigated its role in NO-induced inhibition of proliferation and apoptosis of normal and malignant T cells.

When T lymphoblasts from healthy individuals or malignant ST4 cells were cultured in the presence of SNP (1 mM) or NOC12 (0.1 mM), a drastic inhibition of proliferation and an increase of apoptotic cells were observed (Table IGo). The presence of LY83583 cGMP inhibitor did not modify the kinetic suppressive and proapoptotic effects of NO donors on either normal or malignant T cells (Table IGo).


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  		Table I. Effects of NO and cGMP on proliferation of normal and malignant T lymphocytes

 
Effect of NO on T cell IFN-{gamma}R1 and IFN-{gamma}R2 chain expression

We have shown that interaction between IFN-{gamma} and IFN-{gamma}R is a signal that controls both the growth and the apoptosis of T lymphocytes (5, 6, 7). The outcome of these opposite effects depends upon the density of IFN-{gamma}R chains on the cell membrane. This key feature is under the control of both physiologic environmental signals and stress-inducing conditions (5, 6). Because transient exposure to NO induced malignant T cell apoptosis followed by resumption of proliferation by a fraction of the surviving cells (Figs. 1Go and 4Go), we determined whether NO also modulates IFN-{gamma}R chain expression. ST4 cells were cultured in the absence or presence of NO donors added once at the start, and their IFN-{gamma}R chain expression was monitored by flow cytometry using specific anti-IFN-{gamma}R1 {gamma}R99 and anti-IFN-{gamma}R2 C.11 mAb. Fig. 5Go shows the physical parameters (forward scatter (FSC) and side scatter (SSC)) and IFN-{gamma}R chain expression on ST4 cells cultured 72 h in medium only and in the presence of SNP or NOC12.



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  		FIGURE 5. Expression of IFN-{gamma}R1 and IFN-{gamma}R2 on ST4 cells cultured in the presence of NO donors. ST4 cells were cultured in complete medium, in the absence or presence of SNP (1 mM) or NOC12 (0.1 mM) added once at the start. Cells were recovered after 72 h, stained with {gamma}R99 (middle panels) or C.11 (right panels) mAb, and analyzed by flow cytometry. Bold lines, specific fluorescence for IFN-{gamma}R1 (middle panels) and IFN-{gamma}R2 (right panels); thin lines, nonspecific fluorescence provided by control mouse IgG1 (middle panels) and IgG2a (right panels). Their physical parameters were also evaluated (left panels). On the basis of FSC/SSC values, cells were selected from two regions: viable cells in R1 and death cells in R2, and the percentages of cells in these regions are indicated in each panel. Expression of IFN-{gamma}R1 and IFN-{gamma}R2 was evaluated only in the cells of R1 and is indicated in each panel. Data from one of three experiments are shown.

 
Because viable and dying cells can be distinguished by their size (FSC) and granularity (SSC) (42), ST4 cells were divided into two regions (Fig. 5Go, left panels). Region 1 (R1) delimited viable cells displaying large size and low granularity, and region 2 (R2) delimited dying cells displaying low size and high granularity. Chain expression was evaluated on viable cells only. After 72 h in medium only, most cells were viable (88%, left upper panel): they expressed high IFN-{gamma}R1 (MFI 306, middle upper panel), and almost undetectable levels of IFN-{gamma}R2 (MFI 92, right upper panel). The presence of NO donors decreased the percentage of viable cells (SNP, 62%; NOC12, 53%; left, middle, and lower panels) and significantly increased their surface expression of both chains (Fig. 5Go). The MFI increase induced by the donors on ST4 cells was about 3-fold for IFN-{gamma}R1 (SNP, MFI 871; NOC12, MFI 874) and about 2-fold for IFN-{gamma}R2 (SNP, MFI 176; NOC12, MFI 157).

The kinetics of the viability (evaluated as percentage gated in region R1 cells, Fig. 6Go, upper panel), of MFI of R1-gated IFN-{gamma}R1-positive cells (Fig. 6Go, middle panel), and of R1-gated IFN-{gamma}R2-positive cells (Fig. 6Go, lower panel) of ST4 cells cultured in the presence or absence of SNP and NOC12 was followed. Addition of SNP and NOC12 caused a progressive drop of the percentage of viable cells and progressive enhancement of their expression of both chains from the 24th to the 72nd hour. After 72 h, the percentage of cell viability rose to reach values very similar to those of untreated cells after 120 h. This resumption of viability was accompanied by complete down-modulation of both chains. Similar results were obtained with Jurkat and PF382 cells (data not shown).



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  		FIGURE 6. Kinetics of cell viability and expression of IFN-{gamma}R1 and IFN-{gamma}R2 on ST4 cells cultured in the presence of NO donors. ST4 cells were cultured in complete medium, in the absence or presence of SNP (1 mM) or NOC12 (0.1 mM) added once at the start. Every 24 h, cells were recovered and viable cells (upper panel) and their corresponding expression of IFN-{gamma}R1 (middle panel) and of IFN-{gamma}R2 (lower panel) were evaluated by flow cytometry, as indicated in Fig. 4Go. Results are expressed as percentages of viable cells evaluated on the basis of physical parameters (R1-gated cells) and of MFI evaluated as described in Fig. 5Go. Percentages of cell viability and IFN-{gamma}R chains MFI are expressed as arithmetic means ± SD from three independent experiments.

 
The surface up-regulation of both chains of IFN-{gamma}R by NO donors did not affect T cell mRNA expression. RT-PCR analysis revealed a constitutive expression of both chains on ST4 and PF382. This was not modified by 24-h exposure to either NOC12 or SNP (Fig. 7Go).



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  		FIGURE 7. RT-PCR analysis of IFN-{gamma}R chains mRNA on malignant T cells exposed to NO donors. ST4 and PF382 were cultured in the absence or presence of SNP (1 mM) or NOC12 (0.1 mM). After 24 h, cells were recovered and IFN-{gamma}R1, IFN-{gamma}R2, and ß2-microglobulin (housekeeping gene) mRNA were evaluated. Lane M, marker; lane N, control of contamination-free reaction in which all PCR reagents were present, but there is no cDNA; lane 1, ST4; lane 2, ST4 with SNP; lane 3, ST4 with NOC12; lane 4, PF382; lane 5, PF382 with SNP; lane 6, PF382 with NOC12. The sizes of the PCR fragments are shown on the right.

 
Effects of NO on T cell IL-2R expression

Because it has been reported that NO has no effect on T cell IL2R expression (43), we compared IL-2R{alpha} (CD25) expression with that of the two IFN-{gamma}R chains after normal T lymphoblasts were exposed to SNP (1 mM) or NOC12 (0.1 mM). After 48 h of culture in the presence of IL-2, 45%, 25%, and 87% of T lymphoblasts expressed IFN-{gamma}R1, IFN-{gamma}R2, and CD25, respectively (Table IIGo).


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  		Table II. Effects of NO on IFN-{gamma} and IL-2 receptor expression of normal T lymphocytes1

 
The expression of both IFN-{gamma}R chains was increased by SNP (IFN-{gamma}R1, 75%; IFN-{gamma}R2, 52%) and NOC12 (IFN-{gamma}R1, 56%; IFN-{gamma}R2, 49%). By contrast, neither SNP nor NOC12 significantly modified IL-2R{alpha} expression (CD25 SNP, 88%; NOC12, 77%), as expected (43).

Induction of IRF-1 on T cells by IFN-{gamma}

Because NO induced the up-modulation of IFN-{gamma}R chains on malignant T cells that escaped apoptosis, we evaluated the effect of their exposure to NO on the ability of the entire IFN-{gamma}R complex to transduce IFN-{gamma}-mediated signals. The effectiveness of IFN-{gamma}R was analyzed by examining the nuclear expression of IRF-1, whose transcriptional activation is specifically induced by IFN-{gamma} (44) and requires both chains. ST4 cells were cultured in the presence or absence of SNP or NOC12. After 48 h, each culture was split and cultured in the presence or absence of 1000 U/ml IFN-{gamma} for an additional 24 h. As shown in Fig. 8Go, IRF-1 was always induced by IFN-{gamma}, although in a lesser extent in the presence of NO donors. Similar results were obtained with Jurkat and PF382 cells (data not shown).



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  		FIGURE 8. Induction of IRF-1 in malignant T cells after treatment with NO donors. ST4 cells were cultured in medium only, in medium supplemented with SNP (1 mM), or with NOC12 (0.1 mM). After 48 h, each culture was divided and recultured for an additional 24 h in the absence or presence of 1000 U/ml IFN-{gamma}. Then IRF-1 induction was evaluated by Western blot analysis on cell nuclear extracts.

 
Effect of NO on IFN-{gamma}-mediated T cell proliferation and apoptosis

Because we have shown that T cells expressing high levels of IFN-{gamma}R respond to IFN-{gamma} with a rapid apoptotic death (5, 6, 7), we evaluated the effect of exogenous IFN-{gamma} on the apoptosis and proliferative resumption of malignant T cells exposed to an NO donor. ST4 cells were cultured in medium alone or in the presence of NOC12 added once at the start. After 72 h, each culture was split and cultured for an additional 48 h in the presence or absence of 1000 U/ml IFN-{gamma}. Every 24 h, the cells were recovered, counted with trypan blue to assess cell viability, and stained with FITC-dUTP to assess apoptosis. Fig. 9Go (A) shows that ST4 cells cultured in medium alone grew optimally until 120 h. When IFN-{gamma} was added during the last 48 h, however, their proliferation was significantly augmented. This enhancement was not observed when IFN-{gamma} was admixed with {gamma}R99 anti-IFN-{gamma}R1-blocking mAb (data not shown). By contrast, proliferation of ST4 exposed to NOC12 was blocked until 72 h. The surviving cells proliferated significantly in medium only, although less than the unexposed controls. When IFN-{gamma} was added during the last 48 h of culture, proliferation was abolished. This inhibition was not observed when IFN-{gamma} was admixed with {gamma}R99 anti-IFN-{gamma}R1-blocking mAb (data not shown).



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  		FIGURE 9. Effect of IFN-{gamma} on malignant T cells treated with NOC12 on viability (A), apoptosis (B), and caspase-1 expression (C). ST4 cells were cultured in the absence or presence of NOC12 (0.1 mM) added once at the start. After 72 h, each culture was split and cultured for an additional 48 h in the presence or absence of 1000 U/ml of IFN-{gamma}. Every 24 h, the cells were recovered and the number of viable cells and the percentage of apoptotic cells were determined by trypan blue dye exclusion and TUNEL staining, respectively. Numbers of viable cells and apoptosis percentages are expressed as arithmetic means ± SD from three independent experiments. To evaluate caspase-1 expression, cells were recovered after 24 h of IFN-{gamma} treatment and assayed by Western blot analysis on total cell extracts.

 
ST4 cells cultured in medium alone did not show detectable levels of apoptosis until 120 h, irrespective of the addition or not of IFN-{gamma} alone (Fig. 9GoB) or admixed with {gamma}R99 mAb (data not shown) during the last 48 h. When NOC12 was added at the start, cells reached a peak of apoptosis after 72 h, followed by a rapid decrease to undetectable levels, whereas high levels were still observed when IFN-{gamma} was added in the last 48 h, either alone or admixed with {gamma}R99 (data not shown).

To further analyze the molecules involved in the IFN-{gamma}-dependent death pathway, the expression of the death effector caspase-1 (45, 46, 47), whose expression can be induced by IFN-{gamma} (48), was also evaluated in the same cultures. Western blot analysis performed on ST4 cells recovered after 24 h of IFN-{gamma} treatment revealed that caspase-1 was consistently induced only on cells previously exposed to NOC12 (Fig. 9GoC).

Similar results were obtained with Jurkat and PF382 cells (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Our findings show that NO has dual effects on human malignant T cells: it induces rapid apoptosis of most cells; the few that escape apoptosis resume proliferation, but are tagged by overexpression of IFN-{gamma}R chains.

NO equally inhibits proliferation, up-regulates IFN-{gamma}R chains, and induces apoptosis in normal and malignant T cells, suggesting that its effect on T cell response may be of physiologic significance. Moreover, this is endorsed by the observations that, although with different kinetics, the amounts of NO released by NOC12 and SNP are comparable with those released by LPS- and IFN-{gamma}-stimulated macrophage J774 cells, which represent a realistic in vitro model of physiologic NO release (49).

This rapid apoptosis elicited by transient exposure to NO is cGMP independent, characterized by a decrease in Bcl-2 expression and unaccompanied by a significant change in Fas modulation (data not shown). Moreover, p53 mRNA is induced in malignant T cells exposed to NOC12 (data not shown). These findings suggest that the mechanism causing NO-induced apoptosis of T cells is common to a wide range of cell types. In effect, previous reports have shown that Bcl-2, a negative regulator of cell death (39), is involved in NO-induced apoptosis in B lymphocytes and hypothalamic cells (50, 51).

This NO-induced apoptosis appears to be IFN-{gamma} independent, because the cell lines we used did not produce IFN-{gamma} either constitutively (5) nor after NO exposure (data not shown). However, it is a transient event and wanes after about 4 days, when a fraction of NO-treated malignant T cells resumes growth.

Exposure to NO may select a subpopulation of malignant T cells able to scavenge or detoxify NO through the rapid switch from aerobic to anaerobic respiration, or through p53-mediated DNA repair mechanisms (52). Remarkably, the increase of p53 mRNA was observed in T cells after NO exposure (data not shown). In this study, we show that the fraction of malignant T cells that evaded NO-induced apoptosis up-regulated their membrane expression of both IFN-{gamma}R chains. This up-regulation of IFN-{gamma}R chains, induction of hypoxia, and increased p53 levels, however, may be interconnected events in these surviving cells. Deferoxamine, an iron chelator that mimics hypoxia, induces the accumulation of p53 (53) and up-regulates T cell IFN-{gamma}R (54). Studies addressing the role of these events in regulating the expression of IFN-{gamma}R of NO-exposed T cells are currently in progress in our laboratory.

Brief exposure of T cells to NO induces both their apoptosis and accumulation in the S phase (data not shown). These S phase-arrested T cells may start to proliferate again. However, as a result of their exposure to NO, they overexpress IFN-{gamma}R chains. This is a critical feature that makes them susceptible to IFN-{gamma}-mediated apoptosis. Our data show that the reversibility of this proliferative inhibition is associated with down-modulation of both chains, and T cells are only susceptible to IFN-{gamma}-mediated apoptosis when their progression in the cell cycle is arrested. In effect, we have previously shown that environmental stimuli of various kinds, namely serum and IL-2 deprivation (5, 6, 7), TCR ligation (6, 7), exposure to x-rays (5), negative growth regulators (9), or chemotherapeutic drugs (8), regulate IFN-{gamma}R expression. All these treatments, including NO, induce arrest in S and G2/M phases (55, 56, 57) and make T cells susceptible to IFN-{gamma}-mediated apoptosis (5, 6, 7). Thus, it is likely that up-regulation of IFN-{gamma}R chains by NO is a general event related to the cycle arrest of T cells, because they down-regulate their expression on reentry into the cycle.

We show that NO induces up-regulated expression of membrane IFN-{gamma}R chains without modifying their constitutive mRNA expression. Because both chains are preferentially expressed in the cytoplasmic compartment of T cells (7), their increased membrane expression is probably the results of their recruitment from granule stores, rather than from the synthesis of new protein. By contrast, the IL-2R{alpha}-chain, whose surface up-regulation requires gene transcription (58), was unaffected by NO treatment.

In function of its receptor chain expression, IFN-{gamma} itself promotes either proliferation (5, 59) or rapid apoptotic death (6, 7, 9). Therefore, the fate of a T cell is determined by the interplay between particular cytokines or growth factors and environmental signals (5, 6, 7). The data reported in this work indicate that NO is one of these signals and acts by up-regulating both IFN-{gamma}R chains and thus priming T cells for IFN-{gamma}-induced apoptosis. Addition of IFN-{gamma} to unexposed T cells, in fact, increases their growth and does not induce caspase-1, whereas its addition to T cells that have escaped NO-induced death enhances caspase-1 and completely abolishes their proliferation by triggering rapid apoptosis. However, the IFN-{gamma}-mediated apoptosis is completely independent of NO, because the T cells used in this study did not produce NO endogenously after treatment with IFN-{gamma} (data not shown).

It has been reported that the antiproliferative signal of IFN-{gamma} is switched to growth promoting when IRF-1 expression is inhibited by the antisense technique (60). Our Western blot data show that IFN-{gamma} induces expression of IRF-1 protein in both unexposed and NO-exposed malignant T cells. However, because the expression of IRF-1 appears to be controlled by IRF-2, another transcription factor that counteracts the inhibitory effect of IRF-1 (44), the possibility that an altered expression ratio between the two factors in NO-exposed malignant T cells contributes to the triggering of apoptotic effects of IFN-{gamma} cannot be completely ruled out.

It has been shown in the mouse that Th1, but not Th2 cells, can be activated to produce NO and this NO is a self-regulatory molecule that leads to Th2 expansion through inhibition of IL-2 and IFN-{gamma} production and enhancement of IL-4 production (61, 62, 63, 64). However, it has been reported that human Th cells appear to be equally affected by NO (65).

Modulation of the IFN-{gamma}R2 chain is a critical event during Th1/Th2 differentiation (66, 67, 68). The antiproliferative effect of IFN-{gamma} on Th2 cells (69) is due to their ability to express IFN-{gamma}R2 (66, 67). Lack of this expression would make Th1 cells resistant to IFN-{gamma} by preventing transduction of its signals (66). Present data suggest that NO may also play a physiologic role in human Th1/Th2 differentiation by favoring the IFN-{gamma}-mediated apoptosis of Th cells through high levels of IFN-{gamma}R2 expression.

NO produced by macrophages or tumor cells is involved in tumor-induced immunosuppression (70). Tumor-infiltrating lymphocytes from rat tumors did not proliferate in response to mitogens, whereas addition of a NOS inhibitor restored their proliferation. Moreover, these lymphocytes are more sensitive to the antiproliferative effect of NO than the tumor cells (70).

Furthermore, the severe hypoxia typical of most tumors induces a group of molecular responses in mammalian cells (71, 72). One particularly significant response is the regulation of iNOS expression in macrophages mediated by a hypoxia-responsive enhancer (20, 21). NO produced in hypoxic conditions such as those of a tumor growth area could act on tumor-infiltrating lymphocytes by altering IFN-{gamma}R chain expression and hence favoring apoptotic signals mediated by IFN-{gamma}.

NO-mediated apoptosis seems to be a broad phenomenon, irrespective of their differentiation stage or ability to progress in the cell cycle. Our observations on the role of NO in regulation of the apoptosis of normal ongoing and malignant T cells are germane to those described for thymocytes (73) and TCR-triggered mature lymphocytes (17) and related to the role of IFN-{gamma}R chain modulation during these events.

NO-dependent up-regulation of the two IFN-{gamma}R chains and the subsequent bias of T cells toward IFN-{gamma}-mediated apoptosis define a new way in which the fate of T cells encountering IFN-{gamma} is decided. There is in vivo and in vitro evidence to support the view that IL-12-induced IFN-{gamma} production leads to generation of high levels of NO and that this impairs the proliferation of T cells (25). Moreover, human T cells themselves generate NO upon TCR stimulation, and this leads to the increase of surface expression of FasL and T cell apoptosis (17). As IFN-{gamma} increases FasL in T cells overexpressing IFN-{gamma}R (7), the existence of an interplay between NO, IFN-{gamma}, IFN-{gamma}R, and FasL expression with an important role in down-regulation of T cell effector function can be hypothesized.

In conclusion, the data presented in this work do indeed show that NO produced by activated macrophages or by T cells themselves can be a critical factor that concurs in the IFN-{gamma} switch of the T cell program from proliferation to apoptosis.


   Acknowledgments
 
We thank Dr. J. Iliffe for critically reading the manuscript.


   Footnotes
 
1 This work was supported by grants from the Associazione Italiana per la Ricerca sul Cancro (AIRC), Istituto Superiore di Sanita (special project on AIDS), Associazione Italiana Sclerosi Multipla (AISM), and Fondazione Piemontese per la Ricerca e gli Studi sulle Ustioni (FPRSU). Back

2 Address correspondence and reprint requests to Dr. Francesco Novelli, Dipartimento di Scienze Cliniche e Biologiche, Ospedale S. Luigi Gonzaga, 10043 Orbassano (TO), Italy. E-mail address: Back

3 Abbreviations used in this paper: NOS, NO synthase; cGMP, cyclic GMP; FSC, forward scatter; iNOS, inducible NOS; IRF, IFN regulatory factor; MFI, mean fluorescence intensity; PI, propidium iodide; SNP, sodium nitroprusside; SSC, side scatter; FasL, Fas ligand. Back

Received for publication December 2, 1998. Accepted for publication August 2, 1999.


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