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-Dependent and IFN--Independent Induction of Apoptosis1
Department of Clinical and Biological Sciences, University of Turin, Orbassano, Italy
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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R, and thus
become susceptible to apoptosis in the presence of IFN-. By
contrast, in the presence of IFN-, no apoptosis, but an increase of
proliferation was displayed by control cultures of T cells not exposed
to NO and not overexpressing IFN-R chains. The NO-induced cell
surface overexpression of IFN-R chains did not affect the
transduction of IFN--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- 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-. By inducing the overexpression of IFN-R chains, NO decides
whether IFN- promotes cell proliferation or the induction of
apoptosis. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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- to its cell surface receptor
(IFN-R), which is composed of at least two chains, IFN-R1
(2) and IFN-R2, the latter cloned as accessory factor-1
(3). Although IFN-R1 alone binds IFN- with high
affinity, its interaction with the other chain is required for
IFN--mediated signaling (4).
We have previously shown that IFN- 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-R1 and low IFN-R2 levels (6, 7),
IFN- 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-R chains by T
cells and render them susceptible to IFN--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-R chains is a critical event that influences the cell’s fate
and responsiveness to IFN-, 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- 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- 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- mediates the apoptosis of T lymphocytes
(5, 18). By contrast, when accessory cells are present and
costimulatory signals are provided, IFN- promotes the progression of
T lymphocyte activation (5, 6, 7). NO produced by
monocytes/macrophages following interaction with IFN- or bacterial
products (12, 13), or following a decrease of oxygen
partial pressure (20, 21), may interact with
IFN--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--mediated NO production by macrophages, which suppress T cell
proliferative responses. Thus, the interplay between NO and IFN- 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-, we evaluated its
effect in regulation of IFN-R chain expression.
This study investigates the role of NO on the apoptosis and IFN-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--independent apoptosis, because
these lines did not produce IFN- 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--mediated apoptosis. In effect, the addition of IFN-
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--independent apoptosis of human T cells. They also identify NO
as one of the environmental factors that, by inducing the recruitment
of IFN-R chains from granule stores, increase their surface
expression and convert the signal delivered by IFN- from growth
promoting into apoptotic.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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-- 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- and mAb to IFN-R
IFN- 
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- (31). Mouse mAb R99
is an IgG1 that specifically interacts with the extracellular domain of
human IFN-R1 and inhibits the binding of IFN- (32);
mouse mAb C.11 is an IgG2a that specifically interacts with the
extracellular domain of the human IFN-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 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
-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- 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-R1 and
IFN-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-R1 and IFN-R2 were designed on the basis of published
sequences (2, 3): IFN-R1, 5'-GTCCTCAGTGCCTACACCAACTAA
and 3'-CCACACATGTAAGACTCCTCCTGC (amplified fragment of 594 bp);
IFN-R2, 5'-GCAAGATTCGCCTGTACAACGCA and
3'-GTCACCTCAATCTTTTCTGGAGGC (amplified fragment of 339 bp). Primer
pairs were used in the following conditions: IFN-R1 and IFN-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.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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. 1
). The cells then began to proliferate
again.
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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. 2A).
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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-. Murine J774 cell line releases NO in response to IFN- 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. 2
B, 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-.
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- plus LPS-stimulated J774 produced nitrite amounts
comparable with that released by NOC12 and SNP (see Fig. 2B), the growth of normal and malignant T cells was
significantly inhibited by coculture with J774 cells, irrespective of
their activation with IFN- 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-, 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. 1). 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. 3, 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. 3, 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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, 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. 1). 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. 4), 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. 4).
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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 I). 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 I).
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R1 and IFN-R2 chain expression
We have shown that interaction between IFN- and IFN-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-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. 1 and 4), we determined whether NO also modulates IFN-R chain
expression. ST4 cells were cultured in the absence or presence of NO
donors added once at the start, and their IFN-R chain expression was
monitored by flow cytometry using specific anti-IFN-R1 R99
and anti-IFN-R2 C.11 mAb. Fig. 5
shows the physical parameters (forward scatter (FSC) and side scatter
(SSC)) and IFN-R chain expression on ST4 cells cultured 72 h in
medium only and in the presence of SNP or NOC12.
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, 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-R1 (MFI 306, middle upper
panel), and almost undetectable levels of IFN-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. 5). The MFI
increase induced by the donors on ST4 cells was about 3-fold for
IFN-R1 (SNP, MFI 871; NOC12, MFI 874) and about 2-fold for IFN-R2
(SNP, MFI 176; NOC12, MFI 157).
The kinetics of the viability (evaluated as percentage gated in region
R1 cells, Fig. 6, upper
panel), of MFI of R1-gated IFN-R1-positive cells (Fig. 6, middle panel), and of R1-gated IFN-R2-positive cells
(Fig. 6, 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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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. 7).
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Because it has been reported that NO has no effect on T cell IL2R
expression (43), we compared IL-2R (CD25) expression
with that of the two IFN-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-R1, IFN-R2, and CD25, respectively (Table II).
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R chains was increased by SNP (IFN-R1,
75%; IFN-R2, 52%) and NOC12 (IFN-R1, 56%; IFN-R2, 49%). By
contrast, neither SNP nor NOC12 significantly modified IL-2R
expression (CD25 SNP, 88%; NOC12, 77%), as expected
(43).
Induction of IRF-1 on T cells by IFN-
Because NO induced the up-modulation of IFN-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-R complex to
transduce IFN--mediated signals. The effectiveness of IFN-R was
analyzed by examining the nuclear expression of IRF-1, whose
transcriptional activation is specifically induced by IFN-
(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-
for an additional 24 h. As shown in Fig. 8, IRF-1 was always induced by IFN-,
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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-mediated T cell proliferation and apoptosis
Because we have shown that T cells expressing high levels of
IFN-R respond to IFN- with a rapid apoptotic death
(5, 6, 7), we evaluated the effect of exogenous IFN- 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-. Every 24 h, the cells
were recovered, counted with trypan blue to assess cell viability, and
stained with FITC-dUTP to assess apoptosis. Fig. 9 (A) shows that ST4 cells
cultured in medium alone grew optimally until 120 h. When IFN-
was added during the last 48 h, however, their proliferation was
significantly augmented. This enhancement was not observed when IFN-
was admixed with R99 anti-IFN-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-
was added during the last 48 h of culture, proliferation was
abolished. This inhibition was not observed when IFN- was admixed
with R99 anti-IFN-R1-blocking mAb (data not shown).
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alone (Fig. 9B) or admixed with 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- was added in the last 48 h, either alone or
admixed with R99 (data not shown).
To further analyze the molecules involved in the IFN--dependent
death pathway, the expression of the death effector caspase-1
(45, 46, 47), whose expression can be induced by IFN-
(48), was also evaluated in the same cultures. Western
blot analysis performed on ST4 cells recovered after 24 h of
IFN- treatment revealed that caspase-1 was consistently induced only
on cells previously exposed to NOC12 (Fig. 9C).
Similar results were obtained with Jurkat and PF382 cells (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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R chains.
NO equally inhibits proliferation, up-regulates IFN-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--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- independent, because
the cell lines we used did not produce IFN- 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-R chains. This
up-regulation of IFN-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-R
(54). Studies addressing the role of these events in
regulating the expression of IFN-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-R chains. This is a critical
feature that makes them susceptible to IFN--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--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-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--mediated apoptosis
(5, 6, 7). Thus, it is likely that up-regulation of IFN-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-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-chain, whose surface up-regulation requires gene transcription
(58), was unaffected by NO treatment.
In function of its receptor chain expression, IFN- 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-R chains and thus priming T cells for IFN--induced
apoptosis. Addition of IFN- 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--mediated apoptosis is completely independent of
NO, because the T cells used in this study did not produce NO
endogenously after treatment with IFN- (data not shown).
It has been reported that the antiproliferative signal of IFN- is
switched to growth promoting when IRF-1 expression is inhibited by the
antisense technique (60). Our Western blot data show that
IFN- 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- 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-
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-R2 chain is a critical event during Th1/Th2
differentiation (66, 67, 68). The antiproliferative effect of
IFN- on Th2 cells (69) is due to their ability to
express IFN-R2 (66, 67). Lack of this expression would
make Th1 cells resistant to IFN- 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--mediated apoptosis of Th cells through high levels of IFN-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-R chain expression and hence favoring apoptotic signals mediated
by IFN-.
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-R chain modulation
during these events.
NO-dependent up-regulation of the two IFN-R chains and the
subsequent bias of T cells toward IFN--mediated apoptosis define a
new way in which the fate of T cells encountering IFN- is decided.
There is in vivo and in vitro evidence to support the view that
IL-12-induced IFN- 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- increases
FasL in T cells overexpressing IFN-R (7), the existence
of an interplay between NO, IFN-, IFN-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- switch of the T cell
program from proliferation to apoptosis.
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Acknowledgments |
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Footnotes |
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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:
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.
Received for publication December 2, 1998. Accepted for publication August 2, 1999.
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