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Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 3876-3884
Interleukin-2-Activated Rat Natural Killer Cells Express Inducible
Nitric Oxide Synthase That Contributes to Cytotoxic Function and
Interferon- Production
By
M. Grazia Cifone,
Simona D'Alò,
Raffaella Parroni,
Danilo Millimaggi,
Leda Biordi,
Stefano Martinotti, and
Angela Santoni
From the Department of Experimental Medicine, University of
L'Aquila, L'Aquila; and the Department of Experimental Medicine and
Pathology, University La Sapienza, Rome, Italy.
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ABSTRACT |
Natural killer (NK) cells are large granular lymphocytes capable of
destroying cells infected by virus or bacteria and susceptible tumor
cells without prior sensitization and restriction by major histocompatability complex (MHC) antigens. Their cytotoxic activity could be strongly enhanced by interleukin-2 (IL-2). Previous findings, even if obtained with indirect experimental approaches, have suggested a possible involvement of the inducible nitric oxide (iNOS) pathway in
the NK-mediated target cell killing. The aim of the present study was
first to directly examine the induction of iNOS in IL-2-activated rat
NK cells isolated from peripheral blood (PB-NK) or spleen (S-NK), and
second to investigate the involvement of the iNOS-derived NO in the
cytotoxic function of these cells. Our findings clearly indicate the
induction of iNOS expression in IL-2-activated PB-NK and S-NK cells,
as evaluated either at mRNA and protein levels. Accordingly,
significantly high levels of iNOS activity were shown, as detected by
the L-arginine to L-citrulline conversion in appropriate assay
conditions. The consequent NO generation appears to partially account
for NK cell-mediated DNA fragmentation and lysis of sensitive tumor
target cells. In fact, functional inhibition of iNOS through specific
inhibitors, as well as the almost complete abrogation of its expression
through a specific iNOS mRNA oligodeoxynucleotide antisense,
significantly reduced the lytic activity of IL-2-activated NK
cells. Moreover, IL-2-induced interferon- production appears also to be dependent, at least in part, on iNOS induction.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
NATURAL KILLER (NK) cells are a distinct
subset of CD3 CD16+ CD56+
large granular lymphocytes (LGL), which possess the ability to mediate
major histocompatibility complex (MHC)-unrestricted cytolytic activity
against cells infected by viruses or bacteria, transplanted cells, and
neoplastic cells.1 Cytotoxic factors, contained in the
granules and released from the NK cell after interaction with the
target cell through mutual surface receptors, mainly include perforin
and granzymes, able to induce both membrane and DNA damage in the
target cells.2,3 Moreover, murine NK cells and
FcR-stimulated human NK cells have been reported to express Fas
ligand and kill Fas (CD95/APO-1)-expressing targets.4,5 The
functions of NK cells, as well as their maturation and differentiation, are regulated by various stimuli, including interleukin-2
(IL-2).6 Resting NK cells can respond directly to IL-2 with
enhanced cytotoxic activity and eventually proliferation, with no
additional requirement for other activating stimuli. IL-2 augments mRNA
expression for perforin and interferon- (IFN-),7,8 as
well as synthesis and expression of various adhesion molecules on NK
cells,9 which increase their cytotoxic potential. In vitro
long-term culture of NK cells in the presence of high doses of IL-2
leads to the generation of cells with a broad range of tumor target
cell lysis (lymphokine-activated killer [LAK] activity).6
Interestingly, generation of LAK activity has been previously reported
to depend on nitric oxide (NO) production.10-14 Indeed,
both in vivo and in vitro IL-2 treatment, able to markedly stimulate NK
cell tumoricidal activity, correlates with increased NO production
determined as nitrite levels in serum or culture
medium.10-14 Moreover, the inhibition of NO synthesis
blocks IL-2-induced NO production and significantly reduces
IL-2-induced NK cytotoxicity, thus suggesting that NO synthesis may
contribute to IL-2-induced antitumor responses of NK cells. Moreover,
NKR-P1A triggering, which is known to induce NK cell activation and
mediate reverse antibody-dependent cellular cytotoxicity
(ADCC),15,16 is able to induce nitric oxide synthase (NOS)
activity.10 Of note, evidence has been recently reported that genetic deletion of functional inactivation of NOS2 (inducible NOS
[iNOS]) in mice abolished NK cell responses, thus providing the first
evidence that iNOS-derived NO is a prerequisite for the development of
NK cytotoxicity in vivo.17 Expression of NOS2 seems to be
essential for at least four key elements of the innate immune response:
(1) prevention of the microbial dissemination; (2) responsiveness of NK
cells to the NK cell-activating factor, IL-12; (3) release by NK cells
of IFN-; and (4) suppression by IFN- of the production of
transforming growth factor- (TGF-), a potent NOS2-suppressing
cytokine.17,18
NO, a small lipophilic molecule enzymatically generated from cleavage
of terminal guanidino nitrogen from L-arginine by a family of NOS,
takes part in several biologic events.19,20 Constitutive
and inducible isoforms of the NOS exist, and they differ in structure
and regulation.18,21 All of the isoforms convert arginine
to citrulline and NO and require nicotinamide adenine dinucleotide
phosphate (NADPH) as a cofactor.22 Constitutive isoforms of
NOS (cNOS) produce small amounts of NO over several minutes in response
to agonists that elevate intracellular Ca2+. The
cytokine-inducible, or inflammatory, NOS (iNOS, NOS2) produces NO in a
sustained manner independently of intracellular calcium elevations and
is expressed mainly in inflammatory conditions. Cell-mediated immune
responses are considered as classical NO-mediated actions.18,22 In particular, activation of the NO system
mediates macrophage cytotoxicity as a first line of defense against
invading pathogens or tumor cells and elicits apoptotic cell death in
different cellular systems.23 NO-induced inhibition of
iron-sulphur enzymes involved in cellular energy supply, as well as
NAD(H)-dependent modification of protein thiols24 or direct
DNA damage25,26 have been implicated in the cytotoxic NO
signaling, or in the apoptogenic action of NO. Interestingly, the
susceptibility of iNOS-deficient mice to several bacterial and viral
infections, as well as to the growth of several implantable tumors is
greatly increased compared with wild-type mice.23,27,28
Here, we show direct evidence for the induction of iNOS expression and
activity in IL-2-activated peripheral blood and splenic rat NK cells.
Our findings also indicate that NO represents an effector molecule that
contributes to the tumoricidal activity of IL-2-stimulated NK cells
and is involved in the IFN- production from these cells.
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MATERIALS AND METHODS |
Animals.
Fisher F344 rats (5 to 6 weeks old) were purchased from Charles River
Breeding Laboratories (Calco, Como, Italy).
Reagents.
The following mouse monoclonal antibodies (MoAbs) were used: OX19,
which identifies CD5, a pan-T surface antigen; MCA1427 (Serotec,
Oxford, UK), an IgG1 that recognizes rat CD161 protein, also known as
NKR-P1A, a 60-kD disulfide-linked dimer composed of two 30-kD subunits
expressed on rat NK cells; a F(ab')2 preparation of
goat antiserum to mouse Ig was purchased from Cappel Laboratories (Cochranville, PA); MCA 341, an IgG1, which specifically identifies rat
monocytes and macrophages by recognizing a 97-kD antigen (ED1) predominantly located intracellularly (although membrane expression also occurs), and MCA1427RPE, a mouse anti-rat CD161-R-phycoerythrin (PE)-conjugated MoAb; were purchased from Serotec. Human recombinant IL-2 (rIL-2) was from EuroCetus B.V. (Amsterdam, The Netherlands). Anti-iNOS MoAb purchased from Transduction Laboratories (Lexington, KY)
was directed against a protein fragment (961-1144 aa) of mouse macNOS.
L-nitro-monomethylarginine (L-NMMA), aminoguanidine (AG), and
N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide (W13) were from
Alexis Biochemicals (Laufelfingen, Switzerland). Lipopolysaccharide (LPS) was from Calbiochem (La Jolla, CA) and recombinant rat IFN- was from Endogen (Cambridge, MA). Sodium nitroprussiate (SNP) was
purchased from Sigma (St Louis, MO).
Isolation of peripheral blood NK cells (PB-NK).
Mononuclear cells were separated from cardiac-puncture-obtained
heparinized peripheral blood by Lymphoprep gradient centrifugation (Nicadem, Oslo, Norway), further purified by passage through a nylon
wool column (Cellular Products, Buffalo, NY), plastic adherence to
remove monocytes, Percoll (Pharmacia, Uppsala, Sweden) density gradient
fractionation, and panning on plastic dishes (Falcon, Becton Dickinson,
Mountain View, CA) as previously described.29 Isolated PB-NK were then cultured over 18 hours at 37°C, in 5% CO2 in air in the presence of rIL-2 (100 U/mL) before
analysis of iNOS expression and enzymatic activity, and cytotoxicity studies.
Cytofluorymetric analysis.
The surface phenotype of LGL population was analyzed by indirect
immunofluorescence labeling and cytofluorimetry on FACS II Analyzer
(Becton Dickinson) by using MCA1427 (anti-CD161), OX19, and MCA 341 MoAbs. Unpermeabilized cells were used for MCA1427 and OX19 staining.
Fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG
F(ab')2 fragment was used as secondary antibody (G M). MCA 341 staining was performed on permeabilized cells. Briefly, cells were washed with 10 mmol/L HEPES buffer (pH 7.4), 150 mmol/L NaCl, 4% fetal calf serum (FCS), resuspended at 5 × 106/mL in phosphate-buffered saline (PBS) and permeabilized
with methanol 70% for 5 minutes on ice. After confirming the adequacy of permeabilization by trypan blue uptake, cells were pelletted and
resuspended in PBS containing saturating concentration of MCA 341 MoAb.
The results presented in Fig 1 show that
the NK cell population purified as above described, was >99%
MCA1427+ OX19 MCA
341. The absence of contaminating monocytes
was also confirmed by negative staining for nonspecific esterase
(data not shown).
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| Fig 1.
Phenotypic analysis of PB-NK and S-NK cells. Phenotype of
purified NK cells was analyzed by immunofluorescence and
flow cytometry using the indicated MoAbs to determine the purity of the
cell populations used in this study. White area represents staining
with secondary antibody alone.
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Spleen-NK cell (S-NK) purification.
S-NK cells were obtained as previously described.30
Briefly, spleens were aseptically removed and single-cell suspensions were prepared in RPMI 1640 with 5% FCS. Mononuclear cells obtained by
centrifugation on Ficoll/Hypaque gradients were incubated on plastic
tissue culture flasks in a 5% CO2 atmosphere for 1 hour. The nonadherent cells were further passed through nylon-wool columns and then cultured at 2 × 106/mL in RPMI 1640 medium
supplemented with 10% heat-inactivated FCS, 2 mmol/L glutamine, 5 × 105 mol/L 2-mercaptoethanol, streptomycin,
and penicillin and containing 500 U/mL rIL-2. After 48 hours of
incubation at 37°C, in 5% CO2 in air, the adherent
cells were washed with prewarmed medium, starved for 48 hours (control
cells) and cultured in the absence or presence of IL-2 (100 U/mL) for
an additional 96 hours. The phenotype of S-NK cells was routinely
>99% MCA1427+ OX19
MCA341 as assessed by flow cytofluorimetric analysis
on FACS II Analyzer (Fig 1).
Nitrite levels.
In aqueous solution, NO reacts rapidly with O2 and
accumulates in the culture medium as nitrite and nitrate ions. After
indicated incubation times, 100-µL samples of the culture
supernatants were removed for nitrite measurement. Nitrite
concentrations were quantified by a colorimetric assay based on the
Griess reaction.31 Briefly, 100 µL of culture supernatant
were mixed with an equal volume of Griess reagent in a U-well
microtiter plate and incubated at room temperature for 10 minutes. The
optical density (OD) was measured at 550 nm using a
micro-ELISA reader (MultiSkan Plus; DASIT, Milan, Italy) using
NaNO2 as a standard and Dulbecco's medium supplemented
with 10% FCS as a blank.
Determination of NOS activity.
NOS activity was determined by measuring the conversion of
[3H]-L-arginine to [3H]-L-citrulline as
previously described.32 Cells were homogenized by
sonication in homogenization ice-cold buffer (25 mmol/L Tris-HCl, pH
7.4, 1 mmol/L EDTA, 1 mmol/L EGTA) containing protease inhibitors (0.2 mmol/L phenylmethylsulfonyl fluoride [PMSF], 10 µg/mL aprotinin, 10 µg/mL pepstatin A, 10 µg/mL leupeptin). The homogenates were centrifuged at 4°C for 20 minutes at 15,000 rpm. The supernatants were passed through an AG50WX-8 Dowex resin (Bio-Rad Laboratories, Melville, NY) to remove endogenous arginine. The protein content was
determined by the bicinchoninic acid procedure (BCA; Pierce, Rockford,
IL) using bovine serum albumin (BSA) as
standard. Enzymatic reactions were conducted at 37°C for 30 minutes
in 50 mmol/L Tris-HCl, pH 7.4, containing 1 mmol/L CaCl2, 1 mmol/L MgCl2, 6 µmol/L tetrahydrobiopterin (BH4), 2 µmol/L flavin adenine dinucleotide (FAD), 2 µmol/L flavin adenine mononucleotide (FAM), 1 mmol/L L-citrulline (to
inhibit the catabolism of [3H]-L-citrulline, 60 mmol/L
L-valine, 60 mmol/L L-ornithine, 60 mmol/L L-lysine (to inhibit
nonspecific arginase activity), and 10 µCi/mL of
[3H]-L-arginine (Amersham, Bukinghamshire, UK). Where
indicated, CaCl2 (0.6 mmol/L) and calmodulin (CAM) (0.1 µmol/L) were added to the assay system. The reaction was stopped by
the addition of 0.4 mL of stop buffer (50 mmol/L Hepes, pH 5.5, 5 mmol/L EDTA). Citrulline was determined by applying the samples to
columns containing 0.1 mL of Dowex AG50WX-8, equilibrated resin to the
Na+ form, to bind all of the unreacted, radiolabeled
L-arginine. The columns were vortexed and centrifuged (15,000 rpm for
30 seconds). One hundred microliters of the eluate was counted by
liquid scintillation to quantitate the formation of
[3H]-L-citrulline. This assay measures both the
calcium-dependent (constitutive) and the calcium-independent
(inducible) isoenzymes. Any activity detected in the absence of Ca/CAM
and in the presence of the CAM antagonist W13 (100 µmol/L) (to
inhibit endogenous CAM) represented iNOS activity. iNOS activity was
also considered inhibited by AG.
RNA isolation and reverse transcription-polymerase chain reaction
(RT-PCR) amplification.
Total RNA was isolated from both PB-NK and S-NK cells after incubation
for the indicated times in the absence or presence of IL-2, using the
method of Chomczynski and Sacchi.33 A total of 200 ng RNA
was incubated with 2.5 mmol/L random hexamers, 1 mmol/L each
deoxynucleotide triphosphate (dNTP), 2.5 U/mL Moloney murine leukemia
virus RT (Boehringer Mannheim, Indianapolis, IN), and 5 mmol/L
MgCl2 in 20 mL of RT buffer (50 mmol/L KCl, 10 mmol/L Tris-HCl, pH 8.3) at 42°C for 20 minutes. After denaturation at 95°C for 5 minutes, the synthesized cDNA was subjected to a
35-cycle PCR, each cycle consisting of 1 minute at 94°C, 1 minute
at 60°C, and 2 minutes at 72°C. The 5' antisense primer
corresponded to nucleotides 2698-2720 of the murine macrophage iNOS
cDNA sequence, (Primm USA Inc, Andover, MA) and the 3' sense
primer to nucleotides 2941-2963. For comparison, -actin transcripts
were PCR-amplified from the same sample RNA using primers to
nucleotides 2406-2423 (sense) and to nucleotides 2726-2708 (antisense)
(Primm). The PCR mix contained 20 pmol of each primer, 200 mmol/L of
each dNTP, and 2.5 U Taq polymerase (AmpliTaq; Perkin-Elmer Cetus,
Norwalk, CT) in 100 µL of RT buffer with 2 mmol/L MgCl2.
The PCR products were separated by electrophoresis on a 2% agarose gel
and visualized by ethidium bromide staining.
Analysis of iNOS protein expression.
The expression of iNOS protein was assessed by flow cytometry and
Western blotting using a specific anti-mouse macNOS MoAb. Briefly, for
flow cytometry, the cells were fixed for 5 minutes at room temperature
with paraformaldehyde (4% in PBS), permeabilized with 0.05% Nonidet
P-40 in PBS, and then stained using 1 µg/106 cells of the
relevant primary antibody (20 minutes at 4°C) and a fivefold excess
of FITC-conjugated goat anti-mouse secondary antibody (DAKO, Milan,
Italy). The fluorescence background was calculated in the absence of
the primary antibody. An irrelevant primary antibody was used as
negative control. In some experiments, two-color cytofluorographic
analysis was performed using mouse anti-rat CD161-PE MoAb and mouse
specific anti-mouse macNOS MoAb and FITC-conjugated GM. For Western
blot analysis, cell pellets were washed with ice-cold PBS and then
solubilized for 15 minutes in a lysis buffer containing 1% Triton
X-100 (vol/vol), 10% glycerol, 100 mmol/L NaCl, 1.5 mmol/L
MgCl2, 10 mmol/L Na3VO4, 4 mmol/L PMSF, 20 mmol/L sodium pyrophosphate, 5 mmol/L EGTA, 2 µg/mL
leupeptin, 2 µg/mL aprotinin, 2 µg/mL pepstatin, 50 mmol/L Hepes,
pH 7.5. The lysates were clarified by centrifugation (15,000 rpm for 20 minutes at 4°C), and the protein content assayed by the
bicinchoninic acid procedure (BCA, Pierce). After addition of sodium
dodecyl sulphate and -mercaptoethanol, the samples were boiled and
250 µg of protein/lane were loaded into the slots of 7.5% sodium
dodecyl sulphate polyacrylamide gels, which were run as described
elsewhere.34 As an internal control for iNOS, 70 µg of
cell lysates from rat peritoneal macrophages treated with IFN- (100 U/mL) plus LPS (100 ng/mL) for 18 hours, were loaded into the same gel.
High efficiency transfer of proteins onto nitrocellulose membranes was
performed at 200 mA for 18 hours in a buffer containing 25 mmol/L Tris,
192 mmol/L glycine, 20% methanol, pH 8.3. After transfer, both the
gels and the blots were routinely stained with Ponceau red. The
nitrocellulose sheets were processed at room temperature, first 1 hour
with PBS + 3% BSA, then for 2 hours with the anti-iNOS MoAb (1:500
dilution) in an incubation buffer containing 150 mmol/L NaCl, 50 mmol/L
Tris-HCl, 0.05% Tween 20, 5% powdered milk, pH 7.4. After washing
five times for 5 minutes with the incubation buffer, the blotted bands
were incubated with the relevant horseradish peroxidase-linked IgG for
1 hour in the same buffer. After extensive washing, immunostaining was
recorded photographically, using an enhanced chemiluminescence kit from Amersham.
Antisense oligodeoxynucleotide treatment in vitro.
Antisense (AS) oligodeoxynucleotide targeting a conserved sequence
within the open reading frame of the cDNA encoding iNOS was designed to
produce selective knock-down of this enzyme, as previously
described.35 The published cDNA transcript to the rat
macrophage (GenBank accession no. M84373) was used to synthesize 21-nucleotide oligodeoxynucleotide. The phosphorothioated
oligodeoxynucleotide was synthesized in an automatic solid-phase DNA
synthesizer (Primm). We targeted a conserved sequence, nucleotides
1829-1809, which is homologous in mouse and rat macrophage iNOS (AS:
CTT CAG AGT CTG CCC ATT GCT). To account for possible aspecific effects
of oligodeoxynucleotide, a scrambled sequence was used as control (Scrambled [Scr]: TCT CAG TGA GCC CTC ATT CTG). Both PB-NK cells and
S-NK cells were treated with either oligomer for 18 and 96 hours,
respectively, at the concentration of 20 µmol/L in RPMI medium.
Neither oliogodeoxynucleotide was cytotoxic as determined by trypan
blue exclusion (95% viability).
Tumor cell lines.
The NK-sensitive target cells, YAC-1 cell line, a subclone of a Moloney
leukemia virus-induced tumor in A/Sn mice, and the LAK-sensitive target
cells, P815, a chemically induced NK-resistant mastocytoma, were
maintained by continuous in vitro culture in complete medium, which
consisted of RPMI 1640 supplemented with 10% heat-inactivated FCS, 2 mmol/L L-glutamine, streptomycin, and penicillin.
Cytotoxicity assay
Cytotoxicity assay was performed by incubating serial dilutions of
effector cells with 5 × 103 51Cr-labeled
(Na251CrO4; New England Nuclear
[NEN], Boston, MA) target cells YAC-1 and P815 cells in
triplicate wells of round-bottomed microtiter plates (Sterilin
Teddington, Middlesex, UK) in a final volume of 0.2 mL. After 4 hours
of incubation, the plates were centrifuged, and 0.1 mL supernatant was
removed and counted. The percentage of specific 51Cr
release was calculated as follows: 100 × (experimental release  spontaneous release)/(maximum release spontaneous
release). Where indicated, the cytotoxicity assay was performed with NK cells pretreated with AS or Scr oligonucleotides as above described or
in the presence of the NOS inhibitors, L-NMMA (500 µmol/L) and
aminoguanidine (50 µmol/L). Inhibitors had no effect on target cell
viability (>90% viability by trypan blue exclusion) or spontaneous 51Cr release (not shown).
JAM test.
The analysis of apoptosis in target cells (YAC-1 and P815) after
coculture for 4 hours with PB-NK or S-NK cells, was performed as
previously described.36 Briefly, effectors or targets cells were pulsed for 18 hours with 2 mCi/mL 3H-thymidine (6.7 Ci/mmol; NEN), washed, and then incubated either alone or with
unlabeled target or effector cells, respectively, at effector/target
(E/T) ratios reported in the figure legends. At the end of the culture,
the cells were harvested on a glass fiber filter, and
3H-thymidine present on the filters was measured by liquid
scintillation counting. The results were expressed as the arithmetic
means cpm ± standard deviation (SD) of five replicate cultures. The
percentage of fragmented DNA was calculated as follows: 100 × (mean cpm cells cultured alone mean cpm cells cultured with
target cells/mean cpm cells cultured alone).
DNA agarose gel electrophoresis.
The susceptibility of target cells (YAC-1 and P815) to undergo
apoptosis in the presence of a NO donor at different doses (SNP, 0.1 to
5 mmol/L) was evaluated through DNA laddering detection. Cells (5 × 106), after the indicated treatments, were
harvested, washed, and incubated in 0.3 mL of 10 mmol/L Tris-HCl pH 8.0 containing 25 mmol/L EDTA, 100 mmol/L NaCl, 0.5% SDS, and 0.1 mg/mL
proteinase K at 37°C for 18 hours. After phenol-chloroform
extraction, the DNA was ethanol precipitated and resuspended in
Tris-EDTA (TE: 10 mmol/L Tris-HCl, pH 7.6 and EDTA 1 mmol/L, pH 8),
incubated for 1 hour at 37°C with 1 mg/mL RNAse and applied on a
1.8% agarose gel, and electrophoresed for 2 hours at 100 V.
Enzyme-linked immunosorbent assay (ELISA) of IFN-.
IFN- levels released from treated NK samples were determined with
the BioSource International Cytoscreen rat IFN- ELISA (BioSource
Int, Inc, Camarillo, CA). Wells of ELISA plates were coated with an
antibody specific for rat IFN-, after which 100 µL of supernatants
from purified PB- or S-NK cells (2 × 106/mL) cultured
with or without IL-2 in the presence or absence of NOS inhibitors, were
added to each well. The IFN- content was determined according to the
manufacturer's instructions and expressed as pg/106 cells.
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RESULTS |
Stimulation of NO release by IL-2 treatment is due to the induction of
iNOS enzyme.
A significant induction of NO generation from IL-2-treated NK cells
compared with untreated cells was observed, as assessed by nitrite
determination in culture medium, confirming our original observations10 (and data not shown). To assess whether
stimulation of NO release after IL-2 treatment was due to an induction
of iNOS or to cNOS activity, we performed enzyme activity assays based
on the conversion of L-arginine to L-citrulline in appropriate conditions allowing the detection of both constitutive and inducible isoenzyme activity on cell sonicates. Table
1 illustrates the NOS activity in extracts from PB-NK and S-NK cells
after incubation with rIL-2 for 18 hours or 96 hours, respectively.
IL-2 significantly increased (P < .001) citrulline generation
in both PB-NK and S-NK cells. This event could be attributed to iNOS,
but not cNOS activity, as it was not affected by the absence of Ca/CAM
and the presence of the CAM antagonist W13. Moreover, IL-2-induced
iNOS activity was inhibited by both L-NMMA, a NO synthesis inhibitor
and AG, a relatively specific inhibitor of the inducible form. The
treatment of control cells with NOS inhibitors alone did not influence
the citrulline basal levels. The analysis of iNOS expression at mRNA and protein levels in IL-2-treated NK cells, definitively established that NO generation was due to the induction of iNOS at transcriptional and translational levels (Fig 2). The
RT-PCR of mRNA isolated from both PB-NK and S-NK cells cultured in the
presence of rIL-2 for 18 or 96 hours, respectively, resulted in the
amplification of an iNOS-specific cDNA fragment (Fig 2A, lanes 4 and
6). This fragment corresponded to the 266-bp PCR product obtained after RT-PCR of mRNA from rat macrophages stimulated with IFN- plus LPS
for 18 hours (Fig 2A, lane 2). No evidence of iNOS mRNA was observed in
untreated cells (Fig 2A, lanes 1, 3, and 5). No PCR product was
amplified in PCR reactions from samples not treated with reverse
transcriptase (data not shown). The protein analysis confirmed the
induction of iNOS in IL-2-treated NK cells, indicating that the iNOS
gene transcription was followed by an effective translation of iNOS
mRNA. As shown in Fig 2B, iNOS protein, which was undetectable in
untreated cells, was highly expressed after treatment of both PB-NK and
S-NK cells with IL-2 for 18 and 96 hours, respectively (lanes 1 through
4). Also in this case, lysates from rat macrophages stimulated with
IFN- plus LPS were used as control. The induction of iNOS protein in
IL-2-treated PB-NK cells and S-NK cells was also confirmed by
immunofluorescence and cytofluorimetric analysis staining permeabilized
cells with a specific anti-iNOS MoAb (Fig 2C). The percent of
iNOS-positive cells greatly increased in CD161 positive cells on IL-2
treatment in both PB-NK and S-NK cell populations. In
Fig 3 are also reported the
cytofluorymetric analysis of iNOS after treatment with antisense oligodeoxynucleotide against iNOS mRNA. It is evident that
iNOS-antisense treatment almost completely inhibited iNOS expression in
both IL-2-activated PB-NK and S-NK cells, whereas the scrambled
oligodeoxynucleotide was ineffective.
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| Fig 2.
Analysis of iNOS expression in PB-NK and S-NK cells. (A)
Expression of iNOS mRNA in PB-NK and S-NK cells after treatment with
IL-2. The mRNA of stimulated and unstimulated cells was isolated and
after RT-PCR was performed with specific primers for iNOS and
-actin. Lane 1, untreated rat peritoneal macrophages; lane 2, rat
peritoneal macrophages stimulated with IFN- plus LPS for 18 hours;
lane 3, control S-NK cells; lane 4, S-NK treated with IL-2 for 96 hours; lane 5, untreated PB-NK; lane 6, PB-NK treated with IL-2 for 18 hours. Size marker: DNA molecular weight marker VI (Boehringer) (M).
Data show one representative experiment of three. (B) Western blot
analysis with specific anti-iNOS MoAb in 200 µg of total cell lysates
obtained from PB-NK cells or S-NK. As internal control for iNOS, 50 µg of rat peritoneal macrophage lysates were loaded into the same
gels. Lane 1, untreated PB-NK; lane 2, PB-NK treated with IL-2 for 18 hours, lane 3, control S-NK cells; lane 4, S-NK treated with IL-2 for
96 hours; lane 5, untreated rat peritoneal macrophages; lane 6, rat
peritoneal macrophages stimulated with IFN- plus LPS for 18 hours.
(C) iNOS expression on CD161 positive PB- and S-NK cells after
treatment with IL-2 as above described. Two-color cytofluorographic
analysis was performed using mouse anti-rat CD161-PE MoAb and mouse
specific anti-mouse macNOS MoAb and FITC-conjugated G M. As control
antibodies, anti-human CD56-PE MoAb and GM-FITC MoAb were used.
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| Fig 3.
Flow cytometric analysis of the expression of iNOS in
PB-NK and S-NK cells treated with IL-2 in the presence or absence of
iNOS-antisense. AS, antisense oligodeoxynucleotide against iNOS mRNA;
Scr, scrambled oligodeoxynucleotide. Fluorescence intensity of
permeabilized cells stained with an irrelevant primary antiody plus a
secondary antibody was not significantly different from that incubated
only with the secondary antibody (not shown). Results shown are
representative of three separate experiments.
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iNOS induction is involved in the IL-2-activated NK cytotoxic
functions.
To assess whether iNOS induction could be involved in the cytotoxic
functions of IL-2-activated NK cells, we analyzed the effect of iNOS
inhibition on 51Cr release and DNA fragmentation of target
cells (YAC-1 as NK-sensitive cells and P815 as LAK-sensitive cells)
after 4 hours incubation with PB-NK or S-NK cells at different E/T
ratios at 37°C. With increasing numbers of either PB- or S-effector
cells, there was a concomitant increase in both specific
51Cr release and specific DNA fragmentation of YAC-1 target
cells. The inhibition of iNOS activity through treatment with
pharmacological inhibitors such as L-NMMA or AG or the almost total
abrogation of its expression with a specific AS oligonucleotide,
markedly decreased the cytotoxic activity of IL-2-stimulated-PB-NK
(Table 2) and -S-NK cells against YAC-1
targets (Table 3); the scrambled oligodeoxynucleotide used as control was without effect. Similar results were obtained when P815 cells were used as target sensitive to
IL-2-activated S-NK cell-mediated lysis (not shown). The
susceptibility of target cells to undergo apoptosis in the presence of
the NO donor SNP is shown in Fig 4.
Overall, these results indicate that the IL-2-mediated induction of
iNOS is involved in the cytotoxic functions of NK cells against tumor
target cells.
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| Fig 4.
SNP-induced DNA fragmentation in YAC-1 and P815 cells.
The cells were incubated for 18 hours with SNP at indicated doses,
after which extracted DNA was electrophoresed on 1.8% agarose gel as
described in Materials and Methods.
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IL-2-induced IFN- secretion from NK cells is strongly reduced in
the presence of iNOS inhibitor.
NK cells have a dual role as effector in innate resistance and
regulatory in innate resistance, antigen-specific adaptive immunity and
hematopoiesis.37-39 The regulatory functions of NK cells
are probably more dependent on their ability to produce lymphokines,
particularly IFN-, than on their cytotoxic activity.37 We asked whether the regulation of NK cells by iNOS inhibitors involves
IFN- production by these cells. As shown in
Fig 5, stimulation of purified PB-NK and
S-NK with IL-2 resulted in a marked induction of IFN-, which in turn
was significantly reduced (about 40% to 45%) in the presence of AG or
L-NMMA. These results suggest that IL-2-induced NO generation may be
partly induced in the control of IFN- production triggered by this
cytokine.
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| Fig 5.
Effect of iNOS inhibitors on IL-2-induced IFN-
production by PB- and S-NK cells. PB-NK and S-NK cells were incubated
with or without IL-2 for 18 ( ) or 96 hours ( ), respectively, in
the presence or absence of AG (10 µmol/L) or L-NMMA (500 µmol/L).
After incubation, cell-free supernatants were collected and IFN-
levels quantitated by ELISA.
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DISCUSSION |
Our results provide the first direct evidence that in response to IL-2,
both rat PB- and S-CD161 positive NK cells express iNOS at the mRNA and
protein levels. The induction of iNOS expression was more significant
in S-NK cells when compared with PB-NK cells likely due to the longer
IL-2 activation (96 hours v 18 hours, respectively), in
accordance with our previous findings indicating the time-dependent
increasing NOS activity in IL-2-activated NK cells.10 This
event is associated with a significant NO generation, which partially
mediates cytotoxic activity of IL-2-activated-NK cells indicating that
NK cells may cause target cell killing through both NO-dependent and
-independent processes. Indeed, the functional inactivation of iNOS
through AG or L-NMMA or the abrogation of its expression with a
specific antisense oligonucleotide, partially, but significantly,
decreased the cytotoxic activity of IL-2-stimulated NK cells. The
inhibition of cytotoxic activity of IL-2-activated NK cells was not
due to NO-mediated downmodulation of activation receptors (eg, CD25 and
I-A), adhesion molecules (eg, leukocyte function antigen-1 [LFA-1])
or serine esterase expression. Indeed, the expression of these
molecules, which increased (LFA-1 and serine esterase) or were induced
(CD25 and I-A) in IL-2-treated cells when compared with untreated
cells, was not significantly influenced by the presence of iNOS
inhibitors (not shown). A recent study40 reported that
human NK cells are induced to express iNOS after treatment with
IL-12/tumor necrosis factor- (TNF-), but the generated NO appears
to downregulate stimulation of lytic potential induced by these
cytokines rather than to play a role as cytotoxic effector molecule.
This discrepancy may be due to differences in the experimental
conditions related to NK stimulation and/or to species differences.
Consistent with our findings, an impairment of NK killing in mice
genetically lacking iNOS has recently been
shown.17 The genetic deletion or the
functional inactivation of iNOS in Leishmania major-infected
mice causes an extensive parasite spreading and abolishes the NK cell
cytotoxic function as determined in vitro against
51Cr-labeled YAC-1 targets in a 4-hour chromium release assay.
The NO-dependent toxic events may involve both cytolysis or apoptosis
of tumor target cell, as previously suggested.10-14,41 Our
findings indicate that NO may play a role as one of the mediators of
rat IL-2-activated NK cell-mediated DNA fragmentation and lysis of
tumor cells, in accordance with previous evidence indicating the
involvement of NO in these events induced by murine NK
cells.42
The basis of NO-mediated cytotoxicity of IL-2-activated NK cells
against tumor cells could be different (as a review, see Cifone et al41) and include the combination of NO with
metal-containing moieties in key enzymes of the mitochondrial
respiratory chain leading to the inhibition of mitochondrial electron
transport, inhibition of aconitase activity affecting iron metabolism,
and inhibition of ribonucleotide reductase, the rate-limiting step in
DNA synthesis. NO can also damage DNA directly by deamination of purine
and pyrimidine bases resulting in mutations and strand breaks.
NO-mediated DNA damage has been shown to induce p53 expression that
arrests cell cycle progression in G1 allowing for repair of damaged DNA
or induces apoptotic cell death. In addition, NO-induced DNA damage
activates the nuclear poly (adenosine diphosphate [ADP]-ribose) polymerase accompanied by nicotinamide adenine dinucleotide and adenosine triphosphate depletion leading to cell death by energy deprivation.
Other than an effector (cytotoxic) function, we provide evidence that
NO can play a role also in the regulatory functions of NK cells, which
are dependent on their ability to produce lymphokines, particularly
IFN-.37-39 Indeed, the iNOS inhibition through AG or
L-NMMA significantly reduced the IL-2-induced IFN- release, in
accordance with previous evidence showing that iNOS expression in vivo
is essential for the IFN- release by NK cells.17,18 There is considerable evidence from in vitro experiments that iNOS-derived NO can modulate the cytokine response of several cell
types. This might be due to its ability to activate and inactivate ion
channels, G proteins, protein tyrosine kinases, Janus kinases, redox-sensitive kinases, and transcription factors (for
review, see Lander,43 Duhé et
al,44 and Bogdan45). Two recent studies highlight the possibility that NO assumes a similar regulatory function
also in vivo. In a murine model of hemorrhagic shock, in the absence of
iNOS, the activation of NK-kB and Stat3, as well as the expression of
IL-6 and granulocyte colony-stimulating factor (G-CSF) was
significantly reduced in the lung and liver.46 In the mouse
model of cutaneous leishmaniosis, iNOS was previously identified as a
critical antileishmanial mechanism, which was thought to start
operating only when macrophages become activated by IFN--secreting
CD4+ T cells (for review, see Fang47). A recent
study shows that the expression of iNOS is not restricted to the
T-cell-dependent late phase of infection, but is also an important
component of the innate response of the host, where it is focally
induced by IFN-/ within the first 24 hours of
infection.17 In iNOS/ mouse,
there was a 30-fold reduction of the baseline expression of IL-12 p40
mRNA and an almost complete lack of the upregulation of IFN- in the
Leishmania major-infected skin and/or lymph node. The effect of NO on
IFN- production by IL-2-activated NK cells could not be related to
its involvement in the cytotoxic functions of these cells. Indeed,
recent reports indicate that different stimuli, which are not able
alone to trigger natural cytotoxicity, induce IFN- production from
NK cells48,49 (B. Perussia, personal communication, May
1998), thus suggesting that different signaling events are required to
elicit distinct NK cell functions. Tay and Welsh50 reported
that the antiviral effector mechanisms by which NK cells control murine
cytomegalovirus infection in liver, which did not require NK cell
contact with virus-modified target cells, are abrogated by in vivo
administration of the NOS inhibitor, L-NMMA. The NO-dependent IFN-
production may thus represent a relevant mechanism in the NK
cell-mediated resistance against viruses. Furthermore, IFN- is the
main responsible factor for the hematopoiesis-inhibiting activity
exerted by some NK cell subsets (as a review, see
Trinchieri37 and Murphy and Longo51). On the
other hand, several studies are consistent with a role of NO in the
regulation of hematopoiesis.52,53 Thus, the NO-mediated IFN- release by NK cells could also represent a potential mechanism underlying the NK-mediated inhibition of hematopoiesis.
| |
FOOTNOTES |
Submitted September 15, 1998; accepted January 27, 1999.
Supported by grants from Ministero dell'Università e della
Ricerca Scientifica e Tecnologica (MURST 60% and 40%), the Centro Nazionale delle Ricerche, the Associazione Italiana per la Ricerca sul
Cancro, and Comunità Economica ed Europea Contract
BIO4-CT95-0062.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to M. Grazia Cifone, PhD, Department of
Experimental Medicine, Via Vetoio 10, Coppito 2, 67100 L'Aquila,
Italy; e-mail: cifone{at}univaq.it.
| |
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ABSTRACT
INTRODUCTION