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Originally published online as doi:10.1189/jlb.1007701 on March 13, 2008

Published online before print March 13, 2008
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(Journal of Leukocyte Biology. 2008;83:1440-1450.)
© 2008 by Society for Leukocyte Biology

Cytokines secreted by IL-2-activated lymphocytes induce endogenous nitric oxide synthesis and apoptosis in macrophages

Kyoung-Seong Choi*, Eun-Kee Song{dagger} and Chang-Yeol Yim{dagger},1

* Department of Animal Science, Kyungpook National University, Sangju, Republic of Korea; and
{dagger} Department of Internal Medicine, Chonbuk National University Medical School, Research Institute of Clinical Medicine, and Advanced Research Cancer Center, Chonbuk National University Hospital, Chonju, Chonbuk, Republic of Korea

1Correspondence: Department of Internal Medicine, Chonbuk National University Medical School, San 2-20 Geumam-dong, Chonju, Chonbuk 561-712, Republic of Korea. E-mail: cyyim{at}chonbuk.ac.kr


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ABSTRACT
 
IL-2-activated killer (LAK) cells secrete inflammatory cytokines such as IFN-{gamma} and TNF-{alpha}, which can induce NO synthesis (NOS). In this study, we investigated IL-2-activated lymphocyte-mediated macrophage apoptosis via NOS. LAK cells and their culture supernatants induced NOS in murine macrophages. NOS was markedly inhibited by blocking antibodies to IFN-{gamma} and TNF-{alpha}, suggesting the key role of these lymphocyte cytokines in mediating NOS. Endogenous NO production inhibited macrophage proliferation and induced apoptosis in concordance with p53 accumulation and caspase-3 activation, processes that were inhibited by NG-monomethyl-L-arginine (a NOS inhibitor) and 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (a NO scavenger). Our study demonstrated a novel, noncontact-dependent mechanism of macrophage suppression by IL-2-activated lymphocytes: induction of growth inhibition and apoptosis of macrophages as a result of endogenous NOS induced by cytokines secreted from IL-2-activated lymphocytes.

Key Words: programmed cell death • iNOS • carboxy-PTIO • SNAP • p53 • caspase-3


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INTRODUCTION
 
Macrophages play important roles in the regulation of innate and adaptive immune responses via phagocytosis and intracellular digestion of microorganisms, apoptotic cells, and inflammatory byproducts, as well as secretion of cytokines, chemokines, and reactive oxygen and nitrogen intermediates [1 2 3 ]. Stimulation of macrophages with activating cytokines secreted during various immune responses, such as IFN-{gamma}/TNF-{alpha} or IFN-{gamma}/IL-1, results in a strong NO synthesis (NOS), which leads macrophages to be cytotoxic against microorganisms and tumor cells as well as normal cells [4 5 6 7 ].

Apoptosis plays a key role in the maintenance of homeostasis in various physiological systems including the immune system [8 9 10 ]. NO can induce apoptosis in various cells including tumor cells and immune cells [11 12 13 14 15 ]. Prolonged and excessive production of NO might therefore trigger secondary damage to normal tissues. NO-secreting, activated macrophages should revert to a resting state or die to prevent the normal tissue damage [6 , 7 , 16 ]. Activation-induced apoptosis is a mechanism by which immunologically stimulated cells are removed to limit immune-mediated injury of normal tissues and even to promote a state of immunologic tolerance.

IL-2, a key cytokine secreted from activated lymphocytes during immune or inflammatory responses, can trigger a variety of immune cells, such as lymphocytes and macrophages, to release a plethora of inflammatory cytokines, including IFN-{gamma}, TNF-{alpha}, TNF-β, IL-1{alpha}, IL-1β, and IL-6 [15 , 17 , 18 ]. Many of these cytokines have the potential to induce responding cells, for example, macrophages, to synthesize and release high levels of NO from L-arginine via the action of an enzyme termed inducible nitric oxide synthase (iNOS) [19 20 21 ]. Although many investigators have studied mutual activation between lymphocytes and macrophages, to our best knowledge, none has been reported about the potential for activated lymphocytes to cause apoptosis of macrophages via induction of NOS.

In the present study, we hypothesized that cytokines secreted from IL-2-activated lymphocytes might directly induce endogenous NOS in macrophages, resulting in the activation of apoptosis pathways in macrophages themselves. To better understand this novel mechanism of activated lymphocyte-induced macrophage apoptosis, we examined the production of important cytokines in IL-2-activated killer (LAK)-conditioned medium and their relationship to the induction of NOS and apoptosis in macrophages using lymphocyte/macrophage cocultures stimulated with IL-2 as an in vitro model of immune activation-induced apoptosis.


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MATERIALS AND METHODS
 
Animals
Specific pathogen-free BALB/c and C57BL/6J (B6) mice (6–8 weeks old) were purchased from Daehan Experimental Animal Center (Daejon, Korea). IFN-{gamma} knockout mice (B6; 129S7-Ifngtm1Ts/J) and iNOS knockout mice (B6; 129P2-Nox2tm1Lau/J) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). All mice were maintained and used in strict accordance with the guidelines issued by the Chonbuk National University Hospital Animal Care Committee (Korea), which also approved experimental protocols. All experiments were performed at least three times with highly concordant results. All experiments were performed using BALB/c mice, except the experiments using IFN-{gamma} or iNOS knockout B6 mice. Two to five mice were used in each experiment.

Macrophages
Peritoneal macrophages were obtained from mice by peritoneal lavage 4 days after injection with 2 ml sterile 3% thioglycollate broth (Difco Laboratories, Detroit, MI, USA). Cells were washed and resuspended in RPMI 1640 medium with 5% FCS (HyClone Laboratories, Logan, UT, USA), 100 U/ml penicillin (Sigma Chemical Co., St. Louis, MO, USA), 50 µg/ml streptomycin (Sigma Chemical Co.), and 2 mM L-glutamine (Sigma Chemical Co.; working medium), which were then plated at 2 x 106 cells/ml and incubated for 2 h. Adherent cells were washed with sterile PBS, harvested using a cell scraper (Baxter Health Care Corp., McGraw Park, IL, USA), and resuspended at 2 x 106 cells/ml in working medium for further experiments.

Lymphocyte culture and LAK-conditioned medium
Plastic adherent macrophage-depleted splenocytes were used as a source of lymphocytes. Spleens from mice were prepared by gentle teasing to obtain single cell suspensions. Cells were washed and incubated in Tris-buffered 0.16 M ammonium chloride to remove erythrocytes, and then the remaining cells were washed and resuspended at 2 x 106 cells/ml in working medium. Splenocytes (2x106/ml) were cultured in the presence of 6000 IU/ml recombinant human IL-2 (a generous gift from Chiron, Emeryville, CA, USA; specific activity, 1.8x107 IU/mg) in RPMI-1640 medium with 10% FCS, 100 U/ml penicillin G, 50 µg/ml streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate (Sigma Chemical Co.), and 0.1 mM nonessential amino acids (Sigma Chemical Co.; LAK medium). Following a 4-day culture, the supernatant was harvested and mixed with fresh medium at the ratios shown (maximum, 50% LAK-conditioned medium). LAK-conditioned medium was added to cultures of macrophages to induce NOS. In some experiments, a NOS inhibitor, NG-monomethyl-L-arginine (MLA), or neutralizing anticytokine mAb [anti-mouse IFN-{gamma} mAb (clone XMG1.2, BD PharMingen, San Jose, CA, USA), anti-mouse TNF-{alpha} mAb (clone MP6-XT22, BD PharMingen), anti-mouse IL-1 mAb (clone 1837-01, Genzyme, Cambridge, MA, USA)] and control rat IgG (Sigma Chemical Co.) and hamster IgG (Sigma Chemical Co.) were added. All reagents and media for tissue culture experiments were tested by Limulus amoebocyte lysate assay (detection limit, 10 pg/ml; Whittaker Bioproducts, Walkersville, MD, USA) to exclude LPS contamination.

Cytokine assay
Cytokine levels in the supernatant were determined using murine (m)IFN-{gamma} (Endogen, Woburn, MA, USA), mTNF-{alpha} (BioSource, Camarillo, CA, USA), and mIL-1 (Endogen) ELISA kits, according to the manufacturer’s recommendation. Briefly, standard or samples were dispensed into a 96-well microtiter plate and incubated at 37°C for 2 h. Plates were then washed and reacted with a HRP-conjugated goat polyclonal anti-mouse cytokine antibody at 37°C for 1 h. After washing, captured cytokines were detected by addition of substrate solution. The OD at 450 nm was determined using a microtiter plate reader (THERMOmax, Molecular Devices Corp., Sunnyvale, CA, USA).

Macrophage proliferation assay
Macrophages (2x106 cells/ml) were cultured in 50% LAK-conditioned medium in the presence of varying concentrations of NOS inhibitor MLA. After a 32-h incubation, cells were pulsed with 0.5 µCi/well tritiated TdR ([3H]TdR; 2.0 Ci/mmol, Du Pont, Boston, MA, USA) over an additional 18 h and then harvested onto glass fiber filters using a cell harvester (Cambridge Technology, Cambridge, MA, USA). Samples were suspended in Optifluor® scintillation fluid (Packard Instrument Co., Downers Grove, IL, USA), and the incorporated [3H]TdR was measured in a Packard Tri-Carb 1500R scintillation counter (Packard Instrument Co.). Each assay was performed in triplicate, and the results were presented as mean ± SD cpm.

Separation of lymphocyte subtypes
To separate CD4+ T cells, CD8+ T cells, and CD49b+/CD3– NK cells, splenic lymphocytes were stained with FITC-conjugated anti-mouse CD4 (L3T4; clone 129.19, BD PharMingen), PE-conjugated anti-mouse CD8a (Ly-2; clone 53-6.7, BD PharMingen), FITC-conjugated anti-mouse CD49b (clone DX5, BD PharMingen), and PE-conjugated anti-mouse CD3 (BD PharMingen) for 30 min on ice and resuspended in PBS. Stained cells were sorted with a FACSVantage SE cell sorter system (Becton Dickinson, San Jose, CA, USA). Sorted cells were > 95% pure, as determined by post-sort analysis.

Apoptosis detection by TUNEL assay and annexin-V staining
Macrophages were cultured in six-well plates at 2 x 106 cells/ml in the presence or absence of 50% LAK-conditioned medium, S-nitro-N-acetylpenicillamine (SNAP; NO donor), 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide, sodium salt (carboxy-PTIO; NO scavenger), or MLA (NOS inhibitor). A TUNEL assay (Fluorescein-Frag EL DNA fragmentation detection kit, Oncogene Research Products, Boston, MA, USA) was used to detect apoptotic cell death by enzymatic labeling of DNA strand breaks with fluorescein-terminal deoxynucleotidyl transferase (TdT) [22 ]. Briefly, cells were fixed and permeabilized in 4% formaldehyde in PBS for 10 min at room temperature, resuspended in 80% ethanol, and then stored at 4°C. After fixation, cells were rehydrated with TBS and suspended in permeabilization solution (proteinase K in 10 mM Tris) for 5 min at room temperature. To inactivate endogenous peroxidase, cells were preincubated with 100 µl 3% H2O2 for 5 min. After rinsing with TBS, cells were incubated with 100 µl TdT equilibration buffer for 30 min. The buffer was then replaced with 60 µl TdT labeling reaction mixture, and plates were incubated at 37°C for 1.5 h and rinsed with TBS. The reaction was stopped with 0.5 M EDTA, pH 8.0, and blocked with blocking buffer (4% BSA in TBS) at room temperature for 10 min. The buffer was aspirated, and 100 µl 0.1 mg/ml ABTS and 0.03% H2O2 were added. After 20 min, absorbance was measured at 410 nm using an MR 700R plate reader (Dynatech, Alexandria, VA, USA). A positive control was prepared by preincubating cells with 1 µl/ml DNase I in TBS containing 1 mM MgSO4 for 20 min. The data were expressed as a percentage of the control (mean±SD).

For flow cytometric detection of annexin-V staining, macrophages were harvested, washed in binding buffer, and stained with annexin-V FITC, according to the manufacturer’s recommendations (Oncogene Research Products). Cells stained with annexin-V FITC were identified and quantitated by flow cytometry. Data acquisition and analysis were performed with the CellQuest software (BD PharMingen).

Immunoblot analyses
Cells were pelleted, resuspended in lysis buffer (Pierce, Rockford, IL, USA), and assayed for protein content (bicinchoninic acid protein assay, Pierce). Protein samples (cell lysates) were resolved in 5–12% SDS-PAGE, transferred to nitrocellulose membranes, and then washed in a blocking solution containing 5% nonfat dry milk in a TBS-Tween 20 (Fisher Biotech, Fair Lawn, NJ, USA). After blocking, the membranes were incubated with anti-iNOS (BD Transduction Laboratories, Lexington, KY, USA), anti-p53 (clone PAb 122, BD PharMingen), anti-caspase-3/CPP32 (procaspase-3, BD Transduction Laboratory), and anti-actin (Sigma Chemical Co.) antibodies. The nitrocellulose blots were then washed and incubated with alkaline phosphatase-conjugated secondary antibody to mouse or rabbit IgG (KPL, Gaithersburg, MD, USA). Bands were detected using ECL (Bio-Rad, Hercules, CA, USA) and exposure film (Pierce). Equal loadings of protein in lanes were confirmed in all experiments by stripping and reprobing the blots for actin.

Caspase-3 colorimetric activity assay
A fluorometric immunosorbent enzyme assay (Roche Diagnostics, Switzerland) was performed, according to the manufacturer’s instruction [23 ]. Briefly, cells were washed, resuspended in lysis buffer, incubated on ice, and centrifuged, and supernatants were harvested. Anticaspase-3 solution was added into a 96-well microtiter plate and incubated at 37°C for 1 h. After washing the plate, substrate (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethyl coumarin) was added and incubated for 2 h, and absorbance was measured at 405 nm in a microtiter plate reader (THERMOmax).

Measurement of nitrite
Nitrite production was assayed by a modification of the colorimetric Griess reaction [24 ]. Briefly, 50 µl samples of cell culture supernatants were added to flat-bottomed microtiter plates and incubated with 100 µl of a 1:1 mixture of 1% sulfanilamide in 30% acetic acid and 0.1% N-(1-Naphthyl) ethylenediamine dihydrochloride in 60% acetic acid. Sample absorbance at 570 nm was measured using a microtiter plate reader (THERMOmax). Concentrations were determined from a linear standard curve generated from 6.25–100 µM sodium nitrite in LAK medium. Results were presented as mean ± SD. LAK medium contained <0.5 µM nitrite.

Statistical analysis
Statistical analysis was performed using Student’s t-test.


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RESULTS
 
IL-2 induces NOS in cocultured lymphocytes and macrophages
We tested whether IL-2 had the ability to induce NOS in cocultures of lymphocytes and macrophages. Lymphocytes (2x106 cells/ml) alone, macrophages alone (2x106 cells/ml), and lymphocyte/macrophage cocultures were cultured with IL-2 (6000 IU/ml) in LAK medium in a 96-well microtiter plate (in triplicate). Following a 4-day culture, nitrite accumulation (a metabolite of NO) in culture supernatants was measured (expressed as mean±SD). Nitrite synthesis was markedly increased when IL-2 was added to lymphocyte/macrophage cocultures (42.5±1.8 µM; Fig. 1 ). Lymphocyte and macrophage cocultures in the absence of IL-2 produced little nitrite (<3.0 µM). Lymphocytes alone or macrophages alone also synthesized little nitrite (<3.0 µM), with or without IL-2 added to the culture medium. The latter result indicated that IL-2 did not directly induce NOS in either cell type.


Figure 1
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  		Figure 1. IL-2-induced nitrite production in lymphocyte/macrophage cocultures. Lymphocytes alone, macrophages alone, and lymphocytes/macrophages were cultured with IL-2 (6000 IU/ml). Cells cultured without IL-2 served as controls. Following a 4 day culture, nitrite was measured in culture supernatants. The results were expressed as mean ± SD of triplicate samples.

Role of soluble mediators in regulating NOS by macrophages
To examine the role of soluble mediators (e.g., cytokines) in regulating NOS, lymphocytes alone (2x106 cells/ml) were cultured with IL-2 (6000 IU/ml) for 96 h to generate LAK-conditioned medium. Serial dilutions of conditioned medium (diluted in fresh medium) were added to macrophages (2x106 cells/ml). After 48 h, nitrite was measured in the culture supernatants as a reflection of NOS. Cultures without conditioned medium served as a control. When LAK-conditioned medium was added to macrophages, nitrite production was induced in a dose-dependent manner (Fig. 2A ). The highest concentration of conditioned medium (50%) induced 37.5 ± 1.5 µM nitrite accumulations. In contrast, the addition of an equivalent volume of fresh medium with IL-2 did not increase nitrite production by macrophages (2.2±0.0 µM).


Figure 2
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  		Figure 2. Induction of NOS in macrophages by lymphocyte LAK-conditioned medium, which was prepared by harvesting supernatants from lymphocyte cultures incubated with IL-2 for 4 days. Serial dilutions (0–50% v:v) of conditioned medium with fresh medium were added to macrophages. After 48 h, nitrite was measured in culture supernatants by a colorimetric assay (A). The result was expressed as mean ± SD of triplicate samples. In a separate experiment, conditioned media were harvested at 24, 48, 72, and 96 h following the addition of IL-2 to lymphocytes. The conditioned media were then admixed 1:1 (v:v) with fresh medium and added to macrophages. After 48 h, nitrite was measured in culture supernatants (B).

In the second experiment, conditioned medium was harvested from LAK cell cultures at 24, 48, 72, and 96 h. After admixture of 50% fresh medium, samples from each time-point were evaluated for their ability to induce NOS in macrophages as described above. This experiment demonstrated that incubation of LAK cells with IL-2 for increasing lengths of time resulted in a progressive increase in the capacity of conditioned medium to elicit NOS (Fig. 2B) . These results suggested that mediators or cytokines secreted by LAK cells into the conditioned medium were the inductive signal for NO production, and macrophages appeared to be the responding cell population.

Evaluation of IFN-{gamma}, TNF-{alpha}, and IL-1 as candidate cytokines responsible for NOS
Macrophages (2x106 cells/ml) were cultured in a 96-well microtiter plate (in triplicate wells) containing 50% LAK-conditioned medium, with or without blocking antibodies against IFN-{gamma}, TNF-{alpha}, and IL-1 (final volume, 0.2 ml/well). Rat IgG and hamster IgG served as controls for the blocking antibodies. After a 48-h incubation, nitrite was measured in culture supernatants as a reflection of NOS. Addition of anti-IFN-{gamma} antibody or anti-TNF-{alpha} antibody inhibited nitrite production by macrophages (>70% and >50%, respectively; Fig. 3A ). In contrast, anti-IL-1 antibody and control IgGs had little effect.


Figure 3
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  		Figure 3. Role of cytokines secreted by IL-2-activated lymphocytes in macrophage NOS. Macrophages were cultured in the presence of 50% LAK-conditioned medium (from lymphocyte cultures incubated with IL-2 for 4 days). Serial dilutions of blocking antibodies directed against mouse IFN-{gamma}, TNF-{alpha}, and IL-1 were added to parallel cultures. Cultures incubated with rat and hamster IgGs served as controls. After 48 h, nitrite was measured by a colorimetric assay (mean±SD of triplicate samples; A). Cytokine concentrations (IFN-{gamma}, TNF-{alpha}, and IL-1) were also measured in the conditioned media (mean±SD of triplicate samples) harvested at 24, 48, 72, and 96 h following the addition of IL-2 to lymphocytes (B). Macrophages were also incubated with 50% conditioned media harvested from IFN-{gamma} knockout LAK cell cultures. After a 48 h incubation, nitrite was measured in culture supernatants (mean±SD of triplicate samples). Macrophages incubated with the conditioned media from normal control LAK cell cultures served as a control (C).

Cytokine levels were analyzed in the conditioned media harvested from LAK cell cultures at 0, 24, 48, 72, and 96 h. This experiment demonstrated that incubation of LAK cells with IL-2 for increasing lengths of time resulted in a progressive increase in the levels of IFN-{gamma}, TNF-{alpha}, and IL-1 in the conditioned media (Fig. 3B) .

IFN-{gamma} is a well-known priming cytokine to induce NOS during macrophage activation [25 , 26 ]. To further evaluate whether IFN-{gamma} is a major inductive signal present in the conditioned media to induce macrophage NOS, macrophages were incubated with 50% conditioned media of LAK cell cultures originated from IFN-{gamma} knockout mice. After a 48-h incubation, nitrite was measured in culture supernatants. Macrophage cultures incubated with the conditioned media of LAK cell cultures from normal mice served as a control. In the presence of the conditioned media from IFN-{gamma} knockout mice, macrophages produced little nitrite (1.1±0.1 µM; Fig. 3C ). In contrast, control macrophage cultures produced a large amount of nitrite (34.9±1.3 µM). The above three experimental results indicated that IFN-{gamma} is a major inductive signal present in LAK-conditioned media during the induction of macrophage NOS.

Identification of lymphocyte subsets that induce NOS
To examine cells types in the LAK cell population that contributed to cytokine production, CD4+ T cells, CD8+ T cells, and CD49b+/CD3– NK cells were enriched (>95% homogeneity) by positive selection prior to culture in IL-2-containing medium. After a 4-day incubation with IL-2, culture supernatants were harvested and then tested to establish whether they retained the capacity to induce nitrite production by macrophages. CD4+, CD8+, and CD49b+/CD3– cells all retained the capacity to stimulate nitrite synthesis in macrophages (Fig. 4A ), demonstrating that this mechanism was not restricted to the CD49b+/CD3– NK cells, although these cells are thought to represent the predominant source of cytotoxic activity against target cells during IL-2-induced activation of lymphocytes [27 ].


Figure 4
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  		Figure 4. Identification of subpopulations of IL-2-activated lymphocytes contributing to cytokine secretion. CD4+, CD8+, and CD49b+/CD3– cells were enriched using positive selection techniques. Following a 4 day incubation at 2 x 106 cells/ml with IL-2 (6000 IU/ml), conditioned media were harvested. Macrophages were cultured in the presence of each 50% conditioned medium. After 48 h, nitrite accumulation was measured by a colorimetric assay. Conditioned media from unfractionated lymphocytes cultured in the presence or absence of IL-2 served as positive and negative controls, respectively. Data were presented as mean ± SD of triplicate samples (A). Cytokine concentrations (IFN-{gamma}, TNF-{alpha}, and IL-1) were also measured in the conditioned media harvested from cultures of CD4+, CD8+, and CD49b+/CD3– cells (mean±SD of triplicate samples) incubated in the presence of IL-2 for 4 days (B).

Cytokine levels were also analyzed in the conditioned media harvested from cultures of CD4+, CD8+, and CD49b+/CD3– cells incubated in the presence of IL-2 for 4 days. All the evaluated CD4+, CD8+, and CD49b+/CD3– cells retained the capacity to produce IFN-{gamma}, TNF-{alpha}, and IL-1 (Fig. 4B) .

iNOS induction in macrophages by LAK-conditioned medium
The induction of iNOS protein expression in macrophages by LAK-conditioned medium was evaluated. Macrophages (2x106 cells/ml) were activated by a 2-day exposure to 50% LAK-conditioned medium. Activation of NOS was confirmed by nitrite assay of culture supernatants. Macrophages were harvested and evaluated for iNOS expression by an immunoblot analysis. Parallel control cultures were macrophages incubated with IFN-{gamma} (10 U/ml) and LPS (50 ng/ml; positive control), macrophages incubated with 50% culture supernatants from IL-2-activated macrophages, lymphocytes incubated with 50% LAK-conditioned medium, and lymphocytes incubated with 50% culture supernatants from IL-2-activated macrophages.

In the presence of LAK-conditioned medium, cultures of macrophages produced a large amount of nitrite (38 µM), and these populations strongly expressed iNOS protein (Fig. 5A and 5B ). The positive control cultures also produced a markedly increased amount of nitrite (117 µM) and strongly expressed the iNOS protein. Other control cultures produced little nitrite (<3.0 µM), and iNOS protein expression could not be detected in these cells.


Figure 5
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  		Figure 5. LAK-conditioned medium induced iNOS protein expression in macrophages. Two kinds of conditioned media were prepared by harvesting supernatants from cultures of lymphocytes or macrophages incubated with IL-2 for 4 days. Each conditioned medium was admixed 1:1 (v:v) with fresh medium and added to fresh lymphocytes or macrophages. After 48 h, nitrite was measured in culture supernatants by a colorimetric assay (A), and iNOS protein expression was analyzed in cells by Western blot (B). Macrophages cultured with IFN-{gamma} (10 U/ml) and LPS (50 ng/ml; positive control, lane 1); macrophages cultured with LAK-conditioned medium (lane 2); macrophages cultured with conditioned medium from IL-2-activated macrophages (lane 3); lymphocytes cultured with LAK-conditioned medium (lane 4); lymphocytes cultured with conditioned medium from IL-2-activated macrophages (lane 5).

Effect of NO on macrophage proliferation
One of the major effects of NO on various cells is the inhibition of ribonucleotide reductase, a rate-limiting step in DNA synthesis, resulting in inhibition of proliferation [28 29 30 ]. We tested whether endogenous NOS was capable of inhibiting macrophage proliferation. Macrophages were cultured with increasing concentrations (0–500 µM) of MLA in 96-well microtiter plates in the presence of 50% LAK-conditioned medium. Thymidine incorporation increased in a dose-dependent manner in the presence of increasing concentrations of MLA (Fig. 6A ). The increase in thymidine incorporation was reciprocally related to levels of nitrite detected in aliquots of medium sampled from the same microtiter wells (Fig. 6B) .


Figure 6
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  		Figure 6. Antiproliferative effects of endogenous NOS in macrophages, which were cultured with 50% LAK-conditioned medium in a 96 well microtiter plate for 32 h in the presence of varying concentrations of MLA. Each well was pulsed with tritiated thymidine for an additional 18 h. Thymidine incorporation was analyzed by scintillation counting (A). Nitrite levels in culture supernatants from parallel wells were analyzed by a colorimetric assay (B). The results were expressed as mean ± SD of triplicate wells.

Effects of endogenous NO on macrophage apoptosis
In addition to producing metabolic inhibition, NO can induce various cells to undergo programmed cell death via p53-dependent and -independent pathways [31 ]. To test whether endogenous NO secretion in macrophages could trigger apoptosis, cells (2x106 cells/ml) were cultured with 50% LAK-conditioned medium and increasing concentrations (0–125 µM) of MLA. Following a 48-h incubation, cells were analyzed for programmed cell death using annexin-V-FITC staining. Nitrite synthesis was also measured in cell culture supernatants. Macrophages cultured without LAK-conditioned medium served as a control. Compared with control cultures, an increase in apoptotic cells was observed in cultures incubated with conditioned medium (Fig. 7A ). Addition of MLA to cultures decreased apoptosis in a dose-dependent manner corresponding to the inhibition of nitrite synthesis (Fig. 7B) . Control cultures did not produce a significant amount of nitrite.


Figure 7
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  		Figure 7. Induction of apoptosis in macrophages by endogenous NOS. Macrophages were cultured with 50% LAK-conditioned medium in the presence of varying concentrations of MLA. After 48 h, cells were analyzed for apoptosis using annexin-V FITC staining (A). Cells cultured without conditioned medium served as a negative control. Accumulation of nitrite was measured in supernatants from parallel cultures (B). Apoptosis was also measured by a TdT Frag EL DNA fragmentation detection kit (C), and nitrite was also measured in the culture supernatants from parallel cultures (D). In a separate experiment, conditioned media were harvested at 24, 48, 72, and 96 h following the addition of IL-2 to lymphocytes. The conditioned media were then admixed 1:1 (v:v) with fresh medium and added to macrophages. After 48 h, apoptosis was also measured by a TdT Frag EL DNA fragmentation detection kit (E). The results were expressed as mean ± SD of triplicate samples.

We also measured macrophage apoptosis using the TdT Frag EL DNA fragmentation detection (TUNEL assay) kit in another set of experiments similar to the above. The TUNEL assay detects DNA fragmentation, a late event during apoptosis [22 ]. Addition of MLA also decreased apoptotic cells and nitrite synthesis in macrophage cultures incubated with LAK-conditioned medium (Fig. 7C and 7D) . In a separate experiment, 50% conditioned media harvested from LAK cell cultures at 24, 48, 72, and 96 h were evaluated for their ability to induce nitrite synthesis and apoptosis in macrophages. This experiment demonstrated that incubation of lymphocytes with IL-2 for increasing lengths of time resulted in a progressive increase in the capacity of conditioned media to elicit nitrite synthesis (data not shown; refer to Fig. 2B ) and apoptosis (Fig. 7E) .

In addition, we tested the effect of a NO scavenger, carboxy-PTIO on apoptosis using the TdT Frag EL DNA fragmentation detection kit in macrophage cultures incubated with LAK-conditioned medium. Addition of carboxy-PTIO decreased apoptotic cells in a concentration-dependent manner (Fig. 8 ).


Figure 8
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  		Figure 8. Effect of carboxy-PTIO on macrophage apoptosis induced by endogenous NOS. Macrophages were cultured with 50% LAK-conditioned medium in the presence of varying concentrations of carboxy-PTIO. After 48 h, cells were analyzed for apoptosis using a TdT Frag EL DNA fragmentation detection kit. Results were expressed as mean ± SD of triplicate samples.

We also investigated the role of iNOS in LAK-conditioned, medium-induced apoptosis using peritoneal macrophages from iNOS knockout mice. Following a 48-h incubation of macrophages with or without 50% LAK-conditioned medium, cells were analyzed for programmed cell death using the TUNEL assay. Nitrite synthesis was also measured in cell culture supernatants. Macrophages from normal control mice cultured with or without LAK-conditioned medium served as controls. Addition of LAK-conditioned medium increased apoptotic cells and nitrite production in cultures of normal control macrophages (Fig. 9A and 9B ). In contrast, cultures of iNOS knockout macrophages did not increase any significant amounts of apoptosis or nitrite in the presence of LAK-conditioned medium. These results indicate a key role of the macrophage iNOS-derived NO in the LAK-conditioned media-induced apoptosis.


Figure 9
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  		Figure 9. The role of iNOS in LAK-conditioned media-induced macrophage apoptosis. Following a 48 h incubation of macrophages harvested from iNOS knockout mice with or without 50% LAK-conditioned medium, cells were analyzed for programmed cell death using the TUNEL assay (A). Nitrite synthesis was measured in cell culture supernatants (B). Macrophages from normal control mice cultured with or without LAK-conditioned medium served as controls. The results were expressed as mean ± SD of triplicate samples.

Effects of exogenous NO on macrophage apoptosis
To test whether exogenous NO has the apoptosis-inducing activity similar to endogenous NO, the effect of direct exposure of macrophages to a NO donor, SNAP on apoptosis was examined using annexin-V staining and TUNEL assays. Addition of SNAP was demonstrated to induce a dose-dependent increase in apoptosis of macrophage cultures (Fig. 10A and 10B ).


Figure 10
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  		Figure 10. Effect of SNAP on macrophage apoptosis. Macrophages were cultured in the presence of varying concentrations of SNAP. After 48 h, cells were analyzed for apoptosis using annexin-V FITC staining (A) and a TdT Frag DNA fragmentation detection kit (B). The results were expressed as mean ± SD of triplicate samples.

Expression of p53 protein in macrophages induced by endogenous NO production
p53 suppresses cell growth through two mechanisms: cell-cycle arrest and apoptosis [32 , 33 ]. We next tested whether endogenous NO induces p53 expression in macrophages, which were cultured with 50% LAK-conditioned medium in the presence or absence of 500 µM MLA. After 48h, cells were analyzed for p53 protein expression using Western blot. Accumulation of nitrite was measured in supernatants from parallel cultures. Macrophages cultured without conditioned medium served as a negative control. This experiment demonstrated that addition of LAK-conditioned medium induced p53 expression and nitrite production in macrophage cultures (Fig. 11A and 11B ). Addition of MLA to cultures decreased LAK-conditioned medium-induced p53 expression and nitrite synthesis.


Figure 11
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  		Figure 11. Expression of p53 protein in macrophages by endogenous NO production. Macrophages were cultured with 50% LAK-conditioned medium in the presence or absence of 500 µM MLA. After 48 h, cells were analyzed for p53 protein expression using Western blot (A). Accumulation of nitrite was measured in culture supernatants from parallel cultures (mean±SD of triplicate samples; B). Macrophages cultured without conditioned medium served as a negative control.

Activation of caspase-3 in macrophages induced by endogenous NO production
Caspase-3 is a key enzyme involved in spontaneous apoptosis that reflects the endpoint of activation via mitochondrial and nonmitochondrial pathways. Activation of caspase-3 can be analyzed indirectly by protein immunoblotting that detects proteolytic cleavage of procaspase-3 [34 ]. We also tested whether endogenous NO induces caspase-3 activation in macrophages, which were cultured with 50% LAK-conditioned medium in the presence of varying concentrations of MLA. After 48 h, cells were analyzed for caspase-3 cleavage using Western blots for the 32-kDa precursor of caspase-3. Accumulation of nitrite was measured in culture supernatants. Active forms of caspase-3 were also assayed using a caspase-3 activity assay kit. Addition of 50% LAK-conditioned medium to macrophage cultures was demonstrated to decrease the amount of the precursor form of caspase-3 and to increase nitrite synthesis and caspase-3 activity (Fig. 12A 12B 12C , respectively). In contrast, addition of MLA abrogated the LAK-conditioned medium-induced decrease in precursor forms of caspase-3, and decreased nitrite synthesis and caspase-3 activity in a concentration-dependent manner.


Figure 12
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  		Figure 12. Activation of caspase-3 in macrophages by endogenous NO production. Macrophages were cultured with 50% LAK-conditioned medium in the presence of varying concentrations of MLA. Cells were analyzed for caspase-3 cleavage using Western blot after 48 h (A). Accumulation of nitrite was measured in culture supernatants from parallel cultures (mean±SD of triplicate samples; B). Caspase-3 activity was analyzed by a fluorescence-based enzyme assay (mean±SD of triplicate samples; C).


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DISCUSSION
 
This study demonstrated a novel mechanism of IL-2-activated lymphocyte-induced macrophage apoptosis. Cytokines secreted from IL-2-activated lymphocytes directly induced endogenous NOS in macrophages, resulting in the activation of programmed cell death pathways of macrophages themselves.

NO produced by activated macrophages is cytotoxic, not only to cancer cells, but also to normal cells [6 , 7 , 35 ]. Overproduction and prolonged production of NO might therefore trigger secondary damage in normal tissues. To prevent tissue damage, activated macrophages should revert to a resting state or undergo apoptosis. In this study, we investigated mechanisms of NO production and NO-induced apoptosis of macrophages themselves during immune stimulation by using lymphocyte/macrophage cocultures stimulated with IL-2 as an in vitro model of immune activation-induced apoptosis.

Immune responses are mediated by interactions among a variety of effector cells including T cells, B cells, NK cells, macrophages, dendritic cells, granulocytes, and even epithelial cells and by the soluble molecules, which they secrete [36 ]. We therefore used lymphocyte/macrophage cocultures rather than cultures of a specific subset of immune cells to evaluate the activation-induced apoptosis. IL-2, a key cytokine secreted from lymphocytes during immune responses, can induce T cell activation and proliferation, B cell growth, NK cell activation and proliferation, and enhanced monocyte/macrophage cytolytic activity and trigger release of a plethora of inflammatory cytokines, including IFN-{gamma}, TNF-{alpha}, TNF-β, IL-1{alpha}, IL-1β, and IL-6 [15 , 17 , 18 ]. Many of these cytokines are known to induce iNOS expression in macrophages [15 , 37 38 39 ]. We therefore used IL-2 as an inductive signal of immune activation followed by activation-induced apoptosis mediated by NO.

We first hypothesized that IL-2-activated lymphocytes (LAK cells) could serve as sources of cytokines, such as IFN-{gamma}, TNF-{alpha}, and IL-1, which could in turn activate NOS in macrophages [15 , 37 38 39 ]. To test this concept, splenic lymphocytes and peritoneal exudate macrophages were cocultured in the presence of IL-2. This resulted in substantial nitrite production, consistent with activation of NOS. IL-2 exposure of lymphocyte or macrophage cultures individually did not result in nitrite production. To evaluate whether soluble mediators, such as cytokines, were involved in the induction of NOS, we added serial dilutions of LAK-conditioned medium to macrophages. This experiment demonstrated that LAK-conditioned medium induced a significant nitrite synthesis by macrophages. In contrast, nitrite production by lymphocyte cultures was not detectable following exposure to IL-2-activated, macrophage-conditioned medium. This result indicated that LAK cells act as the major source of soluble mediators (e.g., cytokines), and macrophages are the population producing NO in response to those mediators.

To further establish the nature of the inductive signals present in LAK-conditioned medium, we added serial dilutions of blocking antibodies to IFN-{gamma}, TNF-{alpha}, and IL-1 to macrophage cultures. Monoclonal antibodies to IFN-{gamma} and TNF-{alpha} strongly decreased nitrite production, suggesting a central role for these LAK cell-derived cytokines in the induction of NOS in macrophages. Increases in IFN-{gamma} and TNF-{alpha} were also confirmed by cytokine assays of the LAK-conditioned media. The key role of IFN-{gamma} in the synthesis of NO in macrophages was also supported by the observation that LAK-conditioned media derived from IFN-{gamma}-deficient mice did not produce NOS. However, it remains to be clarified whether the in vitro-measured concentrations of IFN-{gamma} and TNF-{alpha} in the LAK-conditioned media are physiologically relevant for macrophage function. An in vivo close physical contact may be essential between lymphocytes and macrophages to interact through delivery of the cytokines at high local concentrations. Further confirmation that macrophages were the source of NO was derived from identification of iNOS protein expression following exposure to LAK-conditioned medium. This finding was also supported by the observation that LAK-conditioned media did not induce nitric oxide synthase in macrophages of iNOS-deficient mice.

The observation that MLA is a potent, competitive inhibitor of all NOS isoforms provided an additional experimental tool to differentiate effector functions mediated by the L-arginine: NO pathway [6 , 15 , 16 ]. The addition of MLA to macrophage cultures in the presence of LAK-conditioned medium inhibited nitrite production in a dose-dependent manner, confirming the derivation of nitrite from the L-arginine: NO pathway. Endogenously synthesized NO decreased the thymidine incorporation in macrophage cultures. In the presence of increased concentrations of MLA, proliferation increased in an inverse relationship to nitrite levels. This experiment demonstrated that endogenous NOS in macrophages correlated with antiproliferative effects.

A number of recent studies implicated NO in the induction of programmed cell death in various cells [11 , 13 , 40 41 42 ]. Our current data demonstrated that endogenous NOS in macrophages induced by LAK-conditioned medium caused programmed cell death of macrophages themselves. The apoptosis-inducing effects of LAK-conditioned medium-induced NO production were inhibited in a dose-dependent manner by MLA and carboxy-PTIO (a NOS inhibitor and a NO scavenger, respectively). Furthermore, levels of apoptosis induced by LAK-conditioned medium were in the range of those triggered by high concentrations of a chemical NO donor (SNAP). These results indicated that endogenous NOS in macrophages is a potential mechanism of macrophage apoptosis induced by IL-2-activated lymphocytes. These findings were further supported by the observation that LAK-conditioned media-induced apoptosis was abrogated in macrophages of iNOS-deficient mice. Endogenous NOS induced by LAK-conditioned medium caused p53 expression and caspase-3 activation in macrophages.

The role of NO in signaling apoptotic pathways in leukocyte subsets is still being clarified. NO synthesized in cocultures of lymphocytes/macrophages in the presence of IL-2 could have the potential to induce apoptosis of lymphocytes as well as macrophages [14 ]. A mechanism of mutual inhibition might therefore exist between cytokine-secreting lymphocytes and NO-producing macrophages, contributing to immunologic deactivation and even to a state of immunologic tolerance. A number of recent studies have also implicated NO in the induction of programmed cell death in murine macrophages [43 ] and tumor cells [5 , 44 ], as well as during depletion of double-positive (CD4+/CD8+) thymocytes in the murine thymus [45 ].

Our study has identified a noncontact-mediated mechanism of apoptosis of macrophages induced by lymphocytes during IL-2 stimulation. Inflammatory cytokines, such as IFN-{gamma} and TNF-{alpha}, secreted by IL-2-activated lymphocytes, are capable of inducing NOS in macrophages, resulting in growth arrest and apoptosis. Further investigation might address signaling pathways that mediate this immune activation-induced macrophage apoptosis.


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ACKNOWLEDGEMENTS
 
This study was supported by a grant from the National R&D Program for Cancer Control, Ministry of Health and Welfare, Republic of Korea (0620220-1), and by funds of Chonbuk National University Hospital Research Institute of Clinical Medicine.

Received October 21, 2007; revised February 15, 2008; accepted February 18, 2008.


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