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Originally published online as doi:10.1189/jlb.0106012 on July 19, 2006

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(Journal of Leukocyte Biology. 2006;80:744-752.)
© 2006 by Society for Leukocyte Biology

A suppressive role of nitric oxide in MIP-2 production by macrophages upon coculturing with apoptotic cells

Takehiko Shibata, Kisaburo Nagata and Yoshiro Kobayashi1

Department of Biomolecular Science, Faculty of Science, Toho University, Funabashi, Chiba, Japan

1 Correspondence: Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. E-mail: yoshiro{at}biomol.sci.toho-u.ac.jp

ABSTRACT

Macrophages phagocytose apoptotic cells without causing neutrophil infiltration in vivo under physiological conditions. Our recent study, however, showed that macrophages produce IL-8 or MIP-2, a murine IL-8 homologue, upon coculturing with apoptotic cells, indicating that there must be unknown mechanisms for preventing IL-8 or MIP-2 production. As activated macrophages produce NO to regulate inflammation, we examined the NO production by macrophages upon coculturing with apoptotic or necrotic cells and explored the role of NO in MIP-2 production. NO was produced on coculturing with early apoptotic cells much more significantly than with late apoptotic or necrotic cells. On the contrary, MIP-2 was produced on coculturing with late apoptotic or necrotic cells much more significantly than with early apoptotic cells. NG-Nitro-L-arginine methyl ester, an inhibitor of NO synthase, or 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a scavenger of NO, augmented MIP-2 production on coculturing with early apoptotic cells. The addition of N-ethylethanamine:1,1-diethyl-2-hydroxy-2-nitrosohydrazine [1:1], a donor of NO, conversely, caused suppression of MIP-2 production on coculturing with late apoptotic cells. These results suggest an important role of NO for preventing MIP-2 production by macrophages upon coculturing with early apoptotic cells.

Key Words: iNOS • IRF-1 • L-NAME • PTIO • DEA-NOate • ERK • NF-kB

INTRODUCTION

Apoptotic cells are removed quickly by macrophages, which is not associated with neutrophil infiltration in vivo under physiological conditions. Although there have been several papers demonstrating production of anti-inflammatory cytokines by a coculture of macrophages with early apoptotic cells [1 , 2 ], our recent study showed that macrophages produce IL-8 or MIP-2, a murine IL-8 homologue, but not TNF-{alpha} upon coculturing with early or late apoptotic cells [3 , 4 ], suggesting that there must be unknown mechanisms for preventing neutrophil infiltration in vivo. We have identified four mechanisms so far. First, the level of an anti-inflammatory cytokine such as IL-10 or TGF-ß increased when human monocyte-derived macrophages were cocultured with apoptotic cells in the presence of human serum, leading to suppression of IL-8 production [5 ]. Second, we have reported that coculturing of macrophages with apoptotic cells at an early stage is not accompanied by the production of anti-inflammatory or proinflammatory cytokines [6 ], although the mechanism for this is not known at present. Third, when immature dendritic cells were added to a coculture of macrophages and late apoptotic cells, MIP-2 production was suppressed in a cell-to-cell, contact-dependent manner [7 ]. Fourth, we have recently reported that adiponectin suppresses IL-8 production upon coculturing human macrophages with late apoptotic cells [8 ]. However, there may be other mechanisms for preventing IL-8 or MIP-2 production upon coculturing macrophages with apoptotic cells, in particular, at an early stage.

NO, which is produced by a variety of cells, is involved in physiological and pathological processes, including regulation of blood vessel dilatation, and functions as a neurotransmitter [9 , 10 ]. NO produced by inducible NO synthase (iNOS) in macrophages, which is induced by IL-1ß, TNF-{alpha}, IFN-{gamma}, or LPS, plays microbicidal and tumoricidal roles [11 12 13 14 ]. NO also regulates inflammatory responses. For example, NO increased MIP-2 expression and production, causing increased neutrophil migration into murine lung tissue [15 ]. On the contrary, NO is an important inhibitor of vascular inflammation [16 ]. Thus, NO plays proinflammatory and anti-inflammatory roles.

In this study, we investigated whether NO is produced by macrophages upon coculturing with early apoptotic cells, late apoptotic cells, or necrotic cells and how NO regulates MIP-2 production by macrophages.

MATERIALS AND METHODS

Preparation of peritoneal resident macrophages and coculturing with dead cells
Peritoneal resident cells were obtained by lavage of the peritoneal cavities of male ICR mice (5–6 weeks old; Sankyo Lab Service, Tokyo, Japan), male C57BL/6 mice (6 weeks old; Sankyo Lab Service), or male C57BL/6 mice, deficient in iNOS (6 weeks old; purchased from Taconic, Hudson, NY, and bred in our specific pathogen-free facility of Toho University, Japan), with cold PBS (pH 7.4, containing 14 mM Na2PO4 and 6 mM KH2PO4), followed by incubation in RPMI-1640 medium containing 7% FCS (Life Technologies, Gaithersburg, MD) at a cell density of 2 x 105 cells/well in 96-well plates for 1 h at 37°C in a CO2 incubator. Nonadherent cells were removed and washed three times with warm PBS, followed by the addition of RPMI-1640 medium without phenol red containing 7% FCS. The resultant adherent cells include 90.4 ± 2.4% of macrophages, as judged on hematoxylin and eosin staining. Apoptotic cells were added to macrophages in a ratio of 1:1 after washing with PBS three times. Necrotic cells were added to macrophages in a ratio of 1:1 without washing. They were then cocultured for 3, 12, or 24 h. In some experiments, macrophages were pretreated with 1 mM NG–nitro-L-arginine methyl ester (L-NAME; Sigma-Aldrich, St. Louis, MO) or 100 µM 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (PTIO; Sigma-Aldrich) for 15 min, followed by the addition of dead cells and incubation for various times. In other experiments, 0.1–10 µM N-ethylethanamine:1,1-diethyl-2-hydroxy-2-nitrosohydrazine [1:1] (DEA-NOate; Sigma-Aldrich) was added to macrophages after coculturing with late apoptotic cells for 12 h, followed by incubation for a further 12 h.

Induction of apoptosis or necrosis in CTLL-2 cells
To induce apoptosis, an IL-2-dependent CTL line, CTLL-2 cells, was washed with PBS by centrifugation at 1000 rpm for 5 min at room temperature, followed by incubation in IL-2-free RPMI-1640 medium containing 7% FCS for 4–28 h at 37°C at a cell density of 5 x 105 cells/ml. To induce necrosis, IL-2-free CTLL-2 cells were subjected to freezing at –80°C for 30 min and thawing at 37°C for 5 min.

Induction of apoptosis in thymocytes
Thymocytes were prepared from male ICR mice (5–6 weeks old). They were irradiated with X-rays, 12 Gy (MBR-1505R2; Hitachi Medico, Tokyo, Japan), and were then incubated in RPMI-1640 medium containing 7% FCS at a cell density of 2 x 106 cells/ml for 6 or 12 h.

Induction of apoptosis in HL-60 cells
To induce apoptosis, HL-60 cells were irradiated with 12 Gy of X-ray. They were then incubated in RPMI-1640 medium containing 7% FCS at a cell density of 5 x 106 cells/ml for 4 or 48 h. We designate apoptotic HL-60 cells obtained by culturing for 4 h as early apoptotic cells and ones obtained by culturing for 48 h as late apoptotic cells, which is based on comparison of the percentages of HL-60 cells with a decrease in size with those of apoptotic CTLL-2 cells obtained by culturing for 12 or 28 h. To induce necrosis, HL-60 cells were subjected to freezing at –80°C for 30 min and thawing at 37°C for 5 min. When early apoptotic cells, late apoptotic cells, or necrotic cells were cultured for 24 h, IL-8 was detected in the supernatants at the levels of 0.96 ± 0.03, 0.36 ± 0.02, and 0.31 ± 0.00 ng/ml, respectively.

Flow cytometric analysis of apoptosis
The cells were stained with FITC-conjugated Annexin V and/or propidium iodide (PI), followed by flow cytometry using a FACScan and Lysis software (Becton Dickinson, Mountain View, CA). Annexin V single-positive cells are at an early stage of apoptosis, whereas Annexin V and PI double-positive cells are at a late stage of apoptosis (see Table 1 ).


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  		Table 1. Induction of Apoptosis in CTLL-2 Cells or Thymocytesa

Measurement of NO
NO was quantified by means of the Griess reaction as nitrite. Briefly, 100 µl culture supernatant was mixed with an equal volume of Griess reagent (1% sulfanilamide, 0.1% naphthylethylenediamine dihydrochloride, 2.5% phosphoric acid, Sigma-Aldrich) in 96-well plates and then incubated for 10 min at room temperature in the dark. The absorbance at 570 nm was determined with a microplate reader.

Measurement of MIP-2, IL-10, and TGF-ß
After coculturing, the cells were centrifuged, and the supernatants were harvested. Samples were stored at –80°C until the assay. The MIP-2, IL-10, and TGF-ß levels were determined with DuoSet ELISA development systems (R&D Systems, Minneapolis, MN).

Neutralization of IL-10 or TGF-ß
Macrophages were treated with 50 ng/ml antimurine IL-10 antibody (PeproTech, London, UK), 50 ng/ml of murine IgG, 10 µg/ml chicken antimurine TGF-ß antibody (R&D Systems), or 10 µg/ml chicken IgY (R&D Systems) for 30 min, followed by coculturing with apoptotic or necrotic CTLL-2 cells for 24 h. The concentration of each antibody was determined as follows. When IL-10 produced by macrophages in response to 100 ng/ml LPS (Escherichia coli O55B5, Difco, Detroit, MI) was neutralized with 1, 10, 50, or 100 ng/ml anti-IL-10 antibody, the levels of MIP-2 were increased from 14.3 ± 0.6 ng/ml to 16.0 ± 0.1, 17.7 ± 0.6, 19.3 ± 0.5, or 19.4 ± 0.7 ng/ml, respectively. Similarly, when TGF-ß was neutralized by 0.1, 1, 10, or 50 µg/ml anti-TGF-ß antibody, the levels of MIP-2 were increased from 14.3 ± 0.6 ng/ml to 14.3 ± 0.4, 16.3 ± 1.1, 17.0 ± 0.3, or 17.0 ± 0.3 ng/ml, respectively.

Inhibitors of phagocytosis
Phospho-L-serine (PLS), Arg-Gly-Asp-Ser (RGDS), and Arg-Gly-Glu-Ser (RGES) were purchased from Sigma-Aldrich and diluted in RPMI 1640 to 100 mM. Macrophages were pretreated with 1 mM of these inhibitors for 30 min at 37°C. Apoptotic cells were then added, followed by incubation for 24 h at 37°C, after which NO and MIP-2 production was evaluated.

An inhibitor of MEK1/2
U0126 was purchased from Calbiochem (San Diego, CA) and diluted in PBS to 1 mM. Macrophages were preincubated with 10 µM U0126 for 1 h. Then, apoptotic or necrotic CTLL-2 cells were added, followed by coculturing for 3, 12, or 24 h.

RT-PCR
Total RNA was isolated from a coculture, and RT-PCR was performed as described previously [17 ]. The cDNA product (1 µl), produced through the RT reaction, was then amplified in 1x PCR buffer (Toyobo Co., Ltd., Osaka, Japan) containing 0.3 µM each primer, 0.2 mM deoxy-unspecified nucleoside 5'-triphosphates, and 1 unit KOD-Plus-DNA polymerase (Toyobo Co., Ltd.) in a total volume of 50 µl. The primer sequences were as follows: iNOS sense, ACA ACA GGA ACC TAC CAG CTC A; iNOS antisense, GAT GTT GTA GCG CTG TGT GTC A; and the predicted size of the PCR product is 651 bp [18 ]; IFN regulatory factor-1 (IRF-1) sense, GCA AAA CCA AGA GGA AGC; IRF-1 antisense, GCT GCC ACT CAG ACT GTT CA; and the predicted size of the PCR product is 440 bp [19 ]. The predicted sizes of MIP-2 and ß2 microglobulin (ß2m) were reported previously [17 ]. The PCR conditions were 94°C, 15 s; 60°C, 30 s; and 68°C, 40 s for 30 cycles.

Detection of ERK1/2 and I{kappa}B
ERK1/2 was detected as described previously [20 ]. Briefly, macrophages treated with 0–10 µM DEA-NOate were cocultured with late apoptotic CTLL-2 cells for 1 h. After lysis buffer was added to the cells, the samples were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes for 1 h. The membranes were then incubated in Tris-buffered saline/Tween 20 containing 5% skim milk for 1 h. Immunoreactive proteins were detected using rabbit anti-ERK1/2 antibody, antiphospho-ERK1/2 antibody (New England Biolabs, Beverly, MA), or anti-I{kappa}B antibody (Imgenex, San Diego, CA) for 1 h. After washing, the membranes were incubated with HRP goat anti-rabbit anti-IgG antibody. The membranes were then incubated in ECL Western blotting detection reagents (Amersham Biosciences, UK) for 1 min, followed by analysis with an LAS-1000 and dark box (FujiFilm, Tokyo, Japan).

Statistics
The significance of the data was evaluated by means of one-factor ANOVA, followed by Fisher’s protected least significant difference test. A P value of <0.05 was considered statistically significant.

RESULTS

NO and MIP-2 production by macrophages upon coculturing with dead cells
The role of NO produced by macrophages upon coculturing with dead cells had not yet been examined. In this study, we induced apoptosis or necrosis in CTLL-2 cells, which is an IL-2-dependent cell line, and then examined NO and MIP-2 production by macrophages upon coculturing with the dead cells.

CTLL-2 cells were cultured in the absence of IL-2 for 4, 8, 12, or 28 h to induce apoptosis. The cells were then stained with FITC-Annexin V and/or PI, and the results are shown in Table 1 . Hereafter, we designate apoptotic CTLL-2 cells obtained by culturing for 4, 8, or 12 h, as early apoptotic cells, which are rich in Annexin V single-positive cells, and ones obtained by culturing for 28 h as late apoptotic cells, which are rich in Annexin V and PI double-positive cells.

NO was produced significantly by macrophages upon coculturing with early apoptotic CTLL-2 cells in a time-dependent manner, notably at 24 h (Fig. 1A ). Moreover, the further apoptosis proceeded, the less NO was produced. Conversely, NO was produced minimally by macrophages upon coculturing with late apoptotic or necrotic cells. Next, we examined the expression of mRNA of iNOS in macrophages upon coculturing with dead cells. The expression was also higher upon coculturing with early apoptotic cells than with late apoptotic or necrotic cells, this being in good agreement with NO production (Fig. 1C) . Furthermore, NO was not produced when macrophages of mice deficient in iNOS were cocultured with apoptotic or necrotic cells, although MIP-2 was produced (data not shown). These results indicate that NO was produced by macrophages.


Figure 1
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  		Figure 1. NO and MIP-2 production by macrophages upon coculturing with dead cells. CTLL-2 cells were incubated for 4, 8, 12, or 28 h in the absence of IL-2, according to the method described under Materials and Methods. The levels of NO (A) or MIP-2 (B) in the supernatants of cocultures of macrophages with necrotic or apoptotic CTLL-2 cells for 3, 12, or 24 h were determined. Each set of experiments was carried out in triplicate. The data are expressed as the means ± SE for three independent sets of experiments. The differences between macrophages alone (designated as –) and others were analyzed statistically as described under Materials and Methods. The asterisks indicate significant differences (P<0.05). The levels of iNOS, IRF-1, MIP-2, and ß2m mRNAs were determined by RT-PCR (C). The data are representative of two independent sets of experiments.

IRF-1 is known to be a major transcription factor for IFN-{gamma}-mediated iNOS expression [21 ]. We therefore examined the expression of mRNA of IRF-1 in macrophages upon coculturing with dead cells. The expression paralleled that of iNOS mRNA (Fig. 1C) , suggesting the possibility that IRF-1 is involved in iNOS expression in this case.

Contrary to NO, MIP-2 was produced by macrophages upon coculturing with late apoptotic or necrotic cells more significantly than with early apoptotic cells (Fig. 1B) . The time kinetics was also different from that for NO production, the MIP-2 level being significant at 12 h. Moreover, MIP-2 production continued between 12 and 24 h upon coculturing with late apoptotic cells but scarcely increased after 12 h upon coculturing with early apoptotic cells. The level of MIP-2 mRNA in adherent cells (macrophages) paralleled that of MIP-2 protein (Fig. 1B and 1C) , suggesting that MIP-2 was produced by macrophages. Moreover, when mouse macrophages were cocultured with apoptotic or necrotic HL-60 cells, which are human cells, MIP-2 (mouse chemokine) but not IL-8 (human chemokine) was produced by coculturing of macrophages with dead cells, as compared with culturing of dead cells or macrophages (data not shown). These results suggest that MIP-2 was produced by macrophages.

The role of NO in MIP-2 production by macrophages upon coculturing with dead cells
We then examined the role of NO in MIP-2 production upon coculturing with dead cells. The NO production on coculturing with early apoptotic cells was inhibited significantly with L-NAME, an inhibitor of NOS (Fig. 2A ). Furthermore L-NAME or PTIO, a NO scavenger, caused significant augmentation of MIP-2 production on coculturing with early apoptotic cells (Fig. 2B) . In contrast, L-NAME or PTIO caused significant suppression of MIP-2 production on coculturing with late apoptotic cells or necrotic cells (Fig. 2B) . PTIO is known to scavenge NO by converting it to nitrite, and PTIO increased the level of nitrite in culture supernatants as expected (data not shown).


Figure 2
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  		Figure 2. The relationship between NO and MIP-2 production by macrophages on coculturing with dead cells. CTLL-2 cells were incubated for 4, 8, 12, or 28 h in the absence of IL-2 according to the method described under Materials and Methods. The levels of NO (A) or MIP-2 (B) in the supernatants of cocultures of macrophages with necrotic or apoptotic CTLL-2 cells in the presence (solid bars for L-NAME; shaded bars for PTIO) or absence (open bars) of L-NAME (1 mM) or PTIO (100 µM) for 24 h were determined. Each set of experiments was carried out in triplicate. The data are expressed as the means ± SE for three independent sets of experiments. The differences between cont. and L-NAME (A) and cont. and PTIO (B) were statistically analyzed as described under Materials and Methods. The asterisks indicate significant differences (P<0.05).

NO and MIP-2 production by macrophages upon coculturing with apoptotic thymocytes
Next, we examined the production of NO and MIP-2 by macrophages upon coculturing with apoptotic thymocytes, which were obtained by culturing for 6 h (early apoptotic cells) or 12 h (late apoptotic cells) after X-ray irradiation (12 Gy). The cells were then stained with FITC-Annexin V and/or PI, and the results are shown in Table 1 .

As shown in Table 2 , NO was produced more significantly by macrophages upon coculturing with early apoptotic cells than with late apoptotic cells, although the levels of NO were smaller than those in the case of apoptotic CTLL-2 cells. Conversely, MIP-2 was produced more significantly by macrophages upon coculturing with late apoptotic cells than with early apoptotic cells, although the levels of MIP-2 were also smaller than those in the case of apoptotic CTLL-2 cells. Furthermore, when NO production on coculturing with early apoptotic cells was inhibited by L-NAME, MIP-2 production was augmented, whereas when NO production on coculturing with late apoptotic cells was inhibited by an inhibitor, MIP-2 production was also inhibited. These results are consistent with the results described above.


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  		Table 2. NO and MIP-2 Production by Macrophages upon Coculturing with Apoptotic Thymocytesa

The roles of IL-10 and TGF-ß in MIP-2 production by macrophage on coculturing with dead cells
IL-10 and TGF-ß are reportedly produced by macrophages on coculturing with apoptotic cells [1 , 5 ]. We therefore examined the roles of these two anti-inflammatory cytokines in MIP-2 production on coculturing with dead cells. IL-10 was produced significantly by macrophages on coculturing with early apoptotic cells, late apoptotic cells, or necrotic cells but not with apoptotic CTLL-2 cells obtained by culturing for 4 h, as compared with IL-10 produced by CTLL-2 cells (Fig. 3A ). However anti-IL-10 antibody did not affect MIP-2 production (Fig. 3C) . Conversely, TGF-ß was produced by macrophages on coculturing with apoptotic CTLL-2 cells obtained by culturing for 4 h but not other CTLL-2 cells. Anti-TGF-ß antibody also did not affect MIP-2 production. Taken together, it is unlikely that IL-10 and TGF-ß regulate MIP-2 production on coculturing with dead cells under our conditions.


Figure 3
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  		Figure 3. IL-10 and TGF-ß production by macrophages on coculturing with dead cells and their roles in MIP-2 production. CTLL-2 cells were incubated for 4, 8, 12, or 28 h in the absence of IL-2 according to the method described under Materials and Methods. The levels of IL-10 (A) and TGF-ß (B) in the supernatants of cultures of necrotic or apoptotic CTLL-2 cells for 24 h (only) or cocultures of macrophages with necrotic or apoptotic CTLL-2 cells for 24 h (coculturing) were determined. The levels of MIP-2 in the supernatants of cocultures of macrophages with necrotic or apoptotic CTLL-2 cells for 24 h in the presence or absence of anti-IL-10 antibody or control antibody (C) and anti-TGF-ß antibody or control antibody (D) were also determined. Each set of experiments was carried out in triplicate. The data are expressed as the means ± SE for three independent sets of experiments. n.d., Not detected.

The concentration-dependent, suppressive effect of a NO donor on MIP-2 production
To determine whether the suppressive effect of NO on MIP-2 production depends on the concentration of NO, we then investigated the effect of DEA-NOate (0.1–10 µM), which is a NO donor, on MIP-2 production by macrophages upon coculturing with late apoptotic cells. When the concentration of NO in the supernatants of cultures was increased with DEA-NOate, MIP-2 production was inhibited (Fig. 4A and 4B ). For example, when the concentration of NO had increased to almost the same level as that produced on coculturing with 12 h-cultured apoptotic CTLL-2 cells with the addition of 3 µM DEA-NOate, the level of MIP-2 was reduced to almost the same level as that produced on coculturing with 12 h-cultured apoptotic CTLL-2 cells.


Figure 4
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  		Figure 4. The concentration-dependent effect of a NO donor on MIP-2 production. Macrophages were cultured with late apoptotic CTLL-2 cells for 12 h, followed by the addition of DEA-NOate (0.1–10 µM) to the coculture and the further culture for 12 h. For comparison, macrophages were cultured with early apoptotic CTLL-2 cells for 24 h. The levels of NO (A) or MIP-2 (B) in the supernatants of cocultures were then determined. Each set of experiments was carried out in triplicate. The data are expressed as the means ± SE for three independent sets of experiments.

Inhibition of NO and MIP-2 production by PLS, RGDS, or RGES
To investigate the mechanism that early and late apoptotic cells induce different responses in macrophages, we then examined the effects of PLS, RGDS, or RGES on NO and MIP-2 production. RGES was used as a control peptide for RGDS. NO production induced by early apoptotic cells was suppressed with PLS and RGDS less significantly than MIP-2 production induced by late apoptotic cells (Fig. 5A , left, vs. Fig 5B , right). However, PLS and RGDS failed to inhibit these two responses differentially (see Discussion).


Figure 5
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  		Figure 5. Inhibition of NO and MIP-2 production by PLS, RGDS, or RGES. CTLL-2 cells were incubated for 4 or 28 h in the absence of IL-2, according to the method described under Materials and Methods. The levels of NO (A) and MIP-2 (B) in the supernatants of cocultures of macrophages with apoptotic CTLL-2 cells in the presence of PLS (1 mM), RGES (1 mM), or RGDS (1 mM) for 24 h. Each set of experiments was carried out in triplicate. The data are expressed as means ± SE of three independent sets of experiments. The differences between macrophage alone (designated as –) and others were analyzed statistically according to the method described under Materials and Methods. The asterisks indicate significant differences (P<0.05).

Involvement of ERK1/2 and NF-{kappa}B in MIP-2 production and its suppression by NO
Our previous study revealed that activation of ERK1/2 is involved in the production of MIP-2 on coculturing of macrophages with late apoptotic cells [20 ]. We therefore examined the possibility that activation of ERK1/2 is suppressed by NO. As shown in Figure 6A , U0126, a selective inhibitor of MEK, significantly inhibited MIP-2 production induced by early apoptotic cells as well as late apoptotic cells. Moreover, activation of ERK1/2 was inhibited by NO in a dose-dependent manner (Fig. 6B) .


Figure 6
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  		Figure 6. Involvement of ERK1/2 and NF-{kappa}B in MIP-2 production and its suppression by NO. CTLL-2 cells were incubated for 4, 8, 12, or 28 h in the absence of IL-2, according to the method described under Materials and Methods. The levels of MIP-2 in the supernatants of cocultures of macrophages with apoptotic or necrotic CTLL-2 cells in the presence (solid bars) or absence (open bars) of U0126 (10 µM) for 24 h (A). Each experiment was carried out in triplicate. The data are expressed as means ± SE of three independent sets of experiments. The differences between cont. and U0126 were analyzed statistically according to the method described under Materials and Methods. The asterisks indicate significant differences (P<0.05). Macrophages treated with DEA-NOate (0–10 µM) were cocultured with late apoptotic CTLL-2 cells for 1 h. Western blotting analyses were performed with antibodies against ERK1/2 [total ERK1/2 (t-ERK1/2)], phospho-ERK1/2 (p-ERK1/2), or I{kappa}B. The actual images, the ratios of the band intensities of p-ERK to those of t-ERK and the ratios of the band intensities of I{kappa}B to those of t-ERK (cont.) were shown (B, C). The data are expressed as means ± SE of four independent determinations.

Degradation of I{kappa}B causes activation of NF-{kappa}B, which is involved in proinflammatory cytokine production. I{kappa}B was degraded in macrophages on coculturing with late apoptotic cells, and degradation of I{kappa}B was also inhibited by NO in a dose-dependent manner (Fig. 6C) .

DISCUSSION

Macrophages produced NO in larger quantities upon coculturing with early apoptotic CTLL-2 cells, leading to suppression of MIP-2 production, presumably via inhibition of activation of ERK1/2 and NF-{kappa}B. In contrast, macrophages produced NO in smaller quantities upon coculturing with late apoptotic or necrotic CTLL-2 cells, leading to enhancement of MIP-2 production. Early and late apoptotic thymocytes gave similar results, although macrophages produced NO and MIP-2 in smaller quantities. Furthermore, preliminary experiments showed that this was also true for early and late apoptotic HL-60 cells.

Our previous study demonstrated that early and late apoptotic cells are recognized preferentially by human monocyte-derived macrophages through phosphatidylserine and RGD, respectively [3 , 6 ]. However, PLS and RGDS failed to inhibit NO production induced by early apoptotic cells and MIP-2 production induced by late apoptotic cells differentially. Thus, the reason why a significant amount of NO is produced upon coculturing of macrophages with early apoptotic cells is still not known. Indeed much more study is required to elucidate the underlying mechanism.

NO production by macrophages upon coculturing with dead cells appears to be mediated by iNOS, as the level of iNOS mRNA paralleled that of NO (Fig. 1) , and macrophages from iNOS-deficient mice failed to produce NO upon coculturing with dead cells. As the level of IRF-1 mRNA paralleled that of iNOS, it is likely that IRF-1 is involved in iNOS mRNA expression. STAT-1 also appears to mediate the IFN-{gamma}-enhanced iNOS induction in rat aortic smooth muscle cells and human lung epithelial cells [22 , 23 ]. As the regulation of iNOS transcription is complex and dependent on cell types and stimulants, much more work is required to elucidate the underlying mechanism of iNOS mRNA expression on coculturing with early apoptotic cells.

MIP-2 production by macrophages scarcely increased after 12 h on coculturing with early apoptotic CTLL-2 cells, presumably as a result of the large quantity of NO produced between 12 h and 24 h. Conversely, MIP-2 production continued between 12 h and 24 h on culturing with late apoptotic CTLL-2 cells, presumably as a result of the small quantity of NO produced in the period. In support of this, when NO production by macrophages on coculturing with early apoptotic CTLL-2 cells was inhibited by L-NAME or PTIO, MIP-2 production increased. In addition, when DEA-NOate was added to macrophages cocultured with late apoptotic CTLL-2 cells, MIP-2 production decreased with an increase in the level of NO.

Although it is generally known that NO produced by macrophages acts as a microbicidal and a tumoricidal mediator, the relationship between NO production and inflammatory responses has not yet been defined fully. In the last several years, however, there have been many reports that NO produced by macrophages is involved in inflammatory responses. NO produced by macrophages inhibits the rolling and adhesion of neutrophils [24 , 25 ] and furthermore, induces apoptosis of neutrophils [25 ]. NO accelerated the rolling and adhesion of neutrophils in other studies [26 27 28 ] and produced by bone marrow-derived macrophages and dendritic cells, inhibits IL-12 p40 transcription and production through inhibition of NF-{kappa}B activation [29 ]. NO suppresses inflammation in the murine acute lung inflammatory response [30 ]. The level of chemokine production in iNOS-deficient mice is higher than that in wild-type mice, and migration of neutrophils into lung tissue is augmented in the deficient mice [31 ]. Conversely, NO promotes the production of chemokines [32 , 33 ]. However, the relationship between the NO concentration and its mode of action was not considered in those reports.

There has been a recent report that although a NO donor at lower concentrations caused significant promotion of IL-6 or cyclooxygenase-2 production by macrophages treated with LPS through the activation of NF-{kappa}B, the NO donor at higher concentrations caused suppression [34 ], suggesting that a high concentration of NO has an anti-inflammatory effect and that a low concentration of it has a proinflammatory effect. Our study confirmed their findings and extended them by showing that NO regulated MIP-2 production by macrophages positively or negatively, which appears to be determined by the relative level of NO as well as the stage of apoptotic cells cocultured with the macrophages. Such dual roles of NO have also been suggested in tumor biology, and tumor promoting and suppressing actions of NO seem to depend on the local concentration of iNOS [35 ]. Conversely, there is another example for dual roles of NO, which is protective or damaging to the liver, and in this case, redox stress activates an unknown molecular switch that transforms NO, which is hepatoprotective under resting conditions into an agent that induces hepatocyte death [36 ].

As shown in this study, NO suppressed activation of ERK1/2 and NF-{kappa}B, which is induced by coculturing macrophages with late apoptotic cells in a dose-dependent manner. As our previous study revealed that activation of ERK1/2 is involved in MIP-2 production upon coculturing macrophages with late apoptotic cells [20 ], this suggests that activation of ERK1/2 and NF-{kappa}B is critical in regulation of inflammatory responses.

Finally, we suggest a likely scenario in vivo. When apoptotic cells appear in vivo, macrophages may interact with the cells at an early stage, resulting in the production of NO in larger quantities to prevent an inflammatory response. When a large amount of apoptotic cells appears in vivo, however, macrophages may not dispose of all the cells at an early stage, the stage of apoptotic cells becoming late, and a smaller quantity of NO is then produced to enhance an inflammatory response. Thus, during clearance of dead cells in vivo, NO would play a dual role in regulation of inflammatory responses.

Received January 7, 2006; revised April 24, 2006; accepted April 25, 2006.

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