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The Journal of Immunology, 2007, 179, 3407 -3411
Copyright © 2007 by The American Association of Immunologists, Inc.
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Cutting Edge: A Critical Role of Nitrogen Oxide in Preventing Inflammation upon Apoptotic Cell Clearance1

Takehiko Shibata, Kisaburo Nagata and Yoshiro Kobayashi2

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


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Apoptotic cells are removed by phagocytes without causing inflammation. It remains largely unresolved whether anti-inflammatory mediators prevent neutrophil infiltration upon apoptotic cell clearance in vivo. In this study, we showed that, upon induction of apoptosis in the thymus by x-ray, inducible NO synthase knockout (KO) mice exhibited higher levels of neutrophil infiltration and production of MIP-2 and keratinocyte-derived chemokine (KC) in the thymus than wild-type (WT) mice. Furthermore, administration of NG-nitro-L-arginine methyl ester, an inhibitor of NO synthase, to x-irradiated WT mice increased the level of neutrophil infiltration to that of KO mice by the augmentation of MIP-2 and KC production. Additionally, thymic macrophages isolated from x-irradiated KO mice produced more MIP-2 and KC than those from WT mice. Thus, although apoptosis is believed to be noninflammatory, this is actually achieved by the production of immunosuppressive signals such as NO that counteract proinflammatory chemokines such as MIP-2 and KC.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 Disclosures
 References
 
Apoptotic cells are removed by phagocytes such as macrophages without causing inflammation in vivo (1, 2). This has been considered an active process mediated by the production of TGF-beta, IL-10, and PGE2, because macrophages cocultured with early apoptotic cells produce these anti-inflammatory mediators that suppress the production of proinflammatory mediators in vitro (3, 4, 5). In contrast, we have previously shown that macrophages cocultured with very early apoptotic cells produce neither proinflammatory nor anti-inflammatory cytokines (6) but that such macrophages produce a large quantity of NO to suppress an inflammatory response (7). However, until now it remained largely unresolved whether or not any of these anti-inflammatory mediators prevent neutrophil infiltration, a hallmark of acute inflammation, upon apoptotic cell clearance in vivo with the exception of TGF-beta in a LPS-induced inflammation model (8) in which the instillation of apoptotic cells into LPS-stimulated lung caused TGF-beta production and a subsequent decrease in neutrophil infiltration. In this study we show a critical role of NO produced by macrophages to prevent neutrophil infiltration upon the induction of apoptosis in vivo in a whole body x-irradiation model (9, 10, 11) by using inducible NO synthase (iNOS)3 (2) knockout (KO) mice.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 Disclosures
 References
 
Mice

C57BL/6 mice were purchased from Sankyo Lab Service. Six-week-old male C57BL/6 mice deficient in iNOS were purchased from Taconic Farms and bred in our specific pathogen-free facility at Toho University, Chiba, Japan. This experiment has been approved by the animal experiment committee of Toho University.

Coculturing of macrophages with dead cells

Peritoneal resident macrophages were obtained by lavage of the peritoneal cavity and adherence to a plastic surface, whereas thymic macrophages were obtained by the digestion of small blocks of the thymus with collagenase and adherence to a plastic surface (10). Apoptotic CTLL-2 cells or apoptotic thymocytes were then cocultured with peritoneal resident macrophages or thymic macrophages at a ratio of 1:1 for 24 h, followed by determination of the levels of NO, MIP-2, and keratinocyte-derived chemokine (KC) in the supernatant of cocultures.

Whole body x-irradiation

C57BL/6 or iNOS-deficient mice were used throughout this study and irradiated with 0.75 gray (Gy) of x-ray irradiation at 0.20 Gy/min with an MBR-1505R2 apparatus (Hitachi).

Treatment of mice with NG-nitro-L-arginine methyl ester (L-NAME)

L-NAME (Sigma-Aldrich) or PBS at 0.5–2.0 mg/mouse was injected i.p. into C57BL/6 or iNOS-deficient mice. At 2 h after L-NAME injection, mice were irradiated with 0.75 Gy of x-irradiation. MIP-2, KC, and infiltrated neutrophil levels at 9 or 18 h after x-irradiation, respectively, were determined by specific ELISAs or flow cytometric analysis, respectively.

Measurement of NO, MIP-2, and KC produced by thymic macrophages after whole body x-irradiation

C57BL/6 or iNOS-deficient mice were irradiated with 0.75 Gy of x-ray. Thymic macrophages were isolated from these mice followed by in vitro incubation for 3 h. NO, MIP-2, and KC levels in the supernatants of cultures were determined by Griess assay or specific ELISAs, respectively.

RT-PCR

Total RNA was isolated from a coculture and RT-PCR was performed as previously described (12). One microliter of the cDNA product produced through the reverse transcription reaction was then amplified in 1x PCR buffer (Toyobo) containing 0.3 mM each primer, 0.2 mM dNTPs, and 1 unit of KOD Plus DNA polymerase (Toyobo) in a total volume of 50 µl. The primer sequences were 5'-CGGGATCCCCTATCGCCAATGAG-3' for KC sense and 5'-CCGGAATTCTTACTTGGGGACACC-3' for KC antisense and the predicted size of the PCR product was 232 bp. The PCR conditions were 94°C for 15 s, 60°C for 30 s, and 68°C for 18 s for 35 cycles. The predicted sizes and PCR conditions of iNOS, MIP-2, and beta2 -microglobulin were reported previously (7).

Statistics

The significance of the data was evaluated by means of Student’s t test. Values of p < 0.05 were considered statistically significant.


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
Disclosures
 References
 
NO, MIP-2, and KC production by thymic and peritoneal resident macrophages cocultured with apoptotic cells

When peritoneal resident macrophages are cocultured with early apoptotic cells, but not late apoptotic cells, they induce iNOS at the mRNA level and produce a large quantity of NO that suppresses their own MIP-2 production (7). In this study, early apoptotic cells are defined as annexin V-positive and propidium iodide-negative, whereas late apoptotic cells are defined as annexin V-positive and propidium iodide-positive. Because macrophages are heterogeneous in terms of surface markers and function (13), it is important to examine whether this is the case for thymic macrophages, which are involved in apoptotic cell clearance in the thymus (14). We thus compared the response to apoptotic cells of thymic macrophages with that of peritoneal resident macrophages, both of which were prepared from either wild type (WT) or iNOS deficient (KO) mice. We prepared two types of apoptotic cells, namely apoptotic CTLL-2 cells by IL-2 withdrawal and apoptotic thymocytes by x-irradiation, which were then cocultured with macrophages for 24 h followed by determination of the levels of NO, MIP-2, and KC in the supernatants. A large quantity of NO was produced by WT macrophages, but not by KO macrophages, in any combination of macrophages and early apoptotic cells, namely peritoneal resident macrophages (PM{phi}) with apoptotic CTLL-2 cells (Fig. 1A), PM{phi} with apoptotic thymocytes (Fig. 1B), thymic macrophages (TM{phi}) with apoptotic CTLL-2 cells (Fig. 1C), and TM{phi} with apoptotic thymocytes (Fig. 1D). KO macrophages did not produce NO even by stimulation with LPS and IFN-{gamma} (data not shown). On the contrary, although WT macrophages did not produce significant levels of MIP-2 and KC by coculturing with early apoptotic cells, KO macrophages did, the levels of MIP-2 and KC being 1.7- to 10-fold greater as compared with WT macrophages (Fig. 1). These results clearly indicate that our previous results (7) hold true for thymic macrophages. KO macrophages produced significant amounts of KC without coculturing with apoptotic cells whereas WT macrophages produced NO without coculturing with apoptotic cells, raising the possibility that NO suppresses KC production from unstimulated macrophages.


Figure 1
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  		FIGURE 1. NO (Nitrite), MIP-2, and KC production by macrophages upon coculturing with apoptotic cells (Apo). The levels of NO, MIP-2, and KC in the supernatants of cocultures of macrophages from C57BL/6 mice (WT) or iNOS-deficient mice (KO) with early apoptotic cells for 24 h were determined. The levels of NO (left panel), MIP-2 (middle panel), and KC (right panel) in the supernatants of cocultures of PM{phi} cocultured with apoptotic CTLL-2 cells (A), PM{phi} with apoptotic thymocytes (B), TM{phi} with apoptotic CTLL-2 cells (C), and TM{phi} with apoptotic thymocytes (D) were determined. *, p < 0.05; **, p < 0.01 vs WT. Error bars, SE; n = 3.

 
Neutrophil infiltration and MIP-2 and KC production in the thymus upon whole body x-irradiation

We and other groups have previously indicated that the uptake of late apoptotic cells by macrophages causes a proinflammatory response in vitro (15, 16, 17) and that whole body x-irradiation using 1 or 4 Gy of x-ray gives rise to late apoptotic cells in the thymus and concomitantly causes neutrophil infiltration (9, 10). In the latter study (10), apoptotic cells were phagocytosed by F4/80-positive thymic macrophages in the cortex that are presumed to produce MIP-2 and KC. In a whole body x-irradiation model, apoptosis occurs under sterile conditions as it does under a physiological condition. Consequently, the model is pertinent to examining the role of anti-inflammatory mediators such as NO in preventing neutrophil infiltration upon the induction of apoptosis. In this study we used a dose of 0.75 Gy of x-ray by which early apoptotic cells, but not late apoptotic cells, are produced in the thymus. Following whole body x-irradiation with 0.75 Gy of x-ray, the numbers of thymocytes decreased gradually in a time-dependent manner in WT and KO mice (Fig. 2A). We then examined the flow of thymocyte apoptosis cytometrically (Fig. 2, B and C). Early apoptotic cells peaked at 6 h after whole body x-irradiation in WT and KO thymi. On the contrary, late apoptotic cells were hardly detected in WT and KO thymi, although 1 or 4 Gy of x-ray induced late apoptotic cells (data not shown). Nevertheless, in WT mice neutrophil infiltration into the thymus was detected after irradiation with 0.75 Gy of x-ray (Fig. 2D), the neutrophil number being half of that detected after irradiation with 1 Gy of x-ray (data not shown). In KO mice, however, neutrophil infiltration was greater than in WT mice at 15 or 18 h, respectively (Fig. 2, D and E). These results suggest that NO suppresses neutrophil infiltration upon the induction of apoptosis in vivo. We then examined the levels of MIP-2 and KC in the thymus after whole body x-irradiation. There were more of both of these chemokines in the thymus of KO mice than in that of WT mice, in agreement with the extent of neutrophil infiltration (Fig. 2, F and G). This was also true for mRNA levels as shown in Fig. 2H. In unirradiated KO mice, no neutrophils were detected in the thymus (Fig. 2D) even though MIP-2 but not KC was detected for unknown reasons (Fig. 2, F–H). This is probably because MIP-2 and KC synergistically induce neutrophil recruitment in vivo (18). iNOS mRNA was indeed expressed only in the thymus of WT mice after whole body x-irradiation (Fig. 2H). The numbers of thymic macrophages did not differ between WT and KO mice (data not shown). Moreover, the percentages of phagocytosing thymic and peritoneal macrophages and the phagocytic indices were not different between WT and KO mice (data not shown). These results suggest that NO suppresses neutrophil infiltration by inhibiting MIP-2 and KC production at the mRNA and protein levels. NO may also interfere with neutrophil infiltration by inhibiting the rolling and adhesion of neutrophils in vivo (19, 20).


Figure 2
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  		FIGURE 2. Effect of NO in neutrophil infiltration and production of chemokines. WT or KO mice were irradiated with 0.75 Gy of x-ray followed by determination of the total thymic cell number (A), the levels of early (left panel) or late (right panel) apoptosis (B), neutrophil infiltration (D), MIP-2 (F and H), and KC (G and H) in the thymus after the indicated times by flow cytometric analysis, specific ELISAs, or RT-PCR, respectively. C, Representative data of flow cytometric analysis of apoptosis at 6 h after whole body x-irradiation. PI, Propidium iodide. E, The representative data of flow cytometric analysis of neutrophils 18 h after whole body x-irradiation. PI, Propidium iodide. H, The representative data of RT-PCR of iNOS, MIP-2, and KC mRNAs. beta2m, beta2-microglobulin. *, p < 0.05; **, p < 0.01 vs WT. Error bars, SE; n = 3–5.

 
When we examined the apoptosis of thymocytes that were irradiated with 12 Gy of x-ray in vitro, the percentages of early and late apoptotic thymocytes were not significantly different between WT and KO mice (data not shown). This suggests that a deficiency in iNOS does not affect the apoptotic process and supports the likelihood that WT and KO thymic macrophages encounter similar numbers of apoptotic thymocytes in vivo.

When apoptosis was inhibited by a pan-caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK), neutrophil infiltration was significantly suppressed (data not shown), indicating that apoptosis causes neutrophil infiltration. This is in good agreement with our previous result using p53-deficient mice (9).

Effects of L-NAME on neutrophil infiltration and MIP-2 and KC production upon whole body x-irradiation

We then examined the effect of L-NAME, a NOS inhibitor, on neutrophil infiltration in WT mice after x-irradiation. L-NAME is known to inhibit not only iNOS but also endothelial NOS. Treatment of WT mice with L-NAME increased the number of infiltrating neutrophils up to that of KO mice in a dose-dependent manner (Fig. 3A). Furthermore, injection with 2.0 mg of L-NAME also increased the levels of MIP-2 and KC in WT mice to those in KO mice (Fig. 3, B and C). It is of note that the treatment of KO mice with L-NAME did not augment neutrophil infiltration and the levels of MIP-2 and KC. These results provide further support that NO is mainly produced by iNOS in our x-irradiation model and negates the possibility that KO macrophages may have unknown defects other than the deficiency of iNOS to augment MIP-2 and KC production upon interaction with apoptotic cells.


Figure 3
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  		FIGURE 3. Effects of L-NAME on inflammatory responses. WT or KO mice were injected with L-NAME (0, 0.5. 1.0, or 2.0 mg/head) followed by flow cytometric analysis of neutrophil infiltration at 18 h after whole body x-irradiation (A) or by determination of the levels of MIP-2 (B) and KC (C) at 9 h after x-irradiation in the thymus. *, p < 0.05; **, p < 0.01. Error bars, SE; n = 4.

 
NO, MIP-2, and KC production by thymic macrophages isolated from whole body x-irradiated mice

To confirm that thymic macrophages actually produce NO via iNOS, we isolated thymic macrophages from whole body x-irradiated mice at 9 h after x-irradiation and examined the level of NO in their culture supernatant. More was produced by thymic macrophages from x-irradiated WT mice than from unirradiated WT mice (Fig. 4A), whereas NO was not produced at all by thymic macrophages from KO mice. In this model, however, nitrotyrosine, an adduct of NO to tyrosine, was minimally detectable in the lysate of the thymus by means of Western blotting analysis (data not shown), although it was reportedly present in the thymus following irradiation with 4 Gy of gamma-ray by immunohistochemical technique (11). We then determined the levels of MIP-2 and KC in the supernatant. Thymic macrophages from X-irradiated KO mice produced more MIP-2 and KC than those from X-irradiated WT mice, and both produced more MIP-2 than KC (Fig. 4, B and C). The latter finding contrasted with the in vivo data in that more KC was produced than MIP-2 in the thymus (Fig. 2, F and G). This is probably because MIP-2 is mainly produced by myeloid leukocytes such as macrophage, whereas KC is mainly produced by nonmyeloid cells such as stromal, endothelial, or epithelial cells (21).


Figure 4
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  		FIGURE 4. Production of NO, MIP-2, and KC by thymic macrophages isolated from x-irradiated mice. Thymic macrophages were isolated from WT or iNOS KO mice at 9 h after 0.75 Gy of x-irradiation. The levels of NO (Nitrite) (A), MIP-2 (B), and KC (C) in the supernatants of macrophages cultured for 3 h were determined. *, p < 0.05, **, p < 0.01. Error bars, SE; n = 3.

 
It is of note that thymic macrophages from x-irradiated WT mice also produced iNOS but not TGF-beta and IL-10 at the mRNA levels (data not shown), providing further support that NO plays a critical role in a whole body x-irradiation model.

The observation that the instillation of apoptotic cells into LPS-stimulated lung caused TGF-beta production and a subsequent decrease in neutrophil infiltration (8) has been considered strong in vivo evidence that, upon apoptotic cell clearance, macrophages produce TGF-beta to suppress proinflammatory cytokine production (1). This study provides strong in vivo evidence for the involvement of NO in a silent cleanup of apoptotic cells (6) in a whole body x-irradiation model. Thus, not only TGF-beta but also NO plays a critical role in suppressing neutrophil infiltration upon the induction of apoptosis, thereby contributing to the maintenance of tissue homeostasis. Overall, our findings reinforce the idea that, rather than being noninflammatory, anti-inflammatory signals including NO participate in the process of apoptotic cell clearance to counteract the generation of proinflammatory cytokines.


   Acknowledgments
 
We thank Dr. Joost J. Oppenheim for critical reading.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported in part by a grant from the Japan Science Society (to T.S.). Back

2 Address correspondence and reprint requests to Dr. Yoshiro Kobayashi, Division of Molecular Medicine. Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. E-mail address: yoshiro{at}biomol.sci.toho-u.ac.jp Back

3 Abbreviations used in this paper: iNOS, inducible NO synthase; Gy, gray; KC, keratinocyte-derived chemokine; KO, knockout; L-NAME, NG-nitro-L-arginine methyl ester; PM{phi}, peritoneal resident macrophage; TM{phi}, thymic macrophage; WT, wild type. Back

Received for publication May 24, 2007. Accepted for publication July 24, 2007.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 Disclosures
 References
 

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