bs_bs_banner
Geriatr Gerontol Int 2015
ORIGINAL ARTICLE: BIOLOGY
Attenuated phagocytosis of secondary necrotic neutrophils by macrophages in aged and SMP30 knockout mice Rei Takahashi,1 Sayuri Totsuka,1 Akihito Ishigami,2 Yoshiro Kobayashi1 and Kisaburo Nagata1 1 Department of Biomolecular Science, Faculty of Science, Toho University, Chiba, and 2Aging Regulation, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
Aim: Secondary necrotic cells generated in vivo induce inflammatory responses; for example, the production of macrophage inflammatory protein-2 (MIP-2) and subsequent infiltration of neutrophils. The aim of the present study was to elucidate the effect of aging on the phagocytosis of secondary necrotic cells and the inflammatory responses by using either wild-type (WT) young mice, WT aged mice or senescence-accelerated mice (SMP30−/− mice). Methods: The phagocytosis of secondary necrotic neutrophils with resident macrophage from either WT young mice, WT aged mice or SMP30−/− mice was examined by coculturing macrophages with secondary necrotic neutrophils in vitro. To investigate the inflammatory response induced by secondary necrotic cells, time-dependent infiltration of neutrophils and production of MIP-2 were determined in the peritoneal cavity on the injection of secondary necrotic cells. Results: The phagocytosis of secondary necrotic cells by macrophages from WT aged and SMP30−/− mice was significantly reduced as compared with that by macrophages from WT young mice. On peritoneal injection of secondary necrotic cells, the peak time of neutrophil infiltration was earlier in SMP30−/− mice than in WT young mice. The number of neutrophils in SMP30−/− mice at the peak time was also greater than that in WT young mice. Conclusions: Our findings showed that the phagocytosis of secondary necrotic cells was attenuated in aged mice and SMP30−/− mice, and that the MIP-2 production was enhanced and subsequently neutrophil infiltration was exaggerated on peritoneal injection of secondary necrotic cells into those mice. Geriatr Gerontol Int 2015; ••: ••–••. Keywords: aging, apoptosis, chemokines, inflammation, macrophages.
Introduction Apoptotic cells at an early stage are removed quickly by macrophages, which is not associated with neutrophil infiltration, one of the key inflammatory responses.1 In contrast, if such apoptotic cells are not completely removed, then secondary necrotic cells are generated. Of note is that primary necrotic cells are not generated by early apoptotic cells. Secondary necrotic cells generated in vivo cause various acute inflammatory diseases, such as acute lung injury.2 In support of this, we previously showed that many neutrophils infiltrated into the thymus when sec-
Accepted for publication 30 October 2014. Correspondence: Dr Kisaburo Nagata, PhD, Division of Molecular Medicine, Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. Email: [email protected]
© 2015 Japan Geriatrics Society
ondary necrotic cells were acutely generated in the thymus on whole-body X-irradiation.3,4 Furthermore, we previously reported that the expression of a chemokine specific to neutrophils, interleukin-8 or macrophage inflammatory protein-2 (MIP-2), was upregulated on in vitro coculturing of macrophages with secondary necrotic cells, and that injection of secondary necrotic cells into the mouse peritoneal cavity caused the production of MIP-2 and infiltration of neutrophils, and that anti-MIP-2 antibodies as well as anti-CXCR2 antibodies significantly suppressed the neutrophil infiltration, suggesting that secondary necrotic cells induce neutrophil infiltration mainly through MIP-2 in vivo.5–11 Secondary necrotic cells also cause chronic inflammatory diseases, because Su et al. previously reported that repeated injection of secondary necrotic cells caused autoimmune diseases, such as systematic lupus erythematosus.12 In contrast, the function of the immune system decreases with age, leading to increased susceptibility of doi: 10.1111/ggi.12436
|
1
R Takahashi et al.
the elderly to infections by viruses and bacteria.13,14 Previous studies showed that virus-specific T-cell responses are decreased in aged animals, and that the production of inflammatory mediators, such as tumor necrosis factor-α and nitric oxide, and dangerassociated molecular patterns released from secondary necrotic cells, such as high mobility group box-1, was increased in aged mice when lipopolysaccharide was injected into aged animals.15–17 However, few studies have been undertaken on how aging affects the inflammatory responses induced by secondary necrotic cells. In the present study, we investigated the effect of aging on the inflammatory responses induced by injection of secondary necrotic neutrophils into the peritoneal cavity using young (5–7 weeks-of-age) and aged (18–24 months-of-age) C57BL/6 mice, and SMP30/GNLknockout (SMP30−/−) mice fed a vitamin C (VC)-limited diet.
Materials and methods Mice Young C57BL/6 mice (5–7 weeks-of-age) were purchased from Sankyo Lab Service (Tokyo, Japan). Aged C57BL/6 mice (18–24 months-of-age) were bred in the Tokyo Metropolitan Institute of Gerontology. SMP30/ GNL-knockout (SMP30−/−) mice deficient in VC synthesis were provided by Dr Akihito Ishigami, and were bred at Toho University, Chiba, Japan.18 The SMP30−/− mice had free access to water containing a sufficient amount of VC (1.5 g/L) until 4 weeks-of-age. To obtain aged mice, mice had free access to water containing 0.0375 g/L of VC until 7 weeks-of-age.
Preparation of secondary necrotic neutrophils To obtain neutrophils, a thioglycollate broth solution (2 mL) was injected into the peritoneal cavity of C57BL/6 mice. After 6 h, peritoneal exudate cells (PEC) were collected. The PEC comprised more than 95.0% of neutrophils, which were identified as a Gr-1highCD11bhigh population by flow cytometry analysis. The cells were thereafter regarded as neutrophils in the present study, unless otherwise stated. The neutrophils were washed with phosphate-buffered saline (PBS; saline containing 14 mmol/L Na2PO4 and 6 mmol/L KH2PO4, pH 7.4) twice, and then suspended in RPMI 1640 medium containing 7% fetal calf serum at a cell density of 2 × 106 cells/mL. To induce secondary necrosis, the neutrophils were cultured for various times at 37°C. The number of secondary necrotic neutrophils, identified as Annexin V- and propidium iodide (PI)positive cells, peaked at 12 h. The percentage of Annexin V+/PI− neutrophils, early apoptotic neutrophils and that of Annexin V+/PI+ neutrophils, secondary 2
|
Table 1 Annexin V/PI staining of untreated and 12 h-cultured neutrophils
−
−
Annexin V /PI (living cells) Annexin V+/PI− (early apoptotic cells) Annexin V+/PI+ (secondary necrotic cells)
Untreated
12 h-cultured
88.92 ± 2.41
47.07 ± 3.08
1.89 ± 0.39
17.23 ± 1.31
3.85 ± 0.65
27.03 ± 1.70
All results are expressed as means ± standard error (n = 3–4). Untreated and 12 h-cultured neutrophils were stained with Annexin V and PI. The percentages of the indicated neutrophils among total neutrophils were determined by flow cytometry.
necrotic neutrophils, in untreated and 12 h-cultured neutrophils are shown in Table 1. On incubation for over 12 h, many more neutrophils with permeable membranes (Annexin V−/PI+), primary necrotic neutrophils, appeared. Therefore, the neutrophils cultured for 12 h were used as secondary necrotic neutrophils in the present study.
Flow cytometric analysis of macrophages among PEC and preparation of peritoneal resident macrophages For analysis of macrophages among PEC, PEC were obtained by lavage of the peritoneal cavity of wild-type (WT) young, WT aged or SMP30−/− mice fed a VC-limited diet with cold PBS, and they were stained with anti-F4/80 monoclonal antibody (mAb), antiCD11b mAb. The stained cells were analyzed by flow cytometry. For preparation of peritoneal resident macrophages, PEC were obtained from each mouse with cold PBS, and then the cells that were adhered to a plastic plate were collected. The collected cells comprised more than 98% of macrophages, as determined by staining with anti-F4/80 mAb.
Phagocytosis of secondary necrotic neutrophils by resident peritoneal macrophages in vitro Secondary necrotic neutrophils prepared as aforementioned were labeled with PKH26 Red fluorescent dye, followed by coculturing with resident macrophages, which had been allowed to adhere to a plastic plate beforehand. Then we removed non-trapped apoptotic neutrophils by washing, and stained resident macrophages with F4/80 mAb and examined then under a fluorescence microscope. © 2015 Japan Geriatrics Society
Effect of aging on inflammatory responses
Identification of free secondary necrotic neutrophils in the peritoneal cavity Secondary necrotic neutrophils were stained with PKH26 Red fluorescent dye, and then injected into the peritoneal cavity. After various times, PEC were collected with cold PBS. Free secondary necrotic neutrophils, which were not phagocytosed in the peritoneal cavity, were identified as PKH26 Red-highly positive cells, in which the intensity of PKH26 Red staining was similar to that in the injected cells.
Flow cytometric analysis of infiltrated neutrophils PKH26 Red-labeled secondary necrotic neutrophils were injected into the peritoneal cavity of each mouse. At various times, PEC were collected, and stained with anti-Gr-1 mAb and anti-CD11b mAb. The stained cells were analyzed by flow cytometry with a FACSCalibur (BD Biosciences, San Jose, CA, USA) with which PKH26 Red-highly positive cells, free secondary necrotic neutrophils, were excluded by gating.
Measurement of MIP-2 To measure the protein level of MIP-2 in the peritoneal cavity, supernatants of peritoneal lavage fluid were prepared at various times after the injection of secondary necrotic neutrophils. To measure the protein level of MIP-2 in the cocultures of resident peritoneal macrophages with secondary necrotic neutrophils, supernatants of the cocultures were prepared at various times. The MIP-2 levels were determined using an enzymelinked immunosorbent assay development kit.
Results Effects of aging on the total number, percentage and phagocytic capacity of peritoneal resident macrophages To investigate whether or not aging has any effects on the total number and percentage of peritoneal resident macrophages, we obtained resident macrophages from the peritoneal cavity of WT young and WT aged mice. The macrophages were identified as CD11bhigh and F4/80high. As shown in Figure 1a,b, the total numbers of macrophages did not significantly differ between WT young and WT aged mice, whereas the percentage of macrophages in the peritoneal cavity was significantly smaller in WT aged mice than in WT young mice. Next, we investigated the phagocytic capacity of resident peritoneal macrophages as to secondary necrotic cells. On coculturing for 6 h, resident peritoneal macrophages that did not engulf secondary necrotic neutrophils, indicated by arrows, were detected more in WT aged mice © 2015 Japan Geriatrics Society
Figure 1 Flow cytometric analysis of peritoneal resident cells from wild-type (WT) young mice and WT aged mice. (a) Total numbers of peritoneal resident macrophages in WT young (5–7 weeks-of-age) and WT aged (18–24 months-of-age) mice. (b) Percentage of macrophages among peritoneal resident cells. All results are expressed as means ± standard error (n = 3–4). *P < 0.001 versus WT young mice. NS, not significant.
than in WT young mice (Fig. 2a). A time-dependent change in the percentage of phagocytosis during the coculture was shown in Figure 2b. The percentage of phagocytosis in WT aged mice was significantly decreased as compared with that in WT young mice. Among PEC, however, cells other than macrophages were detected more in WT aged mice than in WT young mice. Of note was that the total numbers of these cells differed between individual WT aged mice, and that the percentage of peritoneal resident macrophages varied from 5 to 20% in WT aged mice, whereas the percentage in WT young mice was constantly ∼50% (data not shown). Such variations interfered with analysis of in vivo inflammatory responses induced by dead cells in WT aged mice.
Decline in the phagocytic capacity in senescence-accelerated mice (SMP30−/− mice) fed a VC-limited diet Based on the results described in the previous section, we decided to make use of senescence-accelerated mice (SMP30−/−) in our experiments, because it was reported previously that these mice do not synthesize VC, and that they show accelerated senescence when fed a VC-limited diet as described in the Materials and Methods. |
3
R Takahashi et al.
Figure 2 Phagocytosis of secondary necrotic neutrophils by peritoneal resident macrophages from wild-type (WT) young mice and WT aged mice. (a) Images of phagocytosis of secondary necrotic neutrophils (red) by peritoneal resident macrophages (green). Peritoneal resident macrophages from WT young (5–7 weeks-of-age) and WT aged (18–24 months-of-age) mice were cocultured with PKH26-labeled secondary necrotic neutrophils for 6 h. After staining of macrophages with fluorescein isothiocyanate-labeled anti-F4/80 monoclonal antibody, the macrophages were examined under a fluorescence microscope. The macrophages that did not engulf secondary necrotic cells are indicated by arrows. (b) Percentages of phagocytosis were calculated. All results are expressed as means ± standard error (n = 3–4). *P < 0.001 versus WT young mice.
The CD11b/F4/80 profile of PEC from SMP30−/−mice fed a VC-limited diet was similar to that of WT young mice. The total cell number and percentage of macrophages among PEC of SMP30−/−mice fed the VC-limited diet were similar to those in WT young mice, and a similar number of cells other than macrophages was detected in SMP30−/−mice fed the VC-limited diet as in WT young mice (data not shown). Therefore, we assumed that analysis of in vivo inflammatory responses induced by secondary necrotic neutrophils was possible in SMP30−/−mice fed the VC-limited diet. We then compared the phagocytic capacity of resident peritoneal macrophages from SMP30−/−mice fed the VC-limited diet as to secondary necrotic neutrophils with that of ones from WT young mice. As shown in Figure 3, the percentages of phagocytosis 3 h and 6 h after cocuturing were significantly diminished for macrophages from SMP30−/−mice fed the VC-limited diet relative to those for ones from WT young mice, 4
|
although the percentage of phagocytosis 1 h after coculturing did not differ between the two groups of mice. These results suggested that the phagocytic capacity of resident peritoneal macrophages similarly decreased in SMP30−/−mice fed the VC-limited diet as in WT aged mice.
Inflammatory responses were induced in SMP30−/−mice fed a VC-limited diet more strongly than in WT young mice We then investigated the in vivo responses to secondary necrotic cells in SMP30−/−mice fed a VC-limited diet and WT young mice. PKH26 Red-labeled secondary necrotic neutrophils were injected into the peritoneal cavity of WT young mice and SMP30−/−mice fed the VC-limited diet, and then PEC were collected at various times after injection. Secondary necrotic neutrophils remained in the SMP30−/−mice fed the VC-limited diet for a longer time © 2015 Japan Geriatrics Society
Effect of aging on inflammatory responses
Figure 3 Phagocytosis of secondary necrotic neutrophils by peritoneal resident macrophages from wild-type (WT) young mice and SMP30−/− mice fed a vitamin C (VC)-limited diet. Peritoneal resident macrophages from WT young and SMP30−/− mice fed a VC-limited diet were cocultured with PKH26 Red-labeled secondary necrotic neutrophils for the times shown. After staining of macrophages with fluorescein isothiocyanate-labeled anti-F4/80 monoclonal antibody, the macrophages were examined under a fluorescence microscope. Percentages of phagocytosis were calculated. All results are expressed as means ± standard error (n = 3). *P < 0.05, **P < 0.001 versus WT young mice.
Figure 4 Secondary necrotic neutrophils remained for a longer time in SMP30−/− mice fed a vitamin C (VC)-limited diet as compared with wild-type (WT) young mice. After secondary necrotic neutrophils had been injected into the peritoneal cavity of WT young and SMP30−/− mice fed a VC-limited diet, peritoneal exudate cells were collected at the times shown. The remaining secondary necrotic neutrophils were identified as PKH26 Red-highly positive cells by flow cytometry. The results are expressed as means ± standard error (n = 3). *P < 0.01, **P < 0.001 versus WT young mice.
as compared with in WT young mice (Fig. 4). We then investigated the infiltration of neutrophils into the peritoneal cavity after injection of secondary necrotic cells. In WT young mice, the infiltration of neutrophils reached a peak at 12 h, whereas the infiltration in SMP30−/−mice fed the VC-limited diet occurred earlier than in WT young mice, peaking at 6 h (Fig. 5a). Furthermore, the number of neutrophils in SMP30−/−mice fed the VC-limited diet at the peak time (6 h) was greater © 2015 Japan Geriatrics Society
Figure 5 Inflammatory responses induced by the injection of secondary necrotic cells into wild-type (WT) young mice and SMP30−/− mice fed a vitamin C (VC)-limited diet. (a) A time-dependent change in the number of neutrophils that had infiltrated into the peritoneal cavity on injection of secondary necrotic cells. After secondary necrotic neutrophils had been injected into the peritoneal cavity of WT young mice and SMP30−/− mice fed a VC-limited diet, peritoneal exudate cells were collected at the times shown. The infiltrated neutrophils were identified as Gr-1highCD11bhigh cells by flow cytometry. (b) Time kinetics of MIP-2 production induced by injection of secondary necrotic cells. Peritoneal lavage fluid was collected at the indicated times after injection of secondary necrotic neutrophils, followed by determination of the levels of macrophage inflammatory protein-2 (MIP-2) by a specific enzyme-linked immunosorbent assay. The results are expressed as means ± standard error (n = 3). *P < 0.01 versus WT young mice.
than that in WT young mice at the same time (6 h). We then measured the MIP-2 levels in the peritoneal cavity in order to determine whether or not the difference in neutrophil infiltration was caused by that in MIP-2, a chemoattractant of neutrophils. MIP-2 was produced earlier in SMP30−/−mice fed the VC-limited diet than in WT young mice, and the MIP-2 level at the peak time was greater in SMP30−/−mice fed the VC-limited diet than in WT young mice (Fig. 5b). Furthermore, the number of neutrophils in WT aged mice at the peak time (6 h) was greater that that in WT young mice at the same time (6 h), and the MIP-2 level at the peak time |
5
R Takahashi et al.
Table 2 Inflammatory responses induced by the injection of secondary necrotic cells into the indicated mice
Neutrophils (×105) MIP-2 (ng/mouse)
WT young
WT aged
SMP30−/−
37.09 ± 7.12 0.83 ± 0.27
100.20 ± 13.57* 3.60 ± 0.62*
152.47 ± 61.89** 2.36 ± 0.27*
The results are expressed as mean ± standard error (n = 3). *P < 0.001, **P < 0.05 versus wild-type (WT) young mice. After secondary necrotic neutrophils had been injected into the peritoneal cavity of WT young mice, WT aged mice and SMP30−/− mice fed a vitamin C-limited diet, peritoneal exudate cells and peritoneal lavage fluid were collected at 6 h and 2 h, respectively. The infiltrated neutrophils were identified as Gr-1highCD11b high cells among living cells by flow cytometry. The levels of macrophage inflammatory protein-2 (MIP-2) were measured using a specific enzyme-linked immunosorbent assay.
(2 h) was greater in WT aged mice than in WT young mice (Table 2). These results suggested that when secondary necrotic cells were injected into SMP30−/− mice fed the VC-limited diet and WT aged mice, the inflammatory responses induced were strong, presumably because of the delay in removal of secondary necrotic cells.
Discussion We and other researchers had previously shown that secondary necrotic cells generated in vivo caused strong inflammatory responses and many diseases; for example, autoimmune disease, such as systematic lupus erythematosus.9,12,18,19 It has been known that the function of the immune system decreases with age, leading to increased susceptibility of the elderly to infections by viruses and bacteria.10,14 In the present study, we hypothesize that if secondary necrotic cells remain as a result of a decrease in phagocytic capacity in WT aged mice, secondary necrotic cells might induce strong inflammatory responses in the mice because of continuous stimulation by secondary necrotic cells. First, as shown in Figure 2, the phagocytic capacity of peritoneal resident macrophages prepared from WT aged mice as to secondary necrotic neutrophils was decreased as compared with that of peritoneal resident macrophages from WT young mice. Based on this finding, it was expected that the clearance of secondary necrotic cells decreased, and the inflammatory responses induced by secondary necrotic cells were enhanced in WT aged mice in vivo. We then attempted to determine what effects secondary necrotic cells would have on the induced inflammatory responses in WT aged mice. Among PEC, however, cells other than macrophages were detected more in WT aged mice than in WT young mice. Although the forward scatter/side scatter profiles suggest that the cells might be lymphocytes, their identities are not clear at 6
|
present. Such variations interfered with analysis of in vivo inflammatory responses induced by dead cells in WT aged mice. Based on these results, we decided to make use of SMP30−/−mice fed a VC-limited diet in our experiments. As shown in Figure 3, the phagocytic capacity of resident peritoneal macrophages similarly decreased in SMP30−/−mice fed the VC-limited diet as in WT aged mice. We found that the phagocytic capacity of resident peritoneal macrophages from SMP30−/−mice fed the VC-limited diet was not restored by the addition of VC on the coculturing of secondary necrotic cells. As expected, if SMP30−/− mice were fed a sufficient amount of VC, the phagocytic capacity of resident peritoneal macrophages of such mice was comparable with that of resident peritoneal macrophages from WT young mice (data not shown). Furthermore, it was reported previously that only a small amount of SMP-30 was expressed even in WT peripheral blood cells containing monocytes as compared with non blood cells.18 These findings suggest that the decrease in phagocytic capacity was a result of the acceleration of senescence, but not VC insufficiency caused by the deficiency of SMP-30 and the VC-limited diet. We next investigated whether or not secondary necrotic neutrophils injected into the peritoneal cavity remained in SMP30−/−mice fed a VC-limited diet. As expected, secondary necrotic neutrophils remained for a long time in the SMP30−/−mice fed the VC-limited diet, but they disappeared in the WT young mice immediately in vivo (Fig. 4). Although the percentage of phagocytosis was less than 10% in WT young mice 1 h after coculturing of secondary necrotic neutrophils with resident peritoneal macrophages (in vitro assay), more than 80% of the injected secondary necrotic neutrophils had been removed by 1 h after injection (in vivo assay) in WT young mice. The discrepancy between the in vitro and in vivo results might be explained by the capacity of phagocytosis being lower in vitro than in vivo. Although there is the possibility that the injected secondary necrotic neutrophils diffused to outside of the © 2015 Japan Geriatrics Society
Effect of aging on inflammatory responses
peritoneal cavity, it is hard to imagine that a deficiency of SMP-30 has some effect on such diffusion of secondary necrotic cells. Taken together, the present results showed that more secondary necrotic cells remained as a result of a decrease in the capacity of phagocytosis of resident peritoneal macrophages in SMP30−/−mice fed a VC-limited diet. The remaining secondary necrotic cells would release danger-associated molecular patterns, such as high mobility group box-1 and S100 A8/A9, and thereby induce various inflammatory responses. Indeed, the peak time of neutrophil infiltration was earlier in SMP30−/− mice fed a VC-limited diet than WT young mice, a greater amount of MIP-2 being produced in SMP30−/− mice fed the VC-limited diet. These results suggested that when secondary necrotic cells were injected into SMP30−/− fed the VC-limited diet, the inflammatory responses induced were strong, presumably because of the delay in the removal of secondary necrotic cells. Apoptotic cells at an early stage are removed quickly by macrophages, which is not associated with neutrophil infiltration, one of the key inflammatory responses. In contrast, secondary necrotic cells are generated if such apoptotic cells are not completely removed. Our present results showed that injection of secondary necrotic cells caused inflammatory responses, and that the inflammatory responses were stronger in aged mice than those in young mice. Although we have no evidence yet that apoptotic cells at an early stage become secondary necrotic cells in vivo, it is expected that such apoptotic cells could proceed to a secondary necrotic stage after 1 h in SMP30−/− mice fed the VC-limited diet, because apoptotic neutrophils became secondary necrotic neutrophils within 1 h in our in vitro experiments.9 Phagocytosis of apoptotic cells at an early stage might also be impaired in aged mice. It is possible, therefore, that early apoptotic cells may enhance production of MIP-2 and subsequently exaggerate neutrophil infiltration in aged mice. In contrast, we cannot exclude the possibility that early apoptotic cells are removed in aged mice as efficiently as in young mice. Further studies are required to clarify this point. Finally, why is the capacity of phagocytosis decreased in WT aged mice and SMP30−/−mice fed a VC-limited diet? Because our preliminary results showed that peritoneal resident macrophages in WT aged and SMP30−/−mice fed a VC-limited diet are pre-activated so that MIP-2 production does not require interferon-γ as a costimulant, the phagocytic capacity of macrophages might be decreased as a result of such activation. Another possibility is that a change from M2 type to M1 type of peritoneal resident macrophages of aged mice might result in a decrease in phagocytic capacity, because we previously showed that the phagocytic © 2015 Japan Geriatrics Society
capacity of alveolar macrophages, M1-like macrophages, as to apoptotic cells was lower than that of peritoneal resident macrophages, M2-like macrophages, and because it was also reported that the number of M1 macrophages significantly increased in the peritoneal cavity of WT aged mice.20,21 The mechanism underlying the decrease in phagocytic capacity needs to be studied more extensively. In conclusion, the present study showed that phagocytosis of secondary necrotic cells by macrophages from WT aged and SMP30−/−mice fed a VC-limited diet was significantly reduced as compared with that by macrophages from WT young mice, and that the inflammatory responses were induced strongly by the injection of secondary necrotic cells. If we could identify a way to restore phagocytic activity of resident peritoneal macrophages in aged mice, then we could ameliorate age-associated disorders, such as systematic lupus erythematosus and rheumatoid arthritis.
Acknowledgements This work was supported partly by Grants-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology.
Disclosure statement The authors declare no conflict of interest.
References 1 Shibata T, Nagata K, Kobayashi Y. Cutting edge: A critical role of nitrogen oxide in preventing inflammation upon apoptotic cell clearance. J Immunol 2007; 179: 3402–3406. 2 Manuel T, do Vale A, dos Santos NM. Secondary necrosis in multicellular animal: an outcome of apoptosis with pathogenic implications. Apoptosis 2008; 13: 463–842. 3 Iyoda T, Kobayashi Y. Involvement of MIP-2 and CXCR2 in neutrophil infiltration following injection of late apoptotic cells into peritoneal cavity. Apoptosis 2004; 9: 485–493. 4 Uchimura E, Kodaira T, Kurosaka K, Yang D, Watanabe N, Kobayashi Y. Interaction of phagocytes with apoptotic cells leads to production of pro-inflammatory cytokines. Biochem Biophys Res Commun 1997; 239: 799–803. 5 Kawagishi C, Kurosaka K, Watanabe N, Kobayashi Y. Cytokines production of macrophages in association with phagocytosis of etoposide-treated P388 cells in vitro and in vivo. Biochem Biophys Acta 2001; 1541: 221–230. 6 Kurosaka K, Watanabe N, Kobayashi Y. Production of proinflammatory cytokines by phorbol myristate acetatetreated THP-1 cells and monocyte-derived macrophages after phagocytosis of apoptotic CTLL-2 cells. J Immunol 1998; 161: 6249–6254. 7 Kurosaka K, Watanabe N, Kobayashi Y. Production of proinflammatory cytokines by resident tissue macrophages after phagocytosis of apoptotic CTLL-2 cells. Cell Immunol 2001; 211: 1–7.
|
7
R Takahashi et al. 8 Misawa R, Kawagishi C, Watanabe N, Kobayashi Y. Infiltration of neutrophils following injection of apoptotic cells into the peritoneal cavity. Apoptosis 2001; 6: 411– 417. 9 Fadok VA, Bratton DL, Guthrie L, Henson PM. Differential effects of apoptotic versus lysed cell on macrophage production of cytokines: role of protease. J Immunol 2001; 166: 6847–6854. 10 Miller RA. The aging immune system: primer and prospectus. Science 1996; 273: 70–74. 11 Tanimoto N, Terasawa M, Nakamura M et al. Involvement of KC, MIP-2, and MCP-1 in leukocyte infiltration following injection of necrotic cells into the peritoneal cavity. Biochem Biophys Res Commun 2007; 361: 533–536. 12 Su YJ, Cheng TT, Chen CJ et al. The association among leukocyte apoptosis, autoantibodies and disease severity in systemic lupus erythematosus. J Transl Med 2013; 11: 261– 267. 13 Chorinchath BB, Kong L, Mao L, McCallum RE. Ageassociated differences in TNF-α and nitric oxide production in endotoxic mice. J Immunol 1996; 156: 1525–1530. 14 Effros RB, Walford RL. The effect of age on the antigenpresenting mechanism in limiting dilution precursor cell frequency analysis. Cell Immunol 1984; 88: 531–539. 15 Ito Y, Betsuyaku T, Nasuhara Y, Nishimura M. Lipopolysaccaride-Induced Neutrophilic Inflammation in
8
|
16 17
18 19
20
21
the Lungs Differs with age. Exp Lung Res 2007; 33: 375– 384. Maruyama N, Ishigami A, Kuramoto M et al. Senescence marker protein-30 knockout mouse as an aging model. Ann N Y Acad Sci 2004; 1019: 383–387. Smallwood HS, López-Ferrer D, Squier TC. Aging enhances the production of reactive oxygen species by upregulating classical activation pathways. Biochemistry 2011; 50: 9911–9922. Feng D, Kondo Y, Ishigami A, Kuramoto M, Machida T, Maruyama N. Senescence marker protein-30 as a novel antiaging molecule. Ann N Y Acad Sci 2004; 1019: 360–364. Shibata T, Nagata K, Kobayashi Y. The mechanism underlying the appearance of late apoptotic neutrophils and subsequent TNF-α production at a late stage during Staphylococcus aureus bioparticle-induced peritoneal inflammation in inducible NO synthase-deficient mice. Biochem Biophys Acta 2010; 1802: 1105–1111. Yamazaki T, Nagata K, Kobayashi Y. Cytokine production by M-CSF- and GM-CSF-induced mouse bone marrowderived macrophages upon coculturing with late apoptotic cells. Cell Immunol 2008; 251: 124–130. Jackaman C, Radley-Crabb HG, Soffe Z, Shavlakadze T, Grounds MD, Nelson DJ. Targeting macrophages rescues age-related immune deficiencies in C57BL/6J geriatric mice. Aging Cell 2013; 12: 345–357.
© 2015 Japan Geriatrics Society
Geriatr Gerontol Int 2015
ORIGINAL ARTICLE: BIOLOGY
Attenuated phagocytosis of secondary necrotic neutrophils by macrophages in aged and SMP30 knockout mice Rei Takahashi,1 Sayuri Totsuka,1 Akihito Ishigami,2 Yoshiro Kobayashi1 and Kisaburo Nagata1 1 Department of Biomolecular Science, Faculty of Science, Toho University, Chiba, and 2Aging Regulation, Tokyo Metropolitan Institute of Gerontology, Tokyo, Japan
Aim: Secondary necrotic cells generated in vivo induce inflammatory responses; for example, the production of macrophage inflammatory protein-2 (MIP-2) and subsequent infiltration of neutrophils. The aim of the present study was to elucidate the effect of aging on the phagocytosis of secondary necrotic cells and the inflammatory responses by using either wild-type (WT) young mice, WT aged mice or senescence-accelerated mice (SMP30−/− mice). Methods: The phagocytosis of secondary necrotic neutrophils with resident macrophage from either WT young mice, WT aged mice or SMP30−/− mice was examined by coculturing macrophages with secondary necrotic neutrophils in vitro. To investigate the inflammatory response induced by secondary necrotic cells, time-dependent infiltration of neutrophils and production of MIP-2 were determined in the peritoneal cavity on the injection of secondary necrotic cells. Results: The phagocytosis of secondary necrotic cells by macrophages from WT aged and SMP30−/− mice was significantly reduced as compared with that by macrophages from WT young mice. On peritoneal injection of secondary necrotic cells, the peak time of neutrophil infiltration was earlier in SMP30−/− mice than in WT young mice. The number of neutrophils in SMP30−/− mice at the peak time was also greater than that in WT young mice. Conclusions: Our findings showed that the phagocytosis of secondary necrotic cells was attenuated in aged mice and SMP30−/− mice, and that the MIP-2 production was enhanced and subsequently neutrophil infiltration was exaggerated on peritoneal injection of secondary necrotic cells into those mice. Geriatr Gerontol Int 2015; ••: ••–••. Keywords: aging, apoptosis, chemokines, inflammation, macrophages.
Introduction Apoptotic cells at an early stage are removed quickly by macrophages, which is not associated with neutrophil infiltration, one of the key inflammatory responses.1 In contrast, if such apoptotic cells are not completely removed, then secondary necrotic cells are generated. Of note is that primary necrotic cells are not generated by early apoptotic cells. Secondary necrotic cells generated in vivo cause various acute inflammatory diseases, such as acute lung injury.2 In support of this, we previously showed that many neutrophils infiltrated into the thymus when sec-
Accepted for publication 30 October 2014. Correspondence: Dr Kisaburo Nagata, PhD, Division of Molecular Medicine, Department of Biomolecular Science, Faculty of Science, Toho University, 2-2-1 Miyama, Funabashi, Chiba 274-8510, Japan. Email: [email protected]
© 2015 Japan Geriatrics Society
ondary necrotic cells were acutely generated in the thymus on whole-body X-irradiation.3,4 Furthermore, we previously reported that the expression of a chemokine specific to neutrophils, interleukin-8 or macrophage inflammatory protein-2 (MIP-2), was upregulated on in vitro coculturing of macrophages with secondary necrotic cells, and that injection of secondary necrotic cells into the mouse peritoneal cavity caused the production of MIP-2 and infiltration of neutrophils, and that anti-MIP-2 antibodies as well as anti-CXCR2 antibodies significantly suppressed the neutrophil infiltration, suggesting that secondary necrotic cells induce neutrophil infiltration mainly through MIP-2 in vivo.5–11 Secondary necrotic cells also cause chronic inflammatory diseases, because Su et al. previously reported that repeated injection of secondary necrotic cells caused autoimmune diseases, such as systematic lupus erythematosus.12 In contrast, the function of the immune system decreases with age, leading to increased susceptibility of doi: 10.1111/ggi.12436
|
1
R Takahashi et al.
the elderly to infections by viruses and bacteria.13,14 Previous studies showed that virus-specific T-cell responses are decreased in aged animals, and that the production of inflammatory mediators, such as tumor necrosis factor-α and nitric oxide, and dangerassociated molecular patterns released from secondary necrotic cells, such as high mobility group box-1, was increased in aged mice when lipopolysaccharide was injected into aged animals.15–17 However, few studies have been undertaken on how aging affects the inflammatory responses induced by secondary necrotic cells. In the present study, we investigated the effect of aging on the inflammatory responses induced by injection of secondary necrotic neutrophils into the peritoneal cavity using young (5–7 weeks-of-age) and aged (18–24 months-of-age) C57BL/6 mice, and SMP30/GNLknockout (SMP30−/−) mice fed a vitamin C (VC)-limited diet.
Materials and methods Mice Young C57BL/6 mice (5–7 weeks-of-age) were purchased from Sankyo Lab Service (Tokyo, Japan). Aged C57BL/6 mice (18–24 months-of-age) were bred in the Tokyo Metropolitan Institute of Gerontology. SMP30/ GNL-knockout (SMP30−/−) mice deficient in VC synthesis were provided by Dr Akihito Ishigami, and were bred at Toho University, Chiba, Japan.18 The SMP30−/− mice had free access to water containing a sufficient amount of VC (1.5 g/L) until 4 weeks-of-age. To obtain aged mice, mice had free access to water containing 0.0375 g/L of VC until 7 weeks-of-age.
Preparation of secondary necrotic neutrophils To obtain neutrophils, a thioglycollate broth solution (2 mL) was injected into the peritoneal cavity of C57BL/6 mice. After 6 h, peritoneal exudate cells (PEC) were collected. The PEC comprised more than 95.0% of neutrophils, which were identified as a Gr-1highCD11bhigh population by flow cytometry analysis. The cells were thereafter regarded as neutrophils in the present study, unless otherwise stated. The neutrophils were washed with phosphate-buffered saline (PBS; saline containing 14 mmol/L Na2PO4 and 6 mmol/L KH2PO4, pH 7.4) twice, and then suspended in RPMI 1640 medium containing 7% fetal calf serum at a cell density of 2 × 106 cells/mL. To induce secondary necrosis, the neutrophils were cultured for various times at 37°C. The number of secondary necrotic neutrophils, identified as Annexin V- and propidium iodide (PI)positive cells, peaked at 12 h. The percentage of Annexin V+/PI− neutrophils, early apoptotic neutrophils and that of Annexin V+/PI+ neutrophils, secondary 2
|
Table 1 Annexin V/PI staining of untreated and 12 h-cultured neutrophils
−
−
Annexin V /PI (living cells) Annexin V+/PI− (early apoptotic cells) Annexin V+/PI+ (secondary necrotic cells)
Untreated
12 h-cultured
88.92 ± 2.41
47.07 ± 3.08
1.89 ± 0.39
17.23 ± 1.31
3.85 ± 0.65
27.03 ± 1.70
All results are expressed as means ± standard error (n = 3–4). Untreated and 12 h-cultured neutrophils were stained with Annexin V and PI. The percentages of the indicated neutrophils among total neutrophils were determined by flow cytometry.
necrotic neutrophils, in untreated and 12 h-cultured neutrophils are shown in Table 1. On incubation for over 12 h, many more neutrophils with permeable membranes (Annexin V−/PI+), primary necrotic neutrophils, appeared. Therefore, the neutrophils cultured for 12 h were used as secondary necrotic neutrophils in the present study.
Flow cytometric analysis of macrophages among PEC and preparation of peritoneal resident macrophages For analysis of macrophages among PEC, PEC were obtained by lavage of the peritoneal cavity of wild-type (WT) young, WT aged or SMP30−/− mice fed a VC-limited diet with cold PBS, and they were stained with anti-F4/80 monoclonal antibody (mAb), antiCD11b mAb. The stained cells were analyzed by flow cytometry. For preparation of peritoneal resident macrophages, PEC were obtained from each mouse with cold PBS, and then the cells that were adhered to a plastic plate were collected. The collected cells comprised more than 98% of macrophages, as determined by staining with anti-F4/80 mAb.
Phagocytosis of secondary necrotic neutrophils by resident peritoneal macrophages in vitro Secondary necrotic neutrophils prepared as aforementioned were labeled with PKH26 Red fluorescent dye, followed by coculturing with resident macrophages, which had been allowed to adhere to a plastic plate beforehand. Then we removed non-trapped apoptotic neutrophils by washing, and stained resident macrophages with F4/80 mAb and examined then under a fluorescence microscope. © 2015 Japan Geriatrics Society
Effect of aging on inflammatory responses
Identification of free secondary necrotic neutrophils in the peritoneal cavity Secondary necrotic neutrophils were stained with PKH26 Red fluorescent dye, and then injected into the peritoneal cavity. After various times, PEC were collected with cold PBS. Free secondary necrotic neutrophils, which were not phagocytosed in the peritoneal cavity, were identified as PKH26 Red-highly positive cells, in which the intensity of PKH26 Red staining was similar to that in the injected cells.
Flow cytometric analysis of infiltrated neutrophils PKH26 Red-labeled secondary necrotic neutrophils were injected into the peritoneal cavity of each mouse. At various times, PEC were collected, and stained with anti-Gr-1 mAb and anti-CD11b mAb. The stained cells were analyzed by flow cytometry with a FACSCalibur (BD Biosciences, San Jose, CA, USA) with which PKH26 Red-highly positive cells, free secondary necrotic neutrophils, were excluded by gating.
Measurement of MIP-2 To measure the protein level of MIP-2 in the peritoneal cavity, supernatants of peritoneal lavage fluid were prepared at various times after the injection of secondary necrotic neutrophils. To measure the protein level of MIP-2 in the cocultures of resident peritoneal macrophages with secondary necrotic neutrophils, supernatants of the cocultures were prepared at various times. The MIP-2 levels were determined using an enzymelinked immunosorbent assay development kit.
Results Effects of aging on the total number, percentage and phagocytic capacity of peritoneal resident macrophages To investigate whether or not aging has any effects on the total number and percentage of peritoneal resident macrophages, we obtained resident macrophages from the peritoneal cavity of WT young and WT aged mice. The macrophages were identified as CD11bhigh and F4/80high. As shown in Figure 1a,b, the total numbers of macrophages did not significantly differ between WT young and WT aged mice, whereas the percentage of macrophages in the peritoneal cavity was significantly smaller in WT aged mice than in WT young mice. Next, we investigated the phagocytic capacity of resident peritoneal macrophages as to secondary necrotic cells. On coculturing for 6 h, resident peritoneal macrophages that did not engulf secondary necrotic neutrophils, indicated by arrows, were detected more in WT aged mice © 2015 Japan Geriatrics Society
Figure 1 Flow cytometric analysis of peritoneal resident cells from wild-type (WT) young mice and WT aged mice. (a) Total numbers of peritoneal resident macrophages in WT young (5–7 weeks-of-age) and WT aged (18–24 months-of-age) mice. (b) Percentage of macrophages among peritoneal resident cells. All results are expressed as means ± standard error (n = 3–4). *P < 0.001 versus WT young mice. NS, not significant.
than in WT young mice (Fig. 2a). A time-dependent change in the percentage of phagocytosis during the coculture was shown in Figure 2b. The percentage of phagocytosis in WT aged mice was significantly decreased as compared with that in WT young mice. Among PEC, however, cells other than macrophages were detected more in WT aged mice than in WT young mice. Of note was that the total numbers of these cells differed between individual WT aged mice, and that the percentage of peritoneal resident macrophages varied from 5 to 20% in WT aged mice, whereas the percentage in WT young mice was constantly ∼50% (data not shown). Such variations interfered with analysis of in vivo inflammatory responses induced by dead cells in WT aged mice.
Decline in the phagocytic capacity in senescence-accelerated mice (SMP30−/− mice) fed a VC-limited diet Based on the results described in the previous section, we decided to make use of senescence-accelerated mice (SMP30−/−) in our experiments, because it was reported previously that these mice do not synthesize VC, and that they show accelerated senescence when fed a VC-limited diet as described in the Materials and Methods. |
3
R Takahashi et al.
Figure 2 Phagocytosis of secondary necrotic neutrophils by peritoneal resident macrophages from wild-type (WT) young mice and WT aged mice. (a) Images of phagocytosis of secondary necrotic neutrophils (red) by peritoneal resident macrophages (green). Peritoneal resident macrophages from WT young (5–7 weeks-of-age) and WT aged (18–24 months-of-age) mice were cocultured with PKH26-labeled secondary necrotic neutrophils for 6 h. After staining of macrophages with fluorescein isothiocyanate-labeled anti-F4/80 monoclonal antibody, the macrophages were examined under a fluorescence microscope. The macrophages that did not engulf secondary necrotic cells are indicated by arrows. (b) Percentages of phagocytosis were calculated. All results are expressed as means ± standard error (n = 3–4). *P < 0.001 versus WT young mice.
The CD11b/F4/80 profile of PEC from SMP30−/−mice fed a VC-limited diet was similar to that of WT young mice. The total cell number and percentage of macrophages among PEC of SMP30−/−mice fed the VC-limited diet were similar to those in WT young mice, and a similar number of cells other than macrophages was detected in SMP30−/−mice fed the VC-limited diet as in WT young mice (data not shown). Therefore, we assumed that analysis of in vivo inflammatory responses induced by secondary necrotic neutrophils was possible in SMP30−/−mice fed the VC-limited diet. We then compared the phagocytic capacity of resident peritoneal macrophages from SMP30−/−mice fed the VC-limited diet as to secondary necrotic neutrophils with that of ones from WT young mice. As shown in Figure 3, the percentages of phagocytosis 3 h and 6 h after cocuturing were significantly diminished for macrophages from SMP30−/−mice fed the VC-limited diet relative to those for ones from WT young mice, 4
|
although the percentage of phagocytosis 1 h after coculturing did not differ between the two groups of mice. These results suggested that the phagocytic capacity of resident peritoneal macrophages similarly decreased in SMP30−/−mice fed the VC-limited diet as in WT aged mice.
Inflammatory responses were induced in SMP30−/−mice fed a VC-limited diet more strongly than in WT young mice We then investigated the in vivo responses to secondary necrotic cells in SMP30−/−mice fed a VC-limited diet and WT young mice. PKH26 Red-labeled secondary necrotic neutrophils were injected into the peritoneal cavity of WT young mice and SMP30−/−mice fed the VC-limited diet, and then PEC were collected at various times after injection. Secondary necrotic neutrophils remained in the SMP30−/−mice fed the VC-limited diet for a longer time © 2015 Japan Geriatrics Society
Effect of aging on inflammatory responses
Figure 3 Phagocytosis of secondary necrotic neutrophils by peritoneal resident macrophages from wild-type (WT) young mice and SMP30−/− mice fed a vitamin C (VC)-limited diet. Peritoneal resident macrophages from WT young and SMP30−/− mice fed a VC-limited diet were cocultured with PKH26 Red-labeled secondary necrotic neutrophils for the times shown. After staining of macrophages with fluorescein isothiocyanate-labeled anti-F4/80 monoclonal antibody, the macrophages were examined under a fluorescence microscope. Percentages of phagocytosis were calculated. All results are expressed as means ± standard error (n = 3). *P < 0.05, **P < 0.001 versus WT young mice.
Figure 4 Secondary necrotic neutrophils remained for a longer time in SMP30−/− mice fed a vitamin C (VC)-limited diet as compared with wild-type (WT) young mice. After secondary necrotic neutrophils had been injected into the peritoneal cavity of WT young and SMP30−/− mice fed a VC-limited diet, peritoneal exudate cells were collected at the times shown. The remaining secondary necrotic neutrophils were identified as PKH26 Red-highly positive cells by flow cytometry. The results are expressed as means ± standard error (n = 3). *P < 0.01, **P < 0.001 versus WT young mice.
as compared with in WT young mice (Fig. 4). We then investigated the infiltration of neutrophils into the peritoneal cavity after injection of secondary necrotic cells. In WT young mice, the infiltration of neutrophils reached a peak at 12 h, whereas the infiltration in SMP30−/−mice fed the VC-limited diet occurred earlier than in WT young mice, peaking at 6 h (Fig. 5a). Furthermore, the number of neutrophils in SMP30−/−mice fed the VC-limited diet at the peak time (6 h) was greater © 2015 Japan Geriatrics Society
Figure 5 Inflammatory responses induced by the injection of secondary necrotic cells into wild-type (WT) young mice and SMP30−/− mice fed a vitamin C (VC)-limited diet. (a) A time-dependent change in the number of neutrophils that had infiltrated into the peritoneal cavity on injection of secondary necrotic cells. After secondary necrotic neutrophils had been injected into the peritoneal cavity of WT young mice and SMP30−/− mice fed a VC-limited diet, peritoneal exudate cells were collected at the times shown. The infiltrated neutrophils were identified as Gr-1highCD11bhigh cells by flow cytometry. (b) Time kinetics of MIP-2 production induced by injection of secondary necrotic cells. Peritoneal lavage fluid was collected at the indicated times after injection of secondary necrotic neutrophils, followed by determination of the levels of macrophage inflammatory protein-2 (MIP-2) by a specific enzyme-linked immunosorbent assay. The results are expressed as means ± standard error (n = 3). *P < 0.01 versus WT young mice.
than that in WT young mice at the same time (6 h). We then measured the MIP-2 levels in the peritoneal cavity in order to determine whether or not the difference in neutrophil infiltration was caused by that in MIP-2, a chemoattractant of neutrophils. MIP-2 was produced earlier in SMP30−/−mice fed the VC-limited diet than in WT young mice, and the MIP-2 level at the peak time was greater in SMP30−/−mice fed the VC-limited diet than in WT young mice (Fig. 5b). Furthermore, the number of neutrophils in WT aged mice at the peak time (6 h) was greater that that in WT young mice at the same time (6 h), and the MIP-2 level at the peak time |
5
R Takahashi et al.
Table 2 Inflammatory responses induced by the injection of secondary necrotic cells into the indicated mice
Neutrophils (×105) MIP-2 (ng/mouse)
WT young
WT aged
SMP30−/−
37.09 ± 7.12 0.83 ± 0.27
100.20 ± 13.57* 3.60 ± 0.62*
152.47 ± 61.89** 2.36 ± 0.27*
The results are expressed as mean ± standard error (n = 3). *P < 0.001, **P < 0.05 versus wild-type (WT) young mice. After secondary necrotic neutrophils had been injected into the peritoneal cavity of WT young mice, WT aged mice and SMP30−/− mice fed a vitamin C-limited diet, peritoneal exudate cells and peritoneal lavage fluid were collected at 6 h and 2 h, respectively. The infiltrated neutrophils were identified as Gr-1highCD11b high cells among living cells by flow cytometry. The levels of macrophage inflammatory protein-2 (MIP-2) were measured using a specific enzyme-linked immunosorbent assay.
(2 h) was greater in WT aged mice than in WT young mice (Table 2). These results suggested that when secondary necrotic cells were injected into SMP30−/− mice fed the VC-limited diet and WT aged mice, the inflammatory responses induced were strong, presumably because of the delay in removal of secondary necrotic cells.
Discussion We and other researchers had previously shown that secondary necrotic cells generated in vivo caused strong inflammatory responses and many diseases; for example, autoimmune disease, such as systematic lupus erythematosus.9,12,18,19 It has been known that the function of the immune system decreases with age, leading to increased susceptibility of the elderly to infections by viruses and bacteria.10,14 In the present study, we hypothesize that if secondary necrotic cells remain as a result of a decrease in phagocytic capacity in WT aged mice, secondary necrotic cells might induce strong inflammatory responses in the mice because of continuous stimulation by secondary necrotic cells. First, as shown in Figure 2, the phagocytic capacity of peritoneal resident macrophages prepared from WT aged mice as to secondary necrotic neutrophils was decreased as compared with that of peritoneal resident macrophages from WT young mice. Based on this finding, it was expected that the clearance of secondary necrotic cells decreased, and the inflammatory responses induced by secondary necrotic cells were enhanced in WT aged mice in vivo. We then attempted to determine what effects secondary necrotic cells would have on the induced inflammatory responses in WT aged mice. Among PEC, however, cells other than macrophages were detected more in WT aged mice than in WT young mice. Although the forward scatter/side scatter profiles suggest that the cells might be lymphocytes, their identities are not clear at 6
|
present. Such variations interfered with analysis of in vivo inflammatory responses induced by dead cells in WT aged mice. Based on these results, we decided to make use of SMP30−/−mice fed a VC-limited diet in our experiments. As shown in Figure 3, the phagocytic capacity of resident peritoneal macrophages similarly decreased in SMP30−/−mice fed the VC-limited diet as in WT aged mice. We found that the phagocytic capacity of resident peritoneal macrophages from SMP30−/−mice fed the VC-limited diet was not restored by the addition of VC on the coculturing of secondary necrotic cells. As expected, if SMP30−/− mice were fed a sufficient amount of VC, the phagocytic capacity of resident peritoneal macrophages of such mice was comparable with that of resident peritoneal macrophages from WT young mice (data not shown). Furthermore, it was reported previously that only a small amount of SMP-30 was expressed even in WT peripheral blood cells containing monocytes as compared with non blood cells.18 These findings suggest that the decrease in phagocytic capacity was a result of the acceleration of senescence, but not VC insufficiency caused by the deficiency of SMP-30 and the VC-limited diet. We next investigated whether or not secondary necrotic neutrophils injected into the peritoneal cavity remained in SMP30−/−mice fed a VC-limited diet. As expected, secondary necrotic neutrophils remained for a long time in the SMP30−/−mice fed the VC-limited diet, but they disappeared in the WT young mice immediately in vivo (Fig. 4). Although the percentage of phagocytosis was less than 10% in WT young mice 1 h after coculturing of secondary necrotic neutrophils with resident peritoneal macrophages (in vitro assay), more than 80% of the injected secondary necrotic neutrophils had been removed by 1 h after injection (in vivo assay) in WT young mice. The discrepancy between the in vitro and in vivo results might be explained by the capacity of phagocytosis being lower in vitro than in vivo. Although there is the possibility that the injected secondary necrotic neutrophils diffused to outside of the © 2015 Japan Geriatrics Society
Effect of aging on inflammatory responses
peritoneal cavity, it is hard to imagine that a deficiency of SMP-30 has some effect on such diffusion of secondary necrotic cells. Taken together, the present results showed that more secondary necrotic cells remained as a result of a decrease in the capacity of phagocytosis of resident peritoneal macrophages in SMP30−/−mice fed a VC-limited diet. The remaining secondary necrotic cells would release danger-associated molecular patterns, such as high mobility group box-1 and S100 A8/A9, and thereby induce various inflammatory responses. Indeed, the peak time of neutrophil infiltration was earlier in SMP30−/− mice fed a VC-limited diet than WT young mice, a greater amount of MIP-2 being produced in SMP30−/− mice fed the VC-limited diet. These results suggested that when secondary necrotic cells were injected into SMP30−/− fed the VC-limited diet, the inflammatory responses induced were strong, presumably because of the delay in the removal of secondary necrotic cells. Apoptotic cells at an early stage are removed quickly by macrophages, which is not associated with neutrophil infiltration, one of the key inflammatory responses. In contrast, secondary necrotic cells are generated if such apoptotic cells are not completely removed. Our present results showed that injection of secondary necrotic cells caused inflammatory responses, and that the inflammatory responses were stronger in aged mice than those in young mice. Although we have no evidence yet that apoptotic cells at an early stage become secondary necrotic cells in vivo, it is expected that such apoptotic cells could proceed to a secondary necrotic stage after 1 h in SMP30−/− mice fed the VC-limited diet, because apoptotic neutrophils became secondary necrotic neutrophils within 1 h in our in vitro experiments.9 Phagocytosis of apoptotic cells at an early stage might also be impaired in aged mice. It is possible, therefore, that early apoptotic cells may enhance production of MIP-2 and subsequently exaggerate neutrophil infiltration in aged mice. In contrast, we cannot exclude the possibility that early apoptotic cells are removed in aged mice as efficiently as in young mice. Further studies are required to clarify this point. Finally, why is the capacity of phagocytosis decreased in WT aged mice and SMP30−/−mice fed a VC-limited diet? Because our preliminary results showed that peritoneal resident macrophages in WT aged and SMP30−/−mice fed a VC-limited diet are pre-activated so that MIP-2 production does not require interferon-γ as a costimulant, the phagocytic capacity of macrophages might be decreased as a result of such activation. Another possibility is that a change from M2 type to M1 type of peritoneal resident macrophages of aged mice might result in a decrease in phagocytic capacity, because we previously showed that the phagocytic © 2015 Japan Geriatrics Society
capacity of alveolar macrophages, M1-like macrophages, as to apoptotic cells was lower than that of peritoneal resident macrophages, M2-like macrophages, and because it was also reported that the number of M1 macrophages significantly increased in the peritoneal cavity of WT aged mice.20,21 The mechanism underlying the decrease in phagocytic capacity needs to be studied more extensively. In conclusion, the present study showed that phagocytosis of secondary necrotic cells by macrophages from WT aged and SMP30−/−mice fed a VC-limited diet was significantly reduced as compared with that by macrophages from WT young mice, and that the inflammatory responses were induced strongly by the injection of secondary necrotic cells. If we could identify a way to restore phagocytic activity of resident peritoneal macrophages in aged mice, then we could ameliorate age-associated disorders, such as systematic lupus erythematosus and rheumatoid arthritis.
Acknowledgements This work was supported partly by Grants-in-Aid for Scientific Research of the Ministry of Education, Culture, Sports, Science and Technology.
Disclosure statement The authors declare no conflict of interest.
References 1 Shibata T, Nagata K, Kobayashi Y. Cutting edge: A critical role of nitrogen oxide in preventing inflammation upon apoptotic cell clearance. J Immunol 2007; 179: 3402–3406. 2 Manuel T, do Vale A, dos Santos NM. Secondary necrosis in multicellular animal: an outcome of apoptosis with pathogenic implications. Apoptosis 2008; 13: 463–842. 3 Iyoda T, Kobayashi Y. Involvement of MIP-2 and CXCR2 in neutrophil infiltration following injection of late apoptotic cells into peritoneal cavity. Apoptosis 2004; 9: 485–493. 4 Uchimura E, Kodaira T, Kurosaka K, Yang D, Watanabe N, Kobayashi Y. Interaction of phagocytes with apoptotic cells leads to production of pro-inflammatory cytokines. Biochem Biophys Res Commun 1997; 239: 799–803. 5 Kawagishi C, Kurosaka K, Watanabe N, Kobayashi Y. Cytokines production of macrophages in association with phagocytosis of etoposide-treated P388 cells in vitro and in vivo. Biochem Biophys Acta 2001; 1541: 221–230. 6 Kurosaka K, Watanabe N, Kobayashi Y. Production of proinflammatory cytokines by phorbol myristate acetatetreated THP-1 cells and monocyte-derived macrophages after phagocytosis of apoptotic CTLL-2 cells. J Immunol 1998; 161: 6249–6254. 7 Kurosaka K, Watanabe N, Kobayashi Y. Production of proinflammatory cytokines by resident tissue macrophages after phagocytosis of apoptotic CTLL-2 cells. Cell Immunol 2001; 211: 1–7.
|
7
R Takahashi et al. 8 Misawa R, Kawagishi C, Watanabe N, Kobayashi Y. Infiltration of neutrophils following injection of apoptotic cells into the peritoneal cavity. Apoptosis 2001; 6: 411– 417. 9 Fadok VA, Bratton DL, Guthrie L, Henson PM. Differential effects of apoptotic versus lysed cell on macrophage production of cytokines: role of protease. J Immunol 2001; 166: 6847–6854. 10 Miller RA. The aging immune system: primer and prospectus. Science 1996; 273: 70–74. 11 Tanimoto N, Terasawa M, Nakamura M et al. Involvement of KC, MIP-2, and MCP-1 in leukocyte infiltration following injection of necrotic cells into the peritoneal cavity. Biochem Biophys Res Commun 2007; 361: 533–536. 12 Su YJ, Cheng TT, Chen CJ et al. The association among leukocyte apoptosis, autoantibodies and disease severity in systemic lupus erythematosus. J Transl Med 2013; 11: 261– 267. 13 Chorinchath BB, Kong L, Mao L, McCallum RE. Ageassociated differences in TNF-α and nitric oxide production in endotoxic mice. J Immunol 1996; 156: 1525–1530. 14 Effros RB, Walford RL. The effect of age on the antigenpresenting mechanism in limiting dilution precursor cell frequency analysis. Cell Immunol 1984; 88: 531–539. 15 Ito Y, Betsuyaku T, Nasuhara Y, Nishimura M. Lipopolysaccaride-Induced Neutrophilic Inflammation in
8
|
16 17
18 19
20
21
the Lungs Differs with age. Exp Lung Res 2007; 33: 375– 384. Maruyama N, Ishigami A, Kuramoto M et al. Senescence marker protein-30 knockout mouse as an aging model. Ann N Y Acad Sci 2004; 1019: 383–387. Smallwood HS, López-Ferrer D, Squier TC. Aging enhances the production of reactive oxygen species by upregulating classical activation pathways. Biochemistry 2011; 50: 9911–9922. Feng D, Kondo Y, Ishigami A, Kuramoto M, Machida T, Maruyama N. Senescence marker protein-30 as a novel antiaging molecule. Ann N Y Acad Sci 2004; 1019: 360–364. Shibata T, Nagata K, Kobayashi Y. The mechanism underlying the appearance of late apoptotic neutrophils and subsequent TNF-α production at a late stage during Staphylococcus aureus bioparticle-induced peritoneal inflammation in inducible NO synthase-deficient mice. Biochem Biophys Acta 2010; 1802: 1105–1111. Yamazaki T, Nagata K, Kobayashi Y. Cytokine production by M-CSF- and GM-CSF-induced mouse bone marrowderived macrophages upon coculturing with late apoptotic cells. Cell Immunol 2008; 251: 124–130. Jackaman C, Radley-Crabb HG, Soffe Z, Shavlakadze T, Grounds MD, Nelson DJ. Targeting macrophages rescues age-related immune deficiencies in C57BL/6J geriatric mice. Aging Cell 2013; 12: 345–357.
© 2015 Japan Geriatrics Society
