It is generally believed that the clearance of apoptotic cells does not lead to inflammation. In contrast, we previously found that injection of apoptotic cells into the peritoneal cavity induced the expression of an inflammatory chemokine, MIP-2, and infiltration of neutrophils, and that anti-MIP-2 Abs suppressed the infiltration significantly. Because our previous study showed that whole-body x-irradiation caused neutrophil infiltration into the thymus along with T cell apoptosis, we examined the role of neutrophils in apoptotic cell clearance. Neutrophil infiltration reached a peak 12 h after irradiation with 1 Gy of x-rays. Immunohistological analysis revealed that apoptotic cells disappeared dramatically from 10.5 to 12 h after x-irradiation. As neutrophils moved from an inner area of the cortex to the periphery, apoptotic cells disappeared concomitantly. Either anti-MIP-2 or anti-CXCR2 Abs suppressed neutrophil infiltration significantly, and the suppression of neutrophil infiltration by anti-MIP-2 Abs delayed the disappearance of apoptotic cells. Moreover, macrophage-mediated digestion of apoptotic thymocytes was accelerated in vitro on coculturing with neutrophils, even if neutrophils were separated from macrophages. These results suggest that neutrophils are recruited to the thymus mainly by MIP-2 after whole-body x-irradiation and that such neutrophils may not induce inflammation but rather accelerate complete digestion of apoptotic cells by macrophages.
It is generally believed that the clearance of apoptotic cells does not lead to inflammation. In support of this view, many investigators have reported that macrophages produced an anti-inflammatory cytokine, TGF-α, but not proinflammatory cytokines including TNF-β on coculturing with apoptotic cells (1, 2, 3, 4, 5). However, we reported previously that the expression of a proinflammatory chemokine, IL-8 or MIP-2, but not TNF-β, was up-regulated on coculturing of macrophages with apoptotic cells at the mRNA and protein levels (6, 7, 8). In addition, we reported that injection of late apoptotic cells into the mouse peritoneal cavity caused the production of MIP-2 and infiltration of neutrophils, and that the macrophages ingesting and/or binding to apoptotic cells, which were isolated with a cell sorter, produced a similar amount of MIP-2 as in the case of coculturing of peritoneal exudate cells (PEC)3 with apoptotic cells (9, 10). Moreover, we recently showed that anti-MIP-2 and anti-CXCR2 Abs significantly suppressed the neutrophil infiltration caused by injection of apoptotic cells into the peritoneal cavity, suggesting that the neutrophil infiltration is mainly caused by MIP-2 (11).
In addition to MIP-2, another member of the ELR+ CXC chemokine family, called KC, is also known to be a chemoattractant for mouse neutrophils. Because an IL-8 gene has not been discovered in mouse or rat, these two chemokines are thought to be functional homologues of IL-8 in these rodents (12, 13, 14). Although both MIP-2 and KC bind to CXCR2 (15, 16), recent studies showed that KC is a more potent chemoattractant than MIP-2, and that KC seems to be essential for the initiation stage of inflammation (17, 18). Consequently, we hypothesized that, when massive and acute apoptosis occurs in vivo, neutrophils infiltrate in response to MIP-2 to play an unknown role possibly unrelated to inflammation.
Although neutrophils are generally thought to be an initiator of inflammation, it was recently reported that neutrophils have other effects. For example, neutrophils were found to play a protective role in host defense through an immunomodulatory function but not phagocytic activity (19). In another study, it was found that neutrophils play an important role in recruiting CD8+ T cells to an inflammatory site, thereby contributing to Th1 polarization (20).
When mice are exposed to a modest dose of x-rays, massive and acute apoptosis is induced in lymphoid tissues such as the thymus. Among thymocytes, CD4/8 double-positive cells mainly die after x-irradiation. We previously reported that transient infiltration of neutrophils was induced in association with apoptosis after whole-body x-irradiation (21). In this study, using the model of whole-body x-irradiation, we investigate the role of neutrophils in apoptotic cell clearance in the thymus.
Materials and Methods
Male B10 Thy1.1 mice were bred from colonies at the Animal Production Facility of the National Institute of Radiological Science (Chiba, Japan) and maintained under specific pathogen-free conditions. These mice (7–8 wk old) were used throughout this study and irradiated with x-rays (1 Gy) at 0.24 Gy/min (irradiation parameters: 150 KV, 5 mA) with an MBR-1505R2 (Hitachi). Eight hundred micrograms of anti-MIP-2 polyclonal Abs (pAb), anti-CXCR2 pAb, and anti-GST pAb were injected through a tail vein just before x-irradiation or 9 h after x-irradiation. These Abs were obtained as described previously, and were found to be specific to MIP-2 and CXCR2, respectively (11). At the indicated times, mice were sacrificed and thymi were obtained.
Flow cytometric analysis
Thymocytes were suspended in 5 ml of RPMI 1640 medium containing 10% heat-inactivated FCS (Invitrogen Life Technologies), and then the total cell number and cell viability were determined by trypan blue dye exclusion. The cells were treated with anti-CD32/16 mAb (2.4G2) and mouse IgG1 (MOPC-21; American Type Culture Collection) (5 μg each), followed by staining with FITC-labeled anti-Gr-1 mAb (mouse neutrophil marker; RB6-8C5, provided by Dr. Sendo of Yamagata University, Yamagata, Japan) and anti-HLA mAb (isotype-matched control mAb; HB-152, American Type Culture Collection) as described previously (22). The cells were then washed twice, followed by analysis with FACScan and CellQuest software (BD Biosciences). The number of thymic neutrophils was then calculated by multiplying the total cell number by the percentage of Gr-1-positive cells.
Immunohistology (frozen sections)
Freshly harvested thymi were immersed in Tissue-Tek O.C.T. compound (Sakura Finetechnical), quickly frozen in liquid nitrogen, and then cut into 7-μm-thick sections with a cryostat, CM1850 (Leica). The sections were placed on silane-coated Superfrost micro slide glasses (Matsunami) and then air dried for at least 30 min at room temperature (r.t.), followed by fixation by immersion in acetone for 5 min at 4°C. During the subsequent steps, the tissue sections were kept under humid conditions. After washing the sections with PBS (pH 7.4, containing 14 mM Na2HPO4 and 6 mM KH2PO4) for 5 min twice, they were immersed in 0.3% H2O2, which had been diluted with methanol, for 30 min to block endogenous peroxidase activity. A circle was drawn around a portion of the tissue section using a liquid blocker super pap pen (Daido Sangyo); then the portion was incubated with a 3% BSA solution for 15 min at r.t. to reduce the background staining. After washing with PBS twice, biotinylated anti-F4/80 mAb (Serotec), which was diluted to 2 μg/ml with dilution buffer (PBS containing 0.1% BSA), was directly dropped onto the sections, which were then incubated for 60 min at r.t. The sections were washed as described above and then reacted with the reagent mixture in a VECTASTAIN ABC-AP Kit (Vector Laboratories) for 30 min at r.t., followed by washing with PBS twice. The reagent mixture in a VECTOR-Blue Substrate Kit (Vector Laboratories), to which Levamisol (Vector Laboratories) had been added to inhibit endogenous alkaline phosphatase activity, was then applied to the sections, which were subsequently incubated for 35 min at r.t. and then washed with PBS three times. The sections were then incubated with 0.5 μg/ml anti-ssDNA pAb (DAKO) for 24 h at 4°C. After washing with PBS twice, 5 μg/ml biotinylated anti-rabbit IgG Ab (H&L) F(ab′)2 (American Qualex) was applied to the sections, followed by incubation for 60 min at r.t. After the sections had been washed with PBS twice, the reagent mixture in a VECTASTAIN elite ABC kit was dropped onto them, followed by incubation for 30 min at r.t. After washing the sections twice, the reagent mixture in a Diaminobenzidine Substrate kit (Vector Laboratories) was added, followed by incubation for 3 min. The sections were washed with PBS three times, and then dehydrated by immersion in 95% ethanol for 30 s twice and in 99.5% ethanol for 60 s. The sections were made transparent with Histo-Clear (National Diagnostics) before mounting with VECTAMOUNT mounting medium (Vector Laboratories). In the case of staining with anti-F4/80 mAb, some sections were treated with unlabeled anti-F4/80 mAb (BMA Biomedicals), followed by reaction with biotinylated anti-F4/80 mAb, whereas in the case of staining with anti-ssDNA pAb, some sections were incubated with normal rabbit IgG (Sigma-Aldrich) instead of anti-ssDNA pAb.
Immunohistology (paraffin sections)
Freshly harvested thymi were fixed in 10% neutral buffered formaldehyde at 4°C overnight. The tissues were then dehydrated for 30 min with 70, 85, 90, 95, and 95.5% (twice) ethanol at r.t. They were made transparent with xylene for 30 min at r.t. three times, followed by immersion in Histoparaffin (Wako) at 60°C for 30 min three times. The tissues embedded in Histoparaffin were cut into 7-μm thick sections. The sections were placed on silane-coated Superfrost microslide glasses and then air-dried at 37°C overnight. They were then deparaffinized with xylene for 5 min three times, and rehydrated with 99.5, 95, 85, and 70% ethanol for 5 min. Endogenous peroxidase was quenched as described above, and then the sections were immersed into 10 mM citrate buffer at 95°C for 45 min to retrieve Ags, followed by cooling for 20 min at r.t. During the subsequent steps, the tissue sections were kept under humid conditions. A circle was drawn around a portion of the section using a liquid blocker super pap pen and then the portion was incubated with a 3% BSA solution as described above. After washing with PBS twice, anti-myeloperoxidase (MPO) pAb (Lab Vision) was added directly to the sections, followed by incubation for 30 min at r.t. The sections were then washed and incubated with biotinylated anti-rabbit IgG Ab for 60 min at r.t. After washing, the reagent mixture in a VECTASTAIN elite ABC Kit was dropped onto the sections, followed by incubation for 30 min at r.t. After washing, the reagent mixture in the Diaminobenzidine Substrate kit (Vector Laboratories) was added to the sections, followed by incubation for 3 min. After staining, the sections were immersed in water for 5 min and then counterstained with Mayer’s hematoxylin. The sections were dehydrated through a graded series of ethanol and then made transparent with xylene before mounting with Entellan Neu mounting medium (Merck). As a control, some of the sections were incubated with normal rabbit IgG instead of anti-MPO pAb.
Preparation of cells
To prepare resident peritoneal macrophages, mouse PEC were obtained with 4 ml of PBS. The cells were cultured on a 24-well plate or a plastic dish (35 mm in diameter) in RPMI 1640 medium containing 10% heat-inactivated FCS at the cell density of 5 × 105 cells/ml for 2 h at 37°C. Then plastic adherent cells were obtained as resident peritoneal macrophages by washing with PBS three times, and then incubated in RPMI 1640 medium containing 10% heat-inactivated FCS for 5 h at 37°C. The cells thus obtained included 90.4 ± 0.4% of macrophages, as determined on DiffQuik staining.
To generate apoptotic cells, thymocytes from B10 Thy1.1 mice were irradiated with x-rays (12 Gy, 0.54 Gy/min) and then incubated for 7 h at 37°C at the cell density of 2.5 × 106 cells/ml in RPMI 1640 medium containing 10% heat-inactivated FCS. Under these conditions, ∼51% of the thymocytes were annexin V+/PI− and 29% of them were annexin V+/PI+.
To obtain neutrophils, B10 Thy1.1 mice were injected with 1 ml of thioglycolate broth (TGC) i.p. Six hours after TGC treatment, PEC were collected as described above. The cells were washed with PBS and then resuspended in RPMI 1640 medium containing 10% heat-inactivated FCS at the cell density of 6.25 × 105 cells/ml. The cells thus obtained were 83.9 ± 0.3% positive for Gr-1.
Fluorescence microscopic analysis
Apoptotic thymocytes were labeled with a PKH26 Red Fluorescent Cell Linker kit (Sigma-Aldrich) according to the manufacturer’s instructions, followed by culturing for 30 min at 37°C in a plastic dish (35 mm in diameter) to which macrophages had been adhered. The cells were washed with PBS three times. Then the macrophages, which had phagocytosed apoptotic thymocytes, were incubated with or without neutrophils for 13 h. After incubation, the cells were washed and treated with anti-CD32/16 mAb and mouse IgG for 30 min at 37°C before staining with FITC-anti-F4/80 mAb (Serotec) for 30 min at 37°C. The cells were then examined under a deconvolution microscope. Phagocytic cells were defined as cells containing at least one PKH26-positive cell. The percentage of phagocytosis was determined by examining five randomly chosen fields. The phagocytic index, a relative value for how many PKH26-positive cells one phagocytic cell contained, was determined by multiplying the percentage of phagocytosis by the average pixel area corresponding to apoptotic cells phagocytosed by each macrophage.
Analysis of DNA degradation
Unlabeled apoptotic thymocytes were cultured with resident peritoneal macrophages for 30 min at 37°C in a 24-well plate. The cells were washed with PBS three times. Then a neutrophil suspension was added directly to wells or Millicell-HA Transwells (Millipore). After coculturing for various times, the cells were washed with PBS three times. DNA present in adherent cells was isolated as reported previously (23) and analyzed by 2% agarose gel electrophoresis.
Measurement of MIP-2 expression
To measure the protein level of MIP-2 in thymic macrophages, thymi were harvested from either mice just after whole-body x-ray irradiation, mice 8 h after such irradiation, or unirradiated mice. The thymi were cut into small blocks of 2–3 mm in size. After thymocytes had been teased out with pincettes in PBS, the remaining residue was washed with PBS and then digested with 100 U/ml collagenase (Nitta Gelatin) for 30 min at 37°C, followed by vigorous pipetting and filtration through a nylon mesh (150 nm). The cell suspension was centrifuged (115 × g, 5 min) at r.t. The resulting pellet was suspended in 1 ml of PBS. Five milliliters of FCS were added underneath the cell suspension, followed by centrifugation (65 × g, 5 min) at r.t. The resulting pellet was suspended in 2 ml of RPMI 1640 medium containing 10% heat-inactivated FCS, followed by incubation for 30 min at 37°C in a plastic dish (35 mm in diameter). After washing with PBS three times, the plastic adherent cells were obtained as thymic macrophages. The cells thus obtained were 93 ± 1% positive for F4/80. After culturing in RPMI 1640 medium containing 10% heat-inactivated FCS for 2 h, supernatants were harvested and stored at −80°C until assaying. The mouse MIP-2 levels were determined using a DuoSet ELISA development system (R&D Systems). The detection limit for MIP-2 was 15.6 pg/ml.
To measure the mRNA level of MIP-2 or β2 microglobulin, thymic macrophages were isolated as described above from either mice just after whole-body x-ray irradiation, mice 10 h after the irradiation, or unirradiated mice. Total RNA was isolated from thymic macrophages and RT-PCR was performed as described previously (6). The primers (5′ primer and 3′ primer), annealing temperatures, concentrations of MgCl2, and predicted sizes were reported previously (6).
Differences between experimental groups were analyzed by means of one-way factorial ANOVA (one factor ANOVA) and the post hoc test (Scheffe’s F). When p < 0.05, the difference was considered statistically significant.
Neutrophil infiltration caused by whole-body x-irradiation
Whole-body x-ray irradiation induces massive and acute apoptosis in lymphoid tissues such as the thymus. Our previous study demonstrated that transient infiltration of neutrophils into the thymus was induced by whole-body x-ray irradiation (21).
We harvested thymi at the indicated times after whole-body x-irradiation (1 Gy), and then determined the neutrophil number with a flow cytometer as described in Materials and Methods. As shown in Fig. 1, neutrophils began to infiltrate into the thymus 9 h after x-irradiation, and the infiltration of neutrophils reached a peak 12 h after x-irradiation.
Localization of apoptotic cells and macrophages in the thymus after whole-body x-irradiation
We then examined the localization of apoptotic cells and macrophages by immunohistochemical analysis of frozen sections of thymi after whole-body x-irradiation. In this analysis, apoptotic cells were stained with anti-ssDNA Abs (brown) and macrophages were stained with anti-F4/80 Abs (blue), respectively.
Because the aim of this study was to determine the role of neutrophils in apoptotic cell clearance in the thymus after x-irradiation, we first analyzed apoptotic cells and macrophages in the thymus 9 h after x-irradiation. Apoptotic cells were found throughout the cortex (Fig. 2,A; denoted by C), whereas there were few apoptotic cells in the medulla (Fig. 2,A; encircled by a dotted line and denoted by M). The cortex and medulla were delineated by H&E staining of sequentially sectioned preparations. Apoptotic cells tended to form large clusters containing many cells (Fig. 2,B). Some of the large clusters resided within F4/80+ macrophages, and such clusters were detected mainly in the subcapsular area of the cortex, in particular near the surface (open arrows in Fig. 2,B, ii and iii). In contrast, many clusters in the inner area of the cortex were not in F4/80+ macrophages (thin arrows in Fig. 2 B, i and ii; see Discussion). It should be noted that there were many F4/80+ macrophages without signals of ssDNA in both the cortex and the medulla.
Ten and a half hours after x-irradiation, there was almost no change in the distribution or appearance of clusters (Fig. 3), as compared with in Fig. 2. Many clusters were still detected throughout the cortex (Fig. 3,A; denoted by C), and some of the clusters were negative for staining with anti-F4/80 Abs (thin arrows in Fig. 3 B, i and ii).
However, 12 h after x-irradiation, there were dramatic changes in the distribution and appearance of clusters (Fig. 4). The majority of apoptotic cell clusters disappeared, with a few apoptotic cell clusters only detected near the surface area of the cortex. As compared with Figs. 2 and 3, macrophages were found to contain either a few apoptotic cells (filled arrowheads in Fig. 4,Biii) or vacuole-like structures (open arrowheads in Fig. 4,B). The former may reflect incomplete digestion of apoptotic cells engulfed, while the latter possibly reflects complete digestion of apoptotic cells. Because much more vacuole-like structures were detected in the inner area of the cortex as compared with in the surface region, the digestion of apoptotic cells appeared to start earlier in the inner area of the cortex. It should be noted that during the same period between 10.5 and 12 h after x-irradiation, the neutrophil infiltration into the thymus reached a peak (Fig. 1). Consequently, these results raised the possibility that apoptotic cell digestion may be related to neutrophil infiltration. Table I shows the results of quantification of these immunohistochemical data in Figs. 2–4. When macrophages contain several apoptotic cells stained with anti-ssDNA Abs strongly, they are categorized as ingested/intact, whereas, when macrophages contain fewer apoptotic cells stained with anti-ssDNA Abs weakly, they are categorized as ingested/being digested.
|.||Time after X-Irradiation (Hours) .||.||.||.|
|.||0 .||9 .||10.5 .||12 .|
|Apoptotic cell cluster|
|Uningested||Nda||1.00 ± 0.07b||0.78 ± 0.29||0.14 ± 0.08c|
|Ingested (intact)||Nd||1.50 ± 0.31||1.15 ± 0.29||0.37 ± 0.17c|
|Ingested (degraded)||Nd||0.02 ± 0.02||0.05 ± 0.05||0.54 ± 0.33c|
|.||Time after X-Irradiation (Hours) .||.||.||.|
|.||0 .||9 .||10.5 .||12 .|
|Apoptotic cell cluster|
|Uningested||Nda||1.00 ± 0.07b||0.78 ± 0.29||0.14 ± 0.08c|
|Ingested (intact)||Nd||1.50 ± 0.31||1.15 ± 0.29||0.37 ± 0.17c|
|Ingested (degraded)||Nd||0.02 ± 0.02||0.05 ± 0.05||0.54 ± 0.33c|
Nd, Not detected.
The number of uningested, ingested (intact), and ingested (degraded) apoptotic cell clusters were counted in six randomly chosen areas of three sections prepared from three mice, and the results were expressed as the relative number per a given area, when the number 9 h after X-irradiation was taken as 1.0, and expressed as means ± SE.
A value of p < 0.05 as compared with the data 9 h after X-irradiation.
Localization of neutrophils in the thymus after whole-body x-irradiation
We then examined the localization of infiltrated neutrophils in the thymus after whole-body x-irradiation by detecting neutrophils with anti-MPO Abs in paraffin-embedded formalin-fixed tissue sections.
Nine hours after x-irradiation, a small number of neutrophils were detected near a vein in the inner area of the cortex (Fig. 5, A and Bii, boxed in red), which is consistent with the flow cytometric data (Fig. 1). At this time, apoptotic cells were also detected in paraffin sections as clusters of cells, which were counterstained as deep blue spots as indicated by open arrowheads (Fig. 5 B, i, iii, and iv). Such clusters were not detected in the thymi of unirradiated mice (data not shown).
In contrast, 12 h after x-irradiation, the localization of neutrophils had changed dramatically, with a large number of neutrophils detected in the subcapsular region but not in the inner area of the cortex (Fig. 6), suggesting that neutrophils migrate from the inner area to the subcapsular region of the cortex. At this time, apoptotic cell clusters were decreased in number and were only detected in the subcapsular region of the cortex (open arrowheads in Fig. 6,Bii). In the inner area of the cortex, many vacuole-like structures were detected (filled arrowheads in Fig. 6 B), which is consistent with the results for frozen sections described above. These results support the possibility that apoptotic cell digestion may be associated with infiltration of neutrophils.
Because we showed in another study that transient infiltration of neutrophils was induced by injection of apoptotic cells into the peritoneal cavity of mice, and that the infiltration was caused mainly by MIP-2 (11), we then examined whether or not the neutrophil infiltration induced by x-irradiation was also caused mainly by MIP-2. We first examined the effects of anti-MIP-2 and anti-CXCR2 Abs on the neutrophil infiltration 12 h after x-irradiation by injecting these Abs 9 h after x-irradiation in a tail vein, because neutrophils began infiltrating at this time. As shown in Fig. 7, the anti-MIP-2 Abs reduced neutrophil infiltration by >50%, and the anti-CXCR2 Abs caused similar suppression, suggesting that the neutrophil infiltration was caused mainly by the MIP-2-CXCR2 interaction. Such suppression was not observed when the anti-GST Abs were used. We then examined the effects of anti-MIP-2 Abs on the localization of neutrophils and apoptotic cell clusters in the thymus 12 h after x-irradiation. The neutrophil infiltration was suppressed in the thymus of an anti-MIP-2 Ab-treated mouse (Fig. 8). Apoptotic cells were not decreased in number and retained the cluster morphology even 12 h after x-irradiation (Fig. 8,B, open arrowheads), suggesting that the treatment with anti-MIP-2 Abs delayed apoptotic digestion. It should be noted that without the treatment with anti-MIP-2 Abs, apoptotic cells were detected as clusters 9 h after x-irradiation, but they were decreased in number 12 h after irradiation (Figs. 2 and 4–6). Fig. 9 shows the results of quantification of immunohistochemical data obtained by staining with anti-ssDNA Abs. These results further support that apoptotic cell digestion may be enhanced by neutrophil infiltration.
Acceleration of macrophage-mediated apoptotic cell digestion by neutrophils
We then examined in vitro the possibility that macrophage-mediated apoptotic cell digestion is accelerated by neutrophils. In this experiment, we used thymocytes 7 h after in vitro x-irradiation as apoptotic cells. Under the conditions we used, ∼51% of the thymocytes were annexin V+/PI−, and 29% of them were annexin V+/PI+. These apoptotic cells were then labeled with PKH26 fluorescent dye, followed by coculturing with resident peritoneal macrophages in the ratio of 1:4 (macrophages to apoptotic cells). Then we removed free, nontrapped apoptotic cells by washing, and the macrophages were cultured with or without neutrophils in the ratio of 1:1 (macrophages to neutrophils). The plastic adherent cells we used in this experiment included ∼90% macrophages, as defined morphologically, whereas the TGC-induced peritoneal cells used as neutrophils were ∼85% positive for Gr-1, a marker for neutrophils. The area stained with PKH26 in macrophages significantly decreased when macrophages were incubated with neutrophils (Fig. 10,A). There was no change in the number of adherent cells even after they had been cocultured with neutrophils (8.7 ± 2.2 cells per field without neutrophils vs 8.9 ± 2.0 cells per field with neutrophils), although the number of adherent cells was decreased during the culture (12.1 ± 2.3 cells per field at the starting time of culture). The results are also presented as the phagocytic index (Fig. 10 B). These data suggested that neutrophils accelerate apoptotic cell digestion inside macrophages.
Because PKH26 is reported to stain the cell membrane preferentially, we then examined the fate of fragmented DNA derived from apoptotic cells. The fragmented DNA was digested more rapidly on culturing of macrophages with neutrophils (Fig. 11,A; see the results for 1.5 h after coculture). The asterisks show the positions of DNA with high m.w. derived from macrophages. When apoptotic cells were cultured alone, the fragmented DNA was not further digested within the time period examined (Fig. 11,B). These results suggested that neutrophils enhance the digestion of apoptotic cell DNA in macrophages, so the infiltrated neutrophils play an auxiliary role in apoptotic cell clearance. When we used a double-chamber culture system to separate neutrophils from macrophages, neutrophils still accelerated the digestion of fragmented DNA (Fig. 11 C), suggesting that acceleration of apoptotic cell digestion is mediated by soluble factors from neutrophils.
Production of MIP-2 by thymic macrophages after interaction with apoptotic cells
Although the above observations strongly suggested a novel role of neutrophils, it is essential to show that neutrophils are recruited to macrophages phagocytosing apoptotic cells in vivo. Because we had difficulty in detecting MIP-2 protein in situ, we then examined whether or not thymic macrophages express MIP-2 by RT-PCR and ELISA.
We isolated thymic macrophages from mice 10 or 12 h after x-irradiation for determination of the MIP-2 protein level by ELISA or the MIP-2 mRNA level by RT-PCR, respectively. To determine the MIP-2 protein level, the macrophages were then cultured for 2 h, followed by harvesting the supernatants. The purity of the macrophages was ∼94%. MIP-2 mRNA expression was significantly elevated in thymic macrophages obtained from mice 12 h after x-irradiation. In contrast, thymic macrophages from either unirradiated mice or mice just after x-irradiation did not express MIP-2 mRNA (Fig. 12,A). This was also true for MIP-2 protein expression (Fig. 12 B). Thus, these results revealed that thymic macrophages were one of the sources of MIP-2.
This study provides several lines of evidence supporting that infiltrating neutrophils accelerate apoptotic cell digestion by macrophages after whole-body x-irradiation. First, as neutrophils moved from an inner area of the cortex to the periphery, apoptotic cells disappeared concomitantly. Second, when apoptotic cells were detected either with anti-ssDNA Abs or on H&E staining, they disappeared 12 h after whole-body x-irradiation in untreated but not anti-MIP-2 Ab-treated mice. Third, neutrophils accelerated the digestion of apoptotic cells or fragmented DNA in macrophages in vitro. However, we cannot exclude the possibility that MIP-2 blockade would also affect monocyte recruitment indirectly, thereby causing suppression of apoptotic cell digestion in vivo.
Even when neutrophils were separated from macrophages with a double chamber, neutrophils still accelerated the digestion of fragmented DNA in macrophages, suggesting that acceleration of apoptotic cell digestion is mediated by soluble factors from neutrophils. One of the candidates for such soluble factors would be MCP-1, because MCP-1 is produced not only by monocytes/macrophages but also by neutrophils (24), and because MCP-1 activates monocytes/macrophages to show an anti-tumor effect or to produce superoxide (25, 26, 27, 28, 29). Although MCP-1 protein production by thymic macrophages was significantly elevated 12 h after whole-body x-irradiation (data not shown), it is not known whether or not MCP-1 is also produced by neutrophils to enhance macrophage-mediated digestion of apoptotic cells. In contrast, metabolites of arachidonic acid, in particular lipoxin (LX), would be other candidates for such soluble factors, because the LX family seems to regulate the resolution of inflammation (30). Furthermore, it has been reported that one of the LX family, LXA4, enhanced macrophage-mediated binding and phagocytosis of apoptotic cells, whereas LXA4 suppressed cytokine production (31, 32, 33). Consequently, it is possible that the LX family, in particular LXA4, also affects macrophage-mediated digestion of apoptotic cells.
The neutrophil infiltration into the thymus after x-irradiation was significantly reduced by either anti-MIP-2 or anti-CXCR2 Abs, as previously reported for neutrophil infiltration induced by injection of late apoptotic cells (11), suggesting that the neutrophil infiltration in both cases is mediated mainly by MIP-2. It should be noted that these Abs are specific to MIP-2 and CXCR2, respectively, and that anti-MIP-2 Abs do not bind to KC (11). In man, the neutrophil infiltration is strictly regulated by IL-8, and its receptors, CXCR1 and CXCR2 (34, 35, 36, 37). However, in rat and mouse, instead of IL-8, homologues of the human Gro family, namely KC and MIP-2, are presumed to be the main chemoattractants for neutrophils (12, 13, 14, 15, 16), because an IL-8 gene has not been discovered in these rodents. Moreover, there is only one type of CXC receptor, CXCR2, in mice, and the CXCR2 protein is reported to be the sole receptor for homologues of human Gro family members (15, 16). Although various mediators other than these chemokines are known to be chemoattractants for neutrophils, investigation involving CXCR2 KO mice suggested that the neutrophil infiltration was regulated mainly through the CXCR2 protein in some cases, such as parasitic infection, bacterial infection, and wound healing (38, 39, 40, 41, 42). Under inflammatory conditions, KC seems to play a major role in neutrophil infiltration and MIP-2 seems to act in concert with KC even if MIP-2 production is up-regulated (43, 44, 45, 46, 47, 48, 49, 50, 51, 52). Although the affinity of MIP-2 toward CXCR2 is stronger than that of KC (16), there have been few reports suggesting that MIP-2 is a main player in an inflammatory response. In addition, it has been reported that KC and MIP-2 differ, in that KC is a more potent chemoattractant whereas MIP-2 is more active as a regulator of neutrophil degranulation (17, 18). In brief, the neutrophil infiltration caused by massive and acute apoptosis appears to be regulated differently from regular inflammatory responses.
Histological analysis of thymi until 10.5 h after whole-body x-irradiation revealed many apoptotic cell clusters in the cortex without F4/80 signals and no free, single apoptotic cells, suggesting that the clusters are not phagocytosed by macrophages. As Soga et al. (53) reported, there are three morphologically distinct types of macrophages, namely dendritic, round, and flat types, in the thymus. Dendritic type macrophages are distributed throughout the thymus, and most of them are positive for F4/80 Ag. Round-type macrophages localized in the corticomedullary region and medulla are negative for F4/80, whereas round-type macrophages localized in the cortex express F4/80. Flat-type macrophages are localized in the subcapsular region of the cortex, and they are positive for F4/80 (53). In brief, all types of macrophages in the cortex are positive for F4/80. Therefore, it is unlikely that the apoptotic cell clusters are phagocytosed by F4/80 negative macrophages, because such macrophages are not found in the cortex. However, we cannot exclude the possibility that apoptotic cell clusters are phagocytosed by either other phagocytes, such as thymic nurse cells (54, 55, 56), or F4/80 negative round-type macrophages, which would have moved from the medulla to the cortex.
Apoptotic cells are thought to be the source of autoantigens. Many investigators have reported that impaired clearance of dying cells plays a pathogenic role in the development of autoimmunity (57, 58, 59, 60). Moreover, it has been reported that macrophages of autoimmune-prone mice, such as MRL/Mp or NOD mice, showed reduced phagocytosis of apoptotic cells (61, 62, 63, 64, 65). To prevent apoptotic cells from triggering autoimmunity, there are various firewall systems in our body. One is that immature DCs do not become mature and, therefore, do not present engulfed Ags to T cells when they phagocytose apoptotic cells (22, 66, 67, 68). The other is that macrophages engulf apoptotic cells and then digest them quickly and completely. The mechanism described in this study may also be the way by which apoptotic cells are quickly cleared to prevent them from causing autoimmune diseases. If neutrophils fail to facilitate the clearance of apoptotic cells by macrophages for some reason, the chance for autoimmune diseases might be increased. This possibility is worth further investigation.
In conclusion, this study suggests that, when massive and acute apoptosis occurs in vivo, macrophages phagocytose apoptotic cells and then produce MIP-2 to accumulate neutrophils, and that the neutrophils promote the efficient clearance of apoptotic cells by macrophages. These results thus provide a new mechanism by which an inflammatory reaction is prevented upon massive and acute apoptosis.
The authors have no financial conflict of interest.
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.
This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science and Technology, and from the Faculty of Science, Toho University (to Y.K.).
Abbreviations used in this paper: PEC, peritoneal exudate cell; pAb, polyclonal Ab; MPO, myeloperoxidase; r.t., room temperature; TGC, thioglycolate broth; LX, lipoxin.