Glycobiology Advance Access originally published online on August 11, 2005
Glycobiology 2005 15(12):1368-1375; doi:10.1093/glycob/cwj028
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Retardation of removal of radiation-induced apoptotic cells in developing neural tubes in macrophage galactose-type C-type lectin-1-deficient mouse embryos
3 Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, and 4 Department of Radiation Oncology, University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
1 Dr. Irimura holds stock and conducts research in collaboration with Summit Glycoresearch Inc.
2 To whom correspondence should be addressed; e-mail: irimura{at}mol.f.u-tokyo.ac.jp
Received on June 8, 2005; revised on August 6, 2005; accepted on September 9, 2005
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Abstract |
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MGL1/CD301a is a C-type lectin that recognizes galactose and N-acetylgalactosamine as monosaccharides and is expressed on limited populations of macrophages and dendritic cells at least in adult mice. In this study, pregnant mice with Mgl1+/– genotype were mated with Mgl1+/– or Mgl1–/– genotype males, and the embryos were used to assess a hypothesis that this molecule plays an important role in the clearance of apoptotic cells. After X-ray irradiation at 1 Gy of developing embryos at 10.5 days post coitus (d.p.c.), the number of Mgl1–/– pups was significantly reduced as compared with Mgl1+/+ pups. Distributions of MGL1-positive cells, MGL2-positive cells, and apoptotic cells were histologically examined in irradiated Mgl1+/+ embryos. MGL1-positive cells were detected in the neural tube in which many cells undergo apoptosis, whereas MGL2-positive cells were not observed. Biotinylated recombinant MGL1 bound a significant portion of the apoptotic cells. When Mgl1+/+ and Mgl1–/– embryos were examined for the presence of apoptotic cells, similar numbers of apoptotic cells gave rise, but the clearance of these cells was slower in Mgl1–/– embryos than in Mgl1+/+ embryos. These results strongly suggest that MGL1/CD301a is involved in the clearance of apoptotic cells. This process should be essential in the repair and normal development of X-ray-irradiated embryos.
Key words: apoptosis / galactose / lectin / macrophage / phagocytosis
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Introduction |
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Macrophage galactose-type calcium-type lectin (MGL), also recently assigned as CD301, is a calcium-type (C-type) lectin of
42 kD expressed on macrophages and dendritic cells in adult tissue, at least in humans and rodents (Sato et al., 1992
; Suzuki et al., 1996; Tsuiji et al., 2002). This is a unique C-type lectin, because it recognizes galactose and N-acetylgalactosamine as monosaccharides, whereas other C-type lectins expressed on similar cell populations, DC-SIGN (CD209), macrophage mannose receptor, langerin, and CD205 are known to recognize mannose and related epitopes (Suzuki et al., 1996; Geijtenbeek et al., 2000, 2002; Higashi et al., 2002a,b; Tsuiji et al., 2002; McGreal et al., 2005). Mice have two highly homologous genes, MGL1 and 2 (CD301a/b), whereas humans have a single MGL/CD301 gene which is also termed ClecSF14 or Clec10A (Suzuki et al., 1996; Tsuiji et al., 2002). MGL1, MGL2, and the human MGL are 42-kD lectins having type-II transmembrane configurations. These lectins have one carbohydrate-recognition domain (CRD) having affinity for galactose and N-acetylgalactosamine as monosaccharides (Tsuiji et al., 2002). Among a variety of oligosaccharides, Lewis X trisaccharides were found to bind strongly to MGL1, whereas MGL2 bound to 
- or ß-GalNAc residues linked to soluble polyacrylamide, as far as recombinant forms of the lectins are concerned (Tsuiji et al., 2002). MGL1/2-positive cells, recognized by a monoclonal antibody (mAb) LOM-14 cross reactive between MGL1 and 2, are widely distributed in the connective tissue in adult mice (Mizuochi et al., 1997) and are expressed in mouse embryos at 11 days post coitus (d.p.c.) (Mizuochi et al., 1998). As MGL1/2 lectins have a di-leucine-like motif and YXXØ motifs (Tsuiji et al., 1999, 2002), they were expected to function as endocytotic receptors. Previous reports showed that galactosylated bovine serum albumin (BSA) (Kawakami et al., 1994) and soluble polyacrylamide polymers containing -GalNAc residues were taken up by cells expressing MGL (Denda-Nagai et al., 2002). It was recently shown that antigen-induced tissue remodeling (granulation tissue formation) did not occur in MGL1-deficient mice (Sato et al., 2005).
Apoptotic cells and apoptotic bodies are known to be engulfed by phagocytes (Henson et al., 2001; Savill et al., 2002; Albert, 2004). This process is necessary for the developing embryo to escape from the release of potentially noxious intracellular materials produced by dying cells. Phagocytes are able to distinguish apoptotic cells and healthy cells, suggesting that the apoptotic cells present signals to phagocytes and that phagocytes recognize the signal using specific mechanisms (Fadok et al., 2001; Savill et al., 2002; Albert, 2004). However, the previous investigations on the macrophage recognition of apoptotic cells were mainly based on in vitro systems. Patterns of glycosylations on cell surfaces were reported to change during the induction of apoptosis of a variety of cells in culture (Morris et al., 1984; Rapoport and Pendu, 1999). The involvement of carbohydrates in the recognition of apoptotic cells by phagocytes has long been postulated because the process can be inhibited by glycoconjugates (Duvall et al., 1985; Falasca et al., 1996; Rapoport et al., 2003). As an example, a report showed that mannan-binding lectin (MBL) bound to apoptotic Jurkat T cells and anti-MBL mAb inhibited the uptake of apoptotic cells by macrophages (Ogden et al., 2001).
To investigate the recognition system functioning in vivo, we chose to test apoptosis induced by X-ray irradiation of mouse embryos. This model was previously used to prove the importance of p53-driven apoptosis in situ (Norimura et al., 1996). The report indicated that mice from 9 to 11 d.p.c. were X-ray sensitive, because major organs were generated during these periods (Russell and Russell, 1954). When embryos are irradiated with X-rays, a variety of cells undergo apoptosis. Cells localized in neural tubes seemed to be especially X-ray sensitive (Norimura et al., 1996). An accumulation of macrophages in X-ray-irradiated neural tubes seemed to be prominent, though these data were based on morphological observations (Norimura et al., 1996). Despite that apoptotic cells were abundant in 1-Gy-irradiated embryos (Nomoto et al., 1998), the number of newborn mice and the percentages of malformed mice were similar to those of nonirradiated mice (Norimura et al., 1996). However, when embryos were exposed to X-ray irradiation at a high dose (2 Gy), the number of newborn mice was lower than nonirradiated groups, and the percentages of malformed embryos increased (Russell and Russell, 1954; Norimura et al., 1996), suggesting that damage because of X-ray irradiation was too extensive to be repaired and that damaged embryos were eventually absorbed into mothers. In other studies, the accumulation of macrophages associated with apoptotic cells was observed (Norimura et al., 1996). Also, X-ray-induced malformation was strongly suppressed by intravenous injection of macrophages into pregnant mice (Nomura et al., 1990). However, molecules involved in the recognition of X-ray-induced apoptotic cells were unknown.
In this report, we asked whether Mgl1 contributed to the removal of apoptotic cells from neural tubes by using X-ray-irradiated Mgl1–/– mouse embryos (Onami et al., 2002). The results clearly indicated that the ratio of Mgl1–/– pups to Mgl1+/– pups was skewed after X-ray irradiation. Furthermore, the clearance of apoptotic cells was retarded in Mgl1–/– embryos. This work provides a definitive evidence that macrophages play significant roles in the removal of apoptotic cells at least in part through recognition systems with a galactose-type lectin.
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Results |
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Survival of Mgl1–/– embryos after irradiation
Effects of X-ray irradiation on the survival of embryo with Mgl1+/– and –/– backgrounds were examined. Male Mgl1–/– mice and female Mgl1+/– mice were mated, and pregnant mice at 10.5 d.p.c. were irradiated at 1 Gy, which is the condition previously known to induce extensive apoptosis of cells in neural tubes. It was also known that the damage caused by X-ray irradiation at 1 Gy was repaired, and the embryos normally developed after this treatment. Genotypes of newborn pups were examined, and the ratios of Mgl1+/– mice and Mgl1–/– mice were calculated (Table I). The ratio of newborn mice from nonirradiated pregnant mice with Mgl1+/– and Mgl1–/– genotypes was 1:1.06 as predicted from Menderian’s law. The ratio of the pups from the X-ray-irradiated Mgl1+/– mice and Mgl1–/– mice was 1:0.64. The results strongly suggest that the survival rate of Mgl1–/– embryos was lower than Mgl1+/– embryos when they were X-ray irradiated at 1 Gy at 10.5 d.p.c.
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Induction of apoptotic cells in developing embryos by X-ray irradiation
Induction of apoptosis in wild-type embryos at 10.5 d.p.c. after X-ray irradiation was confirmed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method. Embryos collected at 0, 3, 6, 12, 24, 36, or 48 h after X-ray irradiation at 1 Gy or 2 Gy were examined. Nonirradiated and age-matched embryos were used as controls. In control embryos, the number of apoptotic cells was very small. In X-ray-irradiated embryos at 1 Gy, apoptotic cells were observed in a variety of tissues, especially in neural tubes (Figure 1). Twelve or twenty-four hours after 1-Gy irradiation, a large number of apoptotic cells were observed in the neural tubes. However, 36 h after irradiation, the number of apoptotic cells stained by the TUNEL method significantly decreased. After 2-Gy irradiation, a significant portion of cells within the neural tube underwent apoptosis, and the tissue was disintegrated (data not shown).
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Accumulation of F4/80-positive cells and MGL1-positive cells in the neural tubes after irradiation
As described above, the number of apoptotic cells in neural tubes was the greatest at 12 h and reduced at the later time points, under the applied conditions. The results suggest that apoptotic cells were removed by phagocytosis. Thus, an immunohistochemical approach was taken to identify phagocytotic cells potentially involved in this process. Embryos were removed 24 h after 1-Gy X-ray irradiation, and frozen sections were prepared and examined for the expression of macrophage markers. Binding sites of mAb F4/80, a macrophage marker, were shown in neural tubes of X-ray-irradiated embryos (Figure 2A). mAb LOM-8.7 specific for MGL1 also bound to cells in neural tubes (Figure 2A), whereas mAb URA-1 specific for MGL2 did not bind to any cells. In the age-matched nonirradiated embryos, the numbers of mAb F4/80-positive or mAb LOM-8.7-positive cells were significantly smaller (Figure 2A), suggesting that macrophages expressing these markers infiltrated the neural tubes. Changes in the number of these cells were observed in a time-dependent manner (Figure 2B and C). The results show that these cells were transiently present in neural tubes, having a peak at 36 h after X-ray irradiation.
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Association between cells expressing MGL1 and apoptotic cells
Double staining with mAb LOM-8.7 and mAb F4/80 revealed that most MGL1-positive cells colocalized with F4/80 staining (Figure 3A). Distributions of CD11b were similar to that of MGL1, whereas binding sites of anti-CD31, an endothelial cell marker, or mAb specific for ßIII tubulin, a marker for neural cells, were distinct from the binding sites of mAb LOM-8.7 (data not shown). Double stainings also showed that MGL1-positive cells associated with structures strongly stained by the TUNEL methods, although not all TUNEL-positive structures colocalized with MGL1 (Figure 3B).
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Generation of binding sites for recombinant MGL1 in neural tubes after X-ray irradiation
The association of MGL1-positive cells with TUNEL-positive structures could be owing to the presence of MGL1 ligands on these apoptotic cells or apoptotic bodies. To prove or disprove this hypothesis, we incubated the frozen sections with biotinylated recombinant MGL1 (brMGL1), and distributions of the binding sites of brMGL1 were examined. As a control, brMGL1 was preincubated with galactose (10 mM) before staining. Binding of brMGL1 to sections from X-ray-irradiated embryos was detected, and the staining was absent after preincubation with galactose (Figure 4A). It appeared that brMGL1-binding sites colocalized with TUNEL-positive structures (Figure 4B). Double staining showed that brMGL1-binding sites always colocalized with MGL1-positive cells, although there were MGL1-positive cells without brMGL1 binding (Figure 4C).
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Retardation in the removal of X-ray-irradiation-induced apoptotic cells in neural tubes of Mgl1–/– mice embryos
To examine the role of MGL1 during the induction and clearance of apoptotic cells, Mgl1 gene-deficient embryos were used. The number of apoptotic cells in the embryos was not affected by maternal genotypes (Mgl1+/+, Mgl1+/–, or Mgl1–/–). Furthermore, the number of apoptotic cells in Mgl1–/– embryos under nonirradiated conditions was not significantly different from Mgl1+/+ or Mgl1+/– embryos (data not shown). Female Mgl1+/– mice were mated with male Mgl1+/– mice, and pregnant mice at 10.5 d.p.c. were X-ray irradiated. Twenty-four hours after the X-ray irradiation, no difference in the number of apoptotic cells between Mgl1+/+ embryos and Mgl1–/– embryos was observed. In tissue sections of X-ray-irradiated embryos after 36 and 48 h, a larger number of apoptotic cells was detected in Mgl1–/– embryos than in Mgl1+/+ embryos (Figure 5A). Forty-eight hours after irradiation, apoptotic cells were not observed in wild-type mice. However, in Mgl1–/– mice apoptotic cells were still present in neural tubes. The numbers of TUNEL-positive cells were determined in three sections for each embryo in three embryos and summarized in Figure 5B. Differences in the numbers were statistically significant at 48 h after irradiation. These data suggest that uptake of apoptotic cells was significantly delayed in Mgl1–/– embryos.
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Previous studies indicated that MGL1 was involved in cellular trafficking (Sato et al., 1998; Chun et al., 2000a,b). Therefore, one could hypothesize that lack of MGL1 interfered with macrophage trafficking into neural tubes. If this is the case, the number of F4/80-positive cells in the X-ray-irradiated neural tubes should be reduced in Mgl1–/– embryos because MGL1-positive cells also expressed F4/80, as described above. Thus, the number of F4/80-positive cells in Mgl1–/– embryos was compared with those in Mgl1+/+ embryos. There was no difference in F4/80-positive cells between Mgl1–/– and Mgl1+/+ embryos before or after X-ray irradiation (Figure 5C). Thus, trafficking of macrophages was not likely to be influenced by the deficiency of MGL1. We tentatively concluded that the increase in the number of apoptotic cells in Mgl1–/– embryos was because of the retarded uptake and digestion of apoptotic cells in the neural tubes.
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Discussion |
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In this study, we found that apoptotic cells induced in the neural tube of developing embryos by X-ray irradiation were removed and that this process was impaired in mice lacking MGL1 expression. Phagocytic macrophages expressing MGL1 were likely to be involved in this process through the recognition of carbohydrate chains generated during the apoptotic process. MGL1 was previously shown to function as an endocytosis receptor (Kawakami et al., 1994
; Denda-Nagai et al., 2002) and regulator of cellular trafficking. (Sato et al., 1998; Chun et al., 2000a,b; Kumamoto et al., 2004). Other C-type lectins such as DC-SIGN and the macrophage mannose receptor also had dual functions like MGL1 (Geijtenbeek et al., 2002). Our study demonstrated that the number of F4/80-positive macrophages found in neural tubes after X-ray irradiation in Mgl1–/– mice was similar to that in Mgl1+/+ mice (Figure 5C), yet the clearance of apoptotic cells was delayed (Figure 5A and B). Thus, MGL1 should be directly involved in the phagocytic process but not in the trafficking of macrophages to the sites where damaged cells were localized.
A question remains as to whether MGL1 expression in X-ray-irradiated wild-type or Mgl1+/– mice was induced in cells present in neural tubes or whether MGL1-positive cells migrate into neural tubes after X-ray irradiation. The number of cells in neural tubes expressing F4/80, CD11b, or MGL1 significantly increased after X-ray irradiation (Figure 2). Similar cells found in fetal livers in 10.5 d.p.c. embryos (data not shown) should be able to migrate and reach the neural tubes through the primitive circulation. Previous studies showed that monocyte-derived precursor cells emerged in neural tubes in 12 d.p.c. embryos and differentiated into glial cells (Perry et al., 1985). It is likely that MGL1-positive cells in neural tubes are derived from circulating cells, although MGL1 might not directly be involved in this trafficking. In mouse development, Hox expression was previously reported to be dependent on the segments (Trainor and Krumlauf, 2000). Therefore, time-dependent reduction of apoptotic cells in relation to the distribution of MGL1-positive cells might reflect putative segmental distribution and movement of these cells. However, segmental distribution was not observed in the numbers of apoptotic cells in X-ray-irradiated wild-type or Mgl1–/– embryos.
Other receptors for apoptotic cells were found, and gene-deficient mice were generated (Henson et al., 2001; Savill et al., 2002; Albert, 2004). Mice deficient with Psr or Mfg-e8 had severe phenotypes (Li et al., 2003; Hanayama et al., 2004), whereas mice deficient with some putative genes responsible for clearance of apoptotic cells had no significant defects (Platt et al., 2000). The results seen with Mgl1–/– should be considered as moderate. Apoptotic receptors played a significant role in a specific stage of development as seen with Psr (Li et al., 2003). However, MGL1 seems to contribute to the removal of apoptotic cells induced by exogenous stress but not by spontaneous processes. MGL1-positive cells are distributed throughout the adult body, but no defect has been observed so far under physiological conditions in adult Mgl1–/– mice. Thus, we can tentatively conclude that it is essential to remove apoptotic cells following X-ray irradiation but not during immunological events in adults. The mechanistic basis for such selectivity is currently unknown.
Almost all previous studies on the role of carbohydrate recognition in the clearance of apoptotic cells were performed using in vitro models (Morris et al., 1984; Duvall et al., 1985; Hall et al., 1994; Falasca et al., 1996; Rapoport and Pendu, 1999; Ogden et al., 2001; Rapoport et al., 2003). Therefore, this report is unique because it focuses on in vivo events. To assess whether alterations in glycosylation occurred in X-ray-irradiated neural tubes, we compared these embryos with nonirradiated embryos for their staining profiles with plant lectins. Soybean agglutinin and RCA120 bound to apoptotic cells induced after X-ray irradiation, whereas lectins with similar carbohydrate specificity, peanut agglutinin, or jacalin did not bind to apoptotic cells (data not shown), suggesting that X-ray irradiation induced specific changes in patterns of glycosylations. This might be owing to incomplete glycosylation or to activation of a new biosynthetic or degradation pathway. Previous studies showed that sphingomyelinase was activated by the radiation (Santana et al., 1996), and plasma-membrane-associated ganglioside sialidase (Neu3) was down-regulated in the process of apoptosis in human colon cancer cells (Kakugawa et al., 2002). Recent evidence indicates that another lectin (galectin-1) with specificity for N-acetyllactosamine induces cell surface exposure of phosphatidylserine and phagocytic recognition of these cells (Dias-Baruffi et al., 2003). It remains to be elucidated whether MGL1 has a similar effect. Although galectins were shown to have a direct proapoptotic role (Rubinstein et al., 2004), MGL1 does not seem to have similar functions.
It remains to be elucidated why retardation in the clearance of apoptotic cells leads to embryonic lethality. According to a previous study by the use of DNA ligase IV knockout mice, failure in the repair of DNA double strand breaks lead to death of developing neurons and resulted in embryonic lethality (Barnes et al., 1998). These results as well as the result of this study support the hypothesis that persistence of apoptotic cells in neural tubes negatively affects embryonic development. MGL1-positive cells remove apoptotic cells and may play a crucial role in the prevention of stress-related embryonic death.
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Materials and methods |
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Mice
Mgl1–/– mice generated with 129 background were backcrossed six generations into C57BL/6J mice in Hedrick’s laboratory (Onami et al., 2002
). Females were caged with potent males in pairs overnight, and those with vaginal plugs in the next morning were considered to be in day 0.5 of pregnancy. In some experiments, specific pathogen-free, pregnant, and adult C57BL/6J mice were purchased from SLC Japan (Shizuoka, Japan) and used.
X-ray irradiation to mice
The pregnant mice at 10.5 d.p.c. were exposed to X-rays with a dose of 1 or 2 Gy by Pantak HF350 (Shimadzu, Japan) at a distance of 50 cm, operating at 20 mA and 200 KV, with a filter of 1.0 mm aluminium and 0.5 mm copper. Dose rates were 119.41 ± 0.27 cGy min–1. Dose and time points of irradiation were chosen according to previous reports (Nomura et al., 1990; Norimura et al., 1996; Nomoto et al., 1998).
Survival of embryos after X-ray irradiation
Male Mgl1–/– mice and female Mgl1+/– mice were mated, and pregnant mice at 10.5 d.p.c. were irradiated at 1 Gy. Genotypes of newborn pups were examined by genomic polymerase chain reaction (PCR). A 3' primer between exon3 and exon4 of Mgl1, 5'-CTCAGGTTCCCTCTGTGCCAGC-3', was used for both wild-type and targeted loci. The 5' primers for detecting wild-type and targeted loci are 5'-GCGGTTATTGGATCTCCTGG-3', downstream of the first-coding exon, and 5'-GACGAGTTCTTCTGAGGGG-3' in the neomycin-resistant gene. DNA extracted from the tail of embryos was used. Nonirradiated and female Mgl1+/– mice, which were mated with Mgl1–/– male mice, were used as controls. Mgl1–/– mice were previously shown to be born according to Menderian’s law, if the embryos were not irradiated. Assuming that Mgl1–/– embryos were X-ray sensitive, it was thought that the percentage of newborn Mgl1–/– mice would decrease after irradiation. Forty-one pups from seven parents were examined in the X-ray-irradiated group. Sixty-five pups from nine parents were examined in nonirradiated controls.
Immunohistochemical analysis
Immunohistochemical analysis was performed according to previous reports (Mizuochi et al., 1997, 1998; Sato et al., 1998). Embryos were collected 0, 3, 6, 12, 24, 36, and 48 h after 1-Gy or 2-Gy irradiation. Nonirradiated and age-matched embryos were used as controls. Whole embryos were embedded in O.C.T. compound (Sakura, Tokyo, Japan) and frozen. Sections (10 µm) were made by Cryostat (Sakura, Japan), fixed in acetone, blocked with 2% goat serum (Rockland, Gilbertsville, PA) and 3% BSA (Seikagaku Kogyo, Tokyo, Japan) for 30 min, and incubated overnight with primary antibodies described below at 4°C. For secondary antibodies, biotinylated goat anti-rat IgG (H+L) (Zymed, South San Francisco, CA) was used and applied for 1 h. To visualize the bounded antibodies, we incubated sections with alkaline phosphatase-conjugated streptavidin (Vector, Burlingame, CA) for 30 min and with Histomark Red (Kirkegaard and Perry, Gaithersburg, MD). Nuclei were stained by Mayer’s Hematoxylin (Merck, Darmstadt, Germany). For fluorescence microscopy, streptavidin, Alexa Fluor 488 conjugate (Molecular Probes) or streptavidin, Alexa Fluor 568 conjugate (Molecular Probes, Eugene, OR) was used.
Sources of primary antibodies were as follows: biotinylated antibody against F4/80 (Serotec, Oxford, UK), antibody against CD11b (Caltag, Burlingame, CA), antibody against CD31 (BD Pharmingen, San Jose, CA), and antibody against ßIII tubulin (Promega, Madison, WI). Culture supernatants of rat hybridoma cells producing mAb specific for MGL1 (mAb LOM-8.7; IgG2a) and MGL2 (mAb URA-1; IgG 2a) were used (Kimura et al., 1995). The preparation of mAb URA-1 will be reported in a separate paper. Purified antibodies were used in all experiments. Normal rat IgG (ICN, Costa Mesa, CA) and normal mouse IgG (ICN) were used as negative controls.
For double immunostaining, mAb LOM-8.7 was applied followed by Alexa Fluor 488-labeled goat anti-rat IgG (Molecular Probes), then biotinylated antibody against F4/80 was applied followed by streptavidin, Alexa Fluor 568 conjugate. Sections were observed using a confocal microscope (MRC-1024, BioRad, Hercules, CA) equipped with a krypton/argon laser. Three embryos from three parents (nine) were examined.
TUNEL method
Sections were air-dried and fixed with 2% paraformaldehyde (PFA) and preincubated with 0.1% sodium citrate containing 0.1% Triton X-100. Apoptotic cells were detected by the In-Situ-Cell-Death-detection kit (Roche, Mannheim, Germany), according to the manufacturer’s instructions.
Immunohistochemical staining with recombinant MGL1
Recombinant MGL1 corresponding to its extracellular domain was prepared using Escherichia coli (BL21), as previously described (Sato et al., 1992; Tsuiji et al., 2002). Proteins were purified by affinity chromatography on galactose-Sepharose 4B (Sato et al., 1992). Recombinant proteins were labeled with N-hydroxyl succinimide-biotin (Sigma, St. Louis, MO). Sections were fixed by 2% PFA and incubated with brMGL1 overnight at 4°C. After treatment with alkaline phosphatase-conjugated streptavidin for 30 min, sections were visualized by Histomark Red. For fluorescence microscopy, sections were visualized by streptavidin, Alexa Fluor 568 conjugate. Sections were washed by phosphate-buffered saline (PBS) containing 0.91 mM of CaCl2 and 0.49 mM of MgCl2. To mask the CRD, we pretreated MGL1 with galactose (10 mM) (Wako Pure chemicals, Osaka, Japan) for 30 min on ice, then applied on sections.
Statistical analysis
All results are expressed as mean ± SEM. A student’s t test was used to determine statistical significance.
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Acknowledgments |
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We thank S.M. Hedrick and T.M. Onami for providing Mgl1–/– mice. This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (11557180, 11672162, and 12307054), from the Research Association for Biotechnology and from the Program for Promotion of Fundamental Studies in Health Sciences of the Pharmaceutical and Medical Device Agency.
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Abbreviations |
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brMGL1, biotinylated recombinant MGL1; C-type lectin, calcium-type lectin; d.p.c., days post coitus; mAb, monoclonal antibody; MGL, macrophage galactose-type C-type lectin; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling
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References |
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