Redistributions of macrophages expressing the macrophage galactose-type C-type lectin (MGL) during antigen-induced chronic granulation tissue formation

Kayoko Sato1, Yasuyuki Imai2, Nobuaki Higashi1, Yosuke Kumamoto1, Naofumi Mukaida3 and Tatsuro Irimura1

1 Laboratory of Cancer Biology and Molecular Immunology, Graduate School of Pharmaceutical Sciences, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
2 Department of Microbiology, University of Shizuoka School of Pharmaceutical Sciences, Shizuoka 422-8526, Japan
3 Division of Molecular and Bioregulation, Cancer Research Institute, Kanazawa University, Ishikawa 920-0934, Japan

Correspondence to: T. Irimura; E-mail: irimura{at}mol.f.u-tokyo.ac.jp


    Abstract
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 Abstract
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 Methods
 Results
 Discussion
 References
 
Cell surface lectins are known to regulate trafficking of cells in the immune system, yet the role of macrophage galactose-type C-type lectin 1 and 2 (MGL1/2) is poorly understood. In this study, antigen-specific chronic inflammation was induced in a subcutaneous air pouch model in mice, and distribution of cells expressing MGL1/2 was investigated. Azobenzenearsonate-conjugated acetylated BSA, used as an antigen, was introduced into an air pouch of immunized mice, and tissue formation and distribution of MGL1/2-positive cells in the sub-dermal regions was examined. Thickness of the inflammatory tissue and number of MGL1/2-positive cells simultaneously reached the maximum at day 4 and returned to the control level at day 6 or 8. When additional antigenic challenges were given, a chronic granulation tissue, which had two distinct layers, was generated. In the chronic tissue, CD11b-positive/MGL1/2-negative cells were abundant in the area close to the antigenic stimulus, while the area far from the antigenic stimulus was dominated by MGL1/2-positive/CD11b-negative or -low cells. Flow cytometric analyses of isolated cells from the granulation tissue revealed that MGL1/2-positive cells expressed MHC class II at high levels, CD11b at low levels but no CD11c. MGL1/2-positive and -negative fractions were separated from cells in the granulation tissue and a higher level of IL-1{alpha} messenger RNA than negative populations was detected in the MGL1/2-positive fraction by the semi-quantitative reverse transcription–PCR method. IL-1{alpha} production by MGL1/2-positive cells was also immunohistochemically detected. Results suggest that MGL1/2-positive cells represent a distinct sub-population of macrophages, having unique functions in the generation and maintenance of granulation tissue induced by antigenic stimuli.

Keywords: carbohydrate recognition, cellular immunity, inflammation, tissue remodeling


    Introduction
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Macrophages and related cells of myeloid origin are known to express a variety of lectins, such as galectin-3, sialoadhesin and mannose receptor (1, 2). These lectins expressed on macrophages are involved in cell–cell adhesion (3), hematopoiesis (4), endocytosis and clearance of decayed cells and molecules (5). Among these lectins, macrophage galactose-type C-type lectins (MGL) are unique for the carbohydrate specificity toward galactose and N-acetylgalactosamine as monosaccharides and specific expression on the surfaces of immature dendritic cells and macrophage precursors (6, 7). Localization of cells expressing MGLs is limited to the connective tissue (8). MGLs are composed of type II transmembrane proteins and were shown to function as a cell surface-binding site for glycans with galactose or N-acetylgalactosamine and to participate in the internalization of these glycans. There are two homologous MGL isomers in mice: MGL1 and 2 (9) but a distinct role for each homolog is not defined.

The occurrence of the cells expressing MGL1 or 2 in tissue appears to be prominent during the course of antigenic sensitization. For example, the expression of the rat homolog of MGL was demonstrated in a rat cardiac allograft with arteriosclerosis during the process of chronic rejection using differential messenger RNA (mRNA) display (10, 11). Migration of MGL1/2-positive dermal macrophages has been demonstrated in the sensitization phase of contact hypersensitivity (12), although causal involvement of MGL1/2 molecules was still unknown. Upon epicutaneous application of haptens, MGL1/2-positive cells migrated from the dermis to the draining lymph nodes. The accumulation of MGL1/2-positive cells in the draining lymph nodes appeared to be positively correlated to the efficacy of sensitization.

Inflammatory cytokines, such as IL-1{alpha}, IL-1ß and tumor necrosis factor-{alpha} (TNF-{alpha}), locally available at the site of sensitization, were demonstrated to be responsible for the initiation of emigration of MGL1/2-positive cells, though the cells secreting these cytokines were not identified (13, 14). A blocking mAb against MGL1 inhibited the initiation of dermal macrophage migration, indicating that MGL1 is not just a marker but was actively involved in the inflammatory processes (14). These studies revealed not only the involvement of MGL1/2-positive cells in the initiation phase of antigen-specific inflammation but the ability of these macrophages located in the connective tissue to migrate to lymph nodes and to turn over during the course of the immune response. An important unsolved question was how MGL1/2-positive cells are related to populations defined by other markers of macrophages and related cells involved in the particular process of immune response and inflammation.

In the present study, we focused on the distribution of MGL1/2-positive cells during the tissue remodeling process induced by antigenic stimulation using an air pouch model (15). In this particular model, MGL1/2-positive cells seem to represent a unique sub-population of macrophages. The cells were MHC class II high, CD11b low or negative, CD11c negative and produced IL-1{alpha}. Their distribution in the granulation tissue was different from that of CD11b-positive macrophages.


    Methods
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 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Mice
Female, specific pathogen-free C57BL/6 mice were purchased from Charles River Japan Inc. (Yokohama, Japan) or SLC Japan Inc. (Shizuoka, Japan). All animal experiments were performed in accordance with the guidelines of the Bioscience Committee of the University of Tokyo and were approved by the Animal Care and Use Committee of the Graduate School of Pharmaceutical Sciences of the University of Tokyo.

Antigen
The antigen used was azobenzenearsonate-conjugated acetylated BSA (ABA-AcBSA) prepared in our laboratory according to the method of Tabachnick and Sobotka (16).

Antibody
Rat mAb LOM-14 (IgG2b), which recognizes both MGL1 and MGL2, and rat mAb LOM-8.7 (IgG2a), which binds MGL1 (9), were prepared as described (17). The reactivities of LOM-14 and LOM-8.7 against mouse MGLs have been described previously (9). Rabbit anti-mouse IL-1{alpha} polyclonal antibody without cross-reactivity to other cytokines (18) was also used. The following were purchased from suppliers: rat mAb specific for mouse MHC class II (M5/114/15.2, rat IgG2a, eBioscience, CA, USA), FITC-conjugated mouse mAb specific for rat {kappa} and {lambda} light chains (Sigma Chemical Co., MO, USA), goat polyclonal antibody specific for rat {kappa} light chain immobilized on microbeads (Miltenyi, Bergisch Gladbach, Germany), rat mAb specific for CD11b (M1/70.15, rat IgG2b, Caltag, CA, USA), mAb ER-TR7 (rat IgG2a, Biogenesis, UK), mAb anti-CD31 (MEC-13.3, rat IgG2a, BD Pharmingen, CA, USA), rat mAb specific for mouse CD45 (30-F11, rat IgG2a, BD Pharmingen), rat mAb specific for mouse CD68 (FA-11, rat IgG2a, Serotec, UK), biotin-conjugated mAb F4/80 (Serotec), hamster mAb specific for mouse CD11c (N418, hamster IgG, BD Pharmingen, FITC-conjugated sheep Fab anti-digoxigenin (Vector Laboratories, CA, USA), Cy3.5-conjugated sheep Fab anti-digoxigenin (Vector Laboratories), FITC-conjugated anti-CD14 mAb (rmC5-2, rat IgG1, BD Pharmingen), Cy3.5-conjugated streptavidin (Vector Laboratories), FITC-conjugated anti-rat IgG (Zymed), FITC-conjugated streptavidin (Zymed), Alexa568-conjugated anti-rat IgG (Molecular Probes, OR, USA), Alexa568-conjugated streptavidin (Molecular Probes), purified hamster IgG (BD Pharmingen) and purified rat IgG2b (Caltag).

Immunization and induction of allergic inflammation in mice
Mice were immunized with 200 µg of ABA-AcBSA emulsified 1 : 1 in CFA (DIFCO, MI, USA). The time course of the protocol is described in Fig. 1. Fifty-microliter aliquots (50 µg antigen) of the emulsion were injected subcutaneously into each of the footpads of a mouse (day –10 in Fig. 1). Nine days after immunization, 2 ml of air was subcutaneously injected into the dorsa of the mice under ether anesthesia to produce a regular oval air pouch (day –1 in Fig. 1). The day following the air pouch formation, an antigenic challenge was carried out by injecting 250 µg of ABA-AcBSA dissolved in 1 ml of a sterile 2% (w/v) solution of sodium carboxymethylcellulose in saline into the air pouch (day 0 in Fig. 1) under ether anesthesia. Skin samples from the air pouch were collected after 12 h as well as 1, 2, 3, 4, 5, 6 and 8 days after the challenge. The tissues were embedded in O.C.T. compound (Miles, Elkhart, IN, USA) and frozen in a liquid nitrogen bath.



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Fig. 1. The time course of immunization and the first and the second challenge. An antigenic challenge was performed by introducing ABA-AcBSA into the dorsal air pouch, which was formed 1 day before, on C57BL/6 mice that had been immunized with the same antigen 10 days before. Skin samples from the air pouch were collected after 12 h as well as 1, 2, 3, 4, 5, 6 and 8 days after the first challenge. To induce chronic inflammation, ABA-AcBSA was injected into the air pouch 5 days after the first challenge. At 6, 12 and 27 days after the second challenge (11, 18 and 32 days after the first challenge), skin samples from the air pouch were collected, sectioned and examined under a microscope.

 
Induction of chronic inflammation by secondary antigenic challenge
To induce chronic inflammation, 500 µg of ABA-AcBSA in 1 ml sterile saline was injected into the air pouch 5 days after the first challenge (day 5 in Fig. 1). On days 11, 18 and 32, in Fig. 1 (days 6, 12 and 27 after the second challenge), skin samples from the air pouch were collected and frozen as above.

Preparation of single-cell suspension from granulation tissue
Sub-dermal granulation tissue formed after the secondary challenge at the site of the air pouch was peeled off from the skin. The tissue samples were cut with scissors into 2-mm cubes, and the fragments were incubated in 0.5% collagenase (Clostridium histolyticum, Wako Pure Chemical, Tokyo, Japan) in sterile DMEM/Ham's F-12 media (Nissui Pharmaceutical, Tokyo, Japan) for 30 min at 37°C. Cells released from the tissue were washed and re-suspended in PBS and then analyzed for cell surface markers by flow cytometry.

MGL1/2-positive cells were prepared by magnetic cell sorting as described previously (12). In brief, 2 x 107 cells were incubated with anti-MGL1/2 mAb LOM-14 (3 µg ml–1 in 2 ml of 0.1% BSA/PBS) for 30 min on ice and then incubated with goat anti-rat {kappa} microbeads for 15 min at 8°C. Cell separation was performed using an RSTM column with a magnetic cell sorter I (Miltenyi). Cells in the flow-through fraction were kept as an MGL1/2-negative population. After the column was washed with 0.1% BSA/PBS, cells retained in the column were recovered by washing the column outside the magnetic field. In some experiments, mAb anti-MHC class II (1/10 dilution) or mAb anti-CD11b (1/10 dilution) was used as a primary antibody to separate cells based on the expression of MHC class II or CD11b.

Flow cytometric analysis
Cells (1 x 106) were pre-incubated for 30 min on ice in 100 µl of PBS containing 0.1% BSA and 0.1% NaN3 (F-PBS: PBS for flowcytometry – PBS with 0.1% BSA and 0.1% NaN3). The cells were then incubated for 60 min on ice either with rat anti-mouse MHC class II mAb (1/10 dilution of supernatant), rat anti-mouse CD11b mAb (1/10 dilution of supernatant), or with hamster anti-mouse CD11c mAb (1/10 dilution of supernatant). As a control, purified rat IgG2b or hamster IgG was used at 1 µg ml–1. After washing twice with F-PBS, cells were incubated with FITC-conjugated anti-rat {kappa} and {lambda} light chains (1/100 dilution) or FITC-conjugated anti-hamster IgG (1/100 dilution) for 30 min on ice. After washing twice, the cells were then incubated with 100 µl of PE-conjugated mAb LOM-14 (1 µg ml–1) for 60 min on ice. The cells were washed twice, and then treated with propidium iodide (PI) at 5 µg ml–1 in F-PBS to discriminate dead cells. The cells were analyzed on a flow cytometer (EPICS XL, Beckman Coulter) with an argon laser (488 nm) by collecting data only from PI-unstained cells. A 525–550 nm band pass filter was used for FITC; 575–600 nm for PE; 620–645 nm for PI. Overlaps of fluorescence emission spectra of PE and PI did not elicit any problem.

Immunohistochemistry
MGL1/2-positive cells were immunohistochemically detected on frozen sections of skin using anti-MGL1/2 mAb LOM-14 (culture supernatant, 1/10 dilution). In some experiments, the sections were incubated with anti-CD11b (1/10 dilution), mAb ER-TR7 (1/100 dilution) or anti-CD31 (1/100 dilution) to detect each marker. As a negative control, purified rat IgG2b (1 µg ml–1) was used. The antibody binding was detected using biotinylated mAb mouse anti-rat {kappa} and {lambda} (1/100 dilution) and alkaline phosphatase–streptavidin (1/100 dilution) as described previously (19). For multicolor immunofluorescence, sections were incubated with digoxigenin-conjugated mAb LOM-14 (1/100 dilution) plus either biotinylated anti-CD11b or biotinylated anti-MHC class II for 1 h at 20°C after acetone fixation. The sections were then fixed in 2% PFA in 0.1 M sodium phosphate (pH 7.0). The binding of the digoxigenin-conjugated mAb LOM-14 and that of biotinylated antibodies was detected by incubation with FITC-conjugated sheep Fab anti-digoxigenin (1/100 dilution) and Cy3.5-conjugated streptavidin (1/200 dilution), respectively, for 1 h at 20°C. In some experiments, sections were stained with a combination of digoxigenin-conjugated mAb LOM-14 plus Cy3.5-conjugated sheep Fab anti-digoxigenin (1/2000 dilution), as well as with FITC-conjugated anti-CD14 mAb (1/20 dilution). In other experiments, sections were incubated with a combination of rat anti-mouse CD68 mAb plus FITC-conjugated anti-rat IgG and biotin-conjugated mAb LOM-14 plus Alexa568-conjugated streptavidin. Alternatively, biotin-conjugated F4/80 plus FITC-conjugated streptavidin and rat mAb LOM-14 plus Alexa568-conjugated anti-rat IgGs were used. Staining reaction under these conditions did not show any cross-reactivity (data not shown). To identify the expression of IL-1{alpha} protein in MGL1/2-positive cells, sections were stained with a combination of rabbit anti-mouse IL-1{alpha} antibody (30 µg ml–1) plus biotinylated goat anti-rabbit IgG mAb plus Alexa568-conjugated avidin and rat mAb LOM-14 plus Cy5-conjugated mouse anti-rat IgG. After each incubation or fixation, the sections were gently washed twice in PBS. After the final wash, the sections were mounted in Vectashield (Vector Laboratories) or PermaFlow (Shandon, CA, USA) and were observed under a confocal microscope (MRC-1024, Bio-Rad, Herts, UK).

Cytokine detection by semi-quantitative reverse transcription–PCR
Cells separated by magnetic cell sorting were subjected to total RNA extraction using Ultraspec RNA zol (Biotex, Houston, TX, USA) according to the manufacturer's instructions. First strand cDNA synthesis was carried out using oligo (dT) 12–18 and superscript II reverse transcriptase (Invitrogen). The cDNA was used as a template for PCR. PCR was performed by Ampli Taq Gold polymerase (Applied Biosystems) and with a specific primer combination for mouse IL-1{alpha} (5'-CAAACTGATGAAGCTCGTCA-3', 5'-TCTCCTTGAGCGCTCACGAA-3'), mouse IL-1ß (5'-TCATGGGATGATGATGATAACCTGCT-3', 5'-CCCATACTTTAGGAAGACACGGATT-3'), mouse TNF-{alpha} (5'-TTCTGTCCCTTTCACTCACTGG-3', 5'-TTGGTGGTTTGCTACGACGTGG-3') and mouse GAPDH (5'-CACCATGGAGAAGAAGGCCGGGG-3, 5'-GACGGACACATTGGGGGTAG-3'). Amplification was carried out using 30, 40 and 50 cycles of 94°C for 30 s, 56°C for 30 s and 72°C for 45 s. The PCR products were separated on 0.8% agarose gels, stained with ethidium bromide.

Statistical analysis
Dunnet's multiple comparison test was used to assess the statistical significance of difference after a one-way analysis of variance. A value of P < 0.05 was considered significant.


    Results
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 Methods
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 References
 
Formation of transient granulation tissue
C57BL/6 mice pre-immunized with ABA-AcBSA were challenged by injecting the antigen into an air pouch on each mouse. A transient formation of granulation tissue was observed when a single dose of antigenic challenge was applied at day 10 into the air pouch that had been formed on the dorsa of the pre-immunized mice. Granulation tissue generated in the sub-dermal area between the skeletal muscle layer and the inner surface of the air pouch facing toward the antigenic stimuli contained morphologically heterogeneous cells and stromal tissue (Fig. 2b). Density of cells in the corresponding area in the air pouch was low when the antigenic challenge was not given (Fig. 2a) or when the control antigen (BSA) was given (data not shown). Thus, the tissue formation in the air pouch was found to be induced by specific antigenic stimuli.



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Fig. 2. Immunohistochemical observations of the granulation tissue after a single dose of antigenic challenge in the air pouch. An antigenic challenge was performed by introducing ABA-AcBSA into the dorsal air pouch on C57BL/6 mice that had been immunized with the same antigen 10 days before. Skin samples from the air pouch were recovered 2 days (g) or 4 days (b–f, h–k) after the first challenge. As a control, the skin on the air pouch was collected without antigenic challenge (a). Frozen sections of the skin samples were immunohistochemically stained for MGL1/2 using mAb LOM-14 (a, c, g, h), for CD11b (d, i), for a fibroblast marker using mAb ER-TR7 (e, j) or with normal rat serum as a control (f, k). The antibody binding was visualized by the alkaline phosphatase reaction products (red). Nuclei were counter stained with hematoxylin (blue purple). Hematoxylin–eosin staining of the section was also shown (b). Boxed areas in (c), (d), (e) and (f) are shown at a higher magnification in (h), (i), (j) and (k), respectively. The area shown in (g) corresponds to the area shown in (h) except that the sample was obtained 2 days, not 4 days, after the challenge. Granulation tissue was formed in the hypodermis toward the inner surface of the air pouch. The granulation tissue abundant with MGL1/2-positive cells is seen. (l) Skin on the air pouch was recovered at the indicated number of days after the challenge (abscissa). The thickness of the granulation tissue was microscopically determined by measuring the hypodermis region between the muscle fiber layer (M) and the inner surface of the air pouch (ordinate). The results are mean ± SE. (m) The percentage of MGL1/2-positive cells in the granulation tissue was microscopically determined by counting the number of MGL1/2-positive cells relative to the total cell number within a defined area. Mean of six independent measurements is shown (ordinate). The thickness of the granulation tissue and the number of MGL1/2-positive cells simultaneously increased and reached maximums at 3 or 4 days after the antigenic challenge. The granulation tissue resolved thereafter. E represents epidermis, D represents dermis and M represents the muscle fiber layer (a–f). Scale bars represent 100 µm (a–f) and 10 µm (g–k). Asterisks indicate the statistically significant difference (P < 0.01) against the thickness at day 0; n.s., not significant.

 
To determine the extent of the granulation tissue formation, the distance between the skeletal muscle layer and the inner surface of the air pouch was measured as shown in Fig. 2 (a and b). Nine arbitrary selected fields in five sections independently prepared from each mouse were used for each measurement. The means of 45 measurements for an individual mouse are plotted in Fig. 2l. The thickness of the granulation tissue increased after the antigenic challenge, reached a maximum at day 4 and then decreased (Fig. 2l). At day 8 after the antigenic challenge, the thickness returned to its original level before the antigenic challenge.

MGL1/2-positive cells in granulation tissue at its transient stages
A significant portion of the cells in the granulation tissue seemed to express MGL1 or 2 as revealed by immunohistochemical methods with mAb LOM-14 (Fig. 2b). The number of these cells reached a maximum at days 3 and 4, and then rapidly decreased (Fig. 2m). MGL1/2-positive cells always seemed to be present during the generation of granulation tissue, while a reduction in the number of MGL1/2-positive cells preceded the disappearance of the granulation tissue (Fig. 2c, g, h). Cells expressing CD11b, another macrophage marker, were also observed in the granulation tissue (Fig. 2d and i). At this stage, MGL1/2-positive cells also seemed to express CD11b (data not shown). In addition, cells stained with mAb ER-TR7, a fibroblast marker, were also abundant in the granulation tissue (Fig. 2e and j), suggesting that fibroblasts were providing the stromal component. No CD31-positive cell was observed at this stage. CD3-positive T cells were observed in this area after day 3 (data not shown) and were thought to be involved in the antigen-induced tissue formation.

Generation of chronic granulation tissue and MGL1/2-positive cells in chronic granulation tissue
An additional antigenic challenge was given 5 days after the first challenge in the air pouch. After this treatment, the granulation tissue apparently transformed itself into a chronic phase. The thickness of the tissue further increased, and the granulation tissue persisted for at least 32 days (Fig. 3a). The distribution patterns of MGL1/2-positive cells in the granulation tissue at this chronic phase were different from those observed after a single antigenic challenge. That is, two layers with different cell populations could be distinguished. The MGL1/2-positive cells were localized in an area distant from the inner surface of the air pouch (Fig. 3b and d). In contrast, CD11b-positive cells were found in an area close to the inner surface (Fig. 3c and e). Thus, CD11b-positive inflammatory cells were mainly observed in the area close to the antigenic stimuli, whereas MGL1/2-positive cells were found in an area behind the front line of inflammatory responses. The blood vessels in the granulation tissue were identified by the CD31 expression only after the second challenge (Fig. 3f), indicating that angiogenesis took place in the chronic phase. At this stage, the cells expressing MGL1/2 did not seem to express CD11b. Two-color immunohistological analyses revealed that MGL1/2-positive cells expressed MHC class II (Fig. 4a–d), CD14 (Fig. 4e–h), or both (data not shown) but that MGL1/2 and CD11b were not expressed on a single cell simultaneously (Fig. 4i–n). In addition, MGL1/2-positive cells were also shown to express CD68 (Fig. 4o–q), F4/80 (Fig. 4r–t) and moma-2 (data not shown).



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Fig. 3. Immunohistochemical observations of the granulation tissue after the second antigenic challenge in the air pouch. Antigen was administrated into the air pouch at day 5 after the first challenge. (a) The thickness of granulation tissue (shown by a broken line in the panel b) was measured according to the time course shown in the legend to Fig. 1. Thickness measurements are summarized in which results after the second challenge are shown by a thick line and those after the single challenge (as shown in the Fig. 1) by a dotted line. The results are mean ± SE (n = 5). Skin samples from the air pouch were collected at day 11, and their frozen sections were stained using anti-MGL1/2 mAb LOM-14 (b, d) or an anti-CD11b mAb (c, e). A boxed area in the panel (b) and a dotted boxed area in panel (c) are shown at a higher magnification in (d) and (e), respectively. Vascular endothelial cells are visualized using an anti-CD31 mAb (f). The MGL1/2-positive cells in the granulation tissue are distributed in the area far from the inner surface of the air pouch (b, d), whereas the area close to the inner surface scarcely contains any MGL1/2-positive cells. In contrast, CD11b-positive cells are mainly distributed in the area close to the inner surface (c, e). Blood vessels are clearly seen in the granulation tissue (f). The granulation tissue persisted at least 28 days after the second challenge (a). D and M represents the dermis and the muscle fiber layer (b and c), respectively. Bars represent 100 µm (b, c, f) and 10 µm (d, e). Asterisks indicate the statistically significant difference (P < 0.05) against the thickness at day 5.

 


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Fig. 4. Phenotypic characterization of MGL1/2-positive cells in the granulation tissue after the second antigenic challenge in the air pouch. Skin samples were prepared as described in the legend to Fig. 3. The frozen sections were prepared and stained for MGL1/2 using digoxigenin-conjugated mAb LOM-14. Bound mAb was visualized by FITC-conjugated anti-digoxigenin (a–d, i–n) or Cy3.5-conjugated anti-digoxigenin (e–h). The sections were also stained by biotin-conjugated anti-MHC class II mAb (a–d), FITC-conjugated anti-CD14 mAb (e–h) or biotin-conjugated anti-CD11b mAb (i–n). Biotin-conjugated antibodies were visualized by Cy3.5-conjugated streptavidin (a–d, i–n). Confocal microscopic data representing single-color signals are shown for MGL1/2 (a, e, i, l), MHC class II (b), CD14 (f) and CD11b (j, m). (o–q) Double staining for macrophage markers CD68 was carried out on MGL1/2-positive cells. The sections were stained with a combination of biotin-conjugated mAb LOM-14 plus FITC–streptavidin and rat anti-mouse CD68 mAb plus Alexa568-conjugated anti-rat IgG. (r–t) The sections were stained with a combination of mAb LOM-14 plus FITC–anti-rat IgG and biotin-conjugated mAb F4/80 plus Alexa568-conjugated streptavidin. Profiles with MGL1/2 (green) and MHC class II (red) were merged in panels (c) and (d). Profiles with CD14 (green) and MGL1/2 (red) were merged in panels (g) and (h). Profiles with MGL1/2 (green) and CD11b (red) were merged in panels (k) and (n). Profiles with MGL1/2 (red) and F4/80 (green) were merged in panel (q). Profiles with MGL1/2 (green) and CD68 (red) were merged in panel (t). The majority of MGL1/2-positive cells expressed MHC class II, CD14, CD68 and F4/80. CD11b-positive cells were not positive with mAb LOM-14. Magnifications are x400 except for the panels (a–c) and (e–g) (x100).

 
Flow cytometric analysis of cells from chronic granulation tissue
To determine marker molecules on MGL1/2-positive cells, flow cytometric analyses using cells enzymatically dissociated from the granulation tissue were carried out. Most MGL1/2-positive cells expressed MHC class II (Fig. 5d), whereas a low level of CD11b expression was detected on a portion of the MGL1/2-positive population (Fig. 5c). Although MGL1/2-positive cells did not express CD11b (Fig. 4i–n) in an immunohistochemical analysis, MGL1/2-positive cells were shown to be stained with anti-CD11b mAb at a low level in the flow cytometric analysis (Fig. 5c). The apparent discrepancy is due, at least in part, to the difference in detection sensitivity between flow cytometric analysis and immunohistochemistry. MGL1/2-positive cells did not show expression of CD11c (Fig. 5e), CD86 or DEC205 (data not shown). Thus, these cells were judged to belong to a sub-population of macrophages.



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Fig. 5. Flow cytometric analysis of cells from the chronic granulation tissue. The cells were collected by enzymatic dissociation and were incubated with rat anti-mouse MHC class II (d), rat anti-mouse CD11b (c), hamster anti-mouse CD11c (e) or purified rat IgG2b (a, b). After incubations, bound antibodies were visualized with FITC-conjugated anti-rat {kappa} and {lambda} light chains or FITC-conjugated anti-hamster IgG and were then incubated with PE-conjugated mAb LOM-14 (b–e) or PE-rat IgG. The cells were analyzed on a flow cytometer (EPICS XL, Beckman Coulter). MGL1/2-positive cells were shown to express high levels of MHC class II and low levels of CD11b. MGL1/2-positive cells did not express CD11c.

 
Cytokine profiles of different cell populations in the chronic granulation tissue
MGL1/2-positive and -negative populations were isolated by collagenase digestion from the granulation tissue obtained at day 18 after the secondary challenge with antigen. The single-cell suspension was subjected to magnetic cell sorting to obtain positive and negative populations regarding MGL1/2, CD11b or MHC class II expression. Negative populations contained weakly positive cells because positive cell isolation was conducted using the column with a magnetic cell sorter I (Miltenyi) twice. The mRNA was extracted from these populations, and the relative mRNA levels for IL-1{alpha} were compared by reverse transcription–PCR in several different conditions.

IL-1{alpha} mRNA was detected in MGL1/2-positive populations and was almost absent from the negative populations obtained from the tissue at day 18 (Fig. 6). Although the results shown in this figure were obtained after 40 cycles of amplification, very similar differences were observed after 50 cycles of amplification. Such a difference in IL-1{alpha} mRNA levels was not seen when cells were separated based on high and low expression of MHC class II or CD11b. The MGL1/2-positive and -negative populations were not different from each other in the levels of mRNA for IL-1ß and TNF-{alpha} (Fig. 6). TNF-{alpha} could be detected on day 11 (data not shown) but was not observed on day 18. To confirm the expression of IL-1{alpha} protein in MGL1/2-positive cells, normal skin and granulation tissues were analyzed by two-color immunohistochemical methods. In the normal dermis, IL-1{alpha} was observed associated with both MGL1/2-positive and -negative cells. In the granulation tissue, expression of IL-1{alpha} was associated only with MGL1/2-positive cells (Fig. 7a–i).



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Fig. 6. Characterization of inflammatory cytokine profiles of MGL1/2-positive cells in the granulation tissue. Cells were isolated by collagenase digestion from the granulation tissue at day 18, and then subjected to magnetic cell sorting based on the expression of MGL1/2, MHC class II or CD11b. The mRNA was prepared from each cell population, and reverse transcription (RT)–PCR was carried out to compare the levels of mRNA for IL-1{alpha}, IL-1ß, TNF-{alpha} and GAPDH as a control. The PCR products after 40 cycles were electrophoretically separated on an agarose gel (0.8%), stained with ethidium bromide and visualized using an image analyzer. The RT–PCR product of each lane represents 400 ng of total RNA. MGL1/2-positive cells expressed IL-1{alpha} mRNA, whereas MGL1/2-negative cells did not. Such differential expression was not seen when cells were separated based on the expression of MHC class II or CD11b.

 


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Fig. 7. Immunohisotochemical localization of inflammatory cytokines in relation to the expression of MGL1/2 in granulation tissue. To confirm the association of IL-1{alpha} with MGL1/2-positive cells, the frozen sections were stained with rabbit polyclonal anti-IL-1{alpha} antibody. Biotinylated anti-rabbit IgG and Alexa568-conjugated streptavidin were used in (a–i). The same sections were also stained with mAb LOM-14, followed by staining Cy5-conjugated mouse anti-rat IgG. Confocal microscopic images representing single-color signals are shown for IL-1{alpha} (a, d, g) and MGL1/2 (b, e, h). Data for IL-1{alpha} (red) and MGL1/2 (blue) were merged in the panels (c), (f) and (i). Three or four cells can be seen in the MGL1/2-positive cells in both dermis and granulation tissues were shown to be stained with anti-IL-1{alpha}. In the dermis, IL-1{alpha} was expressed in MGL1/2-positive cells and MGL1/2-negative cells. In the granulation tissue, expression of IL-1{alpha} was associated with MGL1/2-positive cells. Bars represent 10 µm.

 
Because IL-1{alpha} was shown to be involved in the fibroblast activation (20, 21), the present results suggest that the MGL1/2-positive population participate in the tissue remodeling through the production of IL-1{alpha}. Although IL-1ß was also known to be involved in the tissue remodeling process, MGL1/2-positive cells in this model did not seem to secrete IL-1ß (data not shown).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 
Two different stages of antigen-induced inflammation of tissue formation—the transient and chronic phases—were examined for the distribution of cells expressing cell surface MGL. When a single dose of antigen, ABA-AcBSA, was injected into an air pouch of pre-immunized mice, an acute response characterized by infiltration of inflammatory cells took place and granulation tissue was transiently developed in the sub-dermal region toward the inner surface of the air pouch. This granulation tissue resolved within several days after a single antigenic challenge. When the antigenic challenge was repeated twice or more, the nature of the granulation tissue seemed to be different. Chronic inflammation was induced and the granulation tissue persisted for a longer period of at least a month. Similar air pouch models have been used to determine the effects of anti-inflammatory drugs on inflammatory tissue formation (2224), although molecular and cellular events during the processes were poorly understood. Antigen-induced granulation tissue formation was previously characterized with respect to its time-dependent cellular consequences (25, 26). In a model of soluble egg antigen (SEA)-induced granulomatous hyporesponsiveness, sensitization with SEA seemed to enhance the phagocytic activity of macrophages via increasing expression of CD11b (27). However, characteristics and the stages of activation of cellular participants, such as neutrophils, macrophages and related cells and lymphocytes, were poorly understood.

MGL1/2 are C-type lectins specific for galactose and N-acetylgalactosamine as monosaccharides. MGL1/2 are type II transmembrane glycoproteins with a single carbohydrate recognition domain and form clusters on cell surfaces (9, 28). The range of cells expressing this lectin is limited to the intermediate stage of macrophages and immature dendritic cells (6, 29, 30). Langerhans cells and tissue-specific macrophages such as alveolar macrophages and Kupffer cells do not express MGL1/2. Immunohistochemical examination of MGL1/2-positive cells in naive mice showed that their localization was limited to the connective tissues (8). MGL1/2 were suggested to function protectively against the formation of lymph node metastasis (31).

We used MGL1/2 to identify the localization of a sub-population of macrophages and related cells within the granulation tissue. After a single dose of antigenic stimulation, MGL1/2-positive cells distributed throughout the transient granulation tissue (Fig. 2). CD11b-high cells were also found in the same area. Cells with fibroblast markers (ER-TR7) were also observed. When the chronic response was induced, the localization of MGL1/2-positive cells and that of CD11b-high cells was different. CD11b-high cells accumulated in the region close to the antigenic stimuli, whereas MGL1/2-positive cells were found in the region away from the antigenic stimuli (Fig. 3). In this chronic granulation tissue, angiogenesis was observed in all areas by staining with antibodies against CD31, a marker for vascular endothelial cells. Distributions of CD14-positive cells, MHC class II-positive cells, moma-2-positive cells and F4/80-positive cells were also observed in all areas (data not shown). The spatial separation of MGL1/2-positive/CD11b-low and MGL1/2-negative/CD11b-high populations was further supported by two-color immunohistochemical studies. MGL1/2-positive cells were essentially devoid of, or expressed, a very low level of CD11b, and CD11b-positive cells were lacking in MGL1/2. The two-color analyses revealed that MHC class II and CD14 cells were expressed on MGL1/2-positive cells whereas CD11b-high populations contained MHC class II-positive and -negative cells. The co-expressions of MGL1/2 and MHC class II were further supported by flow cytometric analyses of the cells isolated from the granulation tissue using collagenase digestion. In granulation tissue, the number of CD11b-positive cells was reported to increase in parallel with tissue formation (32, 33). However, the role of CD11b-negative macrophages was not previously identified. In the inflammatory tissue at the transient phase, MGL1/2-positive cells also apparently expressed CD11b. In the chronic phase, MGL1/2-positive/CD11b-low cells appeared and showed unique distributions. These MGL1/2-positive/CD11b-low cells apparently expressed CD14, CD68, F4/80 and moma-2 (data not shown). These cells also expressed a high level of MHC class II. Thus, MGL1/2-positive cells were suggested to be a novel sub-population of macrophages. Whether these cells are unique to antigen-induced chronic inflammatory tissue remains to be elucidated.

MGL1/2-positive cells isolated from the granulation tissue were investigated for their ability to produce cytokines. IL-1{alpha} mRNA was expressed in MGL1/2-positive cells, whereas there were no differences in IL-1ß mRNA expression and TNF-{alpha} mRNA was not detected (Fig. 6). In addition, protein expression of IL-1{alpha} on MGL1/2-positive cells was confirmed by immunohistochemical analysis (Fig. 7). Thus, MGL1/2-positive cells seemed to have a high potential of producing IL-1{alpha}. Among cytokines involved in the tissue remodeling, IL-1{alpha} and ß have been reported to activate fibroblasts by enhancing type I collagen biosynthesis and matrix metalloprotease production (20, 26, 3437). Enhanced IL-1{alpha} production in injured skin suggested that IL-1{alpha} was involved in the wound-healing cascade (21, 38). Although MGL1/2-positive cells were not the sole producers of IL-1{alpha}, IL-1{alpha} from MGL1/2-positive cells should strongly contribute to the formation and maintenance of granulation tissue.

In conclusion, cells expressing multiple macrophage markers and a lectin MGL1/2 appear in inflammatory connective tissue after antigenic challenges in an air pouch model. These cells are distinct from CD11b-positive/MGL1/2-negative macrophages in their distribution and are prominent in the production and secretion of IL-1{alpha}. These populations were observed in antigen-induced granulation tissues but not in naive dermis. Cell surface lectins are often involved in cellular trafficking and MGL1/2 may function as a trafficking molecule in the granulation tissue formation.


    Acknowledgements
 
This work was supported by grants-in-aid from the Ministry of Education, Science, Sports and Culture of Japan (11557180, 11672162 and 12307054) and from the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research.


    Abbreviations
 
ABA-AcBSA   azobenzenearsonate-conjugated acetylated BSA
MGL   macrophage galactose-type C-type lectin
mRNA   messenger RNA
PI   propidium iodide
SEA   soluble egg antigen
TNF   tumor necrosis factor

    Notes
 
Transmitting editor: M. Miyasaka

Received 4 September 2004, accepted 7 February 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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