Matrix Metalloproteinase-9 in Macrophages Induces Thymic Neovascularization following Thymocyte Apoptosis
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免疫学杂志 2005年第2期
Abstract
Matrix metalloproteinase-9 (MMP-9) has been implicated in the degradation of the extracellular matrix in a variety of physiological and pathological processes. We found that MMP-9 expression in thymuses of BALB/c mice that had been injected with anti-CD3 Ab to induce thymocyte apoptosis was increased both at mRNA and protein levels. Macrophages are shown to be the principal stromal cells responsible for phagocytosis of dying thymocytes, and macrophages were found to constitutively express MMP-9. The activity of plasmin, which is known as one of the activators for MMP-9, was increased in the thymuses with MMP-9 activation. Binding of Ab HUIV26, which recognizes a cryptic epitope on collagen type IV following proteolytic cleavage, was found to be reduced in MMP-9 knockout mice, suggesting that collagen type IV is a substrate of MMP-9. Although the formation of thymic neovessels was found following thymocyte apoptosis, it was diminished in anti-CD3 Ab-injected MMP-9 knockout mice. In vivo administration of Ab HUIV26 resulted in a reduction of thymic neovascularization. After clearance of apoptotic thymocytes, the number of macrophages in the thymuses was decreased, and this decrease was delayed by blocking of HUIV26 epitope. Taken together, our results suggest that MMP-9 expression in macrophages mediates degradation of collagen type IV and facilitates their migration from the thymus after clearance of apoptotic thymocytes. These studies demonstrate a potential role of macrophage MMP-9 in the remodeling of thymic extracellular matrix following thymocyte apoptosis.
Introduction
Matrix metalloproteinases (MMPs)2 are a group of zinc-dependent endopeptidases that play a key role in promoting tissue remodeling and tumor invasion by inducing proteolysis of several extracellular matrix components (1, 2). MMPs are produced as zymogens with a signal sequence and propeptide segment that is removed during activation.
Among MMPs, MMP-2 and MMP-9 constitute the key proteinases governing the degradation of basement membrane collagens. These metalloproteinases cleave collagen types IV, V, VII, and X as well as denatured collagens or gelatins (3, 4). They have collectively been referred to as type IV collagenases or gelatinases. Overexpression of MMP-9 has been implicated in physiological process of tissue remodeling (5, 6) and wound healing (7), or in the pathogenesis of various diseases, such as atherosclerosis, tumor invasion, and metastasis (8, 9, 10). MMP-9 from macrophages and neutrophils is thought to play a key role in the migration of these cells during inflammatory diseases such as arthritis (11). MMP-9 is responsible for the processing of cytokines, e.g., pro-IL-1 and pro-TNF- into their active form (12, 13). This proteinase also cleaves latent TGF-, which constitutes a novel mechanism of TGF- activation (14). In addition, MMP-9 is involved in the formation of new vessels essential for tumor growth and triggers the angiogenic switch during carcinogenesis (15). However, the mechanisms by which MMP-9 promotes vasculogenesis and tumor invasion are still poorly understood.
It has been established that stimulation of CD4+CD8+ thymocytes by an anti-CD3 Ab in vivo as well as in vitro induces apoptotic cell death, which has been proposed as a mechanism for deletion of autoreactive T cells (16, 17, 18, 19). In murine thymus after administration of anti-CD3 Ab, macrophages are shown to be key players in clearance of apoptotic thymocytes as well as resolving inflammation (20, 21, 22).
In the present study, we showed that MMP-9 expression was increased in murine thymus after administration of anti-CD3 Ab, and that MMP-9 was most likely derived from macrophages. To define the role of MMP-9, we used MMP-9 knockout (KO) mice and found that collagen type IV was a substrate of MMP-9. Exposure of a cryptic site within collagen type IV was associated with thymic neovascularization and macrophage migration from the thymus after clearance of apoptotic cells.
Materials and Methods
Mice
BALB/c mice were bred in our animal facility and used at the age of 7–12 wk. MMP-9-deficient (MMP-9 KO) mice have been described previously (23) and used at the age of 7 wk. All animal procedures described in this study were performed in accordance with the guidelines for animal experiments of our institutes.
Abs and reagents
Anti-mouse CD3 mAb (145-2C11) was purified from ascites fluid by protein A-affinity chromatography. HUIV26 Ab (24) was a gift from Cell-Matrix, and an isotype-matched control Ab was obtained from Medical Biological Laboratories. Oligonucleotide primers were from Sawady Technology. All other reagents were purchased from Sigma-Aldrich, except as specified below.
Injection of anti-CD3 Ab
Seven-week-old mice were injected i.p. with 50 μg of anti-CD3 Ab. At the indicated time, mice were sacrificed. Thymuses were removed, immersed in OCT compound (Miles), rapidly frozen on dry ice, and sectioned at 5 μm for histological analysis. Alternatively, each thymus was excised and used for protein extraction or total RNA isolation.
Treatment of mice with HUIV26 Ab
Seventeen hours postadministration of anti-CD3 Ab, mice were either untreated or treated i.p. with either purified HUIV26 or an isotype-matched control Ab (75 or 150 μg per mouse). An additional 11 or 19 h later, thymuses were excised and used for immunochemistry analysis.
Cell preparations and cell culture
For preparation of murine resident peritoneal macrophages, peritoneal exudate cells were collected from the peritoneal cavity of 9- to 12-wk-old BALB/c mice and washed with RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 μM 2-ME, 20 U/ml penicillin, 20 μg/ml streptomycin, and 10% heat-inactivated FCS (HyClone) (RPMI 1640 complete medium). The cells were then seeded into 10-cm culture dishes (Corning Glass) at 2 x 106 cells per dish. After incubation at 37°C for 2 h, nonadherent cells were removed, and the final cell population was >96% for Mac-1 expression. Thymocytes from thymus glands of 7-wk-old mice were seeded into 24-well culture plates at 5 x 106 cells/well, and treated with 1 μM dexamethasone at 37°C for 20 h to generate apoptotic cells. Populations of apoptotic thymocytes were 80% annexin V-positive and 63% propidium iodide-positive.
Phagocytosis
Apoptotic cells were added to the macrophage monolayers (2 x 106 cells/10-cm plate) at a ratio of 1:30 (apoptotic thymocytes). Cells were incubated at 37°C for 90 min in 3 ml of complete culture medium. Plates were then washed three times with FCS-free complete RPMI 1640 medium, and incubated in 2 ml of FCS-free complete RPMI 1640 medium at 37°C for specified lengths of time. As controls, macrophages were incubated in the presence of LPS (Escherichia coli O55B5; Difco) (100 ng/ml), or apoptotic cells were incubated alone.
Preparation of cell lysates
Thymuses were homogenized in extraction buffer (20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl) following incubation on ice for 30 min. Insoluble material was removed by centrifugation (25,000 x g, 15 min), and protein concentrations of the lysates were determined by the Bio-Rad dye-binding assay using BSA as a standard.
Substrate zymography
Gelatin zymography was performed, as described previously (15). Culture supernatants of macrophages were concentrated 10-fold at 4°C using an Amicon Centricon 10 concentrator (Millipore) and analyzed immediately. The concentrated supernatants (20 μl) or the protein extracts of each thymus (40 μg) were subjected to SDS-PAGE on gelatin-containing acrylamide gels (7.5% acrylamide and 1% gelatin) under nonreducing conditions. After electrophoresis, gels were washed three times with 2.5% Triton X-100 for 3 h and then rinsed briefly with water, followed by incubation overnight at 37°C in reaction buffer containing 50 mM Tris, pH 7.5, 0.15 M NaCl, and 10 mM CaCl2. The gels were then stained with 0.5% (w/v) Coomassie brilliant blue R-250. Gelatinolytic activity was detected as a transparent band against a dark blue background. The 105-kDa and 8385-kDa gelatinolytic bands were confirmed to be pro- and active MMP-9, respectively, by Western blot analysis with anti-MMP-9-specific Ab.
RNA analysis
Total RNA was isolated using SV Total RNA Isolation System (Promega), according to the manufacturer’s directions. Semiquantitative RT-PCR was performed with 1 μg of RNA using One Step RNA PCR kit (Takara Shuzo). After a hot start for 5 min at 94°C, 30 cycles were used for amplification, with a melting temperature of 94°C, an annealing temperature of 58°C, and an extending temperature of 72°C, each for 1 min, followed by a final extension at 72°C for 8 min. The RT-PCR products were separated on a 2% agarose gel and stained with ethidium bromide. Primers used for these analyses are: for MMP-9, forward, 5'-ACTACTCTGAAGACTTGCCG-3' and reverse, 5'-GGTACAGGAAGAGTACTGCT-3', amplifying a 707-bp product (25). Primers for the housekeeping gene -actin were: forward, 5'-ATGGATGACGATATCGCT-3' and reverse, 5'-ATGAGGTAGTCTGTCAGGT-3', amplifying a 587-bp product (26).
Plasmin activity
Plasmin activity was determined, as described previously (27), and assayed in 0.01 M Tris-HCl (pH 7.4), 0.15 M NaCl, 0.1 mM t-butyloxycarbonyl-L-glutamyl-L-lysyl-L-lysine 4-methylcoumary-7-amide (Boc-Glu-Lys-Lys-MCA; Peptide Institute), and 100 μg of thymus extracts in a total volume of 250 μl. After incubation for 90 min at 37°C, the reactions were terminated by addition of 750 μl of 1 M sodium acetate buffer (pH 4.2), and fluorescence intensity was measured at excitation and emission wavelengths of 380 and 460 nm, respectively. Increase in fluorescence was standardized using free 7-amino-4-methyl-coumarin (Peptide Institute). Values are given as release of 7-amino-4-methyl-coumarin in mole/h/mg proteins.
Histological analysis of thymic sections
Five-micrometer-thick sections of each thymus were air dried and fixed by incubation for 20 min in cold acetone. Apoptotic cells were assessed on sections by TUNEL staining, as described previously (28), and visualized with H2O2-activated diaminobenzidine. Staining for MMP-9 or collagen IV was conducted on sections pretreated with 0.04% pepsin (15 min, 37°C). Thymuses were blocked by incubation with 10% goat serum in PBS, followed by incubation with a polyclonal Ab against collagen type IV (PROGEN Biotechnik, a gift of R. Takahashi at University of Kyoto, Kyoto, Japan; 1/50 dilution) for overnight at 4°C. For visualization of MMP-9, sections were incubated with a 1/1000 dilution of anti-mouse MMP-9 Ab (29) (a gift from R. Senior at Washington University, St. Louis, MO). HUIV26 staining was conducted with the Ab at a concentration of 100 μg/ml. Anti-CD31 Ab was done MEC13.3 (BD Bioscience). Tissues were washed five times in PBS for 5 min each, followed by incubation with biotin-conjugated secondary Abs (10 μg/ml; Cappel) for 1 h at 37°C. The sections were then treated with HRP-conjugated streptavidin (10 μg/ml; Jackson ImmunoResearch Laboratories) and 3, 3'-diaminobenzidine substrate. Immunohistological detection of macrophage F4/80 was performed, as previously described (22), and F4/80-positive staining was revealed using alkaline phosphate-conjugated secondary Ab and alkaline phosphate substrate kit (Vector Laboratories). Sections were counterstained with Meyer’s hematoxylin. Slides were mounted and examined using a light microscopy (Nikon). For immunofluorescent detection, either FITC- or rhodamine-conjugated secondary Abs (Jackson ImmunoResearch Laboratories) were used. In some experiments, fluorochrome-conjugated anti-mouse CD31 and anti-F4/80 (eBioscience) were used. All confocal microscopy was conducted on LSM510 Carl Zeiss confocal (Carl Zeiss).
Statistical analysis
Data were analyzed by two-tailed Student’s t test, with p < 0.05 considered significant.
Results
Expression of MMP-9 in murine thymuses after administration of anti-CD3 Ab
BALB/c mice were administered i.p. 50 μg of anti-CD3 Ab (2C11-145), and thymuses were removed at various time points after the administration. Their extracts were analyzed by gelatin zymography for the presence of MMP-9 and MMP-2. MMP-9 was present in normal thymus and increased following administration of anti-CD3 Ab (Fig. 1A). The levels of the latent 105-kDa form of MMP-9 were induced with a peak of the activity at 21 h postinjection, followed by a decline. The active 8385-kDa form of MMP-9 in thymus extracts was also detectable 18–24 h postinjection of anti-CD3 Ab. The activities of both forms were depleted from thymus extracts by immobilized anti-MMP-9 Ab (data not shown). MMP-9 activity was drastically reduced by 28 h. MMP-2 activity was not apparently present in normal thymus, and an increase in latent 72-kDa activity was detectable up to 18 h after the administration, but the gelatinolytic activity of MMP-2 was smaller compared with that of MMP-9. Expression levels of MMP-9 mRNA in thymuses following administration of anti-CD3 Ab were assessed by semiquantitative RT-PCR analysis (Fig. 1B). MMP-9 mRNA expression was up-regulated within 13 h of the injection with peak levels observed at 16–20 h. Thus, MMP-9 expression in thymuses following administration of anti-CD3 Ab was evaluated at both mRNA and protein levels.
FIGURE 1. Expression of MMP-9 in murine thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab (2C11-145), and each thymus was removed at indicated time points after the injection. A, Thymus extracts were prepared, as described in Materials and Methods, and the samples (40 μg/lane) were analyzed by gelatin zymography. Gelatinolytic activity was visualized by staining with Coomassie blue. Positions of molecular mass standards are indicated. Results are representative of seven independent experiments and of four mice for each experiment. B, Total RNA was isolated from each thymus, and semiquantitative RT-PCR was performed for MMP-9 and -actin. Results are representative of three independent experiments.
The cell type expressing MMP-9 in thymuses after administration of anti-CD3 Ab was determined by immunohistochemical analysis (Fig. 2). In previous studies, we have shown that an increase in apoptotic cells is detectable in the cortical region of thymus as early as 13 h after the administration and, within 18 h, a significant number of TUNEL-positive thymocytes is scattered throughout the cortical area of thymus. A massive increase in F4/80-positive macrophages is evident within 16 h (22). TUNEL analysis presented in Fig. 2 showed similar kinetics to our previous studies (22). Thymus tissue harvested immediately following administration of anti-CD3 Ab contained few cells expressing MMP-9. The number of MMP-9-positive cells was increased within 18 h after the injection, and the cells were dispersed evenly throughout the thymus. Because we have previously observed that the majority of infiltrating cells into the thymus after administration of anti-CD3 Ab are macrophages (22), serial sections of the thymuses were stained with Ab F4/80. The distribution patterns of MMP-9-expressing cells were found to be similar to those with F4/80. Colocalization of MMP-9 in F4/80-positive macrophages was observed with most macrophages expressing intracellular MMP-9. Like our previous studies (22), at this time point, double staining with TUNEL and F4/80 Ab revealed that most apoptotic cells were situated with macrophages (data no shown). By 24 h, TUNEL-positive thymocytes were reduced drastically in number. The number of F4/80-positive macrophages was also decreased, and MMP-9-expressing cells were mainly localized in the subcapsular area. These results suggest that MMP-9 expressed in the thymuses is derived from F4/80-positive macrophages.
FIGURE 2. Expression of MMP-9 in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at 0, 18, or 24 h postinjection. Cryostat sections of each thymus were stained by TUNEL (brown; a, e, and i) or with anti-MMP-9 (brown; b, f, and j) or F4/80 (blue; c, g, and k). d, h, and l, Double staining with anti-MMP-9 (brown) and F4/80 (blue). These sections are representative of four mice for each condition. Bars, 100 μm (a–c, e–g, and i–k) and 60 μm (d, h, and l).
MMP-9 expression in macrophages in response to apoptotic cells
MMP-9 is shown to be expressed in murine peritoneal macrophages elicited by i.p. injection of thioglycolate (30, 31). It is, however, unknown whether phagocytosis can regulate the expression of MMP-9 in macrophages. To determine whether macrophage MMP-9 might be up-regulated upon clearance of apoptotic cells, resident peritoneal macrophages of BALB/c mice were prepared and analyzed for their ability to produce MMP-9 in response to apoptotic cells. Murine thymocytes treated with dexamethasone were used as target cells. Macrophage monolayers were incubated for 90 min with target cells, followed by extensive washing, and cultured in a serum-free medium up to an additional 18 h at 37°C. Expression levels of MMP-9 mRNA in peritoneal macrophages were measured by semiquantitative RT-PCR (Fig. 3A). Previous studies have demonstrated that LPS stimulates the production of the latent form of MMP-9 (30). We also detected increased MMP-9 mRNA in resident peritoneal macrophages stimulated with LPS for 15 h. However, transcript levels of MMP-9 in macrophages exposed to apoptotic cells were unchanged up to 15 h. Culture medium collected from cocultures for 18 h contained the latent form of MMP-9, whereas the active form was hardly detectable (Fig. 3B). Thus, exposure of apoptotic thymocytes to macrophages did not induce MMP-9 production. These results indicated that MMP-9 was constitutively expressed in primary macrophages and that clearance of apoptotic cells did not trigger MMP-9 production.
FIGURE 3. MMP-9 expression and production in murine macrophages in response to apoptotic cells. Resident peritoneal macrophages of BALB/c mice were incubated alone, or with apoptotic thymocytes for 90 min. After removal of target cells, macrophages were incubated in fresh FCS-free RPMI 1640 medium for additional times, as indicated. As control, macrophages were incubated in the presence of LPS (100 ng/ml) or apoptotic cells were incubated alone. A, Semiquantitative RT-PCR was performed using total RNA, as described in Materials and Methods. Lower panel, Demonstrates equal loading as defined by the levels of -actin mRNA. B, After 18-h cultivation, the supernatants were collected, concentrated, and subjected to gelatin zymography analysis. Lane 1, Apoptotic cells alone; lane 2, macrophages alone; lane 3, macrophages exposed to apoptotic cells; lane 4, macrophages treated with LPS. An arrow indicates the position of latent form of 105-kDa MMP-9. These results are representative of three similar experiments.
Plasmin activity in thymuses after administration of anti-CD3 Ab
Several reports have demonstrated plasmin activation of MMP-9 (32, 33). Plasmin is an internal fragment of plasminogen consisting of its five kringle domains (34). The conversion of inactive plasminogen into plasmin is known to be mediated by plasminogen activators, especially urokinase-type plasminogen activator (34). MMP-9 activation has been shown to occur in the presence of physiological concentrations of plasminogen and urokinase-type plasminogen activator (35). Because the plasmin system seemed to be an important effector in MMP-9 activation process, we evaluated plasmin activity in thymuses of BALB/c mice after administration of anti-CD3 Ab. Thymic extracts were prepared, and their plasmin activity was assessed by their Boc-Glu-Lys-Lys-MCA-hydrolyzing activity. As shown in Fig. 4, plasmin activity was increased after 18 h of the injection, and remained elevated up to 24 h.
FIGURE 4. Plasmin activity in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at indicated time points after the administration. Thymic extracts were prepared, as described in Materials and Methods, and plasmin activity in each thymus was determined using a fluorescent substrate, Boc-Glu-Lys-Lys-MCA. Results are shown as mean ± SD from four mice, and are representative of three independent experiments. *, p < 0.05; **, p < 0.01 compared with 0-h group. Results are representative of three independent experiments.
Collagen type IV is a substrate of MMP-9 in thymuses after administration of anti-CD3 Ab
To clarify the role of active MMP-9 in the thymus, we compared thymuses of MMP-9 KO mice and those of wild-type BALB/c mice following administration of anti-CD3 Ab. Similar to the results of Fig. 1A, MMP-9 activity in thymuses of wild-type mice was increased 18–24 h postinjection (Fig. 5A). In contrast, the levels of MMP-9 were completely diminished in MMP-9 KO mice. No obvious differences in the extent of thymic apoptosis were observed between MMP-9 KO mice and wild-type littermates, as assessed by caspase-3-like activity and the number of TUNEL-positive cells (data not shown). In addition, the number of thymic macrophages in MMP-9 KO mice was similar to that in wild-type mice at 18 h postadministration (data not shown).
FIGURE 5. Appearance of collagen type IV cryptic sites and neovessel formation in the thymus of wild-type mice nor in that of MMP-9 KO mice. Seven-week-old wild-type (MMP9+/+) and age-matched MMP9-KO (MMP9–/–) mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at indicated time points after the injection. A, MMP-9 expression in the thymus of wild-type or MMP-9 KO mice. Thymus extracts were prepared and analyzed for gelatinolytic activity, as described in Materials and Methods. B, Immunohistochemical analysis of thymus sections. Cryostat sections of each thymus were stained with anti-collagen type IV Ab (a, c, e, and g), or with HUIV26 Ab (b, d, f, and h), or with anti-CD31 Ab (i–l). All primary Ab reaction products were visualized with respective biotinylated secondary Abs, followed with H2O2-activated diaminobenzidine. Data represent the results of a single experiment, which was one of four with similar results. Bars, 100 μm.
MMP-9 is originally found to cleave collagen type IV, which is a major component of the vascular basement membrane (3). Collagen type IV appeared to delineate all thymic vascular beds 0 h postinjection, and no significant changes between both mice were noted (Fig. 5B). Recently, Xu et al. (24) have developed a mAb, HUIV26, directed to a cryptic site of collagen type IV that is normally hidden within its triple helical structure. We examined by immunohistochemistry using HUIV26 whether proteolytic cleavage of collagen type IV by MMP-9 might expose this cryptic site in thymuses of wild-type mice. The thymuses of both mice 0 h postinjection showed little, if any, staining of the HUIV26 cryptic epitope. Remarkably, 24 h postinjection in the thymus of wild-type mice, HUIV26-positive staining was observed within the subendothelial basement membrane of vessels. MMP-9 KO mice revealed a significant reduction in the number of HUIV26 cryptic sites as compared with control wild-type mice. Together, these findings provide evidence for a role of MMP-9 in the exposure of the HUIV26 cryptic epitope on collagen type IV.
Brooks and colleagues (36, 37) have demonstrated that exposure of this cryptic site is associated with angiogenesis in vivo. We examined the relationship between the exposure of HUIV26 cryptic sites and thymic neovascularization in wild-type mice and MMP-9 KO mice 28 h after administration of anti-CD3 Ab. Thymuses were stained with an Ab directed to CD31, which expresses on the surface of endothelial cells. In anti-CD3 Ab-injected wild-type mice, the thymus became highly vascularized, and the number of large vessels in the cortical area was increased, compared with MMP-9 KO mice. Collectively, these data suggest that MMP-9 is involved in degradation of collagen type IV, followed by induction of thymic neovascularization.
Neovascularization and macrophage distribution in thymuses after administration of anti-CD3 Ab
To ascertain whether neovessel formation correlates with appearance of HUIV26 cryptic epitope, thymuses of BALB/c mice following administration of anti-CD3 Ab were stained with anti-CD31 and Ab HUIV26 (Fig. 6). As shown in Fig. 6A, a significant increase in neovascular formation was noted in the cortical region of thymuses at 25 or 28 h postinjection relative to thymuses at 0 h postinjection. Vessels from the thymus at 0 h postinjection showed little, if any, exposure of the HUIV26 cryptic exposure (Fig. 6B). In the thymuses, 25 or 28 h postinjection, exposure of HUIV26 epitope was readily detectable. Costaining with both Abs showed that not all vessels stained positive for HUIV26 epitope, but significant exposure of the epitope was detectable in association with CD31-positive endothelial cells.
FIGURE 6. Neovessel formation is associated with appearance of collagen type IV cryptic sites in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at indicated time points after the injection. A, Vascular morphology. Cryostat sections of each thymus were stained with anti-CD31 Ab (red). These sections are representatives for each time point. B, Appearance of cryptic site of collagen IV. Cryostat sections of each thymus were stained with HUIV26 Ab (green) and with anti-CD31 Ab (red). These sections are representative of four mice for each condition. Bars, 200 μm.
To examine the fate of macrophages after clearance of apoptotic thymocytes, we analyzed macrophage distribution in thymuses of BALB/c mice following administration of anti-CD3 Ab, by staining with Ab F4/80 and anti-CD31 Ab (Fig. 7). A massive increase in F4/80-positive macrophages was evident within 18 h of the injection, as shown in Fig. 2. Twenty-three hours after the injection, a number of F4/80-positive macrophages was still detectable, and many macrophages were found to be localized in perivascular areas. By 28 h, the number of F4/80-positive macrophages was significantly decreased, and some of the macrophages were seen in the site close to or within vessels.
FIGURE 7. Macrophage distribution in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at indicated time points after the injection. Cryostat sections of each thymus were stained with F4/80 Ab (red) and with anti-CD31 Ab (green). These sections are representative of four mice for each condition. Bars, 200 μm.
Exposure of HUIV26 cryptic site within collagen type IV is involved in thymic neovascularization
Systemic administration of HUIV26 Ab is shown to inhibit angiogenesis and tumor growth in multiple animal models (36, 37). To evaluate whether exposure of this cryptic site may be involved in the process by which new vessels were formed in the thymus, we asked whether HUIV26 Ab might inhibit thymic vascularization. Anti-CD3 Ab was injected into BALB/c mice, and 17 h later the mice were either untreated or treated with i.p. injection of either HUIV26 Ab or isotype-matched control Ab (75 or 150 μg). An additional 11 h later, thymuses were removed and assessed for the vascular morphology by immunohistochemical analysis (Fig. 8). Increased density of vessels in the thymus was detectable in control Ab-treated mice and untreated mice (data not shown) by 28 h following anti-CD3 Ab injection. Administration of HUIV26 Ab potently inhibited thymic neovascularization in a dose-dependent manner (data not shown), and the extent of new vessel formation in HUIV26 (150 μg)-treated mice was decreased, as compared with control Ab-treated mice. Inhibition of thymic vascularization was markedly reduced when HUIV26 Ab was administered 22 h after anti-CD3 Ab injection (data not shown). In addition, when the distribution of macrophages in thymuses was analyzed by staining with F4/80, macrophages were found to be located around or within many vessels of thymic cortical area in control Ab-treated mice. HUIV26 Ab-treated mice apparently harbored an increased number of F4/80-positive macrophages in the thymus than did control Ab-injected mice at 28 h after injection of anti-CD3 Ab. Nevertheless, an additional 8 h later, macrophages in the thymus of HUIV26 Ab-treated mice appeared to be reduced in number and accumulated close to or within vessels (data not shown).
FIGURE 8. Effects of HUIV26 Ab on neovascularization and macrophage distribution in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and 17 h later, mice were either untreated or treated i.p. (150 μg/mouse) with either HUIV26 Ab or an isotype-matched control Ab. Each thymus was removed 28 h after administration of anti-CD3 Ab, and thymic sections were stained with anti-CD31 Ab (green) and with F4/80 Ab (red). These sections are representative of four mice for each condition, and experiments were completed three times with similar results. Bars, 200 μm.
Discussion
In this study, we demonstrate that MMP-9 expression was up-regulated in thymuses of BALB/c mice after administration of anti-CD3 Ab. A number of macrophages promptly infiltrated into the thymuses following apoptosis of thymocytes, and the distribution patterns of MMP-9-expressing cells were found similar to those of F4/80-positive cells. By 28 h, the number of macrophages in the thymus was markedly decreased, and the expression levels of MMP-9 were also reduced. Although MMP-9 was present in thymuses of normal mice, the increased expression of MMP-9 seems not to reflect the enrichment of thymic microenvironmental compartments due to thymocyte depletion. The colocalization of MMP-9 in F4/80-positive cells suggests that macrophages are the likely sources of MMP-9. As shown previously (30, 31), primary macrophages from the peritoneal cavity of BALB/c mice were found to express MMP-9. Increased MMP-9 expression in murine macrophages was undetectable in response to apoptotic cells. Although in vitro findings using macrophages from peritoneal cavity do not necessarily correlate with in vivo thymus studies, it is unlikely that macrophages might synthesize and secrete MMP-9 upon clearance of apoptotic thymocytes. Rather, the number of macrophages in the thymus was significantly increased, and the increased MMP-9 expression may be attributed to the presence of macrophages in thymuses following administration of anti-CD3 Ab.
We detected activation of MMP-9 in thymuses 18–24 h after anti-CD3 Ab injection. When untreated macrophages or macrophages exposed to apoptotic cells were cultured for 18 h, only latent MMP-9 was detected in the culture medium. We found the increased activity of plasmin in thymuses 18–24 h after anti-CD3 Ab injection. It is therefore conceivable that macrophages secrete inactive MMP-9, which may be converted to the active form by plasmin. In Western blot analysis, we observed that 85-kDa plasminogen was expressed in thymuses of normal mice (data not shown). Murine peritoneal macrophages are reported to produce and secrete a plasminogen activator (38). The mechanism underlying the generation of plasmin in thymuses following administration of ani-CD3 Ab remains to be determined.
We observed an exposure of collagen type IV cryptic epitope detectable by HUIV26 Ab in murine thymuses upon MMP-9 activation. HUIV26-positive staining was markedly reduced in MMP-9 KO mice. Although MMP-2 and MMP-9 can cleave triple-helical collagen type IV (3, 4), our study indicated that MMP-9, rather than MMP-2, may be critical in the exposure of HUIV26 cryptic sites. Thymic neovascularization in wild-type mice was evident, especially in the cortical region, as early as 23 h after anti-CD3 Ab injection. Recent studies have indicated that TGF- produced by apoptotic cells as well as phagocytes has a major role in anti-inflammatory effects during clearance of apoptotic cells (39, 40). MMP-9 is shown to proteolytically cleave latent TGF-, resulting in promotion of angiogenesis (14). In our previous study, an increase in TGF- levels was detectable in murine thymuses following thymocyte apoptosis, but the amounts of active TGF- were small (22). We therefore assume that TGF- has a minor role in the thymic vascular development. Mice deficient in MMP-2 or MMP-9 exhibit reduced angiogenesis in vivo (41, 42). In this study, new vessel formation was diminished in thymuses of MMP-9 KO mice following anti-CD3 Ab injection. In addition, administration of HUIV26 Ab inhibited thymic vascularization. Together, we concluded that consistent with previous investigations (36, 37), exposure of HUIV26 cryptic epitope within collagen type IV is important for the initiation of neovessel formation. Our findings agree with previous investigations by Brooks and colleagues (37), which demonstrated that MMP-9 is a primary contributor to vascularization process. Although it is known that cortical thymocytes result in apoptotic cell death after whole body irradiation, Huiskamp et al. (43) reported that the density of vessels throughout the thymus increases from day 2 until day 4 after the irradiation. They speculated that the increased thymic vascularization reflects the active tissue response during the regeneration process after irradiation. Little is, however, known concerning the molecular mechanisms controlling thymic neovessel formation. Also, we have detected MMP-9 activation in murine thymuses after injection of dexamethasone, which induces CD4+CD8+ thymocytes to apoptosis (data not shown). Thus, thymic neovascularization appears a prominent event following thymocyte apoptosis, and our evidence may help to understand the molecular mechanisms of vessel development during thymus remodeling following thymocyte apoptosis. Xu et al. (36) have demonstrated that the exposure of HUIV26 cryptic site is associated with a loss of 11 binding and gain in v3 binding and that interaction of HUIV26 epitope with endothelial cells induces their adhesion and migration. Additional experiments are necessary to determine which interactions between endothelial cells and the cryptic site of collagen type IV initiate signaling cascades required for thymic neovascularization.
MMP-9 is also found to participate in cell migration (44). MMP-9 may be expressed on the surface of cells, allowing for precise, localized proteolysis (14, 45) that would create a path for migration (46). The critical role of MMP-9 in transmigration through basement membrane has previously been described for T cells (47). Also, MMP-9 is necessary for the migration of Langerhans cells, which express MMP-9 on their cell surface (48). We observed that the number of macrophages in the thymus was reduced after clearance of apoptotic thymocytes. Macrophages are shown to migrate from tissues or organs to draining lymph nodes (49, 50). The number of TUNEL-positive cells was drastically reduced 24 h postinjection, suggesting that macrophages do not undergo apoptosis after phagocytosis of dying cells. Additionally, a number of macrophages was seen around or within vessels in thymuses after 23 h postinjection of anti-CD3. Based on our observations, we favor the proposal that macrophage clearance from tissues occurs by emigration rather than by local apoptosis (49), although we are unable to present any additional evidence for macrophage emigration from the thymus after clearance of apoptotic thymocytes. We observed a number of macrophages in thymuses of HUIV26 Ab-treated mice even after 28 h of anti-CD3 Ab injection. In addition, the accumulation of F4/80-positive macrophages was detected in thymuses of MMP-9 KO mice 32 h postinjection of anti-CD3 Ab (data not shown). It is conceivable that suppression of thymic neovessel formation interferes with emigration of macrophages from the thymus.
In this study, we provide evidence that MMP-9 in macrophages facilitates their migration from the thymus by induction of neovascularization. Nevertheless, the mechanisms mediating the trafficking of macrophages from thymus are unknown. Identification of chemokine and its receptor that participate in the migration of thymic macrophages after clearance of apoptotic thymocytes is under investigation.
Acknowledgments
We thank Drs. R. Takahashi and R. Senior for providing us with Abs, and Drs. T. Ebel, B. Freimark, and F. Pernaseffi for a gift of Abs and their critical reading of our manuscript. We are grateful to Drs. T. Endo and S. Izumiyama for excellent technical support in the use of microscope.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Address correspondence and reprint requests to Dr. Chikako Odaka, Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan. E-mail address: odaka@nih.go.jp
2 Abbreviations used in this paper: MMP, matrix metalloproteinase; Boc-Glu-Lys-Lys-MCA, t-butyloxycarbonyl-L-glutamyl-L-lysyl-L-lysine 4-methylcoumary-7-amide; KO, knockout.
Received for publication May 26, 2004. Accepted for publication October 22, 2004.
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Matrix metalloproteinase-9 (MMP-9) has been implicated in the degradation of the extracellular matrix in a variety of physiological and pathological processes. We found that MMP-9 expression in thymuses of BALB/c mice that had been injected with anti-CD3 Ab to induce thymocyte apoptosis was increased both at mRNA and protein levels. Macrophages are shown to be the principal stromal cells responsible for phagocytosis of dying thymocytes, and macrophages were found to constitutively express MMP-9. The activity of plasmin, which is known as one of the activators for MMP-9, was increased in the thymuses with MMP-9 activation. Binding of Ab HUIV26, which recognizes a cryptic epitope on collagen type IV following proteolytic cleavage, was found to be reduced in MMP-9 knockout mice, suggesting that collagen type IV is a substrate of MMP-9. Although the formation of thymic neovessels was found following thymocyte apoptosis, it was diminished in anti-CD3 Ab-injected MMP-9 knockout mice. In vivo administration of Ab HUIV26 resulted in a reduction of thymic neovascularization. After clearance of apoptotic thymocytes, the number of macrophages in the thymuses was decreased, and this decrease was delayed by blocking of HUIV26 epitope. Taken together, our results suggest that MMP-9 expression in macrophages mediates degradation of collagen type IV and facilitates their migration from the thymus after clearance of apoptotic thymocytes. These studies demonstrate a potential role of macrophage MMP-9 in the remodeling of thymic extracellular matrix following thymocyte apoptosis.
Introduction
Matrix metalloproteinases (MMPs)2 are a group of zinc-dependent endopeptidases that play a key role in promoting tissue remodeling and tumor invasion by inducing proteolysis of several extracellular matrix components (1, 2). MMPs are produced as zymogens with a signal sequence and propeptide segment that is removed during activation.
Among MMPs, MMP-2 and MMP-9 constitute the key proteinases governing the degradation of basement membrane collagens. These metalloproteinases cleave collagen types IV, V, VII, and X as well as denatured collagens or gelatins (3, 4). They have collectively been referred to as type IV collagenases or gelatinases. Overexpression of MMP-9 has been implicated in physiological process of tissue remodeling (5, 6) and wound healing (7), or in the pathogenesis of various diseases, such as atherosclerosis, tumor invasion, and metastasis (8, 9, 10). MMP-9 from macrophages and neutrophils is thought to play a key role in the migration of these cells during inflammatory diseases such as arthritis (11). MMP-9 is responsible for the processing of cytokines, e.g., pro-IL-1 and pro-TNF- into their active form (12, 13). This proteinase also cleaves latent TGF-, which constitutes a novel mechanism of TGF- activation (14). In addition, MMP-9 is involved in the formation of new vessels essential for tumor growth and triggers the angiogenic switch during carcinogenesis (15). However, the mechanisms by which MMP-9 promotes vasculogenesis and tumor invasion are still poorly understood.
It has been established that stimulation of CD4+CD8+ thymocytes by an anti-CD3 Ab in vivo as well as in vitro induces apoptotic cell death, which has been proposed as a mechanism for deletion of autoreactive T cells (16, 17, 18, 19). In murine thymus after administration of anti-CD3 Ab, macrophages are shown to be key players in clearance of apoptotic thymocytes as well as resolving inflammation (20, 21, 22).
In the present study, we showed that MMP-9 expression was increased in murine thymus after administration of anti-CD3 Ab, and that MMP-9 was most likely derived from macrophages. To define the role of MMP-9, we used MMP-9 knockout (KO) mice and found that collagen type IV was a substrate of MMP-9. Exposure of a cryptic site within collagen type IV was associated with thymic neovascularization and macrophage migration from the thymus after clearance of apoptotic cells.
Materials and Methods
Mice
BALB/c mice were bred in our animal facility and used at the age of 7–12 wk. MMP-9-deficient (MMP-9 KO) mice have been described previously (23) and used at the age of 7 wk. All animal procedures described in this study were performed in accordance with the guidelines for animal experiments of our institutes.
Abs and reagents
Anti-mouse CD3 mAb (145-2C11) was purified from ascites fluid by protein A-affinity chromatography. HUIV26 Ab (24) was a gift from Cell-Matrix, and an isotype-matched control Ab was obtained from Medical Biological Laboratories. Oligonucleotide primers were from Sawady Technology. All other reagents were purchased from Sigma-Aldrich, except as specified below.
Injection of anti-CD3 Ab
Seven-week-old mice were injected i.p. with 50 μg of anti-CD3 Ab. At the indicated time, mice were sacrificed. Thymuses were removed, immersed in OCT compound (Miles), rapidly frozen on dry ice, and sectioned at 5 μm for histological analysis. Alternatively, each thymus was excised and used for protein extraction or total RNA isolation.
Treatment of mice with HUIV26 Ab
Seventeen hours postadministration of anti-CD3 Ab, mice were either untreated or treated i.p. with either purified HUIV26 or an isotype-matched control Ab (75 or 150 μg per mouse). An additional 11 or 19 h later, thymuses were excised and used for immunochemistry analysis.
Cell preparations and cell culture
For preparation of murine resident peritoneal macrophages, peritoneal exudate cells were collected from the peritoneal cavity of 9- to 12-wk-old BALB/c mice and washed with RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 μM 2-ME, 20 U/ml penicillin, 20 μg/ml streptomycin, and 10% heat-inactivated FCS (HyClone) (RPMI 1640 complete medium). The cells were then seeded into 10-cm culture dishes (Corning Glass) at 2 x 106 cells per dish. After incubation at 37°C for 2 h, nonadherent cells were removed, and the final cell population was >96% for Mac-1 expression. Thymocytes from thymus glands of 7-wk-old mice were seeded into 24-well culture plates at 5 x 106 cells/well, and treated with 1 μM dexamethasone at 37°C for 20 h to generate apoptotic cells. Populations of apoptotic thymocytes were 80% annexin V-positive and 63% propidium iodide-positive.
Phagocytosis
Apoptotic cells were added to the macrophage monolayers (2 x 106 cells/10-cm plate) at a ratio of 1:30 (apoptotic thymocytes). Cells were incubated at 37°C for 90 min in 3 ml of complete culture medium. Plates were then washed three times with FCS-free complete RPMI 1640 medium, and incubated in 2 ml of FCS-free complete RPMI 1640 medium at 37°C for specified lengths of time. As controls, macrophages were incubated in the presence of LPS (Escherichia coli O55B5; Difco) (100 ng/ml), or apoptotic cells were incubated alone.
Preparation of cell lysates
Thymuses were homogenized in extraction buffer (20 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl) following incubation on ice for 30 min. Insoluble material was removed by centrifugation (25,000 x g, 15 min), and protein concentrations of the lysates were determined by the Bio-Rad dye-binding assay using BSA as a standard.
Substrate zymography
Gelatin zymography was performed, as described previously (15). Culture supernatants of macrophages were concentrated 10-fold at 4°C using an Amicon Centricon 10 concentrator (Millipore) and analyzed immediately. The concentrated supernatants (20 μl) or the protein extracts of each thymus (40 μg) were subjected to SDS-PAGE on gelatin-containing acrylamide gels (7.5% acrylamide and 1% gelatin) under nonreducing conditions. After electrophoresis, gels were washed three times with 2.5% Triton X-100 for 3 h and then rinsed briefly with water, followed by incubation overnight at 37°C in reaction buffer containing 50 mM Tris, pH 7.5, 0.15 M NaCl, and 10 mM CaCl2. The gels were then stained with 0.5% (w/v) Coomassie brilliant blue R-250. Gelatinolytic activity was detected as a transparent band against a dark blue background. The 105-kDa and 8385-kDa gelatinolytic bands were confirmed to be pro- and active MMP-9, respectively, by Western blot analysis with anti-MMP-9-specific Ab.
RNA analysis
Total RNA was isolated using SV Total RNA Isolation System (Promega), according to the manufacturer’s directions. Semiquantitative RT-PCR was performed with 1 μg of RNA using One Step RNA PCR kit (Takara Shuzo). After a hot start for 5 min at 94°C, 30 cycles were used for amplification, with a melting temperature of 94°C, an annealing temperature of 58°C, and an extending temperature of 72°C, each for 1 min, followed by a final extension at 72°C for 8 min. The RT-PCR products were separated on a 2% agarose gel and stained with ethidium bromide. Primers used for these analyses are: for MMP-9, forward, 5'-ACTACTCTGAAGACTTGCCG-3' and reverse, 5'-GGTACAGGAAGAGTACTGCT-3', amplifying a 707-bp product (25). Primers for the housekeeping gene -actin were: forward, 5'-ATGGATGACGATATCGCT-3' and reverse, 5'-ATGAGGTAGTCTGTCAGGT-3', amplifying a 587-bp product (26).
Plasmin activity
Plasmin activity was determined, as described previously (27), and assayed in 0.01 M Tris-HCl (pH 7.4), 0.15 M NaCl, 0.1 mM t-butyloxycarbonyl-L-glutamyl-L-lysyl-L-lysine 4-methylcoumary-7-amide (Boc-Glu-Lys-Lys-MCA; Peptide Institute), and 100 μg of thymus extracts in a total volume of 250 μl. After incubation for 90 min at 37°C, the reactions were terminated by addition of 750 μl of 1 M sodium acetate buffer (pH 4.2), and fluorescence intensity was measured at excitation and emission wavelengths of 380 and 460 nm, respectively. Increase in fluorescence was standardized using free 7-amino-4-methyl-coumarin (Peptide Institute). Values are given as release of 7-amino-4-methyl-coumarin in mole/h/mg proteins.
Histological analysis of thymic sections
Five-micrometer-thick sections of each thymus were air dried and fixed by incubation for 20 min in cold acetone. Apoptotic cells were assessed on sections by TUNEL staining, as described previously (28), and visualized with H2O2-activated diaminobenzidine. Staining for MMP-9 or collagen IV was conducted on sections pretreated with 0.04% pepsin (15 min, 37°C). Thymuses were blocked by incubation with 10% goat serum in PBS, followed by incubation with a polyclonal Ab against collagen type IV (PROGEN Biotechnik, a gift of R. Takahashi at University of Kyoto, Kyoto, Japan; 1/50 dilution) for overnight at 4°C. For visualization of MMP-9, sections were incubated with a 1/1000 dilution of anti-mouse MMP-9 Ab (29) (a gift from R. Senior at Washington University, St. Louis, MO). HUIV26 staining was conducted with the Ab at a concentration of 100 μg/ml. Anti-CD31 Ab was done MEC13.3 (BD Bioscience). Tissues were washed five times in PBS for 5 min each, followed by incubation with biotin-conjugated secondary Abs (10 μg/ml; Cappel) for 1 h at 37°C. The sections were then treated with HRP-conjugated streptavidin (10 μg/ml; Jackson ImmunoResearch Laboratories) and 3, 3'-diaminobenzidine substrate. Immunohistological detection of macrophage F4/80 was performed, as previously described (22), and F4/80-positive staining was revealed using alkaline phosphate-conjugated secondary Ab and alkaline phosphate substrate kit (Vector Laboratories). Sections were counterstained with Meyer’s hematoxylin. Slides were mounted and examined using a light microscopy (Nikon). For immunofluorescent detection, either FITC- or rhodamine-conjugated secondary Abs (Jackson ImmunoResearch Laboratories) were used. In some experiments, fluorochrome-conjugated anti-mouse CD31 and anti-F4/80 (eBioscience) were used. All confocal microscopy was conducted on LSM510 Carl Zeiss confocal (Carl Zeiss).
Statistical analysis
Data were analyzed by two-tailed Student’s t test, with p < 0.05 considered significant.
Results
Expression of MMP-9 in murine thymuses after administration of anti-CD3 Ab
BALB/c mice were administered i.p. 50 μg of anti-CD3 Ab (2C11-145), and thymuses were removed at various time points after the administration. Their extracts were analyzed by gelatin zymography for the presence of MMP-9 and MMP-2. MMP-9 was present in normal thymus and increased following administration of anti-CD3 Ab (Fig. 1A). The levels of the latent 105-kDa form of MMP-9 were induced with a peak of the activity at 21 h postinjection, followed by a decline. The active 8385-kDa form of MMP-9 in thymus extracts was also detectable 18–24 h postinjection of anti-CD3 Ab. The activities of both forms were depleted from thymus extracts by immobilized anti-MMP-9 Ab (data not shown). MMP-9 activity was drastically reduced by 28 h. MMP-2 activity was not apparently present in normal thymus, and an increase in latent 72-kDa activity was detectable up to 18 h after the administration, but the gelatinolytic activity of MMP-2 was smaller compared with that of MMP-9. Expression levels of MMP-9 mRNA in thymuses following administration of anti-CD3 Ab were assessed by semiquantitative RT-PCR analysis (Fig. 1B). MMP-9 mRNA expression was up-regulated within 13 h of the injection with peak levels observed at 16–20 h. Thus, MMP-9 expression in thymuses following administration of anti-CD3 Ab was evaluated at both mRNA and protein levels.
FIGURE 1. Expression of MMP-9 in murine thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab (2C11-145), and each thymus was removed at indicated time points after the injection. A, Thymus extracts were prepared, as described in Materials and Methods, and the samples (40 μg/lane) were analyzed by gelatin zymography. Gelatinolytic activity was visualized by staining with Coomassie blue. Positions of molecular mass standards are indicated. Results are representative of seven independent experiments and of four mice for each experiment. B, Total RNA was isolated from each thymus, and semiquantitative RT-PCR was performed for MMP-9 and -actin. Results are representative of three independent experiments.
The cell type expressing MMP-9 in thymuses after administration of anti-CD3 Ab was determined by immunohistochemical analysis (Fig. 2). In previous studies, we have shown that an increase in apoptotic cells is detectable in the cortical region of thymus as early as 13 h after the administration and, within 18 h, a significant number of TUNEL-positive thymocytes is scattered throughout the cortical area of thymus. A massive increase in F4/80-positive macrophages is evident within 16 h (22). TUNEL analysis presented in Fig. 2 showed similar kinetics to our previous studies (22). Thymus tissue harvested immediately following administration of anti-CD3 Ab contained few cells expressing MMP-9. The number of MMP-9-positive cells was increased within 18 h after the injection, and the cells were dispersed evenly throughout the thymus. Because we have previously observed that the majority of infiltrating cells into the thymus after administration of anti-CD3 Ab are macrophages (22), serial sections of the thymuses were stained with Ab F4/80. The distribution patterns of MMP-9-expressing cells were found to be similar to those with F4/80. Colocalization of MMP-9 in F4/80-positive macrophages was observed with most macrophages expressing intracellular MMP-9. Like our previous studies (22), at this time point, double staining with TUNEL and F4/80 Ab revealed that most apoptotic cells were situated with macrophages (data no shown). By 24 h, TUNEL-positive thymocytes were reduced drastically in number. The number of F4/80-positive macrophages was also decreased, and MMP-9-expressing cells were mainly localized in the subcapsular area. These results suggest that MMP-9 expressed in the thymuses is derived from F4/80-positive macrophages.
FIGURE 2. Expression of MMP-9 in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at 0, 18, or 24 h postinjection. Cryostat sections of each thymus were stained by TUNEL (brown; a, e, and i) or with anti-MMP-9 (brown; b, f, and j) or F4/80 (blue; c, g, and k). d, h, and l, Double staining with anti-MMP-9 (brown) and F4/80 (blue). These sections are representative of four mice for each condition. Bars, 100 μm (a–c, e–g, and i–k) and 60 μm (d, h, and l).
MMP-9 expression in macrophages in response to apoptotic cells
MMP-9 is shown to be expressed in murine peritoneal macrophages elicited by i.p. injection of thioglycolate (30, 31). It is, however, unknown whether phagocytosis can regulate the expression of MMP-9 in macrophages. To determine whether macrophage MMP-9 might be up-regulated upon clearance of apoptotic cells, resident peritoneal macrophages of BALB/c mice were prepared and analyzed for their ability to produce MMP-9 in response to apoptotic cells. Murine thymocytes treated with dexamethasone were used as target cells. Macrophage monolayers were incubated for 90 min with target cells, followed by extensive washing, and cultured in a serum-free medium up to an additional 18 h at 37°C. Expression levels of MMP-9 mRNA in peritoneal macrophages were measured by semiquantitative RT-PCR (Fig. 3A). Previous studies have demonstrated that LPS stimulates the production of the latent form of MMP-9 (30). We also detected increased MMP-9 mRNA in resident peritoneal macrophages stimulated with LPS for 15 h. However, transcript levels of MMP-9 in macrophages exposed to apoptotic cells were unchanged up to 15 h. Culture medium collected from cocultures for 18 h contained the latent form of MMP-9, whereas the active form was hardly detectable (Fig. 3B). Thus, exposure of apoptotic thymocytes to macrophages did not induce MMP-9 production. These results indicated that MMP-9 was constitutively expressed in primary macrophages and that clearance of apoptotic cells did not trigger MMP-9 production.
FIGURE 3. MMP-9 expression and production in murine macrophages in response to apoptotic cells. Resident peritoneal macrophages of BALB/c mice were incubated alone, or with apoptotic thymocytes for 90 min. After removal of target cells, macrophages were incubated in fresh FCS-free RPMI 1640 medium for additional times, as indicated. As control, macrophages were incubated in the presence of LPS (100 ng/ml) or apoptotic cells were incubated alone. A, Semiquantitative RT-PCR was performed using total RNA, as described in Materials and Methods. Lower panel, Demonstrates equal loading as defined by the levels of -actin mRNA. B, After 18-h cultivation, the supernatants were collected, concentrated, and subjected to gelatin zymography analysis. Lane 1, Apoptotic cells alone; lane 2, macrophages alone; lane 3, macrophages exposed to apoptotic cells; lane 4, macrophages treated with LPS. An arrow indicates the position of latent form of 105-kDa MMP-9. These results are representative of three similar experiments.
Plasmin activity in thymuses after administration of anti-CD3 Ab
Several reports have demonstrated plasmin activation of MMP-9 (32, 33). Plasmin is an internal fragment of plasminogen consisting of its five kringle domains (34). The conversion of inactive plasminogen into plasmin is known to be mediated by plasminogen activators, especially urokinase-type plasminogen activator (34). MMP-9 activation has been shown to occur in the presence of physiological concentrations of plasminogen and urokinase-type plasminogen activator (35). Because the plasmin system seemed to be an important effector in MMP-9 activation process, we evaluated plasmin activity in thymuses of BALB/c mice after administration of anti-CD3 Ab. Thymic extracts were prepared, and their plasmin activity was assessed by their Boc-Glu-Lys-Lys-MCA-hydrolyzing activity. As shown in Fig. 4, plasmin activity was increased after 18 h of the injection, and remained elevated up to 24 h.
FIGURE 4. Plasmin activity in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at indicated time points after the administration. Thymic extracts were prepared, as described in Materials and Methods, and plasmin activity in each thymus was determined using a fluorescent substrate, Boc-Glu-Lys-Lys-MCA. Results are shown as mean ± SD from four mice, and are representative of three independent experiments. *, p < 0.05; **, p < 0.01 compared with 0-h group. Results are representative of three independent experiments.
Collagen type IV is a substrate of MMP-9 in thymuses after administration of anti-CD3 Ab
To clarify the role of active MMP-9 in the thymus, we compared thymuses of MMP-9 KO mice and those of wild-type BALB/c mice following administration of anti-CD3 Ab. Similar to the results of Fig. 1A, MMP-9 activity in thymuses of wild-type mice was increased 18–24 h postinjection (Fig. 5A). In contrast, the levels of MMP-9 were completely diminished in MMP-9 KO mice. No obvious differences in the extent of thymic apoptosis were observed between MMP-9 KO mice and wild-type littermates, as assessed by caspase-3-like activity and the number of TUNEL-positive cells (data not shown). In addition, the number of thymic macrophages in MMP-9 KO mice was similar to that in wild-type mice at 18 h postadministration (data not shown).
FIGURE 5. Appearance of collagen type IV cryptic sites and neovessel formation in the thymus of wild-type mice nor in that of MMP-9 KO mice. Seven-week-old wild-type (MMP9+/+) and age-matched MMP9-KO (MMP9–/–) mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at indicated time points after the injection. A, MMP-9 expression in the thymus of wild-type or MMP-9 KO mice. Thymus extracts were prepared and analyzed for gelatinolytic activity, as described in Materials and Methods. B, Immunohistochemical analysis of thymus sections. Cryostat sections of each thymus were stained with anti-collagen type IV Ab (a, c, e, and g), or with HUIV26 Ab (b, d, f, and h), or with anti-CD31 Ab (i–l). All primary Ab reaction products were visualized with respective biotinylated secondary Abs, followed with H2O2-activated diaminobenzidine. Data represent the results of a single experiment, which was one of four with similar results. Bars, 100 μm.
MMP-9 is originally found to cleave collagen type IV, which is a major component of the vascular basement membrane (3). Collagen type IV appeared to delineate all thymic vascular beds 0 h postinjection, and no significant changes between both mice were noted (Fig. 5B). Recently, Xu et al. (24) have developed a mAb, HUIV26, directed to a cryptic site of collagen type IV that is normally hidden within its triple helical structure. We examined by immunohistochemistry using HUIV26 whether proteolytic cleavage of collagen type IV by MMP-9 might expose this cryptic site in thymuses of wild-type mice. The thymuses of both mice 0 h postinjection showed little, if any, staining of the HUIV26 cryptic epitope. Remarkably, 24 h postinjection in the thymus of wild-type mice, HUIV26-positive staining was observed within the subendothelial basement membrane of vessels. MMP-9 KO mice revealed a significant reduction in the number of HUIV26 cryptic sites as compared with control wild-type mice. Together, these findings provide evidence for a role of MMP-9 in the exposure of the HUIV26 cryptic epitope on collagen type IV.
Brooks and colleagues (36, 37) have demonstrated that exposure of this cryptic site is associated with angiogenesis in vivo. We examined the relationship between the exposure of HUIV26 cryptic sites and thymic neovascularization in wild-type mice and MMP-9 KO mice 28 h after administration of anti-CD3 Ab. Thymuses were stained with an Ab directed to CD31, which expresses on the surface of endothelial cells. In anti-CD3 Ab-injected wild-type mice, the thymus became highly vascularized, and the number of large vessels in the cortical area was increased, compared with MMP-9 KO mice. Collectively, these data suggest that MMP-9 is involved in degradation of collagen type IV, followed by induction of thymic neovascularization.
Neovascularization and macrophage distribution in thymuses after administration of anti-CD3 Ab
To ascertain whether neovessel formation correlates with appearance of HUIV26 cryptic epitope, thymuses of BALB/c mice following administration of anti-CD3 Ab were stained with anti-CD31 and Ab HUIV26 (Fig. 6). As shown in Fig. 6A, a significant increase in neovascular formation was noted in the cortical region of thymuses at 25 or 28 h postinjection relative to thymuses at 0 h postinjection. Vessels from the thymus at 0 h postinjection showed little, if any, exposure of the HUIV26 cryptic exposure (Fig. 6B). In the thymuses, 25 or 28 h postinjection, exposure of HUIV26 epitope was readily detectable. Costaining with both Abs showed that not all vessels stained positive for HUIV26 epitope, but significant exposure of the epitope was detectable in association with CD31-positive endothelial cells.
FIGURE 6. Neovessel formation is associated with appearance of collagen type IV cryptic sites in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at indicated time points after the injection. A, Vascular morphology. Cryostat sections of each thymus were stained with anti-CD31 Ab (red). These sections are representatives for each time point. B, Appearance of cryptic site of collagen IV. Cryostat sections of each thymus were stained with HUIV26 Ab (green) and with anti-CD31 Ab (red). These sections are representative of four mice for each condition. Bars, 200 μm.
To examine the fate of macrophages after clearance of apoptotic thymocytes, we analyzed macrophage distribution in thymuses of BALB/c mice following administration of anti-CD3 Ab, by staining with Ab F4/80 and anti-CD31 Ab (Fig. 7). A massive increase in F4/80-positive macrophages was evident within 18 h of the injection, as shown in Fig. 2. Twenty-three hours after the injection, a number of F4/80-positive macrophages was still detectable, and many macrophages were found to be localized in perivascular areas. By 28 h, the number of F4/80-positive macrophages was significantly decreased, and some of the macrophages were seen in the site close to or within vessels.
FIGURE 7. Macrophage distribution in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and each thymus was removed at indicated time points after the injection. Cryostat sections of each thymus were stained with F4/80 Ab (red) and with anti-CD31 Ab (green). These sections are representative of four mice for each condition. Bars, 200 μm.
Exposure of HUIV26 cryptic site within collagen type IV is involved in thymic neovascularization
Systemic administration of HUIV26 Ab is shown to inhibit angiogenesis and tumor growth in multiple animal models (36, 37). To evaluate whether exposure of this cryptic site may be involved in the process by which new vessels were formed in the thymus, we asked whether HUIV26 Ab might inhibit thymic vascularization. Anti-CD3 Ab was injected into BALB/c mice, and 17 h later the mice were either untreated or treated with i.p. injection of either HUIV26 Ab or isotype-matched control Ab (75 or 150 μg). An additional 11 h later, thymuses were removed and assessed for the vascular morphology by immunohistochemical analysis (Fig. 8). Increased density of vessels in the thymus was detectable in control Ab-treated mice and untreated mice (data not shown) by 28 h following anti-CD3 Ab injection. Administration of HUIV26 Ab potently inhibited thymic neovascularization in a dose-dependent manner (data not shown), and the extent of new vessel formation in HUIV26 (150 μg)-treated mice was decreased, as compared with control Ab-treated mice. Inhibition of thymic vascularization was markedly reduced when HUIV26 Ab was administered 22 h after anti-CD3 Ab injection (data not shown). In addition, when the distribution of macrophages in thymuses was analyzed by staining with F4/80, macrophages were found to be located around or within many vessels of thymic cortical area in control Ab-treated mice. HUIV26 Ab-treated mice apparently harbored an increased number of F4/80-positive macrophages in the thymus than did control Ab-injected mice at 28 h after injection of anti-CD3 Ab. Nevertheless, an additional 8 h later, macrophages in the thymus of HUIV26 Ab-treated mice appeared to be reduced in number and accumulated close to or within vessels (data not shown).
FIGURE 8. Effects of HUIV26 Ab on neovascularization and macrophage distribution in the thymus after administration of anti-CD3 Ab. Seven-week-old BALB/c mice were injected i.p. with 50 μg of anti-CD3 Ab, and 17 h later, mice were either untreated or treated i.p. (150 μg/mouse) with either HUIV26 Ab or an isotype-matched control Ab. Each thymus was removed 28 h after administration of anti-CD3 Ab, and thymic sections were stained with anti-CD31 Ab (green) and with F4/80 Ab (red). These sections are representative of four mice for each condition, and experiments were completed three times with similar results. Bars, 200 μm.
Discussion
In this study, we demonstrate that MMP-9 expression was up-regulated in thymuses of BALB/c mice after administration of anti-CD3 Ab. A number of macrophages promptly infiltrated into the thymuses following apoptosis of thymocytes, and the distribution patterns of MMP-9-expressing cells were found similar to those of F4/80-positive cells. By 28 h, the number of macrophages in the thymus was markedly decreased, and the expression levels of MMP-9 were also reduced. Although MMP-9 was present in thymuses of normal mice, the increased expression of MMP-9 seems not to reflect the enrichment of thymic microenvironmental compartments due to thymocyte depletion. The colocalization of MMP-9 in F4/80-positive cells suggests that macrophages are the likely sources of MMP-9. As shown previously (30, 31), primary macrophages from the peritoneal cavity of BALB/c mice were found to express MMP-9. Increased MMP-9 expression in murine macrophages was undetectable in response to apoptotic cells. Although in vitro findings using macrophages from peritoneal cavity do not necessarily correlate with in vivo thymus studies, it is unlikely that macrophages might synthesize and secrete MMP-9 upon clearance of apoptotic thymocytes. Rather, the number of macrophages in the thymus was significantly increased, and the increased MMP-9 expression may be attributed to the presence of macrophages in thymuses following administration of anti-CD3 Ab.
We detected activation of MMP-9 in thymuses 18–24 h after anti-CD3 Ab injection. When untreated macrophages or macrophages exposed to apoptotic cells were cultured for 18 h, only latent MMP-9 was detected in the culture medium. We found the increased activity of plasmin in thymuses 18–24 h after anti-CD3 Ab injection. It is therefore conceivable that macrophages secrete inactive MMP-9, which may be converted to the active form by plasmin. In Western blot analysis, we observed that 85-kDa plasminogen was expressed in thymuses of normal mice (data not shown). Murine peritoneal macrophages are reported to produce and secrete a plasminogen activator (38). The mechanism underlying the generation of plasmin in thymuses following administration of ani-CD3 Ab remains to be determined.
We observed an exposure of collagen type IV cryptic epitope detectable by HUIV26 Ab in murine thymuses upon MMP-9 activation. HUIV26-positive staining was markedly reduced in MMP-9 KO mice. Although MMP-2 and MMP-9 can cleave triple-helical collagen type IV (3, 4), our study indicated that MMP-9, rather than MMP-2, may be critical in the exposure of HUIV26 cryptic sites. Thymic neovascularization in wild-type mice was evident, especially in the cortical region, as early as 23 h after anti-CD3 Ab injection. Recent studies have indicated that TGF- produced by apoptotic cells as well as phagocytes has a major role in anti-inflammatory effects during clearance of apoptotic cells (39, 40). MMP-9 is shown to proteolytically cleave latent TGF-, resulting in promotion of angiogenesis (14). In our previous study, an increase in TGF- levels was detectable in murine thymuses following thymocyte apoptosis, but the amounts of active TGF- were small (22). We therefore assume that TGF- has a minor role in the thymic vascular development. Mice deficient in MMP-2 or MMP-9 exhibit reduced angiogenesis in vivo (41, 42). In this study, new vessel formation was diminished in thymuses of MMP-9 KO mice following anti-CD3 Ab injection. In addition, administration of HUIV26 Ab inhibited thymic vascularization. Together, we concluded that consistent with previous investigations (36, 37), exposure of HUIV26 cryptic epitope within collagen type IV is important for the initiation of neovessel formation. Our findings agree with previous investigations by Brooks and colleagues (37), which demonstrated that MMP-9 is a primary contributor to vascularization process. Although it is known that cortical thymocytes result in apoptotic cell death after whole body irradiation, Huiskamp et al. (43) reported that the density of vessels throughout the thymus increases from day 2 until day 4 after the irradiation. They speculated that the increased thymic vascularization reflects the active tissue response during the regeneration process after irradiation. Little is, however, known concerning the molecular mechanisms controlling thymic neovessel formation. Also, we have detected MMP-9 activation in murine thymuses after injection of dexamethasone, which induces CD4+CD8+ thymocytes to apoptosis (data not shown). Thus, thymic neovascularization appears a prominent event following thymocyte apoptosis, and our evidence may help to understand the molecular mechanisms of vessel development during thymus remodeling following thymocyte apoptosis. Xu et al. (36) have demonstrated that the exposure of HUIV26 cryptic site is associated with a loss of 11 binding and gain in v3 binding and that interaction of HUIV26 epitope with endothelial cells induces their adhesion and migration. Additional experiments are necessary to determine which interactions between endothelial cells and the cryptic site of collagen type IV initiate signaling cascades required for thymic neovascularization.
MMP-9 is also found to participate in cell migration (44). MMP-9 may be expressed on the surface of cells, allowing for precise, localized proteolysis (14, 45) that would create a path for migration (46). The critical role of MMP-9 in transmigration through basement membrane has previously been described for T cells (47). Also, MMP-9 is necessary for the migration of Langerhans cells, which express MMP-9 on their cell surface (48). We observed that the number of macrophages in the thymus was reduced after clearance of apoptotic thymocytes. Macrophages are shown to migrate from tissues or organs to draining lymph nodes (49, 50). The number of TUNEL-positive cells was drastically reduced 24 h postinjection, suggesting that macrophages do not undergo apoptosis after phagocytosis of dying cells. Additionally, a number of macrophages was seen around or within vessels in thymuses after 23 h postinjection of anti-CD3. Based on our observations, we favor the proposal that macrophage clearance from tissues occurs by emigration rather than by local apoptosis (49), although we are unable to present any additional evidence for macrophage emigration from the thymus after clearance of apoptotic thymocytes. We observed a number of macrophages in thymuses of HUIV26 Ab-treated mice even after 28 h of anti-CD3 Ab injection. In addition, the accumulation of F4/80-positive macrophages was detected in thymuses of MMP-9 KO mice 32 h postinjection of anti-CD3 Ab (data not shown). It is conceivable that suppression of thymic neovessel formation interferes with emigration of macrophages from the thymus.
In this study, we provide evidence that MMP-9 in macrophages facilitates their migration from the thymus by induction of neovascularization. Nevertheless, the mechanisms mediating the trafficking of macrophages from thymus are unknown. Identification of chemokine and its receptor that participate in the migration of thymic macrophages after clearance of apoptotic thymocytes is under investigation.
Acknowledgments
We thank Drs. R. Takahashi and R. Senior for providing us with Abs, and Drs. T. Ebel, B. Freimark, and F. Pernaseffi for a gift of Abs and their critical reading of our manuscript. We are grateful to Drs. T. Endo and S. Izumiyama for excellent technical support in the use of microscope.
Footnotes
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 Address correspondence and reprint requests to Dr. Chikako Odaka, Department of Safety Research on Blood and Biological Products, National Institute of Infectious Diseases, 1-23-1, Toyama, Shinjuku-ku, Tokyo, 162-8640, Japan. E-mail address: odaka@nih.go.jp
2 Abbreviations used in this paper: MMP, matrix metalloproteinase; Boc-Glu-Lys-Lys-MCA, t-butyloxycarbonyl-L-glutamyl-L-lysyl-L-lysine 4-methylcoumary-7-amide; KO, knockout.
Received for publication May 26, 2004. Accepted for publication October 22, 2004.
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