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DOI: 10.1369/jhc.4A6533.2005
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Journal of Histochemistry and Cytochemistry
Volume 53 (5): 643-651, 2005
Copyright ©The Histochemical Society, Inc.

Presence of Anti-cystatin C–positive Dendritic Cells or Macrophages and Localization of Cysteine Proteases in the Apical Bud of the Enamel Organ in the Rat Incisor

Sumio Nishikawa

Department of Biology, Tsurumi University School of Dental Medicine, Yokohama, Japan

Correspondence to: Sumio Nishikawa, Department of Biology, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan. E-mail: nishikawa-s{at}tsurumi-u.ac.jp


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Cystatin C, a cysteine protease inhibitor, was examined in the apical buds of rat incisors by immunohistochemistry, because in transition and maturation zones most of the dendritic cells in the papillary layer are anti-cystatin C–positive. Anti-cystatin C–labeled cells were sparse and localized to the proliferation and differentiation zones, constituting the apical bud of 5-week-old rat incisors. These cells were considered macrophages or dendritic cells, based on their reactivity with OX6 and ED1, as well as their ultrastructure. Basement membrane at the periphery of apical bud was also labeled by anti-cystatin C antibody. The apical buds included a few apoptotic fragments and weak reactivity with antibody to cathepsin L, a cysteine protease. Reactivity to anti-cystatin C and anti-cathepsin L antibodies was also detected in the apical bud of newborn rat incisors. These results suggest that the cystatin C–positive macrophages or dendritic cells are involved in normal incisor formation. They may be related to the clearance of apoptotic cells or protection from putative cysteine protease activity. (J Histochem Cytochem 53:643–651, 2005)

Key Words: rat incisor • apical bud • cystatin C • cathepsins • macrophages • dendritic cells


   Introduction
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AMELOGENESIS in rodent incisors may be divided into several phases resulting in the proliferation, differentiation, secretion, transition, and maturation zones. In the proliferation zone, putative stem cells divide and become enamel epithelial cells (Harada et al. 1999Go). Because of its bulbous morphology, the proliferation zone has been proposed as the apical bud (Ohshima et al. 2003Go), and this latter terminology is used in this report. In more advanced zones of the enamel organ of rat incisor, that is the transition and maturation zones, many dendritic cells invade the epithelial layer (Nishikawa and Sasaki 1996Go; Takano et al. 1996Go). In particular, in the late maturation zone, numerous dendritic cells are localized in the epithelial papillary layer (Takano et al. 1996Go). Dendritic cells in the transition zone ingest apoptotic ameloblast fragments and enamel matrix remnants in the papillary layer and thus may function in clearing unnecessary cells and materials and in acquiring immunotolerance (Nishikawa and Sasaki 1996Go,1999Go). In the late maturation zone, however, enamel matrix resorption is almost complete, and no apoptotic figures are observed. The functional roles of dendritic cells in this zone are still unclear. Dendritic cells from the transition and maturation zones bind anti-MHC Class II antibodies (OX6) (e.g., antigen-presenting cells) and anti-lysosomal membrane protein antibodies (ED1) (e.g., macrophages and dendritic cells). These dendritic cells, however, do not bind ED2, antibody for resident macrophage protein (Takano et al. 1996Go; Nishikawa and Sasaki 2000Go).

Most of the dendritic cells in the enamel organ also bind anti-cystatin C antibodies (Nishikawa and Sasaki 2000Go). Cystatin C is an endogenous cysteine protease inhibitor and is expressed ubiquitously (Chapman 1991Go). In particular, macrophages secrete cystatin C and are thought to regulate proteolytic activity in the extracellular space (Warfel et al. 1987Go; Chapman 1991Go). Some populations of dendritic cells are also reported to exhibit strong expression of cystatin C (El-Sukkari et al. 2003Go). This expression occurs in dendritic cells in the enamel organ of the rat incisor. Anti-cystatin C–labeled OX6– cells are distributed in the secretion zone of the rat incisors (Nishikawa and Sasaki 2000Go). These cells are speculated to be precursor cells of dendritic cells in the transition and maturation zones, invading the enamel organ initially as OX6– cells and becoming OX6+ thereafter.

It is not known whether invasion of dendritic cell precursors begins at the secretion zone or in an earlier zone, that is, in the differentiation zone or even in the proliferation zone. The aim of this study was to determine the pattern of cystatin C expression in the enamel organ and to characterize the cystatin C–positive cells that may play a role in amelogenesis.


   Materials and Methods
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Six 5-week-old male Wistar rats (140–150 g) (Jcl Wistar; Clea Japan, Tokyo, Japan) were used for the immunofluorescence microscopy, following institutional guidelines for animal care. The animals were perfused under sodium pentobarbital (Nembutal; Abbott, North Chicago, IL) anesthesia with 4% paraformaldehyde in 0.1 M PBS, pH 7.2, through the left ventricle at RT for 10 min. The maxillae and mandibles were dissected and were subsequently immersed in the same fixative at 4C for 2 hr. After being washed with 0.1 M PB, the maxillae and mandibles were demineralized by immersion in 5% EDTA solution adjusted to pH 7.3 with sodium hydroxide solution, at 4C for 3 to 4 weeks. Demineralized incisors were cut transversely into two segments. Both segments were immersed in 25% sucrose in PBS overnight, rapidly frozen, and then cut longitudinally (6- to 8-µm thick) using a cryotome.

Three newborn rats (5.8–6.2 g; Clea Japan) were also killed by decapitation. Mandibles were dissected and fixed with 4% paraformaldehyde in 0.1 M PB. They were subsequently immersed in the 25% sucrose solution overnight, rapidly frozen, then cut using a cryotome.

Antibodies used in this study were mouse monoclonal ED1, ED2, and OX6 antibodies (Serotec; Oxford, UK), rabbit polyclonal anti-cystatin C antibodies (Upstate Biotechnology; Lake Placid, NY), and goat polyclonal anti-cathepsin B (E-19), cathepsin L (C-18), and cathepsin S (M-19) antibodies (Santa Cruz Biotechnology; Santa Cruz, CA). ED1, ED2, OX6, and anti-cystatin C have been previously characterized (McMaster and Williams 1979Go; Dijkstra et al. 1985Go; Damoiseaux et al. 1994Go; Pierre and Mellman 1998Go; Nishikawa and Sasaki 2000Go).

For triple labeling, cryosections on glass microscope slides were incubated in 1% BSA-PBS at RT for 30 min. They were then labeled with ED1, ED2, or OX6 antibodies diluted 1:100 (10 µg/ml) with 1% BSA-PBS at RT for 30 min, followed by labeling at RT for 10 min with biotinylated anti-rabbit and anti-mouse immunoglobulins absorbed to abolish cross-reactivity with rat serum proteins (LINK; DAKO LSAB2 Kit, DAKO, Carpinteria, CA). Sections were visualized by labeling with 2 µg/ml Rhodamine Red-X–conjugated streptavidin (Molecular Probes; Eugene, OR) at RT for 10 min. After being washed with PBS and incubated with 1% BSA-PBS, the sections were labeled with anti-cystatin C antibodies diluted 1:100 (10 µg/ml) at RT for 30 min, followed by labeling with a mixture of Alexa 488–conjugated anti-rabbit IgG diluted 1:100 and 0.5 µg/ml Hoechst 33342 (Molecular Probes). For control sections, mouse isotype control IgG (mouse IgG1–negative control, DAKO) and rabbit immunoglobulin fraction (DAKO) were used instead of ED1, ED2, or OX6 and anti-cystatin C, respectively. For some sections, triple labeling was performed using a mixture of anti-cystatin C and ED1 or OX6, followed by labeling with a mixture of Alexa 488–conjugated anti-rabbit IgG, Alexa 647–conjugated anti-mouse IgG (Molecular Probes), and Hoechst 33342. Fluorescent images were obtained using an Olympus A x80 fluorescence microscope equipped with a Charge Coupled Device camera (Quantix KAF1401E; Photometrics, Tucson, AR) and MetaMorph software (Universal Imaging; Downingtown, PA).

For cathepsin immunohistochemistry, sections were incubated at room temperature for 30 min with anti-cathepsin B, L, and S antibodies diluted 1:20, followed by donkey anti-goat IgG conjugated with biotin (Santa Cruz Biotechnology) diluted 1:100 for 10 to 20 min. They were then incubated with streptavidin peroxidase (DAKO LSAB2), and visualized by Simple Stain DAB (Nichirei; Tokyo, Japan).

Procedures for conventional electron microscopy and preembedding immunoelectron microscopy for anti-cystatin C have been described elsewhere (Nishikawa and Sasaki 1995Go,2000Go).


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Discussion
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Labeling the Proliferation Zone of the Rat Incisor Enamel Organ with Anti-cystatin C, OX6, and ED1 Antibodies
Anti-cystatin C–labeled cells were present mainly in the proliferation and differentiation zones (Figures 1A, 1C, 1D, and 1F) and were scattered sparsely in the apical bud of the incisor enamel organ. The anti-cystatin C–labeled cells varied in shape, from a simple ovoid to a dendritic morphology. Basement membrane of the apical bud was also labeled by anti-cystatin C (Figures 1A and 1D). Several anti-cystatin C–labeled cells were also OX6+ (Figures 1A and 1B), whereas ED1 antibodies labeled the majority of anti-cystatin C–labeled cells (Figures 1D–1F). None of the zones of the enamel organ were labeled with ED2 antibodies (Figure 1G). In the secretion zone, anti-cystatin C, but not OX6, antibodies labeled few cells of enamel organ (Figures 1G and 2), whereas in the maturation zone many OX6+ anti-cystatin C–labeled dendritic cells were observed (Figures 1H and 2). Ratios of average OX6+ cell numbers to average anti-cystatin C–positive cell numbers were 0.56, 0.72, 0.03, and 1.06 in the proliferation, differentiation, secretion, and late maturation zones, respectively (Figure 2). Conventional electron microscopy revealed nonepithelial cells lacking both tonofilaments and desmosomes. These cells exhibited small numbers of lysosomal granules, organelle-poor cytoplasm at the cell periphery, and deep invaginations at the plasma membrane, with an appearance of dendritic-precursor cells (Figure 3A). Some nonepithelial cells exhibited more lysosomal inclusions (Figure 3B). Anti-cystatin C immunocytochemistry revealed that cystatin C–labeled cells possessed endoplasmic reticulum including nuclear envelope (Figure 3C).



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  		Figure 1

Immunohistochemical localization of anti-cystatin C (Cys C) (A–D,F–H), OX6 (B,C,G,H), ED1 (E,F), and ED2 (G). (A–C) Apical bud and differentiation zone of enamel organ triple labeled with Cys C (green) (A), OX6 (red) (B), and Hoechst 33,342 (H33342) (blue). Merged image is shown in (C). (D–F) Portion of apical bud labeled with Cys C (green) (D), ED1 (red) (E), and H33342 (blue). Merged image is shown in (F). Double-positive cells are indicated by arrowheads. (G) Secretion zone of enamel organ triple labeled with Cys C (green), OX6 (blue), and ED2 (red). Cys C single-positive cells are seen in the epithelial layer. (H) Late maturation zone of the enamel organ triple labeled with Cys C (green), OX6 (red), and H33342 (blue). Numerous OX6+ dendritic-like cells are located in the papillary layer. Their cytoplasm is labeled with Cys C showing green to yellow color (C,G). Bars: C,G = 100 µm; D,H = 50 µm.

 


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  		Figure 2

Anti-cystatin C–labeled or OX6+ cell numbers per unit area (10,000 mm2) in the rat incisor enamel organ. Mean and standard deviations were obtained from 5 to 12 sections of the proliferation (4 rats), differentiation (3 rats), secretion (2 rats), and late maturation (1 rat) zones. *, p<0.02; **, p<0.01 (Student's t-test).

 


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  		Figure 3

Conventional electron micrographs (A,B,D) and an anti-cystatin C immunoelectron micrograph (C) in the proliferation zone of the apical bud. A nonepithelial cell (arrow) (A), a juvenile macrophage (B), and apoptotic fragments (D) are shown. (C) Anti-cystatin C–labeled nuclear membrane and endoplasmic reticulum of a cell (arrow). Bars: A,C = 2 µm; B,D = 1 µm.

 
Apoptotic Events of Enamel Epithelial Cells in the Rat Incisor Apical Bud
To assess the role of nonepithelial cystatin C–positive dendritic cells or macrophages in the apical bud, the terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) method was employed to determine whether apoptosis occurred in the proliferation zone. In the apical bud of the enamel organ, TUNEL-positive cells were detected, although their frequency was low (Figure 4A). Conventional electron microscopy revealed the apoptotic cell fragments (Figure 3D).



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  		Figure 4

Apical bud labeled using the TUNEL method (A), anti-cathepsin L, (cat L) (B), anti-cathepsin S, (cat S) (C), and anti-cathepsin B (cat B) (D). A TUNEL-positive cell is seen in A. Cells are stained with cat L, but not with cat S or cat B. Bars: A = 25 µm; B–D = 100 µm.

 
Cathepsin B, L, and S Immunocytochemistry
To determine whether cysteine proteases were active in the enamel organ, cysteine protease (cathepsin B, cathepsin L, cathepsin S) immunocytochemistry was performed. Anti-cathepsin L labeled apical bud enamel organ epithelia weakly, and this labeling extended into the differentiation zone (Figure 4B). Anti-cathepsin S labeling was not detected in most specimens (Figure 4C). Anti-cathepsin B labeling was not detected in the enamel organ (Figure 4D), but the odontoblast layer of dental pulp was weakly labeled.

Cystatin C–positive Cells Present in the Apical Bud Epithelia of Neonatal Rat Incisors
To determine whether invasion of cystatin C–positive cells in the apical bud of enamel organ is related to adulthood, apical bud enamel organ of neonatal rat incisors was also examined. Anti-cystatin C–labeled cells were present in the apical bud of the neonatal incisors (Figures 5A–5C), and the basement membrane of apical bud was also labeled with anti-cystatin C (Figures 5A and 5C). Anti-cathepsin L and S, but not B, weakly labeled the apical bud epithelium (Figures 5D–5F).



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  		Figure 5

Apical buds of newborn (day 0) rats labeled with anti-cystatin C (cys C) (A,C) and a control (B), anti-cathepsin L (cat L) (D), anti-cathepsin S (cat S) (E), and anti-cathepsin B (cat B) (F). A, B, and C are first, second, and third sections of an apical bud, respectively. Cystatin C–positive cells (arrows in A,C) and cystatin C–positive basement membrane (arrowheads in A,C) are characteristic. Cells in insets of A and C are enlargements of arrowed cells of A and C, respectively. Apical buds are weakly positive for anti-cathepsin L (asterisk in D) and S (E) but negative for anti-cathepsin B (F). Bars = 100 µm.

 

   Discussion
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 Summary
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 Materials and Methods
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 Discussion
Literature Cited
 
Nishikawa and Sasaki (2000)Go reported that anti-cystatin C–positive dendritic cells are abundant in the epithelial papillary layer of maturation zone and also exist in the secretion and transition zones in the rat incisors. In this study, anti-cystatin C–labeled cells were localized to the differentiation zone and proliferation zones of the incisor apical bud. Some of these anti-cystatin C–labeled cells were also OX6+. As in previously published results (Nishikawa and Sasaki 2000Go), cystatin C–positive cells seem to be distributed in the epithelial enamel organ throughout rat incisor amelogenesis. These cells seem to be dendritic cells or of macrophage lineage, based on their ultrastructure and immunohistochemistry results from the current study. It is not clear, however, whether these cystatin C–positive cells are a uniform population, because in the current study some of them also stained with OX6 antibodies in the proliferation, differentiation, transition, and maturation zones but did not stain with OX6 antibodies in the secretion zone, located between differentiation and transition zones. These results support previous findings (Nishikawa and Sasaki 2000Go).

It has been shown that macrophages or dendritic cells that incorporate apoptotic cell fragments are localized in the transition zone (Nishikawa and Sasaki 1996Go,2000Go). Therefore, in the current study, apoptosis was examined in the apical bud using the TUNEL method and electron microscopy. Both methods revealed the presence of a limited degree of apoptosis in the apical bud. These results are consistent with those of previous studies, in which molar tooth germs exhibited numerous apoptotic events, but apoptosis in incisor tooth germs was less common (Vaahtokari et al. 1996Go; Kieffer et al. 1999Go). The low level of apoptosis in the incisor apical bud may be related to the survival of progenitor cells of inner enamel epithelia and ameloblasts. Cystatin C–positive cells may eliminate apoptotic cell fragments, or conversely they might offer protection from apoptosis by secretion of the cysteine protease inhibitor, cystatin C.

Cystatin C is a normal blood plasma constituent, secreted constitutively by monocytes/macrophages and ubiquitously expressed (Warfel et al. 1987Go; Abrahamson et al. 1990Go). Because cystatin C is an endogenous cysteine protease inhibitor, it may function in regulating cysteine proteases in the enamel organ. Indeed, in the current study, cathepsin L reactivity was detected at the apical bud of 5-week-old rats, and faint cathepsin L and S reactivities were found at the apical bud of newborn rats. Although the primary targets of cathepsins are cellular lysosomes, a fraction of these enzymes are thought to be secreted into the extracellular space (Chapman 1991Go). Proteolytic regulation by cathepsins and cystatin C has been widely reported in embryo implantation (Afonso et al. 1997Go), normal organogenesis (Roth et al. 2000Go; Tobin et al. 2002Go; Wright et al. 2003Go), and some pathological processes (Shi et al. 1999Go; Stypmann et al. 2002Go). In addition, cystatin C is reported to be involved in the proliferation of neural stem cells as a cofactor of FGF2 (Taupin et al. 2000Go). Serine protease and matrix metalloproteinase function in processing and degradation of enamel matrix proteins (Tanabe et al. 1992Go; Bartlett et al. 1996Go; Robinson et al. 1998Go; Simmer et al. 1998Go). This study raises the possibility that cysteine proteases and cysteine protease inhibitor, cystatin C, play a role in tooth morphogenesis and enamel formation. In the current study, anti-cystatin C also labeled basement membrane, but adjacent epithelial or mesenchymal cells lacked reactivity. Basement membrane labeling of anti-cystatin C may reveal the storage of physiologically active substances such as cytokines and enzymes there, as has been reported (Dowd et al. 1999Go; Kawashima et al. 2003Go; Wenzel et al. 2003Go). Although this point remains to be clarified, it is hypothesized that the secreted cystatin C is trapped at the basement membrane and may protect against attack by proteases diffusing from the epithelia, pulp region, and nearby osteoclasts. Cathepsin S-positive macrophages are localized to the early pulp (unpublished observation) and to many osteoclasts on the alveolar bone socket around apical bud. In mice incisors, strong cathepsin K expression has been reported in cells lining bone apically (Yamada et al. 2003Go). Osteoclasts are known to be positive for cysteine protease cathepsin K and L (Nakase et al. 2000Go). It is thus proposed that cystatin C plays a role in the protection from putative cysteine protease activity, but further study is needed to address this possibility.

OX6+, ED1-positive, or ED2-positive dendritic cells or macrophages are scarce in the incisor and molar pulp of newborn rats but increase in number after 2 weeks in the molar and 1 week in the incisor (Jontell et al. 1991Go; Okiji et al. 1996Go). Dendritic cells in the enamel organ of the maturation zone of rat incisors are scarce in juvenile animals, whereas numerous dendritic cells are present in the adult animals (Takano et al. 1996Go). This study showed the presence of round cystatin C–positive cells in the incisor apical bud of the newborn rat. Furthermore, this study showed immunoreactivity of anti-cystatin C at the basement membrane. These results indicate that cystatin C–positive cells are not unique to adult rats.

In conclusion, the results of this study suggest that cystatin C–positive macrophages or dendritic cells are involved in normal incisor morphogenesis. In the apical bud, these cells seem to be related to apoptotic events and/or protection from proteolytic activity of cysteine proteases.


   Acknowledgments
 
This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan to promote multidisciplinary Research Projects from 2001 to 2005.


   Footnotes
 
Received for publication September 22, 2004; accepted December 9, 2004


   Literature Cited
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 Materials and Methods
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 Literature Cited
 

Abrahamson M, Olafsson I, Palsdottir A, Ulvsbäck M, Lundwall Å, Jensson O, Grubb A (1990) Structure and expression of the human cystatin C gene. Biochem J 268:287–294[Medline]

Afonso S, Romagnano L, Babiarz B (1997) The expression and function of cystatin C and cathepsin B and cathepsin L during mouse embryo implantation and placentation. Development 124:3415–3425[Abstract]

Bartlett JD, Simmer JP, Xue J, Margolis HC, Moreno EC (1996) Molecular cloning, and mRNA tissue distribution of a novel matrix metalloproteinase isolated from porcine enamel organ. Gene 183:123–128[CrossRef][Medline]

Chapman HA (1991) Role of enzyme receptors and inhibitors in regulating proteolytic activities of macrophages. Ann NY Acad Sci 624:87–96[Abstract]

Damoiseaux JGMC, Döpp EA, Calame W, Chao D, MacPherson GG (1994) Rat macrophage lysosomal membrane antigen recognized by monoclonal antibody ED1. Immunology 83:140–147[Medline]

Dijkstra CD, Döpp EA, Joling P, Kraal G (1985) The heterogeneity of mononuclear phagocytes in lymphoid organs: distinct macrophage subpopulations in the rat recognized by monoclonal antibodies ED1, ED2 and ED3. Immunology 54:589–599[Medline]

Dowd CJ, Cooney CL, Nugent MA (1999) Heparan sulfate mediates bFGF transport through basement membrane by diffusion with rapid reversible binding. J Biol Chem 274:5236–5244[Abstract/Free Full Text]

El-Sukkari D, Wilson NS, Hakansson K, Steptoe RJ, Grubb A, Shortman K, Villadangos JA (2003) The protease inhibitor cystatin C is differentially expressed among dendritic cell population, but does not control antigen presentation. J Immunol 171:5003–5011[Abstract/Free Full Text]

Harada H, Kettunen P, Jung HS, Mustonen T, Wang YA, Thesleff I (1999) Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling. J Cell Biol 147:105–120[Abstract/Free Full Text]

Jontell M, Jiang W, Bergenholtz G (1991) Ontogeny of class II antigen expressing cells in rat incisor pulp. Scand J Dent Res 99:384–389[Medline]

Kawashima H, Watanabe N, Hirose M, Sun X, Atarashi K, Kimura T, Shikata K et al. (2003) Collagen XVIII, a basement membrane heparin sulfate proteoglycan, interacts with L-selectin and monocyte chemoattractant protein-1. J Biol Chem 278:13069–13076[Abstract/Free Full Text]

Kieffer S, Peterkova R, Vonesch JL, Ruch JV, Peterka M, Lesot H (1999) Morphogenesis of the lower incisor in the mouse from the bud to early bell stage. Int J Dev Biol 43:531–539[Medline]

McMaster WR, Williams AF (1979) Identification Ig glycoproteins in rat thymus and purification from rat spleen. Eur J Immunol 9:426–433[Medline]

Nakase T, Kaneko M, Tomita T, Myoui M, Ariga K, Sugamoto K, Uchiyama Y, et al. (2000) Immunohistochemical detection of cathepsin D, K, and L in the process of endochondral ossification in the human. Histochem Cell Biol 114:21–27[Medline]

Nishikawa S, Sasaki F (1995) DNA localization in nuclear fragments of apoptotic ameloblasts using anti-DNA immunoelectron microscopy: programmed cell death of ameloblasts. Histochem Cell Biol 104:151–159[CrossRef][Medline]

Nishikawa S, Sasaki F (1996) Phagocytotic processing of apoptotic bodies of transitional ameloblasts by MHC class II-expressing macrophages in rat incisor. J Histochem Cytochem 44:1459–1467[Abstract]

Nishikawa S, Sasaki F (1999) Apoptosis of dental pulp cells and their elimination by macrophages and MHC class II-expressing dendritic cells. J Histochem Cytochem 47:303–311[Abstract/Free Full Text]

Nishikawa S, Sasaki F (2000) Detection of immature dendritic cells in the enamel organ of rat incisors by using anti-cystatin C and anti-MHC class II immunocytochemistry. J Histochem Cytochem 48:1243–1255[Abstract/Free Full Text]

Ohshima H, Kenmotsu S, Harada H (2003) Use of the term apical bud to refer to the apical end of the continuously growing tooth. Arch Comp Biol Tooth Enamel 8:45–49

Okiji T, Kosaka T, Kamal AMM, Kawashima N, Suda H (1996) Age-related changes in the immunoreactivity of the monocyte/macrophage system in rat molar pulp. Arch Oral Biol 41:453–460[CrossRef][Medline]

Pierre P, Mellman I (1998) Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93:1135–1145[CrossRef][Medline]

Robinson C, Brookes SJ, Shore RC, Kirkham J (1998) The developing enamel matrix: nature and function. Eur J Oral Sci 106(suppl 1):282–291

Roth W, Deussing J, Botchkarev VA, Pauly-Evers M, Saftig P, Hafner A, Schmidt P, et al. (2000) Cathepsin L deficiency as molecular defect of furless: hyperproliferation of keratinocytes and perturbation of hair follicle cycling. FASEB J 14:2075–2086[Abstract/Free Full Text]

Shi G-P, Sukhova GK, Grubb A, Ducharme A, Rhode LH, Lee RT, Ridker PM, et al. (1999) Cystatin C deficiency in human atherosclerosis and aortic aneurysms. J Clin Invest 104:1191–1197[Medline]

Simmer JP, Fukae M, Tanabe T, Yamakoshi Y, Uchida T, Xue J, Margolis HC, et al. (1998) Purification, characterization, and cloning of enamel matrix serine proteinase 1. J Dent Res 77:377–386[Abstract/Free Full Text]

Stypmann J, Gläser K, Roth W, Tobin DJ, Petermann I, Matthias R, Mönning G, et al. (2002) Dilated cardiomyopathy in mice deficient for the lysosomal cysteine peptidase cathepsin L. Proc Natl Acad Sci USA 99:6234–6239[Abstract/Free Full Text]

Takano Y, Kawahara I, Hoshino M, Takeuchi K, Maeda T, Ohshima H, Hanaizumi Y, et al. (1996) Dendritic cells: a novel cellular component of the rat incisor enamel organ appearing in the late stages of enamel maturation. Adv Dent Res 10:94–104

Tanabe T, Fukae M, Uchida T, Shimizu M (1992) The localization and characterization of proteinases for the initial cleavage of porcine amelogenin. Calcif Tissue Int 51:213–217[CrossRef][Medline]

Taupin P, Ray J, Fischer WH, Suhr ST, Hakansson K, Grubb A, Gage FH (2000) FGF-2-responsive neural stem cell proliferation requires CCg, a novel autocrine/paracrine cofactor. Neuron 28:385–397[CrossRef][Medline]

Tobin DJ, Foitzik K, Reinheckel T, Mecklenburg L, Botchkarev VA, Peters C, Paus R (2002) The lysosomal protease cathepsin L is an important regulator of keratinocyte and melanocyte differentiation during hair follicle morphogenesis and cycling. Am J Pathol 160:1807–1821[Abstract/Free Full Text]

Vaahtokari A, Åberg T, Thesleff I (1996) Apoptosis in the developing tooth: association with an embryonic signaling center and suppression by EGF and FGF-4. Development 122:121–129[Abstract]

Warfel AH, Zucker-Franklin D, Frangione B, Ghiso J (1987) Constitutive secretion of cystatin C ({gamma}-trace) by monocytes and macrophages and its downregulation. J Exp Med 166:1912–1917[Abstract/Free Full Text]

Wenzel SE, Balzar S, Cundall M, Chu HW (2003) Subepithelial basement membrane immunoreactivity for matrix metalloproteinase 9: Association with asthma severity, neutrophilic inflammation, and wound repair. J Allergy Clin Immunol 111:1345–1352[CrossRef][Medline]

Wright WW, Smith L, Kerr C, Charron M (2003) Mice that express enzymatically inactive cathepsin L exhibit abnormal spermatogenesis. Biol Reprod 68:680–687[Abstract/Free Full Text]

Yamada Y, Sun X, Hu JC-C, Simmer JP, Matsumoto K, Bartlet JD (2003) Characterization of cathepsin K and caldecrin expression in mouse postnatal day 28 incisors and day 3 molars. Arch Comp Biol Tooth Enamel 8:90–94


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