Presence of Anti-cystatin Cpositive Dendritic Cells or Macrophages and Localization of Cysteine Proteases in the Apical Bud of the Enamel Organ in the Rat Incisor
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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Summary |
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Key Words: rat incisor apical bud cystatin C cathepsins macrophages dendritic cells
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Introduction |
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Most of the dendritic cells in the enamel organ also bind anti-cystatin C antibodies (Nishikawa and Sasaki 2000). Cystatin C is an endogenous cysteine protease inhibitor and is expressed ubiquitously (Chapman 1991
). In particular, macrophages secrete cystatin C and are thought to regulate proteolytic activity in the extracellular space (Warfel et al. 1987
; Chapman 1991
). Some populations of dendritic cells are also reported to exhibit strong expression of cystatin C (El-Sukkari et al. 2003
). This expression occurs in dendritic cells in the enamel organ of the rat incisor. Anti-cystatin Clabeled OX6 cells are distributed in the secretion zone of the rat incisors (Nishikawa and Sasaki 2000
). 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 Cpositive cells that may play a role in amelogenesis.
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Materials and Methods |
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Three newborn rats (5.86.2 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 1979; Dijkstra et al. 1985
; Damoiseaux et al. 1994
; Pierre and Mellman 1998
; Nishikawa and Sasaki 2000
).
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-Xconjugated 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 488conjugated anti-rabbit IgG diluted 1:100 and 0.5 µg/ml Hoechst 33342 (Molecular Probes). For control sections, mouse isotype control IgG (mouse IgG1negative 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 488conjugated anti-rabbit IgG, Alexa 647conjugated 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 1995,2000
).
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Results |
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Cystatin Cpositive Cells Present in the Apical Bud Epithelia of Neonatal Rat Incisors
To determine whether invasion of cystatin Cpositive 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 Clabeled cells were present in the apical bud of the neonatal incisors (Figures 5A5C), 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 5D5F).
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Discussion |
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It has been shown that macrophages or dendritic cells that incorporate apoptotic cell fragments are localized in the transition zone (Nishikawa and Sasaki 1996,2000
). 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. 1996
; Kieffer et al. 1999
). 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 Cpositive 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. 1987; Abrahamson et al. 1990
). 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 1991
). Proteolytic regulation by cathepsins and cystatin C has been widely reported in embryo implantation (Afonso et al. 1997
), normal organogenesis (Roth et al. 2000
; Tobin et al. 2002
; Wright et al. 2003
), and some pathological processes (Shi et al. 1999
; Stypmann et al. 2002
). In addition, cystatin C is reported to be involved in the proliferation of neural stem cells as a cofactor of FGF2 (Taupin et al. 2000
). Serine protease and matrix metalloproteinase function in processing and degradation of enamel matrix proteins (Tanabe et al. 1992
; Bartlett et al. 1996
; Robinson et al. 1998
; Simmer et al. 1998
). 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. 1999
; Kawashima et al. 2003
; Wenzel et al. 2003
). 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. 2003
). Osteoclasts are known to be positive for cysteine protease cathepsin K and L (Nakase et al. 2000
). 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. 1991; Okiji et al. 1996
). 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. 1996
). This study showed the presence of round cystatin Cpositive 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 Cpositive cells are not unique to adult rats.
In conclusion, the results of this study suggest that cystatin Cpositive 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.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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