ARTICLE |
Correspondence to: Sumio Nishikawa, Dept. of Biology, Tsurumi University School of Dental Medicine, 2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan. Fax: +81-45-573-9599.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dendritic cells in the enamel organ of rat incisors were examined with immunocytochemistry using an anti-cystatin C antibody for immature dendritic cells and macrophages, OX6 for MHC Class II, ED1 for macrophages and dendritic cells, and ED2 for macrophages. Single cells positive for anti-cystatin C appeared in the enamel organ in zones at which ameloblasts secrete enamel matrix proteins. They were also present in transition and enamel maturation zones. In addition, ameloblasts, osteocytes, and osteoclasts were labeled by anti-cystatin C. ED1 and ED2 immunocytochemistry revealed that there was no macrophage population in the enamel organ of secretion, transition, or enamel maturation zone. A double labeling study showed that most anti-cystatin C-positive cells in the enamel maturation zone were also positive for OX6, whereas anti-cystatin C-positive and OX6-negative cells were prevalent in the secretion zone. The results suggest that immature dendritic cells penetrate the enamel organ of the secretion zone and begin to mature in the zones of transition and enamel maturation. (J Histochem Cytochem 48:12431255, 2000)
Key Words: cystatin C, MHC class II, dendritic cells, amelogenesis, rat incisor, immunocytochemistry, electron microscopy
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dendritic cells are professional antigen-presenting cells. They capture exogenous antigens and then move to lymphoid tissues to present their antigen peptide with major histocompatibility complex Class II (MHC Class II) to helper T-cells (
During amelogenesis in rat incisors, ameloblasts secrete enamel matrix proteins, forming an enamel layer. The same ameloblasts also contribute to enamel maturation, which is accompanied by organic matrix loss and increased mineralization. A narrow zone called the transition zone lies between the secretion and maturation zones. A cell kinetic study of rat incisors (bw 100 g and 300 g) revealed that it takes 6.57.5 days for an ameloblast to pass the secretion zone, less than 1 day to pass the transition zone, and 1222 days to pass the maturation zone (
In this study, antibodies against cystatin C known to be endogenous cysteine protease inhibitors were used to address these questions and ascertain the nature of OX6+ cells in the enamel organ. Cystatin C was shown to be abundant in immature dendritic cells, and therefore the proteolytic processing of the MHC Class II protein complex and antigen proteins was inhibited. On the other hand, cytostatin C was decreased in mature dendritic cells where protein processing occurs, and antigenMHC Class II complexes were expressed in the plasma membrane (
![]() |
Materials and Methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three male Wistar rats (280290 g) (Jcl Wistar; Clea Japan, Tokyo, Japan; institutional guidelines were followed) were sacrificed by diethyl ether inhalation and maxillary and mandibular incisors were dissected. The labial side of each tooth was scraped with a razor blade. Tissue pieces containing secretion, transition, and early maturation ameloblasts were immediately frozen in a cryotome (HM505E; Microm, Walldorf, Germany). Cryosections (68 µm thick) were laid on glass microscopic slides and fixed with pure acetone at 4C for 5 min. The immunoperoxidase labeling method was described previously (
Procedures for pre-embedding immunoelectron microscopy for the OX6 antibody were described previously (
For immunocytochemistry of the anti-cystatin C antibody (Upstate Biotechnology; Lake Placid, NY), 11 male Wistar rats (185260 g; Jcl Wistar, Clea Japan) were used. This anti-human cystatin C antibody has been shown to react specifically with murine 1315-kD cystatin C (
For light microscopy, cryosections were labeled with anti-cystatin C antibody diluted 1:100 with 1% BSAPBS at RT for 60 min and then labeled with FITC-conjugated anti-rabbit IgG (Cappel) diluted 1:50 with 1% BSAPBS at RT for 30 min. Most of the cryosections were triply labeled with anti-cystatin C followed by FITC-conjugated anti-rabbit IgG, with OX6 antibody followed by rhodamine-conjugated anti-mouse IgG (Cappel), and with Hoechst 33342 (1 µg/ml in PBS) (Molecular Probes; Eugene OR) for DNA. Some sections were triply labeled with anti-cystatin C, ED1, and Hoechst 33342. For numerical analysis, the number of anti-cystatin C+ cells and OX6+ cells in the enamel organ of the secretion zone or transition plus early maturation zone were counted along the long axis of the incisors using double labeled sections. The length of the secretion zone for counting was 2.74.7 mm (n = 6 sections, maxillary and mandibular incisors from two rats) and that of the transition plus early maturation zone was 2.44.6 mm (n = 6 sections, maxillary and mandibular incisors from two rats). Some sections were labeled with anti-OX62 antibody (MRC OX62, MCA1029G; Serotec) for some cells with dendritic shape and T-cells, and with anti-CD45R (HIS24; Serotec) for pan-B-cells. Some acetone-fixed and undecalcified sections were double labeled with anti-cystatin C antibody and anti-desmoplakin 1 and 2 antibody (Clone DP1&2-2.15; Progen Biotechnik, Heidelberg, Germany) (
For immunoelectron microscopy, thick cryosections fixed with paraformaldehyde and decalcified with EDTA were labeled with anti-cystatin C diluted 1:100 with 1% BSAPBS at 4C overnight, followed by HRP-conjugated anti-rabbit IgG (Cappel) diluted 1:100 with 1% BSAPBS at 4C overnight. After being fixed with 1% glutaraldehyde solution for 1 hr, the sections were incubated in a DAB solution (0.2 mg/ml in 0.05 M Tris-HCl buffer, pH 7.6) at RT for 30 min and then immersed in DAB plus 0.005% H2O2 at RT for 5 min. They were postfixed with 1% osmium tetroxide and embedded in Epon 812. Ultrathin sections were lightly stained with lead citrate and examined with a JEOL 1200 EXII electron microscope (JEOL; Tokyo, Japan). Control sections were processed in the same way as described above, except that the primary antibody was replaced by 1% BSAPBS. Procedures for conventional electron microscopy have been described elsewhere (
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
OX6+ Cells in Transition and Maturation Zones Are Dendritic Cells
OX6+, ED1+, and ED2+ cells were scattered in connective tissues overlying the enamel organ in the secretion, transition, and maturation zones (Fig 1a1i). In the enamel organ of the secretion zone, however, no OX6+, ED1+, and ED2+ cells were present (Fig 1a, Fig 1d, and Fig 1g). OX6+ or ED1+ cells began to appear in the enamel organ of the transition zone (Fig 1b and Fig 1e; and see E2-integrin of some dendritic cells and intraepithelial dendritic T-cells (
|
OX6 immunoelectron microscopy revealed that OX6+ dendritic cells and OX6- non-epithelial cells with a "dendritic shape" that were morphologically indistinguishable from OX6+ cells were observed in the transition zone (Fig 2a and Fig 2b). These OX6- non-epithelial cells are considered to be dendritic cells. OX6+ dendritic cells with a highly complex shape were observed in the maturation zone (Fig 2c and Fig 2d).
|
Anti-cystatin C Labels Immature and Maturing Dendritic Cells in Enamel Organs
In the secretion zone, anti-cystatin C+ and OX6- cells were sparsely distributed in the enamel organ (Fig 3a, Fig 3c, and Fig 3e). These cells had ovoid profiles and were preferentially located in the stellate reticulum (Fig 3a). Most of these cells were distributed in the intercellular spaces of the stellate reticulum and fewer cells were attached to the inner surface of the blood vessels in light microscopy of Epon-embedded semithin sections. To determine the nature of the cells positive for anti-cystatin C, conventional electron and correlated anti-cystatin C immunoelectron microscopy were performed. Conventional electron microscopy revealed that these ovoid cells had a relatively smaller area of cytoplasm, with sparse cytoplasmic organelles and a greater proportion of heterochromatin in the nuclei, and thus have a monocyte-like appearance (Fig 6f). These cells exhibited no desmosomes or tonofilaments (Fig 6f). By Immunoelectron microscopy showed the labeled structures to be endoplasmic reticulum, including the nuclear membrane and vesicles of various sizes (Fig 6a). To confirm the non-epithelial origin of anti-cystatin C-positive cells, double labeling experiments with anti-desmoplakin 1 and 2 for desmosomes and anti-cystatin C antibodies were performed. The results showed that the anti-cystatin C-positive cells lacked anti-desmoplakin 1- and 2-positive fluorescent dots at their periphery (Fig 6g and Fig 6h).
|
|
|
|
In the transition zone, an increased number of anti-cystatin C+ cells were distributed in the papillary layers (Fig 3b and Fig 4a). Some anti-cystatin C+ cells were also positive for the OX6 antibody (Fig 4a, Fig 4d, and Fig 4g), but the labeling intensity of OX6 was weak (Fig 4d) compared with OX6+ cells in the maturation zone (Fig 4e and Fig 4f). Some of the anti-cystatin C+ cells were negative for OX6 (Fig 3b, Fig 3d, and Fig 3f). The structures labeled by anti-cystatin C on an electron microscopic level were endoplasmic reticulum and unknown vesicles (Fig 6b).
In the maturation zone, a number of OX6+ dendritic cells were distributed in the enamel organ. Most were also positive for the anti-cystatin C antibody in double labeled specimens (Fig 4b, Fig 4c, Fig 4e, Fig 4f, Fig 4h, and Fig 4i), although some were singly positive. Double positive cells were present until the late maturation zone. Double labeling of the enamel organ in the maturation zone with anti-cystatin C and ED1 showed that two different antigens were co-localized (Fig 5a and Fig 5b). The anti-cystatin C antibody labeled endoplasmic reticulum and some unknown vesicles of non-epithelial cells in the enamel organ (Fig 6c and Fig 6d). Control sections without anti-cystatin C showed no specific labeling (Fig 5c, Fig 5d, and Fig 6e).
Using double labeled sections with anti-cystatin C and OX6, the number of positive cells per unit length of the enamel organ along the longitudinal axis of the incisor was counted. Based on a comparison between the secretion zone and the transition and maturation zones, anti-cystatin C-positive cells increased moderately in the more advanced zone (p<0.01, Student's t-test), whereas OX6+ cells were rarely seen in the secretion zone but drastically increased in the transition and maturation zones (p<0.001) (Fig 7).
|
Anti-cystatin C Labels Ameloblasts, Osteocytes, and Osteoclasts
Anti-cystatin C+ dots were observed in the supranuclear region of ameloblasts in the late secretion to early maturation zones but not in the late maturation zone (Fig 3 and Fig 4). The organelles labeled were multivesicular bodies (Fig 8). In alveolar bone covering an incisor, older osteocytes and osteoclasts were positive for anti-cystatin C, but osteoblasts and newly formed osteocytes were only faintly labeled (Fig 9). Some unknown vesicles in the osteocytes were labeled by anti-cystatin C in electron microscopy (Fig 9d). Osteoclasts were double labeled with anti-cystatin C and ED1, but osteocytes and osteoblasts were not labeled by ED1 (Fig 9e9h).
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Dendritic cells are the most professional and specialized antigen-presenting cells. They are distributed in a variety of tissues in vivo, stimulate quiescent, naive, and memory B- and T-lymphocytes and induce tolerance by deletion of self-reactive thymocytes and anergy of mature T-cells (
Mature dendritic cells are stellate cells with long spiny or sheet-like extensions and exhibit high surface MHC Class II molecules and low endocytic activity, whereas immature dendritic cells exhibit high intracellular MHC Class II molecules and high endocytic activity in vitro (
Dendritic cells undergo maturation via antigen peptide loading on MHC Class II molecules in specialized MHC Class II-rich compartments, and present the complexes on the cell surface. To form antigenMHC Class II complexes, both antigen and pro-formed MHC Class II must be cleaved by lysosomal proteases, a candidate for which is cathepsin S (
In this study, dendritic cells in the enamel organ of the secretion, transition, and maturation zones were examined by using an anti-cystatin C antibody and an OX6 antibody directed to MHC Class II antigen.
It has been shown that during amelogenesis there are "macrophages" or macrophage-like cells in the enamel organ of secretion, transition, and maturation zones (
To determine whether or not these dendritic cells are a mature form, anti-cystatin C and OX6 double labeling was performed. Anti-cystatin C-positive cells were observed in the enamel organs of the secretion, transition, and maturation zones by light microscopic immunocytochemistry. Unexpectedly, in the enamel organ of the secretion zone anti-cystatin C-positive cells were present and were negative for OX6 antibody, whereas in the enamel organs of the transition and maturation zones some of the anti-cystatin C-positive cells were also double positive for OX6 antibody. Double positive cells in the transition and maturation zones may be maturing dendritic cells instead of fully matured cells, because matured Langerhans cells in epithelia have mostly lost cystatin C antigenicity (
On the basis of conventional electron microscopy and anti-cystatin C immunoelectron microscopy, anti-cystatin C+ cells in the secretion zone also appear to be immature dendritic cells that do not yet express cell surface MHC Class II antigens. They do not contain desmosomes or tonofilaments, thus demonstrating their non-epithelial origin. Double labeling of the enamel organ with anti-cystatin C and anti-desmoplakin 1 and 2 antibodies for desmosomes confirmed that the anticystatin C+ cells were of non-epithelial origin. In anti-cystatin C immunoelectron microscopy, the organelles labeled were the nuclear membrane, the endoplasmic reticulum, and some vesicles. These vesicles should be phagolysosomes, because ED1 co-localized with anti-cystatin C (
During enamel maturation, a considerable number of ameloblasts are lost, as well as the transition zone (
An unexpected result was the presence of anti-cystatin C+ vesicles in the ameloblasts. These vesicles appear to be multivesicular bodies. Considering that ameloblasts develop a lysosomal system during the period of secretion to the maturation zone and therefore exhibit cathepsin B, one of the cysteine proteases (
In conclusion, immature dendritic cells positive for anti-cystatin C may already be present in the enamel organ of the secretion zone, may increase in number in the transition and maturation zones, and may also begin to express MHC Class II antigens at their plasma membranes. Some immunological responses may be strongly related to the duration of amelogenesis, and those remain to be clarified.
![]() |
Acknowledgments |
---|
Supported in part by a Grant-in-Aid for scientific research (no. 11671824 to SN) from the Ministry of Education, Science, Sports and Culture of Japan.
Received for publication December 20, 1999; accepted March 15, 2000.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Albert ML, Sauter B, Bhardwaj N (1998) Dendritic cells acquire antigen from apoptotic cells and induce class I-restricted CTLs. Nature 392:86-89[Medline]
Al Kawas S, Amizuka N, Bergeron JJM, Warshawsky H (1996) Immunolocalization of the cation-independent mannose 6-phosphate receptor and cathepsin B in the enamel organ and alveolar bone of the rat incisor. Calcif Tissue Int 59:192-199[Medline]
Banchereau J, Steinman RM (1998) Dendritic cells and the control of immunity. Nature 392:245-252[Medline]
Brenan M, Rees DJG (1997) Sequence analysis of rat integrin alpha E1 and alpha E2 subunits: tissue expression reveals plenotypic similarities between intraepithelial lymphocytes and dendritic cells in lymph. Eur J Immunol 27:3070-3079[Medline]
Chapman HA, Jr (1991) Role of enzyme receptors and inhibitors in regulating proteolytic activities of macrophages. Ann NY Acad Sci 624:87-96[Abstract]
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]
Jessen H, Moe H (1972) The fine structure of macrophages in the enamel organ, with special reference to the microtubular system. Z Zellforsch 126:466-482[Medline]
Josephsen K, Fejerskov O (1977) Ameloblast modulation in the maturation zone of the rat incisor enamel organ. A light and electron microscopic study. J Anat 124:45-70[Medline]
Kakegawa H, Nikawa T, Tagami K, Kaioka H, Sumitani K, Kawata T, DrobnicKosorok M, Lenarcic B, Turk V, Katunuma N (1993) Participation of cathepsin L in bone resorption. FEBS Letts 321:247-250[Medline]
Kallenbach E (1970) Fine structure of rat incisor enamel organ during late pigmentation and regression stages. J Ultrastruct Res 30:38-63[Medline]
Kallenbach E (1978) Fine structure of the stratum intermedium, stellate reticulum, and outer enamel epithelium in the enamel organ of the kitten. J Anat 126:247-260[Medline]
Lerner UH, Johansson L, Ransjö M, Rosenquist JB, Reinholt FP, Grubb A (1997) Cystatin C, an inhibitor of bone resorption produced by osteoblasts. Acta Physiol Scand 161:81-92[Medline]
Matsuno K, Ezaki T, Kudo S, Uehara Y (1996) A life stage of particle-laden rat dendritic cells in vivo: their terminal division, active phagocytosis, and translocation from the liver to the draining lymph. J Exp Med 183:1865-1878[Abstract]
Mellman I, Turley SJ, Steinman RM (1998) Antigen processing for amateurs and professionals. Trends Cell Biol 8:231-237[Medline]
Nishikawa S, Fujiwara K, Kitamura H (1988) Formation of the tooth enamel rod pattern and the cytoskeletal organization in secretory ameloblasts of the rat incisor. Eur J Cell Biol 47:222-232[Medline]
Nishikawa S, Josephsen K (1987) Cyclic localization of actin and its relationship to junctional complexes in maturation ameloblasts of the rat incisor. Anat Rec 219:21-31[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[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 (1998) Localization of 2,2,7-trimethylguanosine, SC-35 and TIAR in ameloblast nuclei of the rat incisor, and their disappearance during apoptosis. Acta Histochem Cytochem 31:95-104
Nishikawa S, Sasaki F (1999a) Internalization of amelogenin by dendritic cells of the papillary layer during transition and early maturation stages. Histochem Cell Biol 112:301-305[Medline]
Nishikawa S, Sasaki F (1999b) Apoptosis of dental pulp cells and their elimination by macrophages and MHC class II-expressing dendritic cells. J Histochem Cytochem 47:303-311
Pierre P, Mellman I (1998) Developmental regulation of invariant chain proteolysis controls MHC class II trafficking in mouse dendritic cells. Cell 93:1135-1145[Medline]
Rubartelli A, Poggi A, Zocchi MR (1997) The selective engulfment of apoptotic bodies by dendritic cells is mediated by the vß3 integrin and requires intracellular and extracellular calcium. Eur J Immunol 27:1893-1900[Medline]
Sasaki T, Garant PR (1986) Fate of annular gap junctions in the papillary cells of the enamel organ in the rat incisor. Cell Tissue Res 246:523-530[Medline]
Sato T, Yamamoto H, Sasaki C, Wake K (1998) Maturation of rat dendritic cells during intrahepatic translocation evaluated using monoclonal antibodies and electron microscopy. Cell Tissue Res 294:503-514[Medline]
Shortman K, Maraskovsky E (1998) Developmental options. Science 282:424-425
Smith CE (1979) Ameloblasts: secretory and resorptive functions. J Dent Res 58(suppl B):695-706[Medline]
Smith CE, Warshawsky H (1977) Quantitative analysis of cell turnover in the enamel organ of the rat incisor. Evidence for ameloblast death immediately after enamel matrix secretion. Anat Rec 187:63-98[Medline]
Takano Y, Kawahara I, Hoshino M, Takeuchi K, Maeda T, Ohshima H, Hanaizumi Y, Kawano Y (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[Medline]
Tezuka K, Tezuka Y, Maejima A, Sato T, Nemoto K, Kamioka H, Hakeda Y, Kumegawa M (1994) Molecular cloning of a possible cysteine proteinase predominantly expressed in osteoclasts. J Biol Chem 269:1106-1109