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Immunohistochemical detection of prostaglandin I2 synthase in various calcified tissue-forming cells in rat
0003-9969/93 $6.00 + 0.00 Copyright0 1993 PergamonPressLtd
Archs oral Bid. Vol. 38, No. 1, pp. 31-36, 1993 Printedin Great Britain.All rightsreserved
IMMUNOHISTOCHEMICAL DETECTION OF PROSTAGLANDIN I, SYNTHASE IN VARIOUS CALCIFIED TISSUE-FORMING CELLS IN RAT T.
OKIJI,‘-* I. MORITA,~ N. KAWASHIMA,’
T. KOSAKA,’ H. SUDA’
and S.
MUROTA’
‘Department of Endodontics and *Section of Physiological Chemistry, Faculty of Dentistry, Medical and Dental University, 5-45, Yushima I-chome, Bunkyo-ku, Tokyo 113, Japan (Accepted
I8 August
Tokyo
1992)
Summary-Localization of prostaglandin (PC) I, synthase immunoreactivity was examined in demineralized sections of rat pulpal, periodontal and skeletal tissues using isn-1, a monoclonal antibody raised against the enzyme. Various calcified tissue-forming cells, i.e. odontoblasts, osteoblasts, osteocytes, cementoblasts, cementocytes and chondrocytes, were similarly immunoreactive for PGI, synthase, suggesting that they are capable of producing PGI,. In odontoblasts and chondrocytes, the reactivity increased gradually with maturation. Weak immunoreactivity was also observed in endothelial cells and fibroblast-like cells in pulpal and periodontal tissues. However, no reactivity was seen in ameloblasts.
These results suggest the possible involvement of PGI, in the regulation of the metabolism of various calcified tissues. Monoclonal antibodies such as isn-1 may become useful markers of the maturation of calcified
tissue-forming
Key words:
prostaglandin
cells of mesenchymal I, synthase,
origin.
odontoblast,
osteoblast,
INTRODUCUON
calcified
tissue-forming
cells.
munoreactivity for PGI, synthase was distributed, as expected, in endothelial and fibroblast-like cells. Moreover, many reaction products were found within odontoblasts, which was the most unexpected but interesting finding (Okiji et al., 1992). This stimulated a detailed examination on the localization of the immunoreactivity for PGI, synthase in other kinds of calcified tissue-forming cells, and we have now studied the immunohistochemical distribution of PGI, synthase in various kinds of dental and skeletal tissues of rat.
Prostaglandin I, is one of the biologically active metabolites of arachidonic acid produced via the cycle-oxygenase pathway. It is widely accepted that PGI, has several unique and important roles in the regulation of various physiological and pathological phenomena. In the vascular system PGI, is generated by endothelial and smooth-muscle cells, where it is considered an important endogenous substance, essential for maintaining the homeostasis of the cardiovascular system by acting as a potent vasodilator and inhibitor of platelet aggregation (Moncada and Vane, 1979). Prostaglandin I, also modulates acute inflammation because it can potentiate the increase in vascular permeability by increasing local blood flow (Williams, 1979). Recently, synthesis of PGI, in dental pulp has been reported; in normal rat incisor pulp, PGI, was the major cycle-oxygenase metabolite of arachidonic acid (Hirafuji and Ogura, 1983; Okiji et al., 1987, 1989). However, dental pulp is composed of several kinds of functionally heterogeneous cells and it is unclear what types of cells are actually responsible for the PGI, production. Thus the physiological role of PGI, in the pulp remains uncertain. We earlier examined the immunohistochemical localization of PGI, synthase, an enzyme catalysing the conversion of PGH, to PG12, in extirpated rat upper incisor pulp in order to determine the cellular sites of PGI, biosynthesis and to provide further insights into the function of PG12 in the pulp. Im-
MATERIALS AND METHODS
Tissue preparation Male Wistar rats 1, 4, 6,9, 14, 28 and 56 weeks old (n = 6, 3, 8, 2, 4, 3 and 3, respectively) were used. Upper and lower incisors, lower molars, the mandible, femur and calvaria were investigated. After decapitation under light ether anaesthesia, tissue specimens were removed and fixed immediately with PLP at 4°C for 8 h. They were then demineralized with 14% EDTA containing 15% glycerol at -4°C and embedded in 20% polyethylene glycol 20000 (Wako Pure Chemical, Osaka, Japan) according to the method of Mori et al. (1988). Cryostat sections 7 pm thick were then cut and stained immunohistochemically. Sections of rat aorta, processed in the same way, served as positive controls. Immunohistochemistry Sections were first incubated with isn-1, a mouse monoclonal antibody against PGI, synthase (tissueculture supernatant containing at least 20pgg/ml of mouse IgG, ; purchased from Cayman Chemical, Ann Arbor, MI, U.S.A.), undiluted or diluted up to l/l00
*To whom all correspondence should be addressed. Abbreviations: PLP, periodate-lysine-paraformaldehyde; PC, prostaglandin. 31
T. OKIJI et al.
32
in 0.05 M tris-HCl buffer (pH 7.6), at 4°C overnight. Details of the antibody are described by Dewitt and Smith (1982). A second incubation was made with a biotinylated anti-mouse IgG (rat adsorbed, Vector Laboratories, Burlingame, CA, U.S.A.), diluted l/200, for 30min at 24°C. Endogenous peroxidase activity was then blocked by incubating sections with 3% H,O, for 5 min, and the sections were further incubated with preformed avidin-biotin peroxidase complex (Elite ABC, Vector Laboratories) for 30 min at 24°C. These incubations were followed by at least three washes in tris-HCI buffer for 15-30 min. Finally, the peroxidase activity was developed by incubating sections with 0.5 mg/ml of 3,3’-diaminobenzidine.4HCl in tris-HC1 buffer containing 0.01% H20, for 5-10 min. Slides were counterstained with methyl green. As negativestaining controls, sections were incubated in trisHCI buffer instead of the primary or secondary antibody, diaminobenzidine alone was applied, and the primary antibody was replaced with six kinds of unrelated mouse IgG, monoclonal antibodies (e.g. W3/13, anti-T lymphocytes and EDl, anti-macrophages), diluted to almost the same IgG, content as isn-1. At least the omission and the replacement of the primary antibody were provided in every set of stainings.
blasts, osteocytes, cementoblasts and cementocytes, similarly showed an intense immunoreactivity. Fibroblast-like cells, which are mostly located close to the alveolar bone, and endothelial cells in the periodontal ligament, also reacted weakly. The distribution of the immunoreactivity in the periapical tissue was not affected by the animal’s age. We also examined the distribution of PGI, synthase in some skeletal tissues. Osteoblasts and osteocytes in all bones tested were similarly labelled with isn-I. Figure 5 shows the immunostaining of the mandibular condyle of a 6-week-old rat. In addition to osteoblasts in the osteogenic zone, chondrocytes were immunoreactive for isn-1 , and their immunoreactivity increased with maturation, with cells in the hypertrophic zone showing the most intense reactivity. Similar results were obtained in the epiphysis of the femur. The immunoreactivity of above mentioned skeletal cells was not affected by the animal’s age as far as tested.
RESULTS
In negative controls, eosinophils, mast cells and some of bone marrow cells were stained non-specifically. However, there were few of these cells in pulpal, periodontal and skeletal tissues and they were easily identified morphologically. None of the antibodies used in place of isn- 1 recognized cells immunoreactive for isn- 1. In sections of rat aorta used as positive controls, endothelial and smooth-muscle cells were positively stained, as reported by Smith, Dewitt and Allen (1983). An intensely positive immunoreaction for PGI, synthase was found in the cytoplasm of odontoblasts in molar pulp (Fig. 1). As shown in Fig. 2(a), the intensity of staining gradually increased with the maturation of young odontoblasts located at the end of the forming root. However, there was no visible reaction in ameloblasts, even when they became mature [Fig. 2(b)]. Similar results were obtained in incisor pulp (data not shown). In rats of 28 and 56 weeks old, denticles were usually formed in the coronal pulp chamber of the molars. In addition to odontoblasts, osteodentinoblast-like cells surrounding the denticle also showed an intense immunoreactivity (Fig. 3). Fibroblast-like cells in the central portion of the pulp, and venular and capillary endothelial cells, were also immunoreactive with PGI, synthase (Fig. 1). The reactivity was almost similarly distributed irrespective of the animal’s age as far as tested. However, this immunoreactivity was weaker than that of odontoblasts and was somewhat difficult to detect when the primary antibody was diluted more than l/10. The distribution of PGI, synthase immunoreactivity in the apical periodontal tissues is shown in Fig. 4. Various calcified tissue-forming cells, namely osteo-
Fig. 1. Lower first molar pulp of 6-week-old rat stained with isn-I (undiluted). (a) Low-power view. Magnification x 67; bar = 200pm. (b) Higher-power view of the box showing the immunoreaction of odontoblasts (0) and capillary endothelial cells (arrowheads). Magnification x 420; bar = 20 pm.
Localization of PGI, synthase DISCUSSION
We earlier examined the distribution of PGI, synthase immunoreactivity using extirpated, acetonefixed rat incisor pulps (Okiji er al., 1992). Here PLP-fixed and demineralized tissue specimens were successfully stained with isn-1 and thus the localization of the reactivity could be demonstrated while preserving the architecture of surrounding calcified tissues. We show that PGI, synthase immunoreactivity is distributed in various calcified tissue-forming cells,
33
except for ameloblasts, indicating that they are capable of producing PGI,. Moreover, the immunoreactivity for isn-1 increased with the maturation of odontoblasts [Fig. 2(a)] and chondrocytes (Fig. 5). Thus we assume that, under appropriate conditions, isn-1 may be useful as a histochemical marker of the maturation of calcified tissue-forming cells of mesenchymal origin. The presence of PGI,-synthesizing activity in osteoblasts has been shown before. Raisz et al. (1979) reported that rat bone can metabolize arachidonic acid, mainly into PGE, and PGI, Nolan et al. (1983)
Fig. 2. Lower second molar of l-week-old rat stained with isn-I (diluted l/10). (a) The end of the forming root. YO, Young odontoblasts; MO, mature odontoblasts; A, ameloblasts; ED, epithelial diaphragm; AB, alveolar bone; arrow, non-specifically stained mast cells. Magnification x 130; bar = 100 pm. Immunoreactivity of immature odontoblasts gradually increases with maturation. (b) Coronal part of the crown. Magnification x 130; bar = 100 pm. Odontoblasts (0) show an intense immunoreactivity whereas mature ameloblasts (A) show no reaction.
34
Fig. 3. Coronal pulp chamber of the lower first molar of 56-week-old rat stained with isn-1 (diluted I/IO). Osteodentinoblast-like cells (arrowheads) surrounding the denticle (D) show intense immunoreactivity. Magnification x 200; bar = 50 pm.
showed that the major cycle-oxygenase metabolite of arachidonic acid produced from cultured rat osteoblasts is PGI,. Morita, Toriyama and Murota (1989) demonstrated that the major cycle-oxygenase product produced by MC3T3-El, a cloned mouse osteoblastic cell line, was PGE,, followed by PGI,. Moreover, we have found that, in a subclone of MC3T3-El, PGI, synthesizing activity is higher than that of PGE, (unpublished data). There have been substantial observations supporting the involvement of arachidonic acid metabolites, especially PGE,, in the regulation of bone metabolism. Many investigators have suggested that PGE, may regulate the interaction between osteoblasts and osteoclasts during various phases of bone remodelling. Prostaglandin E, reportedly stimulates bone resorption (Klein and Raisz, 1970; Raisz et al., 1979). There are recent reports that PGE, released from osteoblasts may affect osteoclast progenitors and promote their differentiation into osteoclasts by a mechanism involving CAMP production (Morita et al., 1989; Akatsu et al., 1989). On the other hand, PGE, is also known to have stimulatory effects on
bone formation (Chambers and Ali, 1983; Chyun and Raisz, 1984). Recently, Toriyama, Morita and Murota (1992) have demonstrated two classes of PGE, receptors, one that couples to adenylate cyclase system and another that couples to phospholipase C system, in MC3T3-El cells, indicating the possibility that the former may mediate cell differentiation and the latter cell proliferation in osteoblastic cells. However, the effects of PGJ, on bone metabolism have not yet been studied extensively, presumably because of its rapid inactivation, which makes it difficult to estimate its potency. However, some investigators have reported that PGI, can also affect bone metabolism in a similar fashion to PGE,; PGI, stimulates bone resorption (Raisz et al., 1979) as well as inhibiting osteoclast motility (Chambers and Ali, 1983). PGI, is also known to activate adenylate cyclase and to increase CAMP concentration in osteoblasts (Partridge et al., 1981). From these findings, we assume that the effects of PGI, on bone metabolism may be almost similar to those of PGE,. There are limited findings on arachidonic acid metabolism in cells that synthesize calcified tissue
Fig. 4. Apical periodontal tissues of the mandibular first molar of 6-week-old rat stained with isn-I (undiluted). (a) Low-power view. Magnification x 110; bar = 100 pm. (b) Higher-power view showing the immunoreactivity of osteoblasts (large arrowheads), osteocytes (large arrows), cementoblasts (small arrowheads), cementocytes (small arrows) and fibroblast-like cells (open arrowheads). C, Cementum; PL, periodontal ligament; AB, alveolar bone; BM, bone marrow. Magnification x 210; bar = 50 pm.
35
Localization of PGI, synthase other than osteoblasts. However, some investigators have shown that PGE, and PGI, are the major metabolites produced by cultured chondrocytes (Malemud, Moskowitz and Hassid, 1981; Mitrovic, McCall and Dray, 1982). Our present findings on chondrocytes support those reports. It is possible that PGI, may play an important part in regulating the metabolism of dentine and cementurn. Saegusa (1988) reported that aspirin DL-lysine and sodium salicylate, which are known to inhibit cycle-oxygenase, can inhibit dentine formation in rat
incisor. Although he did not conclude that such effects are caused by the inhibition of cyclo-oxygenase activity, we assume that PGI,, the major cyclooxygenase product of normal pulp (Okiji et al., 1987) may possibly be involved in dentine formation. On the other hand, dentine and cementum are not normally resorbed and undergo continual deposition during life. Thus it is unclear whether PGI, regulates their metabolism in a similar fashion to its effect on bone, which undergoes constant remodelling. Acknowledgemem-This study was supported in part by a Grant-in-Aid for Scientific Research (No. 03304043) from the Ministry of Education, Science and Culture, Japan.
REFERENCES Akatsu T., Takahashi N., Debari K., Morita I., Murota S., Nagata N., Takatani 0. and Suda T. (1989) Prostaglandins promote osteoclast-like cell formation by a mechan-
Fig. 5. Mandibular condyle of 6-week-old rat stained with isn-I (diluted I/10). (a) Low-power view. P, Proliferative zone; M, mature zone; H, hypertrophic zone; 0, osteogenic zone; large arrows, hypertrophic chondrocytes; small arrows, mature chondrocytes; arrowheads, osteoblasts. Magnification x 170; bar = 50 pm. (b) Higher-power view showing mature zone (M) and hypertrophic zone (H). Magnification x 420; bar = 20 pm.
ism involving cyclic adenosine 3’, 5’-monophosphate in mouse bone marrow cell cultures. J. Bone Min. Res. 4, 29-35. Chambers T. J. and Ah N. N. (1983) Inhibition of osteoclastic motility by prostaglandins I,, E,, E, and 6-0x0-E,. J. Path. 139, 3X3-397. Chyun Y. S. and Raisz L. G. (1984) Stimulation of bone formation by prostaglandin E,. Prostaglandins 27, 97-103. Dewitt D. L. and Smith W. L. (1982) Monoclonal antibodies against PGI, synthase: an immunoradiometric assay for quantitating the enzyme. Meth. Enzym. 86, 24&246. Hirafuji M. and Ogura Y. (1983) Endogenous biosynthesis of prostaglandin I, and thromboxane A2 by isolated rat dental pulp. Biochem. Pharmac. 32, 29X3-2985. Klein D. C. and Raisz L. G. (1970) Prostaglandins: stimulation of bone resorption in tissue culture. Endocrinology 86, 14361440. Malemud C. J., Moskowitz R. W. and Hassid A. (1981) Prostaglandin biosynthesis by lapine articular chondrocytes in culture. Biochem. biophys. Acfa 663, 480490. Mitrovic D., McCall E. and Dray F. (1982) The in vitro production of prostanoids by cultured bovine articular chondrocytes. Prostaglandins 23, 17-2X. Moncada S. and Vane J. R. (1979) Pharmacology and endogenous roles of prostaglandin endoperoxides, thromboxane AZ and prostacyclin. Pharmac. Rev. 30, 293.-3 1I. Mori S., Sawai T., Teshima T. and Kyogoku M. (1988) A new decalcifying technique for immunohistochemical studies of calcified tissues, especially applicable to cell surface marker demonstration. J. Hktochem. Cyrochem. 36,111&114. Morita I., Toriyama K. and Murota S. (1989) Effects of prostaglandin E, on phenotype of osteoblast-like cells. Adv. PG TX LT Res. 19, 419422. Nolan R. D., Partridge N. C., Godfrey H. M. and Martin T. J. (1983) Cycle-oxygenase products of arachidonic acid metabolism in rat osteoblasts in culture. Calc. Tiss. In/. 35, 294-297. Okiji T., Morita I., Kobayashi C., Sunada I. and Murota S. (1987) Arachidonic-acid metabolism in normal and experimentally-inflamed rat dental pulp. Archs oral Biol. 32, 723-727. Okiji T., Morita I., Sunada I. and Murota S. (1989) Involvement of arachidonic acid metabohtes in increases in vascular permeability in experimental dental pulpal inflammation in the rat. Archs oral Biol. 34, 523-52X. Okiji T., Morita I., Suda H. and Murota S. (1991) Pathophysiological roles of arachidonic acid metabolites in rat dental pulp. Proc. Finn. dent. Sot. 88, (Suppl. I), 43343X.
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T. OKIJI et (II.
Partridge N. C., Kemp B. E., Veroni M. C. and Martin T. .l. (1981) Activation of adenosine 3’, 5’-monophosphate-dependent protein kinase in normal and malignant bone cells by parathyroid hormone, prostaglandin E, and prostacyclin. Endocrinology 108, 22G225. Raisz L. G., Vanderhoek J. Y., Simmons H. A., Kream B. E. and Nicolaou K. C. (1979) Prostaglandin synthesis by fetal rat bone in vitro: evidence for a role of prostacyclin. Prostuglundins 17, 905-914. Saegusa T. (1988) The effects of aspirin nL-lysine and sodium salicylate on dentine formation in the rat incisor. Jap. J. oral Biol. 30, 559-571.
Smith W. L., Dewitt D. L. and Allen M. L. (1983) Bimodal distribution of Prostaglandin I, synthase antigen in smooth muscle cells. J. biol. Chem. 258, 5922-5926. Toriyama K., Morita I. and Murota S. (1992) The existence of distinct classes of prostaglandin E, receptors mediating adenylate cyclase and phospholipase C pathways in osteoblastic clone MC3T3-El. Prostaglandins Leukofrienes Essenfial Fatty Acids 46, 15-20. Williams T. J. (1979) Prostaglandin E,, prostaglandin I, and the vascular change of inflammation. Br. J. Pharmac. 65, 5 1I-524.
Archs oral Bid. Vol. 38, No. 1, pp. 31-36, 1993 Printedin Great Britain.All rightsreserved
IMMUNOHISTOCHEMICAL DETECTION OF PROSTAGLANDIN I, SYNTHASE IN VARIOUS CALCIFIED TISSUE-FORMING CELLS IN RAT T.
OKIJI,‘-* I. MORITA,~ N. KAWASHIMA,’
T. KOSAKA,’ H. SUDA’
and S.
MUROTA’
‘Department of Endodontics and *Section of Physiological Chemistry, Faculty of Dentistry, Medical and Dental University, 5-45, Yushima I-chome, Bunkyo-ku, Tokyo 113, Japan (Accepted
I8 August
Tokyo
1992)
Summary-Localization of prostaglandin (PC) I, synthase immunoreactivity was examined in demineralized sections of rat pulpal, periodontal and skeletal tissues using isn-1, a monoclonal antibody raised against the enzyme. Various calcified tissue-forming cells, i.e. odontoblasts, osteoblasts, osteocytes, cementoblasts, cementocytes and chondrocytes, were similarly immunoreactive for PGI, synthase, suggesting that they are capable of producing PGI,. In odontoblasts and chondrocytes, the reactivity increased gradually with maturation. Weak immunoreactivity was also observed in endothelial cells and fibroblast-like cells in pulpal and periodontal tissues. However, no reactivity was seen in ameloblasts.
These results suggest the possible involvement of PGI, in the regulation of the metabolism of various calcified tissues. Monoclonal antibodies such as isn-1 may become useful markers of the maturation of calcified
tissue-forming
Key words:
prostaglandin
cells of mesenchymal I, synthase,
origin.
odontoblast,
osteoblast,
INTRODUCUON
calcified
tissue-forming
cells.
munoreactivity for PGI, synthase was distributed, as expected, in endothelial and fibroblast-like cells. Moreover, many reaction products were found within odontoblasts, which was the most unexpected but interesting finding (Okiji et al., 1992). This stimulated a detailed examination on the localization of the immunoreactivity for PGI, synthase in other kinds of calcified tissue-forming cells, and we have now studied the immunohistochemical distribution of PGI, synthase in various kinds of dental and skeletal tissues of rat.
Prostaglandin I, is one of the biologically active metabolites of arachidonic acid produced via the cycle-oxygenase pathway. It is widely accepted that PGI, has several unique and important roles in the regulation of various physiological and pathological phenomena. In the vascular system PGI, is generated by endothelial and smooth-muscle cells, where it is considered an important endogenous substance, essential for maintaining the homeostasis of the cardiovascular system by acting as a potent vasodilator and inhibitor of platelet aggregation (Moncada and Vane, 1979). Prostaglandin I, also modulates acute inflammation because it can potentiate the increase in vascular permeability by increasing local blood flow (Williams, 1979). Recently, synthesis of PGI, in dental pulp has been reported; in normal rat incisor pulp, PGI, was the major cycle-oxygenase metabolite of arachidonic acid (Hirafuji and Ogura, 1983; Okiji et al., 1987, 1989). However, dental pulp is composed of several kinds of functionally heterogeneous cells and it is unclear what types of cells are actually responsible for the PGI, production. Thus the physiological role of PGI, in the pulp remains uncertain. We earlier examined the immunohistochemical localization of PGI, synthase, an enzyme catalysing the conversion of PGH, to PG12, in extirpated rat upper incisor pulp in order to determine the cellular sites of PGI, biosynthesis and to provide further insights into the function of PG12 in the pulp. Im-
MATERIALS AND METHODS
Tissue preparation Male Wistar rats 1, 4, 6,9, 14, 28 and 56 weeks old (n = 6, 3, 8, 2, 4, 3 and 3, respectively) were used. Upper and lower incisors, lower molars, the mandible, femur and calvaria were investigated. After decapitation under light ether anaesthesia, tissue specimens were removed and fixed immediately with PLP at 4°C for 8 h. They were then demineralized with 14% EDTA containing 15% glycerol at -4°C and embedded in 20% polyethylene glycol 20000 (Wako Pure Chemical, Osaka, Japan) according to the method of Mori et al. (1988). Cryostat sections 7 pm thick were then cut and stained immunohistochemically. Sections of rat aorta, processed in the same way, served as positive controls. Immunohistochemistry Sections were first incubated with isn-1, a mouse monoclonal antibody against PGI, synthase (tissueculture supernatant containing at least 20pgg/ml of mouse IgG, ; purchased from Cayman Chemical, Ann Arbor, MI, U.S.A.), undiluted or diluted up to l/l00
*To whom all correspondence should be addressed. Abbreviations: PLP, periodate-lysine-paraformaldehyde; PC, prostaglandin. 31
T. OKIJI et al.
32
in 0.05 M tris-HCl buffer (pH 7.6), at 4°C overnight. Details of the antibody are described by Dewitt and Smith (1982). A second incubation was made with a biotinylated anti-mouse IgG (rat adsorbed, Vector Laboratories, Burlingame, CA, U.S.A.), diluted l/200, for 30min at 24°C. Endogenous peroxidase activity was then blocked by incubating sections with 3% H,O, for 5 min, and the sections were further incubated with preformed avidin-biotin peroxidase complex (Elite ABC, Vector Laboratories) for 30 min at 24°C. These incubations were followed by at least three washes in tris-HCI buffer for 15-30 min. Finally, the peroxidase activity was developed by incubating sections with 0.5 mg/ml of 3,3’-diaminobenzidine.4HCl in tris-HC1 buffer containing 0.01% H20, for 5-10 min. Slides were counterstained with methyl green. As negativestaining controls, sections were incubated in trisHCI buffer instead of the primary or secondary antibody, diaminobenzidine alone was applied, and the primary antibody was replaced with six kinds of unrelated mouse IgG, monoclonal antibodies (e.g. W3/13, anti-T lymphocytes and EDl, anti-macrophages), diluted to almost the same IgG, content as isn-1. At least the omission and the replacement of the primary antibody were provided in every set of stainings.
blasts, osteocytes, cementoblasts and cementocytes, similarly showed an intense immunoreactivity. Fibroblast-like cells, which are mostly located close to the alveolar bone, and endothelial cells in the periodontal ligament, also reacted weakly. The distribution of the immunoreactivity in the periapical tissue was not affected by the animal’s age. We also examined the distribution of PGI, synthase in some skeletal tissues. Osteoblasts and osteocytes in all bones tested were similarly labelled with isn-I. Figure 5 shows the immunostaining of the mandibular condyle of a 6-week-old rat. In addition to osteoblasts in the osteogenic zone, chondrocytes were immunoreactive for isn-1 , and their immunoreactivity increased with maturation, with cells in the hypertrophic zone showing the most intense reactivity. Similar results were obtained in the epiphysis of the femur. The immunoreactivity of above mentioned skeletal cells was not affected by the animal’s age as far as tested.
RESULTS
In negative controls, eosinophils, mast cells and some of bone marrow cells were stained non-specifically. However, there were few of these cells in pulpal, periodontal and skeletal tissues and they were easily identified morphologically. None of the antibodies used in place of isn- 1 recognized cells immunoreactive for isn- 1. In sections of rat aorta used as positive controls, endothelial and smooth-muscle cells were positively stained, as reported by Smith, Dewitt and Allen (1983). An intensely positive immunoreaction for PGI, synthase was found in the cytoplasm of odontoblasts in molar pulp (Fig. 1). As shown in Fig. 2(a), the intensity of staining gradually increased with the maturation of young odontoblasts located at the end of the forming root. However, there was no visible reaction in ameloblasts, even when they became mature [Fig. 2(b)]. Similar results were obtained in incisor pulp (data not shown). In rats of 28 and 56 weeks old, denticles were usually formed in the coronal pulp chamber of the molars. In addition to odontoblasts, osteodentinoblast-like cells surrounding the denticle also showed an intense immunoreactivity (Fig. 3). Fibroblast-like cells in the central portion of the pulp, and venular and capillary endothelial cells, were also immunoreactive with PGI, synthase (Fig. 1). The reactivity was almost similarly distributed irrespective of the animal’s age as far as tested. However, this immunoreactivity was weaker than that of odontoblasts and was somewhat difficult to detect when the primary antibody was diluted more than l/10. The distribution of PGI, synthase immunoreactivity in the apical periodontal tissues is shown in Fig. 4. Various calcified tissue-forming cells, namely osteo-
Fig. 1. Lower first molar pulp of 6-week-old rat stained with isn-I (undiluted). (a) Low-power view. Magnification x 67; bar = 200pm. (b) Higher-power view of the box showing the immunoreaction of odontoblasts (0) and capillary endothelial cells (arrowheads). Magnification x 420; bar = 20 pm.
Localization of PGI, synthase DISCUSSION
We earlier examined the distribution of PGI, synthase immunoreactivity using extirpated, acetonefixed rat incisor pulps (Okiji er al., 1992). Here PLP-fixed and demineralized tissue specimens were successfully stained with isn-1 and thus the localization of the reactivity could be demonstrated while preserving the architecture of surrounding calcified tissues. We show that PGI, synthase immunoreactivity is distributed in various calcified tissue-forming cells,
33
except for ameloblasts, indicating that they are capable of producing PGI,. Moreover, the immunoreactivity for isn-1 increased with the maturation of odontoblasts [Fig. 2(a)] and chondrocytes (Fig. 5). Thus we assume that, under appropriate conditions, isn-1 may be useful as a histochemical marker of the maturation of calcified tissue-forming cells of mesenchymal origin. The presence of PGI,-synthesizing activity in osteoblasts has been shown before. Raisz et al. (1979) reported that rat bone can metabolize arachidonic acid, mainly into PGE, and PGI, Nolan et al. (1983)
Fig. 2. Lower second molar of l-week-old rat stained with isn-I (diluted l/10). (a) The end of the forming root. YO, Young odontoblasts; MO, mature odontoblasts; A, ameloblasts; ED, epithelial diaphragm; AB, alveolar bone; arrow, non-specifically stained mast cells. Magnification x 130; bar = 100 pm. Immunoreactivity of immature odontoblasts gradually increases with maturation. (b) Coronal part of the crown. Magnification x 130; bar = 100 pm. Odontoblasts (0) show an intense immunoreactivity whereas mature ameloblasts (A) show no reaction.
34
Fig. 3. Coronal pulp chamber of the lower first molar of 56-week-old rat stained with isn-1 (diluted I/IO). Osteodentinoblast-like cells (arrowheads) surrounding the denticle (D) show intense immunoreactivity. Magnification x 200; bar = 50 pm.
showed that the major cycle-oxygenase metabolite of arachidonic acid produced from cultured rat osteoblasts is PGI,. Morita, Toriyama and Murota (1989) demonstrated that the major cycle-oxygenase product produced by MC3T3-El, a cloned mouse osteoblastic cell line, was PGE,, followed by PGI,. Moreover, we have found that, in a subclone of MC3T3-El, PGI, synthesizing activity is higher than that of PGE, (unpublished data). There have been substantial observations supporting the involvement of arachidonic acid metabolites, especially PGE,, in the regulation of bone metabolism. Many investigators have suggested that PGE, may regulate the interaction between osteoblasts and osteoclasts during various phases of bone remodelling. Prostaglandin E, reportedly stimulates bone resorption (Klein and Raisz, 1970; Raisz et al., 1979). There are recent reports that PGE, released from osteoblasts may affect osteoclast progenitors and promote their differentiation into osteoclasts by a mechanism involving CAMP production (Morita et al., 1989; Akatsu et al., 1989). On the other hand, PGE, is also known to have stimulatory effects on
bone formation (Chambers and Ali, 1983; Chyun and Raisz, 1984). Recently, Toriyama, Morita and Murota (1992) have demonstrated two classes of PGE, receptors, one that couples to adenylate cyclase system and another that couples to phospholipase C system, in MC3T3-El cells, indicating the possibility that the former may mediate cell differentiation and the latter cell proliferation in osteoblastic cells. However, the effects of PGJ, on bone metabolism have not yet been studied extensively, presumably because of its rapid inactivation, which makes it difficult to estimate its potency. However, some investigators have reported that PGI, can also affect bone metabolism in a similar fashion to PGE,; PGI, stimulates bone resorption (Raisz et al., 1979) as well as inhibiting osteoclast motility (Chambers and Ali, 1983). PGI, is also known to activate adenylate cyclase and to increase CAMP concentration in osteoblasts (Partridge et al., 1981). From these findings, we assume that the effects of PGI, on bone metabolism may be almost similar to those of PGE,. There are limited findings on arachidonic acid metabolism in cells that synthesize calcified tissue
Fig. 4. Apical periodontal tissues of the mandibular first molar of 6-week-old rat stained with isn-I (undiluted). (a) Low-power view. Magnification x 110; bar = 100 pm. (b) Higher-power view showing the immunoreactivity of osteoblasts (large arrowheads), osteocytes (large arrows), cementoblasts (small arrowheads), cementocytes (small arrows) and fibroblast-like cells (open arrowheads). C, Cementum; PL, periodontal ligament; AB, alveolar bone; BM, bone marrow. Magnification x 210; bar = 50 pm.
35
Localization of PGI, synthase other than osteoblasts. However, some investigators have shown that PGE, and PGI, are the major metabolites produced by cultured chondrocytes (Malemud, Moskowitz and Hassid, 1981; Mitrovic, McCall and Dray, 1982). Our present findings on chondrocytes support those reports. It is possible that PGI, may play an important part in regulating the metabolism of dentine and cementurn. Saegusa (1988) reported that aspirin DL-lysine and sodium salicylate, which are known to inhibit cycle-oxygenase, can inhibit dentine formation in rat
incisor. Although he did not conclude that such effects are caused by the inhibition of cyclo-oxygenase activity, we assume that PGI,, the major cyclooxygenase product of normal pulp (Okiji et al., 1987) may possibly be involved in dentine formation. On the other hand, dentine and cementum are not normally resorbed and undergo continual deposition during life. Thus it is unclear whether PGI, regulates their metabolism in a similar fashion to its effect on bone, which undergoes constant remodelling. Acknowledgemem-This study was supported in part by a Grant-in-Aid for Scientific Research (No. 03304043) from the Ministry of Education, Science and Culture, Japan.
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Fig. 5. Mandibular condyle of 6-week-old rat stained with isn-I (diluted I/10). (a) Low-power view. P, Proliferative zone; M, mature zone; H, hypertrophic zone; 0, osteogenic zone; large arrows, hypertrophic chondrocytes; small arrows, mature chondrocytes; arrowheads, osteoblasts. Magnification x 170; bar = 50 pm. (b) Higher-power view showing mature zone (M) and hypertrophic zone (H). Magnification x 420; bar = 20 pm.
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