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Lymphatic involvement in the histopathogenesis of mucous retention cyst
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Pathology – Research and Practice 203 (2007) 89–97 www.elsevier.de/prp
ORIGINAL ARTICLE
Lymphatic involvement in the histopathogenesis of mucous retention cyst Sukalyan Kundua,b, Jun Chenga, Satoshi Maruyamaa, Makoto Suzukia,c, Hiroyuki Kawashimab, Takashi Sakua,c, a
Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan b Division of Cell Biology and Molecular Pharmacology, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan c Surgical Pathology Section, Niigata University Hospital, Niigata, Japan Received 8 August 2006; received in revised form 25 October 2006; accepted 3 November 2006
Abstract Mucous retention cyst results from extravasation of saliva. Our intent was to study the role of lymphatics in its pathogenesis. Twenty-three surgical specimens of mucous retention cyst of the lip were examined for involvement of lymphatic vessels by a comparative immunohistochemical demonstration of lymphatic and blood vascular endothelial cells, as well as lymphatic and salivary contents. Mucous retention cysts were histopathologically classified into three stages: early, intermediate, and advanced. In the early stage, there was diffuse extravasation of mucous material in the interstitium of the lamina propria or the submucosal layer of the oral mucosa. In the intermediate stage, lymphatics, which were clearly revealed and immunohistochemically distinguished from blood vessels by monoclonal antibody D2-40, were dilated and finally ruptured, leaving fragments of lymphatic walls in the periphery of mucous pools. In the advanced stage, thick cyst walls of granulation tissue were formed around mucous retention. Lymphatics were no longer involved in the granulation tissue wall, which was actively driven by blood vessel formation. The results suggest that the lymphatic rupture seems to contribute to the enlargement in the pathogenesis of mucous retention cyst. r 2006 Elsevier GmbH. All rights reserved. Keywords: Angiogenesis; D2-40; Immunohistochemistry; Lymphatics; Mucous retention cyst
Introduction Mucous retention cysts (extravasation type, mucoceles) are oral lesions most commonly diagnosed during Corresponding author. Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Niigata University, Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan. Tel.: +81 25 227 2832; fax: +81 25 227 0805. E-mail address: [email protected] (T. Saku).
0344-0338/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2006.11.003
routine surgical pathology service for dental practice [16,22,23]. Although mucous retention cysts are believed to be generated by extravasation of saliva into the extracellular space due to some traumatic damage of salivary ducts, their detailed histopathogenetic processes are poorly understood. The question remains as to how the mucous pool is confined to some areas or how the cyst becomes larger. In a previous study, we demonstrated how the mucous pool is organized by the capillary endothelial cell sprouting with tubule
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formation, and these endothelial cells were recruited from infiltrating monocytes in the extracellular milieu in which tenascin was enriched and produced by the endothelial progenitor cells themselves [24]. Mucous retention cysts of the oral mucosa are formed in the lamina propria and the submucosal layers, where lymphatics are abundantly distributed [9]. However, lymphatics in the pathogenesis of mucous retention cyst have not yet been investigated. This is partly due to the fact that no one has paid much attention to this and that a reliable method demonstrating lymphatic vessels in tissue specimens still needs to be designed. Recently, monoclonal antibody D2-40 has been introduced for immunohistochemical labeling of lymphatic endothelial cells [11]. In this study, we used this valuable antibody to demonstrate the fate of lymphatic vessels in the process of mucous retention cyst formation to better understand the lymphatic involvement in the initial stage of mucous retention cyst formation.
Materials and methods Materials We studied a total of 23 surgical samples of mucous retention cysts (mucoceles) diagnosed at the Division of Oral Pathology, Niigata University Graduate School of Medical and Dental Sciences, between 2004 and 2005. Surgical materials were fixed in 10% formalin solution and embedded in paraffin. Serial sections were cut at a thickness of 5 mm; one of the sets of serial sections was stained with hematoxylin and eosin (HE) and used for re-evaluation of the histologic diagnosis. The other sets were used for mucin histochemistry, such as periodic acid-Schiff (PAS), colloidal iron, or alcian blue (pH 2.5), and for immunohistochemical investigations.
sulfate proteoglycan (core protein), were raised in rabbits as described elsewhere [18].
Immunohistochemistry Immunohistochemical staining was performed using the Chemomate Envision/HRP system (DakoCytomation) as described previously [24]. For perlecan, the deparaffinized sections were pre-treated with 3 mg/ml hyaluronidase (bovine testicular origin, type I-S, 440 U/mg; Sigma Chemical Co., St. Louis, MO, USA) in phosphate buffered saline (PBS) for 30 min at 37 1C. For SC, type IV, and CD31, sections were pre-treated with 0.02% trypsin (type II, Sigma) in 0.5 M Tris–HCl (pH 7.6) containing 1% CaCl2 for 30 min at 37 1C. Prior to incubation with the primary antibodies, all the sections were treated with 0.3% H2O2 in absolute methanol for 30 min at room temperature to block endogenous peroxidase activities. For blocking non-specific protein binding, sections were incubated with 5% skimmed milk for 1 h at 37 1C after rinsing in PBS containing 0.5% Triton X-100 (T-PBS). They were then incubated overnight at 4 1C with the primary antibodies diluted in T-PBS. For visualization of reaction products, sections were treated with 0.02% 3, 30 -diaminobenzidine (DAB) in 0.05 M Tris–HCl buffer (pH 7.4) containing 0.005% H2O2. The sections were counterstained with hematoxylin. For control studies, the primary antibodies were replaced with pre-immune rabbit IgG or mouse IgG1. For double histochemical staining for mucin and lymphatic vessels, sections immunostained with the D2-40 antibody were visualized with DAB, thoroughly washed in water, and then stained for colloidal iron.
Results Histopathology
Antibodies A mouse monoclonal antibody D2-40 (D2-40, IgG1) was purchased from Nichirei Biosciences Inc. (Tokyo, Japan). Rabbit polyclonal antibodies against human IgA, secretory component (SC), and fibrin, as well as a mouse monoclonal antibody against human CD31 (JC70A, IgG1-k) were purchased from DakoCytomation (Copenhagen, Denmark). A mouse monoclonal antibody against human IgG (HP6025, IgG1-k) was obtained from Zymed Laboratories Inc. (South San Francisco, CA, USA). Rabbit polyclonal antibodies against collagen type IV (type IV) were obtained from ICN Biomedicals Inc. (Aurora, OH, USA). Antibodies against perlecan, a basement membrane-type heparan
Having examined the 23 tissue samples of mucous retention cyst histologically, we classified the mucous retention cysts into three different stages of their organizing process: early, intermediate, and advanced (Fig. 1). This classification was done depending on the mode of mucous material distribution and the granulation tissue wall formation separating the mucous pool from the surrounding tissue. In the early stage (Fig. 1(a)), mucous material was diffusely extravasated in the intercellular space of the lamina propria to the submucosal layer, causing the covering epithelium to be thinner and flatter, with the loss of rete ridges. Thus, the epithelium lost its regular shape, and the subepithelial zone showed an edematous appearance. In the peripheral zone of the edematous
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area, markedly dilated lumina of lymphatic vessels were often observed (Fig. 3(a), boxed area in Fig. 1(a)). In the intermediate stage (Fig. 1(b)), there were focal mucous pools containing a small number of macrophages and lymphocytes in the periphery. When such mucous pools were located beneath the mucosal epithelium, the covering epithelia were focally stretched and protruded to the surface. The mucous pools were clearly separated by immature granulation tissue rich in newly formed blood vessels and fine fibrils and denser infiltration of macrophages and neutrophils. Therefore, such less-cellular zones seemed to be related to the dilated lymphatics mentioned above, and it was
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suggested that some lymphatic contents or components may take part in the development of mucous retention cysts. In the advanced stage (Fig. 1(c)), a distinct granulation barrier was formed around the mucous pool as an isolated mass from the submucosal connective tissue. This tissue barrier could be considered a cyst wall. In the cystic lumen, there were many foamy macrophages floating into the background of eosinophilic amorphous material, which was colloidal-iron- (Figs. 4(a) and (b)), alcian-blue-, or PAS-positive (data not shown).
Immunohistochemistry Early stage In the early stage, amorphous eosinophilic materials in the edematous area (Fig. 2(a)) were positive for colloidal iron staining (Fig. 2(b)) or PAS and alcian blue (data not shown). The colloidal iron-positive amorphous materials were immunopositive for SC (Fig. 2(c)) and IgA (Fig. 2(d)) at the same time, indicating the retention of salivary contents in the area. On the other hand, immunopositivity for IgG (Fig. 2(e)) was also revealed, although the immunolocalization modes of IgA and IgG varied from area to area without any definite difference. Deeply eosinophilic strand-like deposits in the edematous area were immunopositive for fibrin (Fig. 2(f)). These deposits were also immunopositive for SC (Fig. 2(c)). This result indicated that the edematous appearance was due to salivary retention and tissue fluid from blood or lymphatic streams. The dilated lymphatic vessels in the periphery of the edematous area (Fig. 1(b), box) were clearly demonstrated by the D2-40 antibody. There was no immunopositivity with the same antibody in blood vascular endothelial cells (Fig. 3(b)). Lymphatic endothelial cells were faintly immunopositive for CD31 (Fig. 3(c)), and Fig. 1. Histopathologic appearance of mucous retention cyst in three stages: early (a), intermediate (b), and advanced (c). Hematoxylin and eosin (HE) stain. a, 20 , b, 30 , c, 20 . The early stage (a) was characterized by diffuse infiltration of mucous material in the lamina propria to the submucosal layer. Fibrous components and blood vessels were separated by mucous material, which resulted in an edematous appearance. At the periphery of the mucous-retained area, there were pronouncedly dilated lumina of lymphatic vessels. In the intermediate stage (b), there were focally condensed mucous retention areas, which seemed to be due to ruptures of lymphatic vessels. Their separated and fragmented vessel walls were stretched and adhered to the lower surface of the overlying epithelium. The advanced stage (c) was distinguished from the above two stages by the presence of cyst walls composed of characteristic granulation tissue layer. The cystic lumen was isolated by the granulation tissue wall from the surrounding connective tissue. Boxed areas are shown at higher magnifications in Figs. 3–5.
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Fig. 2. Immunohistochemistry and color staining for mucous fluid in mucous retention cyst, stage del. HE stain (a), colloidal iron stain (b), and immunoperoxidase stains for secretory component (SC) (c), IgA (d), IgG (e), and fibrin (f). Hematoxylin counterstain, 200 . Abundance of mucous material detected by colloidal iron staining (b) in the mucous retention cyst. Strong SC- and IgAimmunopositive materials were diffusely demonstrated exclusively in the edematous area, indicating that salivary contents were major constituents of the mucous retention (c, d). At the same time, however, IgG (e) was immunolocalized extensively in the mucous-retained area. In addition, fibrin-immunopositive membranous materials (f) were observed within the edematous area. These results suggest that lymphatic or plasma components, in addition to the salivary contents, also take part in the formation of the initial edematous histology of mucous retention cyst.
their basement membranes were revealed by type IV immunopositivity (Fig. 3(d)), while these two antigens were very intensively immunolocalized in the blood capillaries or venules. This result indicated that the D2-40 antibody was a reliable tool for demonstrating lymphatics of various sizes. Moreover, the infiltration of mucous material into lymphatic lumina from the outer mucous retention was demonstrated by double staining
with colloidal iron and D2-40 antibody (Fig. 3(e)). The staining intensity of colloidal iron appeared to be more condensed within the lymphatic lumen, which was also immunohistochemically demonstrated for SC (Fig. 3(f)). This mucous infiltration into lymphatic lumina might be a passive transport, and the lymphatic luminal territory still seemed to be independent from the surrounding mucous retention at this stage.
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Fig. 3. Immunohistochemical demonstration of dilated lymphatic vessels in early stage of mucous retention cyst (high power view of the boxed area in Panel a, Fig. 1). HE stain (a); immunoperoxidase stains with the D2-40 antibody (b) and for CD31 (c), collagen type IV (d), and secretory component (f); and double staining with D2-40 and colloidal iron (e). Hematoxylin counterstain, 125 . The dilated vessels were considered to be lymphatics, because their endothelial cells were labeled with D2-40 (b) but were not positive for CD31 (c), which was definitely demonstrated in small blood vessels around the dilated lymphatic vessels. Collagen type IV (d) was immunolocalized in most of the vascular channels in the figure, although it was more intensely immunolabeled in blood vessels than in lymphatic ones, indicating that all of the blood vessels and larger lymphatic vessels were circumscribed by basement membranes whose primary constituent is collagen type IV. Colloidal iron-positive mucous material (e) as well as SC (f) were rather condensed within the dilated lymphatic vessels, indicating that salivary contents were absorbed into the lymphatics to be dilated.
Intermediate stage At the periphery of mucous retention (Fig. 1(b), box), there were lymphatic vessels or fragments that were open to mucous pools as revealed by colloidal-iron staining (Figs. 4(a) and (b)). The lymphatic vessels were clearly demonstrated to be immunopositive with the D2-40 antibody (Figs. 4(a) and (b), arrows). In the very
peripheral part of mucous retention, lymphatic wall fragments were often stretched toward the lamina propria connective tissue (Fig. 4(b), arrows). There were CD31- or type IV-immunopositive blood capillaries and venules within the mucous pool and in the narrow lamina propria (Figs. 4(c) and (d)). These results indicated that lymphatics were destroyed by mechanical
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Fig. 4. Immunohistochemical demonstration of ruptured lymphatic vessels in intermediate stage of mucous retention cyst. Double stain of immunoperoxidase with the D2-40 antibody and colloidal iron (a and b) and immunoperoxidase stains for CD31 (c) and collagen type IV (d). Hematoxylin counterstain, a, 60 , b–d, 150 . The D2-40 antibody revealed fragments of ruptured lymphatic vessel walls in the mucous background (a). D2-40-immunopositive lymphatic wall fragments were often pushed underneath the lamina propria (b). Under the fragmented lymphatic wall, lymphocytes and macrophages were floating in clear background substances. Neutrophils were scarcely observed in such areas. Small blood vessels around the ruptured lymphatics were definitely demonstrated by immunopositivity for collagen type IV (d) and CD31 (c).
stresses mostly due to the infiltration of extravasated salivary contents (Figs. 3(e) and (f)).
Advanced stage In the thick granulation tissue, presenting as a cyst wall in the advanced stage (Fig. 1(c), box), there were many blood capillaries and postcapillary venules sprouting towards the cystic lumen, which were demonstrated by immunohistochemistry for type IV (Fig. 5(a)). In addition to the basement membrane of capillary or venous endothelial cells, fine fibrillar structures in the intercellular space were also immunopositive for type IV (Fig. 5(b)) and perlecan (Fig. 5(c)). In addition to these endothelial cells, CD31 was immunolocalized in polygonal-shaped cells between blood vessels (Fig. 5(d)). There were no D2-40-immunopositive cells in the cyst wall of thick granulation tissue (data not shown). This result indicated that lymphatics were not involved in the granulation tissue reaction in the mucous retention cyst wall, but that blood vessels and blood endothelial cells or their progenitors instead took an initiative role in it.
Discussion This study is the first to demonstrate that lymphatic vessels are involved in the histopathogenetic process of mucous retention cysts, a finding that has never been mentioned in the literature. We could show that dilated vascular vessels, often observed in the early stage of the lesions, were lymphatic vessels. Later in the intermediate stage, when the extension of the lesion is almost fixed, fragments of ruptured lymphatics were located at the periphery of the lesion. Based on this observation, one might suggest that the dilatation of lymphatics caused by absorbing extravasated saliva plays some role in the enlargement of mucous retention. The involvement of lymphatics is restricted only to the early stages of the lesion, but not in the later stage, which is characterized by the thick granulation barrier around the cystic lumina. The most important tool in the present study was the D2-40 monoclonal antibody. M2A antigen, an oncofetal antigen, was used to raise the D2-40 monoclonal antibody [11], which has similar specificity to another
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Fig. 5. Immunohistochemistry for advanced stage of mucous retention cyst. Immunoperoxidase stains for collagen type IV (a and b), perlecan (c), and CD31 (d). Hematoxylin counterstain, a, 150 , b–d, 600 . The cyst wall in the advanced stage was composed of granulation tissue, which was rich in postcapillary venules demonstrated by immunopositivity for collagen type IV (a and b), perlecan (c), and CD31 (d). CD31 was immunolocalized in the cytoplasm of polygonal cells regarded as endothelial progenitor cells, in addition to blood vascular endothelial cells (d). There were fine mesh-like immunolocalizations for type IV (b) and perlecan (c) in the interstitium. In such a stage of granulation barrier formation around mucous pools, there was no longer participation of lymphatics.
previously described monoclonal antibody (M2A) [1]. This monoclonal antibody has been characterized as a more specific tool for the identification of lymphatic endothelial cells [30] than the other previously used lymphatic endothelium markers such as vascular endothelial growth factor (VEGF) receptor-3 [7] and lymphatic endothelial hyaluronan receptor (LYVE-1) [2]. Recently, another valuable lymphatic marker, podoplanin, a glomerular podocyte-specific molecule [3], first designated as E11 antigen of rat bone [26], has been shown to be also recognized by antibody D2-40 [19]. In addition to podocytes, podoplanin has also been demonstrated in the choroids plexus and ciliary epithelium in the rat brain and eye [27]. Moreover, the significant homology with podoplanin has been reported in other molecules such as gp40 in MDCK cells [31] and RTI40 in rat alveolar epithelial cells [5]. Most recently, Evangelou et al. showed that both rabbit polyclonal antibodies against podoplanin and the D2-40 were suitable for the detection of lymphatic vessels [4]. Therefore, in our study, introducing this antibody D2-40, we were able to demonstrate lymphatic
vessels in the tissue. In the past, when no valuable and stable lymphatic markers were available, we used BSA-I lectin-binding histochemistry [28], type IV collagen or laminin immunohistochemistry for the detection of basement membrane molecules [8] and CD31 or UEA-I lectin-binding immunohistochemistry for blood endothelium [14] and electron microscopy [15], thus excluding lymphatics from blood vessels, for circumstantial judgment. When compared with these indirect techniques, immunohistochemistry using the D2-40 has a remarkable advantage in histological recognition of lymphatics. The extravasation of saliva is believed to be the primary cause of the lesion [16,22,23]. However, an actual trial to immunohistochemically demonstrate salivary components in the muco-edematous stroma of the cyst has not been described in the literature. In the present study, in addition to the conventional histochemical demonstration of mucous materials by colloidal iron and other stainings, we used IgA and SC immunolocalizations to give evidence of salivary contents, because whole saliva specifically contains SC and
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IgA, whose level is higher than that of IgG [13]. In our series, the immunohistochemical localization modes for IgG and IgA/SC were less different from each other. Although it is difficult to give a quantitative discussion on the basis of immunohistochemistry, one might suggest that within the mucous pools, fibrin, which has not been recognized as a salivary content, may have its origin in blood. Therefore, a certain amount of such kind of free IgG may originate from lymphatic or blood streams. This idea may be reasonable because inflammatory reactions due to mucous extravasation could induce blood capillary permeability. The condensation of colloidal-iron-positive and SC-immunopositive materials within dilated lymphatics also indicated that extravasated salivary contents were absorbed into lymphatics together with serum exudates. It is now obvious that the histologic appearance of ‘‘mucous retention’’ in this lesion is not always due to salivary contents but also to exudates from blood vessels or ruptured lymphatic vessels. During the organization process of mucous retention cyst, mucous liberated into the connective tissue was scavenged by inflammatory cells, mainly neutrophils and macrophages. At the same time, the pressure gradient from the interstitium into the nonmuscular initial lymphatics (microlymphatics or capillary lymphatics) was large enough to cause the percolation of interstitial fluid [21]. In the case of mucous retention cyst, the interstitial fluid was shown to be a mixture of plasma and mucous, and it was condensed within the dilated lymphatics. Therefore, initial lymphatics might expand beyond their limit to rupture and deliver its content back again into the mucous pool. Finally, mucous pools were surrounded by a characteristic granulation barrier that, interestingly, was devoid of lymphatics. Recently, Schledzewski et al. [20] demonstrated that there were significant numbers of LYVE-1 (lymphatic endothelium-specific hyaluronan receptor)immunopositive macrophages in wound healing or tumor tissues. Although we did not use LYVE-1 antibodies for the investigation in this study, neither D2-40-immunopositive mature lymphatics nor macrophage-like singular cells were found in the granulation tissue of mucous retention cyst wall. Instead, this granulation tissue barrier contained an abundance of blood vessels, that were demonstrated by immunohistochemistry for type IV and CD31, perpendicularly arranged to the cystic lumen. Regarding the organization process of the cyst wall, collagen type IV and perlecan, basement membrane constituents, were shown to be rich in intercellular space between sprouting blood vessels. In the previous study, we demonstrated that tenascin functioned as a substrate for the differentiation of endothelial progenitor cells in the angiogenesis in mucous pools [24]. The present result suggests that type IV and perlecan also play roles similar to
tenascin. As shown previously, CD31-immunopositive polygonal-shaped cells were endothelial progenitor cells originated from infiltrating monocytes [24], which have also been shown to produce and utilize cytokines such as VEGF [25], fibroblast growth factor (FGF) [17], platelet derived growth factor (PDGF) [6], tenascin [29], collagen type IV [12], and fibronectin [10] in autocrine manners for their differentiation. In conclusion, this study shows that dilatation of lymphatics, caused by absorbing extravasated saliva, plays a role in the enlargement of mucous retention.
Acknowledgement This work was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science.
References [1] D. Bailey, R. Baumal, J. Law, et al., Production of a monoclonal antibody specific for seminomas and dysgerminomas, Proc. Natl. Acad. Sci. USA 83 (1986) 5291–5295. [2] S. Banerji, J. Ni, S.X. Wang, et al., LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan, J. Cell Biol. 144 (1999) 789–801. [3] S. Breiteneder-Geleff, K. Matsui, A. Soleiman, et al., Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis, Am. J. Pathol. 151 (1997) 1141–1152. [4] E. Evangelou, P.A. Kyzas, T.A. Trikalinos, Comparison of the accuracy of lymphatic endothelium markers: Bayesian approach, Mod. Pathol. 18 (2005) 1490–1497. [5] R.F. Gonzalez, L.G. Dobbs, Purification and analysis of RTI40, a type I alveolar epithelial cell apical membrane protein, Biochim. Biophys. Acta 1429 (1998) 208–216. [6] P. Guo, B. Hu, W. Gu, L. Cheng, et al., Platelet derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment, Am. J. Pathol. 162 (2003) 1083–1093. [7] A. Kaipainen, J. Korhonen, T. Mustonen, et al., Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development, Proc. Natl. Acad. Sci. USA 92 (1995) 3566–3570. [8] Y. Kobayashi, T. Nakajima, T. Saku, Loss of basement membranes in the invading front of O-1N, hamster squamous cell carcinoma with high potential of lymph node metastasis: an immunohistochemical study for laminin and type IV collagen, Pathol. Int. 45 (1995) 327–334. [9] M. Kotani, The lymphatics and lymphoreticular tissues in relation to the action of sex hormones, Arch. Histol. Cytol. Rev. 53 (Suppl.) (1990) 1–76. [10] B. Larivie`re, M. Rouleau, S. Picard, et al., Human plasma fibronectin potentiates the mitogenic activity of
ARTICLE IN PRESS S. Kundu et al. / Pathology – Research and Practice 203 (2007) 89–97
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
platelet-derived growth factor and complements its wound healing effects, Wound Repair. Regen. 11 (2003) 79–89. A. Marks, D.R. Sutherland, D. Bailey, et al., Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours, Br. J. Cancer 80 (1999) 569–578. A.G. Marneros, B.R. Olsen, The role of collagen-derived proteolytic fragments in angiogenesis, Matrix Biol. 20 (2001) 337–345. P.P. Mortimer, J.V. Parry, Detection of antibody to HIV in saliva: a brief review, Clin. Diagn. Virol. 2 (1994) 231–243. K. Niimi, M. Yoshizawa, T. Nakajima, T. Saku, Vascular invasion in squamous cell carcinomas of human oral mucosa, Oral Oncol. 37 (2001) 357–364. Y. Ono, T. Nakajima, T. Saku, Vascular invasion of O-1N, hamster squamous cell carcinoma with high potential of lymph node metastasis: ultrastructural comparison between lymphatics and blood vessels, Pathol. Int. 48 (1998) 254–264. J.A. Regezi, J.J. Sciubba, R.C.K. Jordan, Oral Pathology: Clinical Pathologic Correlations, fourth ed, W.B. Saunders, Philadelphia, 2003, pp. 183–187. S. Rogelj, M. Klagsbrun, R. Atzmon, et al., Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells, J. Cell Biol. 109 (1989) 823–831. T. Saku, H. Furthmayr, Characterization of the major heparan sulfate proteoglycan secreted by bovine aortic endothelial cells in culture: homology to the large molecular weight molecule of basement membranes, J. Biol. Chem. 264 (1989) 3514–3523. V. Schacht, S.S. Dadras, L.A. Johnson, et al., Upregulation of the lymphatic marker podoplanin, a mucintype transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors, Am. J. Pathol. 166 (2005) 913–921. K. Schledzewski, M. Falkowski, G. Moldenhauer, et al., Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD1 1b+macrophages in malignant tumours and wound
[21] [22]
[23] [24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
97
healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis, J. Pathol. 209 (1) (2006) 67–77. G.W. Schmid-Scho¨nbein, Microlymphatics and lymph flow, Physiol. Rev. 70 (1990) 987–1028. G. Seifert, A. Miehlke, J. Haubrich, R. Chilla, Diseases of the Salivary Glands, Georg Thieme Verlag, Stuttgart, 1986, pp. 91–98. M. Shear, Cysts of the Oral Regions, third ed, Wright, Oxford, UK, 1992, pp. 212–221. W. Swelam, H. Ida-Yonemochi, T. Saku, Angiogenesis in mucous retention cyst: a human in vivo-like model of endothelial cell differentiation in mucous substrate, J. Oral Pathol. Med. 34 (2005) 30–38. N.S. Taichman, A.T. Cruchley, L.M. Fletcher, et al., Vascular endothelial growth factor in normal human salivary glands and saliva: a possible role in the maintenance of mucosal homeostasis, Lab. Invest. 78 (1998) 869–875. A. Wetterwald, W. Hoffstetter, M.G. Cecchini, et al., Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes, Bone 18 (1996) 125–132. M.C. Williams, Y. Cao, A. Hinds, et al., T1 alpha protein is developmentally regulated and expressed by alveolar type I cells, choroids plexus, and ciliary epithelia of adult rats, Am. J. Respir. Cell Mol. Biol. 14 (1996) 577–585. M. Yoshizawa, S. Shingaki, T. Nakajima, T. Saku, Histopathological study of lymphatic invasion in squamous cell carcinoma (O-1N) with high potential of lymph node metastasis, Clin. Exp. Metastasis 12 (1994) 347–356. D. Zagzag, B. Shiff, G.I. Jallo, et al., Tenascin-C promotes microvascular cell migration and phosphorylation of focal adhesion kinase, Cancer Res. 62 (2002) 2660–2668. Y. Zeng, K. Opeskin, L.G. Horvath, et al., Lymphatic vessel density and lymph node metastasis in prostate cancer, Prostate 65 (2005) 222–230. G. Zimmer, F. Lottspeich, A. Maisner, et al., Molecular characterization of gp40, a mucin-type glycoprotein from the apical plasma membrane of Medin-Darby canine kidney cells (type I), Biochem. J. 326 (1997) 99–108.
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ORIGINAL ARTICLE
Lymphatic involvement in the histopathogenesis of mucous retention cyst Sukalyan Kundua,b, Jun Chenga, Satoshi Maruyamaa, Makoto Suzukia,c, Hiroyuki Kawashimab, Takashi Sakua,c, a
Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan b Division of Cell Biology and Molecular Pharmacology, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan c Surgical Pathology Section, Niigata University Hospital, Niigata, Japan Received 8 August 2006; received in revised form 25 October 2006; accepted 3 November 2006
Abstract Mucous retention cyst results from extravasation of saliva. Our intent was to study the role of lymphatics in its pathogenesis. Twenty-three surgical specimens of mucous retention cyst of the lip were examined for involvement of lymphatic vessels by a comparative immunohistochemical demonstration of lymphatic and blood vascular endothelial cells, as well as lymphatic and salivary contents. Mucous retention cysts were histopathologically classified into three stages: early, intermediate, and advanced. In the early stage, there was diffuse extravasation of mucous material in the interstitium of the lamina propria or the submucosal layer of the oral mucosa. In the intermediate stage, lymphatics, which were clearly revealed and immunohistochemically distinguished from blood vessels by monoclonal antibody D2-40, were dilated and finally ruptured, leaving fragments of lymphatic walls in the periphery of mucous pools. In the advanced stage, thick cyst walls of granulation tissue were formed around mucous retention. Lymphatics were no longer involved in the granulation tissue wall, which was actively driven by blood vessel formation. The results suggest that the lymphatic rupture seems to contribute to the enlargement in the pathogenesis of mucous retention cyst. r 2006 Elsevier GmbH. All rights reserved. Keywords: Angiogenesis; D2-40; Immunohistochemistry; Lymphatics; Mucous retention cyst
Introduction Mucous retention cysts (extravasation type, mucoceles) are oral lesions most commonly diagnosed during Corresponding author. Division of Oral Pathology, Department of Tissue Regeneration and Reconstruction, Niigata University, Graduate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Niigata 951-8514, Japan. Tel.: +81 25 227 2832; fax: +81 25 227 0805. E-mail address: [email protected] (T. Saku).
0344-0338/$ - see front matter r 2006 Elsevier GmbH. All rights reserved. doi:10.1016/j.prp.2006.11.003
routine surgical pathology service for dental practice [16,22,23]. Although mucous retention cysts are believed to be generated by extravasation of saliva into the extracellular space due to some traumatic damage of salivary ducts, their detailed histopathogenetic processes are poorly understood. The question remains as to how the mucous pool is confined to some areas or how the cyst becomes larger. In a previous study, we demonstrated how the mucous pool is organized by the capillary endothelial cell sprouting with tubule
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formation, and these endothelial cells were recruited from infiltrating monocytes in the extracellular milieu in which tenascin was enriched and produced by the endothelial progenitor cells themselves [24]. Mucous retention cysts of the oral mucosa are formed in the lamina propria and the submucosal layers, where lymphatics are abundantly distributed [9]. However, lymphatics in the pathogenesis of mucous retention cyst have not yet been investigated. This is partly due to the fact that no one has paid much attention to this and that a reliable method demonstrating lymphatic vessels in tissue specimens still needs to be designed. Recently, monoclonal antibody D2-40 has been introduced for immunohistochemical labeling of lymphatic endothelial cells [11]. In this study, we used this valuable antibody to demonstrate the fate of lymphatic vessels in the process of mucous retention cyst formation to better understand the lymphatic involvement in the initial stage of mucous retention cyst formation.
Materials and methods Materials We studied a total of 23 surgical samples of mucous retention cysts (mucoceles) diagnosed at the Division of Oral Pathology, Niigata University Graduate School of Medical and Dental Sciences, between 2004 and 2005. Surgical materials were fixed in 10% formalin solution and embedded in paraffin. Serial sections were cut at a thickness of 5 mm; one of the sets of serial sections was stained with hematoxylin and eosin (HE) and used for re-evaluation of the histologic diagnosis. The other sets were used for mucin histochemistry, such as periodic acid-Schiff (PAS), colloidal iron, or alcian blue (pH 2.5), and for immunohistochemical investigations.
sulfate proteoglycan (core protein), were raised in rabbits as described elsewhere [18].
Immunohistochemistry Immunohistochemical staining was performed using the Chemomate Envision/HRP system (DakoCytomation) as described previously [24]. For perlecan, the deparaffinized sections were pre-treated with 3 mg/ml hyaluronidase (bovine testicular origin, type I-S, 440 U/mg; Sigma Chemical Co., St. Louis, MO, USA) in phosphate buffered saline (PBS) for 30 min at 37 1C. For SC, type IV, and CD31, sections were pre-treated with 0.02% trypsin (type II, Sigma) in 0.5 M Tris–HCl (pH 7.6) containing 1% CaCl2 for 30 min at 37 1C. Prior to incubation with the primary antibodies, all the sections were treated with 0.3% H2O2 in absolute methanol for 30 min at room temperature to block endogenous peroxidase activities. For blocking non-specific protein binding, sections were incubated with 5% skimmed milk for 1 h at 37 1C after rinsing in PBS containing 0.5% Triton X-100 (T-PBS). They were then incubated overnight at 4 1C with the primary antibodies diluted in T-PBS. For visualization of reaction products, sections were treated with 0.02% 3, 30 -diaminobenzidine (DAB) in 0.05 M Tris–HCl buffer (pH 7.4) containing 0.005% H2O2. The sections were counterstained with hematoxylin. For control studies, the primary antibodies were replaced with pre-immune rabbit IgG or mouse IgG1. For double histochemical staining for mucin and lymphatic vessels, sections immunostained with the D2-40 antibody were visualized with DAB, thoroughly washed in water, and then stained for colloidal iron.
Results Histopathology
Antibodies A mouse monoclonal antibody D2-40 (D2-40, IgG1) was purchased from Nichirei Biosciences Inc. (Tokyo, Japan). Rabbit polyclonal antibodies against human IgA, secretory component (SC), and fibrin, as well as a mouse monoclonal antibody against human CD31 (JC70A, IgG1-k) were purchased from DakoCytomation (Copenhagen, Denmark). A mouse monoclonal antibody against human IgG (HP6025, IgG1-k) was obtained from Zymed Laboratories Inc. (South San Francisco, CA, USA). Rabbit polyclonal antibodies against collagen type IV (type IV) were obtained from ICN Biomedicals Inc. (Aurora, OH, USA). Antibodies against perlecan, a basement membrane-type heparan
Having examined the 23 tissue samples of mucous retention cyst histologically, we classified the mucous retention cysts into three different stages of their organizing process: early, intermediate, and advanced (Fig. 1). This classification was done depending on the mode of mucous material distribution and the granulation tissue wall formation separating the mucous pool from the surrounding tissue. In the early stage (Fig. 1(a)), mucous material was diffusely extravasated in the intercellular space of the lamina propria to the submucosal layer, causing the covering epithelium to be thinner and flatter, with the loss of rete ridges. Thus, the epithelium lost its regular shape, and the subepithelial zone showed an edematous appearance. In the peripheral zone of the edematous
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area, markedly dilated lumina of lymphatic vessels were often observed (Fig. 3(a), boxed area in Fig. 1(a)). In the intermediate stage (Fig. 1(b)), there were focal mucous pools containing a small number of macrophages and lymphocytes in the periphery. When such mucous pools were located beneath the mucosal epithelium, the covering epithelia were focally stretched and protruded to the surface. The mucous pools were clearly separated by immature granulation tissue rich in newly formed blood vessels and fine fibrils and denser infiltration of macrophages and neutrophils. Therefore, such less-cellular zones seemed to be related to the dilated lymphatics mentioned above, and it was
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suggested that some lymphatic contents or components may take part in the development of mucous retention cysts. In the advanced stage (Fig. 1(c)), a distinct granulation barrier was formed around the mucous pool as an isolated mass from the submucosal connective tissue. This tissue barrier could be considered a cyst wall. In the cystic lumen, there were many foamy macrophages floating into the background of eosinophilic amorphous material, which was colloidal-iron- (Figs. 4(a) and (b)), alcian-blue-, or PAS-positive (data not shown).
Immunohistochemistry Early stage In the early stage, amorphous eosinophilic materials in the edematous area (Fig. 2(a)) were positive for colloidal iron staining (Fig. 2(b)) or PAS and alcian blue (data not shown). The colloidal iron-positive amorphous materials were immunopositive for SC (Fig. 2(c)) and IgA (Fig. 2(d)) at the same time, indicating the retention of salivary contents in the area. On the other hand, immunopositivity for IgG (Fig. 2(e)) was also revealed, although the immunolocalization modes of IgA and IgG varied from area to area without any definite difference. Deeply eosinophilic strand-like deposits in the edematous area were immunopositive for fibrin (Fig. 2(f)). These deposits were also immunopositive for SC (Fig. 2(c)). This result indicated that the edematous appearance was due to salivary retention and tissue fluid from blood or lymphatic streams. The dilated lymphatic vessels in the periphery of the edematous area (Fig. 1(b), box) were clearly demonstrated by the D2-40 antibody. There was no immunopositivity with the same antibody in blood vascular endothelial cells (Fig. 3(b)). Lymphatic endothelial cells were faintly immunopositive for CD31 (Fig. 3(c)), and Fig. 1. Histopathologic appearance of mucous retention cyst in three stages: early (a), intermediate (b), and advanced (c). Hematoxylin and eosin (HE) stain. a, 20 , b, 30 , c, 20 . The early stage (a) was characterized by diffuse infiltration of mucous material in the lamina propria to the submucosal layer. Fibrous components and blood vessels were separated by mucous material, which resulted in an edematous appearance. At the periphery of the mucous-retained area, there were pronouncedly dilated lumina of lymphatic vessels. In the intermediate stage (b), there were focally condensed mucous retention areas, which seemed to be due to ruptures of lymphatic vessels. Their separated and fragmented vessel walls were stretched and adhered to the lower surface of the overlying epithelium. The advanced stage (c) was distinguished from the above two stages by the presence of cyst walls composed of characteristic granulation tissue layer. The cystic lumen was isolated by the granulation tissue wall from the surrounding connective tissue. Boxed areas are shown at higher magnifications in Figs. 3–5.
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Fig. 2. Immunohistochemistry and color staining for mucous fluid in mucous retention cyst, stage del. HE stain (a), colloidal iron stain (b), and immunoperoxidase stains for secretory component (SC) (c), IgA (d), IgG (e), and fibrin (f). Hematoxylin counterstain, 200 . Abundance of mucous material detected by colloidal iron staining (b) in the mucous retention cyst. Strong SC- and IgAimmunopositive materials were diffusely demonstrated exclusively in the edematous area, indicating that salivary contents were major constituents of the mucous retention (c, d). At the same time, however, IgG (e) was immunolocalized extensively in the mucous-retained area. In addition, fibrin-immunopositive membranous materials (f) were observed within the edematous area. These results suggest that lymphatic or plasma components, in addition to the salivary contents, also take part in the formation of the initial edematous histology of mucous retention cyst.
their basement membranes were revealed by type IV immunopositivity (Fig. 3(d)), while these two antigens were very intensively immunolocalized in the blood capillaries or venules. This result indicated that the D2-40 antibody was a reliable tool for demonstrating lymphatics of various sizes. Moreover, the infiltration of mucous material into lymphatic lumina from the outer mucous retention was demonstrated by double staining
with colloidal iron and D2-40 antibody (Fig. 3(e)). The staining intensity of colloidal iron appeared to be more condensed within the lymphatic lumen, which was also immunohistochemically demonstrated for SC (Fig. 3(f)). This mucous infiltration into lymphatic lumina might be a passive transport, and the lymphatic luminal territory still seemed to be independent from the surrounding mucous retention at this stage.
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Fig. 3. Immunohistochemical demonstration of dilated lymphatic vessels in early stage of mucous retention cyst (high power view of the boxed area in Panel a, Fig. 1). HE stain (a); immunoperoxidase stains with the D2-40 antibody (b) and for CD31 (c), collagen type IV (d), and secretory component (f); and double staining with D2-40 and colloidal iron (e). Hematoxylin counterstain, 125 . The dilated vessels were considered to be lymphatics, because their endothelial cells were labeled with D2-40 (b) but were not positive for CD31 (c), which was definitely demonstrated in small blood vessels around the dilated lymphatic vessels. Collagen type IV (d) was immunolocalized in most of the vascular channels in the figure, although it was more intensely immunolabeled in blood vessels than in lymphatic ones, indicating that all of the blood vessels and larger lymphatic vessels were circumscribed by basement membranes whose primary constituent is collagen type IV. Colloidal iron-positive mucous material (e) as well as SC (f) were rather condensed within the dilated lymphatic vessels, indicating that salivary contents were absorbed into the lymphatics to be dilated.
Intermediate stage At the periphery of mucous retention (Fig. 1(b), box), there were lymphatic vessels or fragments that were open to mucous pools as revealed by colloidal-iron staining (Figs. 4(a) and (b)). The lymphatic vessels were clearly demonstrated to be immunopositive with the D2-40 antibody (Figs. 4(a) and (b), arrows). In the very
peripheral part of mucous retention, lymphatic wall fragments were often stretched toward the lamina propria connective tissue (Fig. 4(b), arrows). There were CD31- or type IV-immunopositive blood capillaries and venules within the mucous pool and in the narrow lamina propria (Figs. 4(c) and (d)). These results indicated that lymphatics were destroyed by mechanical
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Fig. 4. Immunohistochemical demonstration of ruptured lymphatic vessels in intermediate stage of mucous retention cyst. Double stain of immunoperoxidase with the D2-40 antibody and colloidal iron (a and b) and immunoperoxidase stains for CD31 (c) and collagen type IV (d). Hematoxylin counterstain, a, 60 , b–d, 150 . The D2-40 antibody revealed fragments of ruptured lymphatic vessel walls in the mucous background (a). D2-40-immunopositive lymphatic wall fragments were often pushed underneath the lamina propria (b). Under the fragmented lymphatic wall, lymphocytes and macrophages were floating in clear background substances. Neutrophils were scarcely observed in such areas. Small blood vessels around the ruptured lymphatics were definitely demonstrated by immunopositivity for collagen type IV (d) and CD31 (c).
stresses mostly due to the infiltration of extravasated salivary contents (Figs. 3(e) and (f)).
Advanced stage In the thick granulation tissue, presenting as a cyst wall in the advanced stage (Fig. 1(c), box), there were many blood capillaries and postcapillary venules sprouting towards the cystic lumen, which were demonstrated by immunohistochemistry for type IV (Fig. 5(a)). In addition to the basement membrane of capillary or venous endothelial cells, fine fibrillar structures in the intercellular space were also immunopositive for type IV (Fig. 5(b)) and perlecan (Fig. 5(c)). In addition to these endothelial cells, CD31 was immunolocalized in polygonal-shaped cells between blood vessels (Fig. 5(d)). There were no D2-40-immunopositive cells in the cyst wall of thick granulation tissue (data not shown). This result indicated that lymphatics were not involved in the granulation tissue reaction in the mucous retention cyst wall, but that blood vessels and blood endothelial cells or their progenitors instead took an initiative role in it.
Discussion This study is the first to demonstrate that lymphatic vessels are involved in the histopathogenetic process of mucous retention cysts, a finding that has never been mentioned in the literature. We could show that dilated vascular vessels, often observed in the early stage of the lesions, were lymphatic vessels. Later in the intermediate stage, when the extension of the lesion is almost fixed, fragments of ruptured lymphatics were located at the periphery of the lesion. Based on this observation, one might suggest that the dilatation of lymphatics caused by absorbing extravasated saliva plays some role in the enlargement of mucous retention. The involvement of lymphatics is restricted only to the early stages of the lesion, but not in the later stage, which is characterized by the thick granulation barrier around the cystic lumina. The most important tool in the present study was the D2-40 monoclonal antibody. M2A antigen, an oncofetal antigen, was used to raise the D2-40 monoclonal antibody [11], which has similar specificity to another
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Fig. 5. Immunohistochemistry for advanced stage of mucous retention cyst. Immunoperoxidase stains for collagen type IV (a and b), perlecan (c), and CD31 (d). Hematoxylin counterstain, a, 150 , b–d, 600 . The cyst wall in the advanced stage was composed of granulation tissue, which was rich in postcapillary venules demonstrated by immunopositivity for collagen type IV (a and b), perlecan (c), and CD31 (d). CD31 was immunolocalized in the cytoplasm of polygonal cells regarded as endothelial progenitor cells, in addition to blood vascular endothelial cells (d). There were fine mesh-like immunolocalizations for type IV (b) and perlecan (c) in the interstitium. In such a stage of granulation barrier formation around mucous pools, there was no longer participation of lymphatics.
previously described monoclonal antibody (M2A) [1]. This monoclonal antibody has been characterized as a more specific tool for the identification of lymphatic endothelial cells [30] than the other previously used lymphatic endothelium markers such as vascular endothelial growth factor (VEGF) receptor-3 [7] and lymphatic endothelial hyaluronan receptor (LYVE-1) [2]. Recently, another valuable lymphatic marker, podoplanin, a glomerular podocyte-specific molecule [3], first designated as E11 antigen of rat bone [26], has been shown to be also recognized by antibody D2-40 [19]. In addition to podocytes, podoplanin has also been demonstrated in the choroids plexus and ciliary epithelium in the rat brain and eye [27]. Moreover, the significant homology with podoplanin has been reported in other molecules such as gp40 in MDCK cells [31] and RTI40 in rat alveolar epithelial cells [5]. Most recently, Evangelou et al. showed that both rabbit polyclonal antibodies against podoplanin and the D2-40 were suitable for the detection of lymphatic vessels [4]. Therefore, in our study, introducing this antibody D2-40, we were able to demonstrate lymphatic
vessels in the tissue. In the past, when no valuable and stable lymphatic markers were available, we used BSA-I lectin-binding histochemistry [28], type IV collagen or laminin immunohistochemistry for the detection of basement membrane molecules [8] and CD31 or UEA-I lectin-binding immunohistochemistry for blood endothelium [14] and electron microscopy [15], thus excluding lymphatics from blood vessels, for circumstantial judgment. When compared with these indirect techniques, immunohistochemistry using the D2-40 has a remarkable advantage in histological recognition of lymphatics. The extravasation of saliva is believed to be the primary cause of the lesion [16,22,23]. However, an actual trial to immunohistochemically demonstrate salivary components in the muco-edematous stroma of the cyst has not been described in the literature. In the present study, in addition to the conventional histochemical demonstration of mucous materials by colloidal iron and other stainings, we used IgA and SC immunolocalizations to give evidence of salivary contents, because whole saliva specifically contains SC and
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IgA, whose level is higher than that of IgG [13]. In our series, the immunohistochemical localization modes for IgG and IgA/SC were less different from each other. Although it is difficult to give a quantitative discussion on the basis of immunohistochemistry, one might suggest that within the mucous pools, fibrin, which has not been recognized as a salivary content, may have its origin in blood. Therefore, a certain amount of such kind of free IgG may originate from lymphatic or blood streams. This idea may be reasonable because inflammatory reactions due to mucous extravasation could induce blood capillary permeability. The condensation of colloidal-iron-positive and SC-immunopositive materials within dilated lymphatics also indicated that extravasated salivary contents were absorbed into lymphatics together with serum exudates. It is now obvious that the histologic appearance of ‘‘mucous retention’’ in this lesion is not always due to salivary contents but also to exudates from blood vessels or ruptured lymphatic vessels. During the organization process of mucous retention cyst, mucous liberated into the connective tissue was scavenged by inflammatory cells, mainly neutrophils and macrophages. At the same time, the pressure gradient from the interstitium into the nonmuscular initial lymphatics (microlymphatics or capillary lymphatics) was large enough to cause the percolation of interstitial fluid [21]. In the case of mucous retention cyst, the interstitial fluid was shown to be a mixture of plasma and mucous, and it was condensed within the dilated lymphatics. Therefore, initial lymphatics might expand beyond their limit to rupture and deliver its content back again into the mucous pool. Finally, mucous pools were surrounded by a characteristic granulation barrier that, interestingly, was devoid of lymphatics. Recently, Schledzewski et al. [20] demonstrated that there were significant numbers of LYVE-1 (lymphatic endothelium-specific hyaluronan receptor)immunopositive macrophages in wound healing or tumor tissues. Although we did not use LYVE-1 antibodies for the investigation in this study, neither D2-40-immunopositive mature lymphatics nor macrophage-like singular cells were found in the granulation tissue of mucous retention cyst wall. Instead, this granulation tissue barrier contained an abundance of blood vessels, that were demonstrated by immunohistochemistry for type IV and CD31, perpendicularly arranged to the cystic lumen. Regarding the organization process of the cyst wall, collagen type IV and perlecan, basement membrane constituents, were shown to be rich in intercellular space between sprouting blood vessels. In the previous study, we demonstrated that tenascin functioned as a substrate for the differentiation of endothelial progenitor cells in the angiogenesis in mucous pools [24]. The present result suggests that type IV and perlecan also play roles similar to
tenascin. As shown previously, CD31-immunopositive polygonal-shaped cells were endothelial progenitor cells originated from infiltrating monocytes [24], which have also been shown to produce and utilize cytokines such as VEGF [25], fibroblast growth factor (FGF) [17], platelet derived growth factor (PDGF) [6], tenascin [29], collagen type IV [12], and fibronectin [10] in autocrine manners for their differentiation. In conclusion, this study shows that dilatation of lymphatics, caused by absorbing extravasated saliva, plays a role in the enlargement of mucous retention.
Acknowledgement This work was supported in part by Grants-in-Aid for Scientific Research from Japan Society for the Promotion of Science.
References [1] D. Bailey, R. Baumal, J. Law, et al., Production of a monoclonal antibody specific for seminomas and dysgerminomas, Proc. Natl. Acad. Sci. USA 83 (1986) 5291–5295. [2] S. Banerji, J. Ni, S.X. Wang, et al., LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan, J. Cell Biol. 144 (1999) 789–801. [3] S. Breiteneder-Geleff, K. Matsui, A. Soleiman, et al., Podoplanin, novel 43-kd membrane protein of glomerular epithelial cells, is down-regulated in puromycin nephrosis, Am. J. Pathol. 151 (1997) 1141–1152. [4] E. Evangelou, P.A. Kyzas, T.A. Trikalinos, Comparison of the accuracy of lymphatic endothelium markers: Bayesian approach, Mod. Pathol. 18 (2005) 1490–1497. [5] R.F. Gonzalez, L.G. Dobbs, Purification and analysis of RTI40, a type I alveolar epithelial cell apical membrane protein, Biochim. Biophys. Acta 1429 (1998) 208–216. [6] P. Guo, B. Hu, W. Gu, L. Cheng, et al., Platelet derived growth factor-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor expression in tumor endothelia and by promoting pericyte recruitment, Am. J. Pathol. 162 (2003) 1083–1093. [7] A. Kaipainen, J. Korhonen, T. Mustonen, et al., Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development, Proc. Natl. Acad. Sci. USA 92 (1995) 3566–3570. [8] Y. Kobayashi, T. Nakajima, T. Saku, Loss of basement membranes in the invading front of O-1N, hamster squamous cell carcinoma with high potential of lymph node metastasis: an immunohistochemical study for laminin and type IV collagen, Pathol. Int. 45 (1995) 327–334. [9] M. Kotani, The lymphatics and lymphoreticular tissues in relation to the action of sex hormones, Arch. Histol. Cytol. Rev. 53 (Suppl.) (1990) 1–76. [10] B. Larivie`re, M. Rouleau, S. Picard, et al., Human plasma fibronectin potentiates the mitogenic activity of
ARTICLE IN PRESS S. Kundu et al. / Pathology – Research and Practice 203 (2007) 89–97
[11]
[12]
[13]
[14]
[15]
[16]
[17]
[18]
[19]
[20]
platelet-derived growth factor and complements its wound healing effects, Wound Repair. Regen. 11 (2003) 79–89. A. Marks, D.R. Sutherland, D. Bailey, et al., Characterization and distribution of an oncofetal antigen (M2A antigen) expressed on testicular germ cell tumours, Br. J. Cancer 80 (1999) 569–578. A.G. Marneros, B.R. Olsen, The role of collagen-derived proteolytic fragments in angiogenesis, Matrix Biol. 20 (2001) 337–345. P.P. Mortimer, J.V. Parry, Detection of antibody to HIV in saliva: a brief review, Clin. Diagn. Virol. 2 (1994) 231–243. K. Niimi, M. Yoshizawa, T. Nakajima, T. Saku, Vascular invasion in squamous cell carcinomas of human oral mucosa, Oral Oncol. 37 (2001) 357–364. Y. Ono, T. Nakajima, T. Saku, Vascular invasion of O-1N, hamster squamous cell carcinoma with high potential of lymph node metastasis: ultrastructural comparison between lymphatics and blood vessels, Pathol. Int. 48 (1998) 254–264. J.A. Regezi, J.J. Sciubba, R.C.K. Jordan, Oral Pathology: Clinical Pathologic Correlations, fourth ed, W.B. Saunders, Philadelphia, 2003, pp. 183–187. S. Rogelj, M. Klagsbrun, R. Atzmon, et al., Basic fibroblast growth factor is an extracellular matrix component required for supporting the proliferation of vascular endothelial cells and the differentiation of PC12 cells, J. Cell Biol. 109 (1989) 823–831. T. Saku, H. Furthmayr, Characterization of the major heparan sulfate proteoglycan secreted by bovine aortic endothelial cells in culture: homology to the large molecular weight molecule of basement membranes, J. Biol. Chem. 264 (1989) 3514–3523. V. Schacht, S.S. Dadras, L.A. Johnson, et al., Upregulation of the lymphatic marker podoplanin, a mucintype transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors, Am. J. Pathol. 166 (2005) 913–921. K. Schledzewski, M. Falkowski, G. Moldenhauer, et al., Lymphatic endothelium-specific hyaluronan receptor LYVE-1 is expressed by stabilin-1+, F4/80+, CD1 1b+macrophages in malignant tumours and wound
[21] [22]
[23] [24]
[25]
[26]
[27]
[28]
[29]
[30]
[31]
97
healing tissue in vivo and in bone marrow cultures in vitro: implications for the assessment of lymphangiogenesis, J. Pathol. 209 (1) (2006) 67–77. G.W. Schmid-Scho¨nbein, Microlymphatics and lymph flow, Physiol. Rev. 70 (1990) 987–1028. G. Seifert, A. Miehlke, J. Haubrich, R. Chilla, Diseases of the Salivary Glands, Georg Thieme Verlag, Stuttgart, 1986, pp. 91–98. M. Shear, Cysts of the Oral Regions, third ed, Wright, Oxford, UK, 1992, pp. 212–221. W. Swelam, H. Ida-Yonemochi, T. Saku, Angiogenesis in mucous retention cyst: a human in vivo-like model of endothelial cell differentiation in mucous substrate, J. Oral Pathol. Med. 34 (2005) 30–38. N.S. Taichman, A.T. Cruchley, L.M. Fletcher, et al., Vascular endothelial growth factor in normal human salivary glands and saliva: a possible role in the maintenance of mucosal homeostasis, Lab. Invest. 78 (1998) 869–875. A. Wetterwald, W. Hoffstetter, M.G. Cecchini, et al., Characterization and cloning of the E11 antigen, a marker expressed by rat osteoblasts and osteocytes, Bone 18 (1996) 125–132. M.C. Williams, Y. Cao, A. Hinds, et al., T1 alpha protein is developmentally regulated and expressed by alveolar type I cells, choroids plexus, and ciliary epithelia of adult rats, Am. J. Respir. Cell Mol. Biol. 14 (1996) 577–585. M. Yoshizawa, S. Shingaki, T. Nakajima, T. Saku, Histopathological study of lymphatic invasion in squamous cell carcinoma (O-1N) with high potential of lymph node metastasis, Clin. Exp. Metastasis 12 (1994) 347–356. D. Zagzag, B. Shiff, G.I. Jallo, et al., Tenascin-C promotes microvascular cell migration and phosphorylation of focal adhesion kinase, Cancer Res. 62 (2002) 2660–2668. Y. Zeng, K. Opeskin, L.G. Horvath, et al., Lymphatic vessel density and lymph node metastasis in prostate cancer, Prostate 65 (2005) 222–230. G. Zimmer, F. Lottspeich, A. Maisner, et al., Molecular characterization of gp40, a mucin-type glycoprotein from the apical plasma membrane of Medin-Darby canine kidney cells (type I), Biochem. J. 326 (1997) 99–108.