Localization of CD44 and hyaluronan in the synovial membrane of the rat temporomandibular joint.код для вставкиСкачать
THE ANATOMICAL RECORD PART A 288A:646 – 652 (2006) Localization of CD44 and Hyaluronan in the Synovial Membrane of the Rat Temporomandibular Joint AKIKO SUZUKI,1* KAYOKO NOZAWA-INOUE,1 NORIO AMIZUKA,1,2 KAZUHIRO ONO,3 AND TAKEYASU MAEDA1,2 1 Division of Oral Anatomy, Department of Oral Biological Sciences, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan 2 Center for Transdisciplinary Research, Niigata University, Niigata, Japan 3 Division of Dental Hygiene and Health Promotion, Faculty of Dentistry, Niigata University, Niigata, Japan ABSTRACT Previous studies have pointed out a lack of adhesion structures in the synovial lining layer of the rat temporomandibular joint (TMJ) despite showing an epithelial arrangement. CD44, a major cell adhesion molecule, plays crucial roles as an anchor between cells and extracellular matrices by binding hyaluronan (HA) for the development of organs or the metastasis of tumors. The present study examined the localization of CD44 in the synovial membrane of the rat TMJ by immunocytochemistry for OX50, ED1, and Hsp25, which are markers for the rat CD44, macrophage-like type A, and ﬁbroblast-like type B synoviocytes, respectively. Histochemistry for HA-binding protein (HABP) was also employed for the detection of HA. OX50 immunoreactions were found along the cell surface and, in particular, accumulated along the surface of the articular cavity. Observations by a double immunostaining and immunoelectron microscopy revealed that all the OX50-immunopositive cells were categorized as ﬁbroblastic type B cells, which had many caveolae and a few vesicles reactive to intense OX50. However, the macrophage-like type A cells did not have any OX50 immunoreaction in the synovial lining layer. A strong HABP reaction was discernable in the extracellular matrix surrounding both OX50-positive and -negative cells in the synovial lining layers, exhibiting a meshwork distribution, but weak in its sublining layer. This localization pattern of CD44 and HABP might be involved in the formation of the epithelial arrangement of the synovial lining layer. Furthermore, OX50 immunonegativity in the type A cells suggests their low phagocytotic activity in the rat TMJ under normal conditions. Anat Rec Part A, 288A:646 – 652, 2006. © 2006 Wiley-Liss, Inc. Key words: ﬁbroblast-like type B cell; macrophage-like type A cell; CD44; hyaluronan; temporomandibular joint The temporomandibular joint (TMJ), which is a bilateral diarthrosis between the temporal bone and the mandibular condyle, develops a synovial membrane. Many researchers agree with the notion that the synovial lining layer of the TMJ consists of macrophage-like type A cells and ﬁbroblast-like type B cells (Graabæk, 1984; NozawaInoue et al., 1998, 2003), as shown in the systemic joints (for review, see Iwanaga et al., 2000). According to their ultrastructural conﬁgurations, the former cells absorb and degrade of various extracellular matrices and cell debris as well as antigens in the synovial ﬂuid and synovial membrane (Okada et al., 1981; Graabæk, 1984; Athanasou, 1995), whereas the latter cells serve in the synthesis and secretion of collagen (Visnapuu et al., 2000), ﬁbronectins (Mapp and Revell, 1985), and hyaluronan (HA) (Roy and Ghadially, 1967), all of which are the components of the synovial ﬂuid as well as the extracellular matrices (ECMs) of the synovial membrane. In addition, biochemical studies have also detected HA, albumins, white blood © 2006 WILEY-LISS, INC. cells, and debris of the synovial membrane in the synovial ﬂuid (Berumen-Nafarrate et al., 2002). This viscous synovial ﬂuid makes smooth jaw movements possible, serving as a lubricant in both the sliding and rotating movement Grant sponsor: Japanese Ministry of Education, Culture, Sports, Science and Technology (MEXT); Grant number: 16659498. *Correspondence to: Akiko Suzuki, Division of Oral Anatomy Department of Oral Biological Science Niigata University Graduate School of Medical and Dental Sciences, 2-5274 Gakkochodori, Niigata 951-8514, Japan. Fax: 81-25-223-6499. E-mail: email@example.com Received 13 December 2005; Accepted 13 February 2006 DOI 10.1002/ar.a.20331 Published online 3 May 2006 in Wiley InterScience (www.interscience.wiley.com). CD44 AND HA IN RAT TMJ of the condyle, in addition to supplying oxygen and nutrition to the articular cartilage. CD44 is a cell surface transmembrane glycoprotein receptor (Aruffo et al., 1990), categorized in hyaladherins. A variety of cells exhibit this molecule, which involves cell adhesion (Lesley and Hyman, 1998), the metastasis of tumors (Alaniz et al., 2002), cell migration (Lesley and Hyman, 1998), and development of almost all organs (Knudson and Knudson, 1993; Ruiz et al., 1995). CD44 binds not only ECMs such as HA (Asari et al., 1995; Knudson, 2003), types I and IV collagens (Faassen et al., 1992; Knudson et al., 1996), and chondroitin sulfate, but also non-ECM ligands including ﬁbronectins, osteopontin, and serglycin (Aruffo et al., 1990; Lesley et al., 1993). Therefore, this molecule has been considered to play a role as a cell-to-cell and cell-to-ECM anchor. The synovial lining cells in TMJ show an epitheliumlike arrangement without a distinct adhesive structure, leading us to a possibility that CD44 is a candidate for a molecule to serve as cell-to-cell adhesion. However, there has been controversy over the distinct localization of CD44 immunoreaction in the synovial membrane. CD44 immunoreaction has been reported in human knee joints under normal (Asari et al., 1995) and pathological conditions (Johnson et al., 1993; Naor and Nedvetzki, 2003). Furthermore, CD44 immunoreaction decreased in number in the synovial tissue in the human knee joint with rheumatoid arthritis (RA) (Henderson et al., 1994). Although both type A and B cells in TMJ were also positive in CD44 immunoreaction (Yamada et al., 2002), these studies failed to identify the types of CD44-positive cells because they did not utilize any speciﬁc cell marker for each synovial lining cells. HA, widely distributed in the extracellular compartment of most tissues, is a large linear glycosaminoglycan (GAG). This GAG has multiple functions as a scaffold for various cells in cell migration, immune reactions, phagocytosis, and adherence (Hakansson et al., 1980; Forrester and Lackie, 1981; Pisko et al., 1983; Hardwick et al., 1992; Termeer et al., 2003) under both physiological and pathological conditions (Knudson and Knudson, 1993; Goebeler et al., 1996). These regulatory functions of HA on cellular activities are due to its ability to interact with the cell surface hyaladherins and matrix hyaladherins. In addition, since HA is a major component of the synovial ﬂuid and cartilage (Pitsillides et al., 1994; Tulamo et al., 1994; Parkkinen et al., 1996), it has been supposed to play crucial roles in joint lubrication and reduction of cartilage attrition during joint movements in synovial arthrosis including TMJ (Ghosh, 1994; Cascone et al., 2002). The present study was therefore undertaken to investigate the localization of CD44 in the normal rat TMJ by employing immunocytochemistry for OX50, which can recognize rat CD44 (Paterson et al., 1987; Wirth et al., 1993) at light and electron microscopic levels. Histochemistry for HA-binding protein (HABP), which speciﬁcally combines with HA (Hascall and Heinegard, 1974; Asari et al., 1995), was carried out to clarify the topographical relationship between the synovial lining cells and HA in the synovial membrane. Furthermore, a double staining with OX50 and several marker proteins for the synovial lining cells was performed for identifying its immunopositive cell types. 647 MATERIALS AND METHODS All experiments were performed under guidelines of the Niigata University Faculty of Dentistry Intramural Animal Use and Care Committee (approval number 150). Tissue Preparation Male Wistar rats (4 weeks of age, 105–120 g, n ⫽ 6; 8 weeks of age, 300 –330 g, n ⫽ 2) were used in this study. Under anesthesia by an intraperitoneal injection of 8% chloral hydrate (400 mg/kg), they were perfused with a ﬁxative containing 4% paraformaldehyde and 0.0125% glutaraldehyde in a 0.1 M phosphate buffer (pH 7.4). The removed heads were decalciﬁed with a 5% ethylene diamine tetra-acetic acid disodium (EDTA-2Na) solution for 4 weeks at 4°C. One side of each head was embedded in a 30% sucrose solution at 4°C overnight for cryoprotection and embedded in an OCT compound (Leica, Nussloch, Germany). Serial sagittal sections were cut at a thickness of 8 or 35 m in a cryostat (CM 3050S; Leica) for immunocytochemistry at the levels of light and electron microscopy, respectively, and mounted onto silane-coated glass slides. Serial parafﬁn sections were sagittally cut at a thickness of 5 m, stained with hematoxylin and eosin (H&E) for histological observations, and processed for immunohistochemistry. Immunohistochemistry Dewaxed sections were predigested with 0.1% trypsin (Wako Chemicals, Tokyo, Japan) in 0.01 M phosphatebuffered saline (PBS; pH 7.4) for 30 min at 37°C as an unmasking procedure according to a report by Parkkinen et al. (1996). After the inhibition of endogenous peroxidase with 0.3% H2O2 in absolute methanol for 30 min, the sections were incubated for 24 hr at 4°C with an OX50monoclonal antibody (1:1200, Serotec, Oxford, U.K.). The reaction sites of the antigen-antibody were localized using biotinylated antimouse IgG (1:100; Vector Lab, Burlingame, CA) for 2 hr at room temperature and subsequently avidin-conjugated peroxidase (ABC kit; Vector Lab) for 90 min at room temperature. Final visualization employed 0.04% 3,3⬘-diaminobenzidine tetrahydrochloride (DAB) and 0.0125% H2O2 in a 0.05 M Tris-HCl buffer (pH 7.6). Some immunostained sections were counterstained with 0.03% methylene blue. For immunocytochemistry at the electron microscopic level, immunostained cryostat sections without counterstaining were postﬁxed in 1% osmium tetroxide reduced with 1.5% potassium ferrocyanide for 90 min at 4°C, dehydrated in ascending ethanols, and ﬁnally embedded in epoxy resin (Epon 812, Taab, Berkshire, United Kingdom). One m thick sections were stained with 0.03% methylene blue in order to check the locations under a light microscope. Ultrathin sections (70 nm thick) were prepared in a Leica Ultracut R (Leica Microsystems, Wetzlar, Germany) with a diamond knife and brieﬂy stained with lead citrate. They were examined in a Hitachi H-7000 transmission electron microscope (Hitachi, Tokyo, Japan). For the detection of HA, deparafﬁnized sections were reacted with biotin-labeled HABP (2 g/ml; Seikagaku, Tokyo, Japan) for 24 hr at 4°C, followed with avidinconjugated peroxidase (ABC kit; Vector Lab) for 90 min at room temperature. They were developed with DAB solution mentioned above. 648 SUZUKI ET AL. Double Labeling With HABP and OX50 Cryostat sections were primarily incubated with a biotinylated HABP at a concentration of 4 g/ml for 24 hr at 4°C and subsequently with ﬂuorescein isothiocyanate (FITC)-conjugated Avidin D (1:300, Vector Lab) for 1 hr at room temperature. After the inhibition of avidin and biotin according to the manufacturer’s instruction (Vector Lab), these reacted sections were incubated with an OX50 antibody in the same way mentioned above. Following two consecutive incubations of biotinylated antimouse IgG and rhodamine isothiocyanate (RITC)-conjugated Avidin D (1:300; Vector Lab), the double-labeled sections were mounted in the Vectashield mounting medium with 4⬘,6diamidino-2-phenylindole (DAPI; Vector Lab). They were examined with a ﬂuorescent microscope (Axiophot, Carl Zeiss, Jena, Germany). Double Labeling Immunocytochemistry for OX50 With Either ED1 or Hsp25 The detection of ED1 or heat shock protein 25 (Hsp25) was carried out by immunoﬂuorescence using FITC-conjugated Avidin D (1:300; Vector Lab). The ED1-monoclonal antibody (1:500; Serotec) and antiserum against Hsp25 (1:5,000; Stressgen Biotechnologies, Victoria, Canada) used here can recognize monocyte/macrophage lineages (Dijkstra et al., 1985) and ﬁbroblastic type B cells (Nozawa-Inoue et al., 1999a; Andoh et al., 2001) in the TMJ, respectively. OX50 immunoreaction was made visible by an incubation of RITC-conjugated Avidin D (1:300; Vector Lab). Immunohistochemical or Histochemical Controls Immuohistochemical controls of OX50 were performed by replacing the primary antibodies with nonimmune sera or PBS and omitting the antimouse or rabbit IgG, or the ABC conjugated with peroxidase. For the HA reaction, negative controls were prepared by treating parafﬁn sections with Streptomyces hyalurolyticus hyaluronidase (400 turbidometric units/ml; Seikagaku) for 2 hr at 60°C prior to incubation with the biotinylated HABP. Immunocontrol experiments with ED1 and Hsp25 have been described in our previous studies (Nozawa-Inoue et al., 1999a; Andoh et al., 2001). Fig. 1. Sagittal sections of the synovium of the rat TMJ. OX50 (a–c), OX50 and Hsp25 (d), and OX50 and ED1 immunoreaction (e) in 4-week(a, b, d, and e) and 8-week-old (c) rats. a: The anterior portion of the synovial membrane is a thick synovial lining cell layer. OX50 immunoreaction is intense around the synovial lining cells (arrowheads). Some OX50-positive cells with slender proﬁles (arrows) are scattered in the sublining layer. b: At the posterior region of the synovial fold, the synovial lining cells form a sparse cell layer and the OX50 immunoreactions are observed as a line along the surface of the synovial membrane. c: The anterior portion of the synovial membrane at 8 weeks. Immunolocalization pattern of CD44 does not differ between 4- (a) and 8-week-old (c) rats. Arrowheads indicate OX50-positive synovial lining cells. d: OX50 immunoreactions (red) appear along the cellular outlines (arrows) while Hsp25 immunoreactions (green) are diffused in the cytoplasm. The articular cavity is lined with the cytoplasmic processes of OX50-positive cells with Hsp25 immunoreaction (arrowheads). e: The red OX50 immunoreaction is detectable at the periphery of the cells, whereas ED1 reactions take on a granular appearance in the cytoplasm, appearing green (arrows). The ED1-positive cells lack OX50 immunoreaction. Scale bars ⫽ 20 m (a– d). RESULTS Immunolocalization of OX50 in Rat TMJ Double Immunostaining With OX50 and ED1 or Hsp25 Immunostaining with the OX50 antibody was able to demonstrate an intense immunoreaction in the synovial lining layer of the rat TMJ (Fig. 1). The immunolocalization pattern in the rats aged 4 weeks (Fig. 1a) was identical to that in 8-week-old animals (Fig. 1c). These immunoreactions were localized along both the upper and lower articular cavities. At the anterior region of the synovial membrane where the synovial lining cells showed a thick cell layer, OX50 immunoreactivity was discernable around the synovial lining cells, assuming a meshwork distribution (Fig. 1a). At the posterior portion, OX50 immunoreactions were observed as a line along the surface of the articular cavities because the synovial lining layer was thin (Fig. 1b). Some slender cells in the sublining layer also reacted with the OX50 antibody (Fig. 1a). A double immunostaining clearly succeeded in distinguishing the synovial lining cells according to their immunostaining patterns: synoviocytes coexpressed Hsp25 and OX50 immunoreactions (Fig. 1d), and ED1-positive synoviocytes lacked OX50 reaction (Fig. 1e). In a double immunoﬂuorescent staining, OX50, ED1, and Hsp25 immunoreactions respectively took on a cellular outline, cytosolic granular appearance, or diffused cytosolic distribution. Those cells double positive for OX50 and Hsp25 extended their cytoplasmic projections toward the intercellular spaces or the surface of the articular cavities (Fig. 1d). Their immunoexpression patterns suggested that the OX50-immunopositive and -negative lining cells were ﬁbroblast-like type B cells and macrophage-like type A cells, respectively. CD44 AND HA IN RAT TMJ 649 Fig. 2. Immunoelectron micrographs showing OX50-positive (a, c, and d) and -negative (b) cells in the synovial lining layer (a– d) and sublining layer (e) of the rat TMJ. Preembedding method using DAB development. a: Two types of the synovial cells are recognizable in the synovial lining layer: cells with (B) and without (A) OX50 immunoreactions and cells. The OX50-positive cells (B) assemble at the upper portion of the synovial membrane. Their cytoplasmic projections cover the surface of the articular cavity. b: The OX50-negative cell in the synovial lining layer has lysosomes (arrowheads), well-developed ﬁlopodia-like pro- cesses (arrows), and a heterochromatin-rich dark nucleus. c: The OX50positive cells in the synovial lining layer have a well-developed rough endoplasmic reticulum and bright large nuclei. d: Higher magniﬁcation of the boxed area in c. The OX50-positive cell has numerous caveolae with intense (arrowheads) and weak (open arrowheads) immunoproducts. In addition to caveolae, there are some immunopositive vesicles (arrows) in its cytoplasm. e: The OX50-positive cell in the sublining layer displays a serrate outline and has a large irregular nucleus and poor organelle in its cytoplasm. Scale bars ⫽ 5 m (a); 2.5 m (b, c, and e); 500 nm (d). Immunoelectron Microscopic Observation for OX50-Immunopositive Cells tained resultant vesicles, which revealed an OX50 immunoreactivity, implying an internalization of the extracellular matrix. From their cell bodies, the OX50-positive cytoplasmic processes extend toward the articular cavity and took a transverse course to align with the articular cavity surface (Fig. 2a). These cellular processes appeared almost continuous; thus, few portions of the sublining layer were exposed to the articular cavity. On the other hand, immunoelectron microscopy conﬁrmed the existence of some OX50-positive cells in the sublining layer. The positive cells in the sublining layer could be divided into two types according to their ultrastructural conﬁgurations. One was a large round cell that had an invaginated cell surface and a large irregular nucleus with rich heterochromatins, suggesting its categorization as a leukocyte (Fig. 2e). The other type was a In immunoelectron microscopy, immunoreactive products for OX50 were observed as electron-dense deposits. They were conﬁned to the area around the cell membrane of the synovial lining cells (Fig. 2a). In particular, the immunoreactive products appeared to gather at the surface of the synovial lining cell facing the articular cavity. The OX50-immunopositive cells in the synovial lining layer possessed well-developed rough endoplasmic reticula, a bright large nucleus with less heterochromatins, and numerous caveolae with small smooth outlines (Fig. 2c and d). Although the OX50 immunoreaction in the caveolae showed variable intensities, no immunonegative caveolae existed in the OX50-positive synovial lining cells (Fig. 2d). Furthermore, the OX50-positive lining cells con- 650 SUZUKI ET AL. therefore, the colocalization of HABP/OX50 was intensely found in the synovial lining layer. The extracellular matrix around some cells in the sublining layer showed a faint reactivity for HABP and OX50. DISCUSSION Fig. 3. HABP reaction (a and b), negative control (c), and double ﬂuorescent reaction with OX50 and HABP (d). a: The synovial membrane has an intense HABP reaction (arrowheads) as compared with the articular disk (D). HABP reactions are diffusely distributed in the articular cavities. b: The higher magniﬁcation boxed in a. The intense HABP reactions are found in synovial lining layer, compared with sublining layer. c: The equivalent portion to b. The sections treated with hyaluronidase lack any HABP reactivity. d: HABP reactions (green) are found in the extracellular matrix around both the OX50-positive (red) and -negative cells in the synovial lining layer while the sublining layer contains weak HABP reactions. T, temporal bone; C, condyle. Scale bars ⫽ 600 m (a); 20 m (b– d). slender cell that possessed a clear ﬂat or ovoid nucleus, well-developed rough endoplasmic reticula, and a long thin cytoplasmic process, indicating a putative synovial ﬁbroblast (data not shown). OX50-immunonegative cells also existed in both the synovial lining and sublining cell layers (Fig. 2a). They were ultrastructurally characterized by the presence of lysosomes, well-developed ﬁlopodia-like processes, and a dark nucleus with rich heterochromatins (Fig. 2b). Taken together with their immunoelectron microscopic observation and immunoexpression patterns, these ﬁndings indicate that the OX50-immunopositive and -negative lining cells were ﬁbroblast-like type B cells and macrophage-like type A cells, respectively. Histochemistry for HABP Immunoexpression pattern and histochemistry for HA did not differ in the TMJ between 4- and 8-week-old rats. Intense HABP reactions were found in the synovial membrane but were weak in the articular disk (Fig. 3a and b). No reactivity for HABP was recognized in the cytoplasm of the synovial lining cells. The sections pretreated with hyaluronidase did not show any speciﬁc reaction (Fig. 3c). Double Staining With HABP and OX50 Reactions Double staining was able to conﬁrm the presence of an HABP reaction (Fig. 3d). The HABP reactions were localized in the extracellular matrix but not in the cytoplasm; they surrounded both the OX50-positive and -negative synoviocytes in the synovial lining layer. On the other hand, weak HABP reactions existed in the sublining layer; The present immunocytochemical study was clearly able to demonstrate a precise localization of CD44 and HA in the synovial membrane of the rat TMJ: the HA reaction was abundantly distributed in the synovial lining layer, while CD44 was localized on the cell membrane of the ﬁbroblast-like type B synoviocytes, not the macrophagelike type A synoviocytes. The cell type expressing the CD44 molecule remains controversial. In the human knee joint, some researchers suggested that both type A and B synoviocytes expressed CD44 in the healthy and inﬂammatory conditions (Asari et al., 1995; Naor and Nedvetzki, 2003). In addition to in vivo studies, the cultured type B cells in RA showed a CD44 immunoreaction (Wibulswas et al., 2002; Tolboom et al., 2004). However, it is well known that the cultured synovial lining cells change their features, unlike the original synoviocytes (Gadher and Woolley, 1987; Vandenabeele et al., 2003). These controversial ﬁndings may be caused by the absence of any speciﬁc cellular markers to identify the cell type of the positive synoviocytes both in vivo and in vitro. Current double immunolabeling and immunoelectron microscopy have revealed an intense CD44 immunoreactivity in the normal ﬁbroblast-like type B synovial cells. As far as we know, this is the ﬁrst report to disclose the presence of CD44 in the ﬁbroblast-like type B cells and not the type A cells. The synovial lining layer shows an epithelium-like arrangement in spite of the absence or incompleteness of adhesive structures between synovial type B cells (Nozawa-Inoue et al.,1998, 1999b), giving rise to the question of how the lining cells form and keep their epitheliumlike arrangement. The present immunostaining showed intense CD44 immunoreaction in these type B cells whose peripheral ECM displayed HABP reaction. Since CD44, known as a primary receptor for HA, mediates both cellto-cell and cell-to-matrix interactions (Aruffo et al., 1990; Underhill, 1992; Lesley et al., 1993; Lesley and Hyman, 1998; Knudson, 2003), it is easily supposed that CD44 serves as an adhesion molecule among the type B cells in the synovial lining layer both to make and to maintain an epithelial-like arrangement. The ﬁbroblast-like type B cells are characteristic of welldeveloped caveolae whose functional signiﬁcance remains unknown (Nozawa-Inoue et al., 1998). Current immunostaining demonstrated the localization of CD44 immunoexpression in numerous caveolae and some vesicles with various intensities in the type B cells. Our careful observations, however, failed to ﬁnd CD44-positive phagosomes and coated pits in both typed synovial lining cells, comparable with a previous study on cultured skin ﬁbroblasts (Culty et al., 1992). Since the uptake of HA is performed by the membranous indentations with CD44 receptors, the CD44-immunopositive caveolae in the type B cells has been considered to participate in this process (Tammi et al., 2001). Taken together with the fact that the type B cells synthesize HA (Vuorio et al., 1982; Smith and Ghosh, 1987; Anggiansah et al., 2003), we can easily suppose the involvement of ﬁbroblast-like type B cells in the HA metabolism. CD44 AND HA IN RAT TMJ It is noteworthy that the macrophage-like type A cells in the TMJ were devoid of CD44 immunoreaction. The expression of CD44 immunoreactivity has been found in other cell lines of ED1-positive monocyte/macrophages lineages such as osteoclasts and alveolar macrophages (Green et al., 1988; Culty et al., 1994; Nakamura et al., 1995; Vivers et al., 2002), which have an ability to phagocytose matrix HA through CD44. This discrepancy may be explained by the difference in the endocytotic activity between these CD44/ED1-positive cells and synovial type A cells. Indeed, type A cells do not develop phagosomes in their cytoplasm and express a very faint reactivity for acid phosphates, a marker for phagocytotic activity, under normal conditions (Nozawa-Inoue et al., 1998; Iwanaga et al., 2000). Under inﬂammatory conditions, on the other hand, the type A cells and macrophages with a CD44 immunoreaction on their cell membrane migrate to the synovial membrane from blood vessels (DeGrendele et al., 1996; Johnson et al., 2000). These migrated type A cells actively phagocytose various ECMs or antigens through the CD44 receptor. Taken together with our current observations, this lack of CD44 immunoreaction in the macrophage-like type A cells indicates lower and/or no phagocytotic activity under normal conditions, although they can absorb and degrade various extracellular components derived from the synovial ﬂuid and synovial membrane under pathological conditions. In conclusion, the cell-speciﬁc expression and distribution of CD44 and HA in the synovial membrane of rat TMJ suggest a close relation between type B cell with CD44 immunoreaction and the epithelial-like arrangement of the synovial lining layer. ACKNOWLEDGMENTS The authors thank M. Hoshino and K. Takeuchi, Division of Oral Anatomy, Department of Oral Biological Science, Niigata University Graduate School of Medical and Dental Sciences, for their technical assistance. 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