ARTICLE |
Correspondence to: Toshihiko Iwanaga, Lab. of Anatomy, Graduate School of Veterinary Medicine, Hokkaido Univ., Kita-18 Nishi-9, Kita-ku, Sapporo 060-0818, Japan.
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Summary |
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Fibroblast-like (Type B) synoviocytes are cells in the synovial membrane that are responsible for production of both synovial fluid and the extracellular matrix in the synovial intima. Immunostaining of the horse synovial membrane for protein gene product (PGP) 9.5, which is a neuron-specific ubiquitin C-terminal hydrolase, demonstrated selective localization of the immunoreactivity in a synoviocyte population different from acid phosphatase-positive Type A synoviocytes. The immunoreactive cells were lined up in the synovial intima and extended dendritic processes towards the joint cavity to form a dense plexus on the surface. Electron microscopic examination clearly identified the PGP 9.5-immunoreactive cells as Type B synoviocytes characterized by developed rough endoplasmic reticulum and free ribosomes. Immunoreactivity for PGP 9.5 was diffusely distributed throughout the cytoplasm, including the tips of fine processes. Western and Northern blot analyses could not distinguish the corresponding protein and mRNA obtained from the brain and synovial membrane. The existence of the neuron-specific PGP 9.5 in Type B synoviocytes suggests a common mechanism regulating the protein metabolism between neurons and synoviocytes, and also provides a new cytochemical marker for identification of the cells. (J Histochem Cytochem 47:343351, 1999)
Key Words: joint, protein gene product 9.5, synovial membrane, immunohistochemistry, horse
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Introduction |
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Synovial intima contains two morphologically different types of cells: macrophages and fibroblast-like cells (
Protein gene product (PGP) 9.5 was originally isolated from the human brain as a brain-specific protein with a molecular weight of approximately 27,000 (
This article reports the selective localization of PGP 9.5 in Type B synoviocytes in the horse joint. Although we cannot present here the neuronal and paraneuronal nature of the horse synoviocytes, immunostaining for PGP 9.5 clearly demonstrated the detailed distribution and unique shape of Type B synoviocytes, which have not been previously documented.
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Materials and Methods |
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Immunohistochemistry for PGP 9.5
Seven male thoroughbred horses without any abnormalities in the joint were used in this study. All experiments were performed under protocols following the Guidelines for Animal Experimentation, Graduate School of Veterinary Medicine, Hokkaido University. The animals were deeply anesthetized with pentobarbital and thiopental sodium and were sacrificed by bloodletting from the cervical artery. Joint capsules at the carpal joints on both sides were quickly removed and fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4) for 24 hr. They were dissected into small pieces (1 x 1 x 1 cm) and dipped in 30% sucrose solution overnight at 4C. Frozen sections about 20 µm thick were prepared in a cryostat (CM 3050; Leica, Nussloch, Germany) and mounted on poly-L-lysine-coated glass slides.
Immunostaining of PGP 9.5 was carried out according to the avidinbiotin complex (ABC) method. The sections were treated with 0.3% Triton X-100 in 0.1 M PBS, pH 7.4, for 1 hr and then with 0.3% H2O2 in methanol for inhibition of endogenous peroxidase activity. After treatment with a blocking serum for 30 min, the sections were incubated with a rabbit polyclonal antiserum against human PGP 9.5 (RA95101; Ultraclone, Isle of Wight, UK) diluted from 1000 to 48,000 overnight at room temperature (RT). The sections were then incubated with biotinylated goat anti-rabbit immunoglobulins and ABC (Histofine kit; Nichirei, Tokyo, Japan). The antigenantibody reaction was visualized by incubation in 0.05 M Tris-HCl buffer (pH 7.6) containing 0.01% 3,3'-diaminobenzidine (DAB) and 0.002% H2O2. In some cases, DAB reactions were enhanced by adding 0.04% nickel ammonium sulfate.
For immunostaining of whole-mount preparations, we used flat-surfaced synovial membrane and small pieces of the villous region, which contained approximately 10 villi. The staining procedure was essentially identical to the method used for frozen sections except for the constant use of Triton X-100-containing PBS for dilution of the antiserum and rinsing of sections. The tissues were always processed in a free-floating manner in glass bottles shaken by a rotator. After all the steps of immunostaining, the flat-surfaced synovial membrane and villi, which were cut off at the base of villi, were mounted on glass slides and sealed by coverslips with glyceringelatin.
Double Staining with Immunohistochemistry and Acid Phosphatase
For detection of acid phosphatase activity, frozen sections were stained according to
Immunoelectron Microscopy
Frozen sections of the synovial membrane 20 µm thick were stained with the PGP 9.5 antiserum as described above except for omitting the treatment with Triton X-100 in PBS. After DAB reaction, stained sections were postfixed with 1% OsO4 for 30 min. They were dehydrated through a graded series of ethanol and embedded directly in Epon 812 on glass slides. After the embedded sections were detached from the glass slides, ultrathin sections were prepared and stained briefly with lead citrate for observation with a transmission electron microscope (H-7100; Hitachi, Tokyo, Japan).
Western Blotting
The horse synovial membrane, brain, jejunum, and liver were homogenized with 50 mM Tris-HCl buffer (pH 7.5) containing 10 mM EDTA and solubilized in the presence of 1 mM phenylmethylsulfonyl fluoride, 10 mM EDTA, and 1% Triton X-100 for 30 min on ice. The samples were reduced with 0.7 M 2-mercaptoethanol and subjected to 12% polyacrylamide electrophoresis. The proteins were electrophoretically transblotted onto a nitrocellulose membrane. The membrane was then blocked with 5% powdered milk in 0.01 M PBS containing 0.05% Tween 20 and incubated with the antiserum against PGP 9.5, followed by incubation with peroxidase-labeled goat anti-rabbit immunoglobulins (DAKO; Carpinteria, CA). The antigenantibody complex was visualized with DAB solution containing H2O2.
Cloning and Sequencing of Horse PGP 9.5 cDNA
Total RNA was extracted from the brain and synovial membrane by the guanidine isothiocyanate method using TRIzol solution (Gibco BRL; Gaithersburg, MD), according to the manufacturer's directions. Poly (A)+ RNA was prepared by use of oligo-d(T) cellulose columns (Clontech; Palo Alto, CA), and used as a template for reverse transcription-polymerase chain reaction (RT-PCR). The forward (5'-tgctgctgctgtttcccctc-3') and reverse (5'-aactggcgccatggttcacc-3') PCR primers were designed on the basis of common sequences of human and rat PGP 9.5 cDNA (GenBank, X04741 and D10699, respectively) (Figure 6). The PCR product was electrophoresed in a 2.0% agarose gel, purified by Geneclean II Kit (Bio 101; Vista, CA) and ligated into a pCR II plasmid vector (Invitrogen; Carlsbad, KY). The sequences of the obtained cDNAs were determined using a dye terminator cycle sequencing kit (Perkin Elmer Applied Biosystems; Foster City, CA).
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Northern Blot Analysis
Sixty µg of total RNA was separated on a 1% agarose/formaldehyde gel and transferred to a nylon membrane (Amersham; Poole, UK). A cDNA probe was labeled with [-32P]-dCTP using a multiprime DNA labeling kit (Amersham). Nylon membranes were hybridized with the labeled cDNA probe, washed, and exposed to X-ray film (X-OMAT AR; Kodak, Rochester, NY) for 1 week.
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Results |
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Light Microscopy
Immunostaining of frozen sections using the antiserum against PGP 9.5 demonstrated cells scattered in the synovial intima that covered the inner surface of the fibrous capsule (Figure 1). The PGP 9.5-immunoreactive cells were small and round, extending a few thin, long cytoplasmic processes. Typical fibroblasts in the subintimal collagenous layer were free of immunoreactivity. In the synovial villi, most of the cells gathering along the surface were intensely immunolabeled (Figure 2 and Figure 3), sometimes displaying an epithelium-like arrangement. The immunoreactive cells in the villi possessed a round cell body and extended branching processes towards the joint cavity. The processes on the surface were regularly and densely arranged to form a superficial plexus, a kind of lamina limitans (Figure 3). Here, again, slender fibroblasts intervening in the subintimal dense connective tissue remained unreactive. At villous tips, subintimal spaces lacked collagen bundles and were occupied by a clear amorphous material, including blood vessels. The nonfibrous core contained PGP 9.5-immunoreactive dendritic or spindle-shaped cells, which appeared to be continuous with intimal immunoreactive cells (Figure 3). Other intense immunoreactivity for PGP 9.5 occurred in nerve fibers dispersed mainly in the villous region, but these were few. The synovial membrane lacked PGP 9.5-immunoreactive neuronal somata. The synoviocytes and nerves showed the same staining for the antiserum diluted in different concentrations. The most intense immunoreactivity was obtained in 1:12,00020,000. When the antiserum was more diluted, the immunoreactivity in the synoviocytes and nerve elements gradually decreased in intensity. The positive staining produced by the rabbit anti-PGP 9.5 serum could be completely eliminated by substitution of the immune serum with a nonimmune rabbit serum.
Whole-mount preparations of the flat-surfaced synovial membrane and villi demonstrated the entire shape of the immunoreactive synoviocytes, especially extensions of the processes. The cells dispersed in the intima of flat synovial membrane extended uni- or bipolar processes which ran in roughly the same direction as those of adjacent cells and which exhibited terminal branching (Figure 4). When approaching a villous region, these processes became more complicated and branched in a dendritic fashion. All immunoreactive cells in the villi extended branching processes towards the joint cavity to form a plexus of processes on the synovial surface (Figure 6).
Histochemistry for acid phosphatase stained round cells scattered on and near the surface of the synovial membrane. Double staining with the PGP 9.5 antiserum showed a complementary distribution of PGP 9.5 immunoreactivity and acid phosphatase activity (Figure 5). No cells were convincingly positive with both markers. Thus, different cells labeled by each method were intermingled in the synovial intima, although acid phosphatase-positive cells were less numerous than PGP 9.5-immunoreactive cells.
Electron Microscopy
By electron microscopy, immunoreactive cells were identified as cells labeled with electron-dense materials (Figure 7 and Figure 8). The immunoreactivity occurred diffusely and homogenously throughout the cytoplasm along the entire extension, including the tips of fine processes. Immunoreactive cells gathered to form a rough cell line close to the surface of the synovial membrane, but there were no specializations for cell-to-cell contacts. These cell bodies were characterized by free ribosomes and well-developed rough endoplasmic reticulum with broad cisternae (Figure 8). Filamentous structures were rich in the cytoplasm, especially in the processes. There were a few lysosomes but no secretory granules, which were reported in synoviocytes of some rodents (
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Western and Northern Blottings
Western blotting yielded a predominant immunoreactive band, at approximately 27 kD, in the extracts of brain, jejunum, liver, and synovial membrane (Figure 9A). The immunoreaction in the synovial membrane was significantly intense, although it was weaker than that in the brain and intestine, which exhibited large numbers of PGP 9.5-containing nerve elements.
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The expression of PGP 9.5 mRNA was examined in the equine brain and synovial membrane. RT-PCR using two conserved regions between human and rat PGP 9.5 cDNA as primers detected a clear band at 404489 base pairs (BP) in both samples. Cloning of the PCR products from the equine brain and synovial membrane demonstrated the identical sequence with 420 BP (registered to GenBank AB013344). Its homology with the corresponding region of human and rat PGP 9.5 was 98% and 97% in amino acid sequence (not shown) and 92% and 89% in cDNA sequence (Figure 10). In Northern blot analysis in which a cDNA fragment of horse brain PGP 9.5 was used as a probe, a significant band of the same size as the brain PGP 9.5 was detected in the synovial membrane at a condition of total RNA 60 µg, although it was less intense than that of the brain (Figure 9B).
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Discussion |
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The existence of PGP 9.5-like immunoreactivity in Type B synoviocytes is surprising from the general view of selective localization of PGP 9.5 in neurons and some ontogenetically and functionally related cells (
Identification of the PGP 9.5-immunoreactive cells as Type B synoviocytes is supported by the present double staining combined with acid phosphatase activity. Histochemistry for acid phosphatase, a representative lysosomal enzyme, can label macrophagic cells with a developed lysosomal system in various tissues.
For cytochemical detection of Type B synoviocytes, the following antisera and markers have been reported to be available. MAb 67 was able to stain the cell surface of synoviocytes, but also reacted with connective tissue fibers and vascular elements in humans (
Previous cytochemical and ultrastructural studies have suggested that Type B synoviocytes possess broad, branching processes that extend towards the joint cavity (
The existence of a neuron-specific substance in Type B synoviocytes can not be explained by ontogenetic events. Earlier ultrastructural observations, which focused on the rich existence of secretory granules, have indicated the endocrine nature of Type B synoviocytes (
PGP 9.5 is a useful marker for cytochemical detection of Type B synoviocytes in the horse. At present, however, this does not hold true for other mammals. Several immunohistochemical studies have used PGP 9.5 antisera for investigation of the innervation in the joint of rat, sheep, and human but did not describe any positive staining in synoviocytes (
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Acknowledgments |
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We thank Dr Hiroyuki Taniyama (Rakuno Gakuen University) for cooperation with the sampling.
Received for publication July 27, 1998; accepted October 27, 1998.
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