The stromal cell reaction of pigmented villonodular synovitisAn electron microscopic study.код для вставкиСкачать
The Stromal Cell Reaction of Pigmented Villonodular Synovitis: An Electron Microscopic Study By JOHN C. WYLLIE,M.D. Synovium obtained from a patient with pigmented villonodular synovitis was studied with the electron microscope. Attention was directed to the stromal cell hyperplasia, a histological feature of this disease. It was shown that the participating cells were of two main types. The P a curious disease of joints, bursae, and tendon sheaths, is characterized by villous and nodular synovial outgrowths, increased vascularity, hyperplasia of stromal cells within the synovium, infiltrates of hemosiderin-containing macrophages, and a serosanguinous joint effusion.13 The etiology and pathogenesis of this disorder are unknown. The nature of the proliferating stromal ce.lls is also uncertain; they have been regarded as potential or actual macrophages,l as connective tissue-forming cells similar to fibroblasts? and as neoplastic cells of vasculals or synoviale origin. This paper describes an electron microscopic study of a case of pigmented villonodular synovitis. Particular attention was paid to the stromal cell reaction in order to determine the nature of the participating cells. IGMENTED VZLLONODULAR SYNOVITIS, more abundant cell resembled the fibroblast of connective tissue in general, and appeared capable of collagen and mucopolysaccharide synthesis. The second cell type was similar t o a macrophage and contained numerous hemosiderin inclusions. joint, but there was no limitation of active and passive movement. The other joints were normal. A chest radiogram was negative; a radiogram of the right knee showed soft tissue swelling. The hemoglobin was 12.8 Gm. per cent, white blood cell count 8750, sedimentation rate 21, C-reactive protein negative, anti-streptolysin 0 titer 166 (subsequently within normal limits), latex fixation negative, and serum protein electrophoresis normal. Serosanguinous fluid, aspirated from the right knee joint, was sterile. A diagnosis of pigmented villonodular synovitis was made, and a surgical synovectomy was performed. At operation, the joint cavity contained viscid serosanguinous fluid, detached villi, and fibrin. The thickened, reddishbrown synovial membrane was thrown into numerous villous and nodular folds. The patient made an uneventful recovery. Twelve months later she was symptom-free; there was slight residual swelling of the soft tissues about the right knee joint. MATERIALSAND METHODS The patient, a 13-year-old girl, was admitted to hospital with pain, recurrent swelling, and instability of her right knee of 10 months’ duration. She gave no history of trauma, infection, or other joint involvement, and her general health was excellent. An effusion was demonstrated in the right knee Specimens of synovium, for light and electron microscopy, were obtained during the surgical synovectomy. For light microscopy, the synovium was &xed for 18 hours in 10 per cent neutral buffered Formalin, dehydrated, and embedded in paraffin. Sections approximately 5 /.in i thickness were stained with hematoxylin, phloxine, and saffron (H.P.S. ), and phosphotungstic acidhematoxylin, and tested for Perls’ Prussian blue reaction for ferric salts. For electron microscopy, representative samples From the Department of Pathology, Queen’s University, and the Kingston General Hospital, Kingston, Ontario, Canada. Supported b y a research grant from the Canadian Arthritis and Rheumatism Society. JOHN C . WYLLIE, M.D.: Associate Professor of Pathology, Queen’s University, Kingston, Ontario, Canada. Reprint requests should be addressed to Dr. Wyllie. ARTHRITISAND RHEUMATISM,VOL. 12, No. 3 1969) CASEREPORT (JUNE 205 206 JOHN C. WYLLIE Fig. 1.-Photomicrograph showing the thickened synovial membrane infiltrated by hemosiderin-containing macrophages (arrows) and stromal cells. Note multinucleated giant cells near the synovial lining cell layer (S). JS, joint space. H.P.S. X 380 (original size reduced). Fig. 2.-Photomicrograph showing proliferating stromal cells in vascular synovial tissue. H.P.S. X 150 (original size reduced). of the diseased synovium were cut into 2 X 2 X 1 mm. blocks, fixed for 90 min. in cold (4OC) 2 per cent buffered (pH 7.4) osmium tetroxide containing 0.045 Gm. of sucrose per milliliter,7 dehy- drated in ascending concentrations of ethanol, and embedded in Maraglas 655-Cardolite NC-513.8 Material from 20 blocks was examined. The blocks were cut on a Huxley Ultramicrotome with glass STROMAL CELL RGACTION OF PIGMENTED VJLLONODULAR SYNOVITIS 207 Fig. 3.-Photomicrograph showing widespread fibrosis within a synovial villus. There are scattered cells in the fibrous tissue. Note the well-formed small blood vessels. H.P.S. X 155 (original size reduced). knives. Thick sections, of approximately 1 p, were stained with toluidine blue for orientation purposes. Blocks containing foci of stromal cell hyperplasia were selected and thin sections prepared. Thin sections were picked up on uncoated copper grids, stained with uranyl acetate9 for 30 min., followed by lead citratelo for 2 min., and examined in an Hitachi HU-11 or RCA EMU3D electron microscope. Electron micrographs were taken at original magnifications of 2500 to 12,000 and enlarged photographically to the desired size. hSULTS Light Microscopy The typical changes of pigmented villonodular synovitis were observed in the synovial membrane. Synovial villi, covered by a layer of synovial lining cells two or three cells in depth, were infiltrated by clusters of macrophages containing granules of golden-brown pigment (Fig. 1).A similar pigment was present in some of the synovial lining cells. This pigment proved to be iron-positive and was regarded as hemosiderin. There was a striking hyperplasia of stromal cells, resulting in the formation of masses of closely packed cells in the loose, well-vascularized connective tissue of the villi (Fig. 2). These cells had large oval nuclei and moderately abundant weakly acidophilic cytoplasm; some contained granules of hemosiderin. The deposition of variable amounts of collagen accompanied the stromal cell reaction; in some areas, fibrosis was marked, partially obliterating the cellular and vascular reaction (Fig. 3). Well-formed small blood vessels ( arterioles, venules, and capillaries) were present in regions of stromal cell hyperplasia and also in the more fibrotic areas (Figs. 1, 2, and 3). Multinucleated giant cells with abundant strongly acidophilic cytoplasm were observed immediately below the synovial lining cell layer ( Fig. 1). There were a few “foam cells,” with finely vacuolated cytoplasm, scattered among the stromal cells. Electron Microscopy of the Stromul Cell HYPerpM The stromal cells were dispersed randomly in a matrix of low electron density 208 JOHN C. WYLLIE Fig. 4.-Electron micrograph from an area of stromal cell hyperplasia showing loosely dispersed stromal cells containing electron-dense inclusions. There are collagen fibers (C) in the intercellular space. m, marginal fold; P, cell process. Uranyl acetate and lead citrate X 3080 (original size reduced). containing fibrillar material and small groups of collagen fibers (Fig. 4).The cells were frequently closely apposed, but lacked a definite orientation and did not form vascular sprouts or channels (Fig. 4). Two principal cell types could be distinguished on the basis of their fine structure. The more abundant cell, which resembled the fibroblast of connective tissues in general, was elongated, with a large nucleus and abundant fibrillar cytoplasm (Figs. 5 and 6). The Golgi complex was highly developed, consisting of the usual parallel arrays of saccules and accompanying vacuoles (Figs. 5 and 6), and was widely dispersed throughout the cell. A moderate quantity of rough-surfaced endoplasmic reticulum was also present, chiefly distributed in the peripheral cytoplasm (Figs. 5 and 6). The cisternae of the roughsurfaced endoplasmic reticulum were mildly dilated, containing granular and fibrillar material. Large mitochondria were numerous (Fig. 5 ) , and a centriole was often present (Fig. 5 ) . These cells contained a few membrane-bound vesicles, some filled with finely granular electrondense material (Figs. 5 and 6). Lipid droplets were also observed (Figs. 5 and 6), but were sparse. The less abundant cell type was similar to a macrophage and had a large indented eccentrically placed nucleus with a moderate quantity of cytoplasm (Fig. 7). This cell frequently contained numerous membrane-bound vesicles (similar in appearance to those observed in fibroblast-like cells), the majority filled with finely granular electron-dense material (Figs. 7 and 8 ) . These cellular inclusions corresponded with the golden-brown iron-positive granules which had been observed in various synovial cells by light microscopy and regarded as hemosiderin. I n addition, these inclusions had an identical fine structure to that of hemosiderin as described by Bessis and Breton-Gorius.ll A few lipid droplets were noted in the cytoplasm. Rough-sur- STROMAL CELL REACIlON OF PIGMENTED VILLONODULAR SYNOVITIS Fig. 5.-Electron micrograph showing a stromal cell with part of its nucleus (N) and an elongated cell body. The Golgi complex (G) is well-developed, and rough-surfaced endoplasmic reticulum (r) is moderate in amount. Note delicate fibrils ( f ) in the cytoplasm, electron-dense inclusions (H), and lipid droplets (L). c, centriole; C, collagen; M, mitochondrion. Uranyl acetate and lead citrate X 10,000 (original size reduced). Fig. 6.-Electron micrograph showing a stromal cell with a Golgi complex (G) and a moderate quantity of rough-surfaced endoplasmic reticulum (r). Electron-dense inclusions (H) and lipid droplets (L) are present. c, centriole; C, collagen; M, mitochondrion. Uranyl acetate and lead citrate X 10,000 (original size reduced). 209 210 JOHN C. WYLLIE Fig. 7.-Electron micrograph showing a stromal macrophage. There are numerous membrane-bound inclusions with electron-dense finely granular contents. Rough-surfaced endoplasmic reticulum (r) is sparse. N, nucleus; f, filopodium. Uranyl acetate and lead citrate X 20,000 (original size reduced). Fig. S.-Electron micrograph showing hemosiderin inclusions enclosed within membrane-bound vesicles in a stromal macrophage. The electron-dense particles are randomly dispersed or arranged in a crystal-like pattern (arrow). Uranyl acetate and lead citrate X 70,000 (original size reduced). STROMAL CELL REACTION OF PIGMENTED VILLONODULAR SYNOVITIS Fig. 9.-Electron micrograph showing a “foam cell” with large numbers of lipid droplets (L) in its cytoplasm. N, nucleus. Uranyl acetate and lead citrate X 8320 (original size reduced). Fig. 10.-Electron micrograph showing a multinucleated giant cell adjacent to the synovial lining cell layer (S). Note the large numbers of mitochondria (M) and small vacuoles (v). Uranyl acetate and lead citrate X 5700 (original size reduced). 211 212 JOHN C. Fig. 11.-Electron micrograph showing an area of marked fibrosis with wide separation of stromal cell processes (P). Collagen fibers (C) are accompanied by many smaller fibrils (s). Uranyl acetate and lead citrate X 7500 (originalsize reduced). faced endoplasmic reticulum was sparse deposited, resulting in wide separation of and the Golgi complex small and confined the stromal cells (Fig. 11).Fibrillar mater(Fig. 7). Short filopodia extended from the ial was often abundant in these sites (Fig. cell surface into the intercellular space 11). (Fig. 7). DISCUSSION The stromal cell reaction included two The stromal cell hyperplasia of pigadditional cell types. There were a few cells with large numbers of lipid droplets mented villonodular synovitis was examin their cytoplasm (Fig. 9). It was not pos- ined by electron microscopy. It was shown sible to determine whether these “foam that the principal cell involved in this reaccells” were macrophages, fibroblasts, or tion had a well-developed and extensive some other cell type because their fine Golgi complex and a moderate quantity of structure was obscured by the lipid. Multi- rough-surfaced endoplasmic reticulum. This nucleated giant cells were noted on the cell had thecfine structure of a fibrobla~t’~J~ edges of the stromal cell masses, usually rather than ‘&at of a macrophagef4or endoadjacent to the synovial lining cell layer. thelial cell.15 The second cell type observed These cells, in addition to their large size was similar to a macrophage,14 and freand increased number of nuclei, were char- quently contained numerous inclusions of acterized by a high content of mitochondria hemosiderin, an indication of its phagocytic (Fig. 10). A Golgi complex and numerous capabilities. The stromal cell reaction of pigmented small vesicles were present, but rough-surfaced endoplasmic reticulum, hemosiderin- villonodular synovitis has aroused interest containing inclusions and lipid droplets in the past, but the nature of the participatwere not seen (Fig. 10). In parts of the ing cells has not been clear. Jaffe et al. conlesion, large amounts of collagen had been cluded that the stromal cells were potential STROMAL CELL REACTION OF PIGMENTED VILLONODULAR SYNOVITIS or actual macrophages, basing their opinion on the apparent phagocytic capacity of the cells for hemosiderin and lipid.l We have shown that macrophages are a component of this reaction but are fewer than the stromal fibroblasts. Stromal fibroblasts also appear capable of phagocytosis, as indicated by the occurrence of small amounts of hemosiderin within them. However, phagocytosis is not limited to the functionally phagocytic cells of the reticuloendothelial system but is a property (to a lesser degree) of such connective tissue cells as the fibrobla@ and the type B synovial lining cell.17918 The possible neoplastic nature of pigmented villonodular synovitis has been discussed by Jaf€el9 and by Lichtenstein.*O Jaffe feels that the histological evidence, centered around the accumulation of stroma1 cells which he regards as macrophages, is suggestive of an inflammatory process rather than a neoplasm. He points out that the usual tendency of the disease towards fibrosis would also point to inflammation. Clark, on the other hand, views the lesion as a neoplasm of vascular origin with the histological features of a sclerosing hemangioma.16 We observed well-formed blood vessels ( arterioles, venules, and capillaries ) in the connective tissue where the stromal cells were proliferating. But the stromal cells, though often lying in close proximity to one another, did not form vascular sprouts or channels. Pigmented villonodular synovitis has been also conside.red a tumor of synovial 213 origin, related to the giant cell tumor of tendon sheath origin (the benign synovioma) .16 Electron microscopic studies of synovial tumors are rare. Luse has described the fine structure of a synovial sarcoma.2l The neoplastic synovial cells had a h e l y vacuolated cytoplasm, an indistinct Golgi complex, and tended to form acinarlike structures. They differed significantly from the synovial fibroblasts we observed. The stromal cell reaction of pigmented villonodular synovitis appears basically reparative and is similar to fibrosis subsequent to injury in connective tissue elsewhere, i.e., it is characterized by macrophage activity, proliferation of fibroblasts, increase in vascularity, and collagen deposition. The nature of the injurious agent was not apparent from our study. The fibroblast of the stroma1 cell reaction is the probable cellular source of protein required for the formation of collagen fibers observed nearby, as it possesses the necessary organelles for protein synthesis ( the rough-surfaced endoplasmic reticulum) and secretion (the Golgi complex). The degree of development of the Golgi complex in these cells is noteworthy in view of the localization of mucopolysaccharide synthesis in this organelle.22 Hence this cell, inferentially, is in addition a likely source of the mucopolysaccharides of the surrounding ground substance. ACKNOWLEDGMENTS The author wishes to thank Dr. H. G. Kelly and Dr. M. A. Simurda for providing the material for this study, and Dr. Nathan Kaufman for his criticisms and comments. SUMMAFUO IN INTERLINGUA Synovio obtenite ab un patiente con pigmentate synovitis villonodular esseva studiate per medio del microscopio electronic. Le attention del observator se concentrava super le stromal hyperplasia cellular que es un characteristica histologic del morbo in question. Esseva monstrate que le cellulas participante esseva de duo typos principal. Le typo le plus abundante resimilava, a generalmente parlar, le fibroblast0 de tissu conjunctive e pareva capace de synthese de collageno e mucopolysaccharida. Le secunde typo cellular resimilava un macrophago e contineva numerose inclusiones de hemosiderina. 214 JOHN C. WYLLIE REFERENCES 1. Jaffe, H. L., Lichtenstein, L., and Sutro, C. J. : Pigmented villonodular synovitis, bursitis and tenosynovitis. A discussion of the synovial and bursa1 equivalents of the tenosynovial lesion commonly denoted as xanthoma, xanthogranuloma, giant cell tumor or myeloplaxoma of the tendon sheath, with some consideration of this tendon sheath lesion itself. Arch. Path. 31:731, 1941. 2. Copeman, W. S . C.: Textbook of the Rheumatic Diseases (ed. 3.). Edinburgh and London, E. & S. Livingstone Ltd., 1964, p. 571. 3. Gardner, D. L.: Pathology of the Connective Tissue Diseases. London, Edward Arnold Publishers Ltd., 1965, p. 409. 4. Robbins, S. L.: Textbook of Pathology (ed. 2 ) . Philadelphia and London, W. B. Saunders Co., 1962, p. 1097. 5. Clark, W. S.: Pigmented villonodular synovitis. Bull. Rheum. Dis. 8:161, 1958. 6. Wright, C. J. E.: Benign giant cell synovioma. Brit. J. Surg. 38:251, 1951. 7. Caulfield, J. B.: Effects of varying the vehicle for OsO, in tissue hation. J. Biophys. Biochem. Cytol. 3:827, 1957. 8. Spurlock, B. O., Kattine, V. C., and Freeman, J. A.: Technical modifications in Maraglas embedding. J. Cell Biol. 17:203, 1963. 9. Stempak, J. G., and Ward, R. T.: An improved staining method for electron microscopy. J. Cell Biol. 22:697, 1964. 10. Reynolds, E. S.,: The use of lead citrate at high pH as an electron-opaque stain in electron microscopy. J. Cell Biol. 17:208, 1963. 11. Bessis, M. C., and Breton-Gorius, J.: Iron metabolism in the bone marrow as seen by electron microscopy: a critical review. Blood 19:635, 1962. 12. Movat, H. Z., and Fernando, N. V. P.: The fine structure of connective tissue. I. The fibroblast. Exp. Molec. Path. 1:509, 1962. 13. Porter, K. R.: Cell h e structure and biosynthesis of intercellular macromolecules. In: Connective Tissue: Intercellular Macromolecules. Boston, Little, Brown and Co., 1964, p. 167. 14. Bessis, M., and Thiery, J. P.: Electron microscopy of human white blood cells and their stem cells. Int. Rev. Cytol. 12:199, 1961. 15. Rhodin, J. A. G.: An Atlas of Ultrastructure. Philadelphia and London, W. B. Saunders Co., 1963, p. 50. 16. Bloom, W., and Fawcett, D. W.: A Textbook of Histology (ed. 8 ) . Philadelphia and London, W. B. Saunders Co., 1962, p. 93. 17. Ball, J., Chapman, J. A., and Muirden, K. D.: The uptake of iron in rabbit synovial tissue following intra-articular injection of iron dextran; a light and electron microscopic study. J. Cell Biol. 22:351, 1964. 18. Wyllie, J. C., More, R. H., and Haust, M. D.: The fine structure of normal guinea pig synovium. Lab. Invest. 13:1254, 1963. 19. Jaffe, H. L.: Tumors and Tumorous Conditions of the Bones and Joints. Philadelphia, Lea & Febiger, 1958, p. 532. 20. Lichtenstein, L.: Bone Tumors (ed. 3). St. Louis, C. V. Mosby, 1965, p. 388. 21. Luse, S. A.: A synovial sarcoma studied by electron microscopy. Cancer 13:312, 1960. 22. Peterson, M., and Leblond, C. P.: Synthesis of complex carbohydrates in the Golgi region, as shown by radioautography after the injection of labelled glucose. J. Cell Biol. 21:143, 1964.