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PATHOLOGY OF THE SYNOVIAL MEMBRANE IN GOUT L I G H T AND ELECTRON MICROSCOPIC STUDIES. INTERPRETATION OF CRYSTALS IN ELECTRON MICROGRAPHS H. RALPH SCHUMACHER T h e ease of obtaining synovial fluid and the recent well-deserved emphasis on demonstration of monosodium urate (MSU) crystals in synovial fluid as a diagnostic aid in gout have focused most attention on the synovial fluid in this disease. Interaction of MSU crystals and polymorphonuclear leukocytes (PMN) i n synovial fluid is clearly important i n pathogenesis of gouty synovitis ( 1 4 ) ) . However, synovial membrane also has a role i n gouty arthritis. This report reviews the synovial membrane findings and some of their implications. Structures possibly confused with MSU i n electron microscopic studies are also discussed.

TOPHI T h e tophus is the most characteristic lesion of gout and can be found in the synovium as well as elsewhere. W e have found synovial tophi by needle biopsy i n 3 of 10 patients with acute gouty arthritis (5). A synovial tophus was found 2 days after onset in 1 man with his first attack of gouty arthritis. Synovial tophi antedating acute attacks have also been From the University of Pennsylvania School of Medicine and the Veterans Administration Hospital, Philadelphia, Pennsylvania. H. Ralph Schumacher, M.D.: Associate Professor of Medicine, University of Pennsylvania School of Medicine, and Chief, Arthritis Section, Veterans Administration Hospital. Address reprint requests to Dr. Schumacher at VA Hospital, Philadelphia, Pennsylvania 19104.

reported by McCarty (6). T h u s at least in some cases there is evidence that urate crystal deposition in the synovium may precede the typical acute attack. T h e synovial tophi as shown in Figure 1 often lie near the joint surface and are weakly encapsulated so that it has been suggested that minor trauma or some change in the crystal equilibrium within the tophus might easily allow release of sufficient crystals into the joint to precipitate attacks. T h e fibrin between this tophus and the joint space is not typical but could have resulted if crystals had recently been released from the tophus with a fibrin clot as part of the process of sealing off what was left of the crystal deposit. Of course it can also be speculated that crystals are initially precipitated i n the synovial fluid and directly initiate the attack in this fashion. Crystals in tophi in synovium and elsewhere are acicular and often are arranged radially in small clusters (7). T h e crystals i n tophi have been identified by Howell et al (8) as monosodium urate (MSU). Water-based fixatives or stains quickly extract these water-soluble crystals, thus only a n amorphous material is retained at the previous site of the crystals. Such a lesion as shown in Figure 1 can raise the suspicion of a tophus, but identical foci can also be seen in occasional rheumatoid synovia (7) and in pseudogout (9). I n the latter disease some calcium pyrophosphate crystals usually remain after waterbased fixatives. These can be identified with polarized light.

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Fig 1. Synovial tophus ( T ) . Note the thin synovial lining layer, sparse collagen ( C ) , and thin rim of fibrin ( F ) between the tophus and the joint space. T h e crystals are dissolved out in Rouin's solution leaving only the amorphous crystal matrix. T h e cells at the margin of the tophus are fibrocytes and histocytes. Note the dramatic fibrin around this tophus. One might speculale tliat crystals Iiad ruptured from here w i t h the fibrin deposition being the first stage in a new walling off of the tophus.

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Fixation of synovium i n absolute alcohol and processing of the tissue preferably without water allow sections to be cut and examined under compensated polarized light for definitive demonstration of the negatively birefringent MSU crystals. T h e De Galantha stain, which can be used if staining is desired, colors the crystals black-brown. This stain is not specific but is useful and simple (10). Tophi i n the synovium are surrounded by surprisingly few cells (mostly fibrocytes) and the thin fibrous rim. Acute inflammatory cell infiltration has not been seen around these tophi. Scattered giant cells and lymphocytes are much more common than neutropliils at the tophus margin. T h e tophus matrix as noted above looks amorphous after dissolution of the crystals. This material has long been considered a possible factor in the initial precipitation of the MSU crystals. Lipid and mucopolysaccharides have both been demonstrated in tophi (7). Sokoloff (7) reported that the matrix substances might conceivably exude into the tophus as

part of the crystal response, but a role for the matrix in precipitating the crystals was and still is more intriguing. Electron microscopy of a synovial tophus is shown in Figure 2. T h e water-based fixatives for EM dissolve the crystals and leave only the crystalshaped clefts that are often in parallel arrays. Electron diffraction attempts confirm that the crystal is not remaining. Between the crystals the finely granular matrix material is demonstrable. Similar material also outlines the crystal clefts and one can wonder if in life it coats the crystal or is incorporated into it. T h e EM appearance of this granular electron-dense material is consistent with protein or mucopolysaccharide. EM histochemical studies have not yet been reported. Cells surrounding the tophus studied by EM were predominantly fibrocytes. Lipid droplets were common in these cells, which also often had lucent patches of cytoplasm suggesting cell degeneration. Crystals were rarely identified in these cells. When seen they were invariably i n phagosomes. T h u s

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Fig 2. Margin of a synovial tophus. Crystals ( C ) are identified only by the clear spaces from which they were dissolved and are surrounded by finely granular material. Most crystals are extracellular. T h e occasional intracellular crystals (arrows) are in phagosomes of the surrounding cells. L = lipid. Electron micrograph. X 22,000.

EM studies give no support for any intracellular synthesis of crystals. Demonstration of tophi in a synovial biopsy is diagnostic of gout, but as noted above tophi are not found in even 50% of blind needle biopsies. Mikkelsen et al ( 1 1) reported urate deposits in synovia of 8 of 23 patients with gout. Rodnan et aE (12) found

tophi in 6 of 14; Basset and coworkers (13) demonstrated microtophi in 4 of 7 gouty synovia with needle biopsy. Certainly some sampling problems account for negative biopsies, but it is also not an established fact that tophi are present in all joints with acute gouty arthritis.

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ACUTE GOUTY SYNOVITIS Tlie recent report by Agudelo and Schumacher (5) is tlie only systematic study of synovium in acute gouty arthritis since crystal identification has allowed definite diagnosis. All 10 specimens studied showed exudate containing polymorpl~onuclear neutrophilic leukocytes and fibrin adhering to the synovial surface. Some areas showed proliferation of synovial lining cells (SLCs). There was diffuse superficial and perivascular infiltration with polymorplionuclear leukocytes (Figure 3). In addition, even in synovia examined during tlie first known attack and as little as 2 days after onset, there were lymphocytes, macrophages, and occasional plasma cells. Although tophi are sometimes demonstrable, crystals do not seem to be present i n the areas of synovial inflammation. Studies to date (5,14) have not shown crystals by either light microscopy or EM in tlie synovial lining cells or in the inflammatory cells in the synovial membrane. Sokoloff (7) reported finding a few isolated crystals in the interstitium in acute gouty synovitis and suggested that they might represent incipient tophus formation. Crystals have been found in synovial effusion large mononuclear cells that may be lining cells (SLC) released into the joint space (5). Also, i n dogs injected with intraarticular monosodium urate crystals, the crystals have been occasionally identified in SLC still attached to the synovial membrane (15). During acute gouty synovitis there is also vascular congestion, some SLC proliferation, cellular debris, and extravasated erythrocytes. Findings vary among several biopsy specimens from the same joint showing the focal nature of the acute synovitis (5). Occasional giant cells are seen among the SLC (Figure

4)Additional findings on EM in the acute synovitis include the predominance of type B and intermediate SLC (5,16). Many SLC liad large lipid cleposits. Degranulated and degenerated PAMN can be seen in the interstitium. Pliagosomes of synovial macrophages contained cell fragments or entire PMN. PMN in vessel lumens were also degranulated (1i) suggesting tlie possible effect of some diffusible substance (not only direct exposure to crystals) i n PMN degranulation (Figure 5). Some venules had gaps between endotlielial cells where PMN and erythrocytes may have emigrated, multilaminated basement membranes, platelet or cellular plugs, and dense protein-like material in the vessel wall but there was no evidence of vascular necrosis. Tlie nature of tlie

dense deposits is not known but they do morpliologically resemble tlie electron-dense deposits in glomerular vessel walls in lupus nephritis. Microtubules (Figure 5) are easily demonstrated in venular endothlium, pericytes, and SLCs in acute gout but synovial membrane PMNs in tlie specimens studied have rarely liad demonstrable microtubules. Colchicine treatment of 1 patient did not alter synovial microtubule numbers (5).

CHRONIC GOUTY SYNOVITIS T h e synovial membrane in interim gout may show tophi as mentioned above (i). SLC proliferation and chronic synovitis (1 8,19) with plasma cell infiltration can also be seen in patients not experiencing acute attacks. Similar chronic changes have also been seen in pseudogout (20,21). Chronic synovitis with SLC proliferation and plasma cell clusters has been produced by repeated intraarticular injection of MSU crystals into dog joints (15). There has been no EM study of tlie interim chronic synovitis in human gout.

PROBLEMS IN ULTRASTRUCTURAL IDENTIFICATION OF URATE CRYSTALS Electron microscopic evaluation of crystals in tissue is complicated by problems that are of interest and important in interpreting papers on this subject. Hard crystals such as calcium pyrophosphate (CPPD) are commonly dislodged so that they distort adjacent structures and may leave only irregular holes (9,22). Other dislodged material can leave crystalshaped holes. Lipid as seen in Figure GA is softer than adjacent tissues and can be broken loose in sections giving crystal-shaped holes. Invaginations of cells as seen in Figure GB can be confusing if as here tlie collagen in tlie invagination and around the cell did not stain well. Nuclear processes may extend far from the main nucleus (Figure 7) and produce cytoplasmic clefts especially i n cells that may have some degenerative changes as a result of varying periods in the joint space. Such nuclear process-produced clefts are a possible explanation for cytoplasmic clefts attributed to intracytoplasmic MSU by Kiddle et n l (22). Eosinophil granules have somewhat lucent crystalloids that especially when free in a phagosome might be confused with MSU crystals (Figure 8). Some animal PMNs have similar crystalloids. MSU crystals as noted above are dissolved by

A

B Fig 3. A. Synouial villi i n acute gouty arthritis. There is dramatic vascular congestion as well as intense inflammatory cell infiltration with PMN and lymphocytes. Some exudate ( E ) below also contains PMN and lymphocytes. H & E stain. )r 90. B. Higher inagnification shows some mild proliferation of synovial lining cells in the area between the arrows. P M N are most evident superficial to the uessels ( V ) whereas lymphocytes surround the uenules. H Q E stain. X 360.

Fig 4. Giant cells (arrows) just beneath the SLC in gouty synovium. Note also the superficial fibrin (F). H Q E stain. X 180.

Fig 5. Degrnnulation of PMN in vascular lumen ( L ) in acute gouty synouitis. Note also some dense material ( D ) between the endothelium and pericyte ( P ) . T h e arrows identify microtubules. Electron micrograph. X 22,000.

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B Fig 6. A. Artefactual lucent spaces (arrows) with suggestion of crystal outlines where lipid ( L ) was dislodged or the softness of the lipid caused tearing of the tissue. Original magnification X 32,000. B. Cross section of a crystal-shaped invagination in a synovial cell. T h e collagen ( C ) is not well stained. Original magnification X 22,000.

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Fig 7 . A. Long nuclear processes can be associated w i t h artefactiial rlefts (arrows). Original nzagnification in the cytoplasm

of

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21,000. B. Siniilar clefts X 32,000.

a patient w i t h rheumatoid arthritis, not definitely associated with nucleus. Original magnification

the water-based fixatives used for EM. Similar dissolution seems to occur with cholesterol and cystine crystals (23,24) so that they may appear very similar in electron micrographs. Needle-like lucent structures similar to the EM appearance of urates in tissue have also been seen in the liver of porphyria cutanea tarda (25) and with oxalate crystals (26). A variety of other materials are crystalline in electron micrographs. Although they should not be confused with MSU, they d o deserve mention. They include immunoglobulins (27,28) and unidentified material in diabetics (29). We have seen other dense crystal-like material in joints by EM that have not been fully cliaracterized i n patients with undiagnosed disease. Some resemble tiny dense hydroxyapatite-like rods in clumps of granular material (Figure 9). Other dense crystallike materials may be lipid (Figure 10). Artefact from

dirt or stain precipitates are often present on EMS and could superficially be confused with dense crystals, but their distribution without relation to cell structures helps in identification (Figure 8).

REFERENCES 1. Weissmann G: T h e molecular basis of acute gout. Hos-

pital Practice 6:43-52, 1971 2. Schumacher HR, Phelps P: Sequential changes in human polymorphonuclear leucocytes after urate crystal phagocytosis. Arthritis Rheum 14:513-526, 1971 3 . Scliumacher HR: Morphologic studies of the unresolved problems in crystal-induced arthritis. InHo 7:3, 1974 4. Brandt K: T h e effect of synovial hyaluronate on the ingestion of monosodium urate crystals by leucocytes. Clin Chim Acta 55:301-315, 1974 5. Agudelo CA, Schumacher HR: T h e synovitis of acute

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Fig 8. Crystalloids from human eosinophil granules (arrows). Note also the very dense stain precipitate artefact. Electron micrograph. X 44,000.

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Fig 9. Clump of granular material and tiny dense rods in phagosome of a synovial fluid P M N in undiagnosed arthritis X 21,000.

gouty arthritis. A light and electron microscopic study. Hum Pathol4:265-279, 1973 6. McCarty DJ: Personal communication 7. Sokoloff L: T h e pathology of gout. Metabolism 6:230241, 1957 8. Howell RR, Eanes ED, Seegmiller JE: X-ray diffraction studies of the tophaceous deposits in gout. Arthritis Rheum 6:97-103, 1963 9. Reginato AJ, Schumacher HR, Martinez VA: T h e articular cartilage in familial chondrocalcinosis. Light and electron microscopic study. Arthritis Rheum 17: 977-992, 1974 10. DeGalantha E: Technic for preservation and microscopic demonstration of nodules in gout. Am J Clin Pathol 5:165-166, 1935 11. Mikkelsen MW, Duff IF, Castor CW, et al: Synovial specimens obtained by knee joint biopsy. Acta Med Scand 341:15-29, 1958 (suppl) 12. Rodnan GP, Yunis EJ, Totten RS: Experience with punch biopsy of synovium in the study of joint disease. Ann Intern Med 53:319-331, 1960 13. Basset F, Coste F, Guiraudon C, et al: La ponctionbiopsie de la synoviale d u genou. Presse Med 72:18811886, 1964 14. Sokoloff L: Pathology of gout. Arthritis Rheum 8:707713, 1965

15. Schumacher H R , Phelps P, Agudelo CA: Urate crystal induced inflammation in dog joints: sequence of synovial changes. J Rheumatol 1 : 102-113, 1974 16. Barland P, Novikoff AB, Hamerman D: Electron microscopy of the human synovial membrane. J Cell Biol 14:207-220, 1962 17. Schumacher HR, Agudelo CA: Intravascular degranulation of neutrophils: an important factor in inflammation? Science 175:1139-1140, 1972 18. Schwartz S, Cooper N: Synovial membrane punch biopsy. Arch Intern Med 108:400-406, 1961 19. Rodnan GP, Golomb MW: Gout in the negro female. Am J Med Sci 236:269, 1958 20. Schumacher HR: T h e synovitis of pseudogout: electron microscopic observations. Arthritis Rheum 11:426435, 1968 21. Moskowitz RW, Harris BK, Schwartz A, et al: Chronic synovitis as a manifestation of calcium crystal deposition disease. Arthritis Rheum 14: 109-116, 1971 22. Riddle JM, Bluhm GB, Barnhart MI: Ultrastructural study of leucocytes and urates in gouty arthritis. Ann Rheum Dis 26:389-401, 1967 23. Chapman AJ, Martin J H , Race GJ: Xanthoma tendinosum, a localized benign reticuloendotheliosis, studied by thin section and electron microscopy. Texas Rep Biol Med 26:297-301, 1968

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Fig 10. Synouial lining cell in rheumatoid arthritis w i t h electron-dense material that suggests lipid because of the waviness produced by cutting. T h e angulated margins suggest the possibility of a crystal. Electron micrograph. X 80,000.

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24. Schulman JD, Wong V, Olson WH, et al: Lysosomal site of crystalline deposits in cystinosis as shown by ferritin uptake. Arch Pathol 90:259-264, 1970 25. Waldo ED, Tobias HT: Needle-like cytoplasmic inclusions in the liver in porphyria cutanea tarda. Arch Pathol 96:368-373, 1973 26. Evans GW, Phillips G, Mukherjee T h l , et al: Identification of crystals deposited in brain and kidney after xylitol administration by biochemical, histochemical and electron diffraction methods. J Clin Pathol 26: 32-36, 1973

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27. Clark C, Rydell RE, Kaplan ME: Frequent association of Ighl with crystalline inclusions in chronic lymphatic leukemia lymphocytes. N Engl J hled 289:113117, 1973 28. RIennemeyer K, Hammar SP, Cathey WJ: Malignant lymphoma with intracytoplasmic IgM crystalline inclusions. N Engl J Ned 291:960-963, 1974 29. Shafiq SA, Milhorat AT, Gorycki MA: Crystals in muscle fibers in patients with diabetic amyotrophy and neuropathy. Neurology 18:785-790, 1968