juvenile rheumatoidarthritis - annals of the rheumatic ... · summary ricebodies obtained...

Annals of the Rheumatic Diseases, 1979, 38, 8-13

Juvenile rheumatoid arthritis with rice bodies: lightand electron microscopic studiesC. ROSALES WYNNE-ROBERTS AND JAMES T. CASSIDY

From the Division of Rheumatology-Immunology, Department of Medicine, Southern Illinois UniversitySchool of Medicine, Springfield, Illinois, and the Rackham Arthritis Research Unit, Department of InternalMedicine, University of Michigan Medical School, Ann Arbor, Michigan, USA

SUMMARY Rice bodies obtained from a young man with juvenile rheumatoid arthritis were foundby light and electron microscopy to contain cells that appeared viable. The majority of thesecells closely resembled type B synovial lining cells. Type A-like cells were also seen. The cellscontained few mitochondria but often much lipid and glycogen, observations which suggested a

dependence on anaerobic metabolic pathways in the avascular synovial fluid environment. Cellswithin-the rice bodies lay in a matrix of collagen fibres, fibrin, and amorphous material. The source

of the collagen appeared to be the cells themselves. The relatively normal appearance of the cellssuggested that they were protected from many of the inflammatory stimuli present in rheumatoidsynovia. This 'reversion' towards a normal appearance suggested that the stimuli inducing chronicrheumatoid inflammation might not originate in the synovial lining.

Rice bodies, so named because they resemble polishedwhite rice, were extensively described almost acentury ago by Riese (1895). In his classic studyhe reported the microscopic characteristics of ricebodies obtained from the synovial fluids of patientswith tuberculous arthritis. There are no reports ofthe frequency of rice bodies in synovial fluids frompatients with chronic synovitis or in association withspecific diseases such as rheumatoid arthritis. Infact our clinical impression is that rice bodies arerelatively uncommon. In 1965 Albrecht et al.(1965) described biochemical and ultrastructuraldetails of rice bodies from adults with rheumatoidarthritis. The rice bodies from these cases were smalland contained only cell remnants; thus, their des-cription of cellular structure was necessarilylimited. The present report describes the light andelectron microscopic appearance of the cells andmatrix of rice bodies obtained from a young manwith juvenile rheumatoid arthritis.

Case report

The patient is a young white man who developedarthritis of the left knee at the age of 15 years in

Accepted for publication 17 April 1978Correspondence to Dr James T. Cassidy, R4633 KresgeMedical Research Building, University of MichiganMedical School, Ann Arbor, Michigan 48109, USA.

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1973. Swelling of the left calf followed, and anarthrogram showed rupture of a popliteal cyst.Numerous rice bodies were removed at synovec-tomy, and the synovium was described as hyper-aemic and inflamed. The knee improved during thenext year. The left ankle became swollen in theautumn of 1974 and the right knee in early 1975. Thelatter joint was aspirated at that time, and 50 ml ofyellow turbid fluid was removed. The white cell countwas 49000/mm3 with 96% polymorphonuclearneutrophils. A few inclusion bodies were present.The mucin clot was poor. Cultures were negative. Aconsistent programme of aspirin was started atthat time.By June of 1975 the left knee had become mas-

sively swollen. A large effusion was aspirated andnumerous rice bodies were evacuated as completelyas possible through a 16 gauge needle before injec-tion of intra-articular steroid. The fluid was nearlycolourless but very turbid. The white cell count was12 500/mm3 with 74% polymorphonuclear neutro-phils. The mucin clot was fair. No crystals wereseen. A few inclusion bodies were present. The ricebodies were cut readily with a razor, a finding whichsuggested a low collagen content.Symptomatic relief was only temporary and a few

rice bodies were obtained intermittently from theleft knee. The right calf became swollen in theautumn of 1975 and a firm popliteal cyst enlarged

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Juvenile rheumatoid arthritis with rice bodies: light and electron microscopic studies 9

behind the right knee. In October of 1976 moderateeffusions of both knees, together with a smallright popliteal cyst were still present. Full extensionwas preserved at both knees but flexion was limitedto approximately 1100.

LABORATORY DATAThe Westergren erythrocyte sedimentation rate hasvaried between 40 and 53 mm/h. Tests for anti-nuclear antibodies and rheumatoid factors have beennegative. The peripheral blood lymphocytes werenegative for HLA B27 antigen. Ophthalmologicalexaminations have given normal results. Radio-graphs of the left knee between 1974 and 1975showed periosteal new bone formation along themedial and lateral femoral condyles and the pos-terior tibial metaphyseal area. Films have continuedto show soft tissue swelling about both knees andthe left ankle. A sub-Achilles bursitis with anerosion of the posterior tibial cortex was seen on theleft in 1976. Frontal stereoscopic views of the sacro-iliac joints have been negative.

Materials and methods

The rice bodies obtained at arthrocentesis werefixed immediately in 2% paraformaldehyde in 0-01M phosphate buffer, pH 7 2. The bodies werediced into 0 5 to 1 0 mm cubes. After 3 washes incold 0-01 M phosphate-saline buffer, the tissueswere postfixed in Millonig's 1% osmium, pH 7*2,for 2 h at 4°C (Millonig, 1961). The specimens weredehydrated in a graded sequence of ethanols to 100%then infiltrated by and embedded in Spurr epoxyresin (Spurr, 1969).

Thick 1 * 0 [um and ultrathin sections were cut on aReichert ultramicrotome. The thick sections werestained with 1I5% aqueous toluidine blue 0. Theultrathin sections were mounted on Formvar andcarbon-coated copper grids and stained with asaturated solution of uranyl acetate in 50% ethanoland with Reynolds's lead citrate solution (Reynolds,1963). The grids were examined in a JEOL 100Belectron microscope at 60 kV.

Results

LIGHT MICROSCOPYThe rice bodies after aspiration appeared as a massofloosely aggregated material (Fig. 1). The individualrice bodies varied from 0 *2 to 1 * 5 cm in length andwere oval to slightly irregular in shape. They werewhite to slightly yellow and were contained inblood tinged synovial fluid. The toluidine blue 0thick sections showed cells scattered in a blotchy,purple-blue amorphous matrix (Fig. 2). Fibrils were

Fig. 1 Above: A mass ofaggregated material containingrice bodies aspiratedfrom a 17-year-old man withjuvenile rheumatoid arthritis ( x 4). Below: Individualrice bodies of sizes 0 3-4 *6 mm. Some are oval andpebble-like (x 14).

seen around the periphery. Cells of sizes 7 to 21 tmappeared viable, and most were surrounded by paleblue or clear zones. Three types of cells were seen.Some resembled synovial lining cells and sometimescontained granules; some were spindle-shaped and



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10 Rosales Wynne-Roberts, Cassidy

Fig. 2 Above: An overview ofa portion of the interiorofa rice body showing the mottled staining matrix.Cells of various shapes and sizes lie within relativelyclear zones. Cell processes andfilopodia can be seenl insome instances. (1*5% aqueous toluidine blue 0, 1-0 (mSpurr embedded thick section, x 1080). Below: Twocell types are shown. The upper elongated cell isfibroblast-like and shows metachromatic stainingsuggesting the presence ofglycogen or mucopol,v-saccharides. Filopodia are seen at the cell surface. Thecell itself lies in a relatively clear zone of rice bodymatrix. The lower cell contains dark granules and hasmarked filopodia formation (I *5% aqueous toluidineblue 0, 10 pAm Spurr embedded thick section, x 3200).

oval cells containing pale blue vacuoles and areas ofpurple staining were present. That these latter cellsmight contain mucopolysaccharides or glycogenwas suggested by their metachromatic staining. Allcells had either long cell processes or short filopodia.Polymorphonuclear leucocytes, macrophages, lym-phocytes, and plasma cells were not found withinany rice bodies, nor were calcium salts observed.

ELECTRON MICROSCOPYThe predominant cell within the rice bodies wasmost like a type B synovial lining cell (Fig. 3)

(Barland et al., 1962). Some of these were spindle-shaped and similar to fibrocytes of deeper areas ofthe synovium. All cells appeared viable and to be inactive protein synthesis as shown by well developedrough endoplasmic reticulum. The lamellae of theendoplasmic reticulum were often dilated, and theircontents were homogeneous in appearance. Nointra-articular collagen fibril formation was noted.Most cells contained glycogen and sometimes largelipid drops (which appeared to correspond to thepale blue vacuoles of the toluidine blue stainedsections). The lipid was nonmembrane bound andoften surrounded by the glycogen granules. Mito-chondria were sparse, markedly fewer than usual, andcentrioles were occasionally seen. Dark staininglysosomal granules were present in moderate num-bers in some cells and were few in others. Darkstaining granules were also seen extracellularly closeto cell surfaces and were often entwined in filo-podia (Fig. 4). Some granules had the appearanceof being phagocytosed; occasionally an intracellulargranule was bound by a double membrane. Groupsof free ribosomes were seen in the cytoplasm, andmicrofilaments were easily observed. Microtubuleswere not noted. Interestingly, Golgi apparatus wasrarely observed within any cells. Nuclei generallywere slightly irregular in shape and contained 1 or2 nucleoli. No particles suggestive of virus or myco-plasma were seen within any cells. Cell membraneactivity was marked as shown by many slenderfilopodia and micropinocytotic vesicles (coatedpits). Around most of the cells was a zone of col-lagen fibres, sometimes with fine whiskery material(Fig. 4, inset A). The collagen fibres were occasionallyaligned in parallel with each other; however, mostwere short and without specific orientation. Theperiodicity of the collagen was 66 nm. The immediatearea around a few cells was almost devoid of anyamorphous material or fibres. Away from the zonesof collagen a mesh of amorphous material and fibrin(sometimes with a periodicity typical of coagulatedfibrin) made up the mass of a rice body (Fig. 4, insetB). One effete polymorphonuclear neutrophil wasseen by electron microscopy. The nucleus washomogeneous and the cytoplasm was partiallydestroyed.

Discussion

Rice body formation occurs in a small number ofpatients with adult rheumatoid arthritis, and in thisreport we note this phenomenon in a young manwith a persistent course of late-onset, oligoarthricjuvenile rheumatoid arthritis. The more usualfinding in rheumatoid synovial fluids is that ofclumps of fibrin, a byproduct of inflammation



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Fig. 3 Part ofa 'synthetic' or B type appearing fibroblast containing very well developed rough endoplasmicreticulum lamellae (rer). No mitochondria can be identified in this view; however, nonmembrane bound lipid (Lip)and glycogen (GL Y) can be seen. The pericellular area contains small collagen fibres (Cob). Fibrin (FIB) is seenwell away from the cell. Filopodia (fil), lysosomes (Ly), nucleus (N), nucleolar material (n). The arrow points to amicropinocytotic vesicle (coated pit) (uranyl acetate, lead citrate, x 9600).

(Cohen et al., 1975). The morphology of the cellswithin these rice bodies suggested their synovialorigin, presumably from sloughing lining cells. Thecytoplasmic appearance of the rice body cells wascloser to that of normal B lining cells than A cells(Barland et al., 1962). This suggested that theenvironment, though porous to nutrients, was aprotected one, that the cells could proceed primarilywith collagen and hyaluronate synthesis, and thatsome of the intense stimuli to primary lysosomeformation which are present in the inflamed rheu-matoid synovial lining were excluded. The differences

from normal lining cells which were seen in thesecells suggested that local environmental factorswithin the rice bodies produced them. The lipid andglycogen observed and the relative paucity of mito-chondria suggested the greater use of anaerobicpathways of energy utilisation in this avascularenvironment of synovial fluid. To some extent thecells' appearances were reminiscent of fibroblastsgrown in tissue culture, especially cultures main-tained for longer than 1 week without trypsinisation(Wynne-Roberts and Castor, 1972).The explanation for the extracellular granules,



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Fig. 4 A more A-like cell showing markedfilopodia formation (fil). The extracellular granules (G) may be in theprocess either ofbeing extruded or ofbeing ingested by the cell. The cell contains little rough endoplasmic reticulum(rer) and pleomorphic dark staining granules, some of which are most likely lysosomes (Ly), while others may bephagolysosomes or perhaps residual bodies. Free ribosomes (Rb) are scattered in the cytoplasm. Well marked micro-pinocytotic vesicles are arrowed. Nucleus (N), nucleolus (n), glycogen (GL Y), microfilaments (mf) (uranyl acetate andlead citrate, x 12 000). A: Small collagen fibres (Col) show periodicity which measures 66 nm (uranyl acetate and leadcitrate x 34000). B: Fibres from a rice body matrix show the appearance and periodicity typical of coagulatedfibrin (FIB) (uranyl acetate and lead citrate, x 34000).

some apparently being phagocytosed, was notobvious. Their appearance was certainly similarto that of some intracellular lysosomes. Reasons forthe pericellular clear zones might include an arte-fact of dehydration or that the cells secreted enzymesinto their immediate vicinity, which destroyed

fibrin. Possibly some of the extracellular granulesmight also play a role in fibrin destruction. Over aperiod of time the cells would then secrete collagenand mucopolysaccharide matrix into the pericellulararea.The rarity of inflammatory cells within these rice



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bodies is of profound interest. Their presence mightbe expected if the mechanism of formation of ricebodies consisted only of chance trapping of cells infibrin in or on the synovial lining layer. The exclusionof these other cell types from rice bodies may berelated to a variety of circumstances. Polymor-phonuclear leucocytes are normally viable for about3 days and are then either phagocytosed at the syn-ovial surface by lining cells or die in the synovialfluid. The absence of lymphocytes and macrophagesmay be partially explained by their fewer numbers inrheumatoid synovial fluids despite their longerviability than neutrophils. It is possible that inflam-matory cells avoid trapping in fibrin if they aremore actively mobile than synovial lining cells.

It is likely that an individual rice body developsover weeks to months. Initially, a nidus of 1 or morelining cells is trapped in fibrin. This cell nidus isprobably joined by other similar islands in the courseof synovial fluid movement within the joint. Theoval appearance of a rice body suggests that it hasbeen subjected to movement, for example, rollingaround within the joint in a manner similar to thepolishing of stones on a sea shore or in a tumbler.This rolling motion would have to occur away fromthe weight or pressure bearing areas of the joint andpresumably within the niches and folds of the syno-vial lining. The formation of a mucopolysaccharidematrix and the secretion of collagen by the entrappedcells would most likely contribute to the cohesive-ness of the rice body.From the available studies it appears as though

there is a spectrum of histological structure and bio-chemical composition of rice bodies. In a recentpublication Berg et al. (1977) examined rice bodiesfrom 7 patients. They were found to consist of afibrous matrix containing fibrin and variable butsmall amounts of collagen. A variety of viable cellswere present: macrophages were most numerous,but fibrocytes, leucocytes, and red blood cells werealso seen. Blood vessels occurred within 2 of the ricebodies indicating a recent attachment to the syn-ovial membrane. The authors concluded from theirstudy that rice bodies represent a nonspecific re-sponse to chronic inflammation.

In comparing our findings with those of Albrechtet al. (1965) we note that their rice bodies containedcellular fragments and lipid globules. Collagen wasnot present within the rice body matrix by mor-phological and chemical analyses. Protein-poly-saccharide complexes, neuraminic acid, fibrin, and

fibrin-like material were found. They concluded thatrice bodies represented an end product of synovialinflammation, proliferation, and secondary de-generation.The fact that the rice bodies we studied were

larger (up to 1 5 cm long) and contained collagenand viable cells suggested that they had been pre-sent for weeks to months and that the collagen wasindeed synthesised by the rice body cells. These cellsappeared to be within a protected environment inthe rice body and to have 'reverted' to a more nearlynormal histological appearance. This observationsuggests that the stimuli inducing chronic rheumatoidinflammation are brought to the synovial lining cellsfrom elsewhere rather than that these stimuli occurprimarily within the synovial lining and attract theinvasive inflammatory response.

The technical assistance in preparation of tissue for electronmicroscopy by Ruth Blumershine, BS, of the ElectronMicroscopy Unit of the Southern Illinois University Schoolof Medicine is gratefully acknowledged.

References

Albrecht, M., Marinetti, G. V., Jacox, R. F., and Vaughan,J. H. (1965). A biochemical and electron microscopy studyof rice bodies from rheumatoid patients. Arthritis andRheumatism, 8, 1053-1063.

Barland, P., Novikoff, A. B., and Hamerman, D. (1962).Electron microscopy of the human synovial membrane.Journal of Cell Biology, 14, 207-220.

Berg, E., Wainwright, R., Barton, B., Puchtler, H., andMcDonald, T. (1977). On the nature of rheumatoid ricebodies. An immunologic, histochemical, and electronmicroscopy study. Arthritis and Rheumatism, 20, 1343-1349.

Cohen, A. S., Brandt, K. D., and Krey, P. R. (1975). Synovialfluid. Laboratory Diagnostic Procedures in the RheumaticDiseases, 2nd edn., pp. 1-64. Edited by A. S. Cohen.Little, Brown: Boston.

Millonig, G. (1961). Advantages of a phosphate buffer forOs 04 solutions in fixation. Journal of Applied Physiology,32, 1637.

Reynolds, E. S. (1963). The use of lead citrate at high pHas an electron opaque stain in electron microscopy. Journalof Cell Biology, 17, 208-212.

Riese, H. (1895). Die Reiskorperchen in tuberculos erkranktenSynovalsacken. Deutsche Zeitschrift fur Chirurgie, 42,1-99.

Spurr, A. R. (1969). A low viscosity epoxy resin embeddingmedium for electron microscopy. Journal of UltrastructureResearch, 26, 31-43.

Wynne-Roberts, C. R., and Castor, C. W. (1972). Ultra-structural comparison of rheumatoid and nonrheumatoidsynovial fibroblasts grown in tissue culture. Arthritis andRheumatism, 15, 65-83.



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