ABSORPTIVE FUNCTIONS OF SYNOVIAL MEMBRANE

Ann. rheum. Dis. (1965), 24, 2.

ABSORPTIVE FUNCTIONS OF THE SYNOVIALMEMBRANE

BY

W. COCHRANE, D. V. DAVIES, AND A. J. PALFREYFrom the Arthritis and Rheumatism Council Electron Microscopy Unit,

St. Thomas's Hospital Medical School, London

Absorption from the joint cavity has been studiedsporadically for a long time, but we are still far froma complete understanding of the mechanismsinvolved in the exchange between the synovial fluid,tissue fluid, synovial membrane, and the vascularspaces. Substances in solution are removed fromsynovial joints mainly through the capillary bed,colloidal particles including proteins are removedfrom the joint more slowly, mainly by the lymphaticstream, while particles of 100 m,u or more in diameterare removed exceedingly slowly and are mainlydeposited in the subsynovial tissues. Some of thelarger particulate matter is taken up by synovial cells,some is trapped in fibrin clots at the joint surfaces,and some enters directly from the joint into thetissue spaces of the subsynovial layer where it isremoved by phagocytes (macrophages).The problem of the transport of substances in

and out of the synovial cavity is of importance inconnexion with the formation and dispersal of jointeffusions, for the nutrition of the articular cartilage,in connexion with the vascular changes which occurin the synovial membrane in rheumatoid conditions,and in respect of the permeability of the synovialmembrane and the accessibility of the joint cavity todrugs (Bianchi, 1953, 1954; Spector and Willoughby,1962; Sharp, 1963).In the present investigation the absorption of

Thorotrast (thorium dioxide) from the joint cavityhas been studied by electron microscopy.

Material and MethodsFour rabbits, 2 males 8 months old and 2 females 8 and

3 months old have been used. Under intraperitonealNembutal anaesthesia, supplemented by a minimalamount of ether, each received an injection of 0 5 ml. of a50 per cent. dilution of Thorotrast in physiological salineinto one knee joint, the opposite joint serving as a control.

The animals were anaesthetized with chloroform, 1, 2,and 24 hours respectively after the injection and 0 5 ml.fixative was injected into both knee joints; portionsof synovial membrane less than 1 mm. diameter wereremoved. The fixative was osmium tetroxide bufferedto a pH of 7-4 (Palade, 1952). The blocks remained infixative for 2 hours, were then dehydrated in alcohol,passed through propylene oxide, and embedded inAraldite (Glauert, Rogers, and Glauert, 1956). Sectionslu thick were cut from the block and stained with AzurII (Richardson, Jarett, and Finke, 1960); thin sectionswere then cut, mounted on uncoated copper grids,stained with a saturated solution of uranyl acetate inabsolute methyl alcohol, and examined in the EM6electron microscope.

Results1 Hour after Injection.-There was much free

Thorotrast in the joint cavity; there was an abun-dance of Thorotrast particles in the synovial cells, insome of the cells of the subsynovial tissue, and inthe interstices of both the intimal and subsynoviallayers of the synovial membrane. The particles,each 50 to 100 A in diameter, quickly penetratedthe gaps between the synovial cells and becamewidely dispersed in the subsynovial tissue.

Vacuolated and non-vacuolated synovial cells,corresponding to Types A and B of Barland,Novikoff, and Hamerman (1962) and Chapman,Muirden, Ball, and Hyde (1962) could be distin-guished, but many transitional forms occurred.The most prominent accumulations of Thorotrastgranules occurred in vacuolated cells; they werecontained in membrane-bound vacuoles, sometimesin association with a flocculent precipitate. Onehour after injection many of the vacuoles werecompletely filled with Thorotrast, others wereincompletely filled, and some contained none(Fig. 1).

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ABSORPTIVE FUNCTIONS OF SYNOVIAL MEMBRANE

Fig. 1.-A surface cell, showing Thorotrast granules adherent to the plasma membrane and some completely filled and manyincompletely filled vacuoles. Note the flocculent precipitate in the large vacuole below. x 33,250.

In the sybsynovial tissue were highly vacuolatedcells, presumably histiocytes, with a considerableaccumulation of Thorotrast in many vacuoles. fnboth synovial cells and histiocytes, a few scatteredgranules of Thorotrast occurred in the generalcytoplasm outside the vacuoles.At the free surface of the synovial cells an abun-

dance of Thorotrast could be seen adherent to theplasma membranes. In several places this lay in

bays and invaginations of the plasma membrane,some of which extended deeply into the cell body.Folds of the plasma membrane or filopodia werepresent but were not a prominent feature. It wouldappear that particles became included in the cyto-plasm by a process of invagination of the plasmamembrane and the cutting of the resulting bay byapproximation of the walls. Some possibly becameenfolded by membranous folds or filopodia. In

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ANNALS OF THE RHEUMATIC DISEASES

Fig. 2.-Part of a synovial cell, showing incorporated Thorotrast granules and injected material adherent to the plasma membrane.Thorotrast particles are passing directly into the cytoplasm without invagination of the plasma membrane which, in these regions,

cannot be defined. x 52,200.

other places Thorotrast particles appeared to enterthe cytoplasm by another method; they adhered tothe cell surface, the plasma membrane underwentsome modification, perhaps dissolution, and thecytoplasm flowed around and incorporated theparticles. In these regions the plasma membranecould not be defined, whereas elsewhere it remainedintact and could be readily resolved in the electronmicrographs (Fig. 2).Even one hour after injection, polymorphonuclear

leucocytes could be seen infiltrating the synovialtissues, but these cells contained no Thorotrastparticles nor had any granules crossed the basementmembrane of blood capillaries and entered theendothelium.Only small accumulations of Thorotrast were

seen in fibrocytes; most of these cells had notingested any particles (Fig. 3). No Thorotrast

entered fat cells, even where the surrounding tissuewas heavily infiltrated with particles.

2 Hours after Injection.-There was still abundantThorotrast in the joint cavity. There was deeperpenetration and an increased amount of it in thesynovial membrane; the individual cells did notdisplay any obvious increase in the amount ingested.Much of the material was contained in membrane-bound vacuoles, some of which were still onlypartially filled. Some vacuoles were devoid ofgranules. Some Thorotrast appeared in densebodies resembling lysosomes; some of these layclose to, and appeared to be derived from, the Golgiapparatus, which now appeared to be more promi-nent and more frequently encountered than at theI-hour stage (Fig. 4).

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5ABSORPTIVE FUNCTIONS OF SYNOVIAL MEMBRANE

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Many cells in the tissue showed some separationof the inner and outer laminae of the nuclear mem-brane. One cell displayed the appearance of theextrusion of nuclear material through the membrane(Fig. 5).

In one case (Fig. 6) the- outer lamina had beenraised into a bleb which contained a flocculentprecipitate.

Several cells in the intimal and subsynovial layerscontained complicated multivesicular bodies. Thesevaried in diameter, the largest measuring up to 4,.

They were bounded by a triple-layered membraneabout 130 A thick, enclosing smaller vesicles whichusually contained a flocculent precipitate; aroundand between the vesicles was an electron-densecoarsely granular material (Fig. 7). These compli-cated bodies occurred in both synovial cells andhistiocytes. Some of the cells were peppered withdiscrete Thorotrast particles, some contained aggre-gations of granules, while still others were devoidof Thorotrast particles, at least in the sectionsshowing these complex bodies.



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Fig. 4.-Part of a synovial cell. Much of the Thorotrast is contained in membrane-bound darkbodies, possibly lysosomes (L). These bodies are particularly numerous close to the Golgi

apparatus (G). Part of the nucleus (N) is seen. x 34,000.

Numbers of polymorphonuclear leucocytes were

present in the subsynovial tissue, especially aroundblood vessels. They contained few or no Thorotrastgranules. The endothelium of the blood capillariesand fat cells was also devoid of Thorotrast.

24 Hours after Injection.-The distribution ofThorotrast in the synovial cells and histiocytesshowed little change. Some small accumulationsof Thorotrast occurred in fibrocytes (Fig. 8), andthere was possibly less in the intercellular matrix,

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ABSORPTIVE FUNCTIONS OF SYNO VIAL MEMBRANE 7

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Fig. 5.-Surface cell containing accumulations of Thorotrast. Note the widened interval separating the inner and outerlaminae of the nuclear membrane (NM) and apparent extrusion of nuclear material through the nuclear pores (NP)

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Fig. 6.-Part of a synovial cell in which the outer lamina of the nuclear membrane has been raised into a bleb containing aflocculent precipitate. x 56,800.

though the medium was still present in the jointcavity and adherent to the free surfaces of the synovialcells.

In most regions, in synovial cells, histiocytes,fibrocytes, and lymphocytes, the endoplasmic reti-culum was more prominent and was studded withR.N.A. granules. The Golgi apparatus was muchenlarged in many cells (Fig. 9).Some synovial cells contained only a minimal

amount of Thorotrast and some none at all. Thelargest accumulations were in the histiocytes in thesubsynovial tissues. Polymorphonuclear leucocyteswere more numerous in the synovial membrane.They did not phagocytose the Thorotrast in anyquantity; a few isolated granules only were seen insome cells. The cellularity of the synovium wasincreased and rough-walled endoplasmic reticulum

was a conspicuous feature in most cells. Lympho-cytes were present but contained an occasionalThorotrast granule.

Cells with multivesicular bodies were morenumerous than at 2 hours. These bodies occurboth in the synovial cells and deeply in the sub-synovial tissues (Fig. 10).

In some synovial cells a fibrous material appearedin association with some of the Thorotrast accumu-lations; elsewhere an electron-dense secretionwas accumulating in the vesicles in these cells(Fig. 11).No lymphatic capillaries were identified with

certainty in these tissues, but at 24 hours there was avessel with electron-dense contents and very vesicu-lated cell lining in which Thorotrast seemed to beaccumulating and passing into the lumen by a

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ABSORPTIVE FUNCTIONS OF SYNOVIAL MEMBRANE 9

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Fig. 7.-Part of a synovial cell containing large multivesicular bodies. The cell is peppered with Thorotrast granules. x 49,700.



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ANNALS OF THE RHEUMATIC Dl

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Fig. 8.-Fibrocyte with small accumulations of Thorotrast. The large mass contains Thorotrast granules amongstmoderately electron-dense material. x 16,000.

process suggestive of apocrine secretion. NoThorotrast was found in blood vessels.

DiscussionThe speed and ease with which particulate matter

was found to enter the intimal and subsynovial layersof the synovial membrane is in agreement with previ-ous investigations with the light microscope. Whiledrawing attention to the presence of filopodia andpinocytotic vesicles in synovial cells, Chapman andothers (1962) add that "evidence is insufficient toindicate with certainty the mechanism of entry".While the engulfment of particulate matter by foldsand filopodia and its entry via pinocytotic vesiclesand bays at the cell surface seems a probability, this

investigation also suggests another mode ofentry, that of "membrane flow" or "rhopheocy-tosis" as suggested by Policard and Bessis (1962).The injected Thorotrast is incorporated principallyin the vacuolated type of synovial cell (Type A ofBarland and others, 1962); this agrees with thefindings of Chapman and others (1962). Almostall the Thorotrast within the cell is aggregated intovacuoles by the 1-hour stage. A few isolated granulescan be identified in the general cytoplasm around thevacuoles and elsewhere. This suggests that thematerial is either incorporated into the cell mainlyas aggregates or that the transport and aggregationof discrete granules in the cytoplasm is extremelyrapid.

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Fig. 9.-A cell in the subsynovial layer 24 hours after injection, with abundant endoplasmic reticulum and a large and diffuseGolgi apparatus (G). Only one accumulation of Thorotrast is seen in the cell. x 30,420.

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Fig. 10.-Histiocyte with a multivesicular body in the subsynovial tissue at 24 hours after injection. There are accumula-tions of Thorotrast granules in vesicles elsewhere in the cytoplasm and some within the electron-dense material in the multi-

vesicular body. x 33,000.

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Fig. 11.-Synovial cell at 24 hours after injection, showing the development of fibrous material close to accumulations ofThorotrast. Note also the electron-dense secretion in some intracytoplasmic vesicles. x 33,000.



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ANNALS OF THE RHEUMATIC DISEASESThe entry of material between the synovial cells

and its dispersal through the subsynovial tissue isalso rapid, even in the animal maintained underanaesthesia from the time of injection until killed.While earlier work suggests that, with particulatematter, movement is an important factor promotingabsorption from the joint cavity, other factors mustbe operative. In the subsynovial tissue the particlesare transported in the interfibrillar matrix fromwhich they are incorporated into histiocytes or enterlymphatics. The state of the matrix, in particularof the mucopolysaccharide component, is importantin this respect. The molecular size of the muco-polysaccharide, the state of the aqueous componentof the tissue, and the electrical charges on the variousconstituents may well be involved.

Thorotrast particles do not cross the basementmembranes of blood vessels though they quicklyevoke a polymorphonuclear leucocytic reaction.They do, however, transgress the basement mem-brane of lymphatics and enter their endothelial cells.From here they appear to be released into the lumenby a method akin to apocrine secretion. Eachparticle, or group of particles, is released in anenvelope of cytoplasm.The identity of the phagocytic cells in the synovial

membrane presents a problem. In the early stagesmost, if not all, of the intimal cells are synovial cellsand the deeper phagocytic cells are histiocytes. Inlater stages the distinction between these two typescannot be made easily on the basis of their topo-graphy, though the subsynovial cells at this stageare principally histiocytes. Histiocytes are alsopresent amongst the intimal cells and display littlemorphological difference from Type A synovialcells. Polymorphonuclear leucocytes play only aminor role in phagocytosis, fibroblasts and lympho-cytes accumulate only small amounts of material,and fat cells play no part in the process.

Interesting changes occur in the cells, probably asa result of phagocytosis. The inner and outerlamellae of the nuclear membrane become morewidely separated than in the normal, and the outerlamella may be raised into a bleb. The Golgiapparatus increases in size, there is an increasedamount of rough-walled endoplasmic reticulum, andmultivesicular bodies appear in many cells. Noneof these changes is described by Chapman and others(1962). They may be due to the toxicity of theinjected Thorotrast, and the question arises whetherintracellular changes of a similar nature occur undersimilar circumstances but with different types ofparticulate matter. Few foreign substances havebeen investigated; in most studies on the absorptionof particulate matter from joints indian ink sus-

pensions have been used. According to Key (1926),using carbon particles in suspensions, the macro-phages never get rid of the carbon particles, whichare said to remain within the cell until it dies ordisintegrates, when the material is again set freeinto the tissues or the joint cavity. The degenera-tion of these macrophages has not been described.A greatly extended study using a variety of differ-

ent materials could materially help to elucidate someof these problems and those of cell pathology ingeneral.

Summary

The fine structure of the synovial membrane of theknee joint of young adult rabbits has been studiedbetween 1 and 24 hours after the injection of asuspension of Thorotrast (thorium dioxide). Within1 hour of the injection, particles were found invacuoles of synovial cells and histiocytes, and therewere free particles in the subsynovial tissue. After2 hours the amount of thorium dioxide had in-creased; some of the cells containing ingested parti-cles now showed a prominent Golgi apparatus,separation of the constituent layers of the nuclearmembrane, and complicated multivesicular bodies;the tissue showed a polymorph infiltration, butthese cells did not absorb the particles. By 24hours after injection the cells showed further en-largement of the Golgi apparatus and the endo-plasmic reticulum was hypertrophied. Themechanism by which synovial cells absorb particu-late matter is discussed; the later changes within thecells may be due to the toxicity of the injectedmaterial.

The authors thank the Arthritis and RheumatismCouncil for the use of the electron microscope, Messrs.J. S. Fenton and G. Maxwell for technical assistance, andMiss F. M. Fildes for secretarial help.

REFERENCESBarland P., Novikoff, A. B., and Hamerman, D. (1962).

J. Cell Biol., 14, 207.Bianchi, C. (1953). Brit. J. Pharmacol., 8, 130.

(1954). Ibid., 9, 166.Chapman, J. A., Muirden, K. D., Ball, J., and Hyde,

P. A. (1962). In "Electron Microscopy: FifthInternational Congress, Philadelphia, 1963", ed.S. S. Breeze, Jr., vol. 2, ss-12. Academic Press,New York.

Glauert, A. M., Rogers, G. E., and Glauert, R. H. (1956).Nature (Lond.), 178, 803.

Key, J. A. (1926). J. Bone Jt Surg., 8, 666.Palade, G. E. (1952). J. exp. Med., 95, 285.

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ABSORPTIVE FUNCTIONS OF SYNO VIAL MEMBRANE

Policard, A., and Bessis, M. (1962). Nature (Lond.), 194,110.

Richardson, K. C., Jarett, L., and Finke, E. H. (1960).Stain Technol., 35, 313.

Sharp, G. W. G. (1963). J. Endocr., 25, 443.Spector, W. G., and Willoughby, D. A. (1962). Nature

(Lond.), 196, 1104.

Fonctions d'absorption de la membrane synoviale

REsuMELa fine structure de la membrane synoviale de I'articu-

lation de genou de jeunes lapins adultes fut etudiee entreune et 24 heures apres l'injection d'une suspension dedioxyde de thorium. Une heure apres l'injection ontrouva des particules dans les vacuoles des cellulessynoviales et des histiocytes et on observa des particuleslibres dans le tissu synovial. Apres 2 heurs la quantitede dioxyde de thorium se trouva augmentee; quelquescellules contenant des particules ingerees revelaient unappareil de Golgi prononce, la separation des couchesconstituantes de la membrane nucleaire et des corpsmultivesiculaires compliques; le tissu etait infiltre pardes polymorphes, mais ces cellules n'absorbaient pas lesparticules. Vers 24 heures apres l'injection I'appareil deGolgi cellulaire etait encore plus grand et le reseau

endoplasmique se trouvait hypertrophie. On discute lemecanisme par lequel les cellules synoviales absorbentde la matiere sous forme de particules; les alterationsintracellulaires tardives peuvent etre dues a la toxicitedu materiel injecte.

Funciones de absorpcion de la membrana sinovial

SUMARIOLa fina estructura de la membrana sinovial de la

articulaci6n de la rodilla de j6venes conejos adultos fueestudiada entre la una y las 24 horas despues de lainyecci6n de una suspensi6n de dioxido le torio. Dentrode una hora de la inyecci6n, particulas fueron halladasen las vacuolas de las celulas sinoviales y de los histiocitos yparticulas libres se vieron en el tejido sinovial. A lasdos horas, la cantidad de dioxido de torio se vi6 aument-ada; algunas celulas con particulas ingeridas revelarondestacadamente el aparato de Golgi, la separaci6n de lascapas constituyentes de la membrana nuclear y cuerposmultivesiculares complicados; el tejido se vi6 infiltradopor polimorfos, pero las celulas ya no absorbian lasparticulas. A las 24 horas de la inyecci6n el aparatocelular de Golgi fue auin mayor que antes y el reticuloendoplasmico fue hipertrofiado. Se discute el mecanismopor el cual las celulas sinoviales absorben materia enforma de particulas; alteraciones intracelulares tardiaspueden deberse a la toxicidad del material inyectado.

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ABSORPTIVE FUNCTIONS OF SYNOVIAL MEMBRANE

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