0022-34I7/8 1/24870147$2.

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THE FORMATION O F A STRUCTURE WITH THE F E A T U R E S O F S Y N O V I A L L I N I N G BY S U B C U T A N E O U S INJECTION OF AIR: AN I N VIVO TISSUE CULTURE SYSTEM

J. C. W. EDWARDS, D. SEDGWICK A. AND D. A. WILLOUGHBY

Experimental Pathology Department, St. Bartholomew's Hospital Medical School, London EC1

SUMMARY. We have attempted to verify the suggestion that synovial membrane is the result of mechanical disruption of connective tissue, and may occur at any site. Mechanical disruption of the subcutaneous connective tissue was achieved in rats and mice by the repeated injection of air. The resulting cavities developed a lining structure with many of the features of synovial membrane as judged by electron microscopy, and light microscopy using haematoxylin and eosin and van Gieson stains, esterase activity and immunofluorescent staining for Ia antigen. A structure closely resembling synovium is formed as early as 6 days, providing a convenient method for studying large quantities of facsimile synovial tissue under a wide variety of easily administered stimuli. INTRODUCTION SINCEthe original electronmicroscopic description by Barland, Novikoff and Hamerman (1962) it has been recognised that the apparently homogeneous layer of synovial lining cells seen with the light microscope contains two cell types. Type A resembles a macrophage and type B a fibroblast. However, synovial lining cells are still commonly regarded as embryologically distinct from macrophages and fibroblasts and some authors consider the two types to be functional variations of a single cell line (Kinsella, Baum and Ziff, 1970). Recent work in our laboratory using radiation chimeric mice suggests that this is not the case (Edwards and Willoughby, in press). Type A cells appear to be bone marrow derived but type B cells do not. In view of the observation that synovium fails to develop in the embryo in the absence of movement (Drachman and Sokoloff, 1966) coupled with our own observation that air pouch granuloma linings (Selye, 1953) bear a superficial resemblance to synovial lining, we proposed that synovial lining is simply an accretion of macrophages and fibroblasts stimulated by mechanical cavitation of
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connective tissue. To test this hypothesis we injected air alone into the subcutaneous tissue of mice and rats and studied the resulting cavity lining. We describe the synovium-like structure which formed.
MATERIALS AND
METHODS

Animals used for air injection were either normal Griineberg brown mice or normal Wistar rats. The two species were studied to avoid errors due to species idiosynchrasy and for reasons of convenience for certain histological techniques. In particular mice were used for the electron microscopic study because full-thickness specimens could be resin embedded. Mice were anaesthetised and 5 ml of air was injected into the subcutaneous tissue of the back in the midline. Injection of air was repeated only as often as required to keep the cavity inflated, without allowing the wall to be re-stretched. This usually meant every 2-3 days. In some cases colloidal carbon as 1 per cent. india ink (Pelikan, Gunther Wagner) in phosphate buffered saline was injected into the cavity 30 min. before removal for histology. Animals were killed 5, 8, 14 and 30 days after initial air injection and the cavity walls removed. Specimens were fixed in 5 per cent. glutaraldehyde in cacodylate buffer, post fixed in osmium tetroxide and resin embedded (Taab). Rats were injected with 20 ml of air under anaesthesia as for the mice. Re-injection was again used every 2-3 days to maintain the open cavity. Rats were killed 6 and 14 days after the initial injection. Cavity walls were dissected out either fresh for snap freezing in isopentane containing solid carbon dioxide, or after injection of 5 per cent. glutaraldehyde into the cavity for rapid fixation. Such specimens were rapidly transferred into formal saline for further fixation and paraffin embedding. Frozen sections were submitted to staining for non-specific esterase and immunofluorescent staining for Ia antigens. Formalin fixed material was stained with haematoxylin and eosin (H and E) and Van Gieson. Staining for non-specific esterase was performed using the alpha naphthyl acetate and hexazotised pararosaniline method with a light counterstain of either light green or haematoxylin. Immunofluorescent staining for Ia antigen was performed using a mouse anti rat Ia monoclonal antibody supplied by Seralab in the form of tissue culture supernatant. This was derived from the clone 0x4. A standard two-layer immunofluorescent technique was used on frozen sections using five microlitres of anti Ia antibody as first regent and five microlitres of goat anti mouse IgG Fc fragment (]/loo) as second reagent. Incubations were for 30 min. at room temperature within a humidified container. Smears made from rat peritoneal washings, frozen sections of rat thymus, spleen and synovium were used as control tissue. Frozen sections of human and rat synovium were used for comparison with the linings for H and E and non-specific esterase staining. Human material was obtained at arthrotomy from knees of patients with osteoarthritis and ruptured meniscus. Specimens were snap frozen as for animal tissue.

RESULTS

Mouse material Macroscopically, as early as 5 days after initial injection, the cavities had a clearly defined pink translucent wall which was progressively firmer at 8 and 14 days. This wall was loosely connected to the surrounding connective tissue in the manner of areolar synovium, which it closely resembled. At 14 days this lining was remarkably strong. The inner surface was smooth and glistening and coated with a minimal amount of viscous fluid. Electron microscopically a lining layer of cells was seen, separated from the surrounding tissue by a loose areolar zone. At 5 days the lining was composed of haphazardly arranged cells about 10

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FIG. 1.-Electron

micrograph of an 8-day-old air cavity lining from a mouse. A fibroblast or type B cell is shown with numerous micropinocytic vesicles. x 25,000.

deep. Cells at the surface often appeared free. By 8 days the cells had taken up a laminated arrangement, about six deep. By 14 days a highly compact lining was present, resembling areolar synovium from the mouse knee. At 30 days the lining was less cellular and much more fibrous with rather intermittent cells in the deeper zones of the lining and a single sometimes interrupted surface layer of cells. The cells were extremely flattened giving an appearance more similar to the fibrous form of synovium. At 5 days polymorphs were common, chiefly in the zones deep to the lining but occasionally in the lining itself. These were much less prominent at 8 days and rare at 14 days. The lining cells proper were of two main types. The commonest type (50-90 per cent.) had prominent rough endoplasmic reticulum, a smooth ovoid outline with occasional blunt processes and minimal vacuolation. These cells were identified as fibroblasts. By 8 days these cells often contained numerous pinocytic vesicles (fig. 1) and, where colloidal carbon had been injected, limited phagocytosis. As such they were indistinguishable from type B cells of the mouse knee synovial lining. The second major cell type (10-50 per cent.) lacked significant amounts of rough endoplasmic reticulim but had prominent Golgi figures, numerous vacuoles and lipid filled vesicles, and prominent membrane lamellation. These cells were also pinocytic and took up

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FIG. 2.-Electron micrograph of a 5-day-old air cavity lining from a mouse. Carbon had been injected prior to removal. A typical macrophage-like or type A cell is present containing phagocytosed carbon. Lamination is minimal and the cell almost free. x 8000.

colloidal carbon avidly. They could equally be described as macrophages or type A synovial lining cells (figs. 2 and 3). The two main cell types were increasingly tightly interlocked over 14 days (fig. 4) although at 30 days they were more sparsely arranged but still markedly flattened in the plane of the lining. Cells were often in contact via cytoplasmic processes. Intermingled with these two types of cell were occasional eosinophils, recognised by the presence of crystalloid granules. A few cells (less than 5 per cent.) of intermediate morphology between the macrophage and fibroblast types were seen. These were intermediate in the sense of having prominent rough endoplasmic reticulum but also prominent vacuolation and Golgi figures. It was not possible to establish whether these were a distinct cell type or a variant of either the macrophage or fibroblast types or both. Such cells would be called intermediate cells in synovial lining (Barland, Novikoff and Hamerman, 1962) or epithelioid cells in granulomata (Papadimitriou and Spector, 197 1). Just deep to the lining layer mast cells appeared at 8 days and by 14 days were arranged in a regular row about 100 microns apart. Blood vessels were prominent in this same area but rare within the lining itself. No basement

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FIG.3.-Detail of a macrophage-like or type A cell from a 14-day-old air cavity lining. Golgi figures are extensive and vacuoles common. The sparse rough endoplasmic reticulum is only irregularly lined with ribosomes, many of which lie free. x 30,000.

membrane was seen at any time. The increasingly dense intercellular matrix contained both amorphous material of moderately low electron density and characteristic collagen bundles. In summary a structure with all the essential features of synovial lining was seen to form over a period of 14 days. At this stage the lining resembled areolar synovium most closely but by 30 days it had taken on a more fibrous appearance. This synovium-like structure appeared to form by the aggregation of macrophages and fibroblasts.

Rat material Macroscopically, rat air cavity linings were similar to those in the mice but thicker. Cavities often contained septa probably due to tracking of air either side of a neurovascular bundle. The surface was smooth and glistening and when fully relaxed drew up into folds in a similar fashion to some joint synovium. No villi were seen. Haematoxylin and eosin-stained paraffin sections showed a well-defined lining structure at 6 days post injection. This was less cellular and more fibrous than the 5-day-old mouse linings. Similarly the 14-day-old rat linings were more fibrous than those in the mouse, more closely resembling the mouse 30-day linings although a lining layer of cells one to two cells deep was preserved. An

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FIG.4.-Electron micrograph of a 14-day-old air cavity lining from a mouse. Two fibroblasts or type B cells with prominent rough endoplasmic reticulum and smooth cell outlines are seen. A macrophage like cell with vacuoles and surface lamellae and part of an eosinophil are seen also at the bottom of the picture. Lamination is now marked. ~ 2 0 , 0 0 0 .

interesting phenomenon was observed in a number of linings. The rather featureless surface layer of extremely flattened cells one or two deep was interspersed with " hillocks " of up to 100 cells in any one cross section lying haphazardly in a shallow mound up to five cells deep. These hillocks were often accompanied by an aggregation of mononuclear cells in the sub-lining connective tissue around a blood vessel. The two areas of iellularity (lining and sub-lining) sometimes being bridged by cells apparently migrating from one to the other. Similar hillocks or mounds of cells have been observed in human synovium taken from knees of patients with osteoarthritis or ruptured meniscus. Sub-lining aggregates were also seen in the sections with occasional cells bridging the two cellular areas. Eosinophils occurred in very variable numbers in the sub-lining zone. The presence of large numbers of eosinophils in some preparations of rat peritoneal cells suggests that the presence of eosinophils may be a rat species idiosynchrasy. As with the mouse, eosinophils only occasionally occurred within the lining layer itself. Blood vessels were prominent in the sublining zone and also common within the lining itself, in contrast to the mouse linings. This may reflect the thickness and metabolic requirement of the two types of lining. Mast cells occurred just deep to the lining as in the mouse. Van Gieson staining confirmed the collagenous nature of the fibrous material

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FIG. 5.-Frozen section of a 6-day-old air cavity lining from a rat, stained for esterase activity. The lining is folded together and shows the contrast between an area of surface on the right almost devoid of esterase positive cells and an area on the left with a " hillock " of esterase positive cells. Esterase positive cells are also present in the deeper tissue. x 1200.

in the air cavity linings. Collagen staining was more prominent at 14 than at 6 days. Frozen sections stained for esterase activity showed the lining to contain two populations of cells. The majority of lining cells showed no significant staining but a minority of cells showed marked granular cytoplasmic staining shared by cells in nearby connective tissue assumed to be mononuclear phagocytes (macrophages). Mast cells were distinguished by the presence of more uniform yellowish (not red) staining granules and occurred as described above. The

FIG. 6.-Frozen section of human synovium from a patient with a ruptured meniscus. Esterase positive cells are seen around a blood vessel in the subintima and in an aggregate in the lining. Positive cells are also seen between the two. x 1200.

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proportion of macrophage-like esterase positive cells in the lining varied considerably between 10 and 40 per cent. overall. The flattened areas of surface cells in 14-day-old linings were often uniformly negative, but the hillocks or mounds of cells described above were made up of apparently uniformly esterase positive cells (fig, 5 ) . In general the more cellular and less flattened the surface layer the higher the proportion of esterase positive cells. Human synovial frozen sections gave a very similar appearance, the flatter cells being esterase negative and the hillocks esterase positive with esterase positive cells making up about 20-30 per cent. of cells in intermediate areas. In both rat and human tissue the sub-lining aggregates of cells associated with lining hillocks were esterase positive (fig. 6). Using the OX4 clone monoclonal antibody to rat l a antigen, membrane fluorescence was seen on 10-30 per cent. of cells in 6-day-old linings as judged using H and E stained control serial sections. These cells were variably elongated with linear projections of the cell membrane. Their overall configuration was similar to that of the esterase positive cells. By 14 days the number of stained cells in the lining layer was reduced (not more than 5 per cent.) and the superficial cells were uniformly negative. Control material stained with the OX4 antibody showed fluorescent staining consistent with the known distribution of Ia antigens. Smears of rat peritoneal cells showed strong membrane fluorescence on 10 per cent. of cells and weaker fluorescence on a further 10-30 per cent. of cells. Rat thymus frozen sections showed large irregular cells in the medullary areas with membrane fluorescence and threadlike staining between the thymocytes. Background staining was relatively high in thymic sections and too high in spleen sections to allow useful interpretation. This probably relates to the cross reactivity between mouse and rat immunoglobulin leading to binding of the anti mouse immunoglobulin fluorescence conjugate to native rat immunoglobulin in the sections. Frozen sections of rat synovium were, however, quite free of background. Large irregular membrane stained cells were seen similar to those in the air pouch linings. These cells occurred in the subintima and in the connective tissue around muscle, but also in small numbers in the deeper lining cells, making up about 2-5 per cent. of total lining cells. Only one fluorescent cell was seen at the free surface in one specimen. Synovium from a rat knee which had been injected with calcium pyrophosphate crystals 48 hr prior to removal showed a marked increase in membrane fluorescent cells in the subintima, particularly around blood vessels, and also in the deeper layers of the lining, which had become thickened. The superficial lining cells remained unstained. This picture closely resembled that seen in the 14-day-old air cavity linings.
DISCUSSION Joint synovial lining is an extremely variable structure, in collagen content, in cellularity and in the proportion of type A and B cells. The surface may be smooth or villous. The junction between the lining and the subintima is indefinite, quite unlike the basement membrane of the true epithelial tissues.

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Although the linings described above in subcutaneous air cavities may not be identical with any particular piece of joint lining, they demonstrate all the essential features of a synovial lining and fall well within the scope of textbook definitions. Moreover these linings have similar cell sub-populations as judged by esterase activity, electron microscopy and I a expression. The presence of a small proportion of intermediate cells on electron microscopy and of sublining mast cells is also similar to joint synovium. The presence of eosinophils is unlike jointsynovium but it seems reasonable to suggest that these cells may represent an active inflammatory phase of development not seen in mature synovium. These findings support the original suggestion that synovium forms by an accretion of macrophages and fibroblasts in response to tissue disruption and may form a t any anatomical site exposed to the appropriate mechanical stimuli. The well-recognised regeneration of synovium following synovectomy may then be seen as a de nouo redevelopment rather than extension of putative synovial remnants. The presence of aggregates of esterase positive cells or " hillocks " in otherwise flat lining, both in joint synovium and in the air cavity linings may suggest an ongoing process of regeneration or repair. Such areas may represent influx of macrophages into areas of recent minor injury. They may, however, merely reflect the proximity of blood vessels since similar aggregates, known as milk spots, are recognised in the peritoneum close to blood vessels. The pattern of Ia staining in the lining can only be interpreted tentatively at present. Ia antigens occur on the surface of B lymphocytes and some monocyte derived cells. The proportion of mature macrophages carrying Ia antigens is still not clear, and nor is the relationship between I a positive and Ia negative monocyte derived cells. Moreover, different sub-populations may express different individual I a antigens of which there are several. The morphology of the Ia positive cells in synovium and air pouch linings described above closely resembled the esterase positive cells in the same tissues. Lymphocytes were not seen in the mouse linings on electron microscopy, suggesting that the few Ia positive lining cells are probably macrophages. The disappearance of Ia staining from the surface layer of cells at 14 days suggests that I a expression may be lost when macrophages mature in the lining. This may be analogous to the absence of Ia expression on many peritoneal macrophages. The immunological significance of Ia expression in synovial and other mesenchymal linings must remain speculative. The few Ia positive cells in mature synovium may behave like the Langerhans cells in the epidermis, but as yet their ultrastructure is unknown, as is their ability to present antigen to lymphocytes. If the proposed model is valid then the synovial lining should perhaps be considered much more as a dynamic cellular process rather than a fixed tissue. Synovial lining formation would be seen as similar to wound formation and chronic inflammation and a s such should be susceptible to those agents which alter wound healing and chronic inflammatory lesions. The effect of a number of drugs on the development of air cavity linings and on synovium are currently under investigation in the department.

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As a by-product of the above experiment, the air cavity lining provides a potentially extremely valuable model for studying lining cell development and function. The cavity can be used as a culture chamber into which agents can be introduced and their effects on morphology, mobility, enzyme activity and other lining cell functions studied. Such a system differs from the traditional air pouch granuloma model in that the effects of tissue disruption and introduced irritants can be separated if the lining is allowed to form prior to addition of such irritants. The air cavity may prove particularly useful in studying cartilage degradation since it provides an in vivo system into which cartilage fragments can be introduced and observed in contact with lining cells. Both its similarities to and differences from joint synovium may provide clues to the importance of mechanical factors to cell movement in lining tissues.
J. C. W. Edwards is supported by the Muir Hambro Trust, administered by the Royal College of Physicians (U.K.). In addition the work was supported by the European Biological Research Association, the Arthritis & Rheumatism Council and The Knights Bachelor Rheumatology Research Fund. This financial support is gratefully acknowledged. We are also grateful to the Joint Research Board of St Bartholomew's Hospital for the support given to A. D. Sedgwick. We wish to thank Mrs G. Carson and Mr S. Jones for technical assistance.

REFERENCES BARLAND, NOVIKOFF,B., AND HAMERMAN, 1962. Electron microscopy of the human P., A. D. synovial membrane. J. Cell Biol., 14,201-220. DRACHMAN, B., AND SOKOLOFF, 1966. The role of movement in embryonic joint D. L. development. Developmental Biol., 14,40 1-420. KINSELLA, D., BAUM, J., AND ZIFF,M. 1970. Studies of isolated synovial lining cells of T. rheumatoid and non-rheumatoid synovial membranes. Arthritis and Rheumatism, 13, 6 , 734-152. PAPADIMITRIOU,M., AND SPECTOR, G. 1971. The origin, properties and fate of epithelioid J. W. cells. J . Path., 105, 187-198. SELYE, . 1953. On the mechanism through which hydrocortisone affects the resistance of H tissue to injury. J . A m . Med. Ass., 152, 1207-1213.