ARTHRITIS PRODUCED BY INTRA-ARTICULAR INJECTIONS OF STREPTOLYSIN S IN RABBITS

JOSEPHINE COOK AND w. J. FINCHAM Department of Pathology, Institute of Orthopaedics, Royal National Orthopaedic

Hospital, Stanmore, Middlesex

PLATES CXXXVIII-CXL

WEISSMAN et al. (1965) produced chronic arthritic changes by injecting streptolysin S (SLS) into the joints of rabbits. The following is an account of similar experiments in which intra-articular SLS was used to produce arthritis in rabbits and goats.

MATERIALS AND METHODS

Rabbits. New Zealand Reds, weighing between 700 and 1000 g. at the start of the experiment, were used.

SLS. We made this by using streptococcus strain C 203 S, provided for us by Bernheimer and treated according to his method (Bernheimer, 1949). As SLS is labile, it was stored freeze-dried and reconstituted in phosphate-buffered saline just before use. It was sterilised by filtration through a membrane filter, the filtrate proving sterile after culture on both conventional media and media capable of supporting the growth of streptococcal L-forms. Cultures taken from the animals’ joints post mortem were invariably sterile.

Batches of SLS were assayed for haemolytic activity (Bernheimer) after freeze- drying. Initially an intra-articular dose of 1000 haemolytic units was used and later 2000 haemolytic units, which is double the dose used by Weissmann. We subsequently found in later series of animals that the smaller dose produced histo- logical changes of the same severity as the larger dose. The volume injected was 0.2 ml.

Intra-articular injections. These were carried out once a week, into one knee only. Rabbits were bled at the beginning of the experiment by cardiac puncture, and again before being killed. Twelve rabbits were divided into 3 groups. One group received 2 intra-articular injections of SLS, the second received 6 injections and the third, 9.

In the first group, the contralateral knees were left uninjected. The second and third groups received respectively 4 and 6 injections of phosphate-buffered saline in the contralateral knee. In each group, 2 rabbits were killed 24 hr after the last injection and 2 were killed one week later.

Three rabbits received injections, intra-articularly, into one knee only, of the reconstituted freezedried dialysate of the sterile medium used for SLS production. These 3 rabbits had respectively 2, 6 and 9 weekly injections, and were killed 24 hr after the last injection.

Two other rabbits, littermates, were given a course of 9 weekly injections of SLS into one knee only, the other knee being left uninjected. Each injection con- tained lo00 haemolytic units of SLS. One of the pair was given chloroquine in its drinking water. It received 100 mg. over a Cday period before the start of the experiment; thereafter it received 25 mg. daily throughout the experiment, except on the day of each injection, when 50 mg. were given. These rabbits were killed 24 hr after the last injection.

J. PATH. BACT.-VOL. 92 (1966) 461 2 0 2

462 JOSEPHINE COOK AND W. J . FINCHAM

Goats. Two adult goats were given 9 weekly intra-articular injections. 0.5 ml. containing 4OOO haemolytic units of SLS was injected into one knee; the same amount of SLS dissolved in phosphate-buffered saline containing 1 per cent. chloroquine was injected each time into the opposite knee. The goats were killed 24 hr after the last injection.

At necropsy, knee-joints were opened and smears of synovial fluid were made and stained with Leishman’s stain. Synovial membranes, patellas and femoral condyles were removed for fixation and sectioning by the paraffin method. The sections were stained with haematoxylin and eosin, and also with Lendrum’s martius scarlet blue (MSB) (Lendrum et a/., 1962).

During the course of injections, the knee-joints were aspirated frequently. Smears were made of the synovial fluid and stained with Leishman’s stain.

Intramuscular injections. Two adult rabbits of the same strain were given a course of intramuscular SLS, the first 2 injections being combined with sodium alginate (Algivant Colab Laboratories Inc.). They received 500 haemolytic units in 0.5 ml. phosphate-buffered saline on days 1, 17, 24, 25, 29, 30, 37, 51 and 53. Seven weeks later, they were skin tested with 0.1 ml. phosphate-buffered saline containing 500 haemolytic units of SLS. Phosphate-buffered saline alone was used as a control. A normal rabbit was similarly treated. The injected areas were later biopsied. The 2 SLS-treated rabbits were bled by cardiac puncture at the start of the experiment and again at 26,45,64 and 134 days. The sera of these rabbits were tested for antibodies together with the sera of the rabbits that had received intra- articular SLS.

Precipitating antibodies. These were tested for by gel diffusion. Azide agar was made up with 1 per cent. Oxoid ionagar no. 2 and 1 per cent. sodium azide in distilled water. Microscope slides (3 x 1 in.) were dipped in the azide agar and allowed to dry in the air, to prevent seepage of reagents between agar and slide. 2.5 ml. of molten azide agar was added to each slide and allowed to set. Holes were cut with a stainless steel gel cutter made in our workshops. The pattern used cut 4 peripheral holes of 5 mm. diameter, each separated from adjacent holes by a distance of 5 mm. between the nearest points on their circumference. The 4 peri- pheral holes surrounded a central hole 3 mm. in diameter and were separated from it by a distance of 3 mm. The central hole was filled with the antigen and the peripheral holes with rabbit sera. All holes were topped up with reagents on a further 2 occasions.

Complementfixation. The method used was adapted from that employed by the Auto-Immunity Unit of the Middlesex Hospital Medical School. The varying dilutions of antigen were titrated in the presence of complement dilutions to deter- mine the optimum strength of antigen used in the test. The tests were carried out in MRC Perspex agglutination trays. All dilutions were made in Kabat and Meyer buffer. The standard dose of complement in the test itself was 2 MHD. Measured volumes of 0.1 ml. were used instead of a dropping technique. Positive control material consisted of liver homogenate from carbon tetrachloride-treated rabbits.

Fractionation of liver subcellular particles. Fresh liver was cut up and washed 3 times in cold 0 . 2 5 ~ sucrose. The tissue was then homogenised in a Potter-type homogeniser with sufficient 0 . 2 5 ~ sucrose to make a 10 per cent. w/v suspension. Subcellular fractionation based on the techniaue of de Duve et al. (1955) . - was carried out by differential centrifugation as described by Ali and Lack (1965). .

Enzyme assays. Acid phosphatase was determined at pH 5.0 by the method of Gianetto and de Duve (1955), using sodium /?-glycerophosphate as substrate. Inorganic phosphate was determined by the method of Fiske and Subbarow (1925). p-Glucuronidase was measured as described by Allison and Sandelin (1963), except that a 60-min. incubation period was used.

Rabbit polymorphonuclear leucocyres. These were isolated by the method of Cohn and Hirsch (1960). Our yields were 3 to 4 x 108 cells per rabbit, in a volume of

STREPTOLYSIN S IN RABBITS 463

200 ml. Granules were isolated by the procedure described by Cohn and Hirxh, with the modification introduced by Weissmann, Becher and Thomas (1964).

Rlmmzatoidfuctor. The sheep cell agglutination test was used for testing sera.

RESULTS Animuls

Behaviour of animals. After the injections, the rabbits were observed to hold the injected limb in an abnormal position of slight external rotation and abduction for several days. They also spent more time lying on the floor of the pen than normal young rabbits. Both these effects were more noticeable in rabbits receiving multiple injections. Swelling was not marked, but towards the end of a long course of injections, swelling did not subside between injections. Erythema over the joints was not seen. Temperature differences over the skin of injected and uninjected knees were insignificant. Rectal temperatures usually showed small rises on the day following injection and remained raised for 2 or 3 days subsequently. Goats developed permanently swollen joints towards the end of the course of injections.

SynoviulJluid. Synovial fluid was scanty, but very cellular. Of 20 fluids examined during the first 24 hours after injections, 17 showed a predominantly polymorph exudate (fig. 1). One, after an injection late in the course, showed mainly large mononuclear cells and two others at a similar stage showed lymphocytes. When joints were aspirated 48 hr after injection, the fluids were found to contain large mononuclear cells and lymphocytes. Aspirations at 72 hr showed predominantly lymphocytic fluids. Fluids examined one week after the last injection showed lymphocytes and plasma cells (fig. 2).

Histological changes. The histological changes for the first 12 rabbits are shown in the table, and are classified under the headings used by Weissmann. The findings agree closely with his. Synovial lining cell proliferation (fig. 3) is present in all animals, but chronic synovitis did not occur in animals that received only 2 injections, whilst it occurred in all those receiving 6 or more injections. Two out of 4 rabbits that received 6 injections showed erosion of cartilage by pannus (fig. 4). All those receiving 9 injections showed pannus. Macroscopic- ally, this was seen clearly as an encroachment of the synovial edge on to the border of the patella and the articular surfaces of the femur and tibia, to which the menisci were usually adherent. Microscopically, the pannus could be seen emerging from the edge of the chronically inflamed synovium at its junction with cartilage and extending up over the bone, replacing cartilage, and even extending sometimes into the bone itself (fig. 5). The remaining cartilage did not show loss of metachromasia.

The joints that were examined within 24 hr after the second injection of SLS show synovia packed with polymorphs (fig. 6). These changes are not permanent and fig. 7 shows synovium from a joint that had

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COOK AND FINCHAM PLATE CXXXVIII

STREPTOLYSIN S IN RABBITS

FIG. 1 .-Synovial fluid 24 hr after 9th injection of SLS: polymorphs. Leishman. x 105.

FIG. 2.-Synovial fluid 3 days after 3rd injection of SLS: lymphocytes and plasma cells. Leishman. x 105.

FIG. 3.-Synovium 24 hr after 6th injection of SLS: synovial lining- cell proliferation. Haematoxylin and eosin. x200.

FIG. 4.-Femoral articular cartilage show- ing erosion by pannus 24 hr after 9th injection of SLS. HE. x 80.

COOK AND FINCHAM

STREPTOLYSIN S IN RABBITS

PLATE CXXXIX

FIG. 5.-A femur from a joint that had received the 9th injection of SLS 24 hr previously. Extensive pannus has replaced the articular cartilage and penetrated to bone. Azure A. x 50. with polymorphs. HE. x 105.

FIG. 6.-Synovium from a joint that had received 2 injections of SLS, 24 hr after the second injection: cellular infiltration

FIG. 7.-Synovium 1 wk after 2nd injection of SLS. The cellular infiltration has largely disappeared. HE. x 65.

FIG. I.--Synovium 24 hr after 9th injection of SLS. Lymphocytes and plasma cells are present, with polymorphs among them. HE. x320.

COOK AND F~NCHAM STREPTOLYSIN S IN RABBITS

PLATE CXL

FIG. 9.-Synovial changes one wk later than in fig. 8. The predominant cell is the lymphocyte. Several lymph follicles can be seen. HE. x50.

FIG. 10.-Synovial lymph follicle. HE. x 200.

FIG. 11.-Perivascular cuffing by lymph- FIG. 12.-Synovium from a joint that re- ocytes in synovium I wk after 9th ceived its 9th injection of SLS 24 hr injection of SLS. HE. x 320. previously. The amorphous material is

fibrin. This animal was chloroquine treated. Lendrum’s martius scarlet blue. x50.

STREPTOLYSIN S IN RABBITS 465

received 2 SLS injections only. This joint was examined 8 days after the second injection. The cellular infiltration has largely disappeared.

However, if the injections are continued, the synovium is unable to revert to normal, and the polymorph response of the k s t 24 hr following each injection takes place against a background of chronic inflammatory cells. Fig. 8 illustrates this. It is synovium from a joint 24 hr after the 9th injection of SLS; lymphocytes and plasma cells are present, but there are polymorphs among them. These later disappear leaving the residual changes shown in fig. 9. The predominant cell is the lympho- cyte. Plasma cells and a few polymorphs are also present. The lymph follicles are well shown, The table shows that follicles were seen in the majority of injected joints. Fig. 10 shows a high-power view of one of these follicles. Perivascular cuffing by lymphocytes was also frequently seen in the chronically inflamed joints (fig. 11).

Weissmann did not include the presence of fibrin as one of the features of his SLS-produced arthritis. We stained all histological sections with Lendrum’s MSB stain. This stains fibrin bright red. We also noticed many areas of amorphous material, sometimes contiguous to and continuous with the bright red-stained fibrin. These areas stained a greyish blue. We believe this to be fibrin that is altered in some way and have called it “ fibrinoid ”. We have noted the presence of fibrin, fibrinoid, or both, in all the synovia from animals receiving 6 or more injections, at whatever stage they were killed; we also saw it in the 2 rabbits that received only 2 injections and were killed during the acute exudative stage of the first 24 hr after injection.

Some of the fibrin and “ fibrinoid ” is lying free in the joint cavity, but a great deal of it is intrasynovial. Fig. 12 shows the persistence of large amounts of fibrin in the synovium of a joint that had received its 9th injection of SLS 24 hr previously. The histology of the contra- lateral knees is invariably normal.

The rabbit that received SLS while being treated with chloroquine by mouth, and its littermate that received SLS only, show the same histological changes in the joints as the rest of the rabbits receiving 9 injections. The synovia show gross infiltration with lymphocytes, plasma cells and fibrin and there is pannus present. These changes are more marked in the chloroquine-treated animal than in the one that received no chloroquine.

In the goats, both knees of each animal show lymphocytic and plasma cell infiltration in the synovia, with intra-articular and intra- synovial deposits of fibrin. Both joints show pannus formation with destruction of cartilage.

The rabbits that received intra-articular injections of sterile SLS medium did not develop a chronic arthritis. Synovia show only slight lining cell proliferation without any cellular infiltration.

Skin tests. Biopsies of areas of skin into which SLS was injected intradermally show acute inflammatory changes during the first 24 hr. The changes are similar in rabbits that have received an intramuscular

466 JOSEPHINE COOK AND W. J. FINCHAM

course of SLS, and in a normal rabbit. The areas show marked cellular infiltration of the dermis. The predominant cell is the polymorph, and there are small abscess-like areas of degenerate polymorphs. Lymphocytes and a few large mononuclear cells are present. Biopsy at 48 hr of lesions in both treated and untreated animals shows much cellular infiltration, the predominant cell being the lymphocyte. No organisms are seen in Gram-stained sections.

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units per ml.). No lines of precipitation were obtained. The tests were then repeated with streptolysin 0, normal rabbit liver lysosomes, a homogenate of rabbit striated muscle and a homogenate of rabbit synovium as antigens. In every case, a negative result was obtained.

Complement-fixing antibodies. We were also unable to demonstrate in the sera of rabbits treated with SLS any complement-fixing antibodies against normal rabbit liver lysosomes, liver homogenate or synovial homogenate.

Rheumatoid factor. Sera from 4 rabbits after treatment with 9 weekly injections of SLS did not contain rheumatoid factor.

The eflect of SLS on enzymes from heavy granule fractions of normal rabbit liver. Weissmann used heavy granule fractions of rabbit liver for his in-vitro experiments (Weissmann and Thomas, 1962; Weiss- mann, Keiser and Bernheimer, 1963). These contain approximately 85 per cent. mitochondria and 15 per cent. lysosomes. We have repeated his work using both this fraction and a " lysosomal " fraction. Fig. 13 shows the result of such an experiment. This may help to

STREPTOLYSIN S IN RABBITS 467

clarify what is really a question of terminology. Weissmann always referred to his heavy granule fraction as lysosomes. It can be seen from fig. 13 that SLS caused appreciable release of p-glucuronidase from a heavy granule fraction of rabbit liver, but had little or no effect on a " lysosomal " fraction.

The effect of sera from SLS-treated rabbits on release of enzymes from heavy granule fractions of normal rabbit liver by progesterone. Sera from rabbits before and after a course of SLS injections were

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FIG. 14.--The effect of a range of SLS concentrations on turbidity of a RPMN granule suspension.

tested for their effect on the release of acid phosphatase and 8- glucuronidase from heavy granule fractions of rabbit liver. Sera were found to inhibit the release of both enzymes from the fraction, but there was no difference in the effect of sera from untreated rabbits and sera from rabbits after they had finished a course of SLS. Both types of sera enhanced the release of j3-glucuronidase by SLS from the same fractions, and to the same extent.

The effect of SLS on rabbit polymorphonuclear leucocytes and their granules. Our in-vitro experiments have shown turbidity changes in

468 JOSEPHINE COOK AND W. J. FiiVCHAM

rabbit white cell granule suspensions treated with Merent concentra- tions of SLS. Fig. 14 shows these results, which confirm Weissmann’s findings (Weissmann, Becher and Thomas, 1964). But there was poor correlation between these turbidity changes and the very small amounts of enzymes released. When whole cells were used in a similar experiment, there was little change in turbidity with SLS, compared with a large change with Triton X-100. However, there was good enzyme release from whole cells treated with SLS, of up to 60 per cent. of the total enzymes when acid phosphatase and ,k?-glucuronidase were measured.

DISCUSSION Although we have confirmed the histological changes that Weiss-

mann reported to have occurred as a consequence of intra-articular injections of SLS, we have not been able to repeat his findings with respect to complement-fixing antibodies to subcellular particles or inhibition of release of enzymes by the sera of rabbits that have had repeated injections. Weissmann postulated a protective effect on lysosomes by sera from rabbits that had been treated with SLS. He called the sera from rabbits before and after treatment normal and “ i m u n e ” sera. He reported that “ immune ” serum from such a rabbit inhibited the release of ,k?-glucuronidase from heavy granule fractions of normal rabbit liver by progesterone, and correlated this result with a high titre of complement-fixing antibody in its serum after SLS injections.

In in-vitro experiments we have not been able to demonstrate release of enzymes from lysosomes by SLS, although we have shown that it releases appreciable amounts of 8-glucuronidase from heavy granule fractions containing lysosomes and mitochondria. p-Glucuroni- dase is not believed to be restricted to an intralysosomal location and is probably associated with mitochondria1 and microsomal mem- branes. It could be argued that the /3-glucuronidase is derived from the mitochondria or from the larger and, therefore, possibly more fragile lysosomes in the heavy granule fraction. If the former is the case, the in-vivo effect of SLS could be brought about by a two-stage reaction, with subsequent release from lysosomes after mitochondrial disruption.

If enzymes from subcellular particles are involved in the production of arthritis by SLS, the fact that SLS has been shown to release enzymes from rabbit white cells could also be relevant. The intensely cellular exudate in the initial response of these joints to the injections provides a substrate of polymorphs from which enzymes could subsequently be released.

We have, however, found very little evidence that the joint damage is caused by release of hydrolytic enzymes. Cartilage, when it is destroyed, is removed by the encroachment of pannus, and although this may be effected by enzymes at the pannus-cartilage junction, the

STREPTOLYSIN S IN RABBITS 469

remaining cartilage matrix does not lose its metachromasia. Moreover, fibrin persists in large amounts in these joints. Had a release of acid hydrolases from polymorphs and synovial cells been an important factor, one would have expected rapid removal of fibrin as well as loss of metachromasia in the cartilage.

Chloroquine is alleged to be a stabiliser of lysosomes (Weissmann, Standish and Bangham, 1964; Zvaifler, 1964), but it did not modify the lesions produced by SLS injections when given systemically, or when given combined with SLS intra-articularly.

Though we have not found auto-antibodies to subcellular particles in the serum, it is possible that they are present in the synovial fluid, which is too scanty to examine. Though there are many plasma cells in the lesions, no antibody to SLS or altered host tissue has been found.

The proliferation of lymphocytes might suggest a delayed-type hypersensitivity reaction, but it has been shown that SLS will cause the division of cultured lymphocytes in vitro (Hirschhorn et al., 1964).

The similarity of these synovial changes to those in the joints of rabbits injected with heterologous fibrin (Dumonde and Glynn, 1962) suggests that synovium can respond in a non-specific way to a variety of different stimuli, one of which is SLS. Probably Ziff had something like this in mind when he said in his Heberden Oration (Ziff, 1965, p. 113) " ' Take this frail membrane . . . and tell me, if you can, why from time to time, it fills with lymphocytes ' ".

SUMMARY Streptolysin S was injected at weekly intervals into the knees of

rabbits. Three groups of rabbits received respectively 2, 6 and 9 injections. Rabbits were killed either 24 hr or one week after the last injection.

Aspirated synovial fluids showed a polymorph cellular exudate during the first 24 hr, followed by an exudate containing plasma cells and lymphocytes. Animals that received 2 injections showed only transitory infiltration of synovium with polymorphs.

The rabbits receiving longer courses of injections developed joint swelling, which did not subside between the later injections. The histological changes consisted of synovial proliferation with dense infiltration of the synovium with lymphocytes and plasma cells, increased vascularity, fibroblastic proliferation, deposition of fibrin and, in the majority of cases, pannus formation with erosion of articular cartilage. The administration of chloroquine did not modify the lesions produced by SLS.

Chronic arthritis was also produced by intra-articular injections of SLS into goats.

These results confirm those of Weissmann et al. (1965), but their serological findings and in-vitro findings with respect to lysosomes have not been confirmed. Their hypothesis has been discussed.

470 JOSEPHINE COOK AND W. J. FINCHAM

We should like to thank Dr C. H. Lack for a great deal of advice and encourage- ment, and Dr H. M. Tyler and Miss Lois Evans for the biochemical estimations. We are grateful to Dr S. G. Anderson of the Department of Biological Standards, National Institute for Medical Research, Mill Hill, for the rheumatoid factor estimations and to Mr D. C. J. Sayers and the Department of Medical Photography for the photographs. Much of this work has been made possible by a grant to Dr Lack from the Nuffield Foundation; we also acknowledge the provision of the Zeiss photomicroscope by the Peel Medical Research Trust.

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