vivo was suggested by the recent observation that streptolysin 0 (SLO) and streptolysin S (SLS), from hemolytic streptococci was capable of disrupting ...
STUDIES ON LYSOSOMES VII. ACUTE AND CHRONIC ARTHRITIS PRODUCED BY INTRA-ARTICULAR INJECTIONS OF STREPTOLYSIN S IN RABBITS GERALD WEISSMANN, M.D.*; BEVERLY BECHER, B.S.; GERHARD WIEDERMANN, M.D., AND ALAN W. BERNHEIMER, Ph.D.
From the Departments of Medicine and Microbiology of the New York University School of Medicine, New York, N.Y.
It has been suggested that some of the manifestations of connective tissue disease may result from the release of injurious enzymes from lysosomes into the cell sap or surrounding tissues.'-3 Although several studies have indicated that lysosomes are disrupted upon cell death,4 and during acute forms of tissue injury and inflammation,5-7 no direct evidence suggests that chronic lesions of connective tissue may be caused by activation of lysosomal enzymes. The purpose of this study was to observe the consequence, in rabbit synovial tissue, of the chronic rupture of lysosomes, with periodic release of lytic enzymes into cell sap and extracellular spaces. That such an effect might be obtained in vivo was suggested by the recent observation that streptolysin 0 (SLO) and streptolysin S (SLS), from hemolytic streptococci was capable of disrupting lysosomes in the living cell 8 as well as in vitro.9"10 SLS appeared to be an appropriate agent with which to injure lysosomes in vivo for the following reasons: (a) it is water-soluble, therefore intra-articular injections may be made without introducing lipid solvents, (b) it is not itself antigenic," therefore any immune responses encountered should not simply represent a local Arthus reaction to the injected material, (c) it is effective upon lysosomes in vitro at concentrations significantly lower than the other hemolysin,9 and (d) in vitro studies suggest that lysosomes are somewhat more susceptible to the effects of SLS than are other organelles, such as mitochondria.9 Finally, the presence of lysosomes in certain lining cells of synovium has been recently documented.'2"13 The studies to be reported below indicate that SLS regularly produces an acute, and finally a chronic, arthritis upon repeated injection Supported by grants from the United States Public Health Service (AM 053I6), the Armed Forces Epidemiology Board, Commission on Streptococcal Disease, and the Life Insurance Medical Research Fund. Presented, in part, at the 24th Annual Meeting of the American Federation for Clinical Research, Atlantic City, May, I964. Accepted for publication, June 23, I964. * Senior Investigator, Arthritis and Rheumatism Foundation. 129
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into the knee joints of rabbits. The histologic features of the lesion resemble some forms of chronic arthritis in the human; the infiltration of hyperplastic synovium by lymphocytes and plasma cells suggests that an immune reaction may have been evoked. Since SLS did not itself prove antigenic under these circumstances, it appeared possible that the lysin may have elicited an immune response directed not against SLS, but against altered tissue constituents of the host. Experiments to support the latter hypothesis will also be presented. MATERIAL AND METHODS Male albino rabbits from commercially obtained hybrid stock were fed a standard laboratory diet; at the beginning of each experiment these weighed no more than i kg nor less than 8oo gm. Streptococcal Products. The preparation and assay of SLO, SLS, erythrogenic toxin, DNAase A, DPNase, streptokinase, and of a product from the SLS-lacking mutant, have been described before.9 Crystalline streptococcal proteinase was a gift of Dr. Randolph Chase of the Rockefeller Institute. Each toxin was sterilized by filtration and dissolved in saline so that o.i ml of solution contained the requisite amount of toxin. Procedure. Animals were given injections into the knee joint with an appropriate amount of test substance dissolved in o.i ml of saline; disposable tuberculin needles and syringes were employed. At intervals indicated below, joints were opened, cultured in broth, aerobically and anaerobically, and on blood agar plates. Broth cultures were observed for up to 2 weeks. Tissue samples were immediately fixed in formalin, buffered to neutrality. Sections of knee joints were stained with hematoxylin and eosin, and with toluidine blue. Serum was obtained by cardiac puncture before any injections were made, and again at the time of sacrifice. Sections of liver, spleen, kidney, and heart were also obtained from several animals. Preparation of Rabbit Liver Lysosomes, and Treatment with Steroids. A lysosomerich fraction of rabbit liver was obtained in 0.25 M sucrose by methods described in detail previously.9"14'15 Suspensions of these granules (s.o ml) were incubated either as controls, or in the presence of 5 X I-4 M etiocholanolone, or progesterone. These steroids partially disrupt lysosomal membranes, and thus test the "fragility" of the particle in vitro.16 Other aliquots were exposed to the steroids in the presence of o.5 ml of serum obtained from experimental and control animals, both before and after the course of intra-articular injections. Following incubation, the granules were centrifuged at 20,000 X g, and the supernatants analyzed for acid phosphatase and beta-glucuronidase activity by methods previously described.14"5 Complement Fixation Using Subcellular Fractions as Antigens. Sera were examined for their capacity to fix complement in the presence of subcellular fractions from rabbit liver as antigens. "Mitochondrial," "lysosomal," "ribosomal," and "cell supernatant" fractions were prepared in 0.25 M sucrose by methods described in detail elsewhere.17-19 The names of these fractions should by no means suggest homogeneity; "mitochondrial" fractions are partially contaminated by lysosomes, and "lysosomal" fractions have a few mitochondria admixed. The other two fractions are perhaps somewhat purer. Following the incubation of inactivated serum (o.i ml of serial dilutions) with subcellular fractions (o.I ml of suspensions containing 0.3 to o.s mg protein per ml), and fresh guinea pig serum (o.I ml containing 2 C',oo units) for 2 hours at 370 C, sensitized erythrocytes were added and incubated for 45 minutes, also at 370 C. The reactions were carried out in the presence of veronal buffer, o.i5 M, pH 7.4, with 5 X Io-4 M Mg++, and i.5 X Io-4 M Ca++. Results
STREPTOLYSIN S
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131
are expressed as the reciprocal of the highest serum dilution capable of fixing complement with the standard concentration of antigen. RESULTS
Acute and Chronic Arthritis Produced by Intra-Articular SLS. Within four to six hours after the introduction of SLS into rabbit knee joints, a gross arthritis developed in each of I43 joints injected (Table I). Swelling, erythema, and warmth were evident; the rabbits limped TABLE I RELATIONSIP OF JOINT ALTERATION TO NUMBER OF INTRA-ARTICULAR INJECTIONS OF STREPTOLYSIN S
No. of SLS No. of injections animals t I 2
I2
I2*
3
3 4 5
4
3*
4 4 6
6
Total *
Number of animals demonstrating: Inflammatory Synovial lining Chronic Pannus Lymphoid Fibrinoid in exudate cell proliferation synovitis formation follicles vessel walls Polys Monos 0 0
4*
12 3
3
2* 2* 2*
4 4 6
0 0
0 0
4
I
4 4 6
3 4 6
I + 2
7
2
0
2
2
2
9
3
I*
3
3
3
143
38
-
_
0 0
2
I
I
3
4
3
3 4 6
3 3 2
I 2
2
0
2
I
- -
Each joint fixed i8 hours after last SLS injections; others in group 72 hours after last SLS injec-
ion.
t Each animal injected only in left knee joint with SLS (65 ,g, or iooo hemolytic units in o.i ml of saline).
noticeably. The arthritis became quite severe i8 to 20 hours after injection, when cloudy synovial exudate was easily aspirated from the swollen joints. By 48 hours, the joints became less swollen, and by 72 hours, all signs of inflammation had subsided. The animals were then able to hop about without gross limitation of motion. After 3 or 4 injections in some animals, but after 5 injections in all animals, the affected joints became chronically swollen, warm, and persistent effusion was observed. Some animals were observed for periods up to 3 months after arthritis had been so induced; the lesions appeared selfperpetuating for this period. It was possible to aspirate fluid from many of the affected joints. Each injection of SLS provoked a new bout of acute arthritis in the chronically inflamed joints; acute heat and redness disappeared after the same interval as after the first SLS injection. There was no sudden intensification of each acute episode of arthritis upon repeated injections, nor did the reactions appear to be lessened. After chronic arthritis had developed, the rabbits persistently limped about, favoring the affected leg. Cultures of all joints were almost in-
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variably negative. However, from 2 joints injected with saline alone an alpha-hemolytic streptococcus was isolated, perhaps as a contaminant; in one joint from an animal injected with SLS, a coagulase negative staphylococcus was obtained. Microscopically, within 4 to 6 hours, sections revealed an intense polymorphonuclear leukocytic infiltrate in the synovium, which extended to the lining cells. Free granulocytes appeared in the synovial fluid, and could be observed in smears of the aspirated synovial exudate (Fig. I). The exudative phase had disappeared by 72 hours after injection. Soft tissue edema and vacuolization of the lining cells of synovium were also evident. After the third or fourth injection of streptolysin, the histologic picture changed considerably. Although after 4 to 6 hours, and frequently after i8 hours, heterophil infiltration was still evident, most of the inflammatory response was now marked by the presence of many lymphocytes, plasma cells and mononuclear cells (Fig. 2, 3 and 7). These surrounded small blood vessels in the synovium (Fig. 4), the walls of which not infrequently were seen to be undergoing fibrinoid necrosis. The lining cells of synovial tissue, which normally constitute a single layer, had become hyperplastic; this layer frequently grew to a depth of 3 to 4 cells (Fig. 3). The hypertrophied synovial lining often became villous and frond-like in appearance; round cell infiltration was quite prominent (Figs. 2, 3, 4 and 7). Areas of extracellular fibrinoid could be seen in the lining cell layer as well as beneath the surface. At times, the aggregates of lymphocytes and plasma cells immediately beneath the synovial lining, and especially in the angles of the joint lining, resembled the germinal centers of lymph nodes (Fig. 5). In most chronically affected joints, true pannus formation was evident, with fibroblastic proliferation; granulation tissue and mononuclear cells invaded cartilage from the lateral margins of articular surfaces (Fig. 6). Underlying the granulation tissue, in most cases, was cartilage which had lost its normal basophilia on hematoxylin and eosin staining, and its metachromasia to toluidine blue (Fig. 6). The Effects of Streptococcal Products Other Than SLS on Rabbit Joints. As may be seen from TABLE II, only streptolysin S had the capacity to induce severe, chronic arthritis in rabbits. The degree of arthritis was judged by the extent of synovial exudate, either granulocytic (if sacrificed within i8 hours after injection) or mononuclear (if sacrificed 72 hours after injection). Streptolysin 0 was almost without effect upon the synovial tissue of rabbits, despite its ability to disrupt lysosomes in vivo and in vitro. This discrepancy may be due to the capacity of minute amounts of normal rabbit serum to inhibit the action
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on isolated lysosomes of SLO, but not of SLS.20 Perhaps the most appropriate control for SLS is the product prepared in the same manner as SLS, but from a mutant streptococcus lacking the ability to elaborate a hemolytic product.21 This material resembles SLS in gross chemical TABLE II ARTHRITIS INDUCED IN RABBIT KNEE JOINTS BY STREPTOCOCCAL PRODUCTS
Agent injected intra-articularly *
Saline Streptolysin S 65 Ag per kg Streptolysin 0 IOOO Ag per kg SLS-less mutant product DNAase A DPNase Streptokinase Activated proteinase Erythrogenic toxin
No. of injections
97
No. of No. with animals gross arthritis
No. with microscopic evidence of synovitis t minimal moderate severe
35 38
0
2
I
0
I43
38
4
I2
22
9
6
0
2
0
0
38 2 2 4
10
0 0 0 0
7
I
0 0
0 0 0 0 0 0
6
2 2 2 4
6
4
I
I 4
0 0 0 0
I
3
0
* Dosage of SLS, SLO as given. All other toxins dissolved to give concentration of 75 ,g per kg. in 0.I ml of saline. t Judged by degree of inflammatory exudate in synovium.
composition, but lacks any effect upon the membranes of lysosomes.9 ""21 When injected repeatedly into joints, to a maximum of 9 times, it occasionally induced a mild inflammatory exudate, composed mainly of heterophils; no gross arthritis was observed in any of IO animals so injected. Such streptococcal products as DNAase A, DPNase, or streptokinase, were not inflammatory. Erythrogenic toxin, and activated crystalline proteinase produced minimal arthritis, the onset of which was IO to I2 hours after injection: microscopically only edema and a mild polymorphonuclear exudate were observed in 7 animals. In animals injected with these toxins, edema was more pronounced than cellular reaction. Subsequent injections of erythrogenic toxin or proteinase did not induce gross arthritis, perhaps because of circulating antibody. Joints injected repeatedly with saline showed only thickening of the lining cell layer, associated with minimal polymorphonuclear response early after injection. Minimal mononuclear cell infiltration was apparent after the fifth to ninth injections in some animals. In each animal, injection was into only one knee joint, in order to evaluate whether a "sympathetic" arthritis could develop after repeated injections of a given agent. In 6 of 38 rabbits injected with SLS,
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a mild, chronic synovitis consisting mainly of mononuclear infiltrate was indeed observed in the uninjected joint: each had been injected more than 5 times with SLS (Fig. 8). One control animal, and 3 animals injected with the mutant product, showed comparable changes in non-injected joints. Evidence Against Hypersensitivity to SLS. Despite the many reports of the non-antigenicity of SLS, it remained possible that the lesions provoked by repeated injections of the toxin might represent local Arthus reactions to trace antigens present in the injected material. Therefore the appearance of immediate or delayed-type hypersensitivity reactions to SLS was studied in the skin of affected animals. At a time when 8 of the animals had developed severe arthritis (5 injections each), o.i ml of SLS was injected intradermally into the shaved abdominal skin. Similar injections were made in control animals. Only mild erythema, lasting I2 to i8 hours, was evident in any of these animals; the area of erythema did not exceed 2.5 mm2 in either arthritic or control animals. The absence of acute reactions would suggest the absence of circulating antibody. By 36 to 38 hours, no evidence of erythema or induration, such as might be expected to result from delayed-type hypersensitivity, was observed in the skin of any of the experimental or control animals. That Arthus reactions were not, in fact, induced locally was also suggested by the finding that repeated injections did not lead to necrosis, nor was each acute arthritic response worsened. To further exclude the presence of circulating antibody, precipitin reactions in agar were performed in Ouchterlony plates: streptolysin S was set in the center wall as "antigen," and allowed to diffuse towards serial dilutions of sera from the 8 rabbits which had developed severe arthritis. No precipitin arcs were observed. Sera from 2 rabbits injected twice with streptolysin 0 in the knee joint showed discrete lines against SLO used as "antigen." These experiments suggested the absence of circulating or cellular antibody directed against SLS preparations. Since, however, the lesions induced were rich in lymphocytes and plasma cells, it remained possible that streptolysin S had induced an immune response directed against constituents of the host's tissues. Complement Fixation Tests Using Subcellular Fractions of Rabbit Liver as Antigens. Sera were obtained before SLS injections and at the time of sacrifice, after arthritis had developed. As may be seen in Table III, significant increases in titer of complement-fixing antibody directed against lysosome-rich fractions of rabbit liver were observed in 6 of 8 sera of rabbits injected with SLS, but in none of the rabbits injected with the mutant product. Such antibody could be removed
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TABLE: III TITER OF COMPLEMENT-FIXING ANTIBODY IN SERA OF RABBITS BEFORE AND AFTER STREPTOLYSIN S-INDUCED ARTHRITIS
No. of streptolysin S injecAnimal tions "Mitochondria"
E4 I7 E2 lB
Ii
2 4 5 6 9
Is
9
Ls
9 7
L
Controls* 2-9 t
Titer against "Ribosomes" "Cell supernatant" "Lysosomes"
Before
After
Before 40
20
8o I60
After I60
Before
QO
20
320
80
320
80
0 10 0 0 0 0 10
20
80
20
320 320
40
8o 8o
40
320
40 20
I60
20
80
40 20 10
160 40
0-20
0-40
o-80
8o
o-8o
(n = 8)
20
0-20
After Before
Severity of synovitis
After
+++
20 40
10
0
0
40
++
20 10
0 IO
+++
5
++++ ++++
40
0 0 0 0 0 0
0-20
0-IO
O-IO
5 0 0
0 0 0
++
++++
+++ 0-+
Injected with streptolysin S-lacking mutant product. t Lowest and highest titers given.
*
from serum by absorption with lysosome-rich fractions of rat or rabbit liver, and of rabbit leukocyte lysosomes, prepared as described elsewhere.10"7-'9 Some increases in titer directed against "mitochondrial" fractions were also found, perhaps because of the contamination of this fraction by lysosomes. The Effect of Sera Rich in Complement-Fixing Titer Against "Lysosomal" Fractions of Rabbit Liver Upon Release of Beta-Glucuronidase from Rabbit Liver Granules. It may be seen in Table IV that the addition of normal rabbit serum to large granule fractions of rabbit liver (rich in lysosomes) resulted in some augmentation of the release of beta-glucuronidase from the granules after incubation for 6o minutes. When, however, serum from rabbits which had developed a high titer of antibody directed against lysosomes was added to the granules, considerable inhibition of this effect upon enzyme release was observed. This inhibition was evident in control samples, and was particularly striking in samples to which etiocholanolone or progesterone had been added in order to test the "fragility" of the particles."6 Neither set of sera inhibited to any degree the activity of beta-glucuronidase after the enzyme had been solubilized by disintegrating the granules in a Waring blendor. The results of these studies indicate that a factor, or factors, present in sera of rabbits which developed high titer antibodies against lysosomes, inhibited release of lysosomal hydrolases from isolated homologous lysosomes. Such factor(s) would appear to be directed against the membranes of lysosomes, rather than against the soluble enzymes contained within the particles.
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TABLE IV RELEASE OF BETA-GLUCURONIDASE FROM RABBIT LIVER GRANULES EXPOSED TO SERA FROM RABBITS BEFORE AND AFTER SLS ARTHRITIS *
Treatment of granules
Serum from rabbit no.
No. of
%
Enzyme activity
injections
released from controls
nil
-o- IO
nil
L Before t After
9
2I6 I55
E2 Before After
5
227 145
Is Before After
6
200
Ia Before After
9
L Before After
-85
nil nil nil nil Progesterone 5 x IO4M cc
is
Etiocholanolone 5 x Io'M Is 99
Disintegration in Waring Blendor 5'
nil I Before After
nil I Before After nil IL Before After
133 220
162
9
I33
9
430 380 324
9
415 405 286
9
820 8I6 827
* Large granule fractions in 0.25 M sucrose incubated at 370 for 6o' (5.0 ml homogenate: 0.5 ml serum + o.i ml steroid in ethanol), then centrifuged 20,000 X g for 20'. Enzyme activities of supernatant determined. Means of two experiments. t Before and after SLS arthritis had developed. All sera stored at -20° C. : Controls released 12.2 per cent of "total" beta-glucuronidase activity of large granule fraction.
DISCUSSION The data presented above indicate that streptolysin S has the capacity to induce acute and chronic arthritis in rabbits after repeated intraarticular injections. Previous reports as to the in vivo effects of streptolysin S have emphasized the capacity of the toxin to injure renal basement membranes,22 and to induce hemolysis and myocardial lesions after intravenous injection.2B Under the conditions of the present studies, no renal lesions were found nor were consistent cardiac lesions, or hemolysis observed in the affected animals. The absence of these findings presumably reflects the minimal quantities of the toxin employed, and the site of injection, which was local. Although the cytotoxicity of streptolysin S for most somatic cells has not been extensively
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STREPTOLYSIN S
I37
studied, several investigators have documented the toxicity of streptolysins for erythrocytes, lymphocytes, platelets and leukocytes." The mechanism whereby SLS injures leukocytes has been studied in some detail 8'10; there is evidence that rupture of the membranes of leukocyte lysosomes is an early, if not perhaps the first, effect observed by phase contrast microscopy. It was reasonable to suppose, therefore, that injury to lysosomes of synovial lining cells, with release of their tissuedamaging enzymes, might be the mechanism whereby chronic synovial injury was induced. This postulate is supported by the observation that rises in titer of complement-fixing antibody directed against the membranes of autologous lysosomes were demonstrable in the sera of animals given repeated injections of SLS. No comparable rises in titer against other subcellular components were found, nor was there any evidence that antibody had been directed against the injected material itself. Recent histochemical and electron microscopic studies have shown that as early as 5 hours after SLS injections into rabbit joints, the staining properties of synovial lysosomes were significantly affected. Chronic SLS injections led to the formation of residual bodies (altered lysosomes) in synovial lining cells.24 Such changes resembled somewhat the changes described by Barland, Novikoff and Hamerman in chronic human rheumatoid arthritis.'3 Thus two independent lines of evidence suggest that at least one effect of SLS in the rabbit knee joint is upon lysosomal membranes. Since soluble lysosomal extracts, prepared from rabbit peritoneal leukocyte lysosomes, can induce an acute arthritis,5 and since enzymes in such extracts can degrade the protein polysaccharides of cartilage matrix in vitro 25 it is possible that substances released from lysosomes are responsible for the inflammatory response to SLS and the degradation of cartilage matrix in vivo. Under the conditions of the present study, it is impossible to be certain whether SLS did not exert other, perhaps more profound, effects upon other organelles, or upon cell membranes; actions of SLS upon mitochondria have also been reported.26 Nor, for that matter, is it possible to exclude actions of the lysin upon other mediators of the inflammatory response, such as the vaso-active amines or polypeptides. Further studies are in order to discover the detailed sequence of tissue injury by this lysin. However, many of the features of this experimental arthritis bear resemblances to the joint lesions of human disease: (a) hyperplasia of synovial lining cells, (b) vacuolization of synovial lining cells, (c) villous synovitis and vasculitis, (d) the appearance of lymphoid follicles in synovium, often with many plasma cells, and (e) the elaboration of pannus which erodes the underlying cartilage. These
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experiments by no means suggest that streptolysin S is responsible for chronic arthritis in man, but appear to demonstrate that an exogenous agent, capable of disrupting lysosomes in vitro and in living cells, can produce an experimental arthritis morphologically resembling the local lesions of human disease. Finally, the appearance of complement-fixing antibodies directed not against the injected material, but against constituents of the host's tissues, raises the possibility that "auto-antibodies" found in the course of natural and experimental disease may not arise as a consequence of a basic defect in the growth and differentiation of immune cells.27 The partial denaturation of "native" constituents of the animal (brought about by the release of hydrolases from lysosomes into the cytoplasm of antigen-preparing cells) may, in fact, elicit a normal immune response to improperly handled antigenic material. Since sera high in titer of anti-lysosomal antibody appeared to stabilize the granules against exogenous disruptive agents, it remains entirely possible that serum "auto-antibody" may have the protective role that Paterson and Harwin28 have postulated. SUMMARY Streptolysin S, when injected into the knee joints of rabbits, regularly elicited an acute arthritis. Upon repeated injections, a chronic arthritis was induced which was characterized by hyperplasia of synovial lining cells, infiltration of synovium by lymphocytes and plasma cells, fibrinoid necrosis, and true pannus formation, with destruction of underlying articular cartilage. No other streptococcal product regularly induced such changes. Despite the non-antigenicity of the injected lysin, 6 of 8 rabbits with chronic, severe arthritis developed complementfixing antibody directed against subcellular particles (lysosomes) obtained from homologous liver homogenates. Sera from such animals had the capacity to stabilize isolated lysosomes against disruption by various agents. The data are compatible with the hypothesis that streptolysin S, an agent which has previously been shown capable of disrupting lysosomes in vitro and in the living cells, disrupts the lysosomes of synovial tissue thereby releasing tissue-damaging substances.
REFERENCES I. WEISSMANN, G., and DINGLE, J. Release of lysosomal protease by ultraviolet irradiation and inhibition by hydrocortisone. Exper. Cell. Res., I96I, 25, 207-2IO.
2. WEISSMANN, G., and THOMAS, L. Steroids, lysosomes and systemic lupus erythematosus. Bull. New York Acad. Med., I962, 38, 779-787.
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3. DINGLE, J. T. Aetiological factors in the collagen diseases. Lysosomal enzymes and the degradation of cartilage matrix. Proc. Roy. Soc. Med., I962, 55, IO9-III.
4. DEDuvE, C. The Lysosome Concept. In: Lysosomes. Ciba Foundation Symposium. DE REUCK, A. V. S., and CAMERON, M. P. (eds.). Little, Brown & Co., Boston, I963, PP. I-35. S. WEISSMANN, G., and THOMAS, L. On A Mechanism of Tissue Damage by Bacterial Endotoxins. In: Bacterial Endotoxins. BRAUN, W., and LANDY, M. (eds.). Rutgers University Press, I964, 6. THOMAS, L. Possible role of leukocyte granules in the Shwartzman and Arthus reactions. Proc. Soc. Exper. Biol. & Med., I964, II5, 235-240. 7. HALPERN, B. N. Inhibition of the local hemorrhagic Shwartzman reaction by a polypeptide possessing potent antiprotease activity. Proc. Soc. Exper. Biol. & Med., I964, II5, 273-276. 8. HIRscH, J. G.; BERNHEIMER, A. W., and WEISSMANN, G. A motion picture study of the toxic action of streptolysins on leucocytes. J. Exper. Med., I963, iI8, 223-228. 9. WEISSMANN, G.; KEISER, H., and BERNHEIMER, A. W. Studies on lysosomes. III. The effects of streptolysins 0 and S on the release of acid hydrolases from a granular fraction of rabbit liver. J. Exper. Med., I963, ii8, 205-222. IO. WEISSMANN, G.; BECHER, B., and THOMAS, L. Studies on lysosomes. V. The effects of streptolysins and other hemolytic agents on isolated leucocyte granules. J. Cell Biol., I964, 22, 115-I26. II. BERNHEIMER, A. W. Streptolysins and Their Inhibitors. In: Streptococcal Infections. MCCARTY, M. (ed.). Columbia University Press, New York, I954,
PP. I9-3412. BARLAND, P.; NOVIKOFF, A. B., and HAMERMAN, D. Electron microscopy of the human synovial membrane. J. Cell Biol., I962, 14, 207-220. 13. BARLAND, P.; NOVIKOFF, A. B., and HAMERMAN, D. Fine structure and cytochemistry of the rheumatoid synovial membrane, with special reference to lysosomes. Am. J. Path., I964, 44, 853-866. I4. WEISSMANN, G., and THOMAS, L. Studies on lysosomes. I. The effects of endotoxin, endotoxin tolerance, and cortisone on the release of acid hydrolases from a granular fraction of rabbit liver. J. Exper. Med., I962, ii6, 433-450. I5. WEISSMANN, G., and THOMAS, L. Studies on lysosomes. II. The effect of cortisone on the release of acid hydrolases from a large granule fraction of rabbit liver induced by an excess of vitamin A. J. Clin. Invest., I963, 42, 66I-669. i6. WEISSMANN, G. Studies on lysosomes. VI. The effect of neutral steroids and bile acids on lysosomes in vitro. Biochem. Pharmacol. (In press) I7. WIEDERMANN, V. G.; DOERNER, M., and MIESCHER, P. A. Autoimmunitare Vorgange gegen Lebergewebe. Schweiz. med. Wchnschr., I964, 94, 257-262. i8. MIESCHER, P. A.; BARKER, L.; VAINIO, I., and WIEDERMANN, G. Immune Mechanisms of Cell and Tissue Damage in Systemic Lupus Erythematosus. In: Injury, Inflammation, and Immunity. THOMAS, L.; UHR, J., and GRANT, L. (eds.). Williams & Wilkins, Baltimore, I964, PP. 346-352. I9. WIEDERMANN, G., and MIESCHER, P. A. Cytoplasmic antibodies in patients with systemic lupus erythematosus. Ann. New York Acad. Sc. (In press) 20. WEISSMANN, G., and BECHER, B. Unpublished observations. 2I. BERNHEIMER, A. W. Formation of a bacterial toxin (streptolysin S) by resting cells. J. Exper. Med., I949, 90, 373-380.
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22. TAN, E. M., and KAPLAN, M. H. Immunological relation of basement membrane and a serum beta globulin in the mouse. Demonstration of renal basement membrane alteration in mice injected with streptolysin S. Immunology, I963, 6, 33I-344. 23. WEISSMANN, G., and THOMAS, L. The actions of cortisol on connective tissue and subcellular particles. Recent Progr. Hormone Res., I964, 20, 2I5-239. 24. BARNARD, W. G., and TODD, E. W. Lesions in the mouse produced by streptolysin 0 and S. J. Path. & Bact., 1940, 51, 43-47. 25. WEISSMANN, G.; BARLAND, P., and WIEDERMANN, G. Role of lysosomes in streptolysin S-induced arthritis. (Abstract) Clin. Res., I964, 12, 240. 26. KEISER, H.; WEISSMANN, G., and BERNHEIMER, A. W. Studies on lysosomes. IV. Solubilization of enzymes during mitochondrial swelling and disruption of lysosomes by streptolysin S and other hemolytic agents. J. Cell Biol., I964, 22, 10I-II3.
27. ZIFF, M. Genetics, hypersensitivity and the connective tissue diseases. Am. J. Med., I96I, 30, I-7. 28. PATERSON, P. Y., and HARWIN, S. M. Suppression of allergic encephalomyelitis in rats by means of antibrain serum. J. Exper. Med., I963, II7, 755-774.
LEGENDS FOR FIGURES Photomicrographs were prepared from sections stained with hematoxylin and eosin. FIG. x. Rabbit synovium, 24 hours after an injection of 65 ,ug of streptolysin S (SLS) into the knee joint. Areas are filled with a dense polymorphonuclear leukocytic exudate, and fibrin fills the extracellular space. Some leukocytes and fibrin are found free in the joint space. This joint was sterile. X iSo. FIG. 2. Rabbit synovium, 24 hours after the last of 4 injections of SLS into the knee joint. The inflammatory exudate is composed of granulocytes, but a considerable number of small and large round cells are also present. The lining cell layer has become thicker, and fibroblastic proliferation is evident. The joint was sterile. X ISo.
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U
2
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Vol. 46, No.
r
FIG. 3. Rabbit synovium, 72 hours after the last of 5 injections of SLS into the knee joint. The synovium has become villous, the lining cell layer has become markedly thickened, and the whole tissue has become infiltrated by small and large mononuclear cells. Fibroblast proliferation is evident; the joint has become the seat of a chronic inflammatory response. This joint was sterile. X I5O. FIG. 4. Higher-power view of rabbit synovium shown in Figure 3. The synovial lining layer is swollen and hyperplastic. Lymphocyte and plasma cell infiltration is evident around blood vessels. X 500.
Jan., I965
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WEISSMANN ET AL.
Vol. 46, No. I
FIG. 5. Higher-power view of synovium shown in Figure 4. Lymphocytes and occasional plasma cells appear in small clusters. X 200. FIG. 6. Area of articular cartilage from a rabbit injected with SLS into the knee joint 7 times, and sacrificed 24 hours after the last injection. Cartilage, upper right, has lost its basophilia and is undergoing destruction by adjacent granulation tissue. X I50.
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Vol. 46, No. I
FIG. 7. High-power view of area shown in Figure 2. An admixture of polymorphonuclear leukocytes and chronic inflammatory cells comprise the acute response to SLS in a joint which has been the seat of a chronic arthritis. X 200. FIG. 8. "Sympathetic" arthritis in an uninjected joint from a rabbit given 6 injections of SLS in the contralateral joint. The joint was sterile. X 200.
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