1295

SECRETION AND LOCALIZATION OF CATHEPSIN D IN SYNOVIAL TISSUES REMOVED FROM RHEUMATOID AND TRAUMATIZED JOINTS AN IMMUNOHISTOCHEMICA L STUDY A. R. POOLE, R. M. HEMBRY, J. T . DINGLE, I.PINDER, E. F. J. RING, and J . COSH

The proteinase cathepsin D which degrades proteoglycan was never demonstrated in extracellular sites in tissues from patients with traumatized meniscoid cartilage, either before or after culture with an antiserum to human cathepsin D. In contrast, in synovia (but not usually cartilage) from the knees of 6 of 11 rheumatoid patients, extracellular cathepsin D was commonly detected by culturing tissues with an antiserum to this enzyme.

From the Tissue Physiology Department, Strangeways Research Laboratory. Wort’s Causeway, Cambridge, England, and the Royal National Hospital for Rheumatic Diseases, Bath, England. Study supported by the Nuffield Foundation, Arthritis and Rheumatism Council, and the Medical Research Council. A . Robin Poole, Ph.D.: Nuffield Foundation Rheumatism Research Fellow, Tissue Physiology Department, Strangeways Research Laboratory. and Director, Joint Diseases Laboratory, Shriner’s Hospital for Crippled Children, Montreal, Quebec, Canada; Rosalind M. Hembry, B.Sc.: Research Assistant, Strangeways Research Laboratory; John T. Dingle, Ph.D., D.Sc.: Member of the external staff of the Medical Research Council, Head of the Tissue Physiology Department, and Deputy Director, Strangeways Research Laboratory; Ian Pinder, F.R.C.S.: Senior Orthopaedic Registrar, Royal National Hospital for Rheumatic Diseases, and Consultant Orthopaedic Surgeon, Royal Victoria Infirmary; E. Francis J. Ring, M A . : Chief Technical Officer, Royal National Hospital for Rheumatic Diseases; John Cosh, M.D., F.R.C.P.: Consultant Rheumatologist, Royal National Hospital for Rheumatic Diseases. Address reprint requests t o A . Robin Poole, Ph.D., Joint Diseases Laboratory, Shriners’ Hospital for Crippled Children, Cedar Avenue, Montreal, Quebec, Canada. Submitted for publication November 10, 1975; accepted July 12, 1976. Arthritis and Rheumatism, Vol. 19, No. 6 (November-December 1976)

Erosion of articular and patellar cartilage is an important feature of chronic joint disease in rheumatoid arthritis. Such cartilage destruction, which leads to a loss of normal joint function, is usually associated with a proliferation of cells of the synovial lining of the joint cavity and by an invasion of the synovium over and into the cartilage. Cartilage matrix erosion is most marked at the edges of the invading pannus tissue. It seems likely that the cells of the pannus are responsible, at least in part, for the destruction of the cartilage. The matrix of cartilage is composed mainly of two high molecular weight molecular species, collagen and proteoglycan. Together they give cartilage its special mechanical properties (1). When cartilage matrix is lost, proteoglycan molecules are degraded at an early stage (2). This loss of proteoglycan is thought to result from the extracellular activity of proteolytic enzymes ( 3 ) , including lysosomal endopeptidases and neutral proteinases. One such enzyme is the lysosomal proteinase cathepsin D, which readily degrades proteoglycan (4) and is involved in the degradation of proteoglycan in cartilage matrix (3,5,6),being present in living cartilage matrix where cartilage proteoglycan is being lost. Although this enzyme has no effect on proteoglycan at pH 7.0, significant degradation occurs at pH 6.0 (7). The pH of the pericellular environment of cells and of cartilage is unknown. As yet there have been no published reports on the secretion of lysosomal proteinases from rheumatoid synovial tissue. Earlier studies (8-1 I ) indicated that the total activities of some lysosomal hydrolases are in-

POOLE ET AL

1296

creased in rheumatoid synovia and that the amount of enzyme activity is related to the clinical activity, t h e degree of inflammation, and the severity of the disease, as shown by the amount of damage to articular cartilage. Granda and coworkers (1 1 ) in particular noted a close correlation between increased cathepsin D activity and an increased severity of the disease. Previously Fell and Dingle (12) a n d Vaes (13) observed with in vitro experiments that an increase in enzyme activity in a tissue is associated with a n increased secretion of lysosoma1 enzymes, including cathepsin D. T h u s the increased activity of lysosomal enzymes seen in rheumatoid synovia may be associated with an increased secretion of cathepsin D and other proteinases. The extracellular localization of this enzyme in tissues removed from traumatized and rheumatoid joints has been examined in the present study. Because extracellular cathepsin D cannot be detected by direct staining of fixed sections, specific antibodies to human cathepsin D have been used in culture t o capture the extracellular enzyme, which was then demonstrated immunohistochemically as described previously (14-16). This work was the subject of a preliminary communication in the form of a brief abstract (17).

MATERIALS AND METHODS Normal rabbit serum (NRS) was a pool of sera collected from 6 New Zealand white rabbits. Species-specific ovine antisera reacting only with human cathepsin D (As-HD) or rabbit cathepsin D (As-RD) were raised in this laboratory and extensively characterized as described previously (5,6,18). These antisera exhibit no cross-reaction whatsoever with other proteins. They are raised against highly purified enzyme preparations, which are devoid of any neutral proteinase activity (7). The precipitating activities of all these antisera were estimated by radial immunodiffusion by means of a method based on that of Mancini et a1 (l9), as described previously (1 5), and are shown in Figure 1. Nonimmune sheep serum (NSS) was a pool of samples taken from 12 animals. Sera used in culture were heated before use to 56°C for 30 minutes to inactivate complement. A n antiserum to sheep IgG was raised in a pig as follows. A 1.5-ml portion of saline containing 5 mg of sheep IgG was emulsified with 1.5 ml of Freund’s complete adjuvant and injected intramuscularly as two 1.5-ml volumes into each hind leg on day 0. These injections were repeated in the foreleg muscles on day 15 and into the hind legs again on day 36. The animal was bled out by intra-carotid catheterization on day 48. IgG was purified in the manner described previously (20). Partially purified pig IgG was prepared from sera by precipitating twice that fraction obtained at 0-50% saturation with ammonium sulphate at 20°C. Pepsin digests containing F(ab’), were prepared from partially purified IgG by digestion of 25 mg/ml IgG at pH 4.5 with 2% of pepsin (Worthington,

twice crystallized) at 40°C for 20 hours. Pepsin was then inactivated at pH 8.6 and digests were dialyzed against phosphate-buffered saline (PBS, 45 mM NaCI, 9 mM Na,HPO,, 1 mM NaH,PO,) to remove low molecular weight digestion products. This method is based on that previously described (21). After reduction for 30 minutes of F(ab’), with 10 mM cysteine to produce monovalent Fab‘ and gel diffusion, in the presence of 2 m M cysteine, pig antibody Fab’ preparations failed to precipitate antigen (sheep IgG) but inhibited the precipitation of antigen with native antibody. F(ab‘), preparations were labeled with fluorescein isothiocyanate (FITC) (22). The protein contents of Fab’ preparations were determined spectrophotometrically assuming El% = 15.0 at 280 nm by analogy with rabbit Fab’ determinations (23). The molar ratios of fluorescein to protein (F/P) in conjugates were recorded by noting the E,,, and E,,, and by

40

-

35 30 25 -u

Ela!

3

D

20-

cn L

2 Q)

15-

E m

5

105-

1

0

I

I

I

I

20

40

60

80

I

100

Antiserum concentration

(2)

Fig 1. Radial immunodiffusion estimarions ofthe precipitating acrivities of antisera to cathepsin D. The relationship between the concentration ofantiserum to cathepsin D and the square of the diameter (in mml o j the precipitation ring is shown. ( a ) Sheep antiserum to rabbit cathepsin D: the gel contained 13.2 pglml or 5.4 unitslml of purified rabbit cathepsin D. Diflusion wells contained 5 pl volumes of antiserum. (b) Sheep antiserum to human cathepsin D: the gels contained 113 pglml or 5.2 unitslml of puri’ed human cathepsin D. Diffusion wells contained 10 p l volumes of antiserum.

1297

SECRETION OF CATHEPSIN D

criteria. All were seropositive for rheumatoid factor. The intensity of inflammation was assessed a ) clinically, as slight, moderate, or very acute according to pain, tenderness, stimness, synovial thickening, and effusion; b) at surgery, by inspection with color photography of the degree of vascularity of the synovium; and c) by preoperative thermography in 9 of the 1 1 . A calibrated isothermal record was made of the temperature patterns over the knee, from which may be calculated (26) a “thermographic index” ranging from 1.0 (cool) to 6.0 (hot). For simplicity, in this study results were graded I (index < 2.5). 2 (index 2.5-4.0), and 3 (index > 4.0). Isolation of Tissues. Small pieces of articular and/or patellar cartilage with synovium or pannus attached were removed at synovectomy of the knee. These samples were no more than 2 mm thick and about 4 mm square. Normally, two samples were removed from each junctional zone. Six patients, each with a traumatized meniscoid cartilage, constituted the control group. A single specimen of fibrocartilage with synovium attached, and of overall size as described previously, was removed from a site close t o the lateral femoral condyle at meniscectomy. All patients were normally anesthetized with halothane. Unless otherwise stated, tissues were explanted at surgery into BGJ, maintenance medium (15) which contained 5% NRS. They were then transported at atmospheric temperature ( I 5 - 2 5 T ) to the laboratory, where they were transferred onto expanded metal grids in the culture vessels.

using the nomogram described by Goldman (24) on the assumption, when calculating molar ratios, that F(ab’), has a molecular weight of 90,000. Previous unpublished work in this laboratory, using radial immunodiffusion (RID) and spectrophotometric analyses of pepsin digests prepared from pig sera with purified pig F(ab’), as a standard, has established that I mg of F(ab’),/mI determined by R I D is equivalent to 1 mg protein/ml estimated spectrophotometrically. Cathepsin D was purified from human and rabbit liver by Dr. A. J . Barrett and was assayed for protein content and enzyme activity a s described previously (25). Pig anti(sheep IgG Fab’,) labeled with FITC (now referred to as P a s Fab’-F) was used at a protein (P) concentration of 0.2-0.4 mg/ml (molar ratio of FITC/P = 1.6/1; mean value for three reagent preparations). Unlabeled Pas Fab’ was used at a protein concentration of 5 mg/ml. Before use, overlabeled Fab’ molecules were removed from FITC-labeled Fab’ by treatment for 30 minutes with 20 mg of acetone-dried powder chicken liver per 5 mg of protein and centrifuged for 2 minutes in a microhematocrit centrifuge (Gelman-Hawksley, Lancing, England) to remove any particulate matter (20). Immediately before use F(ab’), was reduced for 30 minutes with 10 m M cysteine to yield monovalent Fab’, which was used in the presence of 2 mM cysteine. Clinical Assessment of Rheumatoid Patients. The 1 1 patients studied had classic or definite rheumatoid arthritis (Table I)according to the American Rheumatism Association

Table 1. Clinical Data of Patients

Patient

Age

Sex

Duration of Disease (years)

Period Since Previous Synovectomy of Knee (years)

Drugs Received

Rheumatoid patients

RH

41 41 54 58

M

2.5 7

15

F F M F

HJ GW

51 52 56 41

F F F F

15

JB

53

M

3

SL

53

F

14

M F

-

M M M

-

EP BC AB DB AK IH

Menisrectomy patients

DS MK PU

HT PB BH

28 44 28 54 23 36

F

6 13 6 8 5 5

-

Synacthen, aspirin Safapryn. iron (IM). indomethacin Phenylbutazone None Prednisolone, amitryptiline, butobarbitone Prednisolone Synacthen, indomethacin Aloxiprin Indomethacin, phenobarbitone, phenytoin Ibuprofen, myocrysin, aspirin. adcortyl (intraarticular) Prednisolone, aloxiprin, indornethacin

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Detection of Extracellular Cathepsin D. I n view ofthe relative complexity of this method, it will first be outlined before the different stages are described in detail. Tissues were first cultivated for 24 hours, with sheep antibody to cathepsin D to capture extracellular enzyme with the formation of immune complexes; this stage was controlled with nonimmune sheep serum. Tissues were then transferred to culture medium containing nonimmune rabbit serum for 24 hours to permit any "untrapped" sheep IgG to diffuse out of the tissue and to leave behind any insoluble immune complexes. These complexes were detected by freezing the tissue, cutting frozen sections, and localizing sheep IgG with an FITC-labeled Fab fragment of a pig antibody to sheep IgG. Organ Culture. Each piece of cartilage with synovium attached was divided into two or three and cultured as described previously (14.15) with As-HD or control serum in the presence of 1.5 ml BGJ, culture medium per dish per piece of tissue. The medium contained either 5% As-HD or, as a control. As-RD or NSS. In the routine examinations described in detail, culture media were changed after 24 hours and replaced with BGJ, containing 5% NRS. At 48 hours tissues were removed for histochemical analysis. I n more recent experiments, reported separately here, tissues have been established in culture at 37°C in the operating theater with 5% As-HD or 5% NSS i n BGJ, (15) and transferred to culture vessels containing BGJ, and serum and cultured as described previously. In addition, tissues have also been exposed to As-HD or NSS in BGJ, for periods from 3 to 24 hours to remove uncaptured sheep immunoglobulin. Preparation of Tissues for Sectioning. Tissues were removed from culture and immediately immersed at 30°C in a mixture of 7% gelatin (BDH) in 150 mM sodium chloride and 0.15% sodium azide and frozen for 90 seconds in a reservoir of liquid nitrogen. Frozen sections 6-pm thick were cut on a cryostat and fixed immediately on clean microscope slides for 30 minutes in 4% formaldehyde freshly prepared from paraformaldehyde in PBS (27). Sections were washed in PBS containing 5 m M cysteine for 30 minutes before use. Staining Procedures. Sheep IgG was localized by treating sections with Pas Fab'-F for 1 hour. The specific binding of this reagent was controlled by pretreating some sections with unlabeled Pas Fab' for 1 hour. After each treatment with Fab', sections were washed four times by immersion in PBS containing 5 mM cysteine for four 15-minute periods. Some sections were then counterstained with eriochrome black (Difco) as described previously (14). Representative frozen sections were also fixed in formaldehyde and stained with hematoxylin and eosin, or with toluidine blue (15). Sections treated with Pas Fab'-F were immediately examined with darkground fluorescence microscopy (20). Other sections were inspected with brightfield microscopy. A total of 20-30 sections treated with Pas Fab'-F were examined at each of three or four different levels throughout every explant. sulphate in Autoradiography. Carrier-free sodium [TI aqueous solution was acquired from the Radiochemical Centre, Amersham, England. Purified IgG isolated from NSS was labeled with lz5l by using chloramine T (28), which was kindly supplied by Dr. A. J. Barrett. The permeability of synovium to IgG was assessed by culture of explants in BGJ, + 5% NRS containing 'Z51-lgGat 10 pCi/ml. After culture

periods varying from 30 minutes to 24 hours, tissues were fixed in formaldehyde overnight, washed, embedded in wax, and sectioned at 4 pm. 35S042-incorporation studies were also performed in BGJ, with 5% NRS by using IOpCi/ml. Tissues weresimilarly processed after 1 or 2 hours. After dewaxing with xylene, sections were coated with llford emulsion L4. Autoradiographs were developed and stained with carmalum after up to 4 weeks of exposure.

RESULTS Histologic Appearance of Synovia and Cartilage Synovia from traumatized joints examined a t meniscectomy exhibited a thin layer of synovial lining cells up to three cells thick. These cells were underlaid by fibrous capsular tissue through which fibroblast-like cells were scattered in low density. The fibrocartilage of these patients was stained with toluidine blue. In contrast, synovia of patients with rheumatoid arthritis were very villous and exhibited gross hyperplasia of the lining cells. The synovia were characteristically heavily infiltrated with monocytes, lymphocytes, a n d plasma cells. Fibroblast-like cells were common. There was considerable variation in the histology of the synovium in different sites within a n explant or within a joint. A t the junction of synovium or pannus with articular, patellar, or fibrocartilage, t h e predominant cell type resembled either a fibroblast or a monocyte, or a mixture of both was present. The cartilage in these junctions was usually stained much less intensely with toluidine blue, a n d often no staining was observed.

Ex tracellular Localization of Cathepsin D Meniscectomy Patients. In synovium, capsular tissue, and cartilage that had initially been cultured with NSS or As-RD, cytoplasms of cells counterstained with eriochrome black fluoresced red. Cells that had not been counterstained exhibited no fluorescence and were difficult to see. In extracellular sites there was either n o fluorescence or a very weak, diffuse green fluorescence (Figure 2a). T h e matrix of fibrocartilage and articular cartilage normally displayed a moderately intense greenish autofluorescence. Tissues examined after they had been cultured with the specific antiserum to human cathepsin D usually also contained a little intense green particulate staining in the synovial lining layer (Figure 2b). This staining indicates immunoreaction of antibody with

SECRETION OF CATHEPSIN D

cathepsin D (14,15). Because cells in this zone are tightly packed, it was not possible to determine whether this particulate staining was intracellular or extracellular. In some specimens green fluorescent staining for IgG was associated with fibroblast-like cells in the underlying capsular tissue (Figure 2b). This staining served to outline these cells, but in view of their very narrow spindle shape it was again impossible to determine whether this staining was present on the cell surface or within the cell. Extracellular immunoprecipitates between cells were rarely observed in the capsular tissue and were never seen in or at the edge of cartilage. Rheumatoid Patients. As in traumatized joints, synovia, pannus, and cartilage that were isolated from rheumatoid patients and had been initially cultured with As-RD or NSS never exhibited any intense staining for IgG. Extracellular sites either were unstained or showed only weak diffuse green fluorescent staining (Figure 3a). None of the cells displayed surface staining, although some intracellular particulate staining for IgG was seen. In explants originally cultured with specific antiserum to human cathepsin D, 6 of 11 patients (Table 2) commonly showed intense green extracellular staining for IgG in synovia and pannus tissue. This extracellular cathepsin D was detected between cells and at or close to cell surfaces, and thus it partly outlined them (Figures 3b, 5a, 5c). This specific staining for cathepsin D is difficult to show in black and white micrographs, but it is best seen in the high power micrograph, Figure 4a. These “stained” cells, which are shown stained with hematoxylin and eosin in Figure 4b, resembled macrophages rather than lymphocytes, plasma cells, polymorphonuclear leukocytes, or fibroblasts. Not all macrophage-like cells stained for IgG. Although staining was often detectable throughout the explant, it was sometimes only prominent in discrete areas; these areas often contained predominantly macrophage-like cells. A t the cut edges of explants, both cell surfaces and intracellular contents stained for IgG. This staining was almost certainly due to cellular damage caused during the preparation of the cultures. Such staining at cut edges was regarded as an artifact and was ignored. Gross intracellular staining, indicative of dead cells, was rarely observed away from cut edges. Extracellular cathepsin D was essentially restricted to synovia and pannus tissue (Figure 5c), because in only one site in 1 patient (EP) was this enzyme clearly detected in cartilage, and then it was only present in one small zone of partly degraded matrix (Figure 5a). This matrix had lost most of its autofluorescence and exhibited little or no staining with toluidine blue. Carti-

1299

lage with similar properties in other sites in the same joint (Figure 5b) and in other patients (Figure 5c) did not contain detectable extracellular enzyme. Extracellular cathepsin D was never observed in or at the edge of eroded bone adjacent to invading pannus tissue. I n 4 patients extracellular cathepsin D was detected throughout synovial and pannus tissues but was not always observed in these tissue explants at or close to the cartilage junction (Figure 5b). Areas of positive staining for extracellular cathepsin D were sometimes adjacent or close to areas in which extracellular enzyme was not detectable (Figures 5a and 5b). Of 3 patients who had undergone previous synovectomies of the knee, 1 (AB) displayed moderate amounts of cathepsin D in the synovial and pannus tissues, whereas 2 (IH and G W ) exhibited little detectable extracellular enzyme in these sites (Tables 1 and 2). Tables 1 and 2 clearly demonstrate that there was no correlation between the duration of the disease and the amount of extracellular cathepsin D observed in synovium and pannus. Table 2 reveals that there was no close correlation between the amount of extracellular cathepsin D observed and the clinical assessment of the disease, or the operative assessment of the synovium. It was apparent, however, that substantial amounts of extracellular cathepsin D were observed in thermographically “hot” joints and that relatively little enzyme was detected in “warm” or “cold” joints (Table 2). Effect of Shortened Culture Periods on the Detection of Extracellular Cathepsin D. In more recent studies the authors have investigated the effect of shortening the period of culture with sheep antiserum to cathepsin D or NSS. Synovial tissues removed from rheumatoid joints and transported to the laboratory were each divided into several pieces and cultured in NSS or As-HD containing media for 1, 3, 6, 12, or 24 hours before being transferred to BGJ, containing 5% NRS in the usual way. Extracellular staining for ovine IgG was not detected in control explants initially cultured with NSS. After 24 hours of culture in medium containing As-HD, immunoprecipitates were observed throughout the explants. Similar results were obtained at 12, 6, and 3 hours, but at 1 hour the staining was much less intense. In an attempt to see if it was possible to shorten the “wash-out’’ culture period with NRS (used to remove unbound ovine IgG) a further experiment was performed: After a 12-hour culture with As-HD, explants were cultured in medium containing 5% NRS for 3,6, 12, and 24 hours. After 6 and 12 hours, “wash-out” extracellular immunoprecipitates were as clearly visible as at 24 hours. After only 3 hours of culture with NRS,

1300

POOLE ET AL

Fig 2. Periarticular synovium removed from a single site in patient PU at meniscectomy. E = natural edge of synovium. 2a. Cultured with NSS. Only weak diffuse greenjuorescent staining for sheep IgC (arrow) and some weak yellow-brown tissue autofluorescence were ohserved. 2b. Cultured with A s - H D . Stronger diffuse greenjluorescent staining Jor sheep IgC was seen in the synovial lining ( I ) . and more intense particulate staining (not seen in 2a) was associated with cells in the intima and in the capsular tissue (C). It was not possihle to determine whether this staining in menisrectomy patients was extracellular. It was, however. more clearly ohserved in this patient than in others of this group and was classified grade I (Tahle 2). Scales = 20 p n i .

Figs 2-5. Demonstration of extracellular cathepsin D in joint tissues removed from patients at meniscectomy (synovia) or synovectomy (synovia, pannus, and cartilage). Tissues were initially cultured with nonimmune sheep serum (NSS) or with sheep antiserum to human cathepsin D ( As-HD). After initial culture with nonimmune rabbit serum (NRS) to remove most of the unbound sheep IgC. tissues were frozen, sectioned, fixed, and treated with fluorescein-labeled pig antibody F( ab’) to sheep IgG and counterstained with eriochrome black where indicated. Sections were then examined with darkground fluorescence microscopy.

SECRETION OF CATHEPSIN D

Fig 3. Peripatellar synovial tissue removed by synovectomy from a single site in rheumatoid patient DB. 3a. Culture with NSS. Extracellularfluorescence was only occasionally seen, and then it was difluse and very weak. 3b. Culture with As-HD. Intense particulate and diffuse green fluorescent staining for sheep IgG indicates that caihepsin D was both between cells and around them. and thereby outlined them. The assessment of extracellular cathepsin D in this site was grade 3 (Table 2). The natural edge of the synovium (E)is shown. Scales = 20 fim.

F i g 4a. Peripatellar synovial tissue from rheumatoid patient EP shoning iiiore detailed staining at cell surjaces (arrows). See caption to Figure 36. 4b. Fixed frozen section of specimen shown in 36 stained nith hematoxylin and eosin showing the macrophage-like cells (arrows) with which extracellular stuining for cathepsin D wa.? associated. Scales = IOfim.

1301

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POOLE ET AL

Fig 5s. Pannus-articular cartilage junctions Jrom rheumatoid patient EP. Green particulate fluorescent staining for extracellular cathepsin D was detected in zone X of the pannus but not in the adjacent zone Y . It was also seen in partly degraded cartilage (open arrow ), although this was rarely obseraed. The undegraded (or less degraded) cartilage matrix exhibited drffitse green autofluoresence ( * ), which contrasts with the specific particulate staining for cathepsin D. Cells fluoresced red aoer staining with eriochrome black and thereby demonstrated the extracellular localization ofthe immune complexes containing cathepin D. The cartilagelpannus junction is indicated by closed arrows. 5b. Pannus-articular cartilage junctions from rheumatoid patient EP. N o exiracellular cathepsin D was detected in this zone, uhich was a f e w millimeters from that shown in Sa. Pannus cells ( P ) and chondrocytes ( C ) weresimilarly stained with eriochrome black and fluoresced red. The partly degraded cartilage matris ( M ) has lost its autofluorescence. 5c. Pannus-patellar cartilage junction from rheumatoid patient DB. cultured with As-HD and finally counterstained with erichrome black. Extracellular cathepsin D was only detected in the pannus ( P). I t was not obserced in the patellar cartilage matrix ( M ), which sometimes. as shown here, stained with eriochrome black. Articular and fibrocartilage were necer stained by eriochrome black. Scales = 20 pm.

SECRETION OF CATHEPSIN D

1303

Table 2 . Relative Amounts of Exiracellular Cathepsin D in Synovium and Pannus

Cathepsin D in Synovium and Pannus* Patient

Periarticular

Peripatellar

Rheumatoid patients (extracellular staining deJinite) 3 3 RH EP 3 BC 3 AB 2 DB 2 3 AK 2 3 IH I HJ I GW I JB SL I

Clinical Assessment of Disease?

Operative Assessment of Synoviumt

3 2 3 2 2 3

3 3 3 2 2 3 3 3 2 2 2

-

Thermography9

n ,niscectomy patients (extracellt DS MK PU HT PB BH

I I 1 0 0 1

I

N N

N N I

* Relative amounts of extracellular cathepsin D as indicated by the degree of extracellular immunoprecipitation of As-HD examined immunohistochemically: 0. not detectable: 1. very little and rarely observed; 2, moderate and commonly observed; 3. substantial and present throughout most of the explant. t Clinical assessment: I , slight; 2, moderate: 3, very acute. $Operative assessment: N, normal; I (pale pink), small increase in vascularization; 2 (red), moderately vascular: 3 (deep red), very vascular. ij Thermographic index: 1, cool; 2, warm; 3, hot.

some weak diffuse extracellular staining for IgG was also present, but it was not enough to mask the intense specific staining representing accumulation of antibody as a result of interaction with antigen.

operating theater and those set up in the laboratory 4-6 hours later.

Effect of Establishing Cultures in Operating Theater

Permeability of Synovium to IgC. After 30 minutes of incubation, grains present over all parts of the synovia examined indicated that this tissue was freely permeable to IgG (Figure 6 ) . Similar results were found after longer periods in culture extending up to 24 hours. Uptake of 35S0,2-into Chondrocytes. This study was made to determine whether there were any gross differences in chondrocyte biosynthetic activity at or close to the junction with pannus tissue in rheumatoid patients. Such differences would reflect any loss of normal function or the death of these cells. Cathepsin D was rarely detected i n cartilage. and the viability of these junctional chondrocytes in partly degraded cartilage was questioned. After 1 hour of incubation with 35S0,2-,

Because transportation of tissues to the laboratory in medium BGJ, containing 5% NRS at room temperature may have stimulated or retarded the secretion of cathepsin D, and because a delay in culturing tissues at 37°C with As-HD could have prevented the detection of enzyme secretion, it was decided to construct a portable incubator to enable tissues to be established in culture in the operating theater with As-HD or NSS. Comparisons of tissue removed from the same sites in rheumatoid joints revealed that there were n o detectable differences between cultures established in the

Autoradiographic Studies

1304

POOLE ET AL

Fig 6. Autoradiograph oJ periarticular synooia removed from a rheumatoid parienr. Pernieab:liry of synooium 10 nonimmune sheep IgG. Synooiuni was culturedfor 30 minures wirh 1125 I]-lgG in 5% N R S in BGJ,. then ,fixed and processed J i ~ rautoradiography. Grains were detected around cells rhroughoui the explanr. Scale = 20 pm.

grains were concentrated over and around many chondrocytes both in junctional zones and in those remote from the pannus tissue (Figure 7). There was no indication of a gross loss of biosynthetic activity by chondrocytes in junctional zones. A high density of grains was observed around cells in pannus. Similar results were found after 2 hours of incubation. A few visibly necrotic chondrocytes were seen in junctional zones and these, like a small proportion of chondrocytes of normal appearance, showed no association of grains.

with the capacity to degrade proteoglycan, can often be detected in extracellular sites in rheumatoid pannus and synovial tissue and at the junction between cartilage and pannus provides strong evidence in suport of the pathologic secretion of this proteinase from cells in synovium and pannus. It was notable that there was little evidence for cathepsin D secretion from chondrocytes, even in cartilage depleted of proteoglycan, the matrix of which is permeable to the IgG antibodies used to detect the enzyme (3 1 ). The autoradiographic studies revealed that most of the chondrocytes examined at or close to the edge of the pannus were biosynthetically active and not dead, and thus incapable of secreting this enzyme. This observation also suggests that cartilage at the edge of the pannus rnay not always be dead and undergoing replacement by granulation tissue at its junction with the pannus. The viability studies also show that at this advanced stage in cartilage degradation the main contribution to the extracellular pool of cathepsin D is made by pannus and synovial cells. It is important, however, not to preclude the possibility that this enzyme rnay be secreted from chondrocytes earlier in a more “native” matrix. Cartilage is normally relatively impermeable to IgG (31-33) and its characteristic diffuse green autofluorescence is lost only when it is partially degraded; to-

DISCUSSION The apparent capacity of invasive pannus tissue to degrade both proteoglycan and collagen in cartilage, particularly at the edge of this tissue, points to a secretion by the pannus of proteinases that are capable of degrading these macromolecules. Until now there have been no direct data to indicate that proteinases of this kind are being secreted from cells in or close to this zone where degradation is occurring. Harris et a1(29,30) reported ultrastructural studies which indicated that cartilage c o h g e n is most degraded at the edges of pannus cells. This finding points to the release o f collagen&grading enzymes from these cells. The loss of proevent and teoglycan from cartilage is a extends 10 a greater distance from the invading tissue. The present observation that cathepsin D, an enzyme

Fig 7. Autoradiograph ojperiarticuiar synouia remouedfrom a rheumatoid parienr. Cellular uprake and extracellular accumulation o f 3 s S 0 , a by chondrocytes and synouial cells in rhe junctional zone. The tissue was culrured for I hour with 10 p c i l m l 3sS0,a-in 5% N R S in BGJ,. then fixed and processedfor autoradiography. Grains were presenl over and around chrondrocytes up to the juncrion with the pannus. Many grains can also be seen around cells in the pannus. Scale = 10 pm.

SECRETION OF CATHEPSIN D

gether these features limit the early detection of extracellular enzyme by the authors’ techniques. In this respect it is notable that, although necrotic chrondrocytes were sometimes observed in the junctional zone, they did not exhibit any gross reaction with antibody as was observed with other damaged or necrotic cells at the cut edges of these explants. Although the authors have observed cathepsin D on and around cells in rheumatoid synovial and pannus tissue, it cannot be claimed that this enzyme is functional in vivo. It cannot degrade proteoglycan at pH 7.0, but it does exhibit significant activity at pH 6.0 (7). The precise extracellular pH around cells is unknown. The “bulk phase” pH would be expected to be close to neutrality, but it has been argued that the pericellular environment may be acidic and thus favor the local action of acid proteinases (7,34). In previous studies the present authors have clearly observed that this enzyme is present in cartilage from which proteoglycan is being removed (14-16) and that proteoglycan degradation can sometimes be inhibited in living cartilage in culture by the presence of specific antiserum to cathepsin D (35). In the rheumatoid joint where cartilage proteoglycan erosion is taking place, the enzyme was often seen in extracellular sites unlike in the traumatized joint. Neutral proteinases have been detected in human patellar cartilage (36) and in cultures of human (37) synovial cells. The relevance of these enzymes to proteoglycan degradation in vivo demands careful further evaluation. In future work attention should be paid to earlier lesions where proteoglycan degradation is first detected. In the present study the authors examined well-established lesions; that proteoglycan destruction is often well advanced there is evidenced by the lack or reduction of toluidine blue staining of cartilage. Extracellular cathepsin D was associated with cells resembling macrophages that may be responsible for its secretion. Some of those cells seen in pannus which are associated with cartilage collagen erosion are macrophage-like (29), and macrophage-like cells have been detected with electron microscopy at cartilage-pannus junctions (38). The type A synovial lining cell of healthy joints resembles a macrophage (39). It is well established that the lining cells proliferate in rheumatoid arthritis: lining cells in pig synovium in culture infiltrate the underlying capsular tissue (40). Thus these macrophage-like cells may be derived from lining cells. The reasons for the detection of extracellular cathepsin D in some local sites and not others are unknown. Yet is is known that all of the present patients were receiving a variety of antiinflammatory drugs,

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some of which, such as hydrocortisone administered preoperatively, can inhibit the secretion of lysosomal enzymes (41). This fact may explain the low level or absence of extracellular enzyme in some patients. I n the most inflamed joints, however, extracellular cathepsin D was commonly observed. In studies of this kind to detect extracellular enzyme, this enzyme cannot be demonstrated by direct staining of freshly isolated tissues: the technique is not sensitive enough and it is necessary first to trap the enzyme in immune complexes by culturing the tissue with antibody to the enzyme (16). The possibilities of artifacts must be considered, because removing tissues to an in vitro environment may change secretory activity. T o examine this problem, comparable in vitro and in vivo experiments have been performed with rabbits with an experimental arthritis (42). By injecting antibodies into the joints of living rabbits, the authors were able to demonstrate cathepsin D secretion from synovial and pannus cells in vivo. Because similar results were also yielded by the in vitro technique used here for human tissues, it seems reasonable to conclude that the present findings on human joint tissues in vitro are relevant to the in vivo situation in man.

ACKNOWLEDGMENTS The authors thank Ms. Christine Camus, M r . G. Lenney, and Mr. D. Buttle for their skilled technical assistance. We also thank Mr. P. Morrison for supplying some of the tissues used in this work. Mr. R. Ash of the Agricultural Research Council Unit of Animal Physiology, Babraham, very kindly performed the intracarotid catheterizations of pigs and sheep.

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