Biochimica et Biophysica Acta, 1138 (1992) 85-92
85
© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
BBADIS 61105
Cytotoxic effects of rheumatoid arthritis sera on chondrocytes Daniela Mettal, Kay Brune and Juergen Mollenhauer
*
Department of Pharmacology and Toxicology, UniL,ersityof Erlangen-Nuernberg, Erlangen (Germany) (Received 2 May 1991)
Key words: Cell detachment; Cartilage; Chromium release; Autoantibody; Complement
Human sera from patients with rheumatoid arthritis (RA) and also from healthy donors were found to be toxic to cultured chondrocytes. Immunoglobulins were found to bind to the surface of cultured cells and cells in chicken sternal cartilage, as detected by indirect immunofluorescence. In vitro, cell detachment from the substrate was caused by the incubation of chondrocyte monolayers with 5 per cent and less RA serum. Serum treatment caused cytotoxic degradation of the cells. This could be quantified by a chromium release assay. Heat inactivation of the serum abolished the cytotoxicity. The extent of the cytotoxic reaction was related to the complement content of the serum and also to the intensity of the disease, as determined by the Ritchie-index. Other cell types, as chondrosarcoma cells, normal fibroblasts and corneal epithelium, were not affected by RA sera. Introduction
Autoimmunity is assumed to be one dominant factor for the breakdown of joint cartilage in rheumatic diseases, especially in rheumatoid arthritis (RA). Most research groups relate this process to the action of autoreactive T-cell clones which migrate into the joint area, proliferate and destroy the cartilage tissue [1-5]. On the other hand, a humoral response against joint components has been described, too, by many groups. The target epitopes of autoantibodies found in RA patients cover components of the extracellular matrix as well as of cellular elements [6-15]. However, the direct pathophysiological role of such antibodies is still uncertain and rather seen in the role of regulatory element for T-cell activation. Little work has been done to elucidate the consequences of a direct antibody-cartilage interaction. All these studies (mainly whole animal experiments) so far showed evidence for an immediate negative effect of immunoglobulins or immunocomplexes on cartilage tissue, resulting in cartilage degradation within a few hours. Moreover, purified Fc-domains of immunoglobulins can effect a switch to a catabolic metabolism,
* Present address: Department of Biochemistry, Rush-Presbyterian - St. Lukes Medical Center, 1633 West Congress Parkway, Chicago, IL 60612, U.S.A. Abbreviation: RA, rheumatoid arthritis. Correspondence: J. Mollenhauer, Department of Pharmacology and Toxicology, University of Erlangen-Nuernberg, Universitaetstr. 22, D-8520 Erlangen, Germany.
the release of proteolytic enzymes like collagenase, and the subsequent autolysis of the cartilage in the absence of defined antigen-antibody reaction [21,22]. We have been able to demonstrate the presence of chondrocyte-membrane directed antibodies in the blood of rheumatic patients [14,15]. Now we have investigated to which extent such antibodies, respectively the patient's serum, could induce chondrocyte damage in vitro under standardizised conditions. Therefore we analysed the behaviour of cultured primary chondrocytes in comparison to other cell types in cell attachment studies, by chromium release assays and by immune histology. Material and Methods
Materials Fertilized chicken eggs were from Lohmann Tierzucht (Cuxhaven, Germany). Tissue culture media, serum, and reagents were from Gibco. Chemicals, enzymes and detection antibodies were from Sigma (Munich, Germany). Protein concentrations were measured by the Bio-rad assay (Bio-Rad, Munich, Germany). Cr51 was purchased from Amersham-Buchler (Braunschweig, Germany). Tissue culture Sterna from 16-day-old chicken embryos were dissociated with trypsin-collagenase, and the released chondrocytes were cultured as described elsewhere [22]. In indicated experiments, chondrocytes were pretreated
86 by the addition of 0.(105 mg diaza-5-oxo-L-norleucine (DON) to inhibit matrix formation [33]. Instead of fetal bovine serum, aseptically collected calf serum was used. Fibroblasts were prepared from 10-day-old chicken embryos by trypsination of the lung and skin, hepatocytes from 18-day-old chicken embryo liver by trypsination. The corneal epithelium was prepared from the eyes of day 16 chicken embryos by EDTA-induced release of the cell layer and cultured in monolayer culture. The Swarm rat chondrosarcoma was given by J. Kimura (Department of Biochemistry, Rush-Presbyterian St. Luke's Medical Center, Chicago, IL, U.S.A.) [23]. The kidney epithelium was represented by an established strain (LLC-PK- 1). Human sera
Sera were taken from normal human donors and from a patient collective with defined rheumatoid arthritis (RA), being under investigation and treatment at the Department of Clinical immunology and Rheumatology, University Hospital (Erlangen, Germany). Those patients were also donors in recently published T cell studies (Alsalameh et al). Patients were not treated with basic therapeutics or with cytostatica at the time of blood sampling. Ritchie index [25], C-reactive protein and C3 levels, duration of disease, immune complex determination, antinuclear factors, mixed leukocyte reactions, hemoglobin content and sedimentation values were protocolled in each case. Cell detachment assay
24 h primary cultures in 6 cm diameter dishes were exposed to culture medium, supplemented with 1 per cent calf serum, or the indicated human sera. At the end of the incubation time, the cells in the supernatant and the still adherent cells (after trypsination) were counted. Detachment was expressed as the percentage of cells in the supernatant. Chromium release assay
24 h-primary cultures were labeled for 12 h with Cr51 (chromate) and then were trypsinized. Such treated cells did contain about 10000 cpm Cr51 per million cells. To remove free Cr51, cells were layered on top of 50% calf serum/medium and centrifuged at 500 x g for 5 rain. The cell pellet was resuspended in medium, and batches of about 1.5-3.106 cells were
Fig. 1. I m m u n e histology of monolayer cultures of rat chondrosarcoma cells. A h u m a n R A serum was used, which reacted in western blot with plasma membrane proteins (A, E). C is the control, stained only with the fluorescent detection antibody. B, D, and F are the corresponding phase contrast images. Magnifications: A, B, C, D: 5 0 0 x ; E, F: 1500x.
87 incubated with the test media (patient serum, immunoglobulins etc.) for the indicated time intervals. After incubation, ceils were again subjected to the serum gradient centrifugation, and one additional centrifugation in PBS, to remove all released chromium. The pellet and the collected supernatants were then measured in a gamma counter (LKB-WalIac). To allow comparison of different assays, the mean release of control sera from healthy donors was set to 100% in some experiments.
Purification of IgG IgG from human sera was purified by affinity chromatography on Protein A-Sepharose by FPLC chromatography, according to the manufacturer's procedure (Pharmacia-LKB).
Determination of complement C3 C3 was determined by radial immunodiffusion with the C3 Rid kit (The Binding Site, LD Labor-Diagnostika, Heiden, Germany).
Immune histology Indirect immunofluorescence was performed with native material. Air-dried frozen sections of 16-day-old chicken sterna were saturated with 2 per cent normal goat serum in PBS for 20 min. Then the test serum was added at 1:20 dilution in 2% goat s e r u m / P B S and incubated for 20 min. After intensive washing with PBS, 1:20 diluted (in 2% goat serum) FITC-conjugated goat anti-human immunoglobulins were added to the sections for another 20 min. After washing in PBS, sections were mounted in PBS/gelatin. Cultured cells
were submitted to the same procedure, with the exception that all steps were performed on ice, to minimize cell destruction by the sera. Microscopy was performed on a Zeiss Axioscope. Results
Histological obserc'ations Immune histological staining of cultured chondrocytes (Fig. 1) or cartilage tissue sections (Fig. 2) with serum from RA patients resulted in an intense staining of the pericellular area and of the cell surface, and a less pronounced staining of the far extracellular, respectively interstitial, matrix. At high magnificatios, the cell surface staining showed a spotty distribution (Fig. 1E, Fig. 2B). Such binding of immunoglobulins to the cell surface had consequences for the function of cultured chondrocytes. An exposure of cells to the diluted (1:20) serum caused first cell detachment from the substrate (Fig. 3) and led with increasing time to a complete lysis of the chondrocytes, leaving behind only a network of extracellular matrix (Fig. 3D) and few fibroblastic cell bodies (Fig. 3C). If the serum also contained antibodies against molecules of the extracellular matrix, their binding could also be visualized immune histologically (Fig. 3D). Because this effect occurred always when staining was tried with primary chondrocytes, the result presented in Fig. 1 was obtained using rat chondrosarcoma cells. These cells were not cytotoxically affected by the loading with patient serum to their surface (see Fig. 7). Newborn calf serum, fetal calf serum, heat inactivated human serum, or purified immunoglobulins, however, did not affect cells in culture. De-
Fig. 2. I m m u n e histology of cryosections from chicken sterna. Same serum as in Fig. 1. C is negative control. Magnifications: A: 5 0 x ; B, C: 800 X.
88 tachment was quantified in a cell detachment study presented in Fig. 4: within 4 h a high proportion of the ceils lost contact with the substrate and floated into the medium.
Quantification of the cytotoxic reaction The extent of cytolysis caused by the presence of human sera in cultured chondrocytes was measured by the chromium release assay. Despite of problems with some 'unspecific' heavy metal binding to extracellular matrix components, a sufficient incorporation of Cr51 as c h r o m a t e could be achieved: a b o u t 8000 c p m / 1 000000 cells, with an average release of 40006000 cpm. Spontaneous release of radioactivity from the cells during the experiments was low and did not exceed 10 per cent of the total radioactivity incorporated. In the presence of as little as 1 per cent human serum, within 1 h a significant release of Cr51 (up to 80% of all cell bound radioactivity) into the culture medium could be detected (Fig. 5A). Purified immunoglobulins from RA patients also induced cytotoxic Cr51 release (Fig. 5B) occasionally, despite their inability to induce cell detachment (see also Fig. 4), but to a lesser extent than complete sera. Heat inactivation of the serum (2h, 56°C) abolished the Cr-release totally (Fig. 5B).
Role of extracellular matrix As a side observation we found out that intensive production of extracellular matrix during prolonged time in culture inhibited immune histological cell surface staining of the cell surface and prevented the cytotoxic impact of serum to a certain extent. Treatment of the cells with diazo-oxo-norleucine (DON) inhibits specifically the formation of a proteoglycan matrix without a marked influence on cell viability and cell proliferation [33]. Such treated chondrocytes retain
80
= ~ , , ,
B
70 -~
60
50
A
-~ 40 30 20
10 Fig. 3. A, B: Phase contrast of native chicken chondrocytes, A before, B after 4 h incubation with 1 per cent RA serum. Most cells were detached, only flat fibroblastic morphotypes resisted. C, appearance of the culture after 24 h. Note that only fibers were left on the plate, which were associated with human antibodies, visualized in D with secondary fluorescent antibody. Magnifications: 250 ×.
O. 0
1
2 3 Time [h]
4
5
Fig. 4. Detachment experiment: time-course (A: RA serum; B: RA plasmapheresis fluid, C: RA lgG; D: control serum, E: control IgG.
89 |
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-~ 25
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oE 15
IgG
10 0
5 0
~ I 5
6
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time [ h ]
Heat-lnact.
Fig. 5. Chromium release assay with one RA serum. AI time course; B, a comparison of whole RA serum, protein A - affinity purified IgG and heat inactivated serum. The release is shown as the percentage of totally incorporated Cr51.
their polygonal, epitheloid cell shape b u t do not express a halo of extracellular matrix, as it is often seen for d i f f e r e n t i a t e d chondrocytes in culture. Consequently, D O N t r e a t m e n t increased the susceptibility of chondrocytes to the cytotoxic p r o p e r t i e s of h u m a n sera (Fig. 6).
Chromium release of uarious cell types in culture T h e effect of h u m a n control a n d R A sera to chondrocytes was u n i q u e , a n d the q u e s t i o n rose for typical tissue c u l t u r e artefacts. T o r e d u c e the probability of such a n artefact, several cell types were i n c l u d e d into the study. Besides lymphocytes as a n example of plasma-exposed cells, fibroblasts as m e m b e r s of connective tissue cells, e p i t h e l i u m (kidney cells) a n d the b r a d y t r o p h i c (like cartilage tissue) e p i t h e l i u m of the cornea, m o u s e m y e l o m a cells a n d c h o n d r o s a r c o m a cells as a class of neoplastically modified cells were included. W i t h i n the list of cells tested, exclusively normal chondrocytes exhibited significant cytotoxically in-
80 DON - T r e a t e d
70 ®
60
--~
50
d u c e d c h r o m i u m release, as shown in Fig. 7 for an R A serum.
Patient specific cytotoxicity All h u m a n sera showed a toxic effect to c h o n d r o cytcs, b u t sera from R A p a t i e n t s exceeded that ' n o r mal' level (Fig. 8). By searching a reason for this disease-related effect, a c o m p a r i s o n of the Cr51-release with the levels of some s e r u m p r o t e i n s revealed a relation of the release to the a m o u n t of c o m p l e m e n t proteins (C3 and C-reactive p r o t e i n (Fig. 9 A a n d B), c o m p o n e n t s that differ significantly in their c o n c e n t r a tions in R A p a t i e n t s from that of healthy volunteers. T h e correlation coefficient for C3 c o n c e n t r a t i o n was
Cho
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1 O0 0 0
n,1
80
0
60
>
Kid
40
40
Lo t.-
30
0
0
20 10 0
0
Control
n
Fig. 6. The effect of inhibition of matrix formation by a treatment with DON for 48 h before the cytoxicity testing. Control cells were kept for 48 h in culture without DON before assaying (instead of the 24 h standard time, to allow excessive matrix formation). The release is shown as the percentage of totally incorporated Cr51.
Mye
|
n,-
I
Cor
Fib
1
2
3
4
5
6
7
8
Cell Type Fig. 7. Cytotoxicity of human RA sera to various cell types. The chromium release of chondrocytes in one special assay was set to 100 per cent, to allow a comparison of several independant assays. Cho: primary embryonic chicken sternal chondrocytes; Fib: primary embryonic chicken tendon fibroblasts; RCS: rat chondrosarcoma line; Cot: primary embryonic chicken corneal epithelial cells; Kid: rat kidney tubular cell line LLC-PK-1; Lym: human primary peripheral lymphocytes; Mye: mouse myeloma line X63 Ag8.653; Hep: primary embryonic chicken hepatocytes.
90 350
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300
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250
L
m
200
L
o 150 1 2
3
4
S 6
7
8
9 10111213141516171819202122232425262728 1--15:Controls
Fig. 8. Comparison of the cytotoxicity in sera from normal donors or RA patients. Serum No. 9-12 were used as standard sera in the assays and calculated as the 100%-level, to allow the comparison of the results in the different independent assays. The dotted line shows the mean level of all control sera (84.3%).
relatively high (r = 0.8), that of CRP lower (r = 0.6). Interestingly, some correlation (r = 0.63) of Cr51-release was also achieved in comparison with the Ritchie-Index [25] (Fig. 10). Discussion
Components of human sera are able to react with chondrocytes, either in intact tissue (cryosections) or with cultured cells. These components include immunoglobulins, as shown by immune histological staining with patient serum, and complement proteins, detected by their ability to destroy cartilage cells in a cytotoxic reaction in the presence of antibodies. The results of the immune histology suggests a dense spotty deposition of immunoglobulins on the surface of accessible chondrocytes. The nature of this deposition has not been defined, as of yet. True antigen-antibody reactions via Fab-reaction may take place, and aside of this, also Fc-mediated antibody adsorption. However, because of the saturation of the stained samples with
I000
100
200
i
0
i
i
20 40 RITCHIE-Index
60
Fig. 10. Correlation of the relative cytotoxicity of RA sera and the Ritchie - index from the patients at the time of blood sampling. For standardization, see Fig. 8.
unspecific (goat) immunoglobulins during the staining procedure, the latter possibility might be excluded. The binding of immunoglobulins and immune complexes to cartilage in intact tissue in vivo has been shown by Cooke et al. [16-21], with severe impacts in the tissue structure and function after joint surface deposition. On the other hand, the binding of purified immunoglobulins, either cartilage-specific antibodies in the case of RA antibodies, or mock antibodies from controls, to the chondrocyte surface did not dramatically affect the viability of the cells in our experiments. However, experimentally produced and purified rabbit antibodies against anchorin CII, a collagen receptor of chondrocytes, were able to destroy chondrocytes at 37°C by induction of capping [23]. From that one could claim, the antibody titer and the kind of surface antigen would determine from case to case, whether or not antibodies are directly toxic.
A
B
500
400
600
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16-28:R.A
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0 500
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1000
1500
2000
Complement [mg/L]
0
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10
20
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30 CRP
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40
50
Fig. 9. Complement protein content of human sera and anti-chondrocyte cytotoxicity: a correlation. A: Complement C3; B: C-reactive Protein (CRP values are given in titers, with predilution 1:3, and 4 subsequent dilutions 1 : 1, until 1:48). For standardization, see Fig. 8.
91 In in vivo studies, the inoculation of i m m u n o c o m plexes in joints lead to a rapid destruction of the joint surface, and a relationship of this reaction to a r h e u m a t o i d factor - driven event was discussed by many authors [16-21]. T h e presence of activated m a c r o p h a g e s could speed up the pathophysiological destruction [26,27]. T h e additional presence of c o m p l e m e n t in a native, n o n - h e a t - d e n a t u r e d serum or plasma led to an immediate and drastic labilization of the cultured cells. This could be explained by the (antibody-mediated?) activation of c o m p l e m e n t on the surface of chondrocytes. Especially the finding that rat c h o n d r o s a r c o m a cells were not affected by the serum, despite the fact that they bind immunoglobulins at their surface, allows for the speculation of a cell surface c o m p o n e n t being responsible for the c o m p l e m e n t activation and being absent after neoplastic transformation. Recently Ostensen et al. d e m o n s t r a t e d the presence of complem e n t receptors on the surface of R A chondrocytes [38]. This finding supports such an interpretation of our experimental observations. The accumulation of activated c o m p l e m e n t factors like C3c or C3d in diseased joints favoured the idea of an antigen-driven c o m p l e m e n t activation in the tissue [16,28,29]. Interestingly, a link has been described between H L A gene constellation and c o m p l e m e n t C3 allotypes in rheumatic diseases [30]. T h e H L A background is known to be relevant in R A risk estimation in the caucasian population [31,32]. O u r presented findings, with the correlation between the serum (or plasma) complement, the cytotoxicity in vitro and the clinically relevant Ritchie-index, point out that there might be a linkage between the c o m p l e m e n t system and the development of rheumatic diseases. As of yet, it is difficult to explain why healthy persons with intact joints are not affected by their own serum. Cartilage is known to be a bradytrophic tissue, and is therefore not in direct contact with the blood stream (like cornea). The basic mechanism for joint protection may be simply the exclusion of plasma fluid from the cartilage tissue. Additionally, intact cartilage might be protected by the extracellular matrix, as we found it in case of cultured chondrocytes with matrix formation. A change in matrix composition in diseased joints, r e p o r t e d by several authors [34-36] might abolish this protective capacity. A loss of tissue-associated proteinase inhibitors [37] could then allow the onset of cartilage degradation by activitated complement. But these aspects and ideas are rather speculative and have to be analysed in subsequent investigations. Future aspects of the described findings would include the exploitation of the c o m p l e m e n t activation on the chondrocytes, of c o m p l e m e n t binding proteins, and of the question, w h e t h e r c o m p l e m e n t activation is mediated by autoantibodies.
Acknowledgements This work was supported by a grant of the G e r m a n Ministery for R e s e a r c h and Technology (BMFT), grant no. 01VM88072, and by a grant from the G e r m a n Research Society ( D F G ) at the SFB 263, project no. C2. The authors thank for the skillful technical assistance of Beate Layh; Drs. S. Alsalameh and G.R. Burmester, D e p a r t m e n t of Clinical Immunology, Erlangen, for their support with patient data, sera and theoretical advice.
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92 25 Ritchie, D.M., Boyle, J.A., Mclnnos, J.M., Jasani, M.K., Dalakos, T.G., Grieveson, P. and Buchanan, W.W. (1968) Q. J. Med. 147, 393-406. 26 Malejczyk, J. (1990) Cell. Immunol. 127, 420 431. 27 Stevenson, S., Dannucci, G.A., Sharkey, N.A. and Pool, R.R. (1989) J. Bone Joint Surg. Am. 71, 1297-1307. 28 Stannat, S., Peter, H.H., Raspe, H.H. and Deicher, H. (1986) Zeitschr. f. Rheumatologie 45, 47-51. 29 Male, D. and Roitt, I.M. (1980) Clin. Exp. Immunol. 39, 297-306. 30 Puttick, A. H., Briggs, D.C., Welsh, K.I., Williamson E.A., Jacoby, R.K. and Jones, V.E. (1990)Ann. Rheum. Dis. 49, 225-228. 31 Feldman, M. (1987) Autoimmunity and autoimmune disease, Ciba Foundation Symposium 129, Wiley, Chichester, pp. 88-108. 32 Brackertz, D. and Wernet, P. (1980) Arthritis Rheum. 23, 656 661.
33 Gosh, S., Blumenthal, H.J., Davidson, E. and Roseman, S. (1960) J. Biol. Chem. 235, 1265-1273. 34 Adam, M. and Deyl, Z. (1983) Clin. Chim. Acta 133, 25-32. 35 Bayliss, M.T. (1987) in Sudies in osteoarthrosis - pathogenesis, intervention, assessment. (Lott, D.J., Jasani, M.K. and Birdwood, G.F.B. eds), Wiley & Sons, New York, pp. 49-56. 36 Von der Mark, K. and Gliickert, K. (1990) Orthop~ide 19, 2-15. 37 B6hm, B., Deutzmann, R. and Burkhardt, H. (1991) Biochem. J. 274, 269-273. 38 Ostensen, M., Stiansen R., Hetland, G. and Garred, P. (1991) International Workshop on Articular Cartilage and Osteoarthritis, May 12-16, 199l, Wiesbaden, Germany (Abstract).
85
© 1992 Elsevier Science Publishers B.V. All rights reserved 0925-4439/92/$05.00
BBADIS 61105
Cytotoxic effects of rheumatoid arthritis sera on chondrocytes Daniela Mettal, Kay Brune and Juergen Mollenhauer
*
Department of Pharmacology and Toxicology, UniL,ersityof Erlangen-Nuernberg, Erlangen (Germany) (Received 2 May 1991)
Key words: Cell detachment; Cartilage; Chromium release; Autoantibody; Complement
Human sera from patients with rheumatoid arthritis (RA) and also from healthy donors were found to be toxic to cultured chondrocytes. Immunoglobulins were found to bind to the surface of cultured cells and cells in chicken sternal cartilage, as detected by indirect immunofluorescence. In vitro, cell detachment from the substrate was caused by the incubation of chondrocyte monolayers with 5 per cent and less RA serum. Serum treatment caused cytotoxic degradation of the cells. This could be quantified by a chromium release assay. Heat inactivation of the serum abolished the cytotoxicity. The extent of the cytotoxic reaction was related to the complement content of the serum and also to the intensity of the disease, as determined by the Ritchie-index. Other cell types, as chondrosarcoma cells, normal fibroblasts and corneal epithelium, were not affected by RA sera. Introduction
Autoimmunity is assumed to be one dominant factor for the breakdown of joint cartilage in rheumatic diseases, especially in rheumatoid arthritis (RA). Most research groups relate this process to the action of autoreactive T-cell clones which migrate into the joint area, proliferate and destroy the cartilage tissue [1-5]. On the other hand, a humoral response against joint components has been described, too, by many groups. The target epitopes of autoantibodies found in RA patients cover components of the extracellular matrix as well as of cellular elements [6-15]. However, the direct pathophysiological role of such antibodies is still uncertain and rather seen in the role of regulatory element for T-cell activation. Little work has been done to elucidate the consequences of a direct antibody-cartilage interaction. All these studies (mainly whole animal experiments) so far showed evidence for an immediate negative effect of immunoglobulins or immunocomplexes on cartilage tissue, resulting in cartilage degradation within a few hours. Moreover, purified Fc-domains of immunoglobulins can effect a switch to a catabolic metabolism,
* Present address: Department of Biochemistry, Rush-Presbyterian - St. Lukes Medical Center, 1633 West Congress Parkway, Chicago, IL 60612, U.S.A. Abbreviation: RA, rheumatoid arthritis. Correspondence: J. Mollenhauer, Department of Pharmacology and Toxicology, University of Erlangen-Nuernberg, Universitaetstr. 22, D-8520 Erlangen, Germany.
the release of proteolytic enzymes like collagenase, and the subsequent autolysis of the cartilage in the absence of defined antigen-antibody reaction [21,22]. We have been able to demonstrate the presence of chondrocyte-membrane directed antibodies in the blood of rheumatic patients [14,15]. Now we have investigated to which extent such antibodies, respectively the patient's serum, could induce chondrocyte damage in vitro under standardizised conditions. Therefore we analysed the behaviour of cultured primary chondrocytes in comparison to other cell types in cell attachment studies, by chromium release assays and by immune histology. Material and Methods
Materials Fertilized chicken eggs were from Lohmann Tierzucht (Cuxhaven, Germany). Tissue culture media, serum, and reagents were from Gibco. Chemicals, enzymes and detection antibodies were from Sigma (Munich, Germany). Protein concentrations were measured by the Bio-rad assay (Bio-Rad, Munich, Germany). Cr51 was purchased from Amersham-Buchler (Braunschweig, Germany). Tissue culture Sterna from 16-day-old chicken embryos were dissociated with trypsin-collagenase, and the released chondrocytes were cultured as described elsewhere [22]. In indicated experiments, chondrocytes were pretreated
86 by the addition of 0.(105 mg diaza-5-oxo-L-norleucine (DON) to inhibit matrix formation [33]. Instead of fetal bovine serum, aseptically collected calf serum was used. Fibroblasts were prepared from 10-day-old chicken embryos by trypsination of the lung and skin, hepatocytes from 18-day-old chicken embryo liver by trypsination. The corneal epithelium was prepared from the eyes of day 16 chicken embryos by EDTA-induced release of the cell layer and cultured in monolayer culture. The Swarm rat chondrosarcoma was given by J. Kimura (Department of Biochemistry, Rush-Presbyterian St. Luke's Medical Center, Chicago, IL, U.S.A.) [23]. The kidney epithelium was represented by an established strain (LLC-PK- 1). Human sera
Sera were taken from normal human donors and from a patient collective with defined rheumatoid arthritis (RA), being under investigation and treatment at the Department of Clinical immunology and Rheumatology, University Hospital (Erlangen, Germany). Those patients were also donors in recently published T cell studies (Alsalameh et al). Patients were not treated with basic therapeutics or with cytostatica at the time of blood sampling. Ritchie index [25], C-reactive protein and C3 levels, duration of disease, immune complex determination, antinuclear factors, mixed leukocyte reactions, hemoglobin content and sedimentation values were protocolled in each case. Cell detachment assay
24 h primary cultures in 6 cm diameter dishes were exposed to culture medium, supplemented with 1 per cent calf serum, or the indicated human sera. At the end of the incubation time, the cells in the supernatant and the still adherent cells (after trypsination) were counted. Detachment was expressed as the percentage of cells in the supernatant. Chromium release assay
24 h-primary cultures were labeled for 12 h with Cr51 (chromate) and then were trypsinized. Such treated cells did contain about 10000 cpm Cr51 per million cells. To remove free Cr51, cells were layered on top of 50% calf serum/medium and centrifuged at 500 x g for 5 rain. The cell pellet was resuspended in medium, and batches of about 1.5-3.106 cells were
Fig. 1. I m m u n e histology of monolayer cultures of rat chondrosarcoma cells. A h u m a n R A serum was used, which reacted in western blot with plasma membrane proteins (A, E). C is the control, stained only with the fluorescent detection antibody. B, D, and F are the corresponding phase contrast images. Magnifications: A, B, C, D: 5 0 0 x ; E, F: 1500x.
87 incubated with the test media (patient serum, immunoglobulins etc.) for the indicated time intervals. After incubation, ceils were again subjected to the serum gradient centrifugation, and one additional centrifugation in PBS, to remove all released chromium. The pellet and the collected supernatants were then measured in a gamma counter (LKB-WalIac). To allow comparison of different assays, the mean release of control sera from healthy donors was set to 100% in some experiments.
Purification of IgG IgG from human sera was purified by affinity chromatography on Protein A-Sepharose by FPLC chromatography, according to the manufacturer's procedure (Pharmacia-LKB).
Determination of complement C3 C3 was determined by radial immunodiffusion with the C3 Rid kit (The Binding Site, LD Labor-Diagnostika, Heiden, Germany).
Immune histology Indirect immunofluorescence was performed with native material. Air-dried frozen sections of 16-day-old chicken sterna were saturated with 2 per cent normal goat serum in PBS for 20 min. Then the test serum was added at 1:20 dilution in 2% goat s e r u m / P B S and incubated for 20 min. After intensive washing with PBS, 1:20 diluted (in 2% goat serum) FITC-conjugated goat anti-human immunoglobulins were added to the sections for another 20 min. After washing in PBS, sections were mounted in PBS/gelatin. Cultured cells
were submitted to the same procedure, with the exception that all steps were performed on ice, to minimize cell destruction by the sera. Microscopy was performed on a Zeiss Axioscope. Results
Histological obserc'ations Immune histological staining of cultured chondrocytes (Fig. 1) or cartilage tissue sections (Fig. 2) with serum from RA patients resulted in an intense staining of the pericellular area and of the cell surface, and a less pronounced staining of the far extracellular, respectively interstitial, matrix. At high magnificatios, the cell surface staining showed a spotty distribution (Fig. 1E, Fig. 2B). Such binding of immunoglobulins to the cell surface had consequences for the function of cultured chondrocytes. An exposure of cells to the diluted (1:20) serum caused first cell detachment from the substrate (Fig. 3) and led with increasing time to a complete lysis of the chondrocytes, leaving behind only a network of extracellular matrix (Fig. 3D) and few fibroblastic cell bodies (Fig. 3C). If the serum also contained antibodies against molecules of the extracellular matrix, their binding could also be visualized immune histologically (Fig. 3D). Because this effect occurred always when staining was tried with primary chondrocytes, the result presented in Fig. 1 was obtained using rat chondrosarcoma cells. These cells were not cytotoxically affected by the loading with patient serum to their surface (see Fig. 7). Newborn calf serum, fetal calf serum, heat inactivated human serum, or purified immunoglobulins, however, did not affect cells in culture. De-
Fig. 2. I m m u n e histology of cryosections from chicken sterna. Same serum as in Fig. 1. C is negative control. Magnifications: A: 5 0 x ; B, C: 800 X.
88 tachment was quantified in a cell detachment study presented in Fig. 4: within 4 h a high proportion of the ceils lost contact with the substrate and floated into the medium.
Quantification of the cytotoxic reaction The extent of cytolysis caused by the presence of human sera in cultured chondrocytes was measured by the chromium release assay. Despite of problems with some 'unspecific' heavy metal binding to extracellular matrix components, a sufficient incorporation of Cr51 as c h r o m a t e could be achieved: a b o u t 8000 c p m / 1 000000 cells, with an average release of 40006000 cpm. Spontaneous release of radioactivity from the cells during the experiments was low and did not exceed 10 per cent of the total radioactivity incorporated. In the presence of as little as 1 per cent human serum, within 1 h a significant release of Cr51 (up to 80% of all cell bound radioactivity) into the culture medium could be detected (Fig. 5A). Purified immunoglobulins from RA patients also induced cytotoxic Cr51 release (Fig. 5B) occasionally, despite their inability to induce cell detachment (see also Fig. 4), but to a lesser extent than complete sera. Heat inactivation of the serum (2h, 56°C) abolished the Cr-release totally (Fig. 5B).
Role of extracellular matrix As a side observation we found out that intensive production of extracellular matrix during prolonged time in culture inhibited immune histological cell surface staining of the cell surface and prevented the cytotoxic impact of serum to a certain extent. Treatment of the cells with diazo-oxo-norleucine (DON) inhibits specifically the formation of a proteoglycan matrix without a marked influence on cell viability and cell proliferation [33]. Such treated chondrocytes retain
80
= ~ , , ,
B
70 -~
60
50
A
-~ 40 30 20
10 Fig. 3. A, B: Phase contrast of native chicken chondrocytes, A before, B after 4 h incubation with 1 per cent RA serum. Most cells were detached, only flat fibroblastic morphotypes resisted. C, appearance of the culture after 24 h. Note that only fibers were left on the plate, which were associated with human antibodies, visualized in D with secondary fluorescent antibody. Magnifications: 250 ×.
O. 0
1
2 3 Time [h]
4
5
Fig. 4. Detachment experiment: time-course (A: RA serum; B: RA plasmapheresis fluid, C: RA lgG; D: control serum, E: control IgG.
89 |
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-
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35 30
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-~ 25
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g
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I
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oE 15
IgG
10 0
5 0
~ I 5
6
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5 0
time [ h ]
Heat-lnact.
Fig. 5. Chromium release assay with one RA serum. AI time course; B, a comparison of whole RA serum, protein A - affinity purified IgG and heat inactivated serum. The release is shown as the percentage of totally incorporated Cr51.
their polygonal, epitheloid cell shape b u t do not express a halo of extracellular matrix, as it is often seen for d i f f e r e n t i a t e d chondrocytes in culture. Consequently, D O N t r e a t m e n t increased the susceptibility of chondrocytes to the cytotoxic p r o p e r t i e s of h u m a n sera (Fig. 6).
Chromium release of uarious cell types in culture T h e effect of h u m a n control a n d R A sera to chondrocytes was u n i q u e , a n d the q u e s t i o n rose for typical tissue c u l t u r e artefacts. T o r e d u c e the probability of such a n artefact, several cell types were i n c l u d e d into the study. Besides lymphocytes as a n example of plasma-exposed cells, fibroblasts as m e m b e r s of connective tissue cells, e p i t h e l i u m (kidney cells) a n d the b r a d y t r o p h i c (like cartilage tissue) e p i t h e l i u m of the cornea, m o u s e m y e l o m a cells a n d c h o n d r o s a r c o m a cells as a class of neoplastically modified cells were included. W i t h i n the list of cells tested, exclusively normal chondrocytes exhibited significant cytotoxically in-
80 DON - T r e a t e d
70 ®
60
--~
50
d u c e d c h r o m i u m release, as shown in Fig. 7 for an R A serum.
Patient specific cytotoxicity All h u m a n sera showed a toxic effect to c h o n d r o cytcs, b u t sera from R A p a t i e n t s exceeded that ' n o r mal' level (Fig. 8). By searching a reason for this disease-related effect, a c o m p a r i s o n of the Cr51-release with the levels of some s e r u m p r o t e i n s revealed a relation of the release to the a m o u n t of c o m p l e m e n t proteins (C3 and C-reactive p r o t e i n (Fig. 9 A a n d B), c o m p o n e n t s that differ significantly in their c o n c e n t r a tions in R A p a t i e n t s from that of healthy volunteers. T h e correlation coefficient for C3 c o n c e n t r a t i o n was
Cho
120 0
1 O0 0 0
n,1
80
0
60
>
Kid
40
40
Lo t.-
30
0
0
20 10 0
0
Control
n
Fig. 6. The effect of inhibition of matrix formation by a treatment with DON for 48 h before the cytoxicity testing. Control cells were kept for 48 h in culture without DON before assaying (instead of the 24 h standard time, to allow excessive matrix formation). The release is shown as the percentage of totally incorporated Cr51.
Mye
|
n,-
I
Cor
Fib
1
2
3
4
5
6
7
8
Cell Type Fig. 7. Cytotoxicity of human RA sera to various cell types. The chromium release of chondrocytes in one special assay was set to 100 per cent, to allow a comparison of several independant assays. Cho: primary embryonic chicken sternal chondrocytes; Fib: primary embryonic chicken tendon fibroblasts; RCS: rat chondrosarcoma line; Cot: primary embryonic chicken corneal epithelial cells; Kid: rat kidney tubular cell line LLC-PK-1; Lym: human primary peripheral lymphocytes; Mye: mouse myeloma line X63 Ag8.653; Hep: primary embryonic chicken hepatocytes.
90 350
o
300
I
250
L
m
200
L
o 150 1 2
3
4
S 6
7
8
9 10111213141516171819202122232425262728 1--15:Controls
Fig. 8. Comparison of the cytotoxicity in sera from normal donors or RA patients. Serum No. 9-12 were used as standard sera in the assays and calculated as the 100%-level, to allow the comparison of the results in the different independent assays. The dotted line shows the mean level of all control sera (84.3%).
relatively high (r = 0.8), that of CRP lower (r = 0.6). Interestingly, some correlation (r = 0.63) of Cr51-release was also achieved in comparison with the Ritchie-Index [25] (Fig. 10). Discussion
Components of human sera are able to react with chondrocytes, either in intact tissue (cryosections) or with cultured cells. These components include immunoglobulins, as shown by immune histological staining with patient serum, and complement proteins, detected by their ability to destroy cartilage cells in a cytotoxic reaction in the presence of antibodies. The results of the immune histology suggests a dense spotty deposition of immunoglobulins on the surface of accessible chondrocytes. The nature of this deposition has not been defined, as of yet. True antigen-antibody reactions via Fab-reaction may take place, and aside of this, also Fc-mediated antibody adsorption. However, because of the saturation of the stained samples with
I000
100
200
i
0
i
i
20 40 RITCHIE-Index
60
Fig. 10. Correlation of the relative cytotoxicity of RA sera and the Ritchie - index from the patients at the time of blood sampling. For standardization, see Fig. 8.
unspecific (goat) immunoglobulins during the staining procedure, the latter possibility might be excluded. The binding of immunoglobulins and immune complexes to cartilage in intact tissue in vivo has been shown by Cooke et al. [16-21], with severe impacts in the tissue structure and function after joint surface deposition. On the other hand, the binding of purified immunoglobulins, either cartilage-specific antibodies in the case of RA antibodies, or mock antibodies from controls, to the chondrocyte surface did not dramatically affect the viability of the cells in our experiments. However, experimentally produced and purified rabbit antibodies against anchorin CII, a collagen receptor of chondrocytes, were able to destroy chondrocytes at 37°C by induction of capping [23]. From that one could claim, the antibody titer and the kind of surface antigen would determine from case to case, whether or not antibodies are directly toxic.
A
B
500
400
600
L 400
•
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800 o
,i.-m
16-28:R.A
300
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0 500
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200 100
i
i
i
1000
1500
2000
Complement [mg/L]
0
l.-I
I
m
•
I
I
10
20
I
30 CRP
I
I
40
50
Fig. 9. Complement protein content of human sera and anti-chondrocyte cytotoxicity: a correlation. A: Complement C3; B: C-reactive Protein (CRP values are given in titers, with predilution 1:3, and 4 subsequent dilutions 1 : 1, until 1:48). For standardization, see Fig. 8.
91 In in vivo studies, the inoculation of i m m u n o c o m plexes in joints lead to a rapid destruction of the joint surface, and a relationship of this reaction to a r h e u m a t o i d factor - driven event was discussed by many authors [16-21]. T h e presence of activated m a c r o p h a g e s could speed up the pathophysiological destruction [26,27]. T h e additional presence of c o m p l e m e n t in a native, n o n - h e a t - d e n a t u r e d serum or plasma led to an immediate and drastic labilization of the cultured cells. This could be explained by the (antibody-mediated?) activation of c o m p l e m e n t on the surface of chondrocytes. Especially the finding that rat c h o n d r o s a r c o m a cells were not affected by the serum, despite the fact that they bind immunoglobulins at their surface, allows for the speculation of a cell surface c o m p o n e n t being responsible for the c o m p l e m e n t activation and being absent after neoplastic transformation. Recently Ostensen et al. d e m o n s t r a t e d the presence of complem e n t receptors on the surface of R A chondrocytes [38]. This finding supports such an interpretation of our experimental observations. The accumulation of activated c o m p l e m e n t factors like C3c or C3d in diseased joints favoured the idea of an antigen-driven c o m p l e m e n t activation in the tissue [16,28,29]. Interestingly, a link has been described between H L A gene constellation and c o m p l e m e n t C3 allotypes in rheumatic diseases [30]. T h e H L A background is known to be relevant in R A risk estimation in the caucasian population [31,32]. O u r presented findings, with the correlation between the serum (or plasma) complement, the cytotoxicity in vitro and the clinically relevant Ritchie-index, point out that there might be a linkage between the c o m p l e m e n t system and the development of rheumatic diseases. As of yet, it is difficult to explain why healthy persons with intact joints are not affected by their own serum. Cartilage is known to be a bradytrophic tissue, and is therefore not in direct contact with the blood stream (like cornea). The basic mechanism for joint protection may be simply the exclusion of plasma fluid from the cartilage tissue. Additionally, intact cartilage might be protected by the extracellular matrix, as we found it in case of cultured chondrocytes with matrix formation. A change in matrix composition in diseased joints, r e p o r t e d by several authors [34-36] might abolish this protective capacity. A loss of tissue-associated proteinase inhibitors [37] could then allow the onset of cartilage degradation by activitated complement. But these aspects and ideas are rather speculative and have to be analysed in subsequent investigations. Future aspects of the described findings would include the exploitation of the c o m p l e m e n t activation on the chondrocytes, of c o m p l e m e n t binding proteins, and of the question, w h e t h e r c o m p l e m e n t activation is mediated by autoantibodies.
Acknowledgements This work was supported by a grant of the G e r m a n Ministery for R e s e a r c h and Technology (BMFT), grant no. 01VM88072, and by a grant from the G e r m a n Research Society ( D F G ) at the SFB 263, project no. C2. The authors thank for the skillful technical assistance of Beate Layh; Drs. S. Alsalameh and G.R. Burmester, D e p a r t m e n t of Clinical Immunology, Erlangen, for their support with patient data, sera and theoretical advice.
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