Dendritic cells in rheumatoid synovial membrane after total removal of the hyaline articular cartilage
T. F. Li1,2,
J. Mandelin3,
M. Hukkanen2,
J. Lassus1,
J. Sandelin2,
S. Santavirta1,
I. Virtanen3 and
Y. T. Konttinen2,4,5,
1 Department of Orthopaedics and Traumatology, Helsinki University Hospital,
2 ORTON Research Institute, Orthopaedic Hospital of Invalid Foundation, Helsinki,
3 Department of Anatomy, Biomedicum, University of Helsinki,
4 Department of Medicine/Invärtes Medicine, Helsinki University Hospital and
5 Department of Oral Medicine, Institute of Dentistry, University of Helsinki, Finland
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Abstract
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Objective. To investigate the effect of total removal of the hyaline articular cartilage on dendritic cells in synovial membrane in rheumatoid arthritis (RA) or ankylosing spondylitis (AS).
Patients and methods. Immunohistochemical staining for two dendritic cell markers, CD35 and RFD1, was carried out on synovial membrane specimens from arthritis patients undergoing primary (n=10) or revision (n=8) total hip replacement (THR). The results are expressed as the number (mean±standard deviation) of positive cells per 1000 total cells.
Results. CD35-(112±9) and RFD1-(27±5) positive cells were found in all primary RA synovial membrane, while only two out of eight synovial membrane samples from revision THR contained CD35-positive follicular dendritic cells (nine and 12 cells), and no revision samples contained any RFD1-positive interdigitating dendritic cells.
Conclusion. Removal of the hyaline articular cartilage reduces the infiltration and functional differentiation of dendritic cells in synovial membrane. Our findings suggest that the antigen driving chronic arthritis/synovitis is contained in the hyaline articular cartilage.
KEY WORDS: Dendritic cells, Rheumatoid arthritis, Synovial membrane, Hyaline articular cartilage, Total hip replacement.
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Introduction
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Hyaline articular cartilage contains type II, IX, X and XI collagen. Type II collagen is the most abundant fibrillar protein and constitutes 8085% of the total cartilage collagen. Type II collagen is a major histocompatibility complex-restricted, T cell-dependent antigen. Rheumatoid cartilage and synovium contain antibodies to type II collagen at a prevalence far greater than serum, suggesting an intra-articular antigen-driven immune response [1]. In different animal models, cartilage collagens can induce an erosive polyarthritis similar to rheumatoid arthritis (RA) [2].
Dendritic cells are the most potent antigen presenting cells because of their efficient antigen capture and processing functions, migratory and cell surface remodelling ability and potent T cell stimulatory capacity. They are enriched in RA synovial membrane and fluid and play a role in the initiation and perpetuation of RA by presenting the arthritogenic antigen to autoreactive T cells [3, 4].
End-stage hip RA can be successfully treated with total hip replacement (THR). In such an operation, articular hyaline cartilage in the femoral head and acetabulum is completely removed, and the hip is replaced by an artificial joint composed of an acetabular polyethylene cup and a metallic femoral stem. Aseptic loosening of implants is the major complication, often leading to a revision operation [5]. During revision THR, we collected synovial membrane samples from around the artificial joints. The eventual presence and number of dendritic cells in such tissue were compared with those in the RA synovial membranes from primary THR. We chose two monoclonal antibodies, corresponding to the two types of dendritic cells of lymph nodes. RFD1 is thought to mark the dendritic cells of the T cell zone, while CD35 labels the follicular dendritic cells in the cortical zone. RFD1 recognizes a unique class II antigen associated with dendritic cells. It does not react with granulocytes, monocytes, or lymphocytes, with the exception of a small proportion of B cells [6, 7]. Our aim was to investigate the potential effect of cartilage removal on dendritic cell infiltration in RA synovial membrane.
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Patients and methods
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Between 1995 and 1999 eight synovial membrane samples were obtained from RA (n=6) or ankylosing spondylitis (AS; n=2) patients undergoing revision THR due to aseptic loosening of the femoral component of the total hip prostheses. Of these patients, three were men and five were women, the mean age being 48.4 yr (range 4068 yr). The disease which had led to the primary THR operation was RA or AS of the hip joint. The interval from the primary to the revision THR was 9.8 yr (range 615 yr). Ten synovial membrane samples were collected from patients with RA undergoing primary THR. Of these patients, three were men and seven were women, the mean age being 41.6 yr (range 3874 yr). All tissue samples were snap-frozen in isopentane pre-cooled in dry ice and stored at -70°C. Five serial cryostat sections (6 µm thick) were carefully cut from each sample. The first and last sections were stained with haematoxylin. Section numbers 2 and 3 were used for immunochemical staining with different antibodies, and section number 4 was used as a negative staining control.
The sections were fixed in cold acetone at -20°C for 15 min. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in absolute methanol for 30 min. The sections were incubated with the following reagents at room temperature: (1) normal horse serum [Vector Laboratories, Burlingame, CA, USA; diluted 1:50 in Tris-buffered saline (TBS) containing 0.1% bovine serum albumin (BSA)] for 20 min; (2) the following monoclonal mouse anti-human antibodies for 60 min: CD35 (a follicular dendritic cell marker, Dako, Glostrup, Denmark, 2 µg/ml in TBS) and RFD1 (an interdigitating dendritic cell marker, Serotec Ltd, Oxford, UK, 5 µg/ml in TBS); (3) biotinylated horse anti-mouse IgG (Vector Laboratories; diluted 1:100 in TBS containing 0.1% BSA) for 30 min; (4) avidinbiotinperoxidase complex (Vector Laboratories; diluted 1:100 in TBS) for 30 min; (5) a combination of 0.023% diaminobenzidine tetrahydrochloride (Sigma, St Louis, MO, USA) and 0.006% hydrogen peroxide for 5 min. Between the steps, the sections were washed three times in TBS for 5 min each. Finally, the slides were counterstained with haematoxylin, dehydrated in a graded ethanol series, cleared in xylene and mounted using Diatex. Mouse IgG of the same subtype but with the irrelevant specificity was used at the same concentration instead of the primary antibodies as a negative staining control.
A semiquantitative morphometric assessment was carried out with a low light charge screen mounted with a 12-bit PC digital image camera (SensiCam, Kelheim, Germany) on a Leitz Diaplan light microscope (Wetzlar, Germany). The camera was further linked to a semiautomatic Analysis Pro 3.0 image analysis and processing system (Soft Analysis System GmbH, Münster, Germany). The images were visualized underx200 magnification and recorded in digitized form on a computer. After the parameters were set for the detection of positive cells, they were fixed and used during the following analysis procedure. Cell counting was carried out blind. Whole section areas were used for the analysis. The total number of cells for each sample was recorded as the average number of cells in the first and last sections stained with haematoxylin. The positive cells labelled with different antibodies were counted in the sections without haematoxylin counterstaining. The final result for each section was reported as the average number of positive cells per 1000 haematoxylin-stained cells. The mean and standard deviation were calculated with BMDP statistical software.
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Results
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A synovial lining was found in all revision samples. Macrophages were the main cells in the revision RA and AS synovial membrane samples. Although lymphocytes were invariably found, they never formed aggregates, as seen in the primary RA synovial membranes. Wear debris from THR components, such as metal and polymeric particles, was present in the revision RA and AS synovial membranes.
CD35-positive follicular dendritic cells were found in all RA synovial membrane samples collected at primary THR operation. Such cells were plentiful (112±9 positive cells per 1000 haematoxylin-stained cells, range 95124). They appeared in the lining layers, sublining areas and synovialcartilage junction areas (pannus tissue). CD35-positive cells were especially common at the periphery and centre of lymphocyte aggregates. In the deep stroma of primary RA synovial membrane samples, positive cells were often found around small blood vessels (Fig. 1
). Only two out of eight synovial membrane samples collected at revision THR contained CD35-positive dendritic cells, and the number of positive cells in these two revision samples was very low (nine and 12 positive cells per 1000 haematoxylin-stained cells; Fig. 1
). No positive cells were detected in revision samples from these two AS patients.

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FIG. 1. CD35-positive cells in synovial membrane samples from primary and revision THR operations. In the primary samples, positive cells appeared in the lining layer and the sublining areas (A), lymphocyte aggregates (B), perivascular regions in the deep stroma (C), and pannus tissue (D). In the revision samples, positive cells were occasionally found in the lining-like areas (E) and in perivascular regions in the deep stroma (F).
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All primary RA synovial membrane samples contained RFD1-positive interdigitating dendritic cells (27±5 positive cells per 1000 haematoxylin-stained cells, range 2135). They were present in the lining layers and the sublining areas. RFD1-positive cells were invariably detected in lymphocyte aggregates and in the perivascular regions in the deep stroma of primary RA synovial membrane samples (Fig. 2
). Interestingly, such cells were not detected in the pannus tissue. No RFD1-positive cells were found in any of the synovial membrane samples collected at revision operation (Fig. 2
).

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FIG. 2. RFD1-positive cells in synovial membrane samples from primary and revision THR operations. In the primary samples, positive cells appeared in the lining layers (A), lymphocyte aggregates (B) and the deep stroma (C). No positive cells were found in any of the revision samples (D).
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Discussion
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Clinical observations have demonstrated that RA synovitis often reoccurs after synovectomy, while the relapse is rare if the arthritic joint has been treated with THR [8, 9]. We hypothesize that the antigen intrinsic to the cartilage matrix or trapped in cartilage may act as a stimulus, and that removal of the articular cartilage alters the pathophysiological mechanism driving RA. This is not a new hypothesis, but it has not been examined in this way by other investigators. In the present study, 10 samples from primary THR operations performed for RA were compared with eight revision synovial membrane samples also obtained from patients with chronic arthritis of the hip, either RA or AS, but undergoing revision operations for aseptic loosening of THR prosthetic devices. Revision operations of RA patients are not very common at our unit, but between 1995 and 1999 we managed to collect eight synovial membrane samples from patients who had had a chronic arthritis of the hip joint as the primary indication for their THR operation and who were later re-operated on for aseptic loosening of the femoral component.
The infiltration of synovial membrane with dendritic cells is an important step in the initiation of local inflammation. Dendritic cells accumulate in the cytokine-rich RA synovial membrane, where they functionally differentiate and develop a phenotype identical to that of the interdigitating dendritic cells in lymph nodes. Synovial dendritic cells can migrate to draining lymph nodes and stimulate T cell proliferation and B cell help after antigen presentation [10]. In this study, CD35- and RFD1-positive cells were found in all RA synovial membrane samples collected from primary THR. In contrast, only two out of eight RA/AS synovial membrane samples from revision THR contained CD35-positive cells, and no revision RA/AS synovial membrane samples contained RFD1-positive cells. The present findings indicate that the avascular hyaline articular cartilage contains autoantigens which may perpetuate the autoimmune synovitis in RA and in other chronic arthritides [11]. Removal of the cartilage by THR eradicates the cartilage-derived autoantigen pool. Because no antigen is to be presented, the accumulation of dendritic cells is greatly reduced in rheumatoid synovial membrane around artificial joints.
These findings spur the search for the cartilage-derived disease-perpetuating autoantigens, strengthen those treatment protocols aimed at immunomodulation (e.g. induction of tolerance) and suggest that, when possible, joint replacement operations, e.g. of the knee joint, should be total, i.e. three- rather than two-compartment operations.
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Acknowledgments
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The authors thank Ms Outi Rauanheimo for secretarial help. This study was supported by EVO grant TYH 0341, TYH 8307, TYH 1202, and TYH 0215, the Finnish Academy and Group of Excellence grants (SA, TEKES, Ministry of Education and University of Helsinki), The Sigrid Juselius Foundation, Invalid Foundation, and Finska Läkaresällskapet, Helsinki, Finland.
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Notes
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Correspondence to: Y. T. Konttinen, Biomedicum Helsinki, PO Box 700 (Haartmaninkatu 8), 00029-HUS, Helsinki, Finland. 
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Submitted 25 May 2001;
revised version accepted 17 September 2001.