Wnt signaling in rheumatoid synoviocyte activation

Abstract

Rheumatoid arthritis (RA) is a joint-specific disease with complex pathogenesis. It is characterized by synovial inflammation, cartilage loss, and joint destruction. The reasons why joint damage recurs when therapy is discontinued are not clearly understood. Several lines of evidence suggest that cartilage damage is promoted by the transformed and invasive fibroblast-like synoviocytes (FLS) of the rheumatoid joint. It has been demonstrated in several systems that aberrant wnt-mediated signaling causes blockade of cartilage differentiation and malformation of joints. In this review, we have discussed the importance of wnt–frizzled-mediated signaling in the autonomous activation of FLS in patients with RA. Anti-wnt/anti-frizzled antibodies, frizzled receptor antagonists, or small molecule inhibitors of wnt–frizzled signaling might be useful for therapeutic interventions in RA.     

Invasiveness of fibroblast-like synoviocytes is an individual patient characteristic associated with the rate of joint destruction in patients with rheumatoid arthritis

OBJECTIVE: Rheumatoid arthritis (RA) is characterized by inflammation and destruction of synovial joints. Fibroblast-like synoviocytes (FLS) harvested from synovial tissue of patients with RA can invade normal human cartilage in severe combined immunodeficient (SCID) mice and Matrigel basement membrane matrix in vitro. This study was undertaken to investigate the association of these in vitro characteristics with disease characteristics in patients with RA. METHODS: Synovial tissue samples from 72 RA and 49 osteoarthritis (OA) patients were obtained. Samples of different joints were collected from 7 patients with RA. The FLS invasiveness in Matrigel was studied, and the intraindividual and interindividual differences were compared. From the patients with FLS who exhibited the most extreme differences in in vitro ingrowth (most and least invasive FLS), radiographs of the hands and feet were collected and scored according to the Sharp/van der Heijde method to determine the relationship between in vitro invasion data and estimated yearly joint damage progression. RESULTS: FLS from patients with RA were more invasive than FLS from patients with OA (P < 0.001). The mean intraindividual variation in FLS invasion was much less than the mean interindividual variation (mean +/- SD 1,067 +/- 926 and 3,845 +/- 2,367 for intraindividual and interindividual variation, respectively; P = 0.035), which shows that the level of FLS invasion is a patient characteristic. The mean +/- SEM Sharp score on radiographs of the hands or feet divided by the disease duration was 4.4 +/- 1.1 units per year of disease duration in patients with the least invasive FLS (n = 9), which was much lower compared with the 21.8 +/- 3.1 units per year of disease duration in patients with the most invasive FLS (n = 9) (P < 0.001). CONCLUSION: The ex vivo invasive behavior of FLS from RA patients is associated with the rate of joint destruction and is a patient characteristic, given the much smaller intraindividual than interindividual FLS variation.     

Fractalkine is a novel chemoattractant for rheumatoid arthritis fibroblast-like synoviocyte signaling through MAP kinases and Akt

OBJECTIVE: Fibroblast-like synoviocytes (FLS) are a major constituent of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Fractalkine (FKN/CX(3)CL1) expression is up-regulated in RA synovium and RA synovial fluid. While RA FLS express the FKN receptor, CX(3)CR1, the pathophysiologic relevance of FKN stimulation of RA FLS is not understood. This study was undertaken to better characterize the relationship between FKN and the RA FLS that both produce it and express its receptor. METHODS: RA FLS were subjected to chemotaxis and proliferation assays, Western blotting, enzyme-linked immunosorbent assays, and filamentous actin staining to characterize the relationship between FKN and RA FLS. RESULTS: FKN secretion by RA FLS was regulated mainly by tumor necrosis factor alpha. Stimulation of RA FLS with FKN led to significant cytoskeletal rearrangement but no proliferation. Chemotaxis assays revealed that FKN was a novel chemoattractant for RA FLS. Stimulation of RA FLS with FKN resulted in activation of MAP kinases and Akt. JNK, ERK-1/2, and Akt (at both Ser-473 and Thr-308) were each up-regulated in a time-dependent manner. Inhibition of ERK-1/2-mediated signaling, but not JNK or Akt, significantly repressed FKN-induced RA FLS migration. CONCLUSION: These findings indicate a novel role of FKN in regulating RA FLS cytoskeletal structure and migration. FKN specifically induces RA FLS phosphorylation of the MAP kinases JNK and ERK-1/2, as well as full activation of Akt.     

Wnt signalling in rheumatoid arthritis

Rheumatoid arthritis (RA) is a symmetrical polyarticular disease of unknown aetiology that affects primarily the diarthrodial joints. Characteristic features of RA pathogenesis are synovial hyperplasia and inflammation accompanied by cartilage loss and joint destruction. Synovial hyperplasia and inflammation are a consequence of an increase in the macrophage-like and fibroblast-like synoviocytes of the synovial intimal lining associated with infiltration of leucocytes into the subintimal space. Although therapeutic interventions are available, the disease persists despite therapy in a significant fraction of patients. Several lines of evidence have substantiated a crucial role of activated fibroblast-like synoviocytes (FLS) during RA pathogenesis. The hyperplastic FLS population potentially promotes leucocyte infiltration and retention. The rheumatoid synovium eventually transforms into a pannus that destroys articular cartilage and bone. There are no approved drugs that are known to target the FLS in RA, and the underlying mechanisms driving FLS activation remain unresolved. In this review, the importance of Wnt-frizzled (Fz)-mediated signalling in the autonomous activation of FLS is discussed. Anti-Wnt/anti-Fz antibodies, Fz receptor antagonists or small-molecule inhibitors of Wnt-Fz signalling might be useful for therapeutic interventions in refractory RA.     

Association of the -2849 interleukin-10 promoter polymorphism with autoantibody production and joint destruction in rheumatoid arthritis

OBJECTIVE: To analyze the -2849 A/G interleukin-10 (IL-10) promoter polymorphism, which is associated with high (AG/GG) and low (AA) IL-10 production, in a cohort of rheumatoid arthritis (RA) patients and controls in order to gain a better understanding of its role in the incidence and progression of RA. METHODS: Allele frequencies of the promoter polymorphism -2849 A/G and carriage rates were compared in 283 RA patients, 413 patients with other rheumatic diseases, and 1,220 healthy controls. The rate of joint damage and baseline levels of IgG and IgM rheumatoid factors and anti-citrullinated peptide antibodies were measured and were correlated with the IL-10 gene polymorphism. Furthermore, the correlation between the invasiveness of fibroblast-like synoviocytes (FLS) and the -2849 IL-10 genotype was tested. RESULTS: The IL-10 genotype was not associated with the incidence of RA, but instead, correlated with disease progression, as determined by the extent of joint destruction. A higher rate of joint destruction was observed in patients with the genotype associated with high IL-10 production. Since FLS are thought to be involved in joint destruction, we analyzed IL-10 genotypes in conjunction with FLS invasiveness. Although adenoviral gene transfer of IL-10 to FLS inhibited their invasiveness, no differences were observed in vitro in the FLS from RA patients who were -2849 non-G carriers compared with those who were G carriers. Instead, patients with the -2849 AG/GG genotype, which is associated with high IL-10 production, had higher autoantibody titers at baseline. CONCLUSION: The -2849 IL-10 promoter polymorphism is associated with autoantibody production and subsequent joint damage in RA.     

Modulation of fibroblast-mediated cartilage degradation by articular chondrocytes in rheumatoid arthritis

OBJECTIVE: To determine the role of chondrocytes and factors released from chondrocytes in cartilage destruction by fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis (RA). METHODS: RA FLS from 2 patients were implanted into SCID mice, together with fresh articular cartilage or with cartilage that had been stored for 24 hours at 4 degrees C or at 37 degrees C. The invasion of the same RA FLS into the fresh and stored cartilage was compared histologically using a semiquantitative scoring system. In addition, we investigated whether protein synthesis in chondrocytes affects the invasion of RA FLS in vitro. A 3-dimensional cartilage-like matrix formed by cultured chondrocytes was labeled with 35S. After formation of the cartilage-like matrix, protein synthesis was blocked with cycloheximide. The invasion of RA FLS from 6 patients into cycloheximide-treated and untreated matrix was assessed by measuring the released radioactivity in coculture with and without interleukin-1beta (IL-1beta) and tumor necrosis factor alpha (TNFalpha). RESULTS: The SCID mouse experiments showed a significant invasion of RA FLS into the cartilage (overall mean score 3.2) but revealed significant differences when the invasion of the same RA FLS into fresh and stored cartilage was compared. RA FLS that were implanted with fresh articular cartilage showed a significantly higher invasiveness than those implanted with pieces of cartilage that had been stored for 24 hours (overall mean score 2.3). Storage at 37 degrees C and 4 degrees C resulted in the same reduction of invasion (35% and 37%, respectively). In the in vitro experiments, RA FLS rapidly destroyed the cartilage-like matrix. Blocking of chondrocyte protein biosynthesis significantly decreased the invasion of RA FLS, as shown by a decreased release of radioactivity. Addition of IL-1beta, but not TNFalpha, to the cocultures partially restored the invasiveness of RA FLS. CONCLUSION: These data underline the value of the SCID mouse in vivo model of rheumatoid cartilage destruction and demonstrate that chondrocytes contribute significantly to the degradation of cartilage by releasing factors that stimulate RA FLS. Among those, IL-1beta-mediated mechanisms might be of particular importance.     

Geranylgeranylacetone, a non-toxic inducer of heat shock protein, induces cell death in fibroblast-like synoviocytes from patients with rheumatoid arthritis

Fluvastatin (Fluv) is reported to induce apoptosis in rheumatoid arthritis (RA) synoviocytes through the blocking of protein geranylgeranylation. We report here our investigation of whether geranylgeranylacetone (GGA) induces cell death in RA synoviocytes. Synovial tissues were obtained from patients with RA at the time of total knee arthroplasty. Fibroblast-like synoviocytes (FLS) cultured in three passages were used for the experiments. The FLS were then cultured for 48 h in 48-well flat-bottomed plates containing various concentrations of GGA (0.1–4.0 μg/ml) and either 0.1 or 0.5 μM Fluv. We also examined the effect of GGA and Fluv in human fibroblasts from normal skin (CCD-25SK) and FLS from patients with osteoarthritis (OA). Cells demonstrating cell death were counted following trypan blue staining. In the absence of GGA, there was no apparent cell death, as evidence by trypan blue staining. Concentrations of GGA between 0.1 and 4.0 μg/ml induced cell death in RA FLS, but not in skin fibroblasts (CCD-25SK) nor OA FLS. The number of synoviocytes demonstrating cell death induced by 0.1 or 0.5 μM Fluv was significantly higher than that by the medium alone. In summary, we found that GGA induced cell death in RA FLS, suggesting that GGA may be a potential new therapeutic agent for RA as well as osteoporosis.     

Wnt1 inducible signaling pathway protein-3 regulation and microsatellite structure in arthritis

OBJECTIVE: Rheumatoid arthritis (RA) synovial tissue expresses several embryonic gene families, including wingless (wnt) and their receptors, frizzled (fz). The Wnt proteins, including Wnt-1, activate the Wnt inducible signaling pathway proteins (WISP), which are members of the CCN family that regulate cell growth and differentiation. WISP3 is of particular interest because it contains a microsatellite region in its coding region that is susceptible to frameshift mutations and leads to a truncated protein. To investigate the contribution of WISP3 to synovial inflammation, we evaluated its expression and regulation in arthritis. METHODS: mRNA and protein expression of WISP3 were determined by quantitative real-time polymerase chain reaction (PCR) and Western blot analysis, respectively. For mutation analysis, PCR product amplified from genomic DNA of synovial tissue and cultured fibroblast-like synoviocytes (FLS) was subcloned and sequenced. RESULTS: WISP3 mRNA is expressed in synovial tissue, but is 11-fold higher in RA than osteoarthritis (OA) or normal samples. Surprisingly, WISP3 protein levels are similar in RA, OA, and normal synovium samples. Immunohistochemistry of synovial tissue reveals that WISP3 protein is located primarily in the synovial intimal lining. WISP3 mRNA expression is also 6-fold higher in RA FLS compared with OA FLS and 50-fold higher in RA than in normal FLS. When RA FLS are stimulated with interleukin 1 or tumor necrosis factor-a, WISP3 mRNA is significantly increased. The cytokines also increase WISP3 mRNA in OA FLS, but the maximal level in stimulated OA FLS is still less than medium-treated RA FLS. Mutation analysis in the coding region microsatellite of the WISP3 gene in RA and OA synovium and FLS shows a limited number of insertion and deletion mutations. CONCLUSION: WISP3 gene expression is higher in RA synovium and FLS compared with OA and normal synovial tissue and is further induced by proinflammatory cytokines in vitro. Protein levels are not increased, indicating discoordinate regulation of WISP3 protein and mRNA. Although functionally relevant mutations were observed in genomic DNA, they were noted in both OA and RA samples.     

Protein Tyrosine Phosphatase Expression Profile of Rheumatoid Arthritis Fibroblast‐like Synoviocytes: A Novel Role of SH2 Domain–Containing Phosphatase 2 as a Modulator of Invasion and Survival

Objective

The fibroblast‐like synoviocytes (FLS) in the synovial intimal lining of the joint are key mediators of inflammation and joint destruction in rheumatoid arthritis (RA). In RA, these cells aggressively invade the extracellular matrix, producing cartilage‐degrading proteases and inflammatory cytokines. The behavior of FLS is controlled by multiple interconnected signal transduction pathways involving reversible phosphorylation of proteins on tyrosine residues. However, little is known about the role of the protein tyrosine phosphatases (PTPs) in FLS function. This study was undertaken to explore the expression of all of the PTP genes (the PTPome) in FLS.

Methods

A comparative screening of the expression of the PTPome in FLS from patients with RA and patients with osteoarthritis (OA) was conducted. The functional effect on RA FLS of SH2 domain–containing phosphatase 2 (SHP‐2), a PTP that was up‐regulated in RA, was then analyzed by knockdown using cell‐permeable antisense oligonucleotides.

Results

PTPN11 was overexpressed in RA FLS compared to OA FLS. Knockdown of PTPN11, which encodes SHP‐2, reduced the invasion, migration, adhesion, spreading, and survival of RA FLS. Additionally, signaling in response to growth factors and inflammatory cytokines was impaired by SHP‐2 knockdown. RA FLS that were deficient in SHP‐2 exhibited decreased activation of focal adhesion kinase and mitogen‐activated protein kinases.

Conclusion

These findings indicate that SHP‐2 has a novel role in mediating human FLS function and suggest that it promotes the invasiveness and survival of RA FLS. Further investigation may reveal SHP‐2 to be a candidate therapeutic target for RA.

     

Metastatic lymph node 51 and fibroblast-like synoviocyte hyperproliferation in rheumatoid arthritis pathogenesis

One of the varied characteristic features of the pathogenesis of rheumatoid arthritis (RA) is synovial hyperplasia. Fibroblast-like synoviocytes (FLSs) play a key role in the development of sustained inflammation in arthritic joints. We have reported previously that metastatic lymph node 51 (MLN51) is involved in the proliferation of FLSs in the pathogenesis of RA. Interestingly, the overexpression of MLN51 was observed only in RA FLSs, but not in osteoarthritis FLSs, possibly expecting that MLN51 may be a RA-specific marker. Additionally, we found that granulocyte–macrophage colony-stimulating factor signaling activates mitogen-activated protein kinase, followed by the upregulation of MLN51 and FLICE-inhibitory protein, resulting in FLS hyperplasia in RA. Based on these studies, we could be firm that MLN51 is a key factor in FLS hyperplasia of RA patients.     

Cyr61 participates in the pathogenesis of rheumatoid arthritis via promoting MMP-3 expression by fibroblast-like synoviocytes

Objectives: The aim of this study was to investigate the effect and potential mechanism of Cysteine-rich 61 (Cyr61) on stimulating MMP-3 expression by fibroblast-like synoviocytes (FLS) from rheumatoid arthritis (RA) patients.

Methods: Primarily cultured RA FLS were treated with exogenous Cyr61 protein or Cyr61-siRNA, then, MMP-3 expression was analyzed by real-time PCR, western blotting and ELISA. Signal transduction pathways in Cyr61-induced MMP-3 production were examined by real-time PCR, western blotting, confocal microscopy, luciferase reporter assay. Mice with collagen-induced arthritis (CIA) were treated with anti-Cyr61 monoclonal antibodies (mAb), or IgG1 as control and MMP-3 in the joint was detected by IHC, real-time PCR and western blotting.

Results: High expressed MMP-3 and Cyr61 were positively correlated in RA ST; Cyr61 stimulated MMP-3 production in FLS of RA patients in an IL-1β and TNF-α independent manner. Cyr61 induced MMP-3 could further enhance the invasive ability of RA FLS. Mechanistically, we found that Cyr61 promoted MMP-3 production via the P38, JNK-dependent AP-1 signaling pathway. Blockage of Cyr61 function with monoclonal antibody could decrease MMP-3 expression in the joints of CIA mice.

Conclusion: This study provides new evidence that Cyr61 participates in RA pathogenesis not only as a pro-inflammatory factor but also plays a key role in bone erosion via promoting MMP-3 expression. We suggest that targeting of Cyr61 may represent a potential strategy in RA treatment.     

Fractalkine is a novel chemoattractant for rheumatoid arthritis fibroblast‐like synoviocyte signaling through MAP kinases and Akt

Objective

Fibroblast‐like synoviocytes (FLS) are a major constituent of the hyperplastic synovial pannus that aggressively invades cartilage and bone during the course of rheumatoid arthritis (RA). Fractalkine (FKN/CX₃CL1) expression is up‐regulated in RA synovium and RA synovial fluid. While RA FLS express the FKN receptor, CX₃CR1, the pathophysiologic relevance of FKN stimulation of RA FLS is not understood. This study was undertaken to better characterize the relationship between FKN and the RA FLS that both produce it and express its receptor.

Methods

RA FLS were subjected to chemotaxis and proliferation assays, Western blotting, enzyme‐linked immunosorbent assays, and filamentous actin staining to characterize the relationship between FKN and RA FLS.

Results

FKN secretion by RA FLS was regulated mainly by tumor necrosis factor α. Stimulation of RA FLS with FKN led to significant cytoskeletal rearrangement but no proliferation. Chemotaxis assays revealed that FKN was a novel chemoattractant for RA FLS. Stimulation of RA FLS with FKN resulted in activation of MAP kinases and Akt. JNK, ERK‐1/2, and Akt (at both Ser‐473 and Thr‐308) were each up‐regulated in a time‐dependent manner. Inhibition of ERK‐1/2–mediated signaling, but not JNK or Akt, significantly repressed FKN‐induced RA FLS migration.

Conclusion

These findings indicate a novel role of FKN in regulating RA FLS cytoskeletal structure and migration. FKN specifically induces RA FLS phosphorylation of the MAP kinases JNK and ERK‐1/2, as well as full activation of Akt.

     

Rheumatoid arthritis: new insights into the role of synovial inflammation in joint destruction

 Rheumatoid arthritis (RA) is characterized by inflammation and proliferation of synovial tissue, leading to degradation of articular cartilage and bone with functional impairment as a result. It has recently become clear that early suppression of synovial inflammation is essential in preventing progressive joint destruction, although inflammation and destruction are in part uncoupled. New insights into the role of matrix metalloproteinases (MMPs), aggrecanase, granzyme B, receptor activator of nuclear factor κB (RANK)–receptor activator of nuclear factor κB ligand (RANKL) interaction, and other factors involved in joint destruction may lead to the development of novel therapies aimed at specific inhibition of cartilage and bone degradation. Correspondence to:P.P. Tak     

Invasiveness of synovial fibroblasts is regulated by p53 in the SCID mouse in vivo model of cartilage invasion

OBJECTIVE: In vitro data suggest that the tumor suppressor p53 is critically involved in the regulation of proliferation and apoptosis in fibroblast-like synoviocytes (FLS). Based on evidence that abnormalities in p53 expression and function are found in rheumatoid arthritis (RA), we analyzed whether inhibition of p53 using gene transfer with the human papilloma virus type 18 (HPV-18) E6 protein results in an increased cellularity and invasiveness of synovial fibroblasts in vivo. METHODS: RA and normal FLS were transduced with a pLXSN-based construct encoding for the HPV-18 E6 protein or with the pLXSN vector alone. After selection with G418, FLS were coimplanted with normal human cartilage under the renal capsule of SCID mice. Parental, nontransduced cells were used as additional controls. After 60 days, the implants were removed, and FLS invasion into the cartilage, perichondrocytic degradation, and cellularity were assessed. RESULTS: Nontransduced and mock-transduced RA FLS exhibited characteristic invasion into the cartilage (mean +/- SEM scores 2.2 +/- 0.3 and 2.4 +/- 0.2, respectively). Invasion was increased significantly in the E6-transduced RA FLS (mean score 3.1 +/- 0.3; P < 0.05). Inhibition of p53 also resulted in an increase in cellularity. Parental and mock-transduced normal FLS did not exhibit significant invasion (mean score 1.5 +/- 0.1 and 1.4 +/- 0.3, respectively), but transduction with E6 resulted in clear invasiveness (mean score 2.4 +/- 0.4) as well as increased cellularity. CONCLUSIONS: The data suggest that inhibition of endogenous p53 leads to increased invasiveness and cellularity of RA FLS and may also transform normal FLS to cells that display an aggressive, RA FLS-like behavior. Therefore, abnormalities such as somatic mutations in the p53 tumor suppressor may contribute to synovial hyperplasia and invasion in RA.     

Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors

OBJECTIVE: Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. METHODS: Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. RESULTS: Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. CONCLUSION: Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression.     

Effects of Needle-Arthroscopic Lavage with Different Volumes of Fluid on Knee Synovitis in Rheumatoid Arthritis

To determine the appropriate volume of physiological fluid needed to effectively reduce synovitis and to determine the indications for intra-articular lavage of knee joints with rheumatoid arthritis (RA), intra-articular lavage with different volumes of physiological fluid via needle arthroscopy were performed on 102 rheumatoid knees in 98 patients (25 males, 77 females) with an average age of 46 years at the time of operation. Intra-articular lavage of a knee joint with 5 or 10 l of physiological fluid gave better clinical results than did intra-articular lavage with 0.5–1.5 l. There was no difference between the beneficial effects of intra-articular lavage using 5 l or 10 l of fluid. The preoperative conditions (CRP, frequency of the susceptible factors of HLA-DRB1 alleles, radiological change in the knee joint) and the intraoperative chondroscopic assessments were correlated with clinical improvement. Our study demonstrated that intra-articular lavage with 5 l of physiological fluid performed using needle arthroscopy was beneficial. The following points at least should be checked before performing intra-articular lavage: (1) CRP is not elevated to a high level (≥5 mg/dl), (2) the patient does not have susceptible factors in both HLA-DRB1 alleles, (3) the grade of rheumatoid knee is below Larsen II in preoperative X-ray findings, and (4) the degree of cartilage damage is not more than grade 3 in arthroscopic findings. Received: 5 July 2000 / Accepted: 31 March 2001     

T cells, fibroblast-like synoviocytes, and granzyme B+ cytotoxic cells are associated with joint damage in patients with recent onset rheumatoid arthritis

OBJECTIVE: To determine immunohistological markers in synovial tissue of patients with early rheumatoid arthritis (RA) which are associated with unfavourable disease outcome. METHODS: Synovial tissue was obtained from 36 patients with RA within 1 year after the initial symptoms and before starting disease modifying antirheumatic drug treatment. Clinical, laboratory, and radiological assessments (Larsen score) were performed at the time of the biopsy and at the end of follow up (mean 58 months, range 38-72). Immunohistological analysis was performed to detect T cells, B cells, plasma cells, fibroblast-like synoviocytes (FLS), macrophages, and granzyme B+ cytotoxic cells. The sections were evaluated by digital image analysis. RESULTS: Patients were divided into two groups based upon the radiological progression per year of follow up: group I with mild progression (n = 20; Larsen <2 points/year); group II with more severe progression (n = 16; Larsen > or =2 points/year). Regression analysis with a univariate model showed that the numbers of granzyme B+ cytotoxic cells (relative risk (RR) = 12, p = 0.003), T cells (RR = 11, p = 0.013), and FLS (RR = 10, p = 0.020) discriminated between groups I and II. A multivariate model demonstrated that the numbers of T cells (RR = 1.2, p = 0.015) and FLS (RR = 1.4, p = 0.013) were independent discriminators between groups I and II. CONCLUSION: The numbers of granzyme B+ cytotoxic cells, T cells, and FLS in synovial tissue of patients with RA are related to the severity of joint damage. The data suggest a pathogenetic role for these cells in the process of joint damage.