DNA methylation regulates the expression of CXCL12 in rheumatoid arthritis synovial fibroblasts.

In the search for specific genes regulated by DNA methylation in rheumatoid arthritis (RA), we investigated the expression of CXCL12 in synovial fibroblasts (SFs) and the methylation status of its promoter and determined its contribution to the expression of matrix metalloproteinases (MMPs). DNA was isolated from SFs and methylation was analyzed by bisulfite sequencing and McrBC assay. CXCL12 protein was quantified by enzyme-linked immunosorbent assay before and after treatment with 5-azacytidine. RASFs were transfected with CXCR7-siRNA and stimulated with CXCL12. Expression of MMPs was analyzed by real-time PCR. Basal expression of CXCL12 was higher in RASFs than osteoarthritis (OA) SFs. 5-azacytidine demethylation increased the expression of CXCL12 and reduced the methylation of CpG nucleotides. A lower percentage of CpG methylation was found in the CXCL12 promoter of RASFs compared with OASFs. Overall, we observed a significant correlation in the mRNA expression and the CXCL12 promoter DNA methylation. Stimulation of RASFs with CXCL12 increased the expression of MMPs. CXCR7 but not CXCR4 was expressed and functional in SFs. We show here that RASFs produce more CXCL12 than OASFs due to promoter methylation changes and that stimulation with CXCL12 activates MMPs via CXCR7 in SFs. Thereby we describe an endogenously activated pathway in RASFs, which promotes joint destruction.

Functional autoantibodies against serpin E2 in rheumatoid arthritis.

OBJECTIVE: To search for novel autoantibodies in patients with rheumatoid arthritis (RA) in an effort to better understand the processes of joint destruction in this disease. METHODS: Using a modified SEREX technique and complementary DNA derived from RA synovium, serpin E2 was identified as a novel autoantigen and was analyzed by immunohistochemistry. Levels of anti-serpin E2 autoantibodies in serum and synovial fluid from patients with RA, osteoarthritis (OA), psoriatic arthritis, and ankylosing spondylitis, and/or from healthy individuals were assessed by enzyme-linked immunosorbent assay. Since serpin E2 is an inhibitor of serine proteases, we studied the inhibitory activity of serpin E2 toward its target, urokinase plasminogen activator (uPA), in vitro in the presence of isolated anti-serpin E2 autoantibodies and in vivo using the uPA activity assay. RESULTS: We identified autoantibodies against serpin E2 by the SEREX technique. Serpin E2 was overexpressed in RA synovial tissues as compared with OA synovial tissues. Significantly higher levels of anti-serpin E2 autoantibodies were present in samples of synovial fluid (28%) and serum (22%) from RA patients as compared with OA patients (0 and 6%, respectively) or with healthy individuals (6% of sera). Most importantly, anti-serpin E2 autoantibodies isolated from RA sera reversed the inhibitory activity of serpin E2 by 70%. Furthermore, the levels of anti-serpin E2 autoantibodies correlated with the uPA activity in vivo. CONCLUSION: This study characterizes a functional property of a novel autoantibody in RA. Since anti-serpin E2 autoantibodies interfere with the inhibitory activity of serpin E2 toward serine proteases, they might facilitate the joint destruction in RA.

Expression of cathepsin K and matrix metalloproteinase 1 indicate persistent osteodestructive activity in long-standing ankylosing spondylitis.

BACKGROUND: Ankylosing spondylitis (AS) is a common, largely genetically determined, rheumatic disease that is characterised by spinal inflammation and new bone formation. However, the exact pathogenesis and pathology are still not clear. OBJECTIVE: To analyse tissue obtained at spinal surgery by immunohistochemistry and compare the specimen of patients with AS to those with degenerative disc disease (DDD). METHODS: Bony and soft tissue specimens of 30 patients with AS and 20 with DDD were obtained during spinal osteotomy from different anatomic regions including articular and spinous processes, interspinous ligaments and intervertebral disks. Immunohistolochemistry was performed with established markers for cathepsin K, matrix metalloproteinase (MMP)1, MMP3 and receptor activator for nuclear factor kappaB (RANK) ligand. RESULTS: Cathepsin K and MMP1-positive cells were only observed in AS specimens. Cathepsin K-positive multinucleated cells were detected at articular processes adjacent to fibrous tissues. MMP1 was expressed in smaller mononuclear cells attached to bone. Invasion of bone by MMP1 cells was seen at entheseal sites. In the intervertebral disks, most mononuclear cells were cathepsin K-positive. Isolated cells expressing these matrix-degrading enzymes found in DDD never showed signs of invasion. No differences were found for MMP3 between AS and DDD. Clear expression of RANK ligand was only detected in one patient with AS. CONCLUSIONS: Cathepsin K is strongly expressed in different regions of the spine in AS. Cathepsin K was mainly expressed by mononuclear cells, fibroblast-like cells and cells attached to bone and at sites of bone remodelling, suggestive of high osteoclastic activity. This supports the role of persistent inflammation in the pathogenesis of AS. How these changes relate to osteoproliferation remains to be determined.

The dual inhibitor of lipoxygenase and cyclooxygenase ML3000 decreases the expression of CXCR3 ligands.

OBJECTIVE: To find previously unknown properties of ML3000, a competitive inhibitor of the cyclooxygenase and the lipoxygenase (LO) pathway. METHODS: Gene expression of ML3000 treated and untreated rheumatoid arthritis synovial fibroblasts were measured with Affymetrix gene arrays. Downregulation of chemokine (C-X-C motif) ligands CXCL9, CXCL10 and CXCL11 was verified with Real-time polymerase chain reaction, CXCL10 protein levels were determined with ELISA. Rheumatoid arthritis synovial fibroblasts were treated with the cyclooxygenase inhibitor naproxen, the 5-LO inhibitor BWA4C and the 5-lipoxygenase-activating protein (FLAP) inhibitor MK886, and consecutive changes in CXCL10 protein levels measured. 5-LO expression was determined by polymerase chain reaction and Western blot. RESULTS: In synovial fibroblasts and monocyte-derived macrophages ML3000 inhibited the tumour necrosis factor induced expression of CXCL9, CXCL10 and CXCL11, which are all ligands of the chemokine receptor CXCR3. No effect was observed in monocytes. Whereas inhibition of the cyclooxygenase pathway or the FLAP protein showed no effect, blockade of 5-LO significantly downregulated CXCL10 protein levels. 5-LO mRNA was detected in monocytes and in monocyte-derived macrophages. All tested cell types expressed 5-LO protein. CONCLUSIONS: ML3000 effectively downregulates CXCR3 ligands. This study confirms that a thorough analysis of the impact of a drug on its target cells cannot only reveal unexpected properties of a substance, but also helps to understand the underlying molecular mechanisms. Accordingly, our data provide the basis for further clinical studies testing the application of ML3000 in diseases such as rheumatoid arthritis or multiple sclerosis.

The metastasis associated protein S100A4: a potential novel link to inflammation and consequent aggressive behaviour of rheumatoid arthritis synovial fibroblasts.

The metastasis-associated protein S100A4 belongs to the large family of S100 calcium-binding proteins that appear to play regulatory roles in diverse biological activities. Moreover, a prognostic role of S100A4 has been suggested for patients with several types of cancer. Cancer promoting properties for S100A4 have been demonstrated, particularly through its regulation of cell motility, proliferation and apoptosis, as well as by stimulation of angiogenesis and remodelling of the extracellular matrix. Increased expression of S100A4 mRNA has been detected in proliferating synovial fibroblasts in rheumatoid arthritis. Furthermore, strong upregulation of the S100A4 protein in rheumatoid arthritis synovial tissue compared with osteoarthritis and control tissues has been demonstrated recently, especially at sites of joint invasion. Several immune and vascular cells were also identified to be producing S100A4 within the synovium. The local upregulation of S100A4 was accompanied by high plasma and synovial fluid concentrations of the S100A4 protein existing in the bioactive oligomeric form in patients with rheumatoid arthritis. Consistent with data from cancer studies, the extracellular S100A4 oligomer appears to be involved in regulation of several matrix-degrading enzymes and modulation of the transcriptional activation function of the tumour suppressor protein p53 in rheumatoid arthritis synovial fibroblasts. Taken together, one can speculate that increased S100A4 protein in circulation and locally at sites of inflammation, particularly at sites of joint destruction, might be linked to the process of aggressive fibroblast behaviour contributing to the pathogenesis of chronic autoinflammatory diseases such as rheumatoid arthritis.

S100A4 is expressed at site of invasion in rheumatoid arthritis synovium and modulates production of matrix metalloproteinases.

The metastasis-associated protein S100A4 promotes the progression of cancer by regulating the remodelling of the extracellular matrix. The expression of S100A4 in vivo is shown and the functional role of S100A4 in the pathogenesis of osteoarthritis and rheumatoid arthritisis is explored. The expression of S100A4 in rheumatoid arthritis, osteoarthritis and normal synovial tissues was determined by immunohistochemistry. The expression of matrix metalloproteinase (MMP) mRNA was measured in rheumatoid arthritis and osteoarthritis synovial fibroblasts treated and untreated with S100A4 oligomer by real-time polymerase chain reaction. Levels of released MMPs were confirmed by ELISA in cell culture supernatants. S100A4 protein was expressed in rheumatoid arthritis and osteoarthritis synovial tissues, in contrast with normal synovium. S100A4 up regulated MMP-3 mRNA in rheumatoid arthritis synovial fluid, with a peak after 6 h. This resulted in release of MMP-3 protein. MMP-1, MMP-9 and MMP-13 mRNA were also up regulated in synovial fluid, but with different kinetics. MMP-14 mRNA showed no change. Thus, S100A4 protein is expressed in synovial tissues of patients with rheumatoid arthritis and osteoarthritis in contrast with healthy people. It induces the expression and release of MMP-3 and other MMPs from synovial fluid. The data suggest that S100A4-producing cells could be involved in the pathogenesis of osteoarthritis and rheumatoid arthritis, including pannus formation and joint destruction.

Trichostatin A sensitises rheumatoid arthritis synovial fibroblasts for TRAIL-induced apoptosis.

BACKGROUND: Histone acetylation/deacetylation has a critical role in the regulation of transcription by altering the chromatin structure. OBJECTIVE: To analyse the effect of trichostatin A (TSA), a streptomyces metabolite which specifically inhibits mammalian histone deacetylases, on TRAIL-induced apoptosis of rheumatoid arthritis synovial fibroblasts (RASF). METHODS: Apoptotic cells were detected after co-treatment of RASF with TRAIL (200 ng/ml) and TSA (0.5, 1, and 2 micromol/l) by flow cytometry using propidium iodide/annexin-V-FITC staining. Cell proliferation was assessed using the MTS proliferation test. Induction of the cell cycle inhibitor p21Waf/Cip1 by TSA was analysed by western blot. Expression of the TRAIL receptor-2 (DR5) on the cell surface of RASF was analysed by flow cytometry. Levels of soluble TRAIL were measured in synovial fluid of patients with RA and osteoarthritis (OA) by ELISA. RESULTS: Co-treatment of the cells with TSA and TRAIL induced cell death in a synergistic and dose dependent manner, whereas TRAIL and TSA alone had no effect or only a modest effect. RASF express DR5 (TRAIL receptor 2), but treatment of the cells with TSA for 24 hours did not change the expression level of DR5, as it is shown for cancer cells. TSA induced cell cycle arrest in RASF through up regulation of p21Waf1/Cip1. Levels of soluble TRAIL were significantly higher in RA than in OA synovial fluids. CONCLUSION: Because TSA sensitises RASF for TRAIL-induced apoptosis, it is concluded that TSA discloses sensitive sites in the cascade of TRAIL signalling and may represent a new principle for the treatment of RA.

Galectin-3 is induced in rheumatoid arthritis synovial fibroblasts after adhesion to cartilage oligomeric matrix protein.

BACKGROUND: Galectin-3 is expressed in the synovial tissue of patients with rheumatoid arthritis (RA), particularly at sites of joint destruction. OBJECTIVE: To explore the possibilities that galectin-3 is induced either by proinflammatory cytokines or by adhesion to cartilage components. METHODS: Cell culture plates were coated with fibronectin, collagens I-VI, or cartilage oligomeric matrix protein (COMP), and the suspended cells were then added. The medium was changed after 1 hour at 37 degrees C. Adherent cells were further incubated for 18 hours in the presence or absence of tumour necrosis factor alpha (TNF alpha) or interleukin 1 beta. Cells were pretreated with murine IgG1, anti-CD29, -CD51, -CD61 (integrins), or -CD3 monoclonal antibodies and transferred to culture plates coated with COMP. Adherent cells were counted by light microscopy. The expression of intracellular galectin-3, or cell surface CD29, CD51, and CD61 was determined by flow cytometry before and after adhesion. RESULTS: Four times more RA synovial fibroblasts (SF) than osteoarthritis SF adhered to COMP. RA SF presented more cell surface integrins, and monoclonal antibodies against CD51 inhibited the adhesion to COMP by 80%. TNF alpha reduced the expression of CD61 and the adhesion to COMP, but did not reverse the adhesion once it had taken place. The adhesion of RA SF to COMP was found to increase the intracellular level of galectin-3. In contrast, intracellular galectin-3 decreased after exposure to TNF alpha. CONCLUSION: The increase of galectin-3 occurs after adhesion to COMP, and the alpha V beta 3 receptor (CD51/CD61) has a pivotal role in this process.

Targeting cathepsin L (CL) by specific ribozymes decreases CL protein synthesis and cartilage destruction in rheumatoid arthritis.

The present study was undertaken to examine whether ribozymes cleaving specifically cathepsin L (CL) mRNA are able to decrease the synthesis of CL protease in rheumatoid arthritis synovial fibroblasts (RA-SF) and thereby reduce the invasiveness into cartilage both in vitro and in the SCID mouse coimplantation model of RA. Two different ribozymes that cleave CL mRNA specifically at positions 533 (RzCL533) and 790 (RzCL790) were generated. Using retroviral gene transfer, RA-SF were transduced with the ribozyme constructs or the empty vector. To examine the effect of the ribozymes on the mRNA level, quantitative analysis for CL mRNA was performed using real-time PCR. For evaluation on the protein level, ELISA using specific anti-CL antibodies was performed. In addition, transduced RA-SF were examined in vitro in a three-dimensional destruction assay evaluating their ability to degrade extracellular matrix produced by human chondrocytes. Matrix destruction was monitored by the release of soluble glycosaminoglycans (sGAG). Using the in vivo SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF and control cells were coimplanted with human cartilage for 60 days. After being killed, invasion of RA-SF into the cartilage was evaluated by using a semiquantitative score. Transduction of RA-SF with RzCL533 and RzCL790 ribozymes decreased significantly the expression of CL mRNA to 44% (range 25-62%) and 20% (range 1-43%), respectively, when compared to mock-transduced cells. The protein concentration of CL in the cell culture supernatants of transduced RA-SF was decreased from 16.0 ng/ml in the mock constructs to 4.1 and 8.2 ng/ml (mean), respectively. Using the in vitro cartilage destruction assay, the release of sGAG decreased to 46 and 60%, respectively, after 14 days when compared to mock-transduced cells. In the SCID mouse coimplantation model of RA, RzCL533-transduced RA-SF revealed a significant lower cartilage invasion when compared to mock and untransduced cells. Using retroviral gene transfer, ribozymes cleaving CL mRNA inhibit specifically the synthesis of this matrix-degrading enzyme and reduce cartilage destruction in in vitro and in vivo models. Our study therefore suggests that ribozymes targeting CL could be a novel and efficient tool to inhibit joint destruction in RA.

p53 in rheumatoid arthritis synovial fibroblasts at sites of invasion.

OBJECTIVE: To analyse the functional response of p53 in rheumatoid arthritis synovial fibroblasts (RASF) in vitro and in vivo and to investigate whether activation of p53 modulates the destructive process of RASF. METHODS: RASF and controls grown on chamber slides were either directly examined with DO7 anti-p53 antibodies by immunofluorescence or irradiated with 10 Gy x rays and analysed time dependently for the expression of p53. The percentage of positive cells was evaluated by a quantitative scoring system. RASF and normal (N) SF cultured in vitro were co-implanted with human cartilage in SCID mice for 60 days. Consecutively, the invasion score was evaluated, and the number of p53 positive cells was determined at the sites of invasion by immunohistochemistry. In addition, synovial tissues from RA, osteoarthritis, and normal synovia were stained with DO7 antibodies. RESULTS: In vitro the rate of expression of p53 in RASF was low (<5%), but transiently inducible by ionising irradiation (50%). In vitro low p53 expressing RASF disclosed, when invading articular cartilage, a nuclear p53 signal in 20% of the cells, indicating the induction of p53 in a distinct population of RASF during the invasive process. CONCLUSIONS: These data suggest an inductive p53 response at sites of cartilage invasion during the destructive process driven by activated RASF.

Dual inhibition of 5-lipoxygenase and cyclooxygenases 1 and 2 by ML3000 reduces joint destruction in adjuvant arthritis.

OBJECTIVE: To search for potential new therapies to inhibit the progression of joint destruction in patients with rheumatoid arthritis. METHODS: We evaluated the dual acting antiinflammatory drug ML3000 (2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro- H-pyrrolizine-5-yl) acetic acid, a dual inhibitor of 5-lipoxygenase (5-LOX) as well as both cyclooxygenases (COX-1 and COX-2) in the rat model of adjuvant arthritis. On Day 0, female Lewis rats (5 per group) were injected intradermally with complete Freund's adjuvant at base of the tail. Treatment began on Day 2; the rats received ML3000 (20 or 80 mg/kg/day) twice daily 7 h apart for 28 days and were then sacrificed. To reduce pain, the positive control group and 2 treatment groups received paracetamol (3 mg/ml water). Joint histology was scored for synovial cell proliferation, fibroproliferative pannus, and cartilage and bone erosions, as well as diffuse leukocyte infiltrates. RESULTS: Daily doses of 20 or 80 mg/kg ML3000 significantly reduced the arthritis associated deficiency of body growth, the edema/erythema score, and splenomegaly. In the ankle joint, ML3000 significantly reduced the overall histological score, synovial cell proliferation, and bone/cartilage erosions, and inhibited the appearance of fibroproliferative pannus. The addition of paracetamol in the drinking water had no influence. No side effects were noted. CONCLUSION: ML3000 is an antiarthritic drug with a high gastrointestinal tolerability, which can reduce synovial cell proliferation and joint erosion and is capable of markedly suppressing prostaglandin synthesis.

Evidence of 5-lipoxygenase overexpression in the skin of patients with systemic sclerosis: a newly identified pathway to skin inflammation in systemic sclerosis.

OBJECTIVE: Leukotrienes are a family of arachidonic acid derivatives with potent proinflammatory and profibrotic properties, and 5-lipoxygenase (5-LOX) catalyzes two key steps in the leukotriene biosynthetic pathway. Since inflammatory cell infiltrates and excessive fibrosis are hallmarks of systemic sclerosis (SSc) skin lesions, we undertook the present study to investigate the expression of 5-LOX in skin biopsy specimens from patients with SSc. METHODS: Expression of 5-LOX in skin sections from 10 SSc patients and 8 healthy controls was examined by in situ hybridization with specific riboprobes and by immunohistochemistry analysis with 5-LOX monoclonal antibodies. Synthesis of 5-LOX by cultured dermal fibroblasts from 7 patients with SSc and 4 controls was measured by fluorescence-activated cell sorter analysis. In addition, concentrations of leukotriene B4 (LTB4) and LTE4 in fibroblast supernatants after stimulation were determined using enzyme immunoassays. RESULTS: Expression of 5-LOX was found in all skin sections from SSc patients as well as from controls. However, the number and percentage of 5-LOX-positive cells were significantly higher in SSc skin sections compared with control sections. Expression of 5-LOX was seen in cells within perivascular inflammatory infiltrates as well as in fibroblasts throughout the skin. The experiments with cultured skin fibroblasts revealed that 5-LOX was constitutively expressed in these cells, which resulted in the production of leukotrienes after cell stimulation. Whereas no difference was found for LTE4, SSc fibroblasts produced significantly higher amounts of LTB4 after stimulation, compared with healthy control fibroblasts. CONCLUSION: The results of this study suggest that the 5-LOX pathway may be of significance in the pathogenesis of SSc and may represent a target for new treatment strategies.

Prevalence of TTV DNA and GBV-C RNA in patients with systemic sclerosis, rheumatoid arthritis, and osteoarthritis does not differ from that in healthy blood donors.

OBJECTIVE: To determine the prevalence of GB virus-C (GBV-C) RNA and TT virus (TTV) DNA in patients with systemic sclerosis (SSc), rheumatoid arthritis (RA), and osteoarthritis (OA) as well as to compare the autoantibody pattern in patients with SSc with and without evidence of viral infection. PATIENTS AND METHODS: The study included 168 patients (84 SSc, 41 RA, and 43 OA) diagnosed according to the American College of Rheumatology criteria and 122 volunteer blood donors. The presence of GBV-C RNA and TTV DNA in serum was assessed by nested reverse transcriptase-polymerase chain reaction (RT-PCR) and semi-nested PCR, respectively. Autoantibodies in patients with SSc were determined by enzyme linked immunosorbent assay (ELISA) and Hep-2 immunofluorescence. RESULTS: TTV-DNA was detected in 10/84 (12%) patients with SSc, 9/41 (22%) patients with RA, 3/43 (7%) patients with OA, and 16/122 (13%) blood donors. GBV-C RNA was present in 4/84 (5%) patients with SSc, 2/43 (5%) patients with OA, and 5/122 (4%) blood donors. No patient with RA was positive for GBV-C RNA. One patient with SSc and one patient with OA showed a double infection with GBV-C and TTV. 74/84 (88%) patients with SSc were positive for at least one autoantibody species tested: 18/84 (21%) showed anticentromeric autoantibodies, 55/84 (66%) a speckled (36/84 (43%) fine, 19/84 (23%) coarse), and 20/84 (24%) a homogeneous nuclear Hep-2 pattern, and 21/84 (25%) had antinucleolar autoantibodies. Anti-Scl-70 antibodies were found in 31/84 (37%) and anti-RNP antibodies in 5/84 (6%) patients with SSc. No differences in the autoantibody pattern in patients with SSc with or without viral infection could be detected. CONCLUSION: The prevalence of GBV-C RNA and TTV DNA in serum samples from patients with SSc, RA, and OA was low and comparable with that in blood donors. A continuing infection with TTV and or GBV-C was not associated with a significant change in the autoantibody pattern in patients with SSc. These data provide no evidence for an association between GBV-C and/or TTV infections and SSc and/or arthritis (RA and OA).

[Rheumatoid arthritis: new developments in the pathogenesis with special reference to synovial fibroblasts]. / Die Rheumatoide Arthritis: Neuentwicklungen in der Pathogenese unter besonderer Berücksichtigung der synovialen Fibroblasten.

Rheumatoid arthritis (RA) is a chronic inflammatory disease, which is mainly characterized by synovial hyperplasia, pathological immune phenomena and progressive destruction of the affected joints. Various cell types are involved in the pathogenesis of RA including T cells, antigen presenting cells, and endothelial cells. Recent experimental evidence suggests that the CD40/CD154 system might play an important role in the development of RA. Our experimental approach focuses on RA synovial fibroblasts (RA-SF) that are able to destroy articular cartilage independent of inflammation. To elucidate the specific role of those cells in RA pathophysiology the following questions are currently addressed: 1. Which mechanisms do activate the RA-SF? 2. How do the activated RA-SF attach to the cartilage? 3. How do RA-SF destroy cartilage and bone? Which mechanisms do activate the RA-SF? The process of activation is poorly understood. It is unclear, how far the synovial hyperplasia of RA resembles tumor diseases. Along this line some contradictory results exist concerning the role of the tumor suppressor protein p53. Some investigations could show the expression of p53 in the synovial lining including p53 mutations in RA synovium and in RASF, while other research groups could not confirm these data. Our group has demonstrated that the tumor suppressor PTEN was less expressed in the synovial lining of RA than in normal synovium, but no PTEN mutations could be found in the RA-SF. In addition, the in vivo and in vitro expression of the anti-apoptotic molecule sentrin suggests a functional resistance of RA-SF to undergo apoptosis. Although it is still unclear, whether certain viruses or viral elements are involved in the pathogenesis of RA (cause, consequence or coincidence?), certain viruses could play a role in the pathogenesis of RA. The endogenous retroviral element L1 was found to be expressed in the synovial lining, at sites of invasion as well as in RA-SF grown in vitro. Moreover, the data indicate that after the initial activation of L1 downstream molecules such as the SAP kinase 4, the met-protoonocogene and the galectin-3 binding protein are upregulated. How do the activated RA-SF attach to the cartilage? It has been suggested that integrins mediate the attachment of RA-SF to fibronectin rich sites of cartilage. Intriguingly, other adhesion molecules such as the vascular cellular adhesion molecule-1 (VCAM) and CS-1, a splice variant of fibronectin, are synthesized by RA-SF. By binding to these adhesion molecules, lymphocytes that express the integrin VLA-4 could be stimulated and thereby maintain the inflammatory process. Osteopontin is an extracellular matrix protein, which is associated with matrix adhesion and metastasis in tumors. In RA synovium, osteopontin was detectable in the synovial lining and at sites of invasion. How do RA-SF destroy cartilage and bone? The destruction of cartilage and bone in RA is mediated by matrix metalloproteinases (MMPs) and cathepsins. MMPs exist as secreted and as membrane bound forms. In vitro models are being developed to simulate the invasive process of RA-SF. In an in vitro model developed in our laboratory, the treatment of RA-SF with anti-CD44 or anti-interleukin-1 (IL-1) minimized matrix degradation of RA-SF. On the other hand, co-culture of RA-SF and U937 cells as well as application of interleukin-1 beta (IL-1 beta) or tumor necrosis factor alpha (TNF alpha) increased the invasiveness of RA-SF. Gene transfer of bovine pancreas trypsin inhibitor (BPMI) or interleukin-10 (IL-10) reduced the invasion of RA-SF, while transduction of interleukin-1 receptor antagonist (IL-1Ra) was chondroprotective. Double gene transfer of IL-10 and IL-1Ra resulted in both inhibition of invasion and chondroprotection.

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.

Altered diurnal rhythm of prolactin in systemic sclerosis.

OBJECTIVE: Mild hyperprolactinemia has been reported in systemic lupus erythematosus (SLE) and systemic sclerosis (SSc). We investigated whether the elevated serum level of prolactin (Prl) detected in SSc is due to a sustained increase over 24 h and/or a shift in the diurnal rhythm, and whether Prl autoantibodies--originally described in SLE--may interfere in the assay. METHODS: The serum level of Prl was measured by ELISA and compared between 73 patients with SSc and 73 age and sex matched controls (78% women, age 56 +/- 11 years). The diurnal rhythms of Prl and thyrotropin (thyroid stimulating hormone, TSH) were compared between 3 patients with SSc and 10 healthy controls. Blood was taken at 2-3, 6-7, 10-11 a.m., and 2-3, 6-7, 10-11 p.m. The serum level of Prl autoantibodies was measured by ELISA and compared between matched patients with SSc and SLE and controls (n = 42 each). Standard curves of the Prl ELISA were spiked with 10% sera containing high levels of Prl autoantibodies to test interference. RESULTS: Serum levels of Prl measured in the morning (8-10 a.m.) were significantly higher in patients with SSc (17.9 +/- 7.7 ng/ml), compared with controls (9.3 +/- 4.2 ng/ml; p < 0.05). In SSc, 40% of patients had Prl levels > 20 ng/ml, but no correlation was found with Scl-70 or Prl autoantibodies. Younger patients (< 50 years, n = 23/73) showed higher serum levels of Prl than older patients (21.3 +/- 10.3 vs 16.3 +/- 6.2 ng/ml; p < 0.05). The diurnal rhythm of Prl revealed that both a sustained increase over 24 h and some shift occurred in SSc. Peaks of secretion were detected between 6 and 11 a.m., instead of 2-6 a.m. The median levels of TSH over 24 h in patients with SSc ranged within the normal limits. Nevertheless, in SSc, a significant correlation (r = 0.59, p < 0.01) was found between diurnal rhythms of Prl and TSH. The prevalence of Prl autoantibodies in serum was 8% in SSc, 27% in SLE, and < 5% in controls. However, the presence of Prl autoantibodies did not interfere with our assay. CONCLUSION: Our data confirm that mild hyperprolactinemia occurs in a subgroup of patients with SSc, and showed that the elevated serum level of Prl is due to both a sustained increase over 24 h and a shift in the diurnal rhythm. The correlation between diurnal rhythms of Prl and TSH suggests common regulatory mechanisms.

Anti-interleukin-1 and anti-CD44 interventions producing significant inhibition of cartilage destruction in an in vitro model of cartilage invasion by rheumatoid arthritis synovial fibroblasts.

OBJECTIVE: To establish an in vitro model for the investigation of destructive processes in rheumatoid arthritis (RA), to study the interaction between fibroblasts, macrophages, and chondrocytes, and to evaluate strategies to inhibit joint destruction in RA. METHODS: Human and bovine chondrocytes cultured in sponges pretreated with native bovine embryonic extracellular matrix produced a cartilaginous matrix reflected by the incorporation of 35S into proteoglycans. The 3-dimensional culture system was optimized for the number of chondrocytes (10(5) cells/sponge), the timing of 35S incorporation (day 21 after chondrocyte isolation), and the medium (20% fetal calf serum). RA synovial fibroblasts (RASF; 10(5)) were added, and the matrix destruction mediated by these RASF was monitored by the release of 35S. The system was modulated by the addition of monocytes (U937 cells), cytokines (interleukin-1beta [IL-1beta] and tumor necrosis factor alpha [TNFalpha]), interleukin-1 receptor antagonist (IL-1Ra), and monoclonal antibodies against IL-1beta and CD44. RESULTS: RASF destroyed the bovine cartilaginous matrix within 2 weeks (days 5-12) and the human cartilaginous matrix within 3 weeks (days 10-18). Compared with the effect of RASF alone (mean +/- SD 948 +/-180 counts per minute/week), the addition of U937 cells (a monocytic cell line), IL-1beta, or TNFalpha to the incubation medium increased the destruction of human cartilaginous matrix by at least 71% up to 90% (ranging from 1,618+/-204 cpm/week to 1,802+/-307 cpm/week). IL-1Ra and anti-IL-1beta monoclonal antibodies reduced the destruction of human matrix by 45% and 35%, respectively; this was partially reversed by the addition of U937 cells. The pretreatment of RASF with anti-CD44 monoclonal antibody (an adhesion molecule and receptor for hyaluronic acid) inhibited the destruction of the cartilaginous matrix by an average of 41% over 3 weeks. CONCLUSION: This model is envisioned to study distinct aspects of human destructive joint diseases under in vitro conditions and to replace and/or supplement animal experiments in basic research and drug testing. Based on the fact that proinflammatory cytokines enhance destruction whereas IL-1Ra and antibodies against IL-1beta and CD44 inhibit the process, it is concluded that anti-IL-1- and anti-CD44-directed therapies may help prevent cartilage destruction in RA.