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 TNF superfamily member LIGHT contributes to survival and activation of synovial fibroblasts in rheumatoid arthritis.

OBJECTIVES: The TNF superfamily member LIGHT has a T-cell co-stimulatory role and has previously been associated with inflammation and autoimmunity. To investigate its role in rheumatoid arthritis (RA), a disease where activated T cells contribute in a prominent way, we have analysed the expression of LIGHT and its receptors in RA and analysed its effects on synovial fibroblasts in vitro. METHODS: The expression of LIGHT was measured in synovial tissues and fluids and the receptors of LIGHT were detected on synovial fibroblasts derived from patients with RA and osteoarthritis (OA). The effects of recombinant LIGHT on the production of proinflammatory cytokines and proteases and on the apoptosis of synovial fibroblasts was assessed. RESULTS: LIGHT mRNA was present in synovial tissues of patients with RA but not with OA. Correspondingly, soluble LIGHT protein could be detected in RA synovial fluid samples at much higher levels than in synovial fluid from patients with OA. Immunohistochemical detection of LIGHT and analysis of synovial fluid cells by flow cytometry revealed CD4 T cells as the major source of LIGHT in the rheumatoid joint. Synovial fibroblasts from RA patients were found to express the LIGHT receptors HVEM and LTbetaR. Recombinant LIGHT induced RA synovial fibroblasts to upregulate MMP-9 mRNA, CD54 and IL-6 in an NF-kappaB-dependent fashion. In vitro, exposure of cultured synovial fibroblasts to LIGHT reduced FAS-mediated apoptosis significantly, without affecting the rate of spontaneous apoptosis. CONCLUSIONS: The results provide evidence for a novel T-cell-dependent activation of synovial fibroblasts by LIGHT in joints of patients with RA, contributing to an inflammatory and destructive phenotype.

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.

Discrepancy between mRNA and protein expression of tumour suppressor maspin in synovial tissue may contribute to synovial hyperplasia in rheumatoid arthritis.

OBJECTIVE: To investigate the expression of maspin in RA synovial tissue and compare it with the expression in osteoarthritis (OA) and normal synovial tissue (NS). METHODS: Using specific primers for maspin, a 237 bp fragment was amplified from cDNA obtained from cultured RA, OA, and normal synovial fibroblasts (SF) by RT-PCR. Additionally, mRNA expression levels were determined quantitatively by real time PCR. mRNA expression of maspin was investigated on snap frozen and paraffin embedded synovial tissue sections by in situ hybridisation. Immunohistochemistry was used to identify the cell type expressing maspin. SDS-PAGE and western blotting were performed to evaluate the protein expression in cultured SF. To confirm protein synthesis in situ, immunohistochemistry with specific anti-maspin antibodies was performed in synovial tissue sections of patients with RA. RESULTS: RT-PCR showed expression of maspin in all cDNA samples from cultured SF. Maspin mRNA was found to be decreased in RA SF twofold and 70-fold compared with OA SF and NS SF, respectively. Maspin mRNA was expressed in RA, OA, and normal synovial tissue. Importantly, maspin transcripts were also found at sites of invasion into cartilage and bone. At the protein level, maspin could be detected in RA and, less prominently, OA SF. In RA synovial tissue, maspin protein was detected in only a few synovial lining cells. CONCLUSION: Maspin is expressed intensively in RA SF at the mRNA level, but only slightly at the protein level, possibly owing to down regulation of maspin; this may contribute to the hyperplasia of synovial tissue in RA.

The expression of cyclooxygenase-1, cyclooxygenase-2 and 5-lipoxygenase in inflammatory muscle tissue of patients with polymyositis and dermatomyositis.

OBJECTIVE: To describe cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2) and 5-lipoxygenase (5-LOX) expression in muscle tissue in patients with idiopathic inflammatory myopathies (IIM) - dermatomyositis (DM) and polymyositis (PM) and to find out if any differences between affected and non-affected muscles detected by MRI exist. METHODS: Samples of muscle tissue from 7 patients with dermatomyositis (DM) and from 4 with polymyositis (PM) were obtained by needle biopsy from affected and non-affected sites distinguished by magnetic resonance imaging. In situ hybridization with antisense mRNA probes was employed to detect COX-1, COX-2 and 5-LOX mRNA. RESULTS: Expression of COX-1, COX-2, and 5-LOX mRNA was found in all samples - in the muscle cells, inflammatory cells and in vessels. COX-1 mRNA expression predominated in the inflammatory cells and vessels and was higher in affected than in non-affected sites detected by MRI (mean intensity 3.22+/-0.67 vs. 2.0+/-0.87; p = 0.0006). The expression of COX-2 mRNA was high mainly in inflammatory cells and/or vessels and was increased in MRI-detected affected tissues (3.5+/-0.88; 1.9+/-1.1; p = 0.003), as was the expression of COX-2 mRNA in muscle cells (2.1+/-1.0 vs. 1.3+/-1.0; p = 0.021). 5-LOX mRNA was largely expressed in muscle cells from MRI-detected affected sites and the signal intensity was higher in comparison with samples taken from non-affected tissues detected by MRI (3.22+/-0.7 vs. 1.67+/-0.7; p = 0.0007). CONCLUSION: Expression of COX-1, COX-2 and 5-LOX mRNA was observed for the first time in muscle tissues from IIM patients. This expression was increased in affected tissues detected by MRI, which may suggest a role of COX-1, COX-2, and 5-LOX in the pathogenesis of IIM.

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.

Increased DNA fragmentation and ultrastructural changes in fibromyalgic muscle fibres.

OBJECTIVE: To determine whether there is evidence of increased DNA fragmentation and ultrastructural changes in muscle tissue of patients with fibromyalgia (FM) compared with healthy controls. METHODS: Muscle tissues from 10 community residents with FM and 10 age and sex matched healthy controls were examined "blindly" for the presence of DNA fragmentation by two different methods: terminal deoxynucleotidyl transferase (TdT) staining (TUNEL) and the FragEL-Klenow DNA fragmentation detection kit. Ultrastructural analysis of tissue was performed by electron microscopy. RESULTS: DNA fragmentation was detected by both methods in 55.4 (SEM 2.5)% of the nuclei in muscle tissue of patients with FM compared with 16.1 (4.1)% (p<0.001) of the nuclei in healthy controls. Contrary to expectation, no typical features of apoptosis could be detected by electron microscopy. The myofibres and actin filaments were disorganised and lipofuscin bodies were seen; glycogen and lipid accumulation were also found. The number of mitochondria was significantly lower in patients with FM than in controls and seemed to be morphologically altered. CONCLUSION: The ultrastructural changes described suggest that patients with FM are characterised by abnormalities in muscle tissue that include increased DNA fragmentation and changes in the number and size of mitochondria. These cellular changes are not signs of apoptosis. Persistent focal contractions in muscle may contribute to ultrastructural tissue abnormalities as well as to the induction and/or chronicity of nociceptive transmission from muscle to the central nervous system.

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.

Angiogenic and angiostatic factors in the molecular control of angiogenesis.

The vascular system that ensures an adequate blood flow is required to provide the cells with sufficient supply of nutrients and oxygen. Two different mechanisms of the formation of new vessels can be distinguished: vasculogenesis, the formation of the first primitive vascular plexus de novo and angiogenesis, the formation of new vessels from preexisting ones. Both processes are regulated by a delicate balance of pro- and anti-angiogenic factors. Physiologically, angiostatic mediators outweigh the angiogenic molecules and angiogenesis does not occur. Under certain conditions such as tumor formation or wound healing, the positive regulators of angiogenesis predominate and the endothelium becomes activated. Angiogenesis is initiated by vasodilatation and an increased permeability. After destabilization of the vessel wall, endothelial cells proliferate, migrate and form a tube, which is finally stabilized by pericytes and smooth muscle cells. Numerous soluble growth factors and inhibitors, cytokines and proteases as well as extracellular matrix proteins and adhesion molecules strictly control this multi-step process. The properties and interactions of angiogenic molecules such as VEGFs, FGFs, angiopoietins, PDGF, angiogenin, angiotropin, HGF, CXC chemokines with ELR motif, PECAM-1, integrins and VE-cadherin as well as angiostatic key players such as angiostatin, endostatin, thrombospondin, CXC chemokines without ELR motif, PEDF are discussed in this review with respect to their molecular impact on angiogenesis.

Cartilage degradation and invasion by rheumatoid synovial fibroblasts is inhibited by gene transfer of TIMP-1 and TIMP-3.

Matrix metalloproteinases (MMPs) are believed to be pivotal enzymes in the invasion of articular cartilage by synovial tissue in rheumatoid arthritis (RA). Here, we investigated the effects of gene transfer of tissue inhibitors of metalloproteinases (TIMPs) on the invasiveness of RA synovial fibroblasts (RASF) in vitro and in vivo. Adenoviral vectors (Ad) were used for gene transfer. The effects of AdTIMP-1 and AdTIMP-3 gene transfer on matrix invasion were investigated in vitro in a transwell system. Cartilage invasion in vivo was studied in the SCID mouse co-implantation model for 60 days. In addition, the effects of AdTIMP-1 and AdTIMP-3 on cell proliferation were investigated. A significant reduction in invasiveness was demonstrated in vitro as well as in vivo in both the AdTIMP-1- and AdTIMP-3-transduced RASF compared with untransduced SF or SF that were transduced with control vectors. in vitro, the number of invading cells was reduced to 25% (P<0.001) in the AdTIMP-1-transduced cells and to 13% (P<0.0001) in the AdTIMP-3-transduced cells (% of untransduced cells). Cell proliferation was significantly inhibited by AdTIMP-3 and, less, by AdTIMP-1. In conclusion, overexpression of TIMP-1 and TIMP-3 by Ad gene transfer results in a marked reduction of the invasiveness of RASF in vitro and in the SCID mouse model. Apart from the inhibition of MMPs, a reduction in proliferation rate may contribute to this effect. These results suggest that overexpression of TIMPs, particularly TIMP-3 at the invasive front of pannus tissue, may provide a novel therapeutic strategy for inhibiting joint destruction in RA.

Inhibition of cartilage destruction by double gene transfer of IL-1Ra and IL-10 involves the activin pathway.

The objective of the study was to determine the effects and the molecular background of interleukin-1 receptor antagonist (IL-1Ra) and vIL-10 double gene transfer into human synovial fibroblasts from patients with rheumatoid arthritis (RA) using the SCID mouse model for cartilage erosion in RA. RA synovial fibroblasts were transduced with retro- or adenoviruses encoding IL-1Ra and/or viral IL-10 (vIL-10). SCID mice were engrafted subcutaneously with IL-1Ra and vIL-10 transduced human rheumatoid synovial fibroblasts and normal cartilage. In parallel, gene expression analysis before and after gene transfer using RNA arbitrarily primed PCR in combination with cDNA array was performed. vIL-10 and IL-1Ra double gene transfer resulted in inhibition of cartilage invasion and degradation by RA synovial fibroblasts when compared with control transduced and non-transduced implants. Expression of key genes that were altered after double gene transfer were related to the activin pathway. The results demonstrate not only that virus-based gene transfer using a combination of two joint-protective genes is a feasible approach to inhibit cartilage degradation by activated RA synovial fibroblasts, but also that the underlying molecular effects include modulation of the activin pathway.

Chemokines and chemokine receptors in the pathogenesis of systemic sclerosis.

Abstract Activation of the immune system and increased synthesis of extracellular matrix proteins by fibroblasts are hallmarks in the pathogenesis of systemic sclerosis (SSc). The mechanisms that initiate the accumulation of inflammatory cells are still unknown. Chemokines are a family of small molecules that are divided into subfamilies according to the position of NH2-terminal cysteine motif. A new nomenclature for chemokines recently has been introduced in an attempt to overcome the confusion resulting from a number of different names for the same chemokines. Recent data indicate that chemokines, and in particular MCP-1 (CCL2), might be involved in the pathogenesis of SSc at different levels. MCP-1 is highly upregulated in skin specimens from SSc patients compared with those from healthy controls. Dermal fibroblasts release MCP-1, which is able to induce and perpetuate the migration of inflammatory cells into the skin. Interestingly, data from animal models, as well as from in vitro studies, indicate that MCP-1 might also be involved in the increased synthesis of extracellular matrix proteins, by either direct or indirect mechanisms. In conclusion, chemokines represent interesting candidates for target-directed therapies for SSc. This concept has to be confirmed by further studies using animal models for SSc and other fibrotic diseases.

Overexpression of monocyte chemoattractant protein 1 in systemic sclerosis: role of platelet-derived growth factor and effects on monocyte chemotaxis and collagen synthesis.

OBJECTIVE: In addition to its chemotactic properties, recent evidence suggests that monocyte chemoattractant protein 1 (MCP-1) might participate in the fibrotic process by inducing the secretion of extracellular matrix (ECM) components. Since the factors that initiate the accumulation of inflammatory infiltrates and ECM deposits in systemic sclerosis (SSc) skin lesions are still unknown, this study was undertaken to examine the role of MCP-1 in SSc. METHODS: In situ hybridization and immunohistochemistry studies for MCP-1 were performed on skin biopsy specimens from patients with SSc and healthy controls. To identify possible stimulators of MCP-1 overexpression in SSc lesions, cultured dermal fibroblasts were incubated with recombinant platelet-derived growth factor (PDGF) and analyzed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay. The chemotactic effects of SSc fibroblasts were examined using a modified Boyden chamber assay. To analyze the fibrotic potential of MCP-1, cultured dermal fibroblasts were incubated with recombinant MCP-1, and type I procollagen was measured by radioimmunoassay and real-time PCR. RESULTS: MCP-1 was expressed by fibroblasts, keratinocytes, and perivascular infiltrates throughout the skin, in involved as well as uninvolved skin areas, from 10 of 11 SSc patients, whereas no expression of MCP-1 was found in healthy controls. Stimulation with PDGF resulted in a significant increase in MCP-1 messenger RNA and protein, with differences between healthy control fibroblasts and fibroblasts from SSc patients. The chemotactic activity for peripheral blood mononuclear cells of SSc fibroblast supernatants increased in a time-dependent manner. Antibodies blocking MCP-1 decreased the chemotactic activity of SSc fibroblasts by a mean +/- SD of 37 +/- 12%. Despite an increase in type I collagen levels over time, no effect of recombinant MCP-1 on the synthesis of type I collagen was observed. CONCLUSION: These data indicate that MCP-1 might contribute to the initiation of inflammatory infiltrates in SSc. Possible stimuli of MCP-1 in dermal SSc lesions include PDGF, which is known to be expressed in SSc. In contrast to previous findings in fibrotic lung diseases, no effect of MCP-1 on collagen synthesis was observed in SSc dermal fibroblasts in vitro.

Detection of mRNA by non-radioactive direct primed in situ reverse transcription.

There are various techniques to detect mRNA in tissue specimens. Among these in situ hybridization is widely applied, and for the detection of small quantities of RNA in situ reverse transcriptase polymerase chain reaction (in situ RT-PCR) has been applied. Furthermore in situ transcription, where signal is produced by direct incorporation of labeled nucleotides during production of a cDNA by reverse transcription, has been shown by a few investigators. We present a non-radioactive in situ reverse transcriptase (in situ RT) protocol which is at least as sensitive as in situ hybridization but avoids probe production and long procedures of preincubation, incubation, and washing. Digoxigenin-labeled UTP is incorporated into a cDNA produced by in situ reverse transcription of mRNA. This method is combined with the fast and sensitive immunogold-silver detection system allowing demonstration of the mRNA within 7 h compared to days in the case of in situ hybridization. Contrary to in situ RT-PCR this new method of in situ RT has no background problems due to non-specific amplification or diffusion of the reaction product.

Synovial tissue protease gene expression and joint erosions in early rheumatoid arthritis.

OBJECTIVE: To relate the expression of proteases in the lining and sublining layers of the synovial membrane to the rate of joint damage during 1 year in patients with early inflammatory arthritis. METHODS: Samples of synovial membrane were obtained by closed-needle biopsy or needle arthroscopy from inflamed knees of 20 patients with early inflammatory polyarthritis (mean disease duration 9.6 months, range 2 weeks to 18 months). Expression of matrix metalloproteinase 1 (MMP-1), cathepsin B (CB), and cathepsin L (CL) was examined using in situ hybridization. Immunohistochemistry was used to identify infiltrating mononuclear cell populations. Radiographs of the hands and feet, performed at presentation and after 1 year, were evaluated for the development of new erosions. RESULTS: Twelve patients had rheumatoid arthritis (RA), 6 had psoriatic arthritis (PsA), 1 had gout, and 1 had an undifferentiated arthritis. Six patients had erosions at presentation. Eleven patients (10 with RA, 1 with PsA) demonstrated at least 1 new erosion after 1 year of followup. MMP-1, CB, and CL messenger RNA (mRNA) were expressed in the synovial membrane of all patients and were present throughout the lining layer, as well as in perivascular cellular infiltrates and endothelial cells in the sublining layer. In the lining layer, the mean percentages of protease mRNA-positive cells per high-power field were higher in those patients who developed new joint erosions than in those without evidence of joint damage. A similar pattern was observed in the sublining layer, where mean numbers of protease mRNA-positive cells were also greater in patients with new joint erosions. There were significant differences between the two groups in MMP-1 mRNA expression in both the lining and sublining layers (P = 0.0007 and P = 0.0027, respectively), as well as in sublining layer CL mRNA expression (P = 0.017), but not in CB mRNA expression. Numbers of lining layer CD68+ cells correlated positively with lining layer MMP-1 mRNA expression (P = 0.043) and with the development of new joint erosions (P = 0.002). CONCLUSION: The detection of MMP-1, CB, and CL in the synovium soon after the onset of symptoms highlights the potential for early joint destruction in patients with RA. High levels of MMP-1 mRNA expression in the lining layer distinguished patients with more rapidly progressive erosive disease. This is the first study to demonstrate features of early synovial pathophysiology that may identify patients at increased risk of developing new joint erosions.

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.

Development and characteristics of a synovial-like interface membrane around cemented tibial hemiarthroplasties in a novel rat model of aseptic prosthesis loosening.

OBJECTIVE: Aseptic prosthesis loosening (APL) is related to the formation and aggressive growth of a synovial-like interface membrane (SLIM) between prosthesis and bone. However, investigation of the early phases of SLIM development in humans presents major difficulties. This study was undertaken to develop and characterize the usefulness of a novel animal model of APL that is based on an established model of defined exercise in a running wheel by Wistar rats that have been subjected to intracranial self-stimulation (ICSS). METHODS: Cemented tibial hemiarthroplasties were implanted into the left knees of 7 male Wistar rats. After 2 weeks, exercise in a running wheel was started in all rats, with a running-load of 2 hours/day for 5 days/week. Six months postoperatively, the knee joints were removed, decalcified, and embedded in paraffin. Histologic evaluation on hematoxylin and eosin-stained sections was performed to investigate the development of a SLIM and the presence of cement debris particles. To characterize the SLIM on a molecular level and investigate growth-regulating factors, the expression of transforming growth factor beta (TGFbeta) and the anti-apoptotic molecule Bcl-2 was analyzed by immunohistochemistry. RESULTS: Although the prostheses appeared mechanically stable after 6 months, the development of SLIM with areas of bone resorption was seen in all samples. Resembling human SLIM, these membranes consisted of loose fibrous tissue, with cement debris particles located particularly at sites originally attached to the prostheses. Immunohistochemistry studies revealed the expression of TGFbeta and Bcl-2 in all specimens. Interestingly, staining for TGFbeta and Bcl-2 was restricted to areas where the SLIM were attached to bone. In contrast, there was only negligible expression of both proteins at sites adjacent to the prostheses. CONCLUSION: Our findings demonstrate that the ICSS Wistar rat model constitutes a feasible tool for studying early stages of APL, and specifically the effect of defined running exercise on SLIM formation. The results further suggest that both cellular proliferation, as stimulated by TGFbeta, and altered apoptosis contribute to early stages of SLIM formation. The expression patterns of TGFbeta and Bcl-2 indicate that the growth of the SLIM is initiated and promoted from the bone rather than from the prosthesis.