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1.
Circ Res ; 95(1): 109-16, 2004 Jul 09.
Article in English | MEDLINE | ID: mdl-15178641

ABSTRACT

Systemic sclerosis (SSc) skin lesions are characterized by disturbed vessel morphology with enlarged capillaries and an overall reduction in capillary density, suggesting a deregulated, insufficient angiogenic response. It has been postulated that this phenomenon is due to reduced expression of the potent angiogenic factor vascular endothelial growth factor (VEGF). In contrast to this hypothesis, we demonstrate that the expression of both VEGF and its receptors VEGFR-1 and VEGFR-2 is dramatically upregulated in skin specimens of SSc patients throughout different disease stages. Interestingly, upregulation of VEGF was not mediated by hypoxia-inducible transcription factor-1 (HIF-1) as indicated by only a weak expression of the oxygen-sensitive alpha-subunit of HIF-1 in the skin of SSc patients. This was unexpected on measuring low Po2 values in the SSc skin by using a polarographic oxygen microelectrode system. Considering our observation that PDGF and IL-1beta costimulated VEGF expression, we propose that chronic and uncontrolled VEGF upregulation that is mediated by an orchestrated expression of cytokines rather than VEGF downregulation is the cause of the disturbed vessel morphology in the skin of SSc patients. Consequently, for therapeutic approaches aiming to improve tissue perfusion in these patients, a controlled expression and timely termination of VEGF signaling appears to be crucial for success of proangiogenic therapies.


Subject(s)
Receptors, Vascular Endothelial Growth Factor/biosynthesis , Scleroderma, Systemic/metabolism , Skin/blood supply , Vascular Endothelial Growth Factor A/biosynthesis , Cell Hypoxia , Cytokines/pharmacology , DNA-Binding Proteins/biosynthesis , Dermis/cytology , Fibroblasts/metabolism , Humans , Hypoxia-Inducible Factor 1 , Hypoxia-Inducible Factor 1, alpha Subunit , Neovascularization, Physiologic , Nuclear Proteins/biosynthesis , Scleroderma, Systemic/genetics , Scleroderma, Systemic/pathology , Skin/pathology , Transcription Factors/biosynthesis , Up-Regulation , Vascular Endothelial Growth Factor A/genetics
2.
Autoimmunity ; 37(1): 57-65, 2004 Feb.
Article in English | MEDLINE | ID: mdl-15115313

ABSTRACT

We characterized a full length L1 mRNA in a rheumatoid arthritis (RA) synovial tissue and determined the degree of methylation of its 5'-UTR. We asked whether not only intact but also altered L1s can exert biological activities by transfecting RA synovial fibroblasts (SF) with either retrotransposition-competent or incompetent L1s and examined their capacity to induce p38delta. Total RNA was isolated from the synovial tissue of a 35-year-old woman with highly destructive RA. A complete L1 sequence was obtained by 3'/5'-RACE. Methylation of the genomic 5'-UTR was determined by the sodium-disulfide/PCR method. RA-SF were transfected by lipofection with either a functional L1 or an ORF2-mutated L1 element. The expression of p38delta was measured by RT-PCR and Western blot. The full length L1 mRNA included a 5'-UTR, an ORF1 and an ORF2. Three of five CpG islands (60%) of the genomic L1 5'-UTR were hypomethylated and the ORF2 was deactivated by the insertion of stop codons. Both, intact and ORF2-mutated L1 vectors, induced the expression of p38delta. Thus, even an ORF2-mutated L1 element, as expressed in RA, is biologically active and both L1 ORF1 and p38delta transcripts may appear as a consequence of genomic hypomethylation. The induction of p38delta appears to be mediated by an ORF1/p40-dependent process. This is the first indication of a p40 mediated transactivation.


Subject(s)
DNA-Binding Proteins/metabolism , Mitogen-Activated Protein Kinases/metabolism , Retroelements/genetics , Arthritis, Rheumatoid/genetics , Arthritis, Rheumatoid/metabolism , Blotting, Western , DNA-Binding Proteins/genetics , Humans , Immunohistochemistry , In Situ Hybridization , Mitogen-Activated Protein Kinase 13 , Mitogen-Activated Protein Kinases/genetics , Phylogeny , RNA, Messenger/genetics , RNA, Messenger/metabolism
3.
J Immunol ; 172(2): 1256-65, 2004 Jan 15.
Article in English | MEDLINE | ID: mdl-14707104

ABSTRACT

To analyze the role of Toll-like receptors (TLR) in the pathogenesis of rheumatoid arthritis, we have assessed the effects of stimulation of cultured synovial fibroblasts by the TLR-2 ligand bacterial peptidoglycan. By using high density oligonucleotide microarray analysis we identified 74 genes that were up-regulated >2.5-fold. Fourteen CC and CXC chemokine genes were among the genes with the highest up-regulation. Quantitative real-time PCR analysis confirmed up-regulation of granulocyte chemotactic protein (GCP)-2, RANTES, monocyte chemoattractant protein (MCP)-2, IL-8, growth-related oncogene-2, and to a lesser extent, macrophage-inflammatory protein 1alpha, MCP-1, EXODUS, and CXCL-16. GCP-2, RANTES, and MCP-2 were detected in culture supernatants of synovial fibroblasts stimulated with peptidoglycan. Chemokine secretion induced by stimulation of rheumatoid arthritis synovial fibroblasts via TLR-2 was functionally relevant as demonstrated by chemotaxis assays. GCP-2 and MCP-2 expression, which have not been reported previously in rheumatoid arthritis, was demonstrated in synovial tissue sections of patients diagnosed with rheumatoid arthritis but not in those with osteoarthritis. Correspondingly, synovial fluid levels were significantly higher in patients diagnosed with rheumatoid arthritis as compared with osteoarthritis. Thus, we present evidence for an induction of chemokine secretion by activation of synovial fibroblasts via TLR-2, possibly contributing to the formation of inflammatory infiltrates characteristically found in rheumatoid arthritis joints.


Subject(s)
Arthritis, Rheumatoid/immunology , Chemokines/metabolism , Fibroblasts/immunology , Fibroblasts/metabolism , Membrane Glycoproteins/physiology , Receptors, Cell Surface/physiology , Synovial Membrane/immunology , Synovial Membrane/metabolism , Adult , Aged , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/microbiology , Cells, Cultured , Chemokine CCL5/biosynthesis , Chemokine CCL5/metabolism , Chemokine CCL8 , Chemokine CXCL6 , Chemokines, CXC/biosynthesis , Chemokines, CXC/metabolism , Chemotaxis, Leukocyte/immunology , Fibroblasts/pathology , Gene Expression Profiling , Humans , Ligands , Membrane Glycoproteins/antagonists & inhibitors , Membrane Glycoproteins/metabolism , Middle Aged , Monocyte Chemoattractant Proteins/biosynthesis , Monocyte Chemoattractant Proteins/metabolism , NF-kappa B/antagonists & inhibitors , NF-kappa B/metabolism , Oligonucleotide Array Sequence Analysis , Osteoarthritis/immunology , Osteoarthritis/metabolism , Peptidoglycan/pharmacology , Receptors, Cell Surface/antagonists & inhibitors , Receptors, Cell Surface/metabolism , Synovial Fluid/immunology , Synovial Fluid/metabolism , Synovial Membrane/pathology , Toll-Like Receptor 2 , Toll-Like Receptors , Up-Regulation/immunology
4.
Am J Pathol ; 162(5): 1549-57, 2003 May.
Article in English | MEDLINE | ID: mdl-12707039

ABSTRACT

The aim of the study was to investigate the relationship between invasion and proliferation in rheumatoid arthritis synovial fibroblasts (RASFs). In vitro, RASFs, normal synovial fibroblasts (NSFs), and RASFs transformed with SV40 T-antigen (RASF(SV40)) were analyzed for the expression of cell surface markers (Thy1, VCAM-1, ICAM-1, CD40, CD44) and their proliferation by flow cytometry. Furthermore, colony-forming unit assays were performed and the expression of matrix metalloproteinases (MMP)-14 and cathepsin K mRNA were determined by real-time polymerase chain reaction. In vivo, in the severe combined immunodeficiency (SCID) mouse co-implantation model, RASFs, NSFs, and RASF(SV40) were tested for cartilage invasion, cellular density, and for their expression of the cell cycle-associated protein Ki67. In the SCID mouse co-implantation model, RASFs invaded significantly stronger into the cartilage than NSFs and RASF(SV40). Of note, RASF(SV40) cells formed tumor-like tissues, and the cellular density adjacent to the cartilage was significantly higher than in RASFs or NSFs. In turn, the proliferation marker Ki67 was strongly expressed in the SV40-transformed synoviocytes in SCID mice, but not in RASFs, and specifically not at sites of cartilage invasion. Using the colony-forming unit assay, RASFs and NSFs did not form colonies, whereas RASF(SV40) lost contact inhibition. In vitro, the proliferative rate of RASFs was low (4.3% S phase) in contrast to RASF(SV40) (24.4%). Expression of VCAM-1 was significantly higher, whereas of ICAM-1 was significantly lower, in RASFs than in RASF(SV40). CD40 was significantly stronger expressed in RASF(SV40), whereas CD44 and AS02 were present at the same degree in almost all synoviocytes. Expression of cathepsin K and matrix metalloproteinase-14 mRNA was significantly higher in RASFs than in the RASF(SV40). Our data demonstrate clearly that invasion of cartilage is mediated by activated RASFs characterized by increased expression of adhesion molecules, matrix-degrading enzymes, but does not depend on cellular proliferation, suggesting the dissociation of invasion and proliferation in RASFs.


Subject(s)
Arthritis, Rheumatoid/pathology , Cartilage, Articular/pathology , Osteoarthritis/pathology , Synovial Membrane/pathology , Animals , Arthritis, Rheumatoid/genetics , Base Sequence , Biomarkers/analysis , Cathepsin K , Cathepsins/genetics , Cell Division , Cells, Cultured , DNA Primers , Disease Models, Animal , Fibroblasts/pathology , Flow Cytometry , Gene Expression Regulation , Humans , Matrix Metalloproteinases, Membrane-Associated , Metalloendopeptidases/genetics , Mice , Mice, SCID , Osteoarthritis/genetics , Polymerase Chain Reaction , RNA, Messenger/genetics
5.
Arthritis Rheum ; 48(3): 642-50, 2003 Mar.
Article in English | MEDLINE | ID: mdl-12632416

ABSTRACT

OBJECTIVE: To test the hypothesis that bacterial products acting as adjuvants, such as CpG oligodeoxynucleotides (ODNs) and peptidoglycans (PGs), are able to activate synoviocytes, and to determine the involvement of Toll-like receptors (TLRs) in this activation process. METHODS: Cultured synovial fibroblasts obtained from patients with rheumatoid arthritis (RA) or osteoarthritis (OA) were stimulated with CpG ODNs or PGs. The expression of various integrins was determined by fluorescence-activated cell sorting. TLR and matrix metalloproteinase (MMP) messenger RNA (mRNA) was measured by real-time polymerase chain reaction. Additionally, levels of interleukin-6 (IL-6) and IL-8 in the culture supernatants were assessed by enzyme-linked immunosorbent assay. Blocking experiments were performed by adding anti-TLR-2 and anti-TLR-4 monoclonal antibodies to cultures stimulated with bacterial PGs. RESULTS: Incubation of synovial fibroblasts with CpG ODNs resulted in neither up-regulation of the expression of integrins on the cell surface, up-regulation of MMP mRNA expression, nor IL-6 and IL-8 production. However, incubation of RA synovial fibroblasts as well as OA synovial fibroblasts with staphylococcal PGs led to an up-regulation of CD54 (ICAM-1) surface expression and to increased expression of MMP-1, MMP-3, and MMP-13 mRNA. Furthermore, production of the proinflammatory cytokines IL-6 and IL-8 was increased by treatment with PGs. We demonstrated that cultured synovial fibroblasts express low levels of TLR-2 and TLR-9 mRNA. TLR-2 was up-regulated after stimulation with PGs, whereas TLR-9 mRNA remained at baseline levels after stimulation with CpG ODNs. Anti-TLR-2 monoclonal antibodies significantly inhibited production of IL-6 and IL-8 induced by stimulation with PGs. CONCLUSION: We demonstrate that bacterial PGs activate synovial fibroblasts, at least partially via TLR-2, to express integrins, MMPs, and proinflammatory cytokines. Inhibition of TLR signaling pathways might therefore have a beneficial effect on both joint inflammation and joint destruction.


Subject(s)
CpG Islands , Drosophila Proteins , Fibroblasts/metabolism , Membrane Glycoproteins/metabolism , Oligodeoxyribonucleotides/pharmacology , Peptidoglycan/pharmacology , Receptors, Cell Surface/metabolism , Staphylococcus aureus , Synovial Membrane/metabolism , Arthritis, Rheumatoid/metabolism , Arthritis, Rheumatoid/pathology , Cells, Cultured , DNA, Bacterial , Dose-Response Relationship, Drug , Escherichia coli , Fibroblasts/drug effects , Fibroblasts/pathology , Flow Cytometry , Humans , Intercellular Adhesion Molecule-1/biosynthesis , Intercellular Adhesion Molecule-1/genetics , Interleukins/biosynthesis , Membrane Glycoproteins/genetics , Membrane Glycoproteins/immunology , Metalloendopeptidases/biosynthesis , Metalloendopeptidases/genetics , Oligodeoxyribonucleotides/metabolism , Osteoarthritis/metabolism , Osteoarthritis/pathology , Peptidoglycan/isolation & purification , RNA, Messenger/metabolism , Receptors, Cell Surface/genetics , Receptors, Cell Surface/immunology , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Staphylococcus aureus/chemistry , Synovial Membrane/drug effects , Synovial Membrane/pathology , Toll-Like Receptor 2 , Toll-Like Receptor 9 , Toll-Like Receptors
6.
J Rheumatol ; 30(1): 146-50, 2003 Jan.
Article in English | MEDLINE | ID: mdl-12508404

ABSTRACT

OBJECTIVE: To determine if abnormal collagen metabolism is correlated with neurogenic inflammation, a potential activator of collagen metabolism, in patients with fibromyalgia (FM). METHODS: The presence of inflammatory cytokines, interleukin (IL)-1beta, IL-6, and tumor necrosis factor (TNF)-a was investigated in skin tissues by using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Fifty-three skin biopsies from female patients with FM (30-65 years of age) were examined and compared to skin biopsies of 10 age and sex matched healthy controls. Biopsies were obtained from the left deltoid region. Rheumatoid arthritis synovial fibroblasts and tissues were used as positive controls for the expression of cytokines. Total RNA isolated from the tissue samples were reverse transcribed (RT) by random hexamers as the primer for RT followed by PCR amplification using specific primers for IL-1beta, IL-6 or TNF-a. Expression of IL-1beta, and TNF-a protein was investigated in the skin by immunohistochemistry using specific antibodies (avidin-biotin method). RESULTS: Positive signals (RT-PCR) were detected in skin tissues of 19/50 (38%) FM patients for IL-1beta, in 14/51 FM patients (27%) for IL-6, and in 17/53 patients (32%) for TNF-a. None of the cytokines could be detected in healthy control skin. Immunoreactivity for IL-1beta and TNF-a was demonstrated in certain skin tissues of our FM patients. CONCLUSION: The detection of cytokines in FM skin indicates the presence of inflammatory foci (neurogenic inflammation) in the skin of certain patients (about 30% of FM patients), suggesting an inflammatory component in the induction of pain. This may explain the response to nonsteroidal antiinflammatory therapy in a subset of FM patients.


Subject(s)
Cytokines/genetics , Fibromyalgia/physiopathology , Skin Physiological Phenomena/immunology , Adult , Biopsy , Cytokines/analysis , Female , Fibromyalgia/immunology , Fibromyalgia/pathology , Gene Expression/immunology , Humans , Immunohistochemistry , Interleukin-1/analysis , Interleukin-1/genetics , Interleukin-6/analysis , Interleukin-6/genetics , Middle Aged , RNA, Messenger/analysis , Reverse Transcriptase Polymerase Chain Reaction , Skin/chemistry , Skin/immunology , Skin/pathology , Tumor Necrosis Factor-alpha/analysis , Tumor Necrosis Factor-alpha/genetics
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