Showing papers by "Ulf Müller-Ladner published in 2000"

Fibroblast biology. Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis.

Abstract: There is growing evidence that activated synovial fibroblasts, as part of a complex cellular network, play an important role in the pathogenesis of rheumatoid arthritis. In recent years, significant progress has been made in elucidating the specific features of these fibroblasts. It has been understood that although macrophage and lymphocyte secreted factors contribute to their activation, rheumatoid arthritis synovial fibroblasts (RA-SFs) do not merely respond to stimulation by pro-inflammatory cytokines, but show a complex pattern of molecular changes also maintained in the absence of external stimulation. This pattern of activation is characterized by alterations in the expression of regulatory genes and signaling cascades, as well as changes in pathways leading to apoptosis. These together result in the upregulation of adhesion molecules that mediate the attachment of RA-SFs to the extracellular matrix and in the overexpression of matrix degrading enzymes that mediate the progressive destruction of the joints. In addition, activated RA-SFs exert specific effects on other cell types such as macrophages and lymphocytes. While the initiating step in the activation of RA-SFs remains elusive, several key pathways of RA-SF activation have been identified. However, there is so far no single, specific marker for this phenotype of RA-SF. It appears that activated RA-SFs are characterized by a set of specific properties which together lead to their aggressive behavior.

Expression of osteopontin messenger RNA and protein in rheumatoid arthritis: Effects of osteopontin on the release of collagenase 1 from articular chondrocytes and synovial fibroblasts

Abstract: Objective Osteopontin (OPN) is an extracellular matrix protein that has been implicated in the interactions between tumor cells and host matrix, including those involved in invasion and spread of tumor cells. Because joint destruction in rheumatoid arthritis (RA) is mediated by the invasive growth of synovial tissue through its attachment to cartilage, we examined the expression of OPN in the synovia of patients with RA and the effect of OPN on the production of collagenase 1 in rheumatoid synovial fibroblasts and articular chondrocytes. Methods The expression of OPN messenger RNA (mRNA) and protein in synovia from 10 RA patients was examined by in situ hybridization and immunohistochemistry. Synovial fibroblasts from RA patients and articular chondrocytes from patients without joint disease were cultured in the presence of various concentrations of OPN, and levels of collagenase 1 in the culture supernatants were measured by enzyme-linked immunosorbent assay. Results The expression of OPN mRNA and protein was observed in 9 of 10 specimens obtained from patients with RA. OPN was expressed in the synovial lining and sublining layer and at the interface of cartilage and invading synovium. Double labeling revealed that the majority of OPN-expressing cells were positive for the fibroblast-specific enzyme prolyl 4-hydroxylase and negative for the macrophage marker CD68, while only a few, single OPN-expressing cells were positive for CD68 at sites of synovial invasion into cartilage. OPN staining was not observed in lymphocytic infiltrates or leukocyte common antigen (CD45)–positive cells. Three of 3 cultures of human articular chondrocytes secreted detectable basal amounts of collagenase, with a dose-dependent increase upon OPN stimulation, while synovial fibroblast cultures produced much lower levels of collagenase, with only 2 of 4 fibroblast cultures responding in a dose-dependent manner. Conclusion These findings suggest that OPN produced by synovial fibroblasts in the synovial lining layer and at sites of cartilage invasion not only mediates attachment of these cells to cartilage, but also contributes to matrix degradation in RA by stimulating the secretion of collagenase 1 in articular chondrocytes.

HLA-DQA1*0501 is associated with diffuse systemic sclerosis in Caucasian men.

Abstract: Objective Systemic sclerosis (SSc) is uncommon in men, and relatively little is known about factors contributing to its pathogenesis in this population. In the current study, we investigated HLA class II alleles in men with SSc. We also investigated the hypothesis that HLA compatibility of the mother could be a risk factor for SSc in men. Methods Sequence-specific oligonucleotide probe typing was used to determine DQA1, DQB1, and DRB1 alleles of SSc patients (50 men and 36 parous women), healthy controls (59 men and 80 parous women), 26 mothers of men with SSc, and 44 mothers of healthy men. All study subjects were Caucasian, and allele frequencies were compared with those of Caucasian controls from the Eleventh International Histocompatibility Workshop as well as those of local controls. Results The DQA1*0501 allele was significantly increased among men with SSc compared with healthy men (odds ratio [OR] 2.3, P = 0.006, Pcorr = 0.04). DQA1*0501 was associated with diffuse SSc in men (OR 3.0, P = 0.004, Pcorr = 0.03), but not with limited SSc in men. Maternal HLA compatibility was not a risk factor for SSc in men. Conclusion Previous studies have shown associations of DRB1 alleles with SSc, but have rarely determined DQA1 allele frequencies. Our findings indicate that a specific DQA1 allele is associated with SSc, and that DRB1 associations may be due to linkage disequilibrium with DQA1. Moreover, by analyzing genetic susceptibility according to sex, we found that the contribution of HLA genes to the risk of SSc was substantially greater in men than in parous women.

Activation of synoviocytes.

Abstract: The evaluation of molecular pathways has revealed various novel insights into rheumatoid arthritis pathophysiology during the past year. In addition, there is an increasing tendency toward analysis not merely of a single mechanism but rather of data addressing a substantial part of the cascade of events leading to cellular activation. Because synovial fibroblasts are key cells involved in joint destruction, this review outlines the events that trigger or inhibit the crucial pathways leading to their aggressive behavior. Major topics include cellular and humoral interactions (frequently modulated by cytokines), intracellular signaling and upregulation of gene transcription, and the deleterious effects on articular homeostasis.

Activation of the IL-4 STAT pathway in rheumatoid synovium.

Abstract: STATs act as second messenger after binding of a signaling molecule to its receptor. IL-4 STAT is directly involved in the IL-4-dependent gene transcription in the nucleus. We examined the expression and activation of IL-4 STAT and its related kinase Jak-1 in rheumatoid synovium. Rheumatoid arthritis (RA) synovial frozen sections of patients with short-term ( 2 years) were examined using in situ hybridization and immunohistochemistry. IL-4 STAT mRNA could be detected in synovium of patients with short-term and long-term RA. The most intensive expression of IL-4 STAT mRNA could be seen in follicular inflammatory infiltrates. In the synovial lining, both fibroblasts and macrophages expressed IL-4 STAT mRNA. IL-4 STAT and Jak-1 protein was expressed by synoviocytes, and up-regulation could be induced after stimulation with IL-4. Activation of IL-4 STAT was reflected by phosphorylation of IL-4 STAT. The results indicate that IL-4 STAT is involved in key pathomechanisms in RA synovium and that IL-4 STAT-dependent pathways operate in early and late stages of the disease and presumably contribute to inhibitory immune mechanisms in RA synovium.

Detection of activated complement complex C5b-9 and complement receptor C5a in skin biopsies of patients with systemic sclerosis (scleroderma)

Abstract: Objective. Upregulated matrix synthesis is a hallmark of systemic sclerosis (SSc). There are indications that growth factors such as platelet derived growth factor (PDGF) are involved in proliferative pathways in SSc lesions. As activated complement releases PDGF from endothelial cells, we searched for activated complement and the complement receptor for C5a (C5aR) in skin biopsies of patients with SSc. Methods. Snap frozen sections of 8 patients with early SSc and 5 patients with longterm SSc were examined. Using monoclonal antibodies against activated complement complex C5b-9 and the C5aR, skin biopsies derived from both clinically involved and non-involved skin were examined by APAAP immunohistochemistry. Results. A pattern of activated complement C5b-9 and the C5aR could be detected in SSc microvasculature. Eleven of the 13 patients (7/8 patients with early SSc) showed positive staining for C5b-9. The C5aR was detected in 6 of the 8 patients with early SSc. In 3 patients with longterm disease, C5aR expression could also be detected in non-involved skin. Conclusion. Activated complement and complement receptors could be detected in early and late stages of SSc skin lesions. The presence of complement receptors in non-involved skin may indicate preclinical activation of pathways resulting in growth factor dependent matrix synthesis.

p53 in rheumatoid arthritis: friend or foe?

Abstract: The knowledge of transcription factors and proto-oncogenes has influenced the understanding of cell regulation, cell cycle, and apoptotic cell death in rheumatoid arthritis (RA) synovium. In addition, the development of normal synovial fibroblasts into transformed-appearing aggressive synovial fibroblasts may be triggered by the lack of antiproliferative factors, such as p53, p53-associated molecules, other tumor suppressors, as well as by upregulation of anti-apoptotic genes. Therefore, data derived from experiments such as those performed by Tak and colleagues in this issue of Arthritis Research not only enrich the intensive discussion addressing the impact of p 53 on RA pathophysiology, they also may facilitate development of novel therapeutic approaches including p 53-targeted gene therapy.

Microsatellite analysis in rheumatoid arthritis synovial fibroblasts

Abstract: OBJECTIVES—Rheumatoid arthritis (RA) is a chronic disease characterised by irreversible destruction of the affected joints. As aggressive transformed-appearing synovial fibroblasts are commonly found at the site of invasion of the rheumatoid synovium into the adjacent cartilage and bone, the presence of microsatellite instability (MSI) and expression of mismatch repair enzymes as a possible mechanism in the alteration of these cells was examined. METHODS—DNA was extracted from the synovial fibroblasts and blood of 20 patients with long term RA undergoing joint replacement, and the presence of MSI was studied at 10 microsatellite loci. In addition, immunohistochemistry was performed to evaluate the expression of the two major mismatch repair enzymes (hMLH1 and hMSH2) in rheumatoid synovium. RESULTS—MSI could not be detected in any of the fibroblast cell populations derived from the 20 different rheumatoid synovial samples. In addition, strong expression of mismatch repair enzymes could be seen in numerous cells, including fibroblasts, throughout the synovium. CONCLUSIONS—Applying the currently used and established markers for MSI, the data show for the first time that MSI does not appear to have an important role in alteration of rheumatoid synovial fibroblasts into an aggressive phenotype. On the other hand, strong mismatch repair enzyme synthesis in rheumatoid synovium supports the hypothesis of continuing DNA repair, presumably due to long term, inflammation induced DNA damage.

Gene transfer in rheumatoid arthritis: a novel therapeutic approach.

Abstract: © Copyright CLINICAL AND EXPERIMENTAL RHEUMATOLOGY 2000. Rheumatoid arthritis (RA) is a chronic systemic disorder of unknown etiology that is characterized by progressive joint destruction. Several novel strategies in the treatment of RA have evolved during the last few years. New insights into the molecular mechanisms involved in the pathophysiology of RA as well as advances in biotechnology not only have led to the development of highly specific biological agents for the treatment of RA (1), but also have given rise to the concept of gene transfer as a potential therapeutic approach to RA (2). Gene transfer, defined as the insertion of genetic information into living cells, can be applied with experimental or therapeutic aims. While in the past gene transfer was predominantly used to investigate cellular mechanisms involved in the pathogenesis of RA (3), the focus is shifting continuously to therapeutic interventions (2). The feasability of the strategy to selectively inhibit pathways involved in the pathophysiology of RA has been proven by the successful clinical application of biological agents such as inhibitors of tumor necrosis factor alpha (TNFα) (4). However, the clinical effectiveness of such biologics is limited by their protein properties. They are not orally active and tend to be cleared rapidly after injection. The concept of gene transfer offers the opportunity to overcome this disadvantage by the specific delivery and longterm expression of therapeutic molecules in the target tissues. Nevertheless, several questions about the practical use of gene transfer in RA have not been answered sufficiently so far (5). The efficacy of gene transfer is determined predominantly by the delivery strategy used. Therefore, the development of appropriate delivery systems is a critical step toward successful gene transfer. In this context, an interesting article by P.H. Goossens et al. addressing the efficacy of different promotors in the synovium of rhesus monkeys with collagen induced arthritis is published in this issue (6). In general, cells can be transfected either ex vivo or in vitro. For ex vivo transfer, the cells are removed prior to transfection and re-implanted afterwards. For this method, retroviral vectors still constitute the method of choice (Table I). Although transfection with retroviralbased vectors is restricted to dividing cells, the ex vivo procedure permits the selection of transfected cells and the growing of them under selective conditions before re-implantation into the joints. Therefore, the number and properties of the transfected cells can be determined before re-implantation. Another characteristic feature of retroviral veotors is the genomic integration of the transferred gene (Table I). Since the integration is not site-specific, it bears the potential risk of insertional mutagenesis. However, due to the high transfection efficacy on isolated synovial cells and the possibility of analysing the cells in culture before re-implantation, ex vivo retroviral mediated gene transfer has been used for several experimental gene transfer studies in animals (7, 8) as well as for the first clinical trial of gene transfer in RA (9). In contrast to retroviruses, adenoviruses are able to transfect non-dividing cells and show a high transfection efficacy (Table I). Therefore, adenovirus-based vectors have been used frequently for in

Cartilage erosion in rheumatoid arthritis: studies in SCID mouse model

Abstract: In the past few years there has been a growing interest in gene therapy to be used not only for the treatment of inherited, but also acquired, diseases. Although genetic factors have been frequently implicated in the pathogenesis of rheumatoid arthritis (RA) [1], and recent reports suggest mutations of tumor suppressor genes to play a role [2], RA is not caused by a specific genetic mutation. Much more likely it is an acquired disorder with a complex pathogenesis and yet unknown etiology. Gene therapy approaches in diseases such as RA differ from those in defined genetic disorders. Apart from the problem of how to correct a specific genetic abnormality, in RA another question appears to be crucial: that of which pathogenic pathway to modulate. Therefore, a suitable animal model for gene therapy approaches in RA should not only reflect relevant features of human disease but also permit one to analyze alterations in key disease processes as closely as possible to the conditions found in people.