Ulf Müller-Ladner

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

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.

Different effects of adiponectin isoforms in human monocytic cells.

Adiponectin (APM) is an adipocyte‐derived adipokine with immunosuppressive, antidiabetic, and antiatherosclerotic properties. Low molecular weight (LMW)‐ and higher molecular weight (HMW)‐APM circulate in the serum and activate different signaling pathways. We were interested to see whether LMW‐APM exerts different effects on monocytic cells compared with the HMW isoform. Therefore, the effects of recombinant LMW‐APM produced in insect cells and the APM from higher eukaryotic cells containing HMW forms on monocytic cells were investigated with respect to apoptosis and inflammation. LMW‐ and HMW‐APM induce apoptosis in nondifferentiated THP‐1 cells, reduce macrophage scavenger receptor (MSR) A mRNA expression, and stimulate phosphorylation of adenosine monophosphate‐activated protein kinase (AMPK). However, HMW‐APM induces the secretion of interleukin (IL)‐6 in human monocytes and THP‐1 cells but does not suppress lipopolysaccharide (LPS)‐induced IL‐6 secretion. In contrast, LMW‐APM reduces LPS‐mediated IL‐6 release and furthermore, stimulates IL‐10 secretion, most likely by reducing the abundance of inhibitor of nuclear factor (NF)‐κB kinase β, leading to a diminished nuclear translocation of NF‐κB p65. Our data indicate that the different APM isoforms do share common effects on monocytic cells but also induce isoform‐specific responses. Although apoptosis, the activation of AMPK, and the reduction of MSR are mediated by all APM isoforms, only LMW‐APM displays anti‐inflammatory properties.

Albumin-Based Drug Delivery as Novel Therapeutic Approach for Rheumatoid Arthritis

We reported recently that albumin is a suitable drug carrier for targeted delivery of methotrexate (MTX) to tumors. Due to pathophysiological conditions in neoplastic tissue, high amounts of albumin accumulate in tumors and are metabolized by malignant cells. MTX, covalently coupled to human serum albumin (MTX-HSA) for cancer treatment, is currently being evaluated in phase II clinical trials. Because synovium of patients with rheumatoid arthritis (RA) shares various features observed also in tumors, albumin-based drug targeting of inflamed joints might be an attractive therapeutic approach. Therefore, the pharmacokinetics of albumin and MTX in a mouse model of arthritis was examined. Additionally, uptake of albumin by synovial fibroblasts of RA patients and the efficacy of MTX and MTX-HSA in arthritic mice were studied. The results show that when compared with MTX, significantly higher amounts of albumin accumulate in inflamed paws, and significantly lower amounts of albumin are found in the liver and the kidneys. The protein is metabolized by human synovial fibroblasts in vitro and in vivo. MTX-HSA was significantly more effective in suppression of the onset of arthritis in mice than was MTX. In conclusion, albumin appears to be a suitable drug carrier in RA, most likely due to effects on synovial fibroblasts, which might increase therapeutic efficacy and reduce side effects of MTX.

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

OBJECTIVEnIn 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.nnnMETHODSnIn 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.nnnRESULTSnMCP-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.nnnCONCLUSIONnThese 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.

Expression of the thioredoxin-thioredoxin reductase system in the inflamed joints of patients with rheumatoid arthritis

OBJECTIVEnTo examine the expression of the thioredoxin (TRX)-thioredoxin reductase (TR) system in patients with rheumatoid arthritis (RA) and patients with other rheumatic diseases.nnnMETHODSnLevels of TRX in plasma and synovial fluid (SF) were measured using enzyme-linked immunosorbent assay. Cellular distribution of TRX was determined by flow cytometry and histochemistry. Cellular expression of TR was studied by in situ messenger RNA (mRNA) hybridization. The effect of oxidative stress and tumor necrosis factor alpha (TNF alpha) on TRX expression by cultured rheumatoid fibroblast-like synoviocytes was studied.nnnRESULTSnSignificantly increased TRX levels were found in the SF from 22 patients with RA, when compared with plasma levels in the same patients (P < 0.001) and compared with SF TRX levels in 15 patients with osteoarthritis (P < 0.001), 13 patients with gout (P < 0.05), and 9 patients with reactive arthritis (P < 0.0001). The presence of TRX could be demonstrated within the SF-derived mononuclear cells and synovial tissue (ST) of RA patients. Concordantly, expression of TR mRNA was observed in the ST of these patients. Stimulation of synovial fibroblast-like synoviocytes with either H2O2 or TNF alpha induced an increase in the production of TRX.nnnCONCLUSIONnThe data demonstrate significantly increased concentrations of TRX in the SF and ST of RA patients when compared with the levels in patients with other joint diseases. Evidence is presented that the local environment in the rheumatic joint contributes to increased TRX production. Based on its growth-promoting and cytokine-like properties, it is proposed that increased expression of TRX contributes to the disease activity in RA.

Adiponectin represents an independent cardiovascular risk factor predicting serum HDL-cholesterol levels in type 2 diabetes

Low levels of high‐density lipoprotein (HDL)‐cholesterol represent an independent cardiovascular risk factor and, besides reduced physical activity, mechanisms leading to decreased HDL‐cholesterol levels are not known. We aimed to test the hypothesis, that adiponectin provides a missing link between type 2 diabetes and low levels of HDL‐cholesterol, independent from common metabolic risk factors. 523 patients with type 2 diabetes were investigated for adiponectin serum levels and parameters of lipid metabolism. Even after correction for age, gender, BMI and fasting insulin concentration, serum levels of adiponectin were highly significant (P<0.0001) and positively (regression analysis: r=0.86) associated with HDL‐cholesterol levels in type 2 diabetes. Conclusion: adiponectin seems to predict HDL‐cholesterol levels in patients with diabetes mellitus type 2. Low levels of adiponectin are associated with low levels of HDL‐cholesterol independently from common metabolic risk factors and therefore represent an independent cardiovascular risk factor in type 2 diabetes. Thus, adiponectin is a potentially new drug target in the treatment of dyslipidaemia.

Profiling adipocytokine secretion from creeping fat in Crohn's disease

Background: Adipose tissue is recognized as a compartment secreting highly active molecules. Creeping fat represents a characteristic feature of Crohns disease (CD). Proinflammatory or anti‐inflammatory adipose‐derived secretory products, now generally called adipocytokines, may play a role in the pathogenesis of CD. Materials and Methods: Adipose tissue specimens were obtained from creeping fat contiguous to the involved intestine of 10 patients with CD. Mesenteric adipose tissue specimens resected from 13 patients with colon cancer (CC) and from 7 patients with diverticulitis served as controls. Three fat tissue specimen per well and 6 to 8 wells per patient were incubated ex vivo for 24 h. The release of adiponectin, resistin, leptin, interleukin‐6, macrophage colony‐stimulating factor, monocyte chemotactic protein‐1, and migration inhibitory factor was measured by ELISA. The expression of AdipoR1 and AdipoR2 receptors was investigated by real‐time quantitative polymerase chain reaction in a subset of adipose tissues. Results: The secretion of adiponectin and macrophage colony‐stimulating factor, as well as leptin and migration inhibitory factor, was significantly upregulated in CD compared with CC and diverticulitis or CC only, respectively. Resistin, interleukin‐6, and monocyte chemotactic protein‐1 were not specifically induced in CD but were associated with unspecific inflammation. Adiponectin receptor expression was not different in CC, CD, or diverticulitis. Steroid treatment in CD patients had differential effects on the ex vivo secretion of adipocytokines. Conclusions: A specific secretion pattern of proinflammatory and anti‐inflammatory adipocytokines indicates the significance of adipose tissue in intestinal inflammation in CD. Future investigations of this intestinal compartment may provide novelinsights into the pathophysiological role of creeping fat and CD.

Gene transfer of cytokine inhibitors into human synovial fibroblasts in the scid mouse model

OBJECTIVEnTo investigate the effects of retrovirus-based gene delivery of inhibitory cytokines and cytokine inhibitors into human synovial fibroblasts in the SCID mouse model of rheumatoid arthritis (RA).nnnMETHODSnThe MFG vector was used for gene delivery of tumor necrosis factor alpha receptor (TNFalphaR) p55, viral interleukin-10 (IL-10), and murine IL-10 into RA synovial fibroblasts. The effect on invasion of these cells into human articular cartilage and on perichondrocytic cartilage degradation was examined after 60 days of coimplantation into the SCID mouse.nnnRESULTSnTNFalphaR p55 gene transfer showed only a limited effect on inhibition of RA synovial fibroblast invasiveness and cartilage degradation. In contrast, invasion of the RA synovial fibroblasts into the coimplanted cartilage was strongly inhibited by both viral and murine IL-10. Perichondrocytic cartilage degradation was not affected by either form of IL-10.nnnCONCLUSIONnThe data show that cytokines can be successfully inserted into the genome of human RA synovial fibroblasts using a retroviral vector delivery system, and that the SCID mouse model of human RA is a valuable tool for examining the effects of gene transfer. In addition, inhibition of more than one cytokine pathway may be required to inhibit both synovial- and chondrocyte-mediated cartilage destruction in RA.

Identification of differentially expressed genes in rheumatoid arthritis by a combination of complementary DNA array and RNA arbitrarily primed-polymerase chain reaction

OBJECTIVEnThere is increasing evidence that T cell-independent pathways, such as the up-regulation of protooncogenes and the production of growth factors and matrix-degrading enzymes, lead to progressive destruction of affected joints. Therefore, identification of differentially regulated genes restricted to rheumatoid arthritis (RA) synovial fibroblasts is essential. A combination of RNA arbitrarily primed-polymerase chain reaction (RAP-PCR) and complementary DNA (cDNA) array with defined genes was used for a highly sensitive differential screening using small amounts of RNA.nnnMETHODSnRNA was extracted from cultured synovial fibroblasts obtained from 6 patients with RA and 6 patients with osteoarthritis (OA). RAP-PCR was performed using different arbitrary primers for first- and second-strand synthesis. PCRs were hybridized to cDNA array membranes. RA samples were compared with OA samples for differentially expressed genes.nnnRESULTSnIn contrast to standard cDNA array, the identification of 12 differentially expressed genes in RA compared with OA (approximately 6%) was possible. Differentially expressed genes of interest were confirmed using semiquantitative RT-PCR and in situ hybridization.nnnCONCLUSIONnNumerous variants of the differential display method and continuous improvements, including RAP-PCR, have proven to be both efficient and reliable for examining differentially regulated genes. Our results show that RAP-PCR combined with cDNA arrays is a suitable method for identifying differentially expressed genes in rheumatoid synovial fibroblasts, using very small amounts of RNA.

Treatment of chronic sialadenitis in a murine model of Sjögren's syndrome by local fasL gene transfer.

OBJECTIVEnInfection of Fas (Fas/CD95)-mutant C57BL/6 (B6)-lpr/lpr mice with murine cytomegalovirus (MCMV) leads to a chronic sialadenitis similar to that of Sjögrens syndrome (SS). The aim of this study was to evaluate whether chronic sialadenitis would also occur in Fas ligand (FasL/CD95L)-mutant B6-gld/gld mice upon infection with MCMV and whether the expression of FasL by local gene transfer using recombinant adenoviral vectors would be an effective therapeutic strategy.nnnMETHODSnB6-gld/gld mice were infected intraperitoneally with MCMV, and salivary glands were analyzed histologically at different time points. For treatment of sialadenitis, recombinant adenoviral vectors expressing the fasL gene (AdLoxpFasL + AxCANCre) or the lacZ gene (AdCMVLacZ) were locally injected into the salivary glands of MCMV-infected B6-gld/gld mice and uninfected B6-+/+ and B6-gld/gld mice.nnnRESULTSnFollowing MCMV infection, B6-gld/gld mice developed an acute and chronic sialadenitis characterized by multiple foci of infiltrating T cells. After local injection of adenoviral vectors, high levels of lacZ or fasL gene expression could be detected in acinar and ductal cells. Treatment of acute and chronic sialadenitis in B6-gld/gld mice with local fasL gene transfer resulted in a significant reduction in the number of inflammatory foci and tissue destruction in salivary glands compared with mice treated with AdCMVLacZ. Despite high levels of FasL expression after injection of recombinant vectors, <5% of ductal and acinar cells were TUNEL positive, demonstrating that, in this model of SS, acinar and ductal cells were not highly sensitive to FasL-mediated apoptosis.nnnCONCLUSIONnChronic sialadenitis similar to that of SS developed in B6-gld/gld mice after MCMV infection. FasL expression was reconstituted by local gene transfer, resulting in significant reduction of infiltrating mononuclear cells, which indicates that local gene transfer of fasL might be a novel treatment for chronic sialadenitis.