Biomedical subjects

W B van den Berg

Publications and source records attributed to W B van den Berg.

At least 19 recordsLinked to original sources

PubMed2006

Toll-like receptor 4 induced FcgammaR expression potentiates early onset of joint inflammation and cartilage destruction during immune complex arthritis: Toll-like receptor 4 largely regulates FcgammaR expression by interleukin 10.

OBJECTIVE: To study the role of Toll-like receptor (TLR)2 and 4 in the onset of joint inflammation and cartilage destruction during immune complex-mediated arthritis (ICA), and its relationship with FcgammaR expression. MATERIALS AND METHODS: ICA was induced in knee joints of TLR2-/- and TLR4-/- mice and their wild-type controls. Joint inflammation and cartilage destruction were measured in the knee joint using histology. mRNA levels were determined in synovial specimens and macrophages using quantitative polymerase chain reaction and cytokine protein levels in synovial washouts using Bioplex. RESULTS: Joint inflammation and cartilage destruction were not different in arthritic TLR2-/- and wild-type mice. By contrast, at day 1 after ICA induction, joint swelling and proteoglycan depletion in knee joints of TLR4-/- mice were considerably lower (inflammation 68-79% and proteoglycan depletion 27-76%) when compared with wild-type controls. Cytokine production at this time point was markedly reduced in TLR4-/- mice (interleukin (IL)1, IL6, macrophage inflammatory chemokine (MIP)-1alpha and keratinocyte-derived chemokine 49%, 72%, 68% and 84%, respectively). In arthritic synovia of TLR4-/- mice, and also after injection of the antigen poly-l-lysine (PLL) lysozyme alone, mRNA levels of FcgammaR, and the FcgammaR regulating cytokine IL10 were considerably lower. Stimulation of peritoneal macrophages with PLL lysozyme up regulated mRNA levels of FcgammaR and IL10, whereas neutralisation by anti-IL10 antibodies largely blocked FcgammaR up regulation. At day 4, joint inflammation and cartilage destruction were comparable in TLR4-/- mice and wild-type controls. CONCLUSION: TLR4 regulates early onset of joint inflammation and cartilage destruction during ICA arthritis by up regulation of FcgammaR expression and enhanced cytokine production. TLR4-mediated up regulation of FcgammaR is largely mediated by IL10.

Animals↗

PubMed2006

GM-CSF neutralisation suppresses inflammation and protects cartilage in acute streptococcal cell wall arthritis of mice.

OBJECTIVE: The pathogenic involvement of granulocyte-macrophage colony-stimulating factor (GM-CSF) in arthritis has been put forward. We have investigated the therapeutic effect of GM-CSF neutralisation in the streptococcal cell wall (SCW) arthritis model in mice. In this model, the pathogenic contribution of tumour necrosis factor (TNF)alpha is minor and is expressed only on joint swelling, whereas cartilage proteoglycan depletion is independent of this cytokine. METHODS: Acute monarthritis was induced by injection of SCW bacterial extracts to mouse knees. Treatments (mAb 22E9 at 300, 100, 30 microg; or Enbrel 300 microg) were given twice intraperitoneally 2 h before and 3 days after disease induction. Swelling was assessed by (99m)Tc uptake into knees on days 1 and 2. Local cytokine levels were determined in patellae washouts on day one. Proteoglycan loss from cartilage was scored on histological sections at termination on day four. RESULTS: Treatment with anti-GM-CSF mAb 22E9 showed a dose-related efficacy by decreasing swelling that was significant at the 300 and 100 microg doses in comparison to isotype control, and comparable to dexamethasone (5 mg/ml). Proteoglycan loss from cartilage was also significantly reduced by mAb 22E9 300 microg (p=0.001). This reduced proteoglycan loss observed after GM-CSF neutralisation was not seen after TNFalpha-blockade with Enbrel. Similarly, levels of interleukin 1beta in joints were reduced after treatment with 22E9 mAb (p=0.003) but not in mice receiving Enbrel. CONCLUSIONS: Our findings show a pathogenic role for GM-CSF in this arthritis model, support the therapeutic potential of neutralising this cytokine, and may indicate therapeutic activity of an anti-GM-CSF mAb in TNFalpha-independent disease situations.

Acute Disease↗

PubMed2006

Expression of transforming growth factor-beta (TGFbeta) and the TGFbeta signalling molecule SMAD-2P in spontaneous and instability-induced osteoarthritis: role in cartilage degradation, chondrogenesis and osteophyte formation.

BACKGROUND: The primary feature of osteoarthritis is cartilage loss. In addition, osteophytes can frequently be observed. Transforming growth factor-beta (TGFbeta) has been suggested to be associated with protection against cartilage damage and new cartilage formation as seen in osteophytes. OBJECTIVE: To study TGFbeta and TGFbeta signalling in experimental osteoarthritis to gain insight into the role of TGFbeta in cartilage degradation and osteophyte formation during osteoarthritis progression. METHODS: Histological sections of murine knee joints were stained immunohistochemically for TGFbeta3 and phosphorylated SMAD-2 (SMAD-2P). Expression patterns were studied in two murine osteoarthritis models, representing spontaneous (STR/ort model) and instability-associated osteoarthritis (collagenase-induced instability model). RESULTS: TGFbeta3 and SMAD-2P staining was increasingly reduced in cartilage during osteoarthritis progression in both models. Severely damaged cartilage was negative for TGFbeta3. In contrast, bone morphogenetic protein-2 (BMP-2) expression was increased. In chondrocyte clusters, preceding osteophyte formation, TGFbeta3 and SMAD-2P were strongly expressed. In early osteophytes, TGFbeta3 was found in the outer fibrous layer, in the peripheral chondroblasts and in the core. Late osteophytes expressed TGFbeta3 only in the fibrous layer. SMAD-2P was found throughout the osteophyte at all stages. In the late-stage osteophytes, BMP-2 was strongly expressed. CONCLUSION: Data show that lack of TGFbeta3 is associated with cartilage damage, suggesting loss of the protective effect of TGFbeta3 during osteoarthritis progression. Additionally, our results indicate that TGFbeta3 is involved in early osteophyte development, whereas BMP might be involved in late osteophyte development.

Animals↗

PubMed2006

High-resolution multipinhole single-photon-emission computed tomography in experimental and human arthritis.

OBJECTIVE: To image inflammatory arthritic lesions in experimental arthritis and in patients with arthritis, using a newly developed high-resolution multipinhole single-photon-emission computed tomography (MPH-SPECT) technique. METHODS: Six interleukin-1 receptor antagonist-deficient mice with arthritis of the front and back paws and 2 control BALB/c mice were imaged with MPH-SPECT and scored macroscopically for arthritis. SPECT imaging was performed with a conventional gamma camera upgraded with a pyramidal lead collimator affixed with MPH apertures. All images were reconstructed, and uptake in the paws was quantified in counts/weight and injected activity. To transfer the imaging technique to humans we examined the clinically dominant hand of 6 individuals (3 with established rheumatoid arthritis [RA], 1 with early RA, 1 with osteoarthritis, and 1 healthy control). RESULTS: MPH-SPECT images were high-resolution 3-dimensional tomographic images, which allowed exact localization and quantifiable observation of increased bone metabolism. MPH-SPECT counts of inflamed joints in mice correlated with macroscopic scoring and histologic joint analysis postmortem. In humans, MPH-SPECT images depicted a detailed visualization of tracer accumulation in bony structures of hand and finger joints, and were also capable of imaging increased bone metabolism that had appeared normal with other imaging modalities, e.g., magnetic resonance imaging. CONCLUSION: The MPH-SPECT technique represents a new diagnostic tool in the detection of bone pathology in small-animal arthritis research. Compared with macroscopic scoring, this new method provides a more objective and higher-precision quantifiable measurement of bone reaction, allowing visualization of inflammatory processes of the whole skeleton in vivo. These results suggest that MPH-SPECT may be useful as a diagnostic instrument for monitoring experimental arthritis, with further potential for use in human studies of RA.

Aged↗

PubMed2006

A novel role for suppressor of cytokine signaling 3 in cartilage destruction via induction of chondrocyte desensitization toward insulin-like growth factor.

OBJECTIVE: An important mechanism contributing to cartilage destruction in arthritis is chondrocyte desensitization toward its main anabolic factor, insulin-like growth factor 1 (IGF-1). In this study, we sought to determine the role of suppressor of cytokine signaling 3 (SOCS-3) in the induction of IGF-1 desensitization of murine chondrocytes. METHODS: Chondrocyte responsiveness to IGF-1 was assessed by 35S-sulfate incorporation into proteoglycans (PGs), via aggrecan messenger RNA expression, using quantitative real-time polymerase chain reaction or insulin receptor substrate 1 (IRS-1) tyrosine phosphorylation (Western blot analysis). IGF-1 desensitization of patellar chondrocytes was studied in zymosan-induced arthritis. IGF-1 desensitization was induced in patellar cartilage explants or the H4 chondrocyte cell line, exposed to interleukin-1alpha (IL-1alpha). SOCS-3 protein expression was assessed by immunohistochemistry or by Western blot analysis of protein extracts. The role of SOCS-3 in IGF-1 signaling was elucidated by adenoviral overexpression. RESULTS: Exposure of murine articular cartilage to IL-1 caused a significant decrease in IGF-1-induced PG synthesis. This effect also occurred in inducible nitric oxide synthase-knockout mice, revealing the involvement of a secondary IL-1-induced factor other than nitric oxide. We showed that IL-1 significantly up-regulated SOCS-3 transcription and protein synthesis in H4 chondrocytes. In contrast, IL-18 was unable to induce SOCS-3 expression and failed to induce chondrocyte IGF-1 desensitization. Histologic analysis of samples from arthritic knee joints revealed high expression of SOCS-3 in chondrocytes. Through adenoviral overexpression of SOCS-3, we obtained direct evidence that SOCS-3 inhibits IGF-1-mediated cell signaling, since IRS-1 phosphorylation was reduced. CONCLUSION: This study demonstrates that IL-1-induced SOCS-3 expression is a novel mechanism of IGF-1 desensitization in chondrocytes; in conjunction with nitric oxide it can contribute to cartilage damage during arthritis.

Animals↗

PubMed2006

Connective tissue growth factor/CCN2 overexpression in mouse synovial lining results in transient fibrosis and cartilage damage.

OBJECTIVE: Characteristics of osteoarthritis (OA) include cartilage damage, fibrosis, and osteophyte formation. Connective tissue growth factor (CTGF; also known as CCN2), is found in high levels in OA chondrocytes and is frequently involved in fibrosis, bone formation, and cartilage repair. The present study was therefore undertaken to investigate the potential role of CTGF in OA pathophysiology. METHODS: We transfected the synovial lining of mouse knee joints with a recombinant adenovirus expressing human CTGF and measured synovial fibrosis and proteoglycan content in cartilage on days 1, 3, 7, 14, and 28. Messenger RNA (mRNA) expression in synovium and cartilage was measured on days 3, 7, and 21. RESULTS: CTGF induced synovial fibrosis, as indicated by accumulation of extracellular matrix and an increase in procollagen type I-positive cells. The fibrosis reached a maximum on day 7 and had reversed by day 28. Levels of mRNA for matrix metalloproteinase 3 (MMP-3), MMP-13, ADAMTS-4, ADAMTS-5, tissue inhibitor of metalloproteinases 1 (TIMP-1), and transforming growth factor beta were elevated in the fibrotic tissue. TIMP-1 expression was elevated on day 3, while expression of other genes did not increase until day 7 or later. CTGF induced proteoglycan depletion in cartilage as early as day 1. Maximal depletion was observed on days 3-7. Cartilage damage was reduced by day 28. A high level of MMP-3 mRNA expression was found in cartilage. CTGF overexpression did not induce osteophyte formation. CONCLUSION: CTGF induces transient fibrosis that is reversible within 28 days. Overexpression of CTGF in knee joints results in reversible cartilage damage, induced either by the high CTGF levels or via factors produced by the CTGF-induced fibrotic tissue.

Animals↗

PubMed2006

Potential new targets in arthritis therapy: interleukin (IL)-17 and its relation to tumour necrosis factor and IL-1 in experimental arthritis.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterised by chronic joint inflammation and destruction. Interleukin (IL)-17 is a T cell cytokine expressed in the synovium and synovial fluid of patients with RA. IL-17 is a potent inducer of various cytokines such as tumour necrosis factor (TNF) and IL-1. IL-17 has been shown to have additive or even synergistic effects with TNF and IL-1 during the induction of cytokine expression and joint damage in vitro and in vivo. TNFalpha and IL-1 are considered powerful targets in the treatment of RA because of their leading role in driving the enhanced production of cytokines, chemokines, and degradative enzymes. Besides anti-TNF and anti-IL-1 therapies, whose clinical efficacy is now established, new targets have been proposed for RA which is not responding to conventional treatments. This paper discusses the role of IL-17 in experimental arthritis and its interrelationship with TNF and IL-1, currently the most targeted cytokines in the treatment of RA. IL-17 is involved in both initiation and progression of murine experimental arthritis. Studies have shown that IL-17 not only synergises with TNF, but also enhances inflammation and destruction independent of IL-1 and TNF. On the basis of these studies, the authors propose IL-17 as an interesting additional target in the treatment of RA.

Animals↗

PubMed2006

Fc gamma receptor mediated modulation of dendritic cells as a potential strategy in the battle against rheumatoid arthritis.

Autoimmune diseases such as rheumatoid arthritis (RA) result from a deregulation of immune responses culminating in immune-mediated tissue injury. In RA, this tissue injury is mainly reflected by synovitis and subsequent joint damage, although involvement of visceral organs (heart, lungs and kidneys) often leads to severe comorbidity. Accumulating evidence points towards dendritic cells (DC) as the principal regulators of the balance between immunity and tolerance. Recently, a large body of evidence has demonstrated that the balance between activating and inhibitory Fc gamma receptor (Fc gammaR) subtypes is intricately involved in the regulation of DC behaviour. In this overview we summarise recent findings from our group and others that suggest an important role for Fc gammaR in arthritis. Furthermore, we postulate novel mechanisms of how triggering of Fc gammaR might be used to manipulate DC function and combat autoimmunity. When DC are envisaged as useful targets in the light of DC immunotherapy in RA, detailed knowledge on the regulatory pathways of Fc gammaR in RA is of paramount importance.

Arthritis, Rheumatoid↗

PubMed2005

Osteoarthritis cartilage histopathology: grading and staging.

OBJECTIVE: Current osteoarthritis (OA) histopathology assessment methods have difficulties in their utility for early disease, as well as their reproducibility and validity. Our objective was to devise a more useful method to assess OA histopathology that would have wide application for clinical and experimental OA assessment and would become recognized as the standard method. DESIGN: An OARSI Working Group deliberated on principles, standards and features for an OA cartilage pathology assessment system. Using current knowledge of the pathophysiology of OA morphologic features, a proposed system was presented at OARSI 2000. Subsequently, this was widely circulated for comments amongst experts in OA pathology. RESULTS: An OA cartilage pathology assessment system based on six grades, which reflect depth of the lesion and four stages reflecting extent of OA over the joint surface was developed. CONCLUSIONS: The OARSI cartilage OA histopathology grading system appears consistent and simple to apply. Further studies are required to confirm the system's utility.

Animals↗

PubMed2005

Reduced transforming growth factor-beta signaling in cartilage of old mice: role in impaired repair capacity.

Osteoarthritis (OA) is a common joint disease, mainly effecting the elderly population. The cause of OA seems to be an imbalance in catabolic and anabolic factors that develops with age. IL-1 is a catabolic factor known to induce cartilage damage, and transforming growth factor (TGF)-beta is an anabolic factor that can counteract many IL-1-induced effects. In old mice, we observed reduced responsiveness to TGF-beta-induced IL-1 counteraction. We investigated whether expression of TGF-beta and its signaling molecules altered with age. To mimic the TGF-beta deprived conditions in aged mice, we assessed the functional consequence of TGF-beta blocking. We isolated knee joints of mice aged 5 months or 2 years, half of which were exposed to IL-1 by intra-articular injection 24 h prior to knee joint isolation. Immunohistochemistry was performed, staining for TGF-beta1, -2 or -3, TGF-betaRI or -RII, Smad2, -3, -4, -6 and -7 and Smad-2P. The percentage of cells staining positive was determined in tibial cartilage. To mimic the lack of TGF-beta signaling in old mice, young mice were injected with IL-1 and after 2 days Ad-LAP (TGF-beta inhibitor) or a control virus were injected. Proteoglycan (PG) synthesis (35S-sulfate incorporation) and PG content of the cartilage were determined. Our experiments revealed that TGF-beta2 and -3 expression decreased with age, as did the TGF-beta receptors. Although the number of cells positive for the Smad proteins was not altered, the number of cells expressing Smad2P strongly dropped in old mice. IL-1 did not alter the expression patterns. We mimicked the lack of TGF-beta signaling in old mice by TGF-beta inhibition with LAP. This resulted in a reduced level of PG synthesis and aggravation of PG depletion. The limited response of old mice to TGF-beta induced-IL-1 counteraction is not due to a diminished level of intracellular signaling molecules or an upregulation of intracellular inhibitors, but is likely due to an intrinsic absence of sufficient TGF-beta receptor expression. Blocking TGF-beta distorted the natural repair response after IL-1 injection. In conclusion, TGF-beta appears to play an important role in repair of cartilage and a lack of TGF-beta responsiveness in old mice might be at the root of OA development.

Aging↗

PubMed2005

Dendritic cells, Fc{gamma} receptors, and Toll-like receptors: potential allies in the battle against rheumatoid arthritis.

Recent findings suggest an important role for Fcgamma receptors and Toll-like receptors expressed by dendritic cells (DC) in the pathogenesis of rheumatoid arthritis (RA). Possibly, DC behaviour might be tuned to counteract the misbalanced immune system in RA. Understanding the precise mechanisms that determine the skewed immune response in RA may provide new clues for the therapeutic use of DC in RA.

Arthritis, Rheumatoid↗

PubMed2005

Dendritic cells from patients with rheumatoid arthritis lack the interleukin 13 mediated increase of Fc gamma RII expression, which has clear functional consequences.

BACKGROUND: Dendritic cell (DC) function is largely tailored by Fc gamma receptors (Fc gamma R) and is critical for every immune response. OBJECTIVE: To compare interleukin (IL) 13 mediated regulation of Fc gamma RII and its related DC function between healthy controls and patients with rheumatoid arthritis (RA). METHODS: DC were derived from peripheral blood mononuclear cells according to standardised protocols. F cgammaRI, II, and III expression and DC phenotype were assessed by FACS analysis. The level of cytokine production and chemokine expression was measured by Luminex and real time quantitative polymerase chain reaction techniques. Antigen uptake capacity was studied by DC fluorescent heat aggregated immunoglobulins and FACS analysis. RESULTS: Replacement of IL4 by IL13 clearly increased the expression of Fc gamma RII on DC from healthy controls (CDC), but had no effect on DC from patients with RA (RADC). The lower production of inflammatory mediators by IL13 CDC upon Fc gamma R mediated triggering suggests that IL13 induces up regulation of specifically Fc gamma RII. RADC co-cultured with IL4 already displayed an inhibitory DC phenotype, but this inhibitory phenotype was not augmented by the addition of IL13. The defective Fc gamma RII regulation was further substantiated by the finding that IL13 CDC increased antigen uptake capacity, whereas IL13 RADC did not. CONCLUSION: IL13 regulates the expression of inhibitory Fc gamma RII in normal subjects but not in RA, potentially resulting in a chronic proinflammatory immune reaction in RA. Unravelling the underlying mechanisms of Fc gamma RII regulation might lead to new therapeutic targets in RA.

Arthritis, Rheumatoid↗

PubMed2005

Synovial tissue inflammation in early and late osteoarthritis.

OBJECTIVE: To compare selected immunohistological features of inflammation in synovial tissue from patients with early and late osteoarthritis (OA). METHODS: Synovial tissue samples were obtained from 10 patients with knee pain, normal radiographs, and arthroscopic manifestations of OA (early OA), and from 15 patients with OA undergoing knee joint arthroplasty (late OA). Conventional immunohistochemical techniques were used to measure microscopic manifestations of inflammation. The inflammatory cell infiltrate, blood vessel formation, and angiogenic factors, NF-kappaB activation, expression of tumour necrosis factor alpha (TNFalpha) and interleukin 1beta (IL1beta), and the presence of cyclo-oxygenase (COX)-1 and COX-2 were quantified. Fibroblast-like synoviocytes (FLS) were isolated from early and late OA tissue samples to compare in vitro production of prostaglandin E2 (PGE2) RESULTS: Synovial tissue from patients with early OA demonstrated significantly greater CD4+ (p = 0.017) and CD68+ (p<0.001) cell infiltration, blood vessel formation (p = 0.01), vascular endothelial growth factor (p = 0.001), and intercellular adhesion molecule-1 expression (p<0.001). Numbers of cells producing TNFalpha and IL1beta were also significantly greater in early OA (p<0.001). Manifestations of inflammation in early OA were associated with increased expression of the NF-kappaB1 (p<0.001) and RelA (p = 0.015) subunits, and with increased COX-2 expression (p = 0.04). Cytokine-induced PGE2 production by cultured FLS was similar in both groups. CONCLUSION: Increased mononuclear cell infiltration and overexpression of mediators of inflammation were seen in early OA, compared with late OA. Isolated FLS were functionally similar in both groups, consistent with microenvironmental differences in the synovial tissue during different phases of OA. These observations may have important therapeutic implications for some patients during the early evolution of OA.

Aged↗

PubMed2005

Soluble interleukin-1 receptor accessory protein ameliorates collagen-induced arthritis by a different mode of action from that of interleukin-1 receptor antagonist.

OBJECTIVE: To discern the mode of interleukin-1 (IL-1) inhibition of soluble IL-1 receptor accessory protein (sIL-1RAcP) by comparison with IL-1 receptor antagonist (IL-1Ra) in arthritis. METHODS: Adenoviral vectors encoding either sIL-1RAcP or IL-1Ra were administered systemically before onset of collagen-induced arthritis in DBA/1 mice. Anti-bovine type II collagen IgG and IL-6 were quantified in serum. Proliferative response of splenic T cells was determined in the presence of sIL-1RAcP or IL-1Ra. The effect on IL-1 inhibition of recombinant sIL-1RAcP and IL-1Ra was further examined in vitro, using NF-kappaB luciferase reporter cell lines. Quantitative polymerase chain reaction was used to determine the relative messenger RNA expression of the IL-1 receptors. RESULTS: Adenoviral overexpression of both sIL-1RAcP and IL-1Ra resulted in amelioration of the collagen-induced arthritis. Both IL-1 antagonists reduced the circulating levels of antigen-specific IgG2a antibodies, but only IL-1Ra was able to inhibit lymphocyte proliferation. By using purified lymphocyte populations derived from NF-kappaB reporter mice, we showed that sIL-1RAcP inhibits IL-1-induced NF-kappaB activity in B cells but not T cells, whereas IL-1Ra inhibited IL-1 on both cell types. A study in a panel of NF-kappaB luciferase reporter cells showed that the sIL-1RAcP inhibits IL-1 signaling on cells expressing either low levels of membrane IL-1RAcP or high levels of IL-1RII. CONCLUSION: We show that the sIL-1RAcP ameliorated experimental arthritis without affecting T cell immunity, in contrast to IL-1Ra. Our results provide data in support of receptor competition by sIL-1RAcP as an explanation for the different mode of IL-1 antagonism in comparison with IL-1Ra.

Adenoviridae↗

PubMed2005

The expression of toll-like receptors 3 and 7 in rheumatoid arthritis synovium is increased and costimulation of toll-like receptors 3, 4, and 7/8 results in synergistic cytokine production by dendritic cells.

OBJECTIVE: To evaluate the expression of Toll-like receptors (TLRs) 3 and 7 in synovium and to study potential differences in the maturation and cytokine production mediated by TLR-2, TLR-3, TLR-4, and TLR-7/8 by dendritic cells (DCs) from rheumatoid arthritis (RA) patients and DCs from healthy controls. METHODS: Synovial expression of TLR-3 and TLR-7 in RA was studied using immunohistochemistry. Monocyte-derived DCs from RA patients and healthy controls were cultured for 6 days and subsequently stimulated for 48 hours via TLR-mediated pathways (lipoteichoic acid, Pam(3)Cys, and fibroblast-stimulating lipopeptide 1 for TLR-2, poly[I-C] for TLR-3, lipopolysaccharide and extra domain A for TLR-4, and R848 for TLR-7/8). Phenotypic DC maturation was measured using flow cytometry. The secretion of tumor necrosis factor alpha (TNFalpha), interleukin-6 (IL-6), IL-10, and IL-12 was measured using the Bio-Plex system. Cell lines expressing TLR-2 and TLR-4 were used for the detection of TLR-2 and TLR-4 ligands in serum and synovial fluid from RA patients. RESULTS: TLR-3 and TLR-7 were highly expressed in RA synovium. All TLR ligands elicited phenotypic DC maturation equally between DCs from RA patients and those from healthy controls. TLR-2- and TLR-4-mediated stimulation of DCs from RA patients resulted in markedly higher production of inflammatory mediators (TNFalpha and IL-6) compared with DCs from healthy controls. In contrast, upon stimulation of TLR-3 and TLR-7/8, the level of cytokine production was equal between DCs from RA patients and those from healthy controls. Remarkably, both TLR-3 and TLR-7/8 stimulation resulted in a skewed balance toward IL-12. Intriguingly, the combined stimulation of TLR-4 and TLR-3-7/8 resulted in a marked synergy with respect to the production of inflammatory mediators. As a proof of concept, TLR-4 ligands were increased in the serum and synovial fluid of RA patients. CONCLUSION: TLRs are involved in the regulation of DC activation and cytokine production. We hypothesize that various TLR ligands in the joint trigger multiple TLRs simultaneously, favoring the breakthrough of tolerance in RA.

Arthritis, Rheumatoid↗

PubMed2005

Elucidation of IL-1/TGF-beta interactions in mouse chondrocyte cell line by genome-wide gene expression.

OBJECTIVE: To elucidate the antagonism between interleukin-1 (IL-1) and transforming growth factor-beta (TGF-beta) at the gene expression level, as IL-1 and TGF-beta are postulated to be critical mediators of cartilage degeneration/protection in rheumatic diseases. METHODS: The H4 chondrocyte cell line was validated by comparing metalloproteinase expression profile with intact murine cartilage by reverse transcription polymerase chain reaction. Genome-wide gene expression in the H4 cells in response to IL-1 and TGF-beta, alone and in combination, was analyzed by using oligonucleotide arrays negotiating approximately 12,000 genes. RESULTS: The response of cartilage and the H4 cell line to IL-1 and TGF-beta was comparable. Oligonucleotide array analysis demonstrated a mutual but asymmetrical antagonism as the dominant mode of interaction between IL-1 and TGF-beta. Cluster analysis revealed a remarkable selectivity in the mode of action exerted by TGF-beta on IL-1 regulated genes: antagonistic on pro-inflammatory genes whereas additive on growth regulators such as vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF). While the former cluster underlined the protective effect of TGF-beta, the latter underscored the adverse effect of TGF-beta. We further identified potentially novel classes of target genes under control of TGF-beta such as ras family, histones, proteasome components, and ubiquitin family, highlighting the importance of such genes in TGF signaling besides the well-characterized SMAD pathway. CONCLUSIONS: We identified a cluster of genes as potential targets mediating the adverse effect of TGF-beta such as fibrosis. Transcriptional regulation of ras GTPase and ubiquitin/proteasome pathways is likely to be a novel mechanism mediating the effect of TGF-beta and its interaction with IL-1. These down-stream genes and pathways can be targets in future therapy.

Animals↗

PubMed2004

Expression and localisation of the new metalloproteinase inhibitor RECK (reversion inducing cysteine-rich protein with Kazal motifs) in inflamed synovial membranes of patients with rheumatoid arthritis.

OBJECTIVE: To assess the expression and localisation of the new metalloproteinase inhibitor RECK, an inhibitor of matrix metalloproteinase-14 (MMP-14) secretion and activity, in the synovial membrane of patients with rheumatoid arthritis (RA). METHODS: RECK expression in synovium samples from patients with RA, osteoarthritis (OA), and "trauma" were studied by quantitative real time reverse transcription-polymerase chain reaction (Q-PCR). RECK mRNA levels were compared with those of the enzyme MMP-14. RECK expression on cryostat sections of synovium was disclosed by goat-antihuman RECK monoclonal antibody. RECK protein was detected on synovial cryostat sections and measured by western blotting. RECK expression on macrophages was investigated by double staining of CD68 and RECK on cryostat sections and characterised by confocal microscopy. RECK expression on RA monocytes or normal monocytes was further investigated by FACS analysis. RESULTS: RECK expression in the synovial membrane of patients with RA was significantly lower than in OA and controls. MMP-14 mRNA levels were not significantly different between the three groups. In RA synovium, RECK protein was expressed mainly in the lining layer but also by macrophages around blood vessels. Fibroblasts and about 50% of the CD68 positive macrophages expressed RECK. In CD68 positive macrophages, RECK was only expressed in secretory granules and not on the membrane. The same pattern was found in M-CSF cultured macrophages of patients with RA and controls. In contrast, synovial fibroblasts showed a diffuse membrane expression within the synovium similar to cultured RA fibroblasts. RECK expression was low on the membrane of monocytes according to FACS analysis. CONCLUSION: The new MMP inhibitor RECK is expressed in synovial membranes of RA, OA, and controls. RECK mRNA is lowest in RA synovial membranes. In contrast with fibroblasts, macrophages in the synovium express RECK only cytoplasmically and not on their membrane.

Arthritis, Rheumatoid↗

PubMed2004

Increased expression of CCL18, CCL19, and CCL17 by dendritic cells from patients with rheumatoid arthritis, and regulation by Fc gamma receptors.

BACKGROUND: Dendritic cells (DC) have a role in the regulation of immunity and tolerance, attracting inflammatory cells by the production of various chemokines (CK). Fc gamma receptors (Fc gamma R) may be involved in regulation of the DC function. OBJECTIVE: To assess the expression of CK by immature (iDC) and mature DC (mDC) and its regulation by Fc gamma R in patients with RA and healthy donors (HC). METHODS: Expression of CK by DC from patients with RA and from HC was determined by real time quantitative PCR and ELISA. DC were derived from monocytes following standardised protocols. To study the potential regulation by Fc gamma R, iDC were stimulated with immune complexes (IC) during lipopolysaccharide (LPS) induced maturation. The presence of CK was studied in synovial tissue from patients with RA, osteoarthritis, and healthy subjects by RT-PCR and immunohistochemistry. RESULTS: iDC from patients with RA had markedly increased mRNA levels of the CK CCL18 and CXCL8. Upon maturation with LPS, expression of CCL18, CCL19, CXCL8, CCL3, and CCL17 increased dramatically, reaching significantly higher levels in patients with RA. Monocytes failed to express these CK, except for CXCL8 and CCL3. IC-mediated triggering of the Fc gamma R on DC from patients with highly active RA down regulated all CK, whereas the reverse was seen when DC from patients with low disease activity and healthy donors were stimulated. CCL18 was significantly increased in RA synovial tissue. CONCLUSION: Increased CK expression by DC was found in patients with RA. This expression is partly regulated by Fc gamma R triggering and results in an inhibitory DC subtype in RA upon Fc gamma R-mediated triggering.

Arthritis, Rheumatoid↗