Etanercept decreases synovial expression of tumour necrosis factor-α and lymphotoxin-α in rheumatoid arthritis.

OBJECTIVES: Etanercept is an effective tumour necrosis factor (TNF)-α inhibitor drug with the unique ability to block not only TNF-α but also lymphotoxin (LT)-α, at least in vitro. We aimed to investigate the in vivo effect of etanercept on synovial expression of TNF-α and LT-α. METHOD: Synovial biopsies from 12 rheumatoid arthritis (RA) patients started on etanercept and 11 RA patients started on infliximab were obtained at baseline and 8 weeks after treatment initiation. Synovial expression of TNF-α and LT-α was evaluated by immunohistochemistry followed by computer-assisted image analysis. Differences between paired samples were analysed by the Wilcoxon test and between groups by the Mann-Whitney test. A p-value < 0.05 was considered statistically significant. RESULTS: Six out of the 12 of the patients started on etanercept achieved an American College of Rheumatology (ACR)50 response. Macroscopic evaluation of the joints during arthroscopy revealed a significant decrease of local inflammation mainly in good ACR50 responders. Synovial expression of both LT-α and TNF-α decreased but the differences did not reach statistical significance at a group level. By contrast, a significant decrease in both LT-α and TNF-α was observed when only good ACR50 responders were analysed. Despite higher levels of baseline synovial TNF-α in the good responders, neither baseline LT-α nor TNF-α could predict clinical response after 8 weeks. A decreasing trend of the synovial levels of LT-α was also observed in good responders to infliximab, but the difference did not reach statistical significance. CONCLUSIONS: Etanercept treatment modulates the synovial expression of both TNF-α and LT-α in vivo, a mechanism that might partly explain its clinical efficacy in RA.

Genetic variation in the serotonin receptor gene affects immune responses in rheumatoid arthritis.

Many genetic variants associate with the risk of developing rheumatoid arthritis (RA); however, their functional roles are largely unknown. Here, we aimed to investigate whether the RA-associated serotonin receptor 2A (HTR2A) haplotype affects T-cell and monocyte functions. Patients with established RA (n=379) were genotyped for two single-nucleotide polymorphisms (SNPs) in the HTR2A locus, rs6314 and rs1328674, to define presence of the risk haplotype for each individual. Patients with and without the RA-associated TC haplotype were selected and T-cell and monocyte function was monitored following in vitro stimulations with staphylococcal enterotoxin B and lipopolysaccharide (LPS) using multiparameter flow cytometry. Within the cohort, 44 patients were heterozygous for the TC haplotype (11.6%) while none were homozygous. Upon stimulation, T cells from TC-carrier patients produced more proinflammatory cytokines (tumor necrosis factor alpha (TNF-α), interleukin-17 (IL-17) and interferon gamma (IFN-γ)) and monocytes produced higher levels of TNF-α compared with patients carrying the non-TC haplotype (P<0.05 and 0.01, respectively). Such cytokine production could be inhibited in the presence of the selective 5-HT2 receptor agonist (2,5-Dimethoxy-4-iodoamphetamine, DOI); interestingly, this effect was more pronounced in TC carriers. Our data demonstrate that association of RA with a distinct serotonin receptor haplotype has functional impact by affecting the immunological phenotype of T cells and monocytes.

Multinational evidence-based recommendations on how to investigate and follow-up undifferentiated peripheral inflammatory arthritis: integrating systematic literature research and expert opinion of a broad international panel of rheumatologists in the 3E Initiative.

OBJECTIVE: To develop evidence-based recommendations on how to investigate and follow-up undifferentiated peripheral inflammatory arthritis (UPIA). METHODS: 697 rheumatologists from 17 countries participated in the 3E (Evidence, Expertise, Exchange) Initiative of 2008-9 consisting of three separate rounds of discussions and modified Delphi votes. In the first round 10 clinical questions were selected. A bibliographic team systematically searched Medline, Embase, the Cochrane Library and ACR/EULAR 2007-2008 meeting abstracts. Relevant articles were reviewed for quality assessment, data extraction and synthesis. In the second round each country elaborated a set of national recommendations. Finally, multinational recommendations were formulated and agreement among the participants and the potential impact on their clinical practice was assessed. RESULTS: A total of 39,756 references were identified, of which 250 were systematically reviewed. Ten multinational key recommendations about the investigation and follow-up of UPIA were formulated. One recommendation addressed differential diagnosis and investigations prior to establishing the operational diagnosis of UPIA, seven recommendations related to the diagnostic and prognostic value of clinical and laboratory assessments in established UPIA (history and physical examination, acute phase reactants, autoantibodies, radiographs, MRI and ultrasound, genetic markers and synovial biopsy), one recommendation highlighted predictors of persistence (chronicity) and the final recommendation addressed monitoring of clinical disease activity in UPIA. CONCLUSIONS: Ten recommendations on how to investigate and follow-up UPIA in the clinical setting were developed. They are evidence-based and supported by a large panel of rheumatologists, thus enhancing their validity and practical use.

The inhibitor of death receptor signaling, FLICE-inhibitory protein defines a new class of tumor progression factors.

Death receptor-mediated apoptosis can be modulated by several antiapoptotic proteins, such as the FLICE (FADD [Fas-associated death domain]-like IL-1beta-converting enzyme)-inhibitory proteins (FLIPs). The FLIP family includes both cellular and viral members. The Kaposi's sarcoma-associated herpesvirus protein (KSHV)-FLIP is expressed by human herpesvirus 8 (HHV-8), which is associated with malignancies such as Kaposi's sarcoma and certain lymphomas. In this paper, we demonstrate that KSHV-FLIP protects cells from Fas-mediated apoptosis by inhibiting caspase activation and permits clonal growth in the presence of death stimuli in vitro. Furthermore, we show that KSHV-FLIP can act as a tumor progression factor by promoting tumor establishment and growth in vivo. When injected into immunocompetent recipient mouse strains, murine B lymphoma cells (A20) transduced with KSHV-FLIP rapidly develop into aggressive tumors showing a high rate of survival and growth. The tumor-progressive activity of KSHV-FLIP is mediated by prevention of death receptor-induced apoptosis triggered by conventional T cells. Consequently, inhibitors of death receptor signaling can be regarded as a new class of tumor progression factors, and HHV-8-associated tumors may represent naturally occurring examples of the tumorigenic effect of such inhibitors.

FOXP3 expression in blood, synovial fluid and synovial tissue during inflammatory arthritis and intra-articular corticosteroid treatment.

OBJECTIVE: To analyse the distribution of FOXP3+CD25+CD4+ regulatory T cells (Treg) in peripheral blood, synovial fluid and tissue of patients with rheumatic disease during relapse and after local treatment. METHODS: FOXP3 expression was assessed by flow cytometry, immunohistochemistry, immunofluorescence and real-time polymerase chain reaction (RT-PCR). The functional suppressive capacity of Treg was analysed after co-culture with effector CD4+CD25- T cells through assessment of proliferation and cytokine secretion. RESULTS: It was shown that FOXP3 protein and mRNA expression in synovial fluid T cells was not confined solely to CD25(bright) T cells as seen in blood, but included CD25(intermediate) and even CD25(neg) T cells. Indeed, synovial fluid CD25(high) T cells showed similar suppressive capacity as CD25(bright) T cells, indicating the presence of functional Treg in T cells with lower intensity of CD25. In synovial tissue, FOXP3+ cells were present in low numbers within T-cell infiltrates and decreased further after intra-articular glucocorticosteroid administration, in parallel with the general reduction in inflammation. CONCLUSIONS: Identification of synovial fluid FOXP3+ Treg with varying intensities of CD25 opens up possibilities for thorough characterisation of this important T-cell subset in the inflammatory compartment. However, only scarce synovial membrane expression of FOXP3 was found even in the absence of overt inflammation, suggesting that the synovial membrane is a site that would benefit therapeutically from Treg expansion.

Cell specific synovial expression of nicotinic alpha 7 acetylcholine receptor in rheumatoid arthritis and psoriatic arthritis.

Neuroimmune interactions are known to influence several chronic inflammatory and rheumatic diseases, but the underlying mechanisms have been insufficiently elucidated. The cholinergic anti-inflammatory pathway is characterized by neural regulation of systemic inflammation, mediated by the vagus nerve and specific cholinergic stimulation of the nicotinic alpha-7 acetylcholine receptor (alpha7nAChR) on immune cells. Moreover, alpha7nAChR has been shown important for immune regulation also in the absence of nerves, but little is known about these mechanisms in chronic joint inflammation. The expression and localization of alpha7nAChR in synovial biopsies from patients with rheumatoid arthritis and psoriatic arthritis was investigated by immunohistochemistry using monoclonal antibody against alpha7nAChR. Surface staining of alpha7nAChR was observed in synovial tissue of all arthritis patients investigated and could also to a lesser extent be detected in the synovium of healthy individuals. alpha7nAChR positive cells were detected in mainly synovial lining cells and vessels. The alpha7nAChR positively stained cells were by double immunofluorescence identified as primarily macrophages and fibroblasts, with the majority of these cells expressing the receptor. These results indicate the importance of alpha7nAChR and cholinergic mechanisms in arthritis pathogenesis and implicate specific cholinergic modulation as a potential anti-inflammatory therapeutic strategy in joint inflammation.

Smoking increases peptidylarginine deiminase 2 enzyme expression in human lungs and increases citrullination in BAL cells.

OBJECTIVES: A gene-environment interaction between HLA-DR shared epitope genes and smoking in anti-cyclic citrullinated peptide antibody-positive rheumatoid arthritis (RA) has been reported. Identification of citrullinated proteins in bronchoalveolar lavage (BAL) cells from smokers has led to the suggestion that citrullination induced by smoking might be the first step in the pathogenic chain of RA. OBJECTIVE: To confirm and extend these findings. METHODS: Immunohistochemistry was performed on BAL cells and bronchial mucosal biopsy sections obtained through bronchoscopy from 14 healthy smokers and 16 healthy non-smokers. Two antibodies recognising citrullinated proteins, two antibodies recognising peptidylarginine deiminase (PAD)2 enzyme and one recognising PAD4 enzyme were used. RESULTS: Citrullinated proteins are upregulated in BAL cells of healthy smokers compared with healthy non-smokers. This was associated with higher expression of the PAD2 enzyme. The same level of citrullinated proteins was present in bronchial mucosal biopsy specimens of healthy smokers and non-smokers, despite higher expression of PAD2 in smokers. CONCLUSION: This study provides evidence that smoking enhances PAD2 expression in the bronchial mucosal and alveolar compartment, with consequent generation of citrullinated proteins in the latter. Smoking is an environmental factor that may lead to citrulline autoimmunity in genetically susceptible subjects.

A cytotoxic, apoptotic, low-molecular weight factor from pineal gland.

Previous studies suggest that the pineal gland may play a role in tumour growth inhibition. In this respect, melatonin, as the major hormone of this gland, has been extensively studied. However, there is growing evidence for the existence of other yet unknown pineal factors that may have tumour growth inhibiting properties. Here we describe the partial purification of a highly cytotoxic low molecular weight (<400 Da) hydrophilic fraction (designated F2M3R), starting from a porcine pineal extract (PE), via methanol precipitation followed by reverse-phase HPLC. F2M3R is cytotoxic for a highly apoptosis-resistant human erythroleukemia cell line (K562) at a concentration as low as 30 microg/ml. The viability of the cells was not influenced by an identical prepared porcine pituitary extract or by melatonin. PE induces apoptosis in K562 cells as indicated by three different criteria: morphology, in situ TUNEL assay and bi-parametric FACS analysis with annexin V and propidium iodide, but does not influence the viability of stimulated peripheral blood mononuclear cells. These observations warrant further purification and validation of the cytotoxicity in a panel of different human tumour and non-malignant cells.

Proliferation and apoptosis in the evolution of endemic and acquired immunodeficiency syndrome-related Kaposi's sarcoma.

Kaposi's sarcoma (KS) is a multifocal lesion that occurs predominantly in the skin, most frequently in people infected with HIV-1, and that evolves through early stages (patch and plaque) to a tumor-like late stage (nodular). Both, endemic African (EKS) and AIDS-associated (AKS) KS expressed human herpesvirus 8 (HHV-8) as shown by PCR. By immunohistochemistry the expression of cellular Bcl-2 and c-myc was confined in early stages of both EKS and AKS to relatively few endothelial cells (EC) whereas in nodular KS most of spindle cells (SC) strongly expressed both genes. CD40 was usually strongly expressed in SC at all KS stages as well as in EC of non-involved tissue whereas CD40L (CD154) was not demonstrable. Fas (CD95) was moderately to weakly expressed by SC whereas p53 and Waf-1 were found in less than 5% of the SC. In both AKS and EKS at nodular stage almost no apoptotic SC were detected. In most AKS and EKS low levels of cell proliferation were seen but AKS showed consistently higher values compared to EKS. All clinical types and stages of KS showed a diploid cellular DNA content by flow cytometric analysis of microselected lesions. Thus, we conclude that KS during evolution represents diploid, probably reactive, cell proliferation, which progressively increases the expression of strong cellular and also viral (HHV-8) antiapoptotic factors.

Melatonin shortens the survival rate of Ehrlich ascites-inoculated mice.

UNLABELLED: OBJECTIVES Pineal gland may have a role in organism's protection against cancer. Melatonin as well as still unidentified low-weight molecular pineal substance(s) have been reported to have growth inhibitory effect on different tumor cells. We tested the influence of melatonin and of a bovine pineal extract on the survival rate of AKR mice inoculated with Ehrlich ascites. The tumor is known to have an accelerated development after pinealectomy. MATERIAL AND METHODS: Male AKR mice, kept under a 14/10 hours - Light /Dark cycle, were inoculated intraperitoneally (i.p.) with 1.5x 10(6) Ehrlich ascites cells. On day three after inoculation the animals were divided in three groups (n=10). Each animal received i.p. daily (20.00H), until their death, 250 microl of solution containing melatonin (250 microg), pineal extract (equivalent of 1 bovine pineal gland) or saline. RESULTS: The average survival rate of the animals treated with melatonin was shorter (14.8+/-2.23 days) compared to control animals (21.9+/-2.21 days)(p=0.01). The animals treated with the pineal extract had a longer survival rate (22.6 +/- 1.8 days) but not statistically significant. The pineal extract was not available for testing at higher doses. CONCLUSION: In our model, melatonin had a deleterious effect on the survival rate raising the question whether it is correct to assume that the hormone shows lack of adverse reactions.

Citrullination is an inflammation-dependent process.

OBJECTIVES: To study the presence of citrullinated proteins in inflammatory conditions and in clinically non-affected tissues of controls. METHODS: Synovial biopsy specimens from 19 patients with rheumatoid arthritis and 10 healthy controls were investigated by immunohistochemistry. Additionally, muscle tissue from 5 patients with polymyositis and from 7 healthy controls, intestinal tissue from macroscopically affected and non-affected areas from 10 patients with inflammatory bowel disease (IBD) and tonsil tissues from 4 chronically inflamed tonsils were studied. RESULTS: Citrullinated proteins were present in all synovial biopsy specimens from patients with rheumatoid arthritis, whereas only three of 10 healthy synovial biopsy specimens showed scarce amounts of citrullination. Citrullination was also present in all myositis-affected muscles, whereas it was absent in the muscle tissues of controls. All tonsil biopsy specimens studied were positive for citrulline. Even though more frequently detected in the macroscopically affected colonic areas, no marked difference was observed in the pattern or extent of citrullination between the macroscopically affected and non-affected intestinal IBD tissues. CONCLUSION: Citrullination is present in a wide range of inflammatory tissues, suggesting that this process is inflammation dependent rather than disease dependent.

Inhibition by Artocarpus tonkinensis of the development of collagen-induced arthritis in rats.

Extracts of the leaves and roots from the tree Artocarpus tonkinensis A Cheval (family Moraceae) are used in traditional Vietnamese medicine in order to treat backache as well as rheumatic joint diseases. We prepared an ethyl acetate (EtOAc) extract from this plant and tested its anti-inflammatory properties in an experimental arthritis model, collagen-induced arthritis (CIA). CIA was induced in Dark Agouti rats by means of immunization with collagen type II (CII) emulsified in incomplete Freund's adjuvant. Starting at the day of immunization, the rats were treated daily with intraperitoneal injections of Artocarpus extract. Arthritis progression was measured by means of clinical scoring of paws and anti-CII antibody titres were measured by means of ELISA. In vitro, lymph node (LN) cell cultures were treated with Artocarpus extract and the apoptosis-inducing effect was determined with FACS staining by using annexin V and propidium iodide as well as the TUNEL method. Treatment of the rats with Artocarpus extract decreased arthritis incidence and severity and delayed disease onset. When treatment was started after the onset of arthritis, a tendency towards arthritis amelioration was observed. In vitro, Artocarpus extract acted as a T-cell modulator, inhibiting mitogen-induced T-cell proliferation and inducing apoptosis of activated LN-derived lymphocytes. Thus, we have demonstrated that an EtOAc extract of Artocarpus, a plant traditionally used in Vietnamese folk medicine for treating arthritic conditions, has beneficial effects in an experimental arthritis model. This effect is likely to be T cell-dependent and mediated through apoptosis induction in activated cells.

Low levels of apoptosis and high FLIP expression in early rheumatoid arthritis synovium.

OBJECTIVES: To define synovial apoptosis with respect to disease duration, inflammatory cell type, FLIP (FLICE-like inhibitory protein), and cytokines expression in patients with rheumatoid arthritis (RA). METHODS: Synovial biopsy specimens from 11 patients with longstanding RA (median disease duration 21 years) and eight with early RA (median disease duration five months) were investigated. Apoptosis (TUNEL method combined with morphological analysis), cell surface markers (CD3, CD68), cytokines (interleukin (IL) 1alpha, IL1beta, tumour necrosis factor alpha, and IL6), and FLIP expression were evaluated. Computer assisted image analysis was used for quantification. RESULTS: The apoptosis level in RA synovium was significantly higher in the group of patients with longstanding RA than in the patients with early RA (8.8% v 0.6%, p=0.001), while the number of macrophages and FLIP expression were higher in the group with early disease than in the group with longstanding RA (16.2% v 8.3%, p=0.02 and 31.1% v 0.2%, p=0.001 respectively). All three markers correlated significantly with disease duration (R=-0.7, p<0.001 for FLIP, R=0.6, p=0.001 for apoptosis, and R=-0.5, p<0.05 for CD68). Cytokine expression and T cell score were not significantly different in early RA from longstanding RA. No differences were seen between patients treated or not treated with corticosteroids or between patients treated or not treated with disease modifying antirheumatic drugs. CONCLUSIONS: The findings suggest that RA synovial macrophages are resistant to apoptosis in early RA and express high levels of FLIP. During natural or drug modified disease progression the apoptotic mechanism may be restored with a specific increase of synovial apoptosis in patients with longstanding arthritis.

Anti-tumour necrosis factor (TNF)-alpha therapy (etanercept) down-regulates serum matrix metalloproteinase (MMP)-3 and MMP-1 in rheumatoid arthritis.

OBJECTIVES: Matrix metalloproteinases (MMPs) are cytokine-modulated enzymes that play an important role in the pathogenesis of rheumatoid arthritis (RA) by inducing bone resorption and cartilage destruction. This study evaluated the modulation of serum and synovial MMPs and their inhibitor, tissue inhibitor of matrix metalloproteinases (TIMP)-1, by therapy with soluble tumour necrosis factor (TNF) alpha receptor (etanercept). METHODS: Serum samples were collected from 60 RA patients at baseline and after 8 or 12 weeks of treatment. Paired synovial biopsies were obtained from 11 patients at two time points, before and after 8 weeks of treatment. We measured serum levels of MMP-1, MMP-3 and TIMP-1 by ELISA. Immunohistological analysis of synovial tissue was performed using monoclonal antibodies specific for MMP-1, MMP-3 and TIMP-1. RESULTS: Etanercept therapy significantly down-regulated serum levels of MMP-3 and MMP-1 in parallel with the reduction in inflammatory parameters (C-reactive protein concentration and erythrocyte sedimentation rate) in RA patients. Baseline pretreatment serum levels of MMP-3 correlated with changes in clinical disease activity during therapy. No consistent changes in serum level of TIMP-1 were observed, while ratios of MMP-1 and MMP-3 to TIMP-1 were down-regulated following etanercept treatment. Immunohistochemical analyses revealed great interindividual variability, with generally a high level of expression of MMP and low expression of TIMP. No significant change in the pattern or number of positive cells occurred during therapy. CONCLUSIONS: In RA patients, etanercept therapy down-regulates serum levels of MMP-3 and MMP-1 and the ratio between MMPs and TIMP-1. This may be an important mechanism for the prevention of future development of joint damage.