Evaluation of the role of cytokines in autoimmune disease: The importance of TNFα in rheumatoid arthritis

Cytokines and growth factors are involved in all important biological processes. Hence it is anticipated that they will be of importance in autoimmune disease. The pathogenesis of autoimmune diseases involves a number of stages, initiation, perpetuation and tissue damage, each of which involves different cell and molecular interactions. In this review, we will discuss an outline of the cytokine involvement in the various stages of autoimmune development, prior to focusing on the analysis of cytokines in rheumatoid arthritis. Cytokines exert their effect via high affinity cell surface receptors. Thus an understanding of cytokines involves the analysis of receptor expression, and also of cytokine inhibitors. Currently there is only adequate knowledge of these aspects in rheumatoid arthritis (RA), and as such the emphasis of this review is on RA. One of the major reasons for being interested in the role of cytokines in autoimmunity is to define possible therapeutic targets. There is now considerable evidence that TNFα is such a target in RA, and the effect of anti TNFα monoclonal antibody therapy in RA is discussed.     

TNFα expressed on the surface of microparticles modulates endothelial cell fate in rheumatoid arthritis

Background

Rheumatoid arthritis (RA) is associated with a high prevalence of atherosclerosis. Recently increased levels of microparticles (MPs) have been reported in patients with RA. MPs could represent a link between autoimmunity and endothelial dysfunction by expressing tumor necrosis factor alpha (TNFα), a key cytokine involved in the pathogenesis of RA, altering endothelial apoptosis and autophagy. The aim of this study was to investigate TNFα expression on MPs and its relationship with endothelial cell fate.

Methods

MPs were purified from peripheral blood from 20 healthy controls (HC) and from 20 patients with RA, before (time (T)0) and after (T4) 4-month treatment with etanercept (ETA). Surface expression of TNFα was performed by flow cytometry analysis. EA.hy926 cells, an immortalized endothelial cell line, were treated with RA-MPs purified at T0 and at T4 and also, with RA-MPs in vitro treated with ETA. Apoptosis and autophagy were then evaluated.

Results

RA-MPs purified at T0 expressed TNFα on their surface and this expression significantly decreased at T4. Moreover, at T0 RA-MPs, significantly increased both apoptosis and autophagy levels on endothelial cells, in a dose-dependent manner. RA-MPs did not significantly change these parameters after 4 months of in vivo treatment with ETA.

Conclusions

Our data demonstrate that MPs isolated from patients with RA exert a pathological effect on endothelial cells by TNFα expressed on their surface. In vivo and in vitro treatment with ETA modulates this effect, suggesting anti-TNF therapy protects against endothelial damage in patients with RA.

     

TNFα modulates protein degradation pathways in rheumatoid arthritis synovial fibroblasts

Introduction

Rheumatoid arthritis (RA) is a chronic inflammatory and destructive disease of the joint. The synovial lining consists of two main types of cells: synovial fibroblasts and macrophages. The macrophage-derived cytokine TNFα stimulates RA synovial fibroblasts to proliferate and produce growth factors, chemokines, proteinases and adhesion molecules, making them key players in the RA disease process. If proteins are not correctly folded, cellular stress occurs that can be relieved in part by increased degradation of the aberrant proteins by the proteasome or autophagy. We hypothesized that the activity of the protein degradation pathways would be increased in response to TNFα stimulation in RA synovial fibroblasts compared with control fibroblasts.

Methods

Endoplasmic reticulum (ER) stress markers were examined in synovial fibroblasts by immunoblotting and PCR. Use of the autophagy and proteasome protein degradation pathways in response to TNFα stimulation was determined using a combination of experiments involving chemical inhibition of the autophagy or proteasome pathways followed by immunoblotting for the autophagy marker LC3, measurement of proteasome activity and long-lived protein degradation, and determination of cellular viability.

Results

RA synovial fibroblasts are under acute ER stress, and the stress is increased in the presence of TNFα. Autophagy is the main pathway used to relieve the ER stress in unstimulated fibroblasts, and both autophagy and the proteasome are more active in RA synovial fibroblasts compared with control fibroblasts. In response to TNFα, the autophagy pathway but not the proteasome is consistently stimulated, yet there is an increased dependence on the proteasome for cell viability. If autophagy is blocked in the presence of TNFα, an increase in proteasome activity occurs in RA synovial fibroblasts but not in control cells.

Conclusions

TNFα stimulation of synovial fibroblasts results in increased expression of ER stress markers. Survival of synovial fibroblasts is dependent on continuous removal of proteins by both the lysosome/autophagy and ubiquitin/proteasome protein degradation pathways. Both pathways are more active in RA synovial fibroblasts compared with control fibroblasts. These results may provide a better understanding of the mechanism of TNFα on prolonging the survival of synovial fibroblasts in RA tissue.     

Adalimumab regulates intracellular TNFα production in patients with rheumatoid arthritis

Introduction

Adalimumab is a fully human anti–tumor necrosis factor α (anti-TNFα) monoclonal antibody that specifically blocks the interaction of TNFα with its receptors. It binds both soluble and transmembrane TNFα. We hypothesized that blocking these TNFα signals regulates the altered TNFα production in rheumatoid arthritis (RA) patients.

Methods

We compared, by flow cytometry, Toll-like receptor induction levels of membrane and intracellular TNFα in monocytes (iTNFα + CD14+ cells) from 12 patients before and after adalimumab treatment with those from 5 healthy donors.

Results

Before starting the treatment, the percentage of iTNFα+ CD14+ cells in the RA patients was significantly lower than that in healthy donors (mean ± SEM = 33.16 ± 4.82% vs 66.51 ± 2.4%, P < 0.001). When we added in vitro TNFα to healthy donor culture cells, levels of iTNFα+ CD14+ cells decreased, suggesting that the TNFα signal was responsible for the iTNFα+ CD14+ cell downregulation observed in the RA patients. After 2, 6 and 12 adalimumab injections, we observed significant blocking of membrane and soluble TNFα and a progressive increase in iTNFα+ CD14+ cells in ten patients with a good to moderate response as defined by the European League Against Rheumatism (EULAR) criteria. Levels of iTNFα+ CD14+ cells after 12 injections in these 10 patients were comparable to levels in healthy donors. In two patients, iTNFα+ CD14+ cell upregulation was not observed, and their EULAR-defined responses had not improved. The first patient developed antiadalimumab antibodies, explaining why adalimumab was not able to block membrane and soluble TNFα. In the second patient, adalimumab was discontinued because of adverse effects, which led to a decrease in iTNFα+ CD14+ cells to levels measured before treatment.

Conclusions

Our findings suggest that adalimumab treatment in RA patients can return iTNFα levels to those of healthy donors. This effect was not observed in the presence of neutralizing antiadalimumab antibodies.     

Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients

ObjectiveTo investigate the expression of TNFα membrane-bound receptors: the percentage of cells expressing these receptors and the number of molecules expressed on different immune cell subsets, and to evaluate serum concentrations of soluble TNFα and its receptors (sTNFRI and sTNFRII) in patients with rheumatoid arthritis in acute stage and after response to treatment compared to healthy donors.MethodsThe objects of the study are peripheral blood mononuclear cells (PBMC) of healthy donors (n = 150) and RA patients (n = 40) subjected to hospital treatment with either biological agents (Rituximab) or glucocorticosteroids (methylprednisolone). To determine PBMC phenotype antibodies anti-hCD3-APC, anti-hCD19 PECy7, anti-hCD14 FITC (eBioscience), as well as anti-hTNFRI-PE and anti-hTNFRII-PE (R&D Systems) were used. To determine receptor number on the cells Quantibrite PE Beads (BD) were used.ResultsCells obtained from patients who responded to therapy and achieved disease remission exhibited either an increase in the percentage of TNFRI+ cells or elevated expression density of this receptor type.ConclusionSubsets of immunocompetent cells from RA patients show variation in the percentage of membrane-bound receptor positive cells and receptor expression density, which influences the development and progression of the pathological processes in RA. Response to therapy and achievement of disease remission are associated with an increase of TNFRI expression.     

Dipeptidyl peptidase IV activity and/or structure homologs: Contributing factors in the pathogenesis of rheumatoid arthritis?

Several of the proinflammatory peptides involved in rheumatoid arthritis pathogenesis, including peptides induced downstream of tumor necrosis factor-α as well as the monocyte/T cell-attracting chemokines RANTES and stromal cell-derived factor (SDF)-1α and the neuropeptides vasoactive intestinal peptide (VIP) and substance P, have their biological half-lives controlled by dipeptidyl peptidase IV (DPPIV). Proteolysis by DPPIV regulates not only the half-life but also receptor preference and downstream signaling. In this article, we examine the role of DPPIV homologs, including CD26, the canonical DPPIV, and their substrates in the pathogenesis of rheumatoid arthritis. The differing specific activities of the DPPIV family members and their differential inhibitor response provide new insights into therapeutic design.     

Modulating co-stimulation: a rational strategy in the treatment of rheumatoid arthritis?

Rheumatoid arthritis (RA) is a common destructive inflammatory disease that affects 0.5-1% of the population in many countries. Even though several new treatments have been introduced for patients with RA, a considerable proportion of patients do not benefit from these, and the need for alternative treatment strategies is clear. This review explores the potential for a therapy targeting the adaptive immune system by modulating co-stimulation of T cells with a CTLA4-Ig fusion protein (abatacept).     

Chemokine blockade: a new era in the treatment of rheumatoid arthritis?

Blockade of chemokines or chemokine receptors is emerging as a new potential treatment for various immune-mediated conditions. This review focuses on the therapeutic potential in rheumatoid arthritis, based on studies in animal models and patients. Several knockout models as well as in vivo use of chemokine antagonists are discussed. Review of these data suggests that this approach might lead to novel therapeutic strategies in rheumatoid arthritis and other chronic inflammatory disorders.     

Does the order of second-line treatment in rheumatoid arthritis matter?

In a prospective study 88 patients, with rheumatoid arthritis who had stopped taking gold, penicillamine, or levamisole were randomly allocated to one of the alternative drugs and followed up for a minimum of one year. Concurrent studies of the effects of gold, penicillamine, and levamisole prescribed in 123 patients as the first second-line drug were used for comparison. No difference in toxicity or efficacy between primary and secondary use of gold or penicillamine was identified. Variation in the toxicity of levamisole could in part be accounted for by changes in the dose regimen over the four years of study. The length of the treatment-free interval between drugs did not influence subsequent development of toxicity. These results suggest that an adverse reaction to one of the three second-line drugs studied should not prejudice the selection of another.     

CD154: the atherosclerotic risk factor in rheumatoid arthritis?

Atherosclerosis, now regarded as a chronic inflammatory disease of the arterial wall, and its clinical manifestations have increasingly been associated with rheumatoid arthritis (RA), supporting the notion that autoimmune diseases and vascular disorders share common etiological features. Indeed, evidence pertaining to this matter indicates that inflammation and its multiple components are the driving force behind the pathogenesis of these disorders. Interestingly, CD154 and its receptors have emerged as major players in the development of RA and atherosclerosis, which raises the possibility that this axis may represent an important biological link between both complications. Indeed, CD154 signaling elicits critical inflammatory responses that are common to the pathogenesis of both diseases. Here, we provide an overview of the traditional and disease-related interrelations between RA and vascular abnormalities, while focusing on CD154 as a potential mediator in the development of atherosclerotic events in RA patients.     

What is the future of CCR5 antagonists in rheumatoid arthritis?

A clinical trial of lupus patients with nephritis was established to determine any possible role for Atacicept, a biologic drug that blocks two B-cell-activating factors (BLyS and APRIL). The trial was stopped after just six patients had been enrolled because three patients developed serious infections. Initial concerns that the biologic was the main cause of the increased susceptibility to these infections have had to be revised on close inspection of the data. The evidence clearly points to a previously unrecognised capacity for mycophenolate to cause notable drops in immunoglobulin levels as the prime suspect.     

High synovial expression of the inhibitory FcγRIIb in rheumatoid arthritis

Activating Fc gamma receptors (FcγRs) have been identified as having important roles in the inflammatory joint reaction in rheumatoid arthritis (RA) and murine models of arthritis. However, the role of the inhibitory FcγRIIb in the regulation of the synovial inflammation in RA is less known. Here we have investigated synovial tissue from RA patients using a novel monoclonal antibody (GB3) specific for the FcγRIIb isoform. FcγRIIb was abundantly expressed in synovia of RA patients, in sharp contrast to the absence or weak staining of FcγRIIb in synovial biopsies from healthy volunteers. In addition, the expression of FcγRI, FcγRII and FcγRIII was analyzed in synovia obtained from early and late stages of RA. Compared with healthy synovia, which expressed FcγRII, FcγRIII but not FcγRI, all activating FcγRs were expressed and significantly up-regulated in RA, regardless of disease duration. Macrophages were one of the major cell types in the RA synovium expressing FcγRIIb and the activating FcγRs. Anti-inflammatory treatment with glucocorticoids reduced FcγR expression in arthritic joints, particularly that of FcγRI. This study demonstrates for the first time that RA patients do not fail to up-regulate FcγRIIb upon synovial inflammation, but suggests that the balance between expression of the inhibitory FcγRIIb and activating FcγRs may be in favour of the latter throughout the disease course. Anti-inflammatory drugs that target activating FcγRs may represent valuable therapeutics in this disease.     

Dendritic cells: the driving force behind autoimmunity in rheumatoid arthritis?

Dendritic cells (DC) are likely to play a significant role in immune-mediated diseases such as autoimmunity and allergy. To date there are few treatments capable of inducing permanent remission in rheumatoid arthritis (RA) and elucidation of the role of DC may provide specific strategies for disease intervention. Dendritic cells have proven to be powerful tools for immunotherapy and investigations are under way to determine their clinical efficacy in transplantation and viral and tumour immunotherapy. The present review will focus on the current view of DC and their role in autoimmunity, in particular RA. Two possible roles for DC in the pathogenesis of RA will be proposed, based on recent advances in the field.