The C1q binding activity of IgG is modified in vitro by reactive oxygen species: implications for rheumatoid arthritis

IgG can be denatured in vitro by reactive oxygen species (ROS). Native IgG activates the complement cascade through C1q. Using a modified ELISA, C1q binding activity of rheumatoid IgG has been compared to IgG denatured by neutrophil-derived ROS. The C1q binding activity of rheumatoid synovial fluid IgG is greater than the corresponding serum IgG (P < 0.01). Denaturation of IgG by activated polymorphs or the Fenton reaction decreased its C1q binding activity (P < 0.01). In vitro exposure of IgG to OH. and ROO. increased its interaction with C1q (P < 0.01). Hypochlorous acid had no effect. ROS-induced alteration to IgG-C1q binding activity may promote the inflammatory response in rheumatoid arthritis.

ROS as signalling molecules in T cells - Evidence for abnormal redox signalling in the autoimmune disease, rheumatoid arthritis

Reactive oxygen species are recognised as important signalling molecules within cells of the immune system. This is, at least in part, due to the reversible activation of kinases, phosphatases and transcription factors by modification of critical thiol residues. However, in the chronic inflammatory disease rheumatoid arthritis, cells of the immune system are exposed to increased levels of oxidative stress and the T cell becomes refractory to growth and death stimuli. This contributes to the perpetuation of the immune response. As many of the effective therapies used in the treatment of rheumatoid arthritis modulate intracellular redox state, this raises the question of whether increased oxidative stress is causative of T-cell hyporesponsiveness. To address this hypothesis, this review considers the putative sources of ROS involved in normal intracellular signalling in T cells and the evidence in support of abnormal ROS fluxes contributing to T-cell hyporesponsiveness. © W. S. Maney & Son Ltd.

Aberrant reactive oxygen and nitrogen species generation in rheumatoid arthritis (RA):causes and consequences for immune function, cell survival, and therapeutic intervention

The infiltration and persistence of hematopoietic immune cells within the rheumatoid arthritis (RA) joint results in elevated levels of pro-inflammatory cytokines, increased reactive oxygen (ROS) and -nitrogen (RNS) species generation, that feeds a continuous self-perpetuating cycle of inflammation and destruction. Meanwhile, the controlled production of ROS is required for signaling within the normal physiological reaction to perceived "foreign matter" and for effective apoptosis. This review focuses on the signaling pathways responsible for the induction of the normal immune response and the contribution of ROS to this process. Evidence for defects in the ability of immune cells in RA to regulate the generation of ROS and the consequence for their immune function and for RA progression is considered. As the hypercellularity of the rheumatoid joint and the associated persistence of hematopoietic cells within the rheumatoid joint are symptomatic of unresponsiveness to apoptotic stimuli, the role of apoptotic signaling proteins (specifically Bcl-2 family members and the tumor suppressor p53) as regulators of ROS generation and apoptosis are considered, evaluating evidence for their aberrant expression and function in RA. We postulate that ROS generation is required for effective therapeutic intervention.

Proteinase activity in rheumatoid arthritis

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY WITH PRIOR ARRANGEMENT

Interleukin-6 subfamily cytokines and rheumatoid arthritis:role of antagonists

Many cytokines have been implicated in the inflammatory pathways that characterize rheumatoid arthritis (RA) and related inflammatory diseases of the joints. These include members of the interleukin-6 (IL-6) family of cytokines, several of which have been detected in excess in the synovial fluid from RA patients. What makes the IL-6 group of cytokines a family is their common use of the glycoprotein 130 (gp130) receptor subunit, to which they bind with different affinities. Several strategies have been developed to block the pro-inflammatory activities of IL-6 subfamily cytokines. These include the application of monoclonal antibodies, the creation of mutant form(s) of the cytokine with enhanced binding affinity to gp130 receptor and the generation of antagonists by selective mutagenesis of the specific cytokine/gp130 receptor-binding site(s). The rationale for the use of anti-cytokine therapy in inflammatory joint diseases is based on evidence from studies in vitro and in vivo, which implicate major cytokines such as interleukin-1 (IL-1), tumour necrosis factor (TNF)-alpha and IL-6 in RA pathogenesis. In particular, IL-6 subfamily antagonists have a wide range of potential therapeutic and research applications. This review focuses on the role of some of the IL-6 subfamily cytokines in the pathogenesis of the inflammatory diseases of the joints (IJDs), such as RA. In addition, an overview of the recently developed antagonists will be discussed.

Statin use in rheumatoid arthritis in relation to actual cardiovascular risk:evidence for substantial undertreatment of lipid-associated cardiovascular risk?

Background Cardiovascular disease (CVD) is partially attributed to traditional cardiovascular risk factors, which can be identified and managed based on risk stratification algorithms (Framingham Risk Score, National Cholesterol Education Program, Systematic Cardiovascular Risk Evaluation and Reynolds Risk Score). We aimed to (a) identify the proportion of at risk patients with rheumatoid arthritis (RA) requiring statin therapy identified by conventional risk calculators, and (b) assess whether patients at risk were receiving statins. Methods Patients at high CVD risk (excluding patients with established CVD or diabetes) were identified from a cohort of 400 well characterised patients with RA, by applying risk calculators with or without a ×1.5 multiplier in specific patient subgroups. Actual statin use versus numbers eligible for statins was also calculated. Results The percentage of patients identified as being at risk ranged significantly depending on the method, from 1.6% (for 20% threshold global CVD risk) to 15.5% (for CVD and cerebrovascular morbidity and mortality) to 21.8% (for 10% global CVD risk) and 25.9% (for 5% CVD mortality), with the majority of them (58.1% to 94.8%) not receiving statins. The application of a 1.5 multiplier identified 17% to 78% more at risk patients. Conclusions Depending on the risk stratification method, 2% to 26% of patients with RA without CVD have sufficiently high risk to require statin therapy, yet most of them remain untreated. To address this issue, we would recommend annual systematic screening using the nationally applicable risk calculator, combined with regular audit of whether treatment targets have been achieved.

Lack of association between glucocorticoid use and presence of the metabolic syndrome in patients with rheumatoid arthritis:a cross-sectional study

Introduction - Rheumatoid arthritis (RA) associates with excessive cardiovascular morbidity and mortality, attributed to both traditional and novel cardiovascular risk factors. The metabolic syndrome, a cluster of classical cardiovascular risk factors, including hypertension, obesity, glucose intolerance, and dyslipidaemia, is highly prevalent in RA. Reports suggest that long-term glucocorticoid (GC) use may exacerbate individual cardiovascular risk factors, but there have been no studies in RA to assess whether it associates with the metabolic syndrome. We examined whether GC exposure associates with the presence of metabolic syndrome in patients with RA. Methods - RA patients (n = 398) with detailed clinical and laboratory assessments were categorised into three groups according to GC exposure: no/limited (<3 months) exposure (NE), low-dose (<7.5 mg/day) long-term exposure (LE), and medium-dose (greater than or equal to 7.5 mg to 30 mg/day) long-term exposure (ME). The metabolic syndrome was defined using the National Cholesterol Education Programme III guidelines. The association of GC exposure with the metabolic syndrome was evaluated using binary logistic regression. Results - The metabolic syndrome was present in 40.1% of this population and its prevalence did not differ significantly between the GC exposure groups (NE 37.9% versus LE 40.7% versus ME 50%, P = 0.241). Binary logistic regression did not demonstrate any increased odds for the metabolic syndrome when comparing ME with LE (odds ratio = 1.64, 95% confidence interval 0.92 to 2.92, P = 0.094) and remained non significant after adjusting for multiple potential confounders. Conclusions - Long-term GC exposure does not appear to associate with a higher prevalence of the metabolic syndrome in patients with RA. The components of the metabolic syndrome may already be extensively modified by other processes in RA (including chronic inflammation and treatments other than GCs), leaving little scope for additive effects of GCs.

Are lipid ratios less susceptible to change with systemic inflammation than individual lipid components in patients with rheumatoid arthritis?

Rheumatoid arthritis (RA) associates with excess cardiovascular risk and there is a need to assess that risk. However, individual lipid levels may be influenced by disease activity and drug use, whereas lipid ratios may be more robust. A cross-sectional cohort of 400 consecutive patients was used to establish factors that influenced individual lipid levels and lipid ratios in RA, using multiple regression models. A further longitudinal cohort of 550 patients with RA was used to confirm these findings, using generalized estimating equations. Cross-sectionally, higher C-reactive protein (CRP) levels correlated with lower levels of total cholesterol (TC), low-density lipoprotein-cholesterol (LDL-C), and high-density lipoprotein-cholesterol ([HDL-C] P = .015), whereas lipid ratios did not correlate with CRP. The findings were broadly replicated in the longitudinal data. In summary, the effects of inflammation on individual lipid levels may underestimate lipid-associated cardiovascular disease (CVD) risk in RA, thus lipid ratios may be more appropriate for CVD risk stratification in RA.

The effectiveness of leflunomide etanercept and infliximab in the treatment of rheumatoid arthritis:a systematic review

DUE TO COPYRIGHT RESTRICTIONS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY AND INFORMATION SERVICES WITH PRIOR ARRANGEMENT

Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease?

Biomolecules are susceptible to many different post-translational modifications that have important effects on their function and stability, including glycosylation, glycation, phosphorylation and oxidation chemistries. Specific conversion of aspartic acid to its isoaspartyl derivative or arginine to citrulline leads to autoantibody production in models of rheumatoid disease, and ensuing autoantibodies cross-react with native antigens. Autoimmune conditions associate with increased activation of immune effector cells and production of free radical species via NADPH oxidases and nitric oxide synthases. Generation of neo-antigenic determinants by reactive oxygen and nitrogen species ROS and RNS) may contribute to epitope spreading in autoimmunity. The oxidation of amino acids by peroxynitrite, hypochlorous acid and other reactive oxygen species (ROS) increases the antigenicity of DNA, LDL and IgG, generating ligands for which autoantibodies show higher avidity. This review focuses on the evidence for ROS and RNS in promoting the autoimmune responses observed in diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It considers the evidence for ROS/RNS-induced antigenicity arising as a consequence of failure to remove or repair ROS/RNS damaged biomolecules and suggests that an associated defect, probably in T cell signal processing or/or antigen presentation, is required for the development of disease.

Oxygen radical induced alterations in polyclonal IgG

In the sera and synovial fluid of patients with rheumatoid arthritis, part of the IgG fraction is found in an aggregated and fluorescent form. Oxygen-free radicals have been implicated in this denaturation, although the precise radical species responsible is unknown. In this work, oxygen-free radicals generated radiolytically were allowed to attack polyclonal IgG in solution. OH radicals induced aggregation of the monomer and a new fluorescence appeared in the visible region (Ex 360 nm, Em 454 nm). The superoxide radical anion was found to be inert in both these respects, whilst peroxy radicals induced autofluorescence without concomitant aggregation. The results suggest that OH.and/or peroxy radical attack may be an in vivo mechanism for IgG denaturation.

Free radicals and redox signalling in T-cells during chronic inflammation and ageing

During chronic inflammation and ageing, the increase in oxidative stress in both intracellular and extracellular compartments is likely to influence local cell functions. Redox changes alter the T-cell proteome in a quantitative and qualitative manner, and post-translational modifications to surface and cytoplasmic proteins by increased reactive species can influence T-cell function. Previously, we have shown that RA (rheumatoid arthritis) T-cells exhibit reduced ROS (reactive oxygen species) production in response to extracellular stimulation compared with age-matched controls, and basal ROS levels [measured as DCF (2',7'-dichlorofluorescein) fluorescence] are lower in RA T-cells. In contrast, exposing T-cells in vitro to different extracellular redox environments modulates intracellular signalling and enhances cytokine secretion. Together, these data suggest that a complex relationship exists between intra- and extra-cellular redox compartments which contribute to the T-cell phenotype.

The targeting of corticosteroids to inflamed tissues

In recent years, much interest has focused on the beneficial effects of administering potentially harmful therapeutic agents in drug carriers so as to reduce their toxic side effects. Rheumatoid arthritis is a chronic systemic disease with progressive destruction of the Joints and long term patient disability, Corticosteroids have been shown to retard the progression of Joint destruction but are limited in their use due to adverse side effects,This project, following the line of investigation started by other workers, was designed to study the use of microspheres to deliver corticosteroids to inflamed tissues by both the oral and intravenous routes. Hydrocortisone (HC)-loaded albumin microspheres were prepared by three different methods, by direct incorporation of HC within the particles, by indirect incorporation of HC by the enzymatic conversion of hydrocortisone-21-phosphate (H-21-P) to HC within the particles, and by the adsorption of HC onto the surface. HC was also loaded with PLA microspheres. The level of corticosteriod loading and in vitro release from microspheres was determined by HPLC analysis. A reversed-phase, ion-pairing HPLC method was developed to simultaneously measure both HC and H-21-P. The highest level of corticosteroid loading was achieved using the incorporation of H-21-P with enzymatic conversion to HC method. However, HPLC analysis showed only 5% of the incorporated steroid was HC. In vitro release rates of steroid from albumin microspheres showed >95% of incorporated steroid was released within 2 hours of dissolution. Increasing the protein:steroid ratio, and the temperature and duration of microsphere stabilization, had little effect on prolonging drug release. In vivo studies, using the carrageenan-induced rat hind-paw model of inflammation, indicated steroid-incorporated microspheres administered both orally and intraperitoneally were not therapeutically advantageous when compared to equivalent free steroid doses. The ability of orally and intravenously dosed [125I]~albumin microspheres (2.67 μm mean diameter) to accumulate in acutely and chronically inflamed tissues was investigated, The subcutaneous air-pouch was the model of inflammation used, with carrageenan as the inflammatory stimulus. Acute and chronic inflammation was shown to be consistently formed  in pouch tissues in terms of cell infiltration and fluid exudate formation in the pouch cavity. Albumin microspheres were shown to accumulate in the inflamed tissues and pouch fluids after both oral and intravenous administration. Preliminary, confirmatory studies using latex microspheres and quantitation by GPC analysis, also indicated microsphere accumulation in both acutely and chronically inflamed air-pouch tissues. tntl lUr"'poucbtis,sues; The results indicate the uptake and transfer of microspheres across the gastrointestinal tract into the circulation and their migration through disrupted endothelium and basement membranes at the inflamed sites. , .

Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.