Theresa Barnes

'The Many Faces of Interleukin-6: The Role of IL-6 in Inflammation, Vasculopathy, and Fibrosis in Systemic Sclerosis'

Interleukin-6 is currently attracting significant interest as a potential therapeutic target in systemic sclerosis (SSc). In this paper, the biology of interleukin-6 is reviewed, and the evidence for interleukin-6 dysregulation in SSc is explored. The role of inteleukin-6 classical and trans signalling pathways in SSc relevant phenomena such as chronic inflammation, autoimmunity, endothelial cell dysfunction, and fibrogenesis is discussed. The existing evidence that interventions designed to block interleukin-6 signalling are of therapeutic relevance in SSc is evaluated.

Neutrophil apoptosis in rheumatoid arthritis is regulated by local oxygen tensions within joints

Neutrophils are normally short‐lived cells and die by apoptosis, but when recruited into tissues, their apoptosis is delayed, and they survive for much longer time periods. In inflammatory diseases, such as rheumatoid arthritis (RA), this delayed apoptosis may lead to increased tissue damage and a failure of the inflammation to resolve. However, there are conflicting reports in the literature as to whether neutrophil apoptosis is delayed or accelerated in rheumatoid joints. In this report, we show that neutrophils isolated from the ynovial fluid (SF) of patients with RA show accelerated rates of apoptosis when incubated ex vivo and that SF, despite containing a variety of antiapoptotic cytokines, is proapoptotic. Paradoxically, levels of the key neutrophil survival protein Mcl‐1 are elevated in freshly isolated SF neutrophils compared with matched peripheral blood samples from the same patients, indicating that delayed neutrophil apoptosis has been signaled in vivo as the cells enter the joints. However, when SF was added to neutrophils and incubated under hypoxia (1% O2), conditions known to exist in vivo within joints, the SF was antiapoptotic. These data reveal that the rheumatoid synovial joint contains a complex mixture of pro‐ and antiapoptotic factors and that the low, local oxygen tensions that exist within these joints can exert profound effects on neutrophil survival. These experiments also highlight the importance of performing in vitro experiments under laboratory conditions that closely mimic those that occur in vivo; otherwise, misleading conclusions may be drawn.

Dynamic contrast enhanced MRI of bone marrow oedema in rheumatoid arthritis.

Aims: The aim of this work was to assess the feasibility of using dynamic contrast enhanced (DCE) MRI of bone marrow oedema, to compare it with conventional marrow oedema scoring systems, and to determine the effects of anti-tumour necrosis factor (TNF)α therapy. Methods: The wrist and metacarpophalangeal (MCP) joints of 25 patients with rheumatoid arthritis were studied. A total of 14 were imaged before and 1–2 weeks after anti-TNFα therapy. T2-weighted fat-suppressed images were collected. A dynamic series of 24 3D spoiled gradient-echo images were acquired before, during and after the intravenous administration of gadolinium-based contrast medium. Oedema was scored using the conventional Rheumatoid Arthritis MRI Scoring (RAMRIS) system from T2-weighted images. The relative enhancement rate (RER) was calculated using the dynamic series from oedematous bone, bone adjacent to oedema and from an uninvolved bone. Results: A total of 56% of patients showed bone marrow oedema. The RER was significantly increased in and adjacent to areas of marrow oedema. There was a significant reduction in the RER after treatment, but not in the RAMRIS score. Conclusions: Dynamic contrast enhanced MRI of bone marrow oedema yields additional information to RAMRIS scoring and may be a more sensitive marker of inflammatory activity and response to treatment.

Pharmacokinetic modeling of dynamic contrast-enhanced MRI of the hand and wrist in rheumatoid arthritis and the response to anti-tumor necrosis factor-α therapy

Dynamic contrast‐enhanced MRI (DCE‐MRI) of the hand and wrist was performed in 11 patients with rheumatoid arthritis twice before and once 2 weeks after treatment with anti–tumor necrosis factor (TNF)‐α therapy. A rapid, T1‐weighted 3D spoiled gradient echo (SPGR) sequence was used for the dynamic imaging. T1 estimation was performed using similar images obtained at different flip angles. The relative radiofrequency field was estimated from the known T1 of the periarticular fatty marrow. The arterial input function (AIF) was measured at each examination, and normalized to the expected plasma concentration to reduce partial volume effects. Synovial enhancement was modeled to yield values for Ktrans, ve, and vp. Ktrans and ve showed good reproducibility. There was a significant decrease of about 20% in Ktrans after 2 weeks of treatment. This study demonstrates the potential of DCE‐MRI and pharmacokinetic modeling to study early changes in inflammatory activity in rheumatoid arthritis following treatment. Magn Reson Med 58:482–489, 2007.

Endothelial activation and apoptosis mediated by neutrophil-dependent interleukin 6 trans-signalling: a novel target for systemic sclerosis?

Objectives Systemic sclerosis (SSc) is a connective tissue disease associated with significant morbidity and mortality and generally inadequate treatment. Endothelial cell activation and apoptosis are thought to be pivotal in the pathogenesis of this disease, but the mechanisms that mediate this remain unknown. Methods Human dermal microvascular endothelial cells were cultured with healthy control neutrophils in the presence of 25% healthy control or SSc serum for 24 h. Apoptosis was measured by annexin V-FITC binding and endothelial cell activation was measured using an allophycocyanin-conjugated E-selectin antibody. Fluorescence was quantified and localised using confocal microscopy. Results SSc serum resulted in significantly increased apoptosis (p=0.006) and E-selectin expression (p=0.00004) in endothelial cells compared with control serum, effects that were critically dependent on the presence of neutrophils. Recombinant interleukin 6 (IL-6) reproduced these findings. Immunodepletion of IL-6 and the use of an IL-6 neutralising antibody decreased the effect of SSc serum on E-selectin expression. Soluble gp130, which specifically blocks IL-6 trans-signalling, negated the effect of SSc serum on both E-selectin expression and apoptosis. Conclusions SSc serum induces endothelial cell activation and apoptosis in endothelial cell-neutrophil co-cultures, mediated largely by IL-6 and dependent on the presence of neutrophils. Together with other pathologically relevant effects of IL-6, these data justify further exploration of IL-6 as a therapeutic target in SSc.

Neutrophil-derived reactive oxygen species in SSc

OBJECTIVE Reactive oxygen species (ROS) are implicated in the pathogenesis of SSc. Neutrophils constitute a major source of ROS during inflammation. Here, we examined endogenous and stimulated ex vivo ROS production of SSc neutrophils compared with control neutrophils with and without prior priming with TNF-α. METHODS ROS generation was measured using luminol-enhanced chemiluminescence. Neutrophils isolated from SSc patients and healthy controls were unprimed or were primed with TNF-α. ROS production was stimulated in vitro with phorbol 12-myristate 13-acetate (PMA) and formyl-met-leu-phe (fMLP). To examine the effects of serum mediators on ROS generation, control neutrophils were also stimulated with SSc or control serum. RESULTS Neutrophil stimulation with PMA and fMLP resulted in a greater increase in ROS generation in SSc neutrophils compared with controls. However, unstimulated SSc neutrophils generated lower levels of ROS than controls. SSc neutrophils demonstrated an increased response to fMLP in the absence of in vitro TNF-α priming indicating priming of SSc neutrophils in vivo. SSc serum did not stimulate neutrophil ROS generation in vitro. CONCLUSION SSc neutrophils are primed for ROS generation. Neutrophils binding to activated endothelium in SSc, may induce local production of ROS, perpetuating endothelial dysfunction and mediating fibrosis.

Relative α1-anti-trypsin deficiency in systemic sclerosis

Objective. Neutrophil elastase is secreted by neutrophils during activation and circulates in the plasma where it can play a role in inflammation and fibrosis. This study examines the role of neutrophil elastase in SSc, a systemic CTD that is typified by vascular dysfunction, tissue fibrosis and inflammation. Methods. Serum neutrophil elastase and α1-anti-trypsin concentrations were assessed in SSc patients and healthy controls by ELISA. Serum neutrophil elastase activity was assessed by the elastase-dependent conversion of methoxy-succinyl-alanyl-alanyl-prolyl-valyl-p-nitroanilide to p-nitroanilide using a colourimetric assay. Elastase concentration and activity were correlated with clinical disease features. Results. Serum neutrophil elastase concentration and activity were equivalent in patients and controls; however, in SSc serum, there was an increase in elastase activity for each unit of elastase concentration (P = 0.03). This was due to a decrease in serum α1-anti-trypsin concentrations (P = 0.04). Serum elastase concentration (P = 0.03) and activity (P = 0.02) were significantly lower in RNP-positive patients and serum elastase concentrations were lower in ANA-positive patients (P = 0.003). Conclusions. Relative deficiency in serum α1-anti-trypsin concentrations in SSc could have important and pathogenically relevant effects since elastase has pro-inflammatory and pro-fibrotic roles. Elastase inhibitors are available in clinical practice and could represent potential therapeutic options in SSc.

Targeting oncostatin M in the treatment of rheumatoid arthritis

Oncostatin M (OSM) is a pleiotropic cytokine with potential utility as a treatment for inflammatory arthritis. This pro-inflammatory cytokine is increased in the rheumatoid but not in the osteoarthritic joint. Strategies to block the actions of OSM for use in inflammatory arthritis are being developed and these show significant promise in murine models of disease. Targeting OSM may have a beneficial effect by inhibiting some of the mechanisms of joint destruction in rheumatoid arthritis, which may limit long-term disability in patients. The challenge now is to convert this potential into firm compounds that can be tested in the clinic.

Therapeutic targeting of cytokines

Over recent years, with many advances in understanding the pathological processes that underlie many inflammatory conditions, it has become clear that targeting cytokines has proven to be highly effective therapeutically. The second International Cytokine symposium, held recently in Manchester UK, has provided an important forum for bringing basic and clinical scientists together to discuss many aspects of cytokine medicine from the laboratory to the clinic. In this article, we provide an overview of the main issues raised from this important conference and set the scene for more detailed reports that follow in subsequent articles in this journal.

Second International Conference on Cytokine Medicines

The Second International Conference on Cytokine Medicine was held in Manchester, UK and was organised by an international consortium comprising leading clinicians and researchers from healthcare organisations, universities and industry, all with cognate interests in this field. The conference attracted a diverse mix of delegates including clinicians from a wide range of specialities, biomedical scientists, and a barrister with particular expertise in the legal aspects of care provision in healthcare systems. This meeting uniquely focused on cytokine therapies across the spectrum of medical problems, from bench to bedside, including emerging applications for cytokine therapies, issues raised by the delivery of cytokine medicines in clinical practice, and future developments in this broad and exciting area.