Rheumatoid Arthritis: Mageed RA

Mageed RA, Isenberg DA. · Department of Immunology and Molecular Pathology, Royal Free and University College London, The Windeyer Institute, London, UK. · Lupus. · Pubmed #12529050 No free full text.

Abstract: Tumour necrosis factor (TNFalpha) is a cytokine with a wide range of diverse and at times paradoxical effects. These include immunoregulatory, lymphoid organogenesis and pro-inflammatory effects. In recent years, TNFalpha has become a focus of interest more for its inflammatory effects in a number of chronic autoimmune diseases. This interest culminated in the successful treatment of patients with rheumatoid arthritis, Crohn's diseases and ankylosing spondylitis with blocking antibodies or soluble TNFalpha receptors. Paradoxically, however, TNFalpha also has immunomodulatory effects in some autoimmune conditions such as lupus in some mouse models of the disease and in diabetes in the none-obese diabetic mouse. The role TNFalpha plays in human systemic lupus erythematosus is, however, controversial. In this article we review some of the studies carried out to elucidate the effects of TNFalpha in lupus disease and likely mechanisms of action. Further, we discuss recent data on the likely effects of blocking TNFalpha on anti-DNA autoantibody production.

Chernajovsky Y, Dreja H, Daly G, Annenkov A, Gould D, Adams G, Croxford JL, Baker D, Podhajcer OL, Mageed RA. · Bone and Joint Research Unit, St. Bartholomew's and Royal London School of Medicine and Dentistry, Queen Mary and Westfield College, Charterhouse Square, London EC1M 6BQ, UK. · Genes Immun. · Pubmed #11196690 links to  free full text

Abstract: Animal models of autoimmune disease have been developed that mimic some aspects of the pathophysiology of human disease. These models have increased our understanding of possible mechanisms of pathogenesis at the molecular and cellular level and have been important in the testing, development and validation of new immunotherapies. The susceptibility to develop disease in the majority of these models is polygenic as is the case in humans. The exceptions to this rule are gene knock outs and transgenic models of particular genes which, in particular genetic backgrounds, have also contributed to the understanding of single gene function and their possible contribution to pathogenesis. Gene therapy approaches that target immune functions are being developed with encouraging results, despite the polygenic nature of these diseases. Basically this novel immuno-genetic therapy harnesses the knowledge of immunology with the myriad of biotechnological breakthroughs in vector design and delivery. Autoimmune disease is the result of genetic dysregulation which could be controlled by gene therapy. Here we summarize the genetic basis of these human diseases as well as some of the best characterized murine models. We discuss the strategies for their treatment using immuno- and gene therapy.

Rochas C, Hillion S, Saraux A, Mageed RA, Youinou P, Jamin C, Devauchelle V. · Université Européenne de Bretagne, Université de Brest, IFR 148 ScInBioS, and Laboratory of Immunology, Centre Hospitalier Universitaire, Brest Hôpital Morvan and Cavale Blanche, Brest, France. · Arthritis Rheum. · Pubmed #19404965 No free full text.

Abstract: OBJECTIVE: B cells that accumulate in the synovial tissue of rheumatoid arthritis (RA) patients revise their receptors due to coordinate expression of recombination-activating gene 1 (RAG-1) and RAG-2 genes. The aim of this study was to determine the mechanisms that control this re-expression. METHODS: B cells from healthy control subjects were cocultured with fibroblast-like synoviocytes (FLS) from patients with RA and osteoarthritis (OA). Re-expression of RAG messenger RNA (mRNA) and proteins was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and indirect immunofluorescence. Activity of RAG enzymes was evaluated by flow cytometry to measure variations in immunoglobulin kappa and lambda light chain expression and by ligation-mediated-PCR to assess specific DNA breaks. Blocking antibodies, short hairpin RNA, and recombinant cytokine were used to identify the molecules involved in RAG re-expression. RESULTS: RA FLS, but not OA FLS, induced B cells to re-express RAG mRNA and proteins. Enzymes were functional, since the kappa-to-lambda ratios decreased and specific DNA breaks were detectable after coculture with RA FLS. Transmembrane BAFF provided the first signal of RAG re-expression, since its down-regulation in RA FLS prevented RAG gene transcription in B cells. The failure of transmembrane BAFF from OA FLS to induce RAG suggests that a second signal was provided by RA FLS. Interleukin-6 (IL-6) is a candidate, since blockade of its receptors precluded transcription of RAG genes by RA FLS. Unless supplemented with IL-6, OA FLS were unable to induce RAG gene expression in normal B cells. CONCLUSION: Two independent signals are required for the induction of RAG gene expression in B cells that infiltrate the synovium of patients with RA.

Habib HM, Taher TE, Isenberg DA, Mageed RA. · Centre for Rheumatology, University College London School of Medicine, London, UK. · Scand J Rheumatol. · Pubmed #19110660 No free full text.

Abstract: OBJECTIVE: To determine the effect of inflammation through exposure to tumour necrosis factor (TNF)alpha on T lymphocytes in patients with systemic lupus erythematosus (SLE). METHODS: We studied the effect of TNFalpha on T-lymphocyte apoptosis in patients with SLE, rheumatoid arthritis (RA), and in healthy controls. Apoptosis of CD4 and CD8 T lymphocytes and naive and memory subpopulations was determined by flow cytometry using 7-amino-actinomycin D (7AAD) and propidium iodide (PI). In SLE, apoptosis was studied in patients with active and inactive disease and in patients on different medications. RESULTS: TNFalpha enhanced apoptosis of anti-CD3-activated T lymphocytes. The percentage of apoptotic cells was significantly higher in T lymphocytes from patients with SLE than RA patients and healthy controls. After 3 days of culture, 38% of CD4+ and 37% of CD8+ cells from SLE patients underwent apoptosis in the presence of TNFalpha compared with 25% CD4+ and 26% CD8+ T cells from the controls (p<0.001). In healthy controls, more memory than naive T lymphocytes underwent apoptosis. By contrast, in patients with SLE, more naive T cells underwent apoptosis with TNFalpha (p<0.01). Enhanced apoptosis of T cells in SLE was independent of disease activity or medication. Finally, inhibition experiments showed that apoptosis in the presence of TNFalpha was only partly blocked with anti-Fas ligand (FasL) antibody. CONCLUSIONS: This study demonstrates that T lymphocytes in patients with SLE are more prone to apoptosis in the presence of TNFalpha than T lymphocytes from healthy controls. Defects in TNFalpha signalling pathways rather than distribution of TNF receptors (TNFRs) probably explain the enhanced apoptosis in SLE.

Flores-Borja F, Kabouridis PS, Jury EC, Isenberg DA, Mageed RA. · William Harvey Institute, Queen Mary School of Medicine and Dentistry, London, UK. · Arthritis Rheum. · Pubmed #16320343 links to  free full text

Abstract: OBJECTIVE: B lymphocytes from patients with systemic lupus erythematosus (SLE) are hyperactive and produce anti-double-stranded DNA (anti-dsDNA) autoantibodies. The cause or causes of B cell defects in SLE are unknown. In this study, we determined the level and subcellular distribution of Lyn protein, a key negative regulator of B cell receptor signaling, and assessed whether altered Lyn expression is characteristic of B cells in the setting of SLE. METHODS: Negative selection was used to isolate B lymphocytes from blood. Lipid raft signaling domains were purified from B cells obtained from 62 patients with SLE, 15 patients with rheumatoid arthritis, and 31 healthy controls, by gradient ultracentrifugation. The total Lyn protein level was determined by Western blotting, confocal microscopy, and fluorescein-activated cell sorting (FACS). The distribution of Lyn into lipid raft and nonlipid raft domains was determined by Western blotting and confocal microscopy. Lyn content in B cell subpopulations was determined by FACS. In order to assess B lymphocyte activity, we used (3)H-thymidine incorporation and enzyme-linked immunosorbent assay to measure spontaneous proliferation and IgG and cytokine production by B cells. RESULTS: This study revealed that B lymphocytes from a majority of patients with SLE have a reduced level of Lyn and manifest altered translocation to lipid rafts. An investigation into the mechanisms of Lyn reduction suggested that increased ubiquitination is involved. This was evident from increased ubiquitination of Lyn and translocation of c-Cbl into lipid rafts. Studies of B cell responses showed that altered Lyn expression was associated with heightened spontaneous proliferation, anti-dsDNA autoantibodies, and increased interleukin-10 production. CONCLUSION: This study provides evidence for altered Lyn expression in B cells from a majority of patients with SLE. Altered Lyn expression in SLE may influence the B cell receptor signaling and B cell hyperactivity that are characteristic of the disease.

Jury EC, Kabouridis PS, Abba A, Mageed RA, Isenberg DA. · Royal Free and University College Medical School, University College, London, UK. · Arthritis Rheum. · Pubmed #12746907 links to  free full text

Abstract: OBJECTIVE: To explore regulation of proximal signaling and composition of lipid rafts in T lymphocytes from patients with systemic lupus erythematosus (SLE). METHODS: The expression, phosphorylation, and degradation of lipid raft-associated signaling molecules in T lymphocytes from 50 patients with SLE compared with 28 healthy controls and 22 rheumatoid arthritis patients were investigated. Lipid raft and nonraft fractions from T cells were isolated by ultracentrifugation. Proteins in the lipid raft and nonraft fractions were analyzed by Western blotting and probed for phosphotyrosine activity and for LCK, LAT, and CD3 epsilon. Immunoprecipitation experiments were performed to assess protein ubiquitination in T cell lysates. T cell phenotype and levels of intracellular LCK were determined by flow cytometry. RESULTS: LCK, an essential signaling molecule for T cell activation, was significantly reduced in both lipid raft and nonraft fractions of T lymphocytes from patients with active SLE compared with controls, and the reduction was independent of treatment. To identify the likely causes of reduced LCK, we explored the possibility that chronic activation of T lymphocytes underlies LCK degradation. The results revealed an increase in protein ubiquitination, and specifically LCK ubiquitination, in T cells from SLE patients. However, our findings suggest that the increase in ubiquitination is independent of T cell activation. CONCLUSION: LCK is reduced in T cell lipid rafts from patients with SLE. This reduction appears to be independent of activation and may be associated with abnormal ubiquitin-mediated regulation mechanisms.

Vencovský J, Zd'árský E, Moyes SP, Hajeer A, Ruzicková S, Cimburek Z, Ollier WE, Maini RN, Mageed RA. · Laboratory of Gene Expression, Charles University, Prague, Czech Republic. · Rheumatology (Oxford). · Pubmed #11961170 links to  free full text

Abstract: OBJECTIVE: To investigate the contribution of polymorphism in the immunoglobulin heavy chain variable region V1-69 gene set to genetic susceptibility to rheumatoid arthritis (RA) in Czech and British patients. METHODS: We used V1-69 gene sequence-specific polymerase chain reaction (PCR) and restriction enzyme digestion to study polymorphism in the V1-69 gene set in germline DNA of 109 Czech and 159 British RA patients and 164 ethnically matched controls. Polymorphism was further studied by nucleotide sequencing of the V1-69 gene locus in germline DNA. RESULTS: We found that all patients and controls had at least one V1-69 gene copy. In the Czech RA cohort, the dimorphic nucleotide in codon 73 of V1-69 (GAA or AAA) was present in the homozygous form 73(A/A) in 31 of 109 (28.4%) RA patients vs 12 of 79 (15.2%) controls [odds ratio (OR)=2.22, P<0.001]. When the RA patients and controls were classified according to HLA shared epitope (SE) status, 73(A/A) was found in 18 of 76 (23.7%) SE(+) patients compared with 13 of 38 (34.2%) SE(-) patients, four of 12 (18.2) SE(+) controls and eight of 57 (14%) SE(-) controls. This suggests that homozygosity for the dimorphic sequence 73(A) contributed to susceptibility to RA in SE(-) Czech individuals (OR=3.2, P<0.001). The most striking observation was that none of the 38 SE(-) Czech patients, compared with 11 of 76 (14.5%) SE(+) RA patients, three of 22 (13.6%) SE(+) and 11 of 57 (19.3%) SE(-) ethnically matched controls, were homozygous for the alternative dimorphic sequence 73(G/G) (OR=9.1, P<0.05). These data, however, were not replicated in a Caucasoid British RA population. CONCLUSION: The dimorphic sequence at codon 73 (73(A/A)) of the V1-69 gene contributes to genetic susceptibility in SE(-) Czech RA patients.

Vencovský J, Jarosová K, Růzicková S, Nemcová D, Niederlová J, Ozen S, Alikasifoglu M, Bakkaloglu A, Ollier WE, Mageed RA. · Institute of Rheumatology, Prague, Czech Republic. · Arthritis Rheum. · Pubmed #11665981 No free full text.

Abstract: OBJECTIVE: An increased incidence of allele 2 of the interleukin-1 receptor antagonist gene (IL1RN*2) in several inflammatory diseases has recently been reported. The aim of this study was to examine a variable number tandem repeat (VNTR) polymorphism of the IL1RN gene in patients with juvenile idiopathic arthritis (JIA). METHODS: Findings in 185 Czech patients with JIA were compared with those in 168 Czech controls, 50 JIA patients and 52 controls of Turkish origin, and 79 controls from central England. VNTR polymorphism analysis of IL1RN was performed by polymerase chain reaction using 2 flanking primers to amplify an 86-bp tandem repeat region in intron 2. RESULTS: The frequency and carriage rate of IL1RN*2 were significantly increased in Czech JIA patients compared with the Czech controls (frequency 27.6% versus 15.8%; carriage rate 44.3% versus 26.2%). Increased frequency and carriage rate of IL1RN*2 were found in 23.3% and 40.0% of Turkish JIA patients and in 17.3% and 34.6% of ethnically matched controls. The high representation of IL1RN*2 in 52.3% of the 22 patients with extended oligoarthritis, 31.3% of the 56 patients with enthesitis-related arthritis, and 42.9% of the 14 patients with other arthritis was particularly responsible for the increased frequency of IL1RN*2 in the Czech JIA patients. We found no association of IL1RN*2 with disease activity or severity parameters. CONCLUSION: Inheritance of IL1RN*2 may contribute to genetic susceptibility in several forms of autoimmune diseases, including JIA. The IL1RN*2 allele may be useful as a prognostic indicator of the evolution of an extended oligoarticular course of JIA.

Axford JS, Rees DH, Mageed RA, Wordsworth P, Alavi A, Steere AC. · Academic Unit for Musculoskeletal Disease, St George's Hospital Medical School, London. · Ann Rheum Dis. · Pubmed #10577962 links to  free full text

Abstract: OBJECTIVE: To investigate whether autoreactive mechanisms occur in Lyme disease (LD) by determining IgA, IgG and IgM rheumatoid factor (RF) concentrations and RF associated cross reactive idiotype (CRI) expression in the serum of LD patients, with comparison to patients with rheumatoid arthritis (RA). METHODS: The RF isotype profiles were determined in 59 patients with LD; erythema migrans (EM) (n=19), neuroborreliosis (NB) (n=20) and Lyme arthritis (LA) (n=20). Mouse monoclonal antibodies (mAbs) G6 and G8 (V(H)1 gene associated), D12 (V(H)3 gene associated) and C7 (V(kappa)III gene associated) were then used to determine the RF associated CRI expression on IgM antibodies in 16 of these LD patients (eight seropositive for RF); (EM (n=3), NB (n=6), LA (n=7)). RESULTS: Seven (18%) patients with either NB or LA had increased concentrations of IgA RF compared with none with EM. Significant differences in the number of patients with raised concentrations of IgG RF or IgM RF were not found between the LD patient groups. Five (3NB, 1LA and 1 EM) (31%) and three (2NB and 1LA) (19%) of LD patients had raised concentrations of the CRIs recognised by mAbs G6 and G8, respectively. These CRIs were detected in LD sera both with and without raised concentrations of RF and were not demonstrated on anti-Borrelia burgdorferi antibodies using ELISA. No LD sera tested had raised concentrations of the determinants recognised by mAbs C7 or D12. CONCLUSION: Significantly raised concentrations of IgA RF and increased use of V(H)1 germline gene associated CRIs are found on IgM antibodies in the serum of LD patients. These data indicate the recruitment of autoreactive B lymphocytes in some patients with the later stages of LD.

Williams DG, Moyes SP, Mageed RA. · MRC Collaborative Centre, Mill Hill, London, UK. · Immunology. · Pubmed #10469243 links to  free full text

Abstract: The presence of clonally-related B-lymphocyte aggregates within synovial lining tisue of rheumatoid arthritis (RA) patients suggests a germinal centre-like reaction, which may hold implications for disease pathogenesis and the causes of chronic inflammation. We studied 250 rheumatoid factor (RF) heavy-chain sequences cloned from the synovium of three patients with RA, to determine whether they undergo both somatic mutation and isotype switching consistent with this hypothesis. Size analysis of immunoglobulin heavy-chain cDNAs from synovial RF+ B cells revealed oligoclonal RF+ populations and identically-sized VH-D-JH transcripts of different immunoglobulin isotypes. Sequencing of individual inserts selected from cloned immunoglobulin heavy-chain cDNAs demonstrated a clonal relationship between immunoglobulin M (IgM) RF and IgA RF, suggesting that this isotype switch occurred in synovium. Furthermore, most somatic mutations were found to have occurred after this isotype switch. This finding suggests that the RA synovial microenvironment sustains somatic mutation and isotype switching in RF-specific B lymphocytes akin to secondary lymphoid organs.

Mason AN, Harmer IJ, Mageed RA, Mackworth-Young CG. · Kennedy Institute of Rheumatology, London. · Lupus. · Pubmed #10342717 No free full text.

Abstract: BACKGROUND: A human IgM monoclonal anticardiolipin antibody - BH1 - has previously been described, which has characteristics typical of antiphospholipid antibodies in the serum of patients with antiphospholipid syndrome (APS). It appears to be idiotypically distinct from other human monoclonal autoantibodies of different or overlapping ligand-binding specificities derived from patients with related conditions. AIM: To determine whether the idiotype of BH1 is expressed on particular populations of antibodies (antiphospholipid and anti-beta2-glucoprotein I) in the serum of patients with APS and other conditions. METHODS: Sera from patients with APS (9), systemic lupus erythematosus without APS ('uncomplicated SLE' -9), and rheumatoid arthritis (RA 15), and from normal controls (15) were tested by enzyme-linked immunosorbent assay (ELISA) for reactivity with cardiolipin, beta2 glycoprotein I (beta2GPI), and a polyclonal anti-idiotype raised against BH1 (RIdBH1). Absorption experiments were subsequently performed on selected sera using micelles of cardiolipin or phosphatidyl choline. RESULTS: Eight out of nine patients with APS were positive for binding to RIdBH1 (IgG and/or IgM), while only one patient with uncomplicated SLE and none of the patients with RA or the healthy controls were positive. Although all of the patients with APS were positive for binding to beta2GPI, there was poor correlation between these results and levels of binding to cardiolipin and RIdBH1. Absorption of sera from patients with APS by cardolipin micelles resulted in a median reduction in IgG anticardiolipin and anti-beta2GPI activity of 81.6% and 6.3% respectively. For those sera positive for IgG reactivity with RIdBH1 the median reduction in this activity was 79.4%. Antibodies eluted from selected micelles showed activity against cardiolipin, beta2GPI and RIdBH1. Three anticardiolipin-positive sera from patients with RA were similarly absorbed; however the eluted antibodies failed to bind to RIdBH1. Absorption of all these sera with phosphatidyl choline resulted in no significant reduction in any of these activities. CONCLUSIONS: The BH1 idiotype defines a population of serum antibodies associated with features of APS. The antibody response in this condition, though diverse, may include the expression of a restricted group of variable region genes.