Rizgar A. Mageed

IL-10 gene promoter polymorphisms in rheumatoid arthritis

IL-10 is an anti-inflammatory cytokine which may modulate disease expression in RA. Three dimorphic polymorphisms within the IL-10 gene promoter have recently been identified and appear to influence regulation of its expression. The 1082*A allele has been associated with low and the 1082*G allele with high in vitro IL-10 production. We have analysed 117 unrelated Caucasoid RA patients and 119 ethnically matched controls. No significant differences in the allele frequencies of the three polymorphisms were found between controls and RA patients. In contrast, a significant association between the 1082*A allele and the (-1082*A/-819*C/-592*C) haplotype and IgA RF+ve/IgG RF-ve patients was observed. The association of genotypes encoding low IL-10 production with IgA RF in RA is incompatible with its suggested role in antibody isotype switching. IgA RF has been associated with severe RA and may thus be indirectly correlated with a genotype encoding low IL-10 production.

IL-6 Modulates CD5 Expression in B Cells from Patients with Lupus by Regulating DNA Methylation

B lymphocytes from patients with systemic lupus erythematosus (SLE) are characterized by reduced expression levels of membrane CD5. Recent studies from our laboratory have revealed that the level of membrane CD5 is determined by the relative level of two alternative CD5 isoforms; CD5-E1A, which is expressed on the membrane, and CD5-E1B, which is retained in the cytoplasm. Using bisulfite sequencing and methylation-sensitive endonuclease assays we show that the promoter for the alternative CD5-E1B isoform is demethylated in B cells from patients with SLE but not in healthy controls. We go on to show that differential methylation is more pronounced following BCR engagement. As a result of this demethylation, CD5-E1B mRNA is transcribed at the expense of CD5-E1A mRNA transcription. We provide further evidence that production of high IL-6 levels by SLE B cells abrogates the ability of SLE B cells to induce DNA methyl transferase (DNMT1) and then to methylate DNA, an effect that is reversed in the presence of a blocking Ab to the IL-6 receptor. The pattern of demethylation of CpG islands in the CD5-E1B promoter in SLE B cells is similar to those in B cells from healthy controls stimulated in the presence of IL-6, or treated with the methylation inhibitor PD98059. The study reveals that engagement of the BCR with constitutive IL-6 down-regulates the level of membrane CD5, which negatively regulates BCR signaling, in SLE B cells. This altered signaling could, in turn, promote the activation and expansion of autoreactive B cells in SLE patients.

IVIg modulates BCR signaling through CD22 and promotes apoptosis in mature human B lymphocytes

Among various mechanisms for interactions with B cells, intravenous immunoglobulin (IVIg) may operate through the insertion of its Fc part into the Fc-γ receptor, or the binding of its sialic acid (SA)-bearing glycans to the negatively regulating CD22 lectin. It appeared that IVIg reduces B lymphocyte viability in a dose- and time-dependent manner. Furthermore, we show by confocal microscopy that SA-positive IgG, but not SA-negative IgG bind to CD22. This interaction reduces the strength of B-cell receptor-mediated signaling trough down-regulating tyrosine phosphorylation of Lyn and the B-cell linker proteins, and up-regulating phospholipase Cγ2 activation. This cascade resulted in a sustained activation of Erk 1/2 and arrest of the cell cycle at the G(1) phase. These changes may be accounted for the efficacy of IVIg in autoimmune diseases.

Incomplete response of inflammatory arthritis to TNFα blockade is associated with the Th17 pathway

Objectives To establish if changes in Th1/Th17 cell populations previously reported in experimental arthritis occur in patients with rheumatoid arthritis (RA) treated with anti-tumour necrosis factor α (TNFα) agents, and whether the therapeutic response to anti-TNFα is compromised in patients and mice because of elevated Th17/IL-17 levels. Finally, to assess the efficacy of combined blockade of anti-TNFα and anti-IL-17 in experimental arthritis. Methods A longitudinal study of two independent cohorts (cohort 1, n=24; cohort 2, n=19) of patients with RA treated with anti-TNFα biological agents was carried out to assess their Th17/IL-17 levels before and after the start of anti-TNFα therapy. IL-12/23p40 production was assessed in plasma Peripheral blood lymphocytes (PBLs) and monocytes. Mice with collagen-induced arthritis (CIA) were treated with anti-TNFα alone, anti-IL17 alone or a combination of the two. Efficacy of treatment and response was assessed from changes in Disease Activity Score 28–erythrocyte sedimentation rate scores in patients, and in clinical scores and histological analysis in CIA. Results Significant increases in circulating Th17 cells were observed in patients after anti-TNFα therapy and this was accompanied by increased production of IL-12/23p40. There was an inverse relationship between baseline Th17 levels and the subsequent response of patients with RA to anti-TNFα therapy. In addition, PBLs from non-responder patients showed evidence of increased IL-17 production. Similarly, in anti-TNFα-treated mice, there was a strong correlation between IL-17 production and clinical score. Finally combined blockade of TNFα and IL-17 in CIA was more effective than monotherapy, particularly with respect to the duration of the therapeutic effect. Conclusions These findings, which need to be confirmed in a larger cohort, suggest that a Th17-targeted therapeutic approach may be useful for anti-TNFα non-responder patients or as an adjunct to anti-TNFα therapy, provided that safety concerns can be addressed.

Prevention of collagen-induced arthritis by gene delivery of soluble p75 tumour necrosis factor receptor.

Collagen type II-induced arthritis (CIA) in DBA/1 mice can be passively transferred to SCID mice with spleen B- and T-lymphocytes. In the present study, we show that infection ex vivo of splenocytes from arthritic DBA/1 mice with a retroviral vector, containing cDNA for the soluble form of human p75 receptor of tumour necrosis factor (TNF-R) before transfer, prevents the development of arthritis, bone erosion and joint inflammation in the SCID recipients. Assessment of IgG subclass levels and studies of synovial histology suggest that down-regulating the effector functions of T helper-type 1 (Th1) cells may, at least in part, explain the inhibition of arthritis in the SCID recipients. In contrast, the transfer of splenocytes infected with mouse TNF-α gene construct resulted in exacerbated arthritis and enhancement of IgG2a antibody levels. Intriguingly, infection of splenocytes from arthritic DBA/1 mice with a construct for mouse IL-10 had no modulating effect on the transfer of arthritis. The data suggest that manipulation of the immune system with cytokines, or cytokine inhibitors using gene transfer protocols can be an effective approach to ameliorate arthritis.

Recent Progress in the Understanding of B-Cell Functions in Autoimmunity

Our early concepts of the normal role of B cells in immunity focused on their ability to produce antibodies (Ab) and in the case of autoimmune diseases autoAbs, some of which were pathogenic. Over the past 10 years, it has became apparent that B cells display a variety of characteristics, other than Ab production, which could contribute to autoimmunity. They normally play a role in the development of lymphoid architecture, regulating T‐cell subsets and dendritic cell (DC) function through cytokine production, and in activation of T cells. Receptors editing is also important in B cells which aids in immunity to infection and, possibly, prevention of autoimmunity. Transgenic animal models have now shown that B cells are necessary for many autoimmune diseases although their Ab products are not required in some cases. Negative signalling by CD5 and other molecules, such as CD22, in maintaining tolerance through recruitment of src‐homology two domain‐containing protein tyrosine phosphatase‐1 has also been documented. In fact, we have now reached a new era whereby the B cell has returned as an important contributor to autoimmune disorders, so that the race is on to characterize signalling regulation via the B‐cell receptor and coreceptors. Identification of such molecules and their potential defects should lead to effective ways of controlling the immune response and in particular preventing the development of autoimmune states. The classical view of B cells in the biology of immune responses to infectious and self‐antigens (Ag) that they promote immunity primarily by producing Ab turns out to be rather naïve. Indeed, studies over the last few years indicate that this view is far from complete, and suggest that B lymphocytes have extraordinarily diverse functions within the immune system. Furthermore, it is becoming increasingly clear that the pathogenesis of autoimmune diseases cannot solely be accounted for by T cells, and intrinsic abnormalities of B cells have been described in such conditions. In this brief review we highlight some recent observations in the context of B lymphocyte in pathophysiology, and focus on their revival as pivotal players the pathophysiology in autoimmune diseases. Yet, it remains difficult to provide a model of how important B cells are in immunity and autoimmunity.

Immunopathology and the gene therapy of lupus

Lupus is a chronic autoimmune inflammatory disease with complex clinical manifestations. In humans, lupus, also known as systemic lupus erythematosus (SLE), affects between 40 and 250 individuals, mostly females, in each 100 000 of the population. There are also a number of murine models of lupus widely used in studies of the genetics, immunopathology, and treatment of lupus. Human patients and murine models of lupus manifest a wide range of immunological abnormalities. The most pervasive of these are: (1) the ability to produce pathogenic autoantibodies; (2) lack of T- and B-lymphocyte regulation; and (3) defective clearance of autoantigens and immune complexes. This article briefly reviews immunological abnormalities and disease mechanisms characteristic of lupus autoimmunity and highlight recent studies on the use of gene therapy to target these abnormalities.

CD5 Promotes IL-10 Production in Chronic Lymphocytic Leukemia B Cells through STAT3 and NFAT2 Activation

B lymphocytes from chronic lymphocytic leukemia (CLL) display some CD5 transcripts for CD5 containing the known exon 1 (E1A) and other CD5 transcripts containing the new exon 1 (E1B). These malignant B cells, as well as B cell lines transfected with cDNA for E1A-cd5 or with cDNA for E1B-cd5 produce IL-10, raising the possibility that CD5 participates in the secretion of IL-10. We identified transcription factors involved in this production in CD5+ B lymphocytes from CLL patients and in E1A-cd5–transfected or E1B-cd5–transfected Jok cells. STAT3 is activated via phosphorylation of serine 727 but also NFAT2 through its translocation into the nucleus. Chromatin immunoprecipitation experiments confirmed the role of STAT3 and allowed the discovery of a role for NFAT2 in IL-10 production. Both transcription factors bind not only to the enhancer of the Il-10 gene but also to the promoter of the Il-5 and Il-13 genes. Furthermore, transfection of B cell lines with E1A-cd5 or E1B-cd5 established that activation of STAT3 and NFAT2 is regulated by CD5. The same holds true for the production of IL-10, IL-5, and IL-13 and the expression of the receptors for these cytokines. This interpretation was confirmed by two experiments. In the first, downregulation of CD5 by small interfering RNAs lowered the production of IL-10. In the second experiment, transfection of the GFP-NFAT2 gene into B lymphocytes induced nuclear translocation of NFAT2 in CD5+ but not in CD5− B cells. Thus, CD5 expression is associated with NFAT2 activity (and mildly STAT3 activity), indicating that CD5 controls IL-10 secretion.

B Lymphocyte Cytokines and Rheumatic Autoimmune Disease

There is some debate as to which type of lym-phocyte prevails in the pathogenesis of autoimmunity.This controversy is borne out by the fact that T cellshave, for years, been viewed as competent in their ownright to initiate rheumatoid arthritis (RA), primarySjo¨gren’s syndrome (SS), and systemic lupus erythema-tosus (SLE), while B cells were confined to a subservientrole as producers of antibodies to self antigens. Doubtshave, however, been cast when T cell–directed treat-ments proved to be disappointing.B cells have attracted significant interest follow-ing new information on the key roles they play in RA,SS, and SLE. Encouraged by the promising results ofsmall-scale clinical trials of the anti-CD20 antibodyrituximab in patients with RA (1), several groups haveembarked on similar treatment of patients with RA, SS,or SLE (2). The clinical benefits were found to becorrelated with a decline in B cell numbers, but not witha reduction in the levels of most autoantibodies, exceptfor rheumatoid factor and anti–cyclic citrullinated pep-tide (3). This finding implies that B cell depletion mayameliorate these disorders through mechanisms otherthan suppressing autoantibody production. Given the Bcell dependence of T cell activation in the rheumatoidjoint, it makes sense that heavy infiltration of B cells intothe synovium accompanies a modest response to ritux-imab, that patients in whom depletion of circulating Bcells is incomplete have worse outcomes than those inwhom it is complete (4), and that removal of B cellssignificantly reduces the severity of experimental arthri-tis in mice (5).Furthermore, consistent with this view are theresults of experiments in SLE-prone mice geneticallyengineered to be deficient in B cells. While mice with noB lymphocytes do not suffer from end-organ damage,those with B cells incapable of antibody secretion retainmany features of the syndrome (6). Such observationsindicate that B cells promote autoimmunity by interact-ing with any one of several pathways that impact theregulation of immunity to the self. Although a role of Blymphocytes in SLE, RA, and SS has been suspected formany years, recent discoveries have unveiled new in-sights into B cell–derived cytokines, includinginterferon- (IFN ) and interleukin-4 (IL-4), that mod-ulate the response. They are likely to serve as effectorsof some biologic functions of B cells.Early studies of the functions of B lymphocytesfocused on splitting the B cell population into subpopu-lations. For example, B lymphocytes are classified basedon the expression of CD5 as B-1 cells, which express thisT cell marker, and B-2 cells, which do not. The ensuingmodel of the maturation of B lymphocytes asserts thatB-2 cells leave the bone marrow as immature B cells,populate secondary lymphoid organs as transitional type1 (T1) cells, and evolve into type 2 (T2) cells, whichdifferentiate either into marginal zone or follicular Bcells. Upon antigen encounter, these latter B cellsinitiate the development of a germinal center (GC).

Higher frequency of allele 2 of the interleukin-1 receptor antagonist gene in patients with juvenile idiopathic arthritis.

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.