Production Of Pathogenic Autoantibodies Research Articles

Follicular helper T cells are required for systemic autoimmunity

Production of high-affinity pathogenic autoantibodies appears to be central to the pathogenesis of lupus. Because normal high-affinity antibodies arise from germinal centers (GCs), aberrant selection of GC B cells, caused by either failure of negative selection or enhanced positive selection by follicular helper T (TFH) cells, is a plausible explanation for these autoantibodies. Mice homozygous for the san allele of Roquin, which encodes a RING-type ubiquitin ligase, develop GCs in the absence of foreign antigen, excessive TFH cell numbers, and features of lupus. We postulated a positive selection defect in GCs to account for autoantibodies. We first demonstrate that autoimmunity in Roquinsan/san (sanroque) mice is GC dependent: deletion of one allele of Bcl6 specifically reduces the number of GC cells, ameliorating pathology. We show that Roquinsan acts autonomously to cause accumulation of TFH cells. Introduction of a null allele of the signaling lymphocyte activation molecule family adaptor Sap into the sanroque background resulted in a substantial and selective reduction in sanroque TFH cells, and abrogated formation of GCs, autoantibody formation, and renal pathology. In contrast, adoptive transfer of sanroque TFH cells led to spontaneous GC formation. These findings identify TFH dysfunction within GCs and aberrant positive selection as a pathway to systemic autoimmunity.

Ectopic lymphoid structures support ongoing production of class-switched autoantibodies in rheumatoid synovium.

BackgroundFollicular structures resembling germinal centres (GCs) that are characterized by follicular dendritic cell (FDC) networks have long been recognized in chronically inflamed tissues in autoimmune diseases, including the synovium of rheumatoid arthritis (RA). However, it is debated whether these ectopic structures promote autoimmunity and chronic inflammation driving the production of pathogenic autoantibodies. Anti-citrullinated protein/peptide antibodies (ACPA) are highly specific markers of RA, predict a poor prognosis, and have been suggested to be pathogenic. Therefore, the main study objectives were to determine whether ectopic lymphoid structures in RA synovium: (i) express activation-induced cytidine deaminase (AID), the enzyme required for somatic hypermutation and class-switch recombination (CSR) of Ig genes; (ii) support ongoing CSR and ACPA production; and (iii) remain functional in a RA/severe combined immunodeficiency (SCID) chimera model devoid of new immune cell influx into the synovium.Methods and FindingsUsing immunohistochemistry (IHC) and quantitative Taqman real-time PCR (QT-PCR) in synovial tissue from 55 patients with RA, we demonstrated that FDC+ structures invariably expressed AID with a distribution resembling secondary lymphoid organs. Further, AID+/CD21+ follicular structures were surrounded by ACPA+/CD138+ plasma cells, as demonstrated by immune reactivity to citrullinated fibrinogen. Moreover, we identified a novel subset of synovial AID+/CD20+ B cells outside GCs resembling interfollicular large B cells. In order to gain direct functional evidence that AID+ structures support CSR and in situ manufacturing of class-switched ACPA, 34 SCID mice were transplanted with RA synovium and humanely killed at 4 wk for harvesting of transplants and sera. Persistent expression of AID and Iγ-Cμ circular transcripts (identifying ongoing IgM-IgG class-switching) was observed in synovial grafts expressing FDCs/CD21L. Furthermore, synovial mRNA levels of AID were closely associated with circulating human IgG ACPA in mouse sera. Finally, the survival and proliferation of functional B cell niches was associated with persistent overexpression of genes regulating ectopic lymphoneogenesis.ConclusionsOur demonstration that FDC+ follicular units invariably express AID and are surrounded by ACPA-producing plasma cells provides strong evidence that ectopic lymphoid structures in the RA synovium are functional and support autoantibody production. This concept is further confirmed by evidence of sustained AID expression, B cell proliferation, ongoing CSR, and production of human IgG ACPA from GC+ synovial tissue transplanted into SCID mice, independently of new B cell influx from the systemic circulation. These data identify AID as a potential therapeutic target in RA and suggest that survival of functional synovial B cell niches may profoundly influence chronic inflammation, autoimmunity, and response to B cell–depleting therapies.

The differential expressions of 78-kDa glucose-regulated protein of infiltrating plasma cells in peripheral joints with the histopathological variants of rheumatoid synovitis

IntroductionThe local production of pathogenic autoantibodies by plasma cells in synovium is one of the hallmarks of rheumatoid arthritis (RA). There may be a potential link between ectopic lymphoid neogenesis and the local autoimmunity in rheumatoid synovium. The unfolded protein response (UPR) has key roles in the development and maintenance of plasma cells secreting immunoglobulin. This study was designed to explore the potential links between the activation of the UPR of infiltrating plasma cells in inflamed peripheral joints and the histopathological variants of rheumatoid synovitis as well as the local production of pathogenic autoantibodies.MethodsThe variants of rheumatoid synovium were histopathologically classified into follicular and diffuse synovitis. Immunohistochemical and double-immunofluorescent stainings were performed to detect the expression of 78-kDa glucose-regulated protein (GRP78), a marker of activation of the UPR, in infiltrating plasma cells of synovium, and flow cytometry and immunoblotting analyses were performed to quantify GRP78 in plasma cells of synovial fluid in inflamed peripheral joints of RA. The detections were also taken in osteoarthritis (OA) as controls. The synovial fluid levels of anti-cyclic citrullinated peptide antibodies (anti-CCP) (IgG) were quantified with the enzyme-linked immunosorbent assay and corrected to those of total IgG in RA.ResultsExpressions of GRP78 were more intensive in infiltrating plasma cells in RA synovium relative to those in OA synovium (P < 0.001) and in synovium with follicular synovitis relative to that with diffuse synovitis (P < 0.001). Analyses by flow cytometry and immunoblotting showed that there was a significant upregulation of GRP78 of plasma cells from synovial fluid of RA compared with that of OA (P < 0.05) and from synovial fluid of follicular synovitis relative to that of diffuse synovitis (P < 0.05). Moreover, a positive relationship between the expression of GRP78 of plasma cells from synovial fluid and the corrected synovial levels of anti-CCP (IgG) was seen in RA (P < 0.001).ConclusionsThere may be a link between enhanced activation of the UPR of plasma cells and ectopic lymphoid neogenesis as well as the local production of anti-CCP (IgG) in inflamed peripheral joints of RA.

B-Cell Depletion Immunotherapy in Pemphigus: Effects on Cellular and Humoral Immune Responses

Pemphigus are B-cell-mediated autoimmune diseases affecting skin and mucous membranes. They are characterized by the production of pathogenic autoantibodies directed against desmogleins (Dsg). In this prospective study, we treated 21 pemphigus patients with rituximab and analyzed immunological modifications induced by anti-CD20 immunotherapy. The total depletion of peripheral B cells led to a significant decrease of total serum IgM but not IgG levels. The B-cell depletion was followed by a progressive re-emergence of naive blood B lymphocytes, with one-third of them expressing a transitional CD19+CD38(high)CD24(high) phenotype. In most patients, clinical response to rituximab was closely related to the evolution of anti-Dsg autoantibodies that decreased in patients who achieved complete remission, whereas they remained unchanged or reincreased in relapsing patients. In contrast, serum antimicrobial IgG remained stable after rituximab treatment. B-cell repertoire analysis of three patients using immunoscope showed distortions of VH-IgM and VH-IgG immunoscope profiles before treatment, particularly clonal and oligoclonal expansions in some VH families, which were not found after B-cell reconstitution, following anti-CD20 immunotherapy. The depletion of autoreactive B cells leading to the elimination of anti-Dsg autoantibodies in most remitted patients and the restoration of a diverse B-cell repertoire by naive B lymphocytes may provide an explanation for the long-lasting efficacy of rituximab in pemphigus patients.

Btk regulates localization, in vivo activation, and class switching of anti-DNA B cells

The autoimmune disease systemic lupus erythematosus (SLE) is characterized by loss of tolerance to nuclear antigens such as chromatin, DNA, and RNA. This focused autoreactivity is thought to arise from the ability of DNA or RNA specific B cells to receive dual signals from the BCR and TLR9 or TLR7, respectively. The Tec kinase Btk is necessary for the production of anti-DNA antibodies in several murine models of SLE. To assess the role of Btk in the fate of DNA reactive B cells, we generated Btk−/− mice carrying the 56R anti-DNA Ig transgene on the C57BL/6 background. dsDNA specific B cells were present in 56R.Btk−/− mice, although they were not preferentially localized to the marginal zone. These cells were able to proliferate in response to large CpG DNA containing fragments that require BCR-induced internalization to access TLR9. However, anti-DNA antibodies were not observed in the serum of 56R.Btk−/− mice. A transgene expressing a low level of Btk in B cells (Btk lo) restored anti-DNA IgM in these mice. This correlated with partial rescue of proliferative response to BCR engagement and TLR9-induced IL-10 secretion in Btk lo B cells. anti-DNA IgG was not observed in 56R.Btk lo mice, however. This was likely due, at least in part, to a role for Btk in controlling the expression of T-bet and AID in cells stimulated with CpG DNA. Thus, Btk is required for the initial loss of tolerance to DNA and the subsequent production of pathogenic autoantibodies once tolerance is breached.

Molecular mechanisms resulting in pathogenic anti-mouse erythrocyte antibodies in New Zealand black mice.

The New Zealand black (NZB) mouse strain is genetically predisposed to develop, at approximately 6 months of age, a spontaneous and severe autoimmune anaemia caused by production of pathogenic anti-mouse erythrocyte autoantibodies. In order to investigate the molecular mechanisms which lead to anti-mouse erythrocyte autoantibody production we have generated eight anti-mouse erythrocyte MoAbs producing hybridomas from splenocytes of 9- and 12-month-old NZB with spontaneous autoimmune anaemia. IgG2a was the predominant isotype, while IgM, IgG1 and IgG2b were each produced by one hybridoma cell line. All anti-mouse erythrocyte MoAbs were characterized for their antigen specificities. None of the MoAbs cross-reacted with ss- or dsDNA or with other species' erythrocytes, with the exception of one MoAb which cross-reacted with rat erythrocytes. None of the eight hybridomas was demonstrated to express surface or cytoplasmic CD5, suggesting that they derived from CD5- B lymphocytes. All hybridomas when implanted intraperitoneally into BALB/c mice caused anaemia. In order to define the genetic basis and investigate the molecular mechanisms resulting in pathogenic anti-mouse erythrocyte autoantibody production, the pattern of immunoglobulin variable region gene use has been studied. Five of the eight MoAbs whose IgVH genes were sequenced all have functionally rearranged genes from the VH J558 gene family. There is evidence for somatic point mutations in the complementarity-determining regions (CDR) of the IgVH genes in all of these five MoAbs when compared with the closest known germline gene. We suggest that these nucleotide sequence changes are likely to reflect selection by an antigen-driven mechanism. Furthermore, the data indicate that pathogenic anti-mouse erythrocytes are not derived from 'natural' autoantibodies.

IL‐17 Upregulates Regulator of G‐protein Signaling (Rgs)13 and Rgs16 for the Formation of Autoreactive Germinal Centers in BXD2 Mice

The lupus and arthritis prone BXD2 mice develop a large number of spontaneous germinal centers (GCs) in the spleens and dramatically elevated sera levels of autoantibodies. Sera from BXD2 mice exhibited significantly elevated IL‐17, but not IFN‐γ or IL‐4, compared to B6 mice. Under the optimal TH‐17 polarization condition, 9% and 20% of purified CD4 T cells from B6 and BXD2 mice were polarized into TH‐17 cells, respectively, in vitro. Supplementation of IL‐6 and IL‐23 was not required for the development of TH‐17 CD4+ T cells from the unfractionated spleen cells from the BXD2 mice but was required for their development from the spleens of B6 mice. Quantitative real‐time PCR analysis indicated that purified CD4 T cells and purified B cells from BXD2 mice express high levels of Il17 and Il17r, respectively. In situ confocal fluorescent microscopy showed that TH‐17 CD4 T cells and IL‐17R+ IgM+ B cells were localized preferentially in the PNA+ GC region in the spleens of the BXD2 mice. Treatment of BXD2 mice with anti‐IL‐17 neutralization antibody (250 μg/mouse q.3 days × 4) resulted in dissipation of B cells from CD4 T cells, decreased percentage of PNA+Fas+ GC B cells, and decreased number of B cells that produce IgG antibodies against histone, BiP, and DNA. IL‐17 upregulated the expression of Rgs13 and Rgs16 and suppressed the CXCL12‐induced migration response to both CD4 and B cells. Our results demonstrate a novel pathogenic mechanism of IL‐17 independently of its chronic inflammatory effects in autoimmune disease. IL‐17 induced spontaneous formation of GCs provided the optimal microenvironment for the production of pathogenic autoantibodies. This work is supported by Arthritis Foundation, VAMC Merit Review, ACR, and Daiichi‐Sankyo Co. Ltd.

Intravenous immunoglobulins (IVIg) inhibit antigen presentation in vitro via an FcgammaR‐independent mechanism.

Intravenous immunoglobulins (IVIg) are used to treat a variety of autoimmune diseases, but their mechanism of therapeutic action remains unclear. It has been observed that IVIg could reduce autoantibody titers in treated patients, suggesting that IVIg may inhibit the ongoing and future production of pathogenic autoantibodies. Several studies also suggested that the anti‐inflammatory effects of IVIg were mediated by either inhibitory or activating Fc gamma receptors (FcγRs). In the present work, we have studied the possibility that IVIg interfere with the process of antigen presentation by APC to T cells which is essential for antibody production in a sustained immune response, and whether this effect could be FcγRs‐dependent. Using an in vitro antigen presentation assay with different types of murine APC, we observed that IVIg inhibited OVA presentation by more than 50%. Surprisingly, IVIg still significantly inhibited OVA presentation by dendritic cells isolated from mice deficient in either inhibitory FcγRIIb or activating FcγRIII (gamma chain deficient), suggesting that IVIg inhibit antigen presentation by an FcγR‐independent mechanism. In addition, we showed that this inhibition may not be dependent on a modulation of expression of co‐stimulatory molecules such as CD80, CD86 or MHC‐II expression. We are currently investigating other mechanisms by which IVIg could interfere with antigen presentation.

B-Cell-Targeted Therapy for Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a classic autoimmune disease characterized by a myriad of immune system aberrations, most likely resulting from pathogenic autoantibody production, immune complex deposition, and subsequent end-organ damage. B cells play a key role in the pathogenesis; therefore, B-cell-targeted therapies, including B-cell depletion and blockage of B-cell survival factors such as B-lymphocyte stimulator (BLyS), are potential therapeutic targets for SLE. In uncontrolled clinical trials from approximately 20 studies, rituximab--a mouse-human chimeric anti-CD20 monoclonal antibody that effectively depletes B cells--has been demonstrated to reduce disease activity and decrease serum autoantibodies, with a clinical response of 86% in a case series of approximately 400 SLE patients with refractory disease, with or without concomitant use of cyclophosphamide. Epratuzumab, a humanized anti-CD22 monoclonal antibody that partially depletes B cells, has also been shown to reduce disease activity but not to decrease autoantibody levels in patients with moderately active SLE. Randomized controlled phase I/II trials in patients with active SLE have documented that belimumab, a humanized anti-BLyS monoclonal antibody, reduces B-cell numbers, inhibits disease activity and decreases anti-double-stranded DNA autoantibody in SLE patients. All these therapies are well tolerated, but accompanying infectious complications have been observed. Other B-cell-targeted therapies such as 'humanized' monoclonal antibodies to CD20 (e.g. ocrelizumab) and agents that interrupt B-cell/T-cell interactions also have potential, and the efficacy of these, along with rituximab, belimumab and epratuzumab, needs to be determined by randomized controlled trials.

Interleukin 17–producing T helper cells and interleukin 17 orchestrate autoreactive germinal center development in autoimmune BXD2 mice

Interleukin 17 (IL-17) is a cytokine associated with inflammation, autoimmunity and defense against some bacteria. Here we show that IL-17 can promote autoimmune disease through a mechanism distinct from its proinflammatory effects. As compared with wild-type mice, autoimmune BXD2 mice express more IL-17 and show spontaneous development of germinal centers (GCs) before they increase production of pathogenic autoantibodies. We show that blocking IL-17 signaling disrupts CD4+ T cell and B cell interactions required for the formation of GCs and that mice lacking the IL-17 receptor have reduced GC B cell development and humoral responses. Production of IL-17 correlates with upregulated expression of the genes Rgs13 and Rgs16, which encode regulators of G-protein signaling, and results in suppression of the B cell chemotactic response to the chemokine CXCL12. These findings suggest a mechanism by which IL-17 drives autoimmune responses by promoting the formation of spontaneous GCs.

Role of pathogenic auto-antibody production by Toll-like receptor 9 of B cells in active systemic lupus erythematosus

Toll-like receptor 9 (TLR9) is a pattern-associated receptor functioning in innate immunity that may be involved in the recognition of self-antigens and the production of pathogenic auto-antibodies. Therefore, we examined the expression of TLR9 in systemic lupus erythematosus (SLE) to determine whether TLR9 is involved in the production of pathogenic auto-antibodies. B cells were collected from patients with active SLE, and subjected to analysis of the TLR9 molecule using flow cytometry fluorescence activated cell sorting (FACS) and TLR9 mRNA by reverse-transcriptase polymerase chain reaction. SLE B cells were stimulated with CpG-ODN, and subsequent cytokine and anti-dsDNA antibody production was measured by enzyme-linked immunosorbent assay. The expression and mRNA level of TLR9 on B cells was up-regulated in SLE patients, and SLE disease activity index (SLEDAI) and CH50 were correlated with TLR9 expression on CD20+ B cells. Moreover, TLR9-CpG interaction enhanced the production of anti-dsDNA antibody and IL-10. The present study demonstrated that higher expression of TLR9 on peripheral blood B cells from patients with active SLE was significantly correlated with CH50 and SLEDAI to TLR9, and induced the production of anti-dsDNA antibody and IL-10 by TLR9-CpG ligation. These results suggest that an abnormality of innate immunity plays a crucial role in the pathology of SLE, and that blockade of CpG-TLR9 interaction may be a new therapeutic approach for SLE.

Expression of B-cell activating factor of the tumour necrosis factor family (BAFF) in T cells in active systemic lupus erythematosus: the role of BAFF in T cell-dependent B cell pathogenic autoantibody production

To determine whether B cell activating factor of the tumour necrosis factor family (BAFF) is involved in T cell-dependent B cell pathogenic autoantibody production in systemic lupus erythematosus (SLE). Peripheral blood mononuclear cells (PBMCs) from 23 SLE patients were analysed by flow cytometry to examine the intracellular expression of BAFF in CD4+ and CD8+ T cells and the surface expression of BAFF-receptor (R) and TACI on CD20+ B cells. Moreover, peripheral blood was used to determine the level of BAFF messenger RNA (mRNA) in CD4+ and CD8+ T cells and the level of BAFF-R mRNA in CD20+ B cells. Blocking of BAFF function with TACI-Ig measured anti-double-stranded DNA (dsDNA) antibodies by enzyme-linked immunosorbent assay (ELISA). CD4+ and CD8+ T cells from patients with active SLE expressed intracellular BAFF whereas those from normal subjects did not. BAFF-R and TACI were expressed on B cells from both normal controls and patients with active SLE and there was no significant difference. CD4+ and CD8+ T cells from SLE patients expressed BAFF mRNA whereas those from normal controls did not. Expression of BAFF-R mRNA in CD20+ B cells showed no significant difference between SLE patients and normal controls. TACI-Ig suppressed spontaneous in vitro T cell-dependent B cell anti-dsDNA antibodies production on active SLE with kidney involvement. BAFF may play a pathogenic role in SLE by stimulating T cell-dependent B cell autoantibodies production. Blockade of BAFF is a promising therapeutic approach for SLE especially in patients with kidney involvement.

Possible therapeutic applications of single-chain antibodies in systemic autoimmune diseases

B cells participate in the induction and maintenance of systemic autoimmune diseases, such as rheumatoid arthritis and systemic lupus erythematosus, via production of pathogenic autoantibodies, contributing to the formation of immune complexes. Immune complex deposition in the kidney and joints causes inflammation and organ destruction, and chemokine production enhances T cell activation and tissue damage. The development of the disorder depends on several factors, for example, genetic susceptibility, environmental factors or immune dysregulation. Traditional therapies, which aimed at the alleviation of symptoms, are giving way to biological therapies with the potential of disrupting disease progression. This article focuses on antibody therapies, especially on the applications of single-chain antibodies, as new biological agents for the treatment of systemic autoimmune disorders.

Overexpression of Activation-Induced Cytidine Deaminase in B Cells Is Associated with Production of Highly Pathogenic Autoantibodies

Defective receptor editing or defective B cell checkpoints have been associated with increased frequency of multireactive autoantibodies in autoimmune disease. However, Ig somatic hypermutation and/or class switch recombination may be mechanisms enabling the development of pathogenic multireactive autoantibodies. In this study, we report that, in the BXD2 mouse model of autoimmune disease, elevated expression of activation-induced cytidine deaminase (AID) in recirculating follicular CD86(+) subsets of B cells and increased germinal center B cell activity are associated with the production of pathogenic multireactive autoantibodies. CD4 T cells from BXD2 mice that expressed increased levels of CD28 and an increased proliferative response to anti-CD3 and anti-CD28 stimulation are required for this process. Inhibition of the CD28-CD86 interaction in BXD2 mice with AdCTLA4-Ig resulted in normalization of AID in the B cells and suppression of IgG autoantibodies. This treatment also prevented the development of germinal center autoantibody-producing B cells, suggesting that an optimal microenvironment enabling AID function is important for the formation of pathogenic autoantibodies. Taken together, our data indicate that AID expression in B cells is a promising therapeutic target for the treatment of autoimmune diseases and that suppression of this gene may be a molecular target of CTLA4-Ig therapy.

IL-17 promotes germinal center response and AID-regulated pathogenic autoantibody production in autoimmune BXD2 mice (130.21)

Abstract We have found dramatically elevated levels of activation-induced cytidine deaminase (AID) in the B cells of generalized autoimmune BXD2 mice that develop spontaneous erosive arthritis and glomerulonepheritis. Pathogenic autoantibodies cloned from these mice exhibit extensive somatic hypermutation and the majority of them have undergone class-switch recombination, suggesting that AID activity plays an important role in the production of pathogenic autoantibodies. Sera from BXD2 mice exhibited significantly elevated IL-17, but not IFN-γ or IL-4, compared to B6 mice. FACS analysis indicates that CD4 T cells in BXD2 mice are preferentially polarized to Th17 cells and these cells also express the early activation marker, CD69, in vivo in BXD2 mice. The B cells from BXD2 mice express high levels of IL-17 receptor (IL-17R) and immunohistochemistry staining shows that B cells are in close contact with the IL-17+ cells in the spleen. Administration of AdIL-17 to 1-mo-old B6 and BXD2 mice induced the expression of AID, promoted development of GCs, and enhanced the sera titers of antibodies. In contrast, administration of AdIL-17R:Fc to 10-mo-old BXD2 mice suppressed the expression of AID, down-modulated the percentage of PNA+Fas+ GC B cells, and suppressed the sera titers of antibodies. Our results provided a unique pathogenic function of IL-17 in that it positions IL-17 as a critical cytokine that regulates GC B cell response and the production of pathogenic autoantibodies.

IL-21 Has a Pathogenic Role in a Lupus-Prone Mouse Model and Its Blockade with IL-21R.Fc Reduces Disease Progression

Systemic lupus erythematosus is a complex autoimmune disease characterized by dysregulated interactions between autoreactive T and B lymphocytes and the development of anti-nuclear Abs. The recently described pleiotropic cytokine IL-21 has been shown to regulate B cell differentiation and function. IL-21 is produced by activated T lymphocytes and its interactions with IL-21R are required for isotype switching and differentiation of B cells into Ab-secreting cells. In this report, we studied the impact of blocking IL-21 on disease in the lupus-prone MRL-Fas(lpr) mouse model. Mice treated for 10 wk with IL-21R.Fc fusion protein had reduced proteinuria, fewer IgG glomerular deposits, no glomerular basement membrane thickening, reduced levels of circulating dsDNA autoantibodies and total sera IgG1 and IgG2a, and reduced skin lesions and lymphadenopathy, compared with control mice. Also, treatment with IL-21R.Fc resulted in a reduced number of splenic T lymphocytes and altered splenic B lymphocyte ex vivo function. Our data show for the first time that IL-21 has a pathogenic role in the MRL-Fas(lpr) lupus model by impacting B cell function and regulating the production of pathogenic autoantibodies. From a clinical standpoint, these results suggest that blocking IL-21 in systemic lupus erythematosus patients may represent a promising novel therapeutic approach.

Genotypic analysis of Asp299Gly and Thr399Ile polymorphism of Toll-like receptor 4 in systemic autoimmune diseases of Korean population

Dear Editor, Systemic autoimmune diseases such as rheumatoid arthritis and systemic lupus erythematosus are mainly characterized by chronic inXammation ampliWed by large quantities of proinXammatory cytokines released from immune cells including macrophages and dendritic cells. Although the pathogenesis of autoimmune diseases are largely unknown, adaptive immune responses in various systemic autoimmune diseases can be driven by innate immune system as exempliWed in pathogenic autoantibody production triggered by infectious agents in the pathogenesis of rheumatoid arthritis and systemic lupus erythematosus [1, 2]. Toll-like receptors (TLRs) are phylogenetically conserved receptors expressed on various cells, mainly on macrophages and dendritic cells that recognize pathogen-associated molecular patterns and are key players in cellular innate immunity with an emerging role as a bridge between innate and adaptive immune responses [3]. TLRs, especially TLR4, are predominantly activated by bacterial lipopolysaccharides [4] and by endogenous ligands such as heat shock proteins [5], fragments of hyaluronic acid [6] and Wbronectin [7], which are released by damaged cells. Activation of TLRs Wnally leads to activation of nuclear transcription factors such as NFB via signal transduction involving various intracellular signaling systems and to expression of various inXammatory cytokines. There are two variants of TLR4 gene, Asp299Gly and Thr399Ile; both are present in a putative coreceptor-binding region of TLR-4 and in linkage disequilibrium [8]. The polymorphisms at these two TLR4 variants appeared to be correlated with the susceptibility to gram-negative septic shock [9], the development of atherosclerosis [10] and ischemic stroke [11], and with the host susceptibility to autoimmune process in human [12, 13]. Therefore, aberrant TLR functions, especially TLR4 involved in host defense against Gram-negative bacteria, may alter the response of this receptor to stimulation with LPS and probably endogenous ligands and eventually may induce the loss of self-tolerance and triggering of autoimmune diseases. The expression and function of TLR4 in systemic autoimmune diseases have been studied by diVerent groups, but the frequencies of variant alleles and the clinical signiWcance were diVerent depending on diagnosis or on race [9–14]. The frequencies of TLR4 gene polymorphism in these two alleles among Korean population, is not known. Therefore, we report the frequencies of TLR4 Asp299Gly and Thr399Ile polymorphism in E.-S. Kang Division of Diagnostic Immunology, Department of Laboratory Medicine, Ewha Womans University Mokdong Hospital, College of Medicine, Ewha Womans University, Mokdong, Yangcheongu, Seoul 135-710, South Korea

Expansion of toll‐like receptor 9–expressing B cells in active systemic lupus erythematosus: Implications for the induction and maintenance of the autoimmune process

Toll-like receptors (TLRs) are pattern-associated receptors in innate immunity that may be involved in the recognition of self antigens and the production of pathogenic autoantibodies. This study was undertaken to examine the expression and function of various TLRs in subpopulations of peripheral blood mononuclear cells (PBMCs) of patients with systemic lupus erythematosus (SLE). The expression of TLRs in PBMCs from 50 SLE patients with active disease (SLE Disease Activity Index [SLEDAI] score >or=8; n = 26) or inactive disease (SLEDAI score <8; n = 24) and 20 healthy controls was studied by flow cytometry. TLR expression was assessed on various subpopulations of PBMCs (TLR-2 and TLR-4 by membrane staining; TLR-3 and TLR-9 by intracellular staining). TLR function was accessed by stimulating PBMCs with specific ligands. The proportion of B cells and monocytes expressing TLR-9 was higher among patients with active SLE (mean +/- SD 49.5 +/- 24.4% and 30.7 +/- 24.1%, respectively) than among patients with inactive disease (22.8 +/- 19.6% and 14.3 +/- 8.4%, respectively; P = 0.02 and P = 0.03). Among B cells, the proportion of plasma cells and memory B cells expressing TLR-9 was increased in patients with active SLE. Increased percentages of TLR-9-expressing B cells correlated with the presence of anti-double-stranded DNA antibodies (P = 0.007). Treatment with serum from patients with active disease increased the percentage of TLR-9-expressing plasma cells in serum from healthy controls. Enhanced induction of HLA-DR after TLR-9 stimulation was documented in B cells from patients with active disease. In patients with active SLE, the proportion of peripheral blood memory B cells and plasma cells expressing TLR-9 is increased. Endogenous nucleic acids released during apoptotic cell death may stimulate B cells via TLR-9 and contribute to SLE pathogenesis.

Toll‐Like Receptor Activation in the Pathogenesis of Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is characterized by the production of pathogenic autoantibodies to nucleoproteins and DNA. The level of anti-DNA antibodies correlates with disease severity, and the deposition of these immune complexes (ICs) in the kidneys is thought to contribute to disease pathogenesis. Recent evidence suggests that the DNA component of ICs purified from SLE patients (SLE DNA-ICs) contributes to the development of SLE pathology. SLE DNA-ICs induce proliferation of self-reactive B cells and cytokine production by plasmacytoid dendritic cells (PDCs) in a TLR9-dependent manner. One of the cytokines induced by DNA-containing ICs is interferon alpha (IFN-alpha). Elevated serum levels of IFN-alpha and overexpression of interferon-induced genes have been observed in SLE patient blood and shown to correlate with disease severity. We have recently found that the mechanism of IFN-alpha production by PDCs depends on TLR9 and FcgammaRIIa (CD32), and CD32 delivers SLE DNA-ICs to intracellular lysosomes containing TLR9. This CD32-TLR9 pathway, which is operative in PDCs, is distinct from the BCR/TLR9 pathway in B cells and may prove to be a novel target for future SLE therapies. In this article, the role of toll-like receptors, cytokines, and Fc receptors expressed by PDCs in the pathogenesis of SLE is summarized.

933 POSTER Immunotherapy in patients with recurrent epithelial ovarian cancer with the anti-idiotypic monoclonal antibody ACA125 (AGO-OVAR, phase I/II trial)

Idiotypes (Ab1) are the antigenic determinants of immunoglobulin molecules that are located in the variable region of the antibodies. Anti-idiotypic antibodies (Ab2) are directed against the idiotypic determinants. They are classified into (a) Ab2 alpha, which recognize Ab1 framework region antigen, (b) Ab2 beta, which bind the antigen-binding site of the antibody molecule, (c) Ab2 gamma, which refer to antibodies directed against idiotypes close to, rather than within, the antigen-binding site, and (d) Ab3, which are anti-anti-idiotypic antibodies.Under physiological conditions, each antigenic stimulation leads to the production of idiotypes Ab1 and anti-idiotypes (Ab2 and Ab3) as a network of interacting antibodies; this idiotypic network has a crucial role in the regulation of immune response to external and self-antigens.Manipulation of idiotypic network can also be implicated in the production of pathogenic autoantibodies and induction of autoimmune diseases. Experimental autoimmune diseases, including systemic lupus erythematosus, anti-phospholipid syndrome, and others, have been induced by immunization of mice with polyclonal human anti-DNA and anti-cardiolipin antibodies. In subjects susceptible to autoimmune diseases, the immune response to particular antigens extends over weeks to months and ends with production of idiotypic antibodies that recognize pathogenic epitopes and eventually with the development of an overt autoimmune disease.