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B cells and autoimmune liver diseases
Autoimmunity Reviews 5 (2006) 449 – 457 www.elsevier.com/locate/autrev
B cells and autoimmune liver diseases Yuki Moritoki a,b , Zhe-Xiong Lian a , Yoshiyuki Ohsugi c , Yoshiyuki Ueno b , M. Eric Gershwin a,⁎ a
Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, 451 E. Health Sciences Drive, Suite 6510, Davis, CA 95616, United States b Tohoku University Graduate School of Medicine, Sendai, Japan c Chugai Pharmaceutical, Tokyo, Japan Received 12 January 2006; accepted 16 February 2006 Available online 20 March 2006
Abstract Autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are the three major autoimmune diseases affecting the liver. They are all characterized by the presence of a variety of autoantibodies, some of which are found in all three diseases, whereas others are restricted to one or two of them or are even specific for the particular disease. In this review we will first provide details of the serological features of these three autoimmune diseases that target the liver. In addition, we will highlight the possible pathogenic roles of autoreactive B cells, focusing on their immunological functions as autoantibody producing cells and as antigen-presenting cells for T cell priming. As well, we will describe the contribution of toll-like receptor (TLR) signaling to the activation of autoimmune B cells and the putative role of defects in regulatory T cell function in the development of autoimmune liver diseases. © 2006 Elsevier B.V. All rights reserved. Keywords: B cells; Autoimmunity; Autoantibodies; Autoimmune hepatitis; Primary biliary cirrhosis; Primary sclerosing cholangitis; Toll-like receptors
Contents 1. 2.
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Introduction . . . . . . . . . . . Autoimmune hepatitis (AIH) . . . 2.1. Clinical features of AIH. . 2.2. Autoantibodies in AIH . . 2.3. Immunogenetics in AIH. . 2.4. Cellular responses in AIH. PBC . . . . . . . . . . . . . . . 3.1. Clinical features of PBC . 3.2. Serology of PBC . . . . .
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⁎ Corresponding author. Tel.: +1 530 752 2884; fax: +1 530 752 4669. E-mail address: [email protected] (M.E. Gershwin). 1568-9972/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2006.02.006
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3.3. Immunogenetics of PBC . . . . . . . . . . . . . . . . . . 3.4. Cellular responses of PBC . . . . . . . . . . . . . . . . . 4. PSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Clinical features of PSC . . . . . . . . . . . . . . . . . . 4.2. Autoantibodies in PSC. . . . . . . . . . . . . . . . . . . 4.3. Immunogenetics of PSC . . . . . . . . . . . . . . . . . . 4.4. Cellular responses of PSC . . . . . . . . . . . . . . . . . 5. Autoreactive B cells in autoimmune liver diseases . . . . . . . . 6. Toll-like receptors (TLR) . . . . . . . . . . . . . . . . . . . . . 7. Fibrosis in autoimmune liver diseases: possible involvement of B 8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
2.2. Autoantibodies in AIH
Three autoimmune diseases affect the liver: (1) autoimmune hepatitis (AIH), in which hepatocytes are the target of the autoimmune attack; (2) primary biliary cirrhosis (PBC), in which small bile ducts are destroyed; and (3) primary sclerosing cholangitis (PSC), in which medium to large bile ducts are mainly damaged. They are generally considered to be “complex diseases,” their pathogenesis comprising both genetic and environmental factors. Both of these factors, in turn, appear to play a role in the emergence of autoreactive B cells.
ANA are serological markers of type 1 AIH, but are not specific to the disease. Instead, they are also detected in PSC, PBC, Type C hepatitis, drug-induced hepatitis, and alcoholic hepatitis. These antibodies recognize heterogeneous target antigens, such as centromeres, ribonucleoproteins, cyclin A, and histone. The difference in specificity is not a diagnostic or prognostic marker for AIH. SMA, which bind to actin and non-actin components, such as tubulin, vimentin, and desmin, are also characteristic of type 1 AIH. Actin-associated SMA are found in a large majority of SMA-positive AIH patients, but are rare or even absent in other chronic liver diseases. Hence, they provide somewhat lower sensitivity but higher disease specificity than other SMA. Positivity for actin-associated SMA is more frequently seen in patients with the HLA B8 and DR3 haplotypes and is associated with early disease onset and worse prognosis. Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA): Whereas between 65% and 92% of patients with type 1 AIH have pANCA, these antibodies are never observed in type 2 AIH [1]. pANCA are also seen in PSC and ulcerative colitis (UC) as well as a small percentage of patients with chronic hepatitis C. Their titers are higher in AIH and PSC than in UC. There are some indications that anti-neutrophil antibodies in AIH and PSC frequently do not, in fact, recognize a cytoplasmic antigen, but a 50 kDa myeloid-specific nuclear envelope protein. Hence, the designation peripheral antineutrophil nuclear antibodies (p-ANNA) has been proposed. Anti-LKM antibodies are serological markers of type 2 AIH, but can also be found in some patients with chronic hepatitis type C. In type 2 AIH, the majority of anti-LKM antibodies bind to the LKM1 antigen, which has been identified as the cytochrome monooxygenase
2. Autoimmune hepatitis (AIH) 2.1. Clinical features of AIH AIH is an inflammatory chronic liver disease that is characterized by a higher incidence in females, the presence of serum autoantibodies, hyper IgG globulinemia, and plasma cell infiltration into liver tissue, where periportal or interface hepatitis is the characteristic histological finding. AIH can be classified into three different types based on the presence or absence of certain autoantibodies. Whereas type 1 AIH is characterized by antinuclear antibodies (ANA) and/or smooth muscle antibodies (SMA), the typical antibodies of type 2 AIH are directed against liver kidney microsome type 1 (LKM1) and/or liver cytosol type 1 (LC1). The presence of anti-soluble liver antigen/liver pancreas antigen antibodies (anti-SLA/LP antibodies) has been proposed to characterize a third type of AIH, but this is controversial since anti-SLA/LP-positive patients do not exhibit distinct clinical features. ANA, SMA, and antiLKM1 are neither organ- nor disease-specific, whereas anti-LC1 antibodies are both, and anti-SLA/LP antibodies are specific to AIH. For the treatment of AIH, corticosteroids are known to be effective.
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P450 IID6 (CYP2D6) with an MW of 50 kDa consisting of 263–270 amino acids. These antibodies recognize a short linear epitope on CYP2D6 and inhibit its enzymatic activity in vitro, but not in vivo. In addition, this epitope is also targeted by liver-infiltrating CD4+ T cells. Some AIH type 2 patients (∼ 17%), but not those with AIH type 1, were found to develop autoantibodies against uridine diphosphate glucuronosyltransferase (UGT), designated as LKM3 [2]. These antibodies recognize several UGT isoforms, but show the strongest reactivity with UGT1A1, the isoenzyme mainly involved in bilirubin glucuronidation. Although antiKLM3 antibodies inhibit UGT activity in vitro, the serum bilirubin levels of patients with anti-KLM3 antibodies remain within the normal range. Anti-LC1 antibodies are another serologic marker of type 2 AIH, frequently accompanying anti-LKM1 antibodies. They are seen in 30–50% of patients with type 2 AIH, but not in patients with PBC or viral hepatitis. Their target antigen is formiminotransferase cyclodeaminase (FTCD), a liver-specific metabolic enzyme that has a role in converting glutamic acid into histidine. Anti-LC1 antibodies are capable of binding to different epitopes on the antigen molecule, with preferential recognition of the FT region [3]. In contrast to LKM1 concentrations, anti-LC1 antibody levels are associated with disease activity. From 10% to 30% of AIH patients, including some with PBC/AIH overlap syndrome, have anti-SLA/LP antibodies [4]. These antibodies recognize the UGAsuppressor serine tRNA-associated protein [4] and are highly specific for AIH [4]. Anti-SLA-/LP have never been detected in anti-LKM1-positive AIH patients [4]. Though still controversial, these antibodies have been proposed to characterize a third type of AIH. The asialoglycoprotein receptor (ASGPR) is a component of liver specific lipoprotein (LSP), which is a macromolecular complex expressed on hepatocyte membranes. Anti-ASGPR antibodies appear in 50–76% of patients with all types of AIH and in 85–88% of patients with active disease, and frequently accompany ANA, SMA and anti-LKM1 antibodies. Anti-ASGPR antibodies are also observed in PBC, PSC, and viral hepatitis with a variety of antibody titers, but with significantly lower positivity rates than are seen in AIH patients. The titer of anti-ASGPR antibodies correlates with disease activity and poor prognosis of AIH and might serve as a useful marker for assessing therapy outcomes. Although it largely remains to be elucidated whether antibodies in AIH play a pathogenic role, antibodydependent cell-mediated cytotoxicity (ADCC) and/or
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complement-dependent cytotoxicity (CDC) may possibly be involved in the hepatocyte damage in AIH. Recently, an IgM antibody produced by a clone obtained from a type 1 AIH patient was found to induce CDC in human hepatocellular carcinoma cell lines. This monoclonal autoantibody recognized a 190 kDa molecule expressed on hepatocyte membrane, and autoantibodies to this hepatocyte membrane antigen were also found in other AIH patients, but not in type C hepatitis patients. This antibody cross-reacted with a 190-kDa molecule on murine hepatocytes and, upon injection into mice, resulted in deposition of IgM and C3 on hepatocyte membranes [5]. This was followed by acute liver injury as evidenced by histological changes in periportal and pericentral areas and by elevated ALT and AST levels. 2.3. Immunogenetics in AIH HLA-DR3 (DRB1⁎0301) and DR4 (DRB1⁎0401) are associated with type 1 AIH [6]. In particular, the HLA-A1-B8-DR3 haplotype shows strong associations with early onset of disease, relapse, and the necessity for liver transplantation. In contrast, susceptibility to type 2 AIH is associated with carriage of HLA-DR7 [7]. Genes outside the HLA loci also appear to contribute to disease susceptibility and/or severity. Thus, deficiency of complement C4 due to a gene deletion is related to increased disease severity in both types of AIH. Cytotoxic Tlymphocyte antigen 4 (CTLA-4, also known as CD152) gene polymorphism is related to the development of type 1 AIH, but not type 2 AIH [8]. CTLA-4 is one of the major negative regulators in T cell activation [9]. Expression of CTLA-4 on T cells is induced by their activation, but there is a subset of regulatory CD4+ T cells (Treg) that constitutively express this molecule. These findings suggest that there are immunogenetic differences in the pathogenesis of type 1 and type 2 AIH. 2.4. Cellular responses in AIH Th2 cytokines, which activate B cells and induce their differentiation into antibody-producing cells, dominate in AIH. CD4+ T cell clones established from AIH livers and stimulated with lectin exhibited markedly lower IFN-γ/IL-4 ratios compared to clones from livers from patients with non-AIH diseases. Moreover, whereas a small number of Th2 clones was detected among the AIH clones, none were found in the non-AIH ones. Since CTLA-4 regulates the activation of T cells and their differentiation into Th2 cells, it is possible that the CTLA-4 allele associated with AIH might be involved in this shift of the AIH cytokine
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pattern towards Th2 predominance. In fact, serum levels of IL-4 (involved in the differentiation of B cells) and soluble CD23 (a marker for humoral immunity) are high in AIH and correlate positively with each other. It was suggested more than two decades ago that defective suppressor cell activity contributes to the development of AIH. More recently, AIH patients were found to have significantly fewer peripheral CD4+CD25+ Treg than healthy controls, and their Treg exhibited decreased proliferative activity in response to CD3/CD28 stimulation [10]. Their regulatory function, measured as the ability to suppress intracellular IFN-γ levels of CD4+CD25− T cells, was not affected. In contrast, Treg obtained from AIH at diagnosis, but not in remission, failed to inhibit CD8+ T cell proliferation and cytokine production. Of note, the number of Treg negatively correlated with the amount of anti-LKM or anti-SLA antibodies [10]. CTLA4 is normally expressed in Treg, and an association between CTLA-4 polymorphism and AIH has been reported [8]. This raises the possibility that the CTLA-4 polymorphism causes the Treg abnormality, which then allows the appearance of autoreactive B cells producing autoantibodies. 3. PBC 3.1. Clinical features of PBC PBC is a chronic cholestatic liver disease, in which interlobular bile ducts are damaged. It is characterized by AMA (90–95% of patients) and hyper IgM globulinemia, and affects predominantly females [11]. 3.2. Serology of PBC AMA are detected in 90–95% of PBC patients and are virtually diagnostic or presage the development of PBC within the following 5–10 years. They are directed against members of the 2-oxoacid dehydrogenase complexes (2-OADC) existing in the inner membrane of mitochondria. Among them, the major autoantigen is the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) [12]. The epitopes for this antibody localize to three domains of PDC-E2 component: (1) the inner/ outer lipoyl domain; (2) the E3 binding domain; and (3) the catalytic and E2-binding domain [12–14]. AntiPDC-E2 antibodies are capable of inhibiting PDC-E2 enzyme activity [12,13]. Targeting of enzymes is a common feature of autoantibodies detected in patients with autoimmune diseases, as is the inhibition of their activity by these autoantibodies. It has been shown that CD4+ and CD8+ T cell clones from both liver-
infiltrating lymphocytes and peripheral blood of PBC patients also recognize PDC-E2 [12,15,16]. Importantly, the B and T cell epitopes overlap, which not only suggests that the immune response to this enzyme is of pathological relevance, but also underscores the importance of interactions between these cell types and subtypes in the pathogenesis of PBC. Approximately 50% of PBC patients develop ANA. Certain targets of these antibodies are highly specific for PBC, in particular gp210 and p62 of the nuclear pore complex, which result in a perinuclear pattern in indirect immunofluorescence studies; and Sp100, which yields a multiple nuclear dot pattern. The presence of anti-gp210 antibodies correlates with disease activity and severity. Immunoreactive gp210 could be detected in the nuclear envelope of biliary epithelial cells (BEC) in small bile ducts of almost all (98.6%) PBC liver specimens, but in significantly fewer AIH and viral hepatitis specimens. Of note, the gp210 staining intensity in PBC biopsy samples correlated with the degree of interface hepatitis and lobular inflammation. One of the central questions in PBC research is why the autoimmune attack is focused on the biliary epithelium, even though every cell has mitochondria. In cholangiocytes, unlike other somatic cells, apoptosis does not induce attachment of glutathione to the lysinelipoyl domain of mitochondria's inner lipoyl domain. This prevents cleavage of PDC-E2 by caspases and allows its continued recognition by autoantibodies [11,17]. Mucosal epithelial cells are also distinguished functionally from other epithelial cells in that they transcytose IgA. Transcytosis of this Ig subclass is mediated by the polymeric immunoglobulin receptor (pIgR), and co-localization of PDC-E2 and IgA has been observed in epithelial cells transfected with this receptor [18]. This raises the possibility that IgA-PDC-E2 immune complexes could form during transcytosis of IgA and be transported to the bile duct lumen. Furthermore, there are data indicating that transcytosis of AMA-IgA causes caspase activation, resulting in apoptosis of bile duct epithelium [19]. Recent findings have shown that TLR signaling is important in B cell autoimmunity [20]. We recently reported that PBMC from PBC patients produced significantly more IgM than healthy controls when stimulated with CpG-B, a ligand of TLR9 [21]. Moreover, the amount of IgM produced per memory B cell was higher in PBC patients than in controls. The expression not only of TLR9 but also of CD86 (B7-2; an activation marker for B cells) and CD38 (a differentiation marker for B cells) increased on these memory B cells. This suggests that enhanced responsiveness of memory B
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cells to bacterial products like CpG may be one of the reasons for elevated serum IgM levels in PBC. Of note, CpG-B-stimulated PBMC from PBC patients produced markedly larger amounts of AMA compared to unstimulated controls, implicating the TLR9 signaling pathway in increased autoantibody production. 3.3. Immunogenetics of PBC A genetic component to PBC is suggested by the familial clustering of the disease and its high concordance rate (63%) in monozygotic twins. Despite considerable efforts, however, no consistent associations have been detected between susceptibility to PBC and carriage of particular HLA alleles [22]. Few associations with genes outside the MHC have emerged, although polymorphisms in the vitamin D receptor and the CTLA-4 genes have been implicated in disease susceptibility. X-chromosome monosomy of PBMC may explain the female preponderance of the disease [11]. 3.4. Cellular responses of PBC Eosinophilia is a characteristic finding in PBC patients. Whereas 90% of stage 3 or 4 PBC patients
Low frequency of Treg
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express IL-5 mRNA in the liver, AIH patients and healthy controls express none [23]. The deposit of major basic protein (MBP), in particular, is observed within the portal inflammatory infiltrate. MBP is released from eosinophils, which are activated by IL-5, and has the ability to cause epithelial cell lysis [23]. Serum levels of TNF-α and TGF-β were found to increase with increasing severity of PBC and to decrease after treatment with ursodeoxycholic acid, suggesting a role for both of these cytokines in the progression of the disease. The proportion of Treg in PBMC is lower in PBC patients compared to type C hepatitis patients, as is the FoxP3/CD3+T ratio in liver-infiltrating lymphocytes (R. Lan et al., in press). Of note, the liver infiltrate was found to contain larger numbers of PDC-specific B cells than PBMC. Together, these findings may suggest that a decrease in the number of Treg in the liver might contribute to the induction of autoreactive B cells, resulting in increased AMA production. As in the case of AIH, it has been suggested that CTLA-4 polymorphism contributes to impaired Treg function in PBC, resulting in a decreased B cell regulation and, thus, enhancement of B cell autoimmunity. A possible pathophysiological model for PBC focusing on the roles of B cells is shown in Fig. 1.
Treg Autoreactive B Cell
AMA and ANA production
B
B
Molecular mimicry
NKT
B Interlobular Bile Duct BEC apoptosis
B B B
Transcytosis of dimeric IgA
APC
B
Autoreactive CD4+ T Cell proliferation
CTL
Th
CTL
Th
Th
Autoreactive CD8+ T Cell
BacteriaI or Xenobiotic molecules Mitochondrial autoantigen
PBC Liver Treg Fig. 1. A model of immunopathology of PBC.
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4. PSC 4.1. Clinical features of PSC PSC is a chronic inflammatory disease of large bile ducts, often associated in patients with inflammatory bowel disease, with the vast majority (87%) having UC and only 13% having Crohn's disease [24]. In contrast, only 5% of UC patients have PSC. The development and progression of PSC are not influenced by the concomitant presence of UC. 4.2. Autoantibodies in PSC As many as 88% of PSC patients exhibit ANCA [25]. ANCA are not specific for PSC, since they are also observed in a substantial portion of patients with UC and AIH, a smaller percentage of patients with Crohn's disease and about 5% of patients with PBC. Whether these antibodies have a pathological role is still not known. Targets of ANCA in PSC include h-lamp-2, proteinase-3, and bactericidal/permeability increasing protein and only rarely myeloperoxidase. Indirect immunofluorescence staining for the detection of antineutrophil antibodies in PSC frequently does not yield the homogeneous rim-like pattern typical for classical pANCA, but an atypical heterogeneous rim-like staining of the nuclear periphery and multiple intra-nuclear spots. This and other evidence indicates that the antigen (s) recognized by these atypical p-ANCA or p-ANNA is probably located in the nucleus and is most likely a neutrophil-specific 50 kDa nuclear envelope protein. As in AIH and PBC, ANA can be detected in a substantial portion of PSC patients (53%), whereas AMA are absent. In addition, almost two thirds of PSC patients exhibit anti-cardiolipin antibodies, and their concentrations correlate positively with histological changes and disease severity. 4.3. Immunogenetics of PSC As in AIH, HLA-B8 and DR3 confer susceptibility to PSC, especially B8-MICA5.1-MICB24-DR3. However, it remains controversial whether CTLA-4 polymorphism is associated with disease susceptibility. 4.4. Cellular responses of PSC Blood levels of TNF-α (a pro-inflammatory cytokine), IL-10 (an anti-inflammatory cytokine), and IL8 (a neutrophil activation marker) were found to be significantly higher in PSC compared to patients with
alcoholic cirrhosis or normal controls. In contrast, IFNγ (an inducer of cell-mediated immunity) and GM-CSF (a marker of monocyte/macrophage function) were within the normal range. No correlation was found between these cytokines and positivity for, or titer of, ANCA. 5. Autoreactive B cells in autoimmune liver diseases In PBC liver, the number of infiltrating B cells is higher than that of PSC patients and normal controls. Moreover, it has been reported that the proportion of CD19+CD69+ activated B cells was markedly higher in the liver than in peripheral blood of PBC patients, and that the number of AMA-producing cells was 5 times in the liver than in PBMC. It has been proposed that such IgA-AMA-producing cells might be directly involved in causing damage to bile ducts in PBC livers [18,19]. It has not yet been clarified how the homing of B-1 and B-2 cells into the liver is regulated; however, CXCL13/CXCR5 is known to play a critical role. In SLE-prone NZB/NZW F1 mice, for example, the expression level of CXCL13 increased with age and correlated strongly with the number of B220+ B cells infiltrating the thymus and kidney. The major sources of CXCL13 are CD11b+CD11c+ cells, which are myeloid dendritic cells (mDC). In the liver of B6 mice, about 10% of NK1.1−CD11c+DC consist of mDC [26]. LPS can induce CXCL13 production in human monocytederived DC. Stimulation with LPS + IL-10 of mDC and plasmacytoid DC (pDC) enhances the production of CXCL13 [27]. The expression of the CXCL13 ligand, CXCR5, is higher in B-1 cells than in B-2 cells. It has been reported that B-1a cells can differentiate into splenic plasma cells when the cells are transferred into T cell and B cell-deficient RAG knockout mouse [28]. Together, these findings suggest that stimulation of mDC + pDC with bacterial products (possibly in the presence of IL-10) present in the liver of patients with autoimmune diseases and the resulting CXCL13 production could lead to the accumulation of CXCR5bearing autoantibody-producing B-1 cells in the target organ. 6. Toll-like receptors (TLR) TLRs and their ligands are mediators of innate immune responses that may be involved in the pathogenesis of autoimmune liver diseases. Both human and murine hepatocytes, targeted in AIH, and cholangiocytes, targeted in PBC and PSC, express TLRs on their cell surface (Table 1). Hepatocytes in AIH and
Y. Moritoki et al. / Autoimmunity Reviews 5 (2006) 449–457 Table 1 Expression of TLRs in B cells, hepatocytes and cholangiocytes Receptor B cells
Hepatocytes
Humans Mice Humans TLR1 TLR2 TLR3 TLR4
+ − − −
− + + +
TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 TLR11 Ref.
− + + + + (PBC) + ND [21,32]
− + + ND + ND ND [33]
+ + + (PBC) + (Periportal, PBC) + + + + + ND ND [34,35]
Cholangiocytes Mice
Humans
Mice
ND + ND +
+/− + + + (PBC)
ND + + +
ND ND ND ND + ND ND [36,37]
+ +/− +/− +/− +/− +/− ND [35,38,39]
+ ND ND ND ND ND ND [38]
ND: not determined.
cholangiocytes in PBC and PSC all express MHC-class II molecules, whereas normal hepatocytes and cholangiocytes do not. These cells also show aberrant expression of CD80/CD86, which is a co-stimulatory molecule required for antigen presentation [29]. Based on these findings, it has been speculated that these cells may acquire antigen-presenting activity via TLRs signaling pathways. In fact, it was recently shown that B6 mice who received multiple intraperitoneal injections of the TLR3 ligand, poly I:C, developed PBC, including its characteristic serological markers, AMA and ANA. In these animals, mononuclear cells infiltrated the portal areas and caused damage to bile ducts, although ALP level remained normal. In liver parenchymal and portal vein areas of PBC patients, expression of both TLR3-and type I IFNmRNA is higher than in those of AIH or chronic hepatitis type C patients. The expression levels of these two genes correlated closely, and a positive association was also observed between type I IFN-mRNA and serum levels of ALP. This suggests that in PBC, some viruses might be
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triggering TLR gene expression. Immunohistochemical studies indicate that BEC as well as periportal hepatocytes of PBC livers express TLR4. Moreover, the expression level of TLR4 showed a positive correlation with the intensity of fibrosis in the liver. BEC are much more responsive to bacterial components compared to hepatocytes. It was found that TLR signaling appeared when human BEC cells obtained from chronic hepatitis C and PBC patients were stimulated with LPS or Escherichia coli, although none was observed in the HepG2 cell line [30]. In addition, stimulation of murine BEC and human intrahepatic cholangiocarcinoma cell lines with LPS or E. coli induced NF-κB activation and TNF-α production. These findings may be particularly relevant to PSC liver, which is always exposed to bacterial components derived from the intestine. This repeated stimulation with LPS and other bacterial products may result in the enhancement of the production of pro-inflammatory cytokines such as TNF-α. In fact, liver T cells in PSC produce higher levels of TNF-α than those of PBC or AIH patients, and this may be the cause of periductular fibrosis in PSC. 7. Fibrosis in autoimmune liver diseases: possible involvement of B cells Fibrosis and cirrhosis are associated with the progression of autoimmune liver diseases, particularly with PBC and PSC. Excessive production of extracellular matrix by periductular and periportal fibroblasts causes these pathological changes. Cytokines such as TNF-α and TGF-β, as well as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved in fibrogenesis. Some recent studies have indicated that B cells play a critical role in the development of fibrosis. In B cell-depleted mice, fibrosis did not develop subsequent to liver injury induced by carbon tetrachloride (CCl4). Similarly, in α-naphthylisothiocyanate (ANIT)-induced liver injury, B cell
Table 2 Autoantibodies and identified autoantigen epitopes in autoimmune liver diseases [40]
Auto-Abs
B cell infiltrate in the liver
Type I AIH
Type II AIH
PBC
PSC
ANA: centromere, ribonucleoprotein, cyclin A, histone; SMA: F actin, anti-ASGPR, anti-SLA/LP;pANCA: myeloid-specific nuclear envelop protein + (with plasma cells)
Anti-LKM-1: CYP450 2D6; anti-LC-1: formiminotransferase cyclo-deaminase
AMA: PDC-E2, BCOADC-E2, OGDC-E2, PDC-E3BP, PDC-E1α; ANA: nuclear pore complex, anti-gp210, anti-p62, nuclear dot pattern, anti-Sp100, anti-PML, anti-SUMOs
pANCA: myeloid-specificnuclear envelopprotein; ANA: anti-cardiolipin, thyroperoxidase, rheumatoid factor, H. pylori IgG
+ (with plasma cells)
+ (with plasma cells)
+/−
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depletion inhibited the development of fibrosis [31]. Moreover, in tight skin mice, an animal model for systemic sclerosis, CD19 deficiency suppressed skin fibrosis, although it was not clarified if this was antibody-dependent. Together, these findings suggest that liver-infiltrating autoreactive B cells are involved in the fibrogenesis of autoimmune liver diseases. The mechanism of suppressing fibrinosis by B cell depletion is independent of antibodies or T cells, raising the possibility that cytokines, as well as MMPs and TIMPs, produced or induced by autoimmune B cells, are responsible for fibrosis in autoimmune diseases targeting the liver. 8. Summary Although the mechanisms of cell damage in AIH, PBC, or PSC are not yet fully understood, there is increasing evidence that autoimmunity may be responsible for the pathogenesis of at least AIH and PBC [7,14,24]. The variety of autoantibodies that develop in these three diseases is listed in Table 2. It seems plausible that autoantibodies are involved in the pathogenesis of AIH, but direct evidence is largely lacking. In PBC, it has been suggested that IgA autoantibodies may contribute to liver cell damage and that autoreactive B cells also acts as APC for T cell priming. As for PSC, although various immunological abnormalities have been reported, there is not enough evidence to support the hypothesis that this disease is dependent on autoimmunity. The possibility that autoantibodies contribute to the progression of PBC and AIH is supported by the fact that ursodeoxycholic acid (UDCA) and prednisolone, which are the currently available first choice drugs, are able to suppress the production of AMA and/or immunoglobulin in a dose-dependent manner in the culture of PBMC. This suggests that immunosuppressive therapy regulating B cell activation would be useful in treating patients with PBC and AIH. Take-home messages • Antibody-dependent cell-mediated cytotoxicity and/ or complement-dependent cytotoxicity (CDC) may contribute to the pathogenesis of AIH. • Anti-PDC-E2 of the IgA subclass as well as the priming of autoreactive T cells by autoreactive B cells may contribute to the pathogenesis of PBC. • Despite the presence of serum autoantibodies and other humoral abnormalities, the involvement of B cell autoimmunity in the PSC pathogenesis is still unclear.
• Autoreactive B cells may accumulate in the target organ via CXCL13/CXCR5-dependent mechanisms. • TLRs signaling pathways appear to play an important role in autoimmune liver diseases. • Activation of B cells via TLRs and impairment of Treg numbers and function may exert additive effects on the expansion of autoreactive B cells in autoimmune liver diseases. References [1] Zauli D, Ghetti S, Grassi A, Descovich C, Cassani F, Ballardini G, et al. Anti-neutrophil cytoplasmic antibodies in type 1 and 2 autoimmune hepatitis. Hepatology 1997;25:1105–7. [2] Bachrich T, Thalhammer T, Jager W, Haslmayer P, Alihodzic B, Bakos S, et al. Characterization of autoantibodies against uridinediphosphate glucuronosyltransferase in patients with inflammatory liver diseases. Hepatology 2001;33:1053–9. [3] Muratori L, Sztul E, Muratori P, Gao Y, Ripalti A, Ponti C, et al. Distinct epitopes on formiminotransferase cyclodeaminase induce autoimmune liver cytosol antibody type 1. Hepatology 2001;34:494–501. [4] Wies I, Brunner S, Henninger J, Herkel J, Kanzler S, Meyer zum Buschenfelde KH, et al. Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis. Lancet 2000;355:1510–5. [5] Yamauchi K, Yamaguchi N, Furukawa T, Takatsu K, Nakanishi T, Ishida K, et al. A murine model of acute liver injury induced by human monoclonal autoantibody. Hepatology 2005;42:149–55. [6] Czaja AJ, Donaldson PT. Genetic susceptibilities for immune expression and liver cell injury in autoimmune hepatitis. Immunol Rev 2000;174:250–9. [7] Vergani D, Mieli-Vergani G. Autoimmune hepatitis. Autoimmun Rev 2003;2:241–7. [8] Djilali-Saiah I, Ouellette P, Caillat-Zucman S, Debray D, Kohn JI, Alvarez F. CTLA-4/CD 28 region polymorphisms in children from families with autoimmune hepatitis. Hum Immunol 2001;62:1356–62. [9] Teft WA, Kirchhof MG, Madrenas J. A molecular perspective of CTLA-4 function. Annu Rev Immunol 2006;24:3.1–.33. [10] Longhi MS, Ma Y, Bogdanos DP, Cheeseman P, Mieli-Vergani G, Vergani D. Impairment of CD4(+)CD25(+) regulatory T-cells in autoimmune liver disease. J Hepatol 2004;41:31–7. [11] Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med 2005;353:1261–73. [12] Van de Water J, Gershwin ME, Leung P, Ansari A, Coppel RL. The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase. J Exp Med 1988;167:1791–9. [13] Surh CD, Coppel R, Gershwin ME. Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis. Implication for a conformational autoepitope. J Immunol 1990;144:3367–74. [14] Ishibashi H, Shimoda S, Gershwin ME. The immune response to mitochondrial autoantigens. Semin Liver Dis 2005;25:337–46. [15] Van de Water J, Ansari AA, Surh CD, Coppel R, Roche T, Bonkovsky H, et al. Evidence for the targeting by 2-oxo-dehydrogenase enzymes in the T cell response of primary biliary cirrhosis. J Immunol 1991;146:89–94. [16] Shimoda S, Nakamura M, Ishibashi H, Hayashida K, Niho Y. HLA DRB4 0101-restricted immunodominant T cell autoepitope
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of pyruvate dehydrogenase complex in primary biliary cirrhosis: evidence of molecular mimicry in human autoimmune diseases. J Exp Med 1995;181:1835–45. Odin JA, Huebert RC, Casciola-Rosen L, LaRusso NF, Rosen A. Bcl-2-dependent oxidation of pyruvate dehydrogenase-E2, a primary biliary cirrhosis autoantigen, during apoptosis. J Clin Invest 2001;108:223–32. Fukushima N, Nalbandian G, Van De Water J, White K, Ansari AA, Leung P, et al. Characterization of recombinant monoclonal IgA anti-PDC-E2 autoantibodies derived from patients with PBC. Hepatology 2002;36:1383–92. Matsumura S, Van De Water J, Leung P, Odin JA, Yamamoto K, Gores GJ, et al. Caspase induction by IgA antimitochondrial antibody: IgA-mediated biliary injury in primary biliary cirrhosis. Hepatology 2004;39:1415–22. Pasare C, Medzhitov R. Control of B-cell responses by toll-like receptors. Nature 2005;438:364–8. Kikuchi K, Lian ZX, Yang GX, Ansari AA, Ikehara S, Kaplan M, et al. Bacterial CpG induces hyper-IgM production in CD27(+) memory B cells in primary biliary cirrhosis. Gastroenterology 2005;128:304–12. Selmi C, Invernizzi P, Zuin M, Podda M, Gershwin ME. Genetics and geoepidemiology of primary biliary cirrhosis: following the footprints to disease etiology. Semin Liver Dis 2005;25:265–80. Martinez OM, Villanueva JC, Gershwin ME, Krams SM. Cytokine patterns and cytotoxic mediators in primary biliary cirrhosis. Hepatology 1995;21:113–9. Aoki CA, Bowlus CL, Gershwin ME. The immunobiology of primary sclerosing cholangitis. Autoimmun Rev 2005;4:137–43. Terjung B, Worman HJ. Anti-neutrophil antibodies in primary sclerosing cholangitis. Best Pract Res Clin Gastroenterol 2001;15:629–42. Lian ZX, Okada T, He XS, Kita H, Liu YJ, Ansari AA, et al. Heterogeneity of dendritic cells in the mouse liver: identification and characterization of four distinct populations. J Immunol 2003;170:2323–30. Perrier P, Martinez FO, Locati M, Bianchi G, Nebuloni M, Vago G, et al. Distinct transcriptional programs activated by interleukin-10 with or without lipopolysaccharide in dendritic cells: induction of the B cell-activating chemokine, CXC chemokine ligand 13. J Immunol 2004;172:7031–42. Wen L, Shinton SA, Hardy RR, Hayakawa K. Association of B-1 B cells with follicular dendritic cells in spleen. J Immunol 2005;174:6918–26.
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[29] Tsuneyama K, Harada K, Yasoshima M, Kaji K, Gershwin ME, Nakanuma Y. Expression of co-stimulatory factor B7-2 on the intrahepatic bile ducts in primary biliary cirrhosis and primary sclerosing cholangitis: an immunohistochemical study. J Pathol 1998;186:126–30. [30] Harada K, Ohba K, Ozaki S, Isse K, Hirayama T, Wada A, et al. Peptide antibiotic human beta-defensin-1 and -2 contribute to antimicrobial defense of the intrahepatic biliary tree. Hepatology 2004;40:925–32. [31] Novobrantseva TI, Majeau GR, Amatucci A, Kogan S, Brenner I, Casola S, et al. Attenuated liver fibrosis in the absence of B cells. J Clin Invest 2005;115:3072–82. [32] Bourke E, Bosisio D, Golay J, Polentarutti N, Mantovani A. The toll-like receptor repertoire of human B lymphocytes: inducible and selective expression of TLR9 and TLR10 in normal and transformed cells. Blood 2003;102:956–63. [33] Applequist SE, Wallin RP, Ljunggren HG. Variable expression of Toll-like receptor in murine innate and adaptive immune cell lines. Int Immunol 2002;14:1065–74. [34] Liu S, Gallo DJ, Green AM, Williams DL, Gong X, Shapiro RA, et al. Role of toll-like receptors in changes in gene expression and NF-kappa B activation in mouse hepatocytes stimulated with lipopolysaccharide. Infect Immun 2002;70:3433–42. [35] Wang AP, Migita K, Ito M, Takii Y, Daikoku M, Yokoyama T, et al. Hepatic expression of toll-like receptor 4 in primary biliary cirrhosis. J Autoimmun 2005;25:85–91. [36] Matsumura T, Ito A, Takii T, Hayashi H, Onozaki K. Endotoxin and cytokine regulation of toll-like receptor (TLR) 2 and TLR4 gene expression in murine liver and hepatocytes. J Interferon Cytokine Res 2000;20:915–21. [37] Sanchez-Campillo M, Chicano A, Torio A, Martin-Orozco E, Gamiz P, Hernandez-Caselles T, et al. Implication of CpG-ODN and reactive oxygen species in the inhibition of intracellular growth of Salmonella typhimurium in hepatocytes. Microbes Infect 2004;6:813–20. [38] Harada K, Ohira S, Isse K, Ozaki S, Zen Y, Sato Y, et al. Lipopolysaccharide activates nuclear factor-kappaB through tolllike receptors and related molecules in cultured biliary epithelial cells. Lab Invest 2003;83:1657–67. [39] Mueller T, Kobayashi N, Shibolet O, Podolsky D. TH1 cytokines promote toll-like receptor signaling in human biliary epithelial cells. Hepatology 2005;42:202A. [40] Kita H, Mackay IR, Van De Water J, Gershwin ME. The lymphoid liver: considerations on pathways to autoimmune injury. Gastroenterology 2001;120:1485–501.
Cell-penetrating anti-native DNA antibodies trigger apoptosis through both the neglect and programmed pathways It has been previously shown that certain antibodies to dsDNA are capable to transgress the cell membrane, translocate to the cell nucleus and react with their antigens. When so doing, they signal the penetration cells to undergo apoptosis. In this study, Rivadeneyra-Espinoza L and Ruize-Arguelles A. (J Autoimmun 2006; 26: 52-6) dissected the two main-streams of apoptotic triggering and found that the neglect, mitochondrial pathway, is definitely involved, but activation of caspases 2 and 8, classically known to be associated to membrane pathway, also occurs in a small proportion of cells. These results suggest that penetration anti-dsDNA antibodies, besides activating neglect apoptosis, may also trigger programmed cell death, perhaps initiated at the cell membrane level.
B cells and autoimmune liver diseases Yuki Moritoki a,b , Zhe-Xiong Lian a , Yoshiyuki Ohsugi c , Yoshiyuki Ueno b , M. Eric Gershwin a,⁎ a
Division of Rheumatology, Allergy and Clinical Immunology, University of California at Davis School of Medicine, 451 E. Health Sciences Drive, Suite 6510, Davis, CA 95616, United States b Tohoku University Graduate School of Medicine, Sendai, Japan c Chugai Pharmaceutical, Tokyo, Japan Received 12 January 2006; accepted 16 February 2006 Available online 20 March 2006
Abstract Autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are the three major autoimmune diseases affecting the liver. They are all characterized by the presence of a variety of autoantibodies, some of which are found in all three diseases, whereas others are restricted to one or two of them or are even specific for the particular disease. In this review we will first provide details of the serological features of these three autoimmune diseases that target the liver. In addition, we will highlight the possible pathogenic roles of autoreactive B cells, focusing on their immunological functions as autoantibody producing cells and as antigen-presenting cells for T cell priming. As well, we will describe the contribution of toll-like receptor (TLR) signaling to the activation of autoimmune B cells and the putative role of defects in regulatory T cell function in the development of autoimmune liver diseases. © 2006 Elsevier B.V. All rights reserved. Keywords: B cells; Autoimmunity; Autoantibodies; Autoimmune hepatitis; Primary biliary cirrhosis; Primary sclerosing cholangitis; Toll-like receptors
Contents 1. 2.
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Introduction . . . . . . . . . . . Autoimmune hepatitis (AIH) . . . 2.1. Clinical features of AIH. . 2.2. Autoantibodies in AIH . . 2.3. Immunogenetics in AIH. . 2.4. Cellular responses in AIH. PBC . . . . . . . . . . . . . . . 3.1. Clinical features of PBC . 3.2. Serology of PBC . . . . .
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⁎ Corresponding author. Tel.: +1 530 752 2884; fax: +1 530 752 4669. E-mail address: [email protected] (M.E. Gershwin). 1568-9972/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.autrev.2006.02.006
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3.3. Immunogenetics of PBC . . . . . . . . . . . . . . . . . . 3.4. Cellular responses of PBC . . . . . . . . . . . . . . . . . 4. PSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Clinical features of PSC . . . . . . . . . . . . . . . . . . 4.2. Autoantibodies in PSC. . . . . . . . . . . . . . . . . . . 4.3. Immunogenetics of PSC . . . . . . . . . . . . . . . . . . 4.4. Cellular responses of PSC . . . . . . . . . . . . . . . . . 5. Autoreactive B cells in autoimmune liver diseases . . . . . . . . 6. Toll-like receptors (TLR) . . . . . . . . . . . . . . . . . . . . . 7. Fibrosis in autoimmune liver diseases: possible involvement of B 8. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Take-home messages . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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1. Introduction
2.2. Autoantibodies in AIH
Three autoimmune diseases affect the liver: (1) autoimmune hepatitis (AIH), in which hepatocytes are the target of the autoimmune attack; (2) primary biliary cirrhosis (PBC), in which small bile ducts are destroyed; and (3) primary sclerosing cholangitis (PSC), in which medium to large bile ducts are mainly damaged. They are generally considered to be “complex diseases,” their pathogenesis comprising both genetic and environmental factors. Both of these factors, in turn, appear to play a role in the emergence of autoreactive B cells.
ANA are serological markers of type 1 AIH, but are not specific to the disease. Instead, they are also detected in PSC, PBC, Type C hepatitis, drug-induced hepatitis, and alcoholic hepatitis. These antibodies recognize heterogeneous target antigens, such as centromeres, ribonucleoproteins, cyclin A, and histone. The difference in specificity is not a diagnostic or prognostic marker for AIH. SMA, which bind to actin and non-actin components, such as tubulin, vimentin, and desmin, are also characteristic of type 1 AIH. Actin-associated SMA are found in a large majority of SMA-positive AIH patients, but are rare or even absent in other chronic liver diseases. Hence, they provide somewhat lower sensitivity but higher disease specificity than other SMA. Positivity for actin-associated SMA is more frequently seen in patients with the HLA B8 and DR3 haplotypes and is associated with early disease onset and worse prognosis. Perinuclear anti-neutrophil cytoplasmic antibodies (pANCA): Whereas between 65% and 92% of patients with type 1 AIH have pANCA, these antibodies are never observed in type 2 AIH [1]. pANCA are also seen in PSC and ulcerative colitis (UC) as well as a small percentage of patients with chronic hepatitis C. Their titers are higher in AIH and PSC than in UC. There are some indications that anti-neutrophil antibodies in AIH and PSC frequently do not, in fact, recognize a cytoplasmic antigen, but a 50 kDa myeloid-specific nuclear envelope protein. Hence, the designation peripheral antineutrophil nuclear antibodies (p-ANNA) has been proposed. Anti-LKM antibodies are serological markers of type 2 AIH, but can also be found in some patients with chronic hepatitis type C. In type 2 AIH, the majority of anti-LKM antibodies bind to the LKM1 antigen, which has been identified as the cytochrome monooxygenase
2. Autoimmune hepatitis (AIH) 2.1. Clinical features of AIH AIH is an inflammatory chronic liver disease that is characterized by a higher incidence in females, the presence of serum autoantibodies, hyper IgG globulinemia, and plasma cell infiltration into liver tissue, where periportal or interface hepatitis is the characteristic histological finding. AIH can be classified into three different types based on the presence or absence of certain autoantibodies. Whereas type 1 AIH is characterized by antinuclear antibodies (ANA) and/or smooth muscle antibodies (SMA), the typical antibodies of type 2 AIH are directed against liver kidney microsome type 1 (LKM1) and/or liver cytosol type 1 (LC1). The presence of anti-soluble liver antigen/liver pancreas antigen antibodies (anti-SLA/LP antibodies) has been proposed to characterize a third type of AIH, but this is controversial since anti-SLA/LP-positive patients do not exhibit distinct clinical features. ANA, SMA, and antiLKM1 are neither organ- nor disease-specific, whereas anti-LC1 antibodies are both, and anti-SLA/LP antibodies are specific to AIH. For the treatment of AIH, corticosteroids are known to be effective.
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P450 IID6 (CYP2D6) with an MW of 50 kDa consisting of 263–270 amino acids. These antibodies recognize a short linear epitope on CYP2D6 and inhibit its enzymatic activity in vitro, but not in vivo. In addition, this epitope is also targeted by liver-infiltrating CD4+ T cells. Some AIH type 2 patients (∼ 17%), but not those with AIH type 1, were found to develop autoantibodies against uridine diphosphate glucuronosyltransferase (UGT), designated as LKM3 [2]. These antibodies recognize several UGT isoforms, but show the strongest reactivity with UGT1A1, the isoenzyme mainly involved in bilirubin glucuronidation. Although antiKLM3 antibodies inhibit UGT activity in vitro, the serum bilirubin levels of patients with anti-KLM3 antibodies remain within the normal range. Anti-LC1 antibodies are another serologic marker of type 2 AIH, frequently accompanying anti-LKM1 antibodies. They are seen in 30–50% of patients with type 2 AIH, but not in patients with PBC or viral hepatitis. Their target antigen is formiminotransferase cyclodeaminase (FTCD), a liver-specific metabolic enzyme that has a role in converting glutamic acid into histidine. Anti-LC1 antibodies are capable of binding to different epitopes on the antigen molecule, with preferential recognition of the FT region [3]. In contrast to LKM1 concentrations, anti-LC1 antibody levels are associated with disease activity. From 10% to 30% of AIH patients, including some with PBC/AIH overlap syndrome, have anti-SLA/LP antibodies [4]. These antibodies recognize the UGAsuppressor serine tRNA-associated protein [4] and are highly specific for AIH [4]. Anti-SLA-/LP have never been detected in anti-LKM1-positive AIH patients [4]. Though still controversial, these antibodies have been proposed to characterize a third type of AIH. The asialoglycoprotein receptor (ASGPR) is a component of liver specific lipoprotein (LSP), which is a macromolecular complex expressed on hepatocyte membranes. Anti-ASGPR antibodies appear in 50–76% of patients with all types of AIH and in 85–88% of patients with active disease, and frequently accompany ANA, SMA and anti-LKM1 antibodies. Anti-ASGPR antibodies are also observed in PBC, PSC, and viral hepatitis with a variety of antibody titers, but with significantly lower positivity rates than are seen in AIH patients. The titer of anti-ASGPR antibodies correlates with disease activity and poor prognosis of AIH and might serve as a useful marker for assessing therapy outcomes. Although it largely remains to be elucidated whether antibodies in AIH play a pathogenic role, antibodydependent cell-mediated cytotoxicity (ADCC) and/or
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complement-dependent cytotoxicity (CDC) may possibly be involved in the hepatocyte damage in AIH. Recently, an IgM antibody produced by a clone obtained from a type 1 AIH patient was found to induce CDC in human hepatocellular carcinoma cell lines. This monoclonal autoantibody recognized a 190 kDa molecule expressed on hepatocyte membrane, and autoantibodies to this hepatocyte membrane antigen were also found in other AIH patients, but not in type C hepatitis patients. This antibody cross-reacted with a 190-kDa molecule on murine hepatocytes and, upon injection into mice, resulted in deposition of IgM and C3 on hepatocyte membranes [5]. This was followed by acute liver injury as evidenced by histological changes in periportal and pericentral areas and by elevated ALT and AST levels. 2.3. Immunogenetics in AIH HLA-DR3 (DRB1⁎0301) and DR4 (DRB1⁎0401) are associated with type 1 AIH [6]. In particular, the HLA-A1-B8-DR3 haplotype shows strong associations with early onset of disease, relapse, and the necessity for liver transplantation. In contrast, susceptibility to type 2 AIH is associated with carriage of HLA-DR7 [7]. Genes outside the HLA loci also appear to contribute to disease susceptibility and/or severity. Thus, deficiency of complement C4 due to a gene deletion is related to increased disease severity in both types of AIH. Cytotoxic Tlymphocyte antigen 4 (CTLA-4, also known as CD152) gene polymorphism is related to the development of type 1 AIH, but not type 2 AIH [8]. CTLA-4 is one of the major negative regulators in T cell activation [9]. Expression of CTLA-4 on T cells is induced by their activation, but there is a subset of regulatory CD4+ T cells (Treg) that constitutively express this molecule. These findings suggest that there are immunogenetic differences in the pathogenesis of type 1 and type 2 AIH. 2.4. Cellular responses in AIH Th2 cytokines, which activate B cells and induce their differentiation into antibody-producing cells, dominate in AIH. CD4+ T cell clones established from AIH livers and stimulated with lectin exhibited markedly lower IFN-γ/IL-4 ratios compared to clones from livers from patients with non-AIH diseases. Moreover, whereas a small number of Th2 clones was detected among the AIH clones, none were found in the non-AIH ones. Since CTLA-4 regulates the activation of T cells and their differentiation into Th2 cells, it is possible that the CTLA-4 allele associated with AIH might be involved in this shift of the AIH cytokine
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pattern towards Th2 predominance. In fact, serum levels of IL-4 (involved in the differentiation of B cells) and soluble CD23 (a marker for humoral immunity) are high in AIH and correlate positively with each other. It was suggested more than two decades ago that defective suppressor cell activity contributes to the development of AIH. More recently, AIH patients were found to have significantly fewer peripheral CD4+CD25+ Treg than healthy controls, and their Treg exhibited decreased proliferative activity in response to CD3/CD28 stimulation [10]. Their regulatory function, measured as the ability to suppress intracellular IFN-γ levels of CD4+CD25− T cells, was not affected. In contrast, Treg obtained from AIH at diagnosis, but not in remission, failed to inhibit CD8+ T cell proliferation and cytokine production. Of note, the number of Treg negatively correlated with the amount of anti-LKM or anti-SLA antibodies [10]. CTLA4 is normally expressed in Treg, and an association between CTLA-4 polymorphism and AIH has been reported [8]. This raises the possibility that the CTLA-4 polymorphism causes the Treg abnormality, which then allows the appearance of autoreactive B cells producing autoantibodies. 3. PBC 3.1. Clinical features of PBC PBC is a chronic cholestatic liver disease, in which interlobular bile ducts are damaged. It is characterized by AMA (90–95% of patients) and hyper IgM globulinemia, and affects predominantly females [11]. 3.2. Serology of PBC AMA are detected in 90–95% of PBC patients and are virtually diagnostic or presage the development of PBC within the following 5–10 years. They are directed against members of the 2-oxoacid dehydrogenase complexes (2-OADC) existing in the inner membrane of mitochondria. Among them, the major autoantigen is the E2 subunit of the pyruvate dehydrogenase complex (PDC-E2) [12]. The epitopes for this antibody localize to three domains of PDC-E2 component: (1) the inner/ outer lipoyl domain; (2) the E3 binding domain; and (3) the catalytic and E2-binding domain [12–14]. AntiPDC-E2 antibodies are capable of inhibiting PDC-E2 enzyme activity [12,13]. Targeting of enzymes is a common feature of autoantibodies detected in patients with autoimmune diseases, as is the inhibition of their activity by these autoantibodies. It has been shown that CD4+ and CD8+ T cell clones from both liver-
infiltrating lymphocytes and peripheral blood of PBC patients also recognize PDC-E2 [12,15,16]. Importantly, the B and T cell epitopes overlap, which not only suggests that the immune response to this enzyme is of pathological relevance, but also underscores the importance of interactions between these cell types and subtypes in the pathogenesis of PBC. Approximately 50% of PBC patients develop ANA. Certain targets of these antibodies are highly specific for PBC, in particular gp210 and p62 of the nuclear pore complex, which result in a perinuclear pattern in indirect immunofluorescence studies; and Sp100, which yields a multiple nuclear dot pattern. The presence of anti-gp210 antibodies correlates with disease activity and severity. Immunoreactive gp210 could be detected in the nuclear envelope of biliary epithelial cells (BEC) in small bile ducts of almost all (98.6%) PBC liver specimens, but in significantly fewer AIH and viral hepatitis specimens. Of note, the gp210 staining intensity in PBC biopsy samples correlated with the degree of interface hepatitis and lobular inflammation. One of the central questions in PBC research is why the autoimmune attack is focused on the biliary epithelium, even though every cell has mitochondria. In cholangiocytes, unlike other somatic cells, apoptosis does not induce attachment of glutathione to the lysinelipoyl domain of mitochondria's inner lipoyl domain. This prevents cleavage of PDC-E2 by caspases and allows its continued recognition by autoantibodies [11,17]. Mucosal epithelial cells are also distinguished functionally from other epithelial cells in that they transcytose IgA. Transcytosis of this Ig subclass is mediated by the polymeric immunoglobulin receptor (pIgR), and co-localization of PDC-E2 and IgA has been observed in epithelial cells transfected with this receptor [18]. This raises the possibility that IgA-PDC-E2 immune complexes could form during transcytosis of IgA and be transported to the bile duct lumen. Furthermore, there are data indicating that transcytosis of AMA-IgA causes caspase activation, resulting in apoptosis of bile duct epithelium [19]. Recent findings have shown that TLR signaling is important in B cell autoimmunity [20]. We recently reported that PBMC from PBC patients produced significantly more IgM than healthy controls when stimulated with CpG-B, a ligand of TLR9 [21]. Moreover, the amount of IgM produced per memory B cell was higher in PBC patients than in controls. The expression not only of TLR9 but also of CD86 (B7-2; an activation marker for B cells) and CD38 (a differentiation marker for B cells) increased on these memory B cells. This suggests that enhanced responsiveness of memory B
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cells to bacterial products like CpG may be one of the reasons for elevated serum IgM levels in PBC. Of note, CpG-B-stimulated PBMC from PBC patients produced markedly larger amounts of AMA compared to unstimulated controls, implicating the TLR9 signaling pathway in increased autoantibody production. 3.3. Immunogenetics of PBC A genetic component to PBC is suggested by the familial clustering of the disease and its high concordance rate (63%) in monozygotic twins. Despite considerable efforts, however, no consistent associations have been detected between susceptibility to PBC and carriage of particular HLA alleles [22]. Few associations with genes outside the MHC have emerged, although polymorphisms in the vitamin D receptor and the CTLA-4 genes have been implicated in disease susceptibility. X-chromosome monosomy of PBMC may explain the female preponderance of the disease [11]. 3.4. Cellular responses of PBC Eosinophilia is a characteristic finding in PBC patients. Whereas 90% of stage 3 or 4 PBC patients
Low frequency of Treg
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express IL-5 mRNA in the liver, AIH patients and healthy controls express none [23]. The deposit of major basic protein (MBP), in particular, is observed within the portal inflammatory infiltrate. MBP is released from eosinophils, which are activated by IL-5, and has the ability to cause epithelial cell lysis [23]. Serum levels of TNF-α and TGF-β were found to increase with increasing severity of PBC and to decrease after treatment with ursodeoxycholic acid, suggesting a role for both of these cytokines in the progression of the disease. The proportion of Treg in PBMC is lower in PBC patients compared to type C hepatitis patients, as is the FoxP3/CD3+T ratio in liver-infiltrating lymphocytes (R. Lan et al., in press). Of note, the liver infiltrate was found to contain larger numbers of PDC-specific B cells than PBMC. Together, these findings may suggest that a decrease in the number of Treg in the liver might contribute to the induction of autoreactive B cells, resulting in increased AMA production. As in the case of AIH, it has been suggested that CTLA-4 polymorphism contributes to impaired Treg function in PBC, resulting in a decreased B cell regulation and, thus, enhancement of B cell autoimmunity. A possible pathophysiological model for PBC focusing on the roles of B cells is shown in Fig. 1.
Treg Autoreactive B Cell
AMA and ANA production
B
B
Molecular mimicry
NKT
B Interlobular Bile Duct BEC apoptosis
B B B
Transcytosis of dimeric IgA
APC
B
Autoreactive CD4+ T Cell proliferation
CTL
Th
CTL
Th
Th
Autoreactive CD8+ T Cell
BacteriaI or Xenobiotic molecules Mitochondrial autoantigen
PBC Liver Treg Fig. 1. A model of immunopathology of PBC.
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4. PSC 4.1. Clinical features of PSC PSC is a chronic inflammatory disease of large bile ducts, often associated in patients with inflammatory bowel disease, with the vast majority (87%) having UC and only 13% having Crohn's disease [24]. In contrast, only 5% of UC patients have PSC. The development and progression of PSC are not influenced by the concomitant presence of UC. 4.2. Autoantibodies in PSC As many as 88% of PSC patients exhibit ANCA [25]. ANCA are not specific for PSC, since they are also observed in a substantial portion of patients with UC and AIH, a smaller percentage of patients with Crohn's disease and about 5% of patients with PBC. Whether these antibodies have a pathological role is still not known. Targets of ANCA in PSC include h-lamp-2, proteinase-3, and bactericidal/permeability increasing protein and only rarely myeloperoxidase. Indirect immunofluorescence staining for the detection of antineutrophil antibodies in PSC frequently does not yield the homogeneous rim-like pattern typical for classical pANCA, but an atypical heterogeneous rim-like staining of the nuclear periphery and multiple intra-nuclear spots. This and other evidence indicates that the antigen (s) recognized by these atypical p-ANCA or p-ANNA is probably located in the nucleus and is most likely a neutrophil-specific 50 kDa nuclear envelope protein. As in AIH and PBC, ANA can be detected in a substantial portion of PSC patients (53%), whereas AMA are absent. In addition, almost two thirds of PSC patients exhibit anti-cardiolipin antibodies, and their concentrations correlate positively with histological changes and disease severity. 4.3. Immunogenetics of PSC As in AIH, HLA-B8 and DR3 confer susceptibility to PSC, especially B8-MICA5.1-MICB24-DR3. However, it remains controversial whether CTLA-4 polymorphism is associated with disease susceptibility. 4.4. Cellular responses of PSC Blood levels of TNF-α (a pro-inflammatory cytokine), IL-10 (an anti-inflammatory cytokine), and IL8 (a neutrophil activation marker) were found to be significantly higher in PSC compared to patients with
alcoholic cirrhosis or normal controls. In contrast, IFNγ (an inducer of cell-mediated immunity) and GM-CSF (a marker of monocyte/macrophage function) were within the normal range. No correlation was found between these cytokines and positivity for, or titer of, ANCA. 5. Autoreactive B cells in autoimmune liver diseases In PBC liver, the number of infiltrating B cells is higher than that of PSC patients and normal controls. Moreover, it has been reported that the proportion of CD19+CD69+ activated B cells was markedly higher in the liver than in peripheral blood of PBC patients, and that the number of AMA-producing cells was 5 times in the liver than in PBMC. It has been proposed that such IgA-AMA-producing cells might be directly involved in causing damage to bile ducts in PBC livers [18,19]. It has not yet been clarified how the homing of B-1 and B-2 cells into the liver is regulated; however, CXCL13/CXCR5 is known to play a critical role. In SLE-prone NZB/NZW F1 mice, for example, the expression level of CXCL13 increased with age and correlated strongly with the number of B220+ B cells infiltrating the thymus and kidney. The major sources of CXCL13 are CD11b+CD11c+ cells, which are myeloid dendritic cells (mDC). In the liver of B6 mice, about 10% of NK1.1−CD11c+DC consist of mDC [26]. LPS can induce CXCL13 production in human monocytederived DC. Stimulation with LPS + IL-10 of mDC and plasmacytoid DC (pDC) enhances the production of CXCL13 [27]. The expression of the CXCL13 ligand, CXCR5, is higher in B-1 cells than in B-2 cells. It has been reported that B-1a cells can differentiate into splenic plasma cells when the cells are transferred into T cell and B cell-deficient RAG knockout mouse [28]. Together, these findings suggest that stimulation of mDC + pDC with bacterial products (possibly in the presence of IL-10) present in the liver of patients with autoimmune diseases and the resulting CXCL13 production could lead to the accumulation of CXCR5bearing autoantibody-producing B-1 cells in the target organ. 6. Toll-like receptors (TLR) TLRs and their ligands are mediators of innate immune responses that may be involved in the pathogenesis of autoimmune liver diseases. Both human and murine hepatocytes, targeted in AIH, and cholangiocytes, targeted in PBC and PSC, express TLRs on their cell surface (Table 1). Hepatocytes in AIH and
Y. Moritoki et al. / Autoimmunity Reviews 5 (2006) 449–457 Table 1 Expression of TLRs in B cells, hepatocytes and cholangiocytes Receptor B cells
Hepatocytes
Humans Mice Humans TLR1 TLR2 TLR3 TLR4
+ − − −
− + + +
TLR5 TLR6 TLR7 TLR8 TLR9 TLR10 TLR11 Ref.
− + + + + (PBC) + ND [21,32]
− + + ND + ND ND [33]
+ + + (PBC) + (Periportal, PBC) + + + + + ND ND [34,35]
Cholangiocytes Mice
Humans
Mice
ND + ND +
+/− + + + (PBC)
ND + + +
ND ND ND ND + ND ND [36,37]
+ +/− +/− +/− +/− +/− ND [35,38,39]
+ ND ND ND ND ND ND [38]
ND: not determined.
cholangiocytes in PBC and PSC all express MHC-class II molecules, whereas normal hepatocytes and cholangiocytes do not. These cells also show aberrant expression of CD80/CD86, which is a co-stimulatory molecule required for antigen presentation [29]. Based on these findings, it has been speculated that these cells may acquire antigen-presenting activity via TLRs signaling pathways. In fact, it was recently shown that B6 mice who received multiple intraperitoneal injections of the TLR3 ligand, poly I:C, developed PBC, including its characteristic serological markers, AMA and ANA. In these animals, mononuclear cells infiltrated the portal areas and caused damage to bile ducts, although ALP level remained normal. In liver parenchymal and portal vein areas of PBC patients, expression of both TLR3-and type I IFNmRNA is higher than in those of AIH or chronic hepatitis type C patients. The expression levels of these two genes correlated closely, and a positive association was also observed between type I IFN-mRNA and serum levels of ALP. This suggests that in PBC, some viruses might be
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triggering TLR gene expression. Immunohistochemical studies indicate that BEC as well as periportal hepatocytes of PBC livers express TLR4. Moreover, the expression level of TLR4 showed a positive correlation with the intensity of fibrosis in the liver. BEC are much more responsive to bacterial components compared to hepatocytes. It was found that TLR signaling appeared when human BEC cells obtained from chronic hepatitis C and PBC patients were stimulated with LPS or Escherichia coli, although none was observed in the HepG2 cell line [30]. In addition, stimulation of murine BEC and human intrahepatic cholangiocarcinoma cell lines with LPS or E. coli induced NF-κB activation and TNF-α production. These findings may be particularly relevant to PSC liver, which is always exposed to bacterial components derived from the intestine. This repeated stimulation with LPS and other bacterial products may result in the enhancement of the production of pro-inflammatory cytokines such as TNF-α. In fact, liver T cells in PSC produce higher levels of TNF-α than those of PBC or AIH patients, and this may be the cause of periductular fibrosis in PSC. 7. Fibrosis in autoimmune liver diseases: possible involvement of B cells Fibrosis and cirrhosis are associated with the progression of autoimmune liver diseases, particularly with PBC and PSC. Excessive production of extracellular matrix by periductular and periportal fibroblasts causes these pathological changes. Cytokines such as TNF-α and TGF-β, as well as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are involved in fibrogenesis. Some recent studies have indicated that B cells play a critical role in the development of fibrosis. In B cell-depleted mice, fibrosis did not develop subsequent to liver injury induced by carbon tetrachloride (CCl4). Similarly, in α-naphthylisothiocyanate (ANIT)-induced liver injury, B cell
Table 2 Autoantibodies and identified autoantigen epitopes in autoimmune liver diseases [40]
Auto-Abs
B cell infiltrate in the liver
Type I AIH
Type II AIH
PBC
PSC
ANA: centromere, ribonucleoprotein, cyclin A, histone; SMA: F actin, anti-ASGPR, anti-SLA/LP;pANCA: myeloid-specific nuclear envelop protein + (with plasma cells)
Anti-LKM-1: CYP450 2D6; anti-LC-1: formiminotransferase cyclo-deaminase
AMA: PDC-E2, BCOADC-E2, OGDC-E2, PDC-E3BP, PDC-E1α; ANA: nuclear pore complex, anti-gp210, anti-p62, nuclear dot pattern, anti-Sp100, anti-PML, anti-SUMOs
pANCA: myeloid-specificnuclear envelopprotein; ANA: anti-cardiolipin, thyroperoxidase, rheumatoid factor, H. pylori IgG
+ (with plasma cells)
+ (with plasma cells)
+/−
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depletion inhibited the development of fibrosis [31]. Moreover, in tight skin mice, an animal model for systemic sclerosis, CD19 deficiency suppressed skin fibrosis, although it was not clarified if this was antibody-dependent. Together, these findings suggest that liver-infiltrating autoreactive B cells are involved in the fibrogenesis of autoimmune liver diseases. The mechanism of suppressing fibrinosis by B cell depletion is independent of antibodies or T cells, raising the possibility that cytokines, as well as MMPs and TIMPs, produced or induced by autoimmune B cells, are responsible for fibrosis in autoimmune diseases targeting the liver. 8. Summary Although the mechanisms of cell damage in AIH, PBC, or PSC are not yet fully understood, there is increasing evidence that autoimmunity may be responsible for the pathogenesis of at least AIH and PBC [7,14,24]. The variety of autoantibodies that develop in these three diseases is listed in Table 2. It seems plausible that autoantibodies are involved in the pathogenesis of AIH, but direct evidence is largely lacking. In PBC, it has been suggested that IgA autoantibodies may contribute to liver cell damage and that autoreactive B cells also acts as APC for T cell priming. As for PSC, although various immunological abnormalities have been reported, there is not enough evidence to support the hypothesis that this disease is dependent on autoimmunity. The possibility that autoantibodies contribute to the progression of PBC and AIH is supported by the fact that ursodeoxycholic acid (UDCA) and prednisolone, which are the currently available first choice drugs, are able to suppress the production of AMA and/or immunoglobulin in a dose-dependent manner in the culture of PBMC. This suggests that immunosuppressive therapy regulating B cell activation would be useful in treating patients with PBC and AIH. Take-home messages • Antibody-dependent cell-mediated cytotoxicity and/ or complement-dependent cytotoxicity (CDC) may contribute to the pathogenesis of AIH. • Anti-PDC-E2 of the IgA subclass as well as the priming of autoreactive T cells by autoreactive B cells may contribute to the pathogenesis of PBC. • Despite the presence of serum autoantibodies and other humoral abnormalities, the involvement of B cell autoimmunity in the PSC pathogenesis is still unclear.
• Autoreactive B cells may accumulate in the target organ via CXCL13/CXCR5-dependent mechanisms. • TLRs signaling pathways appear to play an important role in autoimmune liver diseases. • Activation of B cells via TLRs and impairment of Treg numbers and function may exert additive effects on the expansion of autoreactive B cells in autoimmune liver diseases. References [1] Zauli D, Ghetti S, Grassi A, Descovich C, Cassani F, Ballardini G, et al. Anti-neutrophil cytoplasmic antibodies in type 1 and 2 autoimmune hepatitis. Hepatology 1997;25:1105–7. [2] Bachrich T, Thalhammer T, Jager W, Haslmayer P, Alihodzic B, Bakos S, et al. Characterization of autoantibodies against uridinediphosphate glucuronosyltransferase in patients with inflammatory liver diseases. Hepatology 2001;33:1053–9. [3] Muratori L, Sztul E, Muratori P, Gao Y, Ripalti A, Ponti C, et al. Distinct epitopes on formiminotransferase cyclodeaminase induce autoimmune liver cytosol antibody type 1. Hepatology 2001;34:494–501. [4] Wies I, Brunner S, Henninger J, Herkel J, Kanzler S, Meyer zum Buschenfelde KH, et al. Identification of target antigen for SLA/LP autoantibodies in autoimmune hepatitis. Lancet 2000;355:1510–5. [5] Yamauchi K, Yamaguchi N, Furukawa T, Takatsu K, Nakanishi T, Ishida K, et al. A murine model of acute liver injury induced by human monoclonal autoantibody. Hepatology 2005;42:149–55. [6] Czaja AJ, Donaldson PT. Genetic susceptibilities for immune expression and liver cell injury in autoimmune hepatitis. Immunol Rev 2000;174:250–9. [7] Vergani D, Mieli-Vergani G. Autoimmune hepatitis. Autoimmun Rev 2003;2:241–7. [8] Djilali-Saiah I, Ouellette P, Caillat-Zucman S, Debray D, Kohn JI, Alvarez F. CTLA-4/CD 28 region polymorphisms in children from families with autoimmune hepatitis. Hum Immunol 2001;62:1356–62. [9] Teft WA, Kirchhof MG, Madrenas J. A molecular perspective of CTLA-4 function. Annu Rev Immunol 2006;24:3.1–.33. [10] Longhi MS, Ma Y, Bogdanos DP, Cheeseman P, Mieli-Vergani G, Vergani D. Impairment of CD4(+)CD25(+) regulatory T-cells in autoimmune liver disease. J Hepatol 2004;41:31–7. [11] Kaplan MM, Gershwin ME. Primary biliary cirrhosis. N Engl J Med 2005;353:1261–73. [12] Van de Water J, Gershwin ME, Leung P, Ansari A, Coppel RL. The autoepitope of the 74-kD mitochondrial autoantigen of primary biliary cirrhosis corresponds to the functional site of dihydrolipoamide acetyltransferase. J Exp Med 1988;167:1791–9. [13] Surh CD, Coppel R, Gershwin ME. Structural requirement for autoreactivity on human pyruvate dehydrogenase-E2, the major autoantigen of primary biliary cirrhosis. Implication for a conformational autoepitope. J Immunol 1990;144:3367–74. [14] Ishibashi H, Shimoda S, Gershwin ME. The immune response to mitochondrial autoantigens. Semin Liver Dis 2005;25:337–46. [15] Van de Water J, Ansari AA, Surh CD, Coppel R, Roche T, Bonkovsky H, et al. Evidence for the targeting by 2-oxo-dehydrogenase enzymes in the T cell response of primary biliary cirrhosis. J Immunol 1991;146:89–94. [16] Shimoda S, Nakamura M, Ishibashi H, Hayashida K, Niho Y. HLA DRB4 0101-restricted immunodominant T cell autoepitope
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Cell-penetrating anti-native DNA antibodies trigger apoptosis through both the neglect and programmed pathways It has been previously shown that certain antibodies to dsDNA are capable to transgress the cell membrane, translocate to the cell nucleus and react with their antigens. When so doing, they signal the penetration cells to undergo apoptosis. In this study, Rivadeneyra-Espinoza L and Ruize-Arguelles A. (J Autoimmun 2006; 26: 52-6) dissected the two main-streams of apoptotic triggering and found that the neglect, mitochondrial pathway, is definitely involved, but activation of caspases 2 and 8, classically known to be associated to membrane pathway, also occurs in a small proportion of cells. These results suggest that penetration anti-dsDNA antibodies, besides activating neglect apoptosis, may also trigger programmed cell death, perhaps initiated at the cell membrane level.