Cofactor-dependent conformational heterogeneity of GAD65 and its role in autoimmunity and neurotransmitter homeostasis.

The human neuroendocrine enzyme glutamate decarboxylase (GAD) catalyses the synthesis of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) using pyridoxal 5'-phosphate as a cofactor. GAD exists as two isoforms named according to their respective molecular weights: GAD65 and GAD67. Although cytosolic GAD67 is typically saturated with the cofactor (holoGAD67) and constitutively active to produce basal levels of GABA, the membrane-associated GAD65 exists mainly as the inactive apo form. GAD65, but not GAD67, is a prevalent autoantigen, with autoantibodies to GAD65 being detected at high frequency in patients with autoimmune (type 1) diabetes and certain other autoimmune disorders. The significance of GAD65 autoinactivation into the apo form for regulation of neurotransmitter levels and autoantibody reactivity is not understood. We have used computational and experimental approaches to decipher the nature of the holo → apo conversion in GAD65 and thus, its mechanism of autoinactivation. Molecular dynamics simulations of GAD65 reveal coupling between the C-terminal domain, catalytic loop, and pyridoxal 5'-phosphate-binding domain that drives structural rearrangement, dimer opening, and autoinactivation, consistent with limited proteolysis fragmentation patterns. Together with small-angle X-ray scattering and fluorescence spectroscopy data, our findings are consistent with apoGAD65 existing as an ensemble of conformations. Antibody-binding kinetics suggest a mechanism of mutually induced conformational changes, implicating the flexibility of apoGAD65 in its autoantigenicity. Although conformational diversity may provide a mechanism for cofactor-controlled regulation of neurotransmitter biosynthesis, it may also come at a cost of insufficient development of immune self-tolerance that favors the production of GAD65 autoantibodies.

Chemokine (C-X-C motif) ligand 13 promotes intrahepatic chemokine (C-X-C motif) receptor 5+ lymphocyte homing and aberrant B-cell immune responses in primary biliary cirrhosis.

UNLABELLED: The serological hallmark of primary biliary cirrhosis (PBC) is the presence of high titer and specific antimitochondrial antibodies (AMAs). Although there is no global immune defect in patients with PBC, there is widespread dysregulated B-cell function, including increased sera levels of immunoglobulin M and enhanced B-cell responses to cytosine-phosphate-guanine stimulation. The mechanisms involved in this B-cell dysfunction have remained unknown. To address this issue, we focused on identifying the frequencies of B-cell subsets in patients with PBC and the mechanisms that lead to B-cell dysregulation, including the relationships with chemokine (C-X-C motif) receptor 5 (CXCR5)(+) CD4(+) T cells. Herein, we report that elevations of both serum and intrahepatic interleukin-21 (IL-21) were found in patients with PBC and, in particular, promoted B-cell proliferation, signal transducer and activator of transcription 3 phosphorylation and AMA production in vitro. More important, upon stimulation with recombinant E2 subunit of pyruvate dehydrogenase complex, CXCR5(+) CD4(+) T cells in PBC produced higher levels of IL-21 than healthy controls. Additionally, sorted CXCR5(+) CD4(+) T cells increased production of AMAs by autologous CD19(+) B cells. Indeed, elevated expression of intrahepatic chemokine (C-X-C motif) ligand 13 (CXCL13), a key chemokine of CXCR5(+) cells, was uniquely found within the portal tracts in PBC, accompanied by infiltrates of CD4(+) , CXCR5(+) , CD19(+) , and CD38(+) cells. CONCLUSION: CXCL13 promotes aggregation of CD19(+) B cells and CXCR5(+) CD4(+) T cells, which directs the aberrant AMA response by IL-21. These data have implications for potential immunotherapy and also reflect the unique lymphoid biology in liver of PBC.

Ongoing activation of autoantigen-specific B cells in primary biliary cirrhosis.

UNLABELLED: The serologic hallmark of primary biliary cirrhosis (PBC), the antimitochondrial response to the E2 component of the pyruvate dehydrogenase complex (PDC-E2), has unique features, including continuous high titers of immunoglobulin M (IgM) and IgG reactivity throughout all stages of disease, capable not only of target enzyme inhibition, but also crossreactive with chemical xenobiotics that share molecular homology with the inner lipoyl domain of PDC-E2; such chemicals have been proposed as potential etiological agents. We used flow cytometry and enzyme-linked immunospot assay (ELISPOT) to examine B-cell subsets in 59 subjects, including 28 with PBC, 13 with primary sclerosing cholangitis (PSC), and 18 healthy controls. Strikingly, in PBC, although there were no significant differences in B-cell phenotype subpopulations, 10% of the total IgG and IgA plasmablast population and 23% of the IgM plasmablast population were uniquely reactive with PDC-E2, detected in the CXCR7+ CCR10low plasmablast population. In contrast, plasmablast reactivity to a control antigen, tetanus toxoid, was minimal and similar in all groups. Additionally, we isolated plasmablast-derived polyclonal antibodies and compared reactivity with plasma-derived antibodies and noted a distinct noncirculating tissue source of xenobiotic crossreacting antibodies. The high levels of autoantigen specific peripheral plasmablasts indicate recent activation of naive or memory B cells and a continuous and robust activation. The presence of CXCR7+ CCR10low PDC-E2-specific ASCs suggests a mechanistic basis for the migration of circulating antigen specific plasmablasts to the mucosal epithelial ligands CXCL12 and CCL28. CONCLUSION: Our findings suggest a sustained rigorous B-cell response in PBC, likely activated and perpetuated by cognate autoantigen.

NOD.c3c4 congenic mice develop autoimmune biliary disease that serologically and pathogenetically models human primary biliary cirrhosis.

Primary biliary cirrhosis (PBC) is an autoimmune disease with a strong genetic component characterized by biliary ductular inflammation with eventual liver cirrhosis. The serologic hallmark of PBC is antimitochondrial antibodies that react with the pyruvate dehydrogenase complex, targeting the inner lipoyl domain of the E2 subunit (anti-PDC-E2). Herein we demonstrate that NOD.c3c4 mice congenically derived from the nonobese diabetic strain develop an autoimmune biliary disease (ABD) that models human PBC. NOD.c3c4 (at 9-10 wk, before significant biliary pathology) develop antibodies to PDC-E2 that are specific for the inner lipoyl domain. Affected areas of biliary epithelium are infiltrated with CD3+, CD4+, and CD8+ T cells, and treatment of NOD.c3c4 mice with monoclonal antibody to CD3 protects from ABD. Furthermore, NOD.c3c4-scid mice develop disease after adoptive transfer of splenocytes or CD4+ T cells, demonstrating a central role for T cells in pathogenesis. Histological analysis reveals destructive cholangitis, granuloma formation, and eosinophilic infiltration as seen in PBC, although, unlike PBC, the extrahepatic biliary ducts are also affected. Using a congenic mapping approach, we define the first ABD (Abd) locus, Abd1. These results identify the NOD.c3c4 mouse as the first spontaneous mouse model of PBC.

BAFF/BLyS inhibitors: A new prospect for treatment of systemic lupus erythematosus.

In November 2009, Human Genome Sciences and Glaxo-Smith Kline [HGS (Rockville, Maryland) and GSK, respectively] announced that Belimumab, a neutralizing antibody to the tumour necrosis factor (TNF)-like ligand, B-cell activating factor (BAFF belonging to the TNF family, also named BLyS), met the primary endpoints in two phase III clinical trials in systemic lupus erythematosus (SLE, lupus). In March 2011, Belimumab was approved by the US Federal Drug Agency for treatment of SLE patients; this was followed in May with approval by the European Medicines Agency for use in the European Union. This is an exciting development as it is the first successful late-stage clinical trial in SLE in over 40 years. In the light of this breakthrough, we review the key data and research outcomes and examine how blocking BAFF in patients with SLE significantly improves clinical outcomes.

Autoimmune hepatitis: what must be said.

Autoimmune hepatitis (AIH) was first studied under its earlier name of "chronic active hepatitis" (CAH) from the 1950s, coincident with a renaissance of interest in autoimmunity. The definition of autoimmune serum reactants in disease, including CAH, gave new insights into chronic hepatitis and liver cirrhosis, and led to refinements of Burnet's clonal selection theory of acquired immunity, 1957-59. Various discoveries including serological reactants in CAH prompted its designation in 1965 as autoimmune hepatitis, and treatment with immunosuppressive drug regimens transformed outcomes and survival. Serological observations further indicated that AIH could exist as either of two types, clinically similar but genetically different: Type 1 aligned more with the non-organ-specific multisystem diseases, and the infrequent Type 2 more with the organ-specific diseases. However, events in either type that could explain the onset of autoimmunity in the normally tolerogenic milieu of the liver have not been discerned. In the genetically predisposed individual, initiation may depend on non-specific death of hepatocytes after which fragments derived from disordered apoptosis acquire the capacity for ongoing auto-immunogenic stimulation. Insufficiency in numbers and function of Treg populations appears important in the promotion of this autoimmune process.

GABA production by glutamic acid decarboxylase is regulated by a dynamic catalytic loop.

Gamma-aminobutyric acid (GABA) is synthesized by two isoforms of the pyridoxal 5'-phosphate-dependent enzyme glutamic acid decarboxylase (GAD65 and GAD67). GAD67 is constitutively active and is responsible for basal GABA production. In contrast, GAD65, an autoantigen in type I diabetes, is transiently activated in response to the demand for extra GABA in neurotransmission, and cycles between an active holo form and an inactive apo form. We have determined the crystal structures of N-terminal truncations of both GAD isoforms. The structure of GAD67 shows a tethered loop covering the active site, providing a catalytic environment that sustains GABA production. In contrast, the same catalytic loop is inherently mobile in GAD65. Kinetic studies suggest that mobility in the catalytic loop promotes a side reaction that results in cofactor release and GAD65 autoinactivation. These data reveal the molecular basis for regulation of GABA homeostasis.

The autoimmunity of primary biliary cirrhosis and the clonal selection theory.

Primary biliary cirrhosis (PBC) is a chronic cholestatic liver disease in which an immune-mediated injury targets the small intrahepatic bile ducts. PBC is further characterized by highly specific serum antimitochondrial autoantibodies (AMAs) and autoreactive T cells, a striking female predominance, a strong genetic susceptibility and a plethora of candidate environmental factors to trigger the disease onset. For these reasons, PBC appears ideal to represent the developments of the clonal selection theory over the past decades. First, a sufficiently potent autoimmunogenic stimulus in PBC would require the coexistence of numerous pre-existing conditions (mostly genetic, as recently illustrated by genome-wide association studies and animal models) to perpetuate the destruction of the biliary epithelium by the immune system via the persistence of forbidden clones. Second, the proposed modifications of mitochondrial autoantigens caused by infectious agents and/or xenobiotics well illustrate the possibility that peculiar changes in the antigen structure and flexibility may contribute to tolerance breakdown. Third, the unique apoptotic features shown for cholangiocytes are the ideal setting for the development of mitochondrial autoantigen presentation to the immune system through macrophages and AMA; thus, turning the non-traditional mitochondrial antigen into a traditional one. This article will review the current knowledge on PBC etiology and pathogenesis in light of the clonal selection theory developments.

Stiff-person syndrome (SPS) and anti-GAD-related CNS degenerations: protean additions to the autoimmune central neuropathies.

Stiff Person Syndrome (SPS) is a rare autoimmune neurological disease attributable to autoantibodies to glutamic acid decarboxylase (anti-GAD) more usually associated with the islet beta cell destruction of autoimmune type 1 diabetes (T1D). SPS is characterized by interference in neurons with the synthesis/activity of the inhibitory neurotransmitter gamma amino butyric acid (GABA) resulting in the prototypic progressive spasmodic muscular rigidity of SPS, or diverse neurological syndromes, cerebellar ataxia, intractable epilepsy, myoclonus and several others. Remarkably, a single autoantibody, anti-GAD, can be common to widely different disease expressions, i.e. T1D and SPS. One explanation for these data is the differences in epitope engagement between the anti-GAD reactivity in SPS and T1D: in both diseases, anti-GAD antibody reactivity is predominantly to a conformational epitope region in the PLP- and C-terminal domains of the 65 kDa isoform but, additionally in SPS, there is reactivity to conformational epitope(s) on GAD67, and short linear epitopes in the C-terminal region and at the N-terminus of GAD65. Another explanation for disease expressions in SPS includes ready access of anti-GAD to antigen sites due to immune responsiveness within the CNS itself according to intrathecal anti-GAD-specific B cells and autoantibody. Closer study of the mysterious stiff-person syndrome should enhance the understanding of this disease itself, and autoimmunity in general.

CX3CL1 (fractalkine): a signpost for biliary inflammation in primary biliary cirrhosis.

UNLABELLED: Improvements in the treatment of primary biliary cirrhosis (PBC) may depend upon dissection of mechanisms that determine recruitment of mononuclear cells to intralobular bile ducts, including the role of the chemokine-adhesion molecule CX3CL1 (fractalkine). We submit that there are unique interactions between intrahepatic biliary epithelial cells (BECs), endothelial cells (ECs), liver sinusoidal endothelial cells (LSECs), and liver-infiltrating mononuclear cells (LMCs), and that such interactions will in part dictate the biliary-specific inflammatory response. To address this, we studied fresh explanted livers from pretransplantation patients with PBC and with inflammatory liver disease due to viral infection (disease controls) and biopsy material from patients with a discrete liver tumor (normal controls). Using this clinical material, we isolated and stimulated BECs, ECs, LSECs, and LMCs with a panel of Toll-like receptor ligands. We also studied the interactions of these cell populations with LMCs with respect to adhesion capability and production of tumor necrosis factor alpha (TNF-alpha). Finally, we used fresh biopsy samples to evaluate mononuclear cells around intrahepatic biliary ductules using monoclonal antibodies specific to CD68 or CD154, markers for monocytes/macrophages, and activated T cells, respectively. CONCLUSION: There are common properties of ECs, LSECs, and BECs, whether derived from PBC or viral hepatitis, but there are also significant differences, particularly in the potential in PBC for LMCs to adhere to ECs and BECs and to produce TNF-alpha; such properties were associated with augmented CX3CL1 production by BEC from PBC liver. The processes defined herein suggest potential novel biotherapies for biliary specific inflammation.

B cells suppress the inflammatory response in a mouse model of primary biliary cirrhosis.

BACKGROUND & AIMS: Mice that express a dominant-negative form of transforming growth factor-beta receptor restricted to T cells (dnTGF-betaRII) develop antimitochondrial antibodies and liver inflammation similar to human primary biliary cirrhosis. METHODS: To address the role of B cells in this model of primary biliary cirrhosis, we bred B cell-deficient mice (Igmu(-/-)) with dnTGF-betaRII mice, creating Igmu(-/-)dnTGF-betaRII mice, and compared the resulting disease phenotype with that of dnTGF-betaRII mice (controls). We also performed adoptive transfer of dnTGF-betaRII CD8(+) splenocytes, with or without B cells, to 8-week-old female Rag-1(-/-) mice to assess the role of B cells in the inflammatory response. RESULTS: The B cell-deficient Igmu(-/-)dnTGF-betaRII mice unexpectedly developed a more severe form of cholangitis than controls (dnTGF-betaRII mice) and had a significantly greater frequency of activated CD4(+) and CD8(+) T cells in the liver. They also had reduced frequency of Foxp3(+) regulatory T cells in the hepatic CD4(+) T-cell population and natural killer (NK) T cells (NK1.1(+) CD3(+)) in hepatic inflammatory cell infiltrates. The Igmu(-/-)dnTGF-betaRII mice had increased levels of proinflammatory cytokines (tumor necrosis factor-alpha and interleukin-6) and developed a more severe form of colitis than controls. Adoptive transfer of CD8(+) splenocytes from dnTGF-betaRII mice and peritoneal cavity-derived, but not spleen-derived, CD19(+) B cells into Rag-1(-/-) mice resulted in decreased amounts of liver inflammation and bile duct damage, compared with Rag-1(-/-) mice in which only CD8(+) splenocytes were transferred. CONCLUSION: B cells have a suppressive effect on the inflammatory response in the dnTGF-betaRII model of primary biliary cirrhosis.

The odd couple: a fresh look at autoimmunity and immunodeficiency.

The paradoxical relationship between immunodeficiency (under-responsiveness) and autoimmunity (over-responsiveness) affecting the immune system was debated at the Fourth AARDA Colloquium on cross-disciplinary issues in autoimmunity. Immunodeficiency disease and autoimmune disease, far from being mutually exclusive, share profound dysregulation of the immune system. Among the most keenly discussed issues were: i) the remarkably high number of molecularly identified primary immunodeficiencies with autoimmune expressions; ii) the homeostasis of immune function such that deficiency in any one given compartment can result in over activity in the same or another compartment; iii) whilst some immune deficiency states are essentially monogenic, each of them can exhibit striking variability in autoimmune outcome, indicative of epigenetic or environmental influences on phenotypic expression; iv) innate immunity, particularly complement defects, as well as adaptive immunity, is complicit in the immunodeficiency-autoimmunity axis; v) features of certain of the disorders discussed at the meeting forced a reappraisal of what actually is meant by 'autoimmune disease'. It was concluded that genes that determine inherited immunodeficiencies, hitherto rather neglected by autoimmunologists, compel attention to consideration of molecular genetic anomalies critical for emergence of autoimmune disease in humans and animals.

Apotopes and the biliary specificity of primary biliary cirrhosis.

UNLABELLED: Primary biliary cirrhosis (PBC) is characterized by antimitochondrial antibodies (AMAs), directed to the E2 component of the pyruvate dehydrogenase complex (PDC-E2). Notwithstanding the presence of mitochondria in virtually all nucleated cells, the destruction in PBC is limited to small intrahepatic bile ducts. The reasons for this tissue specificity remain unknown, although biliary epithelial cells (BECs) uniquely preserve the PDC-E2 epitope following apoptosis. Notably, PBC recurs in an allogeneic transplanted liver, suggesting generic rather than host PBC-specific susceptibility of BEC. We used cultured human intrahepatic BECs (HIBECs) and other well-characterized cell lines, including, HeLa, CaCo-2 cells, and nontransformed human keratinocytes and bronchial epithelial cells, to determine the integrity and specific localization of PDC-E2 during induced apoptosis. All cell lines, both before and after apoptosis, were tested with sera from patients with PBC (n = 30), other autoimmune liver and rheumatic diseases (n = 20), and healthy individuals (n = 20) as well as with a mouse monoclonal antibody against PDC-E2 and AMA with an immunoglobulin A isotype. PDC-E2 was found to localize unmodified within apoptotic blebs of HIBECs, but not within blebs of various other cell lineages studied. The fact that AMA-containing sera reacted with PDC-E2 on apoptotic BECs without a requirement for permeabilization suggests that the autoantigen is accessible to the immune system during apoptosis. CONCLUSION: Our data indicate that the tissue (cholangiocyte) specificity of the autoimmune injury in PBC is a consequence of the unique characteristics of HIBECs during apoptosis and can be explained by exposure to the immune system of intact immunoreactive PDC-E2 within apoptotic blebs.