ABSTRACT
Autoantibodies targeting epitopes contained within the intracellular domain (IC) of the protein phosphatase-like islet antigen 2 (IA-2) are a common marker of autoimmune type 1 diabetes (T1D), however the isolation of genuine, serum derived anti-IA-2 autoantibodies has proven challenging due to a lack of suitable bioassays. In the current study, an ELISA format was developed for affinity purification of human anti-IA-2ic autoantibodies utilizing a fusion protein (FP) incorporating maltose binding protein and the full-length IA-2IC domain. Using a T1D patient cohort validated for anti-IA-2ic autoantibodies by commercial ELISA, we demonstrate the MBP-IA-2ic FP ELISA detects serum anti-IA-2IC autoantibodies from 3 of 9 IA-2 positive patients. Further to this, a multi-plate MBP-IA-2ic FP ELISA protocol specifically affinity purifies IgG enriched for anti-IA-2ic autoantibodies. Interestingly, serum derived autoantibodies immobilised on the MBP-IA-2ic FP ELISA demonstrate increased Kappa light chain usage when compared to the respective total IgG derived from donor patients, suggesting a clonally restricted repertoire of anti-IA-2ic autoantigen specific B plasma cells is responsible for autoantibodies detect by the MBP-IA-2ic FP ELISA. This study is the first to demonstrate the generation of specific, genuine human derived anti-IA-2ic autoantibodies, thereby facilitating further investigation into the origin and functional significance of IA-2 autoantibodies in T1D.
ABSTRACT
Recent studies have postulated the presence of functional autoantibodies (Abs) against L-type voltage gated calcium channels (VGCCs) in the serum of patients with type 1 diabetes, with various proposed physiological consequences, both islet cell associated and extra-glandular. Arguably, the most potentially damaging effect reported for these Abs is induction of apoptosis in pancreatic beta (ß) cells, yet a convincing pathogenic mechanism remains to be demonstrated. In the current study, we report an assay of reactive oxygen species (ROS) stress induction in the rat insulinoma cell line Rin A12, as determined by 2', 7'-Dichlorofluorescein diacetate (DCF-DA) fluorescence detection by flow cytometry. We demonstrate that incubation of Rin A12 cells with immunoglobulin G (IgG) containing anti-VGCC activity from patients with T1D mediates a significant increase in ROS, with subsequent induction of apoptosis, as determined by positivity for annexin V expression. Neither T1D patient-derived IgG lacking anti-VGCC activity or IgG from healthy donors altered ROS or annexin V expression, indicating the new assay is specific for the detection of functional anti-VGCC Abs. Subsequent screening of IgG samples derived from individual patients indicated a prevalence of approximately 75% in a cohort of 20 patients with T1D. The new cell-based assay provides, for the first time, experimental evidence supporting a plausible pathophysiological mechanism underlying anti-VGCC Ab-mediated apoptosis induction in ß cells. Additionally, the assay is a considerable advance on previously published methods for detecting and characterising the functional activity of anti-VGCC Abs in patient-derived samples.
Subject(s)
Biological Assay , Calcium Channels, L-Type/immunology , Diabetes Mellitus, Type 1 , Immunoglobulin G , Insulin-Secreting Cells , Adolescent , Adult , Aged , Animals , Apoptosis/immunology , Autoantibodies/blood , Autoantibodies/immunology , Calcium Channels, L-Type/metabolism , Cell Line, Tumor , Diabetes Mellitus, Type 1/blood , Diabetes Mellitus, Type 1/immunology , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Insulin-Secreting Cells/immunology , Insulin-Secreting Cells/metabolism , Male , Middle Aged , Rats , Reactive Oxygen Species/blood , Reactive Oxygen Species/immunologyABSTRACT
We have used high-resolution mass spectrometry to sequence precipitating anti-Ro60 proteomes from sera of patients with primary Sjögren's syndrome and compare immunoglobulin variable-region (IgV) peptide signatures in Ro/La autoantibody subsets. Anti-Ro60 were purified by elution from native Ro60-coated ELISA plates and subjected to combined de novo amino acid sequencing and database matching. Monospecific anti-Ro60 Igs comprised dominant public and minor private sets of IgG1 kappa and lambda restricted heavy and light chains. Specific IgV amino acid substitutions stratified anti-Ro60 from anti-Ro60/La responses, providing a molecular fingerprint of Ro60/La determinant spreading and suggesting that different forms of Ro60 antigen drive these responses. Sequencing of linked anti-Ro52 proteomes from individual patients and comparison with their anti-Ro60 partners revealed sharing of a dominant IGHV3-23/IGKV3-20 paired clonotype but with divergent IgV mutational signatures. In summary, anti-Ro60 IgV peptide mapping provides insights into Ro/La autoantibody diversification and reveals serum-based molecular markers of humoral Ro60 autoimmunity.
Subject(s)
Autoantibodies/immunology , Autoantigens/immunology , Immunoglobulin Variable Region/immunology , RNA, Small Cytoplasmic/immunology , Ribonucleoproteins/immunology , Sjogren's Syndrome/immunology , Autoantibodies/blood , Humans , Immunoglobulin G/blood , Immunoglobulin G/immunology , Peptide Mapping , Proteome , Sjogren's Syndrome/bloodABSTRACT
The structures of epitopes bound by autoantibodies against RNA-protein complexes have been well-defined over several decades, but little is known of the clonality, immunoglobulin (Ig) variable (V) gene usage and mutational status of the autoantibodies themselves at the level of the secreted (serum) proteome. A novel proteomic workflow is presented based on affinity purification of specific Igs from serum, high-resolution two-dimensional gel electrophoresis, and de novo and database-driven sequencing of V-region proteins by mass spectrometry. Analysis of anti-Ro52/Ro60/La proteomes in primary Sjögren's syndrome (SS) and anti-Sm and anti-ribosomal P proteomes in systemic lupus erythematosus (SLE) has revealed that these antibody responses are dominated by restricted sets of public (shared) clonotypes, consistent with common pathways of production across unrelated individuals. The discovery of shared sets of specific V-region peptides can be exploited for diagnostic biomarkers in targeted mass spectrometry platforms and for tracking and removal of pathogenic clones.
Subject(s)
Autoantibodies/immunology , Lupus Erythematosus, Systemic/immunology , Sjogren's Syndrome/immunology , Animals , Humans , Proteome/immunology , Proteomics , Ribosomes/immunologyABSTRACT
Inhibitory autoantibodies acting at the muscarinic acetylcholine receptor type 3 (M3R) are postulated to mediate autonomic dysfunction, including decreased salivary and lacrimal gland output and extra-glandular manifestations, in patients with primary Sjögren's syndrome. However, the contention that anti-M3R antibodies are pathogenic in patients remains untested, due to a lack of assays both sophisticated enough to detect inhibitory anti-M3R antibodies yet suitable for screening large patient cohorts. In the current study, we have established a cell-based bioassay of M3R activity, based on dual transfection of the M3R and a luciferase reporter gene. The bioassay is capable of capturing real-time agonist-mediated signalling of the M3R, which is inhibited specifically by patient IgG that have previously been demonstrated to have anti-M3R activity. The assay can be run in multi-well culture plates, and analysed using simple luminescence readers. As such, the new bioassay incorporating M3R-mediated luciferase transduction is the first assay adaptable to common diagnostic platforms that is capable of determining the presence in patient serum of functionally active anti-M3R autoantibodies. The new bioassay should prove useful for large cohort screening studies aiming to correlate the presence in patients of inhibitory anti-M3R antibodies with symptoms of both glandular and extra-glandular autonomic dysfunction.
Subject(s)
Autoantibodies/blood , Immunoassay/methods , Receptor, Muscarinic M3/immunology , Sjogren's Syndrome/immunology , Autoantigens/immunology , HEK293 Cells , Humans , TransfectionABSTRACT
Recent advances in mass spectrometry-based proteomic methods have allowed variable (V)-region peptide signatures to be derived from human autoantibodies present in complex serum mixtures. Here, we analysed the clonality and V-region composition of immunoglobulin (Ig) proteomes specific for the immunodominant SmD protein subunit of the lupus-specific Sm autoantigen. Precipitating SmD-specific IgGs were eluted from native SmD-coated ELISA plates preincubated with sera from six patients with systemic lupus erythematosus (SLE) positive for anti-Sm/RNP. Heavy (H)- and light (L)-chain clonality and V-region sequences were analysed by 2-dimensional gel electrophoresis and combined de novo database mass spectrometric sequencing. SmD autoantibody proteomes from all six patients with SLE expressed IgG1 kappa restricted clonotypes specified by IGHV3-7 and IGHV1-69 H-chains and IGKV3-20 and IGKV2-28 L-chains, with shared and individual V-region amino acid replacement mutations. Clonotypic sharing and restricted V-region diversity of systemic autoimmunity can now be extended from the Ro/La cluster to Sm autoantigen and implies a common pathway of anti-Sm autoantibody production in unrelated patients with SLE.
Subject(s)
Autoantibodies/immunology , Immunoglobulin Variable Region/immunology , Peptides/immunology , Proteome/immunology , snRNP Core Proteins/immunology , Adult , Aged , Amino Acid Sequence , Antibodies, Antinuclear/genetics , Antibodies, Antinuclear/immunology , Autoantibodies/blood , Autoantibodies/genetics , Electrophoresis, Gel, Two-Dimensional , Female , Humans , Immunoglobulin G/genetics , Immunoglobulin G/immunology , Immunoglobulin Variable Region/genetics , Lupus Erythematosus, Systemic/blood , Lupus Erythematosus, Systemic/genetics , Lupus Erythematosus, Systemic/immunology , Mass Spectrometry , Middle Aged , Molecular Sequence Data , Mutation , Peptides/genetics , Proteome/genetics , Proteomics/methods , Sequence Homology, Amino AcidABSTRACT
Impairment of gastrointestinal tract (GI) function, including delayed gastric emptying and colonic dysmotility, are common features of primary Sjögren's syndrome (SS). However, the pathogenesis remains largely unknown. The aim of the current study was to investigate the role of functional autoantibodies to the muscarinic receptor in mediating GI dysfunction associated with primary SS. The effect of SS or normal immunoglobulin G (IgG) on smooth muscle (SM) motility was assessed by comparing the amplitude of carbachol (CCh) or electrical field stimulation (EFS) - induced muscle contraction before and after IgG application. Muscarinic receptor type 3 (M3R) played a dominant role in both colon and gastric SM contraction, while M2R was partly involved in gastric smooth muscle contraction. Preincubation for 1h of the colon and gastric SM strips with 1mg/ml purified IgG from the sera of four primary SS patients (SS IgG) significantly inhibited carbachol-induced smooth muscle contraction (CISC) over a range of CCh concentrations, whereas IgG from healthy controls had little effect. Incubation of the colon SM strips with SS IgG also inhibited EFS-induced colon muscle contraction, which was mimicked by the M3R-selective blocker, 4-DAMP. SR1403330, an NK1 antagonist, had little effect on EFS-mediated colonic SM contraction. The results suggest that autoantibodies isolated from primary SS patients' sera inhibit muscarinic receptor-mediated cholinergic neurotransmission in mouse colon and stomach, which may provide clues for explaining the GI dysfunction seen in patients with primary SS.
Subject(s)
Autoantibodies/pharmacology , Gastrointestinal Motility/drug effects , Muscarinic Antagonists/pharmacology , Sjogren's Syndrome/immunology , Animals , Autoantibodies/blood , Autoantibodies/immunology , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Colon/drug effects , Colon/immunology , Colon/physiology , Diamines/pharmacology , Dose-Response Relationship, Drug , Gastrointestinal Motility/immunology , Humans , In Vitro Techniques , Male , Mice , Mice, Inbred BALB C , Muscle Contraction/drug effects , Muscle Contraction/immunology , Muscle, Smooth/drug effects , Muscle, Smooth/immunology , Muscle, Smooth/physiology , Piperidines/pharmacology , Receptor, Muscarinic M2/agonists , Receptor, Muscarinic M2/antagonists & inhibitors , Receptor, Muscarinic M2/immunology , Receptor, Muscarinic M3/agonists , Receptor, Muscarinic M3/antagonists & inhibitors , Receptor, Muscarinic M3/immunology , Sjogren's Syndrome/blood , Stomach/drug effects , Stomach/immunology , Stomach/physiologyABSTRACT
Long-term humoral autoimmunity to RNA-protein autoantigens is considered a hallmark of systemic autoimmune diseases. We use high resolution Orbitrap mass spectrometric autoantibody sequencing to track the evolution of a Ro60-specific public clonotypic autoantibody in 4 patients with primary Sjögren's syndrome. This clonotype is specified by a VH3-23/VK3-20 heavy and light chain pairing. Despite apparent stability by conventional immunoassay, analysis of V-region molecular signatures of clonotypes purified from serum samples collected retrospectively over 7years revealed sequential clonal replacement. Prospective longitudinal studies confirmed clonotype loss and replacement at approximately three-monthly intervals. Levels of secreted anti-Ro60 clonotypes fluctuated markedly over time, despite minimal changes in clonal affinity. Our novel findings indicate a relentless turnover of short-lived clonotypic variants, masquerading as long-lived Ro60 humoral autoimmunity.
Subject(s)
Autoantigens/immunology , RNA, Small Cytoplasmic/immunology , Ribonucleoproteins/immunology , Sjogren's Syndrome/immunology , Adult , Aged , Autoantigens/blood , Autoimmunity/immunology , Clone Cells , Female , Humans , Longitudinal Studies , Mass Spectrometry , Middle Aged , Prospective Studies , RNA, Small Cytoplasmic/blood , Retrospective Studies , Ribonucleoproteins/blood , Sequence Analysis, Protein , Sjogren's Syndrome/blood , Sjogren's Syndrome/pathologyABSTRACT
Ro/SSA and La/SSB comprise a linked set of autoantigens that are clinically important members of the extractable nuclear antigen family and key translational biomarkers for lupus and primary Sjögren's syndrome. Autoantibodies directed against the Ro60 and La polypeptide components of the Ro/La ribonucleoprotein complex, and the structurally unrelated Ro52 protein, mediate tissue damage in the neonatal lupus syndrome, a model of passively acquired autoimmunity in humans in which the most serious manifestation is congenital heart block (CHB). Recent studies have concentrated on two distinct pathogenic mechanisms by which maternal anti-Ro/La autoantibodies can cause CHB: by forming immune complexes with apoptotic cells in developing fetal heart; and/or by acting as functional autoantibodies that cross-react with and inhibit calcium channels. Although the precise role of the individual autoantibodies is yet to be settled, maternal anti-Ro60 and anti-Ro52 remain the most likely culprits. This article will discuss the molecular pathways that culminate in the development of CHB, including the recent discovery of ß2 glycoprotein I as a protective factor, and present a proteomic approach based on direct mass spectrometric sequencing, which may give a more representative snapshot of the idiotype repertoire of these autoantibodies than genomic-based technologies.
Subject(s)
Autoantibodies/immunology , Autoantigens/immunology , Heart Block/congenital , Immunity, Maternally-Acquired , Ribonucleoproteins/immunology , Animals , Antigen-Antibody Complex/immunology , Cross Reactions , Female , Heart Block/immunology , Humans , Infant, Newborn , Lupus Erythematosus, Systemic/congenital , Lupus Erythematosus, Systemic/immunology , Proteomics , SS-B AntigenABSTRACT
Mackay and Burnet's Autoimmune diseases, published in 1962, marked the beginning of autoimmunity as a clinical science and led to the future acceptance of the existence of autoimmunity. While there is still controversy regarding the mechanisms of autoimmunity, the authors' insightful hypothesis based on clonal selection theory and the emergence of "forbidden clones", due to somatic mutations, is still current, with recent evidence giving further credence to this hypothesis. We salute Mackay and Burnet on the 50th anniversary of this seminal publication. It is particularly pleasing that it has an iconic Australian origin.
Subject(s)
Autoimmune Diseases/history , Periodicals as Topic/history , Australia , History, 20th Century , HumansABSTRACT
OBJECTIVE: This study was undertaken to determine the molecular characteristics of clonotypic autoantibodies in the sera of patients with primary Sjögren's syndrome (SS). This characterization is hampered by the presence of mixed anti-Ro/La specificities that may conceal clonotypic species. In order to narrow clonotypic diversity, a positive selection step was performed on a peg-like determinant of Ro 60 (termed Ro 60-peg) prior to analysis of the autoantibody proteome. METHODS: Monospecific anti-Ro 60-peg IgG were isolated by affinity purification from the sera of 7 patients with primary SS and anti-Ro/La and subjected to 2-dimensional gel electrophoresis and high-resolution orbitrap mass spectrometric sequencing. V regions of heavy and light chains were analyzed by combined database and de novo amino acid sequencing. RESULTS: Proteomic analysis revealed a Ro 60-peg-specific IgG1κ-restricted monoclonal autoantibody that was present in the sera of all patients and specified by a V(H) 3-23 heavy chain paired with a V(κ) 3-20 light chain. The public anti-Ro 60-peg clonotype was specified further by common mutations in the heavy-chain and light-chain complementarity-determining regions. Titers and relative affinities of clonotypic IgG did not vary over the course of the disease. CONCLUSION: The expression of a Ro 60-reactive public B cell clonotype in a subset of patients with primary SS as a long-lived, class-switched circulating autoantibody implies a common breach of B cell tolerance checkpoints in these patients. The unique heavy chain/light chain signature opens the possibility of tracking the development of a "forbidden" clone against a bona fide systemic autoantigen in human disease.
Subject(s)
Autoantibodies/genetics , Autoimmunity/genetics , Sjogren's Syndrome/genetics , Adult , Aged , Antibodies, Antinuclear/genetics , Antibodies, Antinuclear/immunology , Autoantibodies/immunology , Autoimmunity/immunology , Epitopes/genetics , Epitopes/immunology , Female , Humans , Middle Aged , Proteomics , Sjogren's Syndrome/immunologyABSTRACT
OBJECTIVE: In primary Sjögren's syndrome (SS), impairment of the gastrointestinal (GI) tract is common, and includes reduced esophageal motor function, delayed gastric emptying, and abnormalities in colonic motility; the pathogenesis is as yet unknown. We undertook this study to investigate the role of functional antibodies to the type 3 muscarinic receptor (M3R) in GI dysfunction associated with primary SS. METHODS: Muscle strip and whole-organ functional assays were used to determine whether IgG with anti-M3R activity from patients with primary SS disrupted neurotransmission in tissue from throughout the mouse GI tract. Specificity of the autoantibody for the M3R was determined using knockout mice that were deficient in the expression of muscarinic receptor subtypes. RESULTS: Functional antibodies to the M3R inhibited neuronally mediated contraction of smooth muscle from throughout the GI tract and disrupted complex contractile motility patterns in the colon. The autoantibodies were not active on tissue from mice that lacked the M3R, providing compelling evidence of the direct interaction of patient autoantibodies with the M3R. CONCLUSION: Our results indicate that anti-M3R autoantibodies have the potential to mediate multiple dysfunctions of the GI tract in primary SS, ranging from reduced esophageal motor activity to altered colonic motility. We hypothesize that altered GI motility forms part of a broader autonomic dysfunction mediated by pathogenic anti-M3R autoantibodies in primary SS.
Subject(s)
Autoantibodies/immunology , Gastrointestinal Motility/immunology , Receptor, Muscarinic M3/immunology , Sjogren's Syndrome/immunology , Synaptic Transmission/immunology , Acetylcholine/metabolism , Analysis of Variance , Animals , Antineoplastic Combined Chemotherapy Protocols , Carbachol/pharmacology , Cholinergic Agonists/pharmacology , Cisplatin , Gastrointestinal Motility/drug effects , Humans , Ifosfamide , Male , Mice , Mice, Knockout , Mitomycin , Muscle Contraction/drug effects , Muscle Contraction/immunology , Muscle, Smooth/drug effects , Muscle, Smooth/immunology , Receptor, Muscarinic M3/metabolism , Sjogren's Syndrome/metabolism , Sjogren's Syndrome/physiopathology , Synaptic Transmission/drug effectsABSTRACT
We have recently postulated that functional autoantibodies (Abs) against L-type voltage-gated calcium channels (VGCCs) contribute to autonomic dysfunction in type 1 diabetes (T1D). Previous studies based on whole-organ assays have proven valuable in establishing the mechanism of anti-VGCC Ab activity, but are complex and unsuitable for screening large patient cohorts. In the current study, we used real-time dynamic monitoring of cell impedance to demonstrate that anti-VGCC Abs from patients with T1D inhibit the adherence of Rin A12 cells. The functional effect of the anti-VGCC Abs was mimicked by the dihydropyridine agonist, Bay K8644, and reversed by the antagonist, nicardipine, providing a pharmacological link to the whole-organ studies. IVIg neutralized the effect on cell adhesion of the anti-VGCC Abs, consistent with the presence of anti-idiotypic Abs in IVIg that may prevent the emergence of pathogenic Abs in healthy individuals. The cell impedance assay can be performed in a 96 well plate format, and represents a simple method for detecting the presence of anti-VGCC activity in patient immunoglobulin (IgG). The new cell assay should prove useful for further studies to determine the prevalence of the Ab and its association with symptoms of autonomic dysfunction in patients with T1D.
Subject(s)
Autoantibodies/immunology , Calcium Channels, L-Type/immunology , Cell Adhesion/immunology , Diabetes Mellitus, Type 1/immunology , Electric Impedance , Immunoglobulin G/immunology , 3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-(trifluoromethyl)phenyl)-, Methyl ester/pharmacology , Animals , Autoantibodies/analysis , Calcium Channel Agonists/pharmacology , Calcium Channel Blockers/pharmacology , Cell Line , Humans , Nicardipine/pharmacology , RatsABSTRACT
OBJECTIVE: Analysis of B cell determinants of Ro 60 exposed on the surface of apoptotic cells (apotopes) or intracellular epitopes provides insight into the structural forms of the autoantigen that break immune tolerance. This study was initiated to compare anti-Ro 60 responses in systemic lupus erythematosus (SLE) and primary Sjögren's syndrome (SS) against membrane-bound and intracellular forms of Ro 60. METHODS: The reactivity of autoantibodies from patients with SLE and primary SS to Ro 60 apotopes and epitopes was assessed by multiparameter flow cytometry and solid-phase immunoassay. Anti-Ro 60 IgG was eluted from early apoptotic cells or recombinant Ro 60 immobilized on nitrocellulose, and binding to membrane-bound and intracellular forms of Ro 60 was quantitated by flow cytometry. RESULTS: An immunodominant apotope, which was recognized by IgG from a subset of SLE patients with anti-Ro, but not anti-La, autoantibodies, was mapped to a region forming a helix-loop-helix at the apical tip of the Ro 60 molecule. Immobilization of this region to the solid phase exposed an epitope that was recognized by IgG from primary SS and SLE patients whose sera had both anti-Ro and anti-La autoantibodies. Autoantibodies eluted from either the surface of apoptotic cells or the Ro 60 epitope on the solid phase were non-cross-reactive and specifically recognized membrane-bound or cytoplasmic forms of Ro 60. CONCLUSION: This is the first example of a dichotomy of human autoantibody responses against mutually exclusive determinants linked to a single domain of a systemic autoantigen and supports a model in which tolerance is broken by different immunogenic forms of Ro 60.
Subject(s)
Autoantibodies/immunology , Autoantigens/chemistry , Autoantigens/immunology , Lupus Erythematosus, Systemic/immunology , RNA, Small Cytoplasmic/chemistry , RNA, Small Cytoplasmic/immunology , Ribonucleoproteins/chemistry , Ribonucleoproteins/immunology , Sjogren's Syndrome/immunology , Cross Reactions/immunology , Epitope Mapping , Flow Cytometry , Humans , Immunodominant Epitopes/chemistry , Immunodominant Epitopes/immunology , Immunoglobulin G/immunology , Jurkat Cells , Protein Structure, Secondary , Protein Structure, TertiaryABSTRACT
We have recently reported the presence of an immunoglobulin G (IgG) autoantibody (Ab) in patients with narcolepsy with cataplexy that abolishes spontaneous colonic migrating motor complexes (CMMCs) and increases smooth muscle tension and atropine-sensitive phasic contractions in a physiological assay of an isolated colon. In this study, we used the cholinesterase inhibitor, neostigmine, to explore the mechanism of the narcoleptic IgG-mediated disruption of enteric motor function in four patients with narcolepsy with cataplexy and to identify a pharmacological mimic of the Ab. Neostigmine potentiated the narcoleptic IgG-mediated increase in smooth muscle resting tension and phasic smooth muscle contractions by an atropine-sensitive mechanism but exerted no effect on resting tension in the presence of control IgG. Decreased frequency of CMMCs mediated by IgG with anti-M3R activity was reversed by neostigmine. Therefore, a challenge with a cholinesterase inhibitor improves the specificity of the CMMC assay for narcoleptic IgG. Tetrodotoxin (TTX), a neuronal sodium channel blocker, also abolished CMMCs and increased resting tone, and a similar potentiation was observed with neostigmine; thus, TTX is a mimic of the functional effects of the narcoleptic IgG in this bioassay. These findings provide a link to pharmacological studies of canine narcolepsy and are consistent with a functional blockade of both excitatory and inhibitory motor neurons by the narcoleptic Ab, similar to the TTX mimic, presumably by binding to an autoantigenic target expressed in both populations of neurons.
Subject(s)
Autoantibodies/physiology , Cholinesterase Inhibitors/pharmacology , Enteric Nervous System/physiopathology , Myoelectric Complex, Migrating , Narcolepsy/physiopathology , Neostigmine/pharmacology , Adult , Aged , Aged, 80 and over , Animals , Atropine , Case-Control Studies , Colon/physiopathology , Female , Humans , Immunoglobulin G/physiology , Male , Mice , Mice, Inbred BALB C , Middle Aged , Muscarinic Antagonists , Muscle Tonus , Muscle, Smooth/physiopathology , Narcolepsy/immunology , Sodium Channel Blockers , Synaptic Transmission , Tetrodotoxin/pharmacology , Young AdultABSTRACT
Multiple sclerosis (MS) is a relatively common disease of young adults. Patients with MS can have a wide range of symptoms and may develop significant disability. The cause of MS is unknown, but immunological mechanisms are important. In MS, the pathological features include prominent demyelination and inflammation, but there is also evidence of neurodegeneration. Bladder symptoms are common in MS. The bladder is under neural control, and bladder disturbance is usually attributed to demyelination or loss of axons from the neural pathways, particularly those in the spinal cord, that control the bladder. However, as with other symptoms in MS, the presence of bladder disturbance does not always correlate well with MRI lesions. We speculate that other possible causes of bladder dysfunction in MS might include the effects of circulating toxic factors. Urgency of micturition is prominent in MS, and better understanding of the receptors involved in bladder sensation suggests possible treatment strategies through inhibiting these receptors.
Subject(s)
Multiple Sclerosis/complications , Urinary Bladder Diseases/etiology , Expert Testimony , Humans , Urinary Bladder Diseases/pathology , Urinary Bladder Diseases/therapyABSTRACT
OBJECTIVE: The autoantigens 60-kd Ro/SSA (Ro 60) and beta(2)-glycoprotein I (beta(2)GPI) are both displayed on the surface membrane of apoptotic cells. Epitope-spreading experiments have suggested that these autoantigens may be present as a complex on the apoptotic cell surface. This study was undertaken to investigate whether beta(2)GPI interacts with Ro 60 on apoptotic cells and alters the binding of anti-Ro 60 IgG. METHODS: The interaction between soluble recombinant Ro 60 fragments and beta(2)GPI was investigated in vitro by direct and saturation binding assays using native human beta(2)GPI and recombinant domain deletion mutants. Binding of beta(2)GPI to early and late apoptotic cells was assessed by multiparameter flow cytometry, and specificity of binding was determined by competitive inhibition with soluble recombinant Ro 60 and anti-Ro 60 IgG. RESULTS: The Ro 60 fragment expressing a surface-exposed epitope (apotope) bound with high affinity (K(d) = approximately 15 nM) to domain V of beta(2)GPI in vitro. Beta(2)-glycoprotein I bound to the surface of apoptotic cells in a dose-dependent manner and was blocked by the Ro 60 apotope fragment. In reciprocal competitive inhibition studies, beta(2)GPI blocked the binding of anti-Ro 60 autoantibodies to apoptotic cells in a dose-dependent manner, and anti-Ro 60 IgG inhibited the binding of beta(2)GPI. Moreover, beta(2)GPI showed a 2-fold increase in binding to apoptotic cells that overexpress Ro 60 on the surface. CONCLUSION: These results demonstrate that Ro 60 functions as a novel receptor for beta(2)GPI on the surface of apoptotic cells. The formation of Ro 60-beta(2)GPI complexes may protect against anti-Ro 60 autoantibody-mediated tissue injury.
Subject(s)
Apoptosis/physiology , Autoantigens/metabolism , Epithelial Cells/metabolism , Epithelial Cells/pathology , RNA, Small Cytoplasmic/metabolism , Receptors, Cell Surface/metabolism , Ribonucleoproteins/metabolism , beta 2-Glycoprotein I/metabolism , Antigen-Antibody Complex/immunology , Apoptosis/immunology , Autoantibodies/immunology , Autoantibodies/metabolism , Autoantigens/immunology , Cell Line , Epitopes/immunology , Epitopes/metabolism , Humans , Jurkat Cells , Protein Binding/immunology , Protein Binding/physiology , RNA, Small Cytoplasmic/immunology , Receptors, Cell Surface/immunology , Ribonucleoproteins/immunology , beta 2-Glycoprotein I/immunologyABSTRACT
Despite strong circumstantial evidence for the autoimmune hypothesis of narcolepsy, conventional immunological methods have failed to detect an autoantibody. This study investigated the real-time effects of narcoleptic immunoglobulins on a spontaneous colonic migrating motor complex (CMMC) preparation. IgG from patients with narcolepsy with cataplexy or healthy controls was added directly to isolated mouse colons undergoing CMMC activity to test for autoantibodies that disrupt colonic motility. The effect of immunoglobulins prepared for clinical intravenous treatment (IVIg) on autoantibody-mediated colonic disruption was also assessed. Narcoleptic IgGs markedly reduced the frequency of CMMCs or irreversibly abolished them. Abrogation of CMMCs was followed by an increase in the resting tension of the colon preparation and appearance of atropine-sensitive phasic smooth muscle contractions. IVIg partially neutralized the inhibitory effect of narcoleptic IgG on the CMMCs. The dramatic effect of narcoleptic IgG on CMMC generation is consistent with an autoantibody-mediated disruption of enteric neural pathways. The ex vivo whole-organ approach allows real-time examination of the physiological effects of the narcoleptic autoantibody and offers a new avenue for exploring the autoimmune basis of narcolepsy. The neutralizing effect of IVIg on the autoantibody provides a rationale for the reported clinical improvement in cataplexy when IVIg are given at disease onset.
Subject(s)
Autoantibodies/immunology , Colon/immunology , Colon/physiopathology , Narcolepsy/immunology , Peristalsis/immunology , Adult , Aged , Aged, 80 and over , Animals , Atropine/pharmacology , Autoantibodies/toxicity , Colon/drug effects , Drug Interactions/immunology , Enteric Nervous System/drug effects , Enteric Nervous System/immunology , Enteric Nervous System/physiopathology , Female , Humans , Immunoglobulins/immunology , Immunoglobulins/toxicity , Immunoglobulins, Intravenous/pharmacology , Immunoglobulins, Intravenous/therapeutic use , Male , Mice , Mice, Inbred BALB C , Middle Aged , Muscarinic Antagonists/pharmacology , Muscle Contraction/drug effects , Muscle Contraction/immunology , Muscle, Smooth/drug effects , Muscle, Smooth/immunology , Muscle, Smooth/physiopathology , Narcolepsy/drug therapy , Narcolepsy/physiopathology , Neuromuscular Junction/drug effects , Neuromuscular Junction/immunology , Neuromuscular Junction/physiopathology , Peristalsis/drug effects , Young AdultABSTRACT
Apoptosis has been proposed to influence the initiation and diversification of autoimmunity to the Ro (SSA)/La (SSB) ribonucleoprotein (RNP) particle and serve as a target for autoantibody-mediated tissue injury. We have developed a new approach to B cell epitope mapping which identifies "apotopes," defined as epitopes expressed on the surface of apoptotic cells. Preliminary studies support a role for apotopes as diagnostic markers in systemic lupus erythematosus (SLE) and primary Sjögren's syndrome. For example, apotopes within the NH(2)-terminal and central regions of La react with the majority of sera from mothers of infants with congenital heart block. Furthermore, a Ro60 apotope is specific for a subset of SLE with isolated anti-Ro60 responses. The mapping of B cell apotopes may prove superior to standard epitope mapping by suggesting novel pathways of autoantibody production and identifying pathogenic species of autoantibodies.
Subject(s)
Apoptosis/immunology , Autoantigens/immunology , B-Lymphocytes/immunology , Epitopes, B-Lymphocyte/immunology , RNA, Small Cytoplasmic/immunology , Ribonucleoproteins/immunology , Animals , Autoantibodies/immunology , Autoimmunity , Epitope Mapping , Humans , SS-B AntigenABSTRACT
Autoantibodies (Abs) directed against L-type voltage-gated calcium channels (VGCCs) have been shown to contribute to autonomic dysfunction of the gastrointestinal tract and bladder in patients with Type 1 diabetes mellitus (T1D). We used a passive transfer model to determine whether the functional activity of the Ab requires crosslinking of channels in colon and bladder and can be neutralized by intravenous immunoglobulin (IVIg). Mice were injected with mono- and divalent F(ab) fragments of patient IgG with anti-VGCC activity and tested for gut and bladder function using a colonic migrating motor complex (MMC) assay and bladder-filling cystometry. The ability of IVIg to neutralize anti-VGCC IgG-mediated autonomic dysfunction was investigated by injection of mice with an equimolar concentration of IVIg prior to T1D IgG injection, or by injection with T1D IgG passed over a sepharose 4B column coupled with F(ab')(2) from IVIg. Passive transfer of T1D IgG and its F(ab')(2) or F(ab) fragments reduced the amplitude of spontaneous colonic motility. In contrast, intact IgG and F(ab')(2,) but not F(ab), produced the urodynamics features of an overactive bladder. T1D IgG-mediated colonic and bladder dysfunction was neutralized in vivo by prior injection of animals with equimolar IVIg. Moreover, anti-VGCC activity was depleted by preabsorption of patient IgG on a IVIg F(ab')(2) column. The activity of anti-VGCC IgG is mediated by the antigen-binding site consistent with a true functional Ab. The pathogenic effect on the bladder requires crosslinking of the channel, whereas monovalent binding of Ab is sufficient for disruption of colon motility. The anti-VGCC Abs are neutralized by antiidiotypic antibodies present in IVIg that may prevent the emergence of these Abs in healthy individuals.