Identification of a unique anti-Ro60 subset with restricted serological and molecular profiles.

Anti-Ro60 is one of the most common and clinically important serum autoantibodies that has a number of diagnostic and predictive capabilities. Most diagnostic laboratories report this simply as a qualitative positive/negative result. The objective of this study was to examine the clinical and serological relevance of a novel subset of anti-Ro60 in patients who display low levels of anti-Ro60 (anti-Ro60low ). We retrospectively identified anti-Ro60 sera during a 12-month period at a major immunopathology diagnostic laboratory in Australia. These all were anti-Ro60-precipitin-positive on the diagnostic gold standard counter-immuno-electrophoresis (CIEP). Lineblot immunoassay was used to stratify patients into either anti-Ro60low or anti-Ro60high subsets. We compared the medical and laboratory parameters associated with each group. Enzyme-linked immunosorbent assay (ELISA) and mass spectrometry techniques were used to analyse the serological and molecular basis behind the two subsets. Anti-Ro60low patients displayed less serological activity than anti-Ro60high patients with less intermolecular spreading, hypergammaglobulinaemia and less tendency to undergo anti-Ro60 isotype-switching than anti-Ro60high patients. Mass spectrometric typing of the anti-Ro60low subset showed restricted variable heavy chain subfamily usage and amino acid point mutations. This subset also displayed clinical relevance, being present in a number of patients with systemic autoimmune rheumatic diseases (SARD). We identify a novel anti-Ro60low patient subset that is distinct from anti-Ro60high patients serologically and molecularly. It is not clear whether they arise from common or separate origins; however, they probably have different developmental pathways to account for the stark difference in immunological maturity. We hence demonstrate significance to anti-Ro60low and justify accurate detection in the diagnostic laboratory.

Precipitating anti-dsDNA peptide repertoires in lupus.

Anti-double-stranded (ds)DNA autoantibodies are prototypical serological markers of systemic lupus erythematosus (SLE), but little is known about their immunoglobulin variable (IgV) region composition at the level of the secreted (serum) proteome. Here, we use a novel proteomic workflow based on de novo mass spectrometric sequencing of anti-dsDNA precipitins to analyse IgV subfamily expression and mutational signatures of high-affinity, precipitating anti-dsDNA responses. Serum anti-dsDNA proteomes were oligoclonal with shared (public) expression of immunoglobulin (Ig)G heavy chain variable region (IGHV) and kappa chain variable region (IGKV) subfamilies. IgV peptide maps from eight subjects showed extensive public and random (private) amino acid replacement mutations with prominent arginine substitutions across heavy (H)- and light (L)-chains. Shared sets of L-chain complementarity determining region 3 (CDR3) peptides specified by arginine substitutions were sequenced from the dominantly expressed IGKV3-20 subfamily, with changes in expression levels of a clonal L-chain CDR3 peptide by quantitative multiple reaction monitoring (MRM) paralleling the rise and fall of anti-dsDNA levels by Farr radioimmunoassays (RIA). The heavily mutated IgV peptide signatures of precipitating anti-dsDNA autoantibody proteomes reflect the strong selective forces that shape humoral anti-dsDNA responses in germinal centres. Direct sequencing of agarose gel precipitins using microlitre volumes of stored sera streamlines the antibody sequencing workflow and is generalizable to other precipitating serum antibodies.

Lupus anti-ribosomal P autoantibody proteomes express convergent biclonal signatures.

Lupus-specific anti-ribosomal P (anti-Rib-P) autoantibodies have been implicated in the pathogenesis of neurological complications in systemic lupus erythematosus (SLE). The aim of the present study was to determine variable (V)-region signatures of secreted autoantibody proteomes specific for the Rib-P heterocomplex and investigate the molecular basis of the reported cross-reactivity with Sm autoantigen. Anti-Rib-P immunoglobulins (IgGs) were purified from six anti-Rib-P-positive sera by elution from enzyme-linked immunosorbent assay (ELISA) plates coated with either native Rib-P proteins or an 11-amino acid peptide (11-C peptide) representing the conserved COOH-terminal P epitope. Rib-P- and 11-C peptide-specific IgGs were analysed for heavy (H) and light (L) chain clonality and V-region expression using an electrophoretic and de-novo and database-driven mass spectrometric sequencing workflow. Purified anti-Rib-P and anti-SmD IgGs were tested for cross-reactivity on ELISA and their proteome data sets analysed for shared clonotypes. Anti-Rib-P autoantibody proteomes were IgG1 kappa-restricted and comprised two public clonotypes defined by unique H/L chain pairings. The major clonotypic population was specific for the common COOH-terminal epitope, while the second shared the same pairing signature as a recently reported anti-SmD clonotype, accounting for two-way immunoassay cross-reactivity between these lupus autoantibodies. Sequence convergence of anti-Rib-P proteomes suggests common molecular pathways of autoantibody production and identifies stereotyped clonal populations that are thought to play a pathogenic role in neuropsychiatric lupus. Shared clonotypic structures for anti-Rib-P and anti-Sm responses suggest a common B cell clonal origin for subsets of these lupus-specific autoantibodies.

An immunodominant La/SSB autoantibody proteome derives from public clonotypes.

The La/SSB autoantigen is a major target of long-term humoral autoimmunity in primary Sjögren's Syndrome (SS) and systemic lupus erythematosus. A majority of patients with linked anti-Ro60/Ro52/La responses target an NH2-terminal epitope designated LaA that is expressed on Ro/La ribonucleoprotein complexes and the surface membrane of apoptotic cells. In this study, we used high-resolution Orbitrap mass spectrometry to determine the clonality, isotype and V-region sequences of LaA-specific autoantibodies in seven patients with primary SS. Anti-LaA immunoglobulin (Ig)Gs purified from polyclonal sera by epitope-specific affinity chromatography were analysed by combined database and de-novo mass spectrometric sequencing. Autoantibody responses comprised two heavily mutated IgG1 kappa-restricted monoclonal species that were shared (public) across unrelated patients; one clonotype was specified by an IGHV3-30 heavy chain paired with IGKV3-15 light chain and the second by an IGHV3-43/IGKV3-20 pairing. Shared amino acid replacement mutations were also seen within heavy and light chain complementarity-determining regions, consistent with a common breach of B cell tolerance followed by antigen-driven clonal selection. The discovery of public clonotypic autoantibodies directed against an immunodominant epitope on La, taken together with recent findings for the linked Ro52 and Ro60 autoantigens, supports a model of systemic autoimmunity in which humoral responses against protein-RNA complexes are mediated by public sets of autoreactive B cell clonotypes.

Prevalence of sicca symptoms in a South Australian cohort with systemic sclerosis.

BACKGROUND: The presence of sicca symptoms is a frequent finding in patients with systemic sclerosis (SSc). The aim of this study was to examine the prevalence of sicca symptoms in a South Australian cohort of SSc patients and correlate this to a number of parameters, including autoantibody status, use of anticholinergic medication, age and the presence of functional anti-muscarinic-3 receptor (M3R)-blocking antibodies. METHODS: A screening questionnaire was sent out to all patients on the South Australian Scleroderma Register from the years 1998-2006 to determine the prevalence of sicca symptoms. A subset of patients on the register had ocular sicca symptoms tested by use of Schirmer's strips to validate the accuracy of the questionnaire. Eight patients were tested for anti-M3R-blocking antibodies using a functional physiological assay. RESULTS: One hundred and ninety-three SSc patients took part in this study. Sicca symptoms were present in 59% of patients with the limited form of SSc, compared with 49% of patients with the diffuse form and 40% of patients with the overlap syndrome. The use of anticholinergic medication or thyroxine was associated with higher sicca scores in SSc patients. SS-A and SS-B autoantibodies (seen in Sjögren's syndrome) were detected in eight patients in this study. The detection of anti-M3R-blocking antibodies correlated well to presence of sicca. CONCLUSION: This study confirmed that sicca symptoms are found in a high proportion of patients with SSc, especially those with the limited variant. Further testing of larger numbers of SSc patients with sicca for anti-M3R-blocking antibodies will be needed before more definitive conclusions can be drawn. Physicians should be made aware that sicca symptoms are a frequent cause of morbidity for SSc patients*.

Different temporal expression of immunodominant Ro60/60 kDa-SSA and La/SSB apotopes.

Opsonization of apoptotic cardiocytes by maternal anti-Ro/SSA and anti-La/SSB antibodies contributes to tissue injury in the neonatal lupus syndrome. The objective of the current study was to quantify the surface membrane expression of Ro/La components during different phases of apoptosis and map the Ro/La apotopes (epitopes expressed on apoptotic cells) bound by cognate antibodies. Multi-parameter flow cytometry was used to define early and late apoptotic populations and their respective binding by monospecific anti-Ro and anti-La IgGs. Anti-Ro60 bound specifically to early apoptotic Jurkat cells and remained accessible on the cell surface throughout early and late apoptosis. In contrast, anti-La bound exclusively to late apoptotic cells in experiments controlled for non-specific membrane leakage of IgG. Ro52 was not accessible for antibody binding on either apoptotic population. The immunodominant NH2-terminal and RNA recognition motif (RRM) epitopes of La were expressed as apotopes on late apoptotic cells, confirming recent in vivo findings. An immunodominant internal epitope of Ro60 that contains the RRM, and is recognized by a majority of sera from mothers of children with congenital heart block (CHB) and patients with primary Sjögren's syndrome, was also accessible as an apotope on early apoptotic cells. The distinct temporal expression of the immunodominant Ro60 and La apotopes indicates that these intracellular autoantigens translocate independently to the cell surface, and supports a model in which maternal antibody populations against both Ro60 and La apotopes act in an additive fashion to increase the risk of tissue damage in CHB.

Autoantibody-mediated bladder dysfunction in type 1 diabetes.

Bladder dysfunction is a common complication of diabetic autonomic neuropathy; however, its cause remains uncertain. We have recently identified a novel IgG autoantibody (Ab) in patients with type 1 diabetes that acts as an agonist at the dihydropyridine (DHP) site of L-type voltage-gated calcium channels (VGCC), disrupting neuronal regulation of visceral smooth muscle. In the present study, passive transfer to mice of IgG from patients with type 1 diabetes was used to investigate the role of anti-VGCC Abs in mediating diabetic bladder dysfunction. Injection of mice with diabetic immunoglobulin (IgG) with anti-VGCC activity induced features of an overactive bladder, including phasic detrusor contractions and a loss of bladder wall compliance. The bladder overactivity is mimicked by the DHP agonist Bay K8644, reversed by the DHP antagonist nicardipine, but is insensitive to the motor nerve blocker tetrodotoxin, indicating that the anti-VGCC Ab acts at the level of the bladder detrusor itself. This study reports the first evidence of Ab-mediated bladder dysfunction in type 1 diabetes, which may be part of a wider spectrum of smooth muscle and cardiac abnormalities.

Neo-epitopes are required for immunogenicity of the La/SS-B nuclear antigen in the context of late apoptotic cells.

Mechanisms responsible for the induction of anti-nuclear autoantibodies (ANA) following exposure of the immune system to an excess of apoptotic cells are incompletely understood. In this study, the immunogenicity of late apoptotic cells expressing heterologous or syngeneic forms of La/SS-B was investigated following subcutaneous administration to A/J mice, a non-autoimmune strain in which the La antigenic system is well understood. Immunization of A/J mice with late apoptotic thymocytes taken from mice transgenic (Tg) for the human La (hLa) nuclear antigen resulted in the production of IgG ANA specific for human and mouse forms of La in the absence of foreign adjuvants. Preparations of phenotypically healthy cells expressing heterologous hLa were also immunogenic. However, hLa Tg late apoptotic cells accelerated and enhanced the apparent heterologous healthy cell-induced anti-La humoral response, while non-Tg late apoptotic cells did not. Subcutaneous administration of late apoptotic cells was insufficient to break existing tolerance to the hLa antigen in hLa Tg mice or to the endogenous mouse La (mLa) antigen in A/J mice immunized with syngeneic thymocytes, indicating a requirement for the presence of heterologous epitopes for anti-La ANA production. Lymph node dendritic cells (DC) but not B cells isolated from non-Tg mice injected with hLa Tg late apoptotic cells presented immunodominant T helper cell epitopes of hLa. These studies support a model in which the generation of neo-T cell epitopes is required for loss of tolerance to nuclear proteins after exposure of the healthy immune system to an excess of cells in late stages of apoptosis.

Antibodies raised against the second extracellular loop of the human muscarinic M3 receptor mimic functional autoantibodies in Sjögren's syndrome.

Functional antimuscarinic M3 receptor (M3R) autoantibodies have been shown to inhibit cholinergic neurotransmission at the postsynaptic level and appear to mediate parasympathetic dysfunction, including sicca symptoms in Sjögren's syndrome (SS). The precise epitope(s) involved in the inhibition of M3R-mediated cholinergic neurotransmission has not been defined. In this study, an active immunization approach to raise antibodies with functional activity against the second extracellular loop of the M3R was used and their functional properties were compared with those of human autoantibodies. Peptides corresponding to the second extracellular loop of the M3R were used as immunogens in rabbits, and antisera were tested for inhibition of carbachol-evoked colon smooth muscle contraction in parallel with immunoglobulin G from a patient with SS. Anti-M3R antibodies were affinity purified on a peptide representing a dominant functional epitope at the COOH terminus of the second extracellular loop of the M3R and tested for concentration-dependent inhibition. Experimentally raised anti-M3R antibodies, like the human autoantibodies, showed concentration-dependent and noncompetitive inhibition of carbachol-evoked colon contractions. Inhibitory activity was detected by functional assays at concentrations as low as 3 ng/ml, which was below the threshold of detection of antibody by peptide enzyme-linked immunosorbent assay. It is concluded that the experimentally raised anti-M3R antibodies share the functional properties of autoantibodies in patients with SS.

Increased expression of the B-cell-regulatory molecule CD72 in primary Sjögren's syndrome.

To determine whether there is an intrinsic abnormality of B-cell signaling in primary Sjögren's syndrome (pSS), the expression of B-cell coreceptors was determined in patients with primary Sjögren's syndrome and healthy and disease controls. Peripheral blood mononuclear cells were labeled with monoclonal antibodies to CD21, CD22, or CD72, and then the pan B-cell marker CD19. The expression of these coreceptors on the total CD19(+) population was determined. There was a significant increased expression of CD72 on the B cells of pSS patients (MFI, 215 +/- 6) compared to normal controls (MFI, 141 +/- 6). The increased CD72 expression was disease specific for pSS, as it was not observed in systemic lupus erythematosus or rheumatoid arthritis. The effect of B-cell stimulation on coreceptor expression was determined by culturing cells with B-lymphocyte-activating factor (BAFF) and/or pokeweed mitogen (PWM) or without either. Following culture, CD72 expression was decreased in both pSS and normal controls, regardless of the presence of BAFF or PWM. The upregulation of CD72 in pSS might be a compensatory response to increased B-cell receptor stimulation or a primary abnormality leading to uncontrolled B-cell activation.

Fas gene promoter polymorphisms in primary Sjögren's syndrome.

BACKGROUND: Fas mediated apoptosis may be important in the pathogenesis of primary Sjögren's syndrome (pSS). OBJECTIVE: To examine genetic variation in the promoter region of the Fas gene in pSS. METHODS: Two single nucleotide polymorphisms at positions -1377(G/A) and -670(G/A) in the Fas gene promoter were genotyped by PCR-SSP in 101 patients with pSS and 108 Caucasoid controls. RESULTS: No significant differences in allele or genotype frequencies were detected between the patients with pSS and controls. However, significant associations were observed with Ro/La autoantibody negative patients, who display milder and later onset disease. The -670A allele was more frequent in Ro/La autoantibody negative patients than in Ro/La autoantibody positive patients (p = 0.04). CONCLUSION: This study does not confirm an earlier report of an association between pSS and the Fas promoter -670G allele. However, the results suggest that genetically determined variability in Fas expression may modulate Ro/La autoantibody responses in patients with pSS.

Association of stress proteins with autoantigens: a possible mechanism for triggering autoimmunity?

Patterns of autoantibody production are diagnostic of many autoimmune disorders; the recent observation of additional autospecificities towards stress-induced proteins may also provide insight into the mechanisms by which such responses arise. Grp78 (also known as BiP) is a target of autoaggressive B and T cell responses in our murine model of anti-Ro (SS-A) autoimmunity and also in rheumatoid arthritis. In this report we demonstrate reciprocal intermolecular spreading occurs between Ro52 and Grp78 in immunized mice, reflecting physiological association of these molecules in vivo. Moreover, we provide direct biochemical evidence that Grp78 associates with the clinically relevant autoantigen, Ro52 (SS-A). Due to the discrete compartmentalization of Ro52 (nucleocytoplasmic) and Grp78 (endoplasmic reticulum; ER) we propose that association of these molecules occurs either in apoptotic cells, where they have been demonstrated indirectly to co-localize in discrete apoptotic bodies, or in B cells themselves where both Ro52 and Grp78 are known to bind to immunoglobulin heavy chains. Tagging of molecules by association with Grp78 may facilitate receptor mediated phagocytotsis of the complex; we show evidence that exogenous Grp78 can associate with cell surface receptors on a subpopulation of murine splenocytes. Given the likelihood that Grp78 will associate with viral glycoproteins in the ER it is possible that it may become a bystander target of the spreading antiviral immune response. Thus, we propose a model whereby immunity elicited towards Grp78 leads to the selection of responses towards the Ro polypeptides and the subsequent cascade of responses observed in human disease.

Restricted specificity of intermolecular spreading to endogenous La (SS-B) and 60 kDa Ro (SS-A) in experimental autoimmunity.

Intermolecular spreading of humoral autoimmunity to different components of the Ro (SS-A) and La (SS-B) ribonucleoprotein (RNP) complex has been reported following immunization with a single component of the complex. Although the immune response to the immunizing antigen is polyclonal and diversified, little is known about the specificity of the recruited autoimmune responses to the endogenous Ro and La antigens which drive B-cell spreading. To determine the specificity of intermolecular spreading to La, we examined sera from 52 kDa Ro (Ro52)- and 60 kDa Ro (Ro60)-immunized C3H/HeJ mice for reactivity with recombinant fragments spanning endogenous mouse (m)La by enzyme-linked immunosorbent assay (ELISA) and immunoblotting. Sera from mice primed and boosted with recombinant Ro52 and Ro60 showed reactivity restricted to the COOH-terminal fragment of mLa (aa361-415). The recruited anti-La response was species-specific, cross-reacting weakly with the corresponding region on the human La molecule, and was abrogated by the preabsorption of the Ro-immune sera with mLa 361-415. Analogous experiments using recombinant mRo60 fragments spanning the mRo60 molecule revealed a similar pattern of oligoclonality in the specificity of anti-Ro60 autoimmunity following active immunization with La and Ro52. These results suggest that intermolecular-intrastructural T-B help is limiting in this model, and reveal unsuspected immunodominance of selected Ro-La epitopes in the spreading of the autoantibody response to these structures. The focusing of the recruited autoantibody response to these COOH-terminal regions of the Ro and La polypeptides may also reflect the surface accessibility of these regions in La-Ro RNP.

Subcellular distribution of aquaporin 5 in salivary glands in primary Sjögren's syndrome.

Secretions from salivary and lacrimal glands are reduced in patients with primary Sjögren's syndrome (PSS). Since aquaporin 5 is involved in transport of water and is present in salivary and lacrimal glands, this protein was thought to have a major role in the pathogenesis of PSS. We used indirect immunofluorescence and an immunoperoxidase technique to assess expression and subcellular localisation of aquaporin 5 in patients and controls. Our results suggest that the distribution and density of aquaporin 5 in salivary glands does not differ between patients with PSS and those without. Thus, the role of aquaporin 5 in the pathogenesis of PSS needs to be reassessed and alternative pathogenetic mechanisms investigated.

Can B cell epitopes of 60 kDa Ro distinguish systemic lupus erythematosus from Sjögren's syndrome?

Antibodies binding components of the Ro/La (or SSA/SSB) ribonucleoprotein particle are found in the sera of patients with systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS) as well as mothers who give birth to babies with neonatal lupus. Anti-La occurs in a subset of sera that contain anti-Ro, and anti-La is found more commonly in sera of patients with SS than in sera from SLE patients. The fine specificity of autoantibodies binding 60 kDa has been studied extensively. Recent data have suggested that there are disease-specific epitopes which identify patients with either SLE or SS. Alternatively, other data suggest that the B cell epitopes of 60kDa Ro vary according to the presence of anti-La. The present study was undertaken to determine whether binding of putative disease-specific 60 kDa Ro epitopes is associated with the diagnosis of SLE vs SS, or instead associated with the presence of anti-La. Anti-60 kDa Ro positive sera from 24 SLE patients and 44 SS patients were studied for antibodies binding two epitopes of 60 kDa Ro. We find the epitope defined by residues 171-190 is associated with anti-60 kDa Ro without anti-La, regardless of diagnosis. Meanwhile, binding of the epitope defined by residues 215-232 is not commonly found in anti-60 kDa Ro sera, especially in those sera with both anti-60 kDa Ro and anti-La. Thus, the fine specificity of antibody binding to 60 kDa Ro varies according to the presence of anti-La, not to the diagnosis of either SLE or SS.

Cognate T cell help is sufficient to trigger anti-nuclear autoantibodies in naive mice.

The mechanisms involved in the initiation of anti-nuclear autoantibodies are unknown. In this study, we show that one factor allowing anti-nuclear autoantibodies to develop is the incomplete nature of immune tolerance to many of these proteins. Immune responses in mice toward the ubiquitous nuclear autoantigen La/SS-B are much weaker than responses to the xenoantigen, human La (hLa; 74% identical). However, in transgenic (Tg) mice expressing hLa, the Ab response to this neo-autoantigen was reduced to a level resembling the weak autoimmune response to mouse LA: Partial tolerance to endogenous La autoantigen was restricted to the T compartment because transfer of CD4(+) T cells specific for one or more hLa determinants into mice bearing the hLa transgene was sufficient to elicit production of anti-hLa autoantibodies. Notably, only hLa- specific T cells from non-Tg mice, and not T cells from hLa Tg mice, induced autoantibody production in hLa Tg mice. These findings confirm partial Th tolerance to endogenous La and indicate the existence in normal animals of autoreactive B cells continuously presenting La nuclear AG: Therefore, the B cell compartment is constitutively set to respond to particular nuclear autoantigens, implicating limiting Th responses as a critical checkpoint in the development of anti-nuclear autoantibodies in normal individuals.