Autoantigens and Autoantibodies in the Pathogenesis of Sjögren’s Syndrome

1 Introduction

It is well established that environmental factors in concert with an appropriate genetic background play a fundamental role in triggering Sjögren’s syndrome (SS) leading to chronic inflammation of the target organs through different mechanisms, among which the molecular mimicry between infectious agents and autoantigens has been the most studied. An aberrant autoimmune response due to T- and B-lymphocyte hyperactivity and autoantibody production is known to be a crucial mechanism for the induction of autoimmune epitheliitis and the perpetuation of inflammation. In particular, histopathological damage ranges from mild to diffuse cell infiltrates characterized by a predominance of T and B cells. B-lymphocyte hyperactivity and the development of B-cell follicles containing germinal center–like structures represent a multistep process known as ectopic lymphoid neogenesis, which typically involves salivary glands and other SS target organs. As far as the T-cell compartment is concerned, different T-lymphocyte subpopulations participate in the development and maintenance of glandular inflammation. Indeed, T helper (Th) 1 lymphocytes and their soluble products, particularly interferons (IFNs), have long been considered to be the main players in the induction of chronic tissue damage, but the identification of Th17, Th22, T regulatory cells, and Th follicular cells has recently changed the view on the role played by the different T-cell subpopulations. Moreover, the interleukin (IL)-17-axis can favor B/T lymphocyte survival, protecting T cells from apoptosis and, as a consequence, potentiating aberrant autoimmune responses.

B-cell hyperactivity and the production of autoantibodies against different autoantigens are typical features of SS. Nevertheless, these autoantibodies are non–organ specific and their role in the pathogenesis of the disease is not clearly understood or defined ( Table 9.1 ). However, immunoglobulins (Igs) such as rheumatoid factor (RF), antinuclear antigens (ANAs), antiextractable nuclear and cytoplasmic antigens, particularly anti-Ro/SSA and anti-La/SSB, which have all been included in the diagnostic criteria of the disease, and anti-α-fodrin, are present in 80% to 90% of patients. Indeed, 10% to 20% of SS patients are considered seronegative and this condition seems to be associated with a potential delay in diagnosis, but also with a milder form of the disease, similar to what happens in other seronegative autoimmune disorders. Besides the autoantibodies associated to SS described so far, recently the presence of novel autoantibodies has been investigated with the aim of identifying new autoantigens, which may be specific for the disease. Such novel autoantibodies may have different characteristics and functions: be present in a percentage of seronegative patients; play a role in the pathogenesis of the disease; have a prognostic role in case of a correlation with specific organ damage; be a marker of response to therapy and important.

Table 9.1

Prevalence of Autoantibodies in SS Patients

Autoantibodies Against	Prevalence (%)
Nuclear antigens	59–85
Ro52/TRIM21	66.7
Ro60/TROVE2	52.1
La/SSB	49
U1RNP	2
Rheumatoid factor	36–74
Cryoglobulins	9–15
Centromere	4–17
Mitochondria	1.7–27
Smooth muscle	30
Cyclic citrullinated peptides	3–10
Calreticulin	20
Muscarinic 3 receptors	11
Carbonic anhydrase II	12.5–20.8
α-Fodrin	50–90

Several players may participate in the development of the disease. Firstly, genetic background plays a fundamental role, including genes encoding molecules implicated in B-cell activation [such as B-cell activating factor (BAFF)], lymphotoxins α and β, and tumor necrosis factor (TNF), together with genes involved in increased production of type I IFNs, known as IFN-signature, and with genes associated with specific human leukocyte antigens (HLAs), such as HLA-B8, HLA-Dw3, HLA-DR3, and DRw52. Secondly, infectious agents have been implicated in the pathogenesis of the disease including, Epstein–Barr virus (EBV), human T-cell virus type 1, cytomegalovirus, and hepatitis C virus, all characterized by selective tropism for epithelial and immune cells and by ability to induce neurohormonal disturbances which may interfere with sex hormone ratios affecting steroid-dependent cells such as epithelial and autoreactive cells, both involved in the pathogenesis of the disease.

The interplay between genetic and environmental factors may facilitate an autoimmune response against self-antigens, leading to tissue infiltration by immune cells and tissue damage with exposure of novel autoantigens and to further autoantibody production.

In this chapter, we will analyze both identified and proposed autoantigens and autoantibodies with the aim of clarifying the possible link between these molecules and the pathogenic mechanisms of the disease.

2 Pathogenesis and Autoantigens

The aberrant autoimmune response with T- and B-lymphocyte hyperactivity and autoantibody production has been described as the main mechanism for the induction of autoimmune damage typically related to the onset of SS. It has also been suggested that there is a cooperative role played by macrophages, natural killer (NK) cells, and dendritic cells (DCs), which have also been described in inflamed salivary glands. Particularly, based on their ability to participate in peripheral tolerance, DCs play an important role in the maintenance of autoimmune aggression by both interfering with autoreactive T-cell functions and acting as a putative source of IFNγ. This cytokine plays an important role in immune regulatory processes and is strongly associated with SS, because high concentrations have been found in the biopsy of target organs, particularly at the salivary gland level. Moreover, IFNγ is functionally linked to some of the established autoantigens of the disease, such as Ro/SSA and La/SS.

In this setting, the comprehension of the possible role played by self-antigens in the pathogenesis of the disease is crucial.

Indeed, various molecules have been reported as possible autoantigens. However, a causal connection has not been established for all the proposed molecules and in the majority of cases, we may only describe the presence of autoantibodies directed against such antigens as a mirror of the autoimmune aggression without any proof of pathogenic effect.

2.1 Ro/SSA and La/SSB

The Ro/La ribonucleoprotein (RNP) complexes are protein–RNA complexes formed by the association of the Ro52 kDa, Ro60 kDa, and La proteins with small cytoplasmic RNA.

In particular, Ro52 is an IFN-inducible protein belonging to the tripartite motif (TRIM) protein family, acting as intracellular Fc-receptor, and is implicated in the regulation of cell proliferation and apoptosis. Conversely, Ro60/TROVE2 protein is a ring-shaped RNA-binding protein participating in recognition and leading to degradation of misfolded defective RNAs. Similar to Ro60/TROVE2, La/SSB protein appears to be involved in RNA metabolism and, in particular, in the regulation of micro RNA (miRNA) expression by protecting and stabilizing precursor miRNA from nuclease activity.

Ro52 messenger RNA transcript may be spliced into a common or alternative form in relation to the presence or deletion of exon 4 in the process of transcription. The alternative form has been demonstrated in a variety of tissues, including the fetal heart and salivary glands, but the significance of this alternative splicing is still unclear because the demonstration of the corresponding Ro52 isoform has never been identified at the protein level in vivo.

The Ro52 protein has an E3 ligase activity and plays a role in the ubiquitination of proteins, leading to the regulation of cellular levels and activity of specific proteins. In particular, several proteins have been suggested as substrate for Ro52-mediated ubiquitination, including several members of the IFN-regulatory factor (IRF) transcription factor family. In a deficient Ro52 mouse model, it has been demonstrated that the lack of Ro52-mediated ubiquitination of IRF transcription factors lead to an aberrant expression of type I IFNs and proinflammatory cytokines, such as IL-6, IL-12/IL-23, and TNF-α, confirming a central role for Ro52 as negative regulators of IRFs and proinflammatory cytokines. Of relevance, Ro52 resides in the cytoplasm of unstimulated cells and translocates into the nucleus upon INF stimulation after viral infection. It seems that upregulation and nuclear translocation of Ro52 might be a functional part of the negative feedback loop suppressing IFN-mediated immune activation.

Ro60, a component of Ro/La RNP complex, is involved in quality control of RNA. Several reports indicate a sequence homology between Ro60 and viral protein, such as Coxsackie virus 2B protein and EBV nuclear antigen 1, suggesting that Ro60 may be an antigen responsible for a molecular mimicry mechanism. This hypothesis is in accordance with data which has reported Ro60 autoantibodies months before the appearance of Ro52 and La autoantibodies in systemic lupus erythematosus (SLE) patients, but unfortunately similar data are not available in SS.

The phosphoprotein La/SSB is a member of the RNA-recognition motif protein family and is a part of the Ro/La RNP complex that associates with small cytoplasmic RNAs and viral RNAs. Several studies have reported that viral infections induce miRNA and La complexes able to induce TLR3-related secretion of type-1 IFN and of TNF.

In this setting, it may be hypothesized that at the time of viral infection of salivary gland epithelial cells, Ro52 is overexpressed as a defensive mechanism both to suppress viral replication and to protect cells from prolonged activation of the type-1 IFN system. Simultaneously, the viral pathogen may use La protein to escape from the immune response, leading to a La/SSB overexpression. Nevertheless, both Ro and La antigens contribute to the amplification of an exuberant immune response, leading to aggression toward target organs.

2.2 Calreticulin

Calreticulin is an endoplasmic reticulum resident protein of approximately 46 kDa molecular weight with multiple functions, including control of cellular adhesiveness, gene expression, calcium homeostasis regulation, and molecular chaperoning. It acts as a multifunctional protein that behaves as a molecular chaperone and contributes to CD91-mediated antigen presentation. The major receptor of calreticulin is CD91. This receptor is involved in the cross-presentation of chaperoned peptides within classical antigen-presenting cells, leading to specific innate and adaptive immune responses, and it is also able to mediate a variety of cellular functions because it is expressed in other cell types, including hepatocytes, adipocytes, fibroblasts, neuronal cells, and epithelial cells of salivary glands. Of relevance, interaction of CD91 with calreticulin seems to be related to the clearance of apoptotic cells, a keystone in SS pathogenesis. It has been also demonstrated that calreticulin specifically binds to a linear chemically synthesized epitope of Ro60 and induces conformation-dependent recognition by autoantibodies obtained from human autoimmune patients’ sera. Moreover, the complex calreticulin-Ro60 can be internalized by salivary glands epithelial cells by CD91. These cells can act as nonprofessional APCs and trigger specific cellular autoimmune response and autoantibody production, thus amplifying the autoimmune response against Ro60.

In summary, calreticulin has been shown to be implicated in SS pathogenesis. It serves as molecular chaperone, has the ability to bind and induce processing of antigenic peptides, and colocalizes with Ro60 antigen in apoptotic blebs, leading to autoimmune response.

2.3 Muscarinic 3 Receptors

Five different subtypes of muscarine acetylcholine receptors (M1R–M5R) have been identified and, among all of them, muscarinic 3 receptor (M3R) is selectively expressed in exocrine glands and plays an important role in exocrine secretion. Acetylcholine binds to and activates M3R on salivary gland cells, increasing intracellular Ca ²⁺ concentration and leading to an activation of apical Cl ⁻ channels and consequent up-stimulation of salivary secretion. Activation of M3R also induces trafficking of aquaporin-5 from the cytoplasm to the apical membrane, causing a rapid transport of water across the cell membrane.

M3R has four extracellular domains, known as the N-terminal region and three extracellular loops. The second extracellular loop plays an important role in intracellular signaling and is critical for receptor activation by agonists. Moreover, the second extracellular domain of M3R seems to act as autoantigen both for T and B cells leading to an excess of INFγ secretion by autoreactive T lymphocytes in glandular infiltrates and to autoantibody production. Indeed, M3R reactive T cells were detected in a variable percentage of SS patients and it has been demonstrated in animal models that M3R-reactive Th1 and Th17 cells are essential for the development of sialadenitis. Similarly, detection of autoantibodies against the second loop of M3R is present in about 50% of SS patients and the same antibodies seem to be pathogenetic, because they are able to induce a variation in intracellular Ca ²⁺ influx.

Taken together these data provide evidence for a pathogenic role of M3R and suggest the potential for both T- and B-cell targeted therapy in SS.

2.4 Carbonic Anhydrase II

Carbonic anhydrases form a family of enzymes classified as metalloproteases, because their active site contains a zinc ion and is able to catalyze the reversible hydration of carbon dioxide to generate a proton and a bicarbonate ion. Carbonic anhydrase II (CAII) is the only soluble form of the enzyme and regulates the acid–base homeostasis in erythrocytes and the aqueous chambers of the eyes and renal tubules. CAII can be shown in the cytosol of tubular renal cells of both proximal and distal tubules and also in the salivary gland epithelial cells of animal models. Moreover, in a mouse model, immunization of mice with CAII leads to a systemic exocrine gland inflammation and infiltration, similar to that observed in human SS. There are no reports regarding the potential pathogenic role of CAII in inflammation of salivary gland epithelial cells and renal tubule cells, but only a strong association between anti-CAII antibodies and a high risk of renal tubular acidosis development.

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