Inhibitory effects of autoantibodies on the muscarinic receptors in Sjögren's syndrome

Sjogren's syndrome (SS) is a systemic autoimmune disease that involves reduced salivary secretions. Recently, circulating autoantibodies from SS patients against the type 3 muscarinic cholinergic receptor (M3R) has been reported in the sera of SS patients. However, the role of these autoantibodies in the development of SS has not been elucidated. In this study, purified IgG was obtained from the sera of 11 SS patients, and its inhibitory effect on the M3R of the salivary glands was evaluated using RT-PCR, microspectrofluorimetry, immunohistochemistry, and Western blot analysis. Stimulation with carbachol (CCh) evoked a [Ca2+]i transient in the fura-2 loaded HSG cells. However, pretreatment of the cells with SS IgG (0.5 mg/ml) for 12 or 24 h significantly reduced the magnitude of the CCh-induced [Ca2+]i transient (CICT). We found that the magnitude of CICT was decreased by 62-45% when cells were pretreated with the SS IgG. However, the [Ca2+]i response to ATP was not altered by the pretreatment of SS IgG. The effect of SS IgG on CICT was abrogated by the inclusion of excessive competitive peptides that encode the amino-acid sequence of M3R, which was not recapitulated by nonspecific peptides. The inhibitory effect of SS IgG on the aquaporin (AQP)-5 expression was also examined. After confirming the apical localization of AQP-5 along with its increase by pilocarpine (10(-5) M), we examined whether SS IgG had an effect on pilocarpine-induced AQP-5 trafficking to the apical membrane (APM) using rat parotid acinar cells. After incubating the cells with SS IgG for 12 h, the amount of pilocarpine-induced AQP-5 significantly decreased compared to the control groups. In conclusion, autoantibodies from the SS patients inhibit the function of the human M3R that is mediated by Ca2+ mobilization and AQP-5 trafficking. Our results could partly explain the underlying mechanisms of glandular dysfunction and associated features of impaired autonomic function in SS patients.     

Polarization of B effector cells in Sjögren's syndrome

Analysis of salivary glands of patients with primary Sj?gren's syndrome has yielded conflicting results with respect to T helper (Th)1/Th2 polarization. This balance might parallel the progress of the local lesions. B-cells are now taking center stage in this disease. They can also be primed to differentiate into two cytokine-production pathways, dubbed B effector (Be) 1 and Be2 cells. This is discussed in the light of our recent finding that Be1 accompany Th1, while Be2 accompany in the tissue lesions.     

Impaired expression of Act1mRNA in B cells of patients with Sjögren's syndrome

Sj?gren's syndrome (SS) is a systemic autoimmune disorder characterized by profound lymphocytic infiltration into the lacrimal and salivary glands, thereby diminished secretory function. B cell hyper-activation is a predominant feature of SS related to hypergammaglobulinemia and production of autoantibodies. The adaptor molecule NF-kB activator 1 (Act1) plays an important role in the homeostasis of B cells by attenuating CD40 and B cell-activating factor belonging to the tumor necrosis factor family receptor (BAFFR) signaling. Act1-deficient mice develop autoimmune manifestations similar to SS, which are hypergammaglobulinemia, high levels of anti-SSA and anti-SSB autoantibodies. In this study, to investigate the role of Act1 in the pathogenesis of SS, we examined Act1mRNA expressions in B cells from patients with SS and discussed the association of Act1 with parameters and clinical manifestations of SS. We showed the low level of Act1mRNA expression in patients with SS and reciprocal association of Act1 with serum IgG level. Diminished Act1mRNA expression in SS may be associated with B cell hyperactivity and elevated immunoglobulin production in SS by uncontrolled B cell activation signal through CD40 and BAFFR.     

Clinical significance of autoantibodies recognizing Sjögren's syndrome A (SSA), SSB, calpastatin and alpha-fodrin in primary Sjögren's syndrome

The aim of our study was (i) to compare the clinical and biological characteristics of 148 (137 women, 11 men) primary Sjögren's syndrome (pSS) patients at diagnosis as a function of their sex and (ii) to assess the prognostic value of anti‐calpastatin and anti‐alpha‐fodrin autoantibodies. In addition, the presence of anti‐nuclear antibodies (ANA), anti‐52‐ and 60‐kDa Sjögren's syndrome A (SSA), anti‐Sjögren's syndrome B (SSB), anti‐cyclic citrullinated peptide (CCP) antibodies and rheumatoid factors (RF) of IgA, IgG and IgM isotypes was sought in sera collected at pSS onset. Raynaud's syndrome, significantly more frequent in women, was the only systemic manifestation of pSS whose frequency differed significantly as a function of the patient's sex (P = 0·02). ANA (P = 0·001) and anti‐60‐kDa SSA autoantibodies (P = 0·03) were significantly more common in women, while men never synthesized detectable levels of anti‐SSB, anti‐calpastatin or IgG anti‐alpha‐fodrin autoantibodies. In addition, anti‐CCP autoantibodies were found in low percentages of pSS patients (4% F/18% M). The absence of autoantibodies does not exclude the diagnosis of pSS in men that will be based mainly on the anatomopathological findings of a minor salivary gland biopsy. Positivity of anti‐60‐kDa SSA, anti‐SSB, anti‐calpastatin, IgA and IgG anti‐alpha‐fodrin antibodies is not associated with pSS clinical and biological severity.     

Circulating cytokines in primary Sjögren's syndrome determined by a multiplex cytokine array system

Modulation of cytotoxic responses by viral immunoevasins plays an important role in the establishment of latent and persistent viral infections. Together with MHC class-I-restricted CD8T-lymphocytes, non-MHC-restricted natural killer (NK) and lymphokine-activated killer (LAK) cells participate in this anti-viral control. The Us3 protein kinase of herpes simplex virus-1 (HSV-1) inhibits CD8T-cell cytotoxicity, which correlates with the inhibition of granzyme-B (GrB)-induced activation of pro-apoptotic Bid. We have investigated the effect of Us3 on NK and LAK cytotoxicity, because these effectors are believed to share common mechanisms for inducing cell death. We show that, in contrast to their lower sensitivity to CD8T-cell lysis, HSV-1-infected cells are lysed by NK cells or LAK cells as efficiently as the uninfected controls. Both CD8T and NK/LAK effectors were dependent on the activity of GrB and were efficiently blocked by means of treatment with a GrB inhibitor. However, unlike CD8T cells, LAK cells and NK cells failed to induce Bid cleavage, suggesting that various GrB downstream targets be involved in the induction of cell lysis. This finding explains their various sensitivities to viral modulation, which is likely to be important for the respective role of MHC-restricted and non-restricted effectors in the control of HSV-1 infection.     

Role of dendritic cells in Sjögren's syndrome

Sjögren's syndrome (SS) is a chronic inflammatory and lymphoproliferative autoimmune disease of unknown aetiology. It is characterised by progressive mononuclear cell infiltration of the salivary and lacrimal glands and a decreased glandular secretion, resulting in dryness of the mouth and eyes (xerostomia and keratoconjunctivitis sicca, respectively). Dendritic cells (DC) are considered to be the most potent antigen‐presenting cells. Because of their central role in initiating an immune response while maintaining tolerance, impaired function of these cells might lead to the break of peripheral tolerance and initiation of immune responses to self‐antigens. This review will focus on the possible role of DC in SS.     

Sensitive and robust luminescent profiling of anti-La and other autoantibodies in Sjögren's syndrome

Sjögren's syndrome (SjS) patients often have a variety of extraglandular manifestations including neurological and gastrointestinal involvement. In this study we evaluated the diagnostic performance of luciferase immunoprecipitation system (LIPS) that employs mammalian cell-produced recombinant antigens for analyzing SjS autoantibody responses. LIPS testing of mammalian cell-produced La, Ro60 and Ro52 recombinant antigens with defined commercial antibodies demonstrated highly specific immunoprecitation of each antigen without cross-reactivity. Next, sera from 57 SjS and 25 volunteers were evaluated by LIPS against a panel of human autoantigens. LIPS detected robust anti-La antibody levels in 43/57 SjS patients (75% sensitivity) and markedly outperformed an ELISA (46% sensitivity). Profiling of other SjS-associated autoantigens revealed the presence of anti-Ro60, anti-Ro52 in 63% and 61%, of SjS patients, respectively. Interestingly, a C-terminal fragment of Ro52 (Ro52-Δ2), a protein fragment not previously found to be antigenic by ELISA, also showed positive immunoreactivity in 42/57 SjS patients (65% sensitivity). Additional profiling of other autoantigens revealed that certain SjS patients also showed positive immunoreactivity with thyroid peroxidase (14%), AQP-4 (12%) and the H⁺/K⁺ gastric ATPase (16%) suggesting potential autoantibody attack of thyroid, neuronal and gastric parietal cells, respectively. These heterogeneous autoantibody responses detected by LIPS in SjS will likely be useful for diagnosis and for evaluating extraglandular manifestations.     

Autoantigen-specific memory B cells in primary Sjögren's syndrome

Sjögren’s syndrome (SS) is a systemic rheumatic autoimmune disease affecting the exocrine glandular function and is characterized by the presence of autoantibodies against the ribonucleoprotein particles, SS‐A/Ro and SS‐B/La, and mononuclear cell infiltration of exocrine tissues. Our aim is to characterize memory B cell pattern and function in relation to the progression of the disease, by analysing samples from a well‐defined cohort of patients with primary SS. We have measured the number of Ro/La‐specific plasma cells in peripheral blood mononuclear cells (PBMC) from 23 patients and 20 healthy controls by direct enzyme‐linked immunospot (ELISPOT) assay. Furthermore, we quantified the Ro‐ and La‐specific memory B cells in these individuals by a 6‐day in vitro polyclonal stimulation of PBMC followed by an antigen‐specific ELISPOT assay for the detection of memory B cells. In addition to this, ELISA profiling of autoantibodies was carried out using patients’ plasma and supernatant, collected post‐mitogen stimulation of PBMC. The average Ro60‐, Ro52‐ and La48‐specific plasma cells in PB was 9, 17 and 13 cells in 10⁵ PBMC, respectively. After in vitro stimulation, these numbers increased to 43, 50 and 26 for Ro60, Ro52 and La48, correspondingly. However, the fraction of memory B cells activated into antibody‐secreting cells was lower than the overall IgG B cell population. We conclude that these lower Ro/La‐specific memory B cell levels may indicate that a greater portion of the Ro‐ and La‐specific B cells are in an activated stage. This is in tune with previous reports.     

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 Sj?gren's syndrome (pSS), the expression of B-cell coreceptors was determined in patients with primary Sj?gren'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.     

Discerning the kinetics of autoimmune manifestations in a model of Sjögren's syndrome

Ectopic follicles are non-encapsulated organized lymphoid structures that form at sites of inflammation and presumably contribute to the activation and differentiation of cells with autoreactive potential within target tissues. As such, directed targeting of ectopic follicles in settings of autoimmunity may provide a means to specifically inhibit the activation of autoreactive cells without impairing protective immune responses ongoing in peripheral lymphoid tissues. NOD·H2h4 mice are a non-diabetic strain of NOD mice which develop a Sjögren's syndrome-like disease which includes the formation of ectopic follicles in the salivary gland and characteristic Sjögren's autoantibodies. The goal of these studies was to better characterize the formation of ectopic follicles in this model and to explore their contribution to autoimmunity. Our studies show that by 8 weeks of age, young NOD·H2h4 mice spontaneously develop an abundance of splenic germinal centers, prior to the emergence of lymphocyte infiltration in the salivary gland tissue. Ectopic follicle formation in the salivary gland begins to appear in these mice between 12 and 16 weeks of age. Interestingly, anti-Ro and anti-La autoantibodies precede the development of ectopic follicles in young NOD·H2h4 mice. In contrast, production of anti-dsDNA antibodies is delayed and largely coincides with the formation of ectopic follicles in these mice. These data suggest that tertiary lymphoid structures may arise from the trafficking of activated T and B cells to sites of inflammation in non-lymphoid tissues. Furthermore, local presentation of autoantigens may then promote the expansion of autoreactive cells with specificities distinct from those generated in the splenic micro-environment.     

Fas-induced apoptosis is a rare event in Sjögren's syndrome

The aim of this study was to perform a controlled in situ analysis on the incidence of apoptosis, investigate the expression of apoptosis-mediating proteins, and determine the frequency of apoptotic CD4+ and CD8+ T cells in Sj?gren's syndrome (SS). The study was extended to patients with atrophy-fibrosis (AF) not related to SS, as well as to a control group. Immunohistochemistry and the terminal deoxynucleotidyl transferase mediated dUTP digoxigenin nick end labeling (TUNEL) method were applied to study the Fas and FasL expression and the incidence of apoptosis in salivary glands (SG) from patients with primary and secondary SS, AF, and controls. These methods were also combined to enable simultaneous detection of apoptotic and CD4+ or CD8+ T cells. Despite abundant expression of Fas and FasL in SS SG, apoptotic cells were not exceeding 1% in the foci of infiltrating mononuclear cells (IMC). Double staining showed that the frequency of apoptosis was low among both CD4+ and CD8+ T cells. Only a few TUNEL+ epithelial cells were found in all patient groups. Fas was expressed predominantly on SS IMC, single SS epithelial cells, and a few normal acinar cells, but not in AF SG. Although FasL was present on SS and AF IMC and epithelial cells, it was rarely detected in normal tissue. Consequently we demonstrate that Fas-induced apoptosis among SS SG is a rare event. Our findings support an earlier hypothesis indicating that IMC seem to be able to escape apoptosis, resulting in foci of inflammatory cells. Notably, however, no obvious correlation can be drawn to previous studies where a high incidence of apoptosis of epithelial cells was proposed as an important mechanism leading to decreased glandular function, which is a hallmark of SS.     

Pathophysiologic role of Interleukin-33/ST2 in Sjögren's syndrome

Interleukin-33 (IL-33) is a member of the IL-1 family and has dual functions as a nuclear factor as well as a cytokine. The pivotal role of IL-33 as an active player contributing to aberrant local and systemic damage has been highlighted in several inflammatory and autoimmune diseases. Primary Sjögren's syndrome (pSS) is an autoimmune disease characterized by dry eyes and mouth syndrome due to local dysfunctions of exocrine glands, but also accompanied with systemic manifestations. The pathophysiology of pSS has been advocated as a conjecture of activated B and T cells as well as the production of inflammatory cytokines and autoantibodies, driving epithelial tissue damage and disease progression. In pSS, IL-33 is released in the extracellular space from damaged salivary cells upon pro-inflammatory stimuli and/or dysfunction of epithelial barrier. Counter-regulatory mechanisms are initiated to limit the pro-inflammatory actions of IL-33 as portrayed by an increase in the decoy receptor for IL-33, the soluble form of ST2 (sST2). In pSS and associated diseases, the levels of IL-33 are significantly elevated in the serum or tears of patients. Mechanistically, IL-33 acts in synergy with IL-12 and IL-23 on NK and NKT cells to boost the production of IFN-γ contributing to inflammation. TNF-α, IL-1β and IFN-γ in turn further increase the activation of IL-33/ST2 pathway, thereby constituting a vicious inflammatory loop leading to disease exacerbation. IL-33/ST2 axis is involved in Sjögren's syndrome and opens new perspectives as therapeutic target of one of the culprits in the inflammatory perpetuation.     

Distinct profiles of Sjögren's syndrome patients with ectopic salivary gland germinal centers revealed by serum cytokines and BAFF

The formation of ectopic germinal centers (GC) has been described in Sjögren's syndrome (SS), although little is known about the molecular basis of this phenomenon. These structures are a focus of in situ autoantibody production and have been hypothesized to be involved in lymphomagenesis in SS patients. Serum cytokines also play an important role in SS pathogenesis in part via immune dysregulation and may therefore contribute to ectopic GC formation. Herein, highly multiplex cytokine screening of SS patients with (SSGC+) and without (SSGC?) GC formation was done to identify cytokine profiles that correlate with this phenomenon. Serum levels of B-cell activating factor (BAFF) were also screened as a potential biomarker of immune dysregulation in SS and SSGC formation. Univariate analysis demonstrated that serum levels of a broad spectrum of immune and inflammatory modulating cytokines are upregulated in SSGC+ and SSGC? patients relative to unaffected controls IL-1β, IL?2, IL-6, IL-15, IFN-γ and CCL4 (MIP-1β). SSGC+ patients were distinguished from healthy individuals by higher levels of IL-4, IL-10, GM-CSF, IFN-α, CCL3 (MIP-1α), CCL11 (Eotaxin) and BAFF, while SSGC+ and SSGC? patients differed in CCL2 (MCP-1) expression. Discriminant function analysis (DFA), a multivariate discrimination method that uses observed differences to characterize groups when casual relationships are not well understood, was employed to identify a subset of these biomarkers that maximally discriminate among SSGC+, SSGC? and unaffected individuals. The biomarker having the strongest discriminatory power identified by DFA besides CCL11 (Eotaxin) and IFN-γ was BAFF. The variables identified by DFA are interdependent and are often of mechanistic significance to the pathologic states they distinguish, suggesting that these factors modulate SS pathology and SSGC formation in a synergistic manner.     

Attenuated apoptosis of B cell activating factor-expressing cells in primary Sjögren's syndrome

B cell activating factor (BAFF) is known to be a powerful regulator of B-cell differentiation and proliferation. The aim of this study was to assess the incidence of apoptosis among BAFF-expressing cells in Sj?gren's syndrome (SS) salivary gland tissue. We performed double stainings of BAFF together with one of the markers, CD21, CD68, CD40, Fas, Bcl-2 or Bax, and monitored apoptosis among BAFF expressing cells by using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-digoxigenin nick-end labeling method. A significantly lower level of apoptosis among the BAFF-expressing cells was detected in salivary glands from patients with SS compared with controls (p = 0.03). Furthermore, no difference in the coexpression of Fas or CD40 together with BAFF was detected between patients and controls. Coexpression of the pro apoptotic molecule Bax together with BAFF was nonsignificantly decreased in patients with SS compared with controls. Our results suggest that a reduced level of apoptosis among BAFF-expressing cells might lead to longer-existing BAFF expression within these cells and thereby maintain signaling for tissue-infiltrating B cells to proliferate and mature.     

Identification of tear lipocalin as a novel autoantigen target in Sjögren's syndrome

Sj?gren's syndrome (SS) is an autoimmune disease characterized by lymphocytic infiltration and tissue damage mainly confined to the salivary and lacrimal glands, resulting in dryness of mouth and eyes. Since different epithelial cells of exocrine and non-exocrine tissues are primarily affected, an autoimmune reaction against antigens commonly expressed in epithelial cells is believed to play a pathogenic role. To identify novel autoantigen targets associated with the systemic involvement in SS, we screened a random peptide library with pooled IgG immunoglobulins derived from patients with primary SS. Among the identified peptides, one was recognized by the majority of patients' sera, but not by sera of normal donors and of patients with other autoimmune diseases. The peptide showed homology with an Epstein-Barr Virus (EBV) derived protein and with tear lipocalin, a protein highly expressed in tears and saliva, and with alpha-fodrin, a cytoskeleton protein considered an important autoantigen target in SS. Anti-peptide antibodies affinity purified from patients' sera recognize the viral protein, tear lipocalin and alpha-fodrin. Our findings suggest that EBV infection may be linked to the pathogenesis of SS and that tear lipocalin can be considered a novel and yet unidentified autoantigen in SS.     

Neurophysiological assessment of peripheral neuropathy in primary Sjögren's syndrome

Peripheral nervous system complications are rare in patients with primary Sjögren's syndrome. We investigated a group of six women aged 43–64 years who complained of pain and sensory symptoms. Conventional neurophysiological tests reflecting large nerve fiber function revealed normal motor conduction in all patients, whereas sensory nerve action potentials were absent in two. On the other hand, quantitative thermometry and autonomic nerve function tests indicating small nerve fiber function were more sensitive in the assessment of nerve dysfunction; these showed abnormalities in all cases. Vibrametry showed dysfunctions in four patients. The latter methods possess great sensitivity in discovering sensory disturbances. Neurophysiological assessment of the sensory and autonomic nervous system demonstrating sensory neuropathy contributes to early diagnosis of primary Sjögren's syndrome.Abbreviations HRV heart rate variation - PNS peripheral nervous system - pSS primary Sjögren's syndrome - SSEP short-latency somatosensory evoked potentials - SSR sympathetic skin response - SuCV sudomotor nerve conduction velocity     

Genetics of Sjögren's syndrome in the genome-wide association era

While Sj?gren's syndrome (SS) is more common than related autoimmune disorders, such as systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA), scientific and medical research in SS has lagged behind significantly. This is especially true in the field of SS genetics, where efforts to date have relied heavily on candidate gene approaches. Within the last decade, the advent of the genome-wide association (GWA) scan has altered our understanding of disease pathogenesis in hundreds of disorders through the successful identification of novel risk loci. With strong evidence for a genetic component in SS as evidenced by familial aggregation of SS as well as similarities between SS and SLE and RA, the application of GWA approaches would likely yield numerous novel risk loci in SS. Here we review the fundamental scientific principles employed in GWA scans as well as the limitations of this tool, and we discuss the application of GWA scans in determining genetic variants at play in complex disease. We also examine the successful application of GWA scans in SLE, which now has more than 40 confirmed risk loci, and consider the possibility for a similar trajectory of SS genetic discovery in the era of GWA scans. Ultimately, the GWA studies that will be performed in SS have the potential to identify a myriad of novel genetic loci that will allow scientists to begin filling in the gaps in our understanding of the SS pathogenesis.