Increased serum levels of macrophage migration inhibitory factor in patients with primary Sj?gren's syndrome

The objective of this study was to analyse levels of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) in patients with primary Sj?gren's syndrome (pSS) and to examine associations of MIF with clinical, serological and immunological variables. MIF was determined by ELISA in the sera of 76 patients with pSS. Further relevant cytokines (IL-1, IL-6, IL-10, IFN-γ and TNF-α) secreted by peripheral blood mononuclear cells (PBMC) were determined by ELISPOT assay. Lymphocytes and monocytes were examined flow-cytometrically for the expression of activation markers. Results were correlated with clinical and laboratory findings as well as with the HLA-DR genotype. Healthy age- and sex-matched volunteers served as controls. We found that MIF was increased in patients with pSS compared with healthy controls (p < 0.01). In particular, increased levels of MIF were associated with hypergammaglobulinemia. Further, we found a negative correlation of MIF levels with the number of IL-10-secreting PBMC in pSS patients (r = -0.389, p < 0.01). Our data indicate that MIF might participate in the pathogenesis of primary Sj?gren's syndrome. MIF may contribute to B-cell hyperactivity indicated by hypergammaglobulinemia. The inverse relationship of IL-10 and MIF suggests that IL-10 works as an antagonist of MIF in pSS.     

Increased serum levels of macrophage migration inhibitory factor in patients with primary Sjögren's syndrome

The objective of this study was to analyse levels of the proinflammatory cytokine macrophage migration inhibitory factor (MIF) in patients with primary Sj?gren's syndrome (pSS) and to examine associations of MIF with clinical, serological and immunological variables. MIF was determined by ELISA in the sera of 76 patients with pSS. Further relevant cytokines (IL-1, IL-6, IL-10, IFN-gamma and TNF-alpha) secreted by peripheral blood mononuclear cells (PBMC) were determined by ELISPOT assay. Lymphocytes and monocytes were examined flow-cytometrically for the expression of activation markers. Results were correlated with clinical and laboratory findings as well as with the HLA-DR genotype. Healthy age- and sex-matched volunteers served as controls. We found that MIF was increased in patients with pSS compared with healthy controls (p < 0.01). In particular, increased levels of MIF were associated with hypergammaglobulinemia. Further, we found a negative correlation of MIF levels with the number of IL-10-secreting PBMC in pSS patients (r = -0.389, p < 0.01). Our data indicate that MIF might participate in the pathogenesis of primary Sj?gren's syndrome. MIF may contribute to B-cell hyperactivity indicated by hypergammaglobulinemia. The inverse relationship of IL-10 and MIF suggests that IL-10 works as an antagonist of MIF in pSS.     

Mild autonomic dysfunction in primary Sj?gren's syndrome: a controlled study

Introduction  

The aim of this study was to compare cardiovascular autonomic nervous system function in patients with primary Sj?gren's syndrome (pSS) with that in control individuals, and to correlate the findings with autonomic symptoms and the presence of exocrine secretory dysfunction.     

Novel variant of p230 trans-Golgi network protein identified by serum from Sjögren's syndrome patient

Trans-Golgi network (TGN) protein p230 is a peripheral membrane protein associated with the cytoplasmic face of the TGN. TGNp230 is an extensively coiled-coil protein with flexible amino- and carboxyl-terminal ends, associates with non-clathrin-coated vesicles arising from the TGN, and is implicated in vesicle biogenesis. Here we used an autoimmune serum from a patient with S ogren's syndrome to clone partial cDNAs from a human hepatoma HepG2 expression library. The partial cDNAs encoded a novel amino-terminal splice variant of TGNp230. Specific reactivity of the autoimmune serum for p230 is supported by immunofluorescene staining of the Golgi apparatus, immunoblotting of a > 200-kDa HeLa cell protein, and reactivity with a bacterially expressed GST-p230 fusion protein. The alternative splicing occurs within the first proline-rich domain of p230. It comprises a deletion of 30 bp followed immediately by an additional 66 bp absent in the published sequence. RT-PCR analysis indicated that the splicing occurs independently of previously reported carboxyl-terminal splicing, and that this novel splice variant is more frequent than the previously reported p230. The novel splice variant of p230 is also located at the TGN. We propose that p230 splice variants may be implicated in selection of cargo molecules for vesicles arising from the TGN.     

What have we learned from clinical trials in primary Sj?gren's syndrome about pathogenesis?

In vitro and in vivo experimental data have pointed to new immunopathogenic mechanisms in primary Sj?gren's syndrome (pSS). The availability of targeted treatment modalities has opened new ways to selectively target these mechanistic pathways in vivo. This has taught us that the role of proinflammatory cytokines, in particular TNFα, is not crucial in the immunopathogenesis of pSS. B cells appear to play a major role, as depletion of B cells leads to restoration of salivary flow and is efficacious for treatment of extraglandular manifestations and mucosa-associated lymphoid tissue lymphoma. B cells also orchestrate T-cell infiltration and ductal epithelial dearrangement in the salivary glands. Gene profiling of salivary gland tissue in relation to B-cell depletion confirms that the axis of IFNα, B-cell activating factor, B-cell activation, proliferation and survival constitutes a major pathogenic route in pSS.     

Health-related quality of life in patients with primary Sjögren's syndrome and xerostomia: a comparative study

Objective: To compare the health status of groups of Primary Sjögren's and Xerostomia patients, using the Medical Outcomes Short Form 36 (SF‐36). The SF‐36 is a generic measure, divided into eight domains, used in the assessment of health‐related quality of life. Patients and methods : The SF‐36 was given to 2 groups: Group 1 comprised 43 patients diagnosed with Primary Sjögren's Syndrome (1SS) and an unstimulated whole salivary flow rate (UFR) of <0.1 ml/min). Group 2 (n = 40) reported Xerosiomia but had an UFR >0.2 ml/min. Sub groups of patients in Groups 1 and 2 were compared with community normative data, for the SF‐36 Results: There were trends to suggest lower SF36 scores for 1SS patients but there were no significant differences between the mean domain scores of Groups 1 and 2. 1SS and Xerostomia patients registered lower mean scores across all 8 domains, compared with normative community data. Conclusion: The SF‐36 was unable to detect significant differences between subjects with 1SS and Xerostomia but a larger sample size is required to confirm these findings. The results of this limited study suggest that a disease‐specific measure is required to assess the impact 1SS on health‐related Quality of life (QOL).     

In vitro suppression of immune responses using monocyte-derived tolerogenic dendritic cells from patients with primary Sj?gren's syndrome

Introduction

Therapeutic vaccination with antigen-specific tolerogenic dendritic cells (tolDC) might become a future option of individualized therapy for patients with autoimmune diseases. In this study, we tested the possibility of generating monocyte-derived tolDC from patients with primary Sjögren''s syndrome (pSS). We analyzed phenotype, cytokine production and ability to suppress Ro/La-specific immune responses.

Methods

Monocyte-derived tolDC from patients with pSS were generated in the presence of dexamethasone, vitamin D3 and lipopolysaccharide (DexVD3 DC). The phenotype was analyzed by flow cytometry and the cytokine profile was investigated using a 25-plex Luminex assay and ELISA. The capacity to both stimulate Ro/La-specific T cells and suppress this response was evaluated by autologous mixed lymphocyte reaction (MLR).

Results

DC generated from patients with pSS had a similar phenotype and cytokine profile to those from healthy controls. DexVD3 DC from pSS patients induced little antigen-specific T cell proliferation, but DexVD3 DC-primed lymphocytes successfully suppressed Ro/La-specific T cell responses.

Conclusions

DexVD3 DC presenting Ro/La antigens might be a promising new therapeutic option for patients with pSS.     

Proteomic analysis of saliva: a unique tool to distinguish primary Sjögren's syndrome from secondary Sjögren's syndrome and other sicca syndromes

Introduction

A growing interest has arisen in salivary proteomics as a tool for the identification of biomarkers for primary Sjögren's syndrome (pSS). Nonetheless, only a limited number of preclinical validation studies have been performed, limiting the possibility of translating proteomic results into clinical practice. The primary aim of this study was to refine the diagnostic power of a panel of candidate salivary biomarkers described in pSS with respect to both healthy volunteers and pathological controls. We also explored the pathogenetic function of the detected putative biomarkers both in the local exocrinopathy and in the systemic inflammatory processes of SS.

Methods

One hundred and eighty patients were included in the study overall. In the first "exploratory phase", we enrolled 40 females with pSS, 40 sex- and age-matched healthy volunteers, 10 patients with sicca non-SS and 15 secondary SS (sSS) patients. The testing cohort of the second "challenge phase" of the study was represented by 75 unselected, consecutive subjects: 19 pSS, 21 healthy volunteers, 10 sicca non-SS and 25 sSS patients. Salivary proteomic analysis was performed combining two-dimensional electrophoresis (2DE) and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). Western blot (WB) analysis and enzyme-linked immunosorbent assay (ELISA) were employed to validate 2DE results. Ingenuity Pathway Analysis (IPA) Knowledge base was adopted to associate candidate biomarkers in a signalling pathogenetic network.

Results

A total of 28, 6, 7 and 12 protein spots were found to be significantly different in pSS samples with respect to healthy volunteers, non-SS sicca syndrome, SSc-sSS and rheumatoid arthritis-sSS, leading to the identification of 15 differently expressed proteins. Among them, α-amylases precursor, carbonic anhydrase VI, β-2 microglobulin, glyceraldehydes-3-phosphate dehydrogenase (G3PDH), epidermal fatty acid binding protein (E-FABP) and immunoglobulin k light chain (IGK-light chain) apparently showed the most significant differences in pSS when compared to healthy volunteers and non-SS pathological controls. On the other hand, as expected, pSS and sSS salivary profiles shared a great number of similarities.

Conclusions

This study demonstrated that salivary fluid might represent a novel ideal milieu for the detection of a diagnostic panel of candidate biomarkers for pSS, and to gain an insight into the pathogenetic processes underlying glandular and systemic autoimmune disorders.     

What have we learned from clinical trials in primary Sjögren's syndrome about pathogenesis?

In vitro and in vivo experimental data have pointed to new immunopathogenic mechanisms in primary Sj?gren's syndrome (pSS). The availability of targeted treatment modalities has opened new ways to selectively target these mechanistic pathways in vivo. This has taught us that the role of proinflammatory cytokines, in particular TNFα, is not crucial in the immunopathogenesis of pSS. B cells appear to play a major role, as depletion of B cells leads to restoration of salivary flow and is efficacious for treatment of extraglandular manifestations and mucosa-associated lymphoid tissue lymphoma. B cells also orchestrate T-cell infiltration and ductal epithelial dearrangement in the salivary glands. Gene profiling of salivary gland tissue in relation to B-cell depletion confirms that the axis of IFNα, B-cell activating factor, B-cell activation, proliferation and survival constitutes a major pathogenic route in pSS.     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sjögren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (Vλ2E) and variable kappa chain genes (VκA27). Moreover, clonally related V_L chain rearrangements were identified; namely, VκA27–Jκ5 and VκA19–Jκ2 in the parotid gland, and Vλ1C–Jλ3 in the parotid gland and the peripheral blood. Vκ and Vλ rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V_L chain genes caused by selection and clonal expansion of B cells expressing particular V_L genes. In addition, the data document an accumulation of B cells bearing mutated V_L gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.     

Analysis of immunoglobulin light chain rearrangements in the salivary gland and blood of a patient with Sjögren's syndrome

Patients with Sj?gren's syndrome (SS) have characteristic lymphocytic infiltrates of the salivary glands. To determine whether the B cells accumulating in the salivary glands of SS patients represent a distinct population and to delineate their potential immunopathologic impact, individual B cells obtained from the parotid gland and from the peripheral blood were analyzed for immunoglobulin light chain gene rearrangements by PCR amplification of genomic DNA. The productive immunoglobulin light chain repertoire in the parotid gland of the SS patient was found to be restricted, showing a preferential usage of particular variable lambda chain genes (V lambda 2E) and variable kappa chain genes (V kappa A27). Moreover, clonally related V(L) chain rearrangements were identified; namely, V kappa A27-J kappa 5 and V kappa A19-J kappa 2 in the parotid gland, and V lambda 1C-J lambda 3 in the parotid gland and the peripheral blood. V kappa and V lambda rearrangements from the parotid gland exhibited a significantly elevated mutational frequency compared with those from the peripheral blood (P < 0.001). Mutational analysis revealed a pattern of somatic hypermutation similar to that found in normal donors, and a comparable impact of selection of mutated rearrangements in both the peripheral blood and the parotid gland. These data indicate that there is biased usage of V(L) chain genes caused by selection and clonal expansion of B cells expressing particular V(L) genes. In addition, the data document an accumulation of B cells bearing mutated V(L) gene rearrangements within the parotid gland of the SS patient. These results suggest a role of antigen-activated and selected B cells in the local autoimmune process in SS.     

Changes of glycosylation of serum proteins in Sjögren's syndrome: correlation with interleukin-6 and soluble interleukin-2 receptor

A strong increase of the affinity for concanavalin A (Con A) of serum alpha 2-macroglobulin, a non-acute-phase protein, was observed by lectin blotting in patients with Sj?gren's syndrome (SS). On the contrary, the total Con A reactivity of serum proteins, measured by enzyme-linked lectin assay, was not augmented in SS, compared with normal donors, probably because positive changes of certain proteins were balanced by negative changes of others, as suggested by lectin blotting analysis. However, a significant increase of total Con A reactivity occurred in subjects with increased serum concentrations of soluble interleukin (IL)-2 receptor, compared with patients with normal concentrations of this marker of disease activity. On the other hand, the same parameter did not appear to be different in patients with normal or increased serum concentrations of IL-6, indicating that this cytokine was not probably responsible for the changes of glycosylation described here.     

Idiotypic analysis of a B cell clone with anti-intermediate filament specificity in a patient with Sj?gren's syndrome: involvement of five subpopulations producing different immunoglobulin isotypes

An IgM paraprotein from patient LP with Sj?gren's syndrome exhibited an antibody activity to intermediate filaments (IMF) of cells from all vertebrates examined, and appeared to recognize several classes of IMF (i.e., vimentin, desmin, and keratin). A mouse monoclonal anti-idiotype (Id) antibody, K4A, was prepared against the IgMk (LP) and used as a specific probe in two-color immunofluorescence to examine the extent of clonal involvement in the patient's blood and bone marrow mononuclear cells (MNC). Twenty to 30% of MNC in her blood samples were IgMk+ plasmablasts with morphologic similarity to Waldenstr?m's macroglobulinemia cells. IgG+ and IgA+ plasmablasts were demonstrated in lower frequencies (approximately 2%). Almost all of the IgM+ cells and approximately 80% of the IgG+ cells and IgA+ cells in the blood were reactive with the K4A anti-Id antibody. Immunoglobulin (Ig) subclass analysis revealed that the K4A Id was expressed by IgG1+, IgG3+, IgA1+ and IgA2+ plasmablasts. Similar observations were obtained with bone marrow samples, although the proportion of Id+ cells among IgG+ or IgA+ cells was lower in marrow than in blood. IgG and IgA fractions isolated from the patient's serum were also shown to contain anti-IMF activity. Ig biosynthetic analysis of blood MNC revealed that the K4A anti-Id antibody precipitated not only IgM but also IgG and IgA. Because cells simultaneously producing two different Ig isotypes were not detected, these results indicate the presence of five separate subpopulations of the K4A Id+ neoplastic clone. The data thus suggest the occurrence of a neoplastic or pre-neoplastic transformation event before the switching of Ig heavy chain isotypes, and imply a role for the IMF antigen in the exaggerated proliferation and differentiation along five of the nine potential intraclonal pathways.     

The significance of salivary and serum interleukin 6 and basic fibroblast growth factor levels in patients with Sjögren's syndrome

Role of various cytokines have been implicated in the development and perpetuation of Sjogren's syndrome (SS), but no specific cytokine could be determined as a major contributor to the SS. Salivary and serum interleukin 6 (IL-6) levels have been studied previously in patients with SS, but data upon salivary and serum basic fibroblast growth factor (bFGF) in SS are lacking. The aim of this study was to evaluate levels of salivary and serum IL-6 and bFGF in 18 patients with SS, age range 32-79, mean 54.05 years. Control group consisted of 23 healthy participants, mean age 25 years. Serum IL-6 and bFGF levels were not significantly different between patients with SS and healthy controls. Elevated levels of salivary IL-6 and bFGF in patients with SS when compared to the healthy controls were found (p<0.001). We might speculate that higher levels of salivary IL-6 and bFGF in patients with SS might originate from local production probably having source in the salivary glands.     

A case of systemic lupus erythematosus (SLE) and Sj?gren's syndrome associated with anti-T3 autoantibodies

A case of systemic lupus erythematosus (SLE) associated with Sj?gren's syndrome had extremely low serum triiodothyronine (T3) with normal levels of serum thyroxine (T4) measured by single antibody radioimmunoassays (RIAs) and thyroid stimulating hormone (TSH) during steroid treatment. Measurement of serum T3 and T4 with double antibody RIAs showed unusually high T3 and normal T4 concentrations. Examination of her serum revealed the presence of IgG class anti-T3 autoantibodies whose Scatchard plot was analyzed in two components; one with a higher associate constant (8.6 X 10(8)M-1) and a lower binding capacity (5.6 X 10(-7) mol/ml serum); the other a lower associate constant (3.5 X 10(7)M-1) and a higher binding capacity (2.1 X 10(-6) mol/ml serum). Antithyroglobulin (Tg) autoantibody has been positive throughout the seven year observation period. A significant positive correlation between titers of anti-Tg autoantibodies indicated that the antigen of anti-T3 antibodies in the patient could be T3 containing antigenic site(s) on the Tg molecule.     

Epistasis with HLA DR3 implicates the P2X7 receptor in the pathogenesis of primary Sj?gren's syndrome

Introduction

The aim of this study was to examine the association between functional polymorphisms in the pro-inflammatory P2X7 receptor and the Ro/La autoantibody response in primary Sjögren''s syndrome (pSS).

Methods

Twelve functional P2RX7 polymorphisms were genotyped in 114 pSS patients fulfilling the Revised American-European Consensus Criteria for pSS, and 136 controls. Genotyping of the A1405G (rs2230912) polymorphism was performed on a replication cohort consisting of 281 pSS patients and 534 controls. P2X7 receptor function in lymphocytes and monocytes was assessed by measurement of ATP-induced ethidium+ uptake. Serum IL-18 levels were determined by ELISA.

Results

The minor allele of P2RX7 A1405G is a tag for a common haplotype associated with gain in receptor function, as assessed by ATP-induced ethidium+ uptake. A positive association between 1405G and anti-Ro±La seropositive pSS patients was observed in Cohort 1. Although not replicated in Cohort 2, there was a consistent, significant, negative epistatic interaction effect with HLA-DR3 in seropositive pSS patients from both cohorts, thereby implicating this gain of function variant in the pathogenesis of pSS. Serum IL-18 was elevated in seropositive pSS patients, but was not influenced by P2RX7 A1405G.

Conclusions

The P2RX7 1405G gain-of-function haplotype may be a risk factor for seropositive pSS in a subset of subjects who do not carry HLA risk alleles, but has no effect in subjects who do (epistasis). Potential mechanisms relate to autoantigen exposure and inflammatory cytokine expression. The observed elevation of IL-18 levels is consistent with P2X7 receptor activation in seropositive pSS patients. Collectively these findings implicate P2X7 receptor function in the pathogenesis of pSS.     

Efficacy and safety of rituximab treatment in early primary Sj?gren's syndrome: a prospective,multi-center,follow-up study

Introduction

Primary Sjögren’s syndrome (pSS) is an autoimmune disorder affecting exocrine glands; however, a subgroup of pSS patients experience systemic extra-glandular involvement leading to a worsening of disease prognosis. Current therapeutic options are mainly empiric and often translated by other autoimmune diseases. In the last few years growing evidence suggests that B-cell depletion by rituximab (RTX) is effective also in pSS. Patients with early active disease appear to be those who could benefit the most from RTX. The aim of this study was to investigate the efficacy and safety of RTX in comparison to disease modifying anti-rheumatic drugs (DMARDs) in early active pSS patients.

Methods

Forty-one patients with early pSS and active disease (EULAR Sjogren’s syndrome disease activity index, ESSDAI ≥ 6) were enrolled in the study. Patients were treated with either RTX or DMARDs in two different Rheumatology centers and followed up for 120 weeks. Clinical assessment was performed by ESSDAI every 12 weeks up to week 120 and by self-reported global disease activity pain, sicca symptoms and fatigue on visual analogic scales, unstimulated saliva flow and Schirmer’s I test at week 12, 24, 48, 72, 96, and 120. Laboratory assessment was performed every 12 weeks to week 120. Two labial minor salivary gland (MSG) biopsies were obtained from all patients at the time of inclusion in the study and at week 120.

Results

Our study demonstrated that RTX treatment results in a faster and more pronounced decrease of ESSDAI and other clinical parameters compared to DMARDs treatment. No adverse events were reported in the two groups. We also observed that RTX is able to reduce glandular infiltrate, interfere with B/T compartmentalization and consequently with the formation of ectopic lymphoid structures and germinal center-like structures in pSS-MSGs.

Conclusions

To our knowledge, this is the first study performed in a large cohort of early active pSS patients for a period of 120 weeks. We showed that RTX is a safe and effective agent to be employed in pSS patients with systemic, extra-glandular involvement. Furthermore, our data on pSS-MSGs provide additional biological basis to employ RTX in this disease.