Anne-Kristine Halse

Detection of anti-Ro/SSA and anti-La/SSB autoantibody-producing cells in salivary glands from patients with Sjögren's syndrome

OBJECTIVE To investigate and identify the presence of cells producing anti-Ro/SSA and anti-La/SSB autoantibodies in salivary glands from patients with Sjögrens syndrome (SS). METHODS Submucosal salivary gland biopsy samples from 10 SS patients (8 with and 2 without circulating Ro and La autoantibodies) and 14 control subjects were evaluated. Frozen tissue sections were immunostained by an avidin-biotin complex technique, using biotinylated recombinant Ro and La proteins as detection reagents. Autoantibody levels in SS patient sera were analyzed by enzyme-linked immunosorbent assay. RESULTS Cells producing autoantibodies to the Ro 52-kd, Ro 60-kd, and La proteins were recorded in 8, 6, and 7 of the 10 SS patient biopsy samples, respectively. Samples from the 2 SS patients without circulating Ro and La autoantibodies were negative for these autoantibody-producing cells, as were all control biopsy samples. A strong positive correlation between the presence of autoantibodies in sera and the presence of autoantibody-producing cells in glandular biopsy tissues was evident. The number of autoantibody-producing cells and the serum autoantibody levels were also correlated (r(s)=0.94, P < 0.0001). CONCLUSION Using a novel technique, we have demonstrated the presence of Ro and La autoantibody-producing cells in salivary gland biopsy tissues from patients with SS. These findings indicate that anti-Ro/ SSA and anti-La/SSB autoantibodies are produced and are present at sites of inflammation and indicate their potential involvement in the autoimmune exocrinopathy of this disease.

Ro/SS-A-reactive B lymphocytes in salivary glands and peripheral blood of patients with Sjögren's syndrome

The aim of this study was to investigate the production of anti‐Ro/SS‐A antibodies in labial salivary glands (LSG) and peripheral blood (PB) of Sjögrens syndrome (SS) patients. The ELISPOT method was performed to quantify the frequency of LSG lymphocytes and PB lymphocytes spontaneously secreting anti‐Ro/SS‐A antibodies. The total number of IgG‐, IgA‐ and IgM‐producing cells was also quantified. The bovine Ro 60‐kD protein was used as target antigen. Six of six primary SS patients had LSG B cells producing anti‐bovine Ro 60 kD of the IgG isotype, and two of two primary SS patients had in addition PB lymphocytes producing anti‐bovine Ro 60 kD of the IgG isotype. The six patients who had IgG antibodies against the Ro/SS‐A antigen in LSG all had focus scores of ≥ 7 in biopsies of LSG. The results indicate that SS patients with a high degree of local inflammation in LSG have B cells producing anti‐Ro/SS‐A antibodies in both LSG and PB. Thus, the anti‐Ro/SS‐A antibodies may have pathogenic importance in the progression of the exocrinopathy of SS.

Associations of MHC Class II Alleles in Norwegian Primary Sjogren's Syndrome Patients: Implications for Development of Autoantibodies to the Ro52 Autoantigen

Sjögrens syndrome (SS) is a chronic autoimmune disease characterized by dryness of the eyes and mouth. Currently, the highly polymorphic major histocompatibility complex (MHC) genes are the best documented genetic risk factor for the development of autoimmune disease. We examined the MHC class II alleles DRB1, DRB3, DRB4, DRB5, DQA1 and DQB1 in a group of Norwegian pSS patients and compared with a group of healthy controls. Because a number of studies have shown that some of the MHC class II alleles are not associated with the disease as a whole, but rather to the development of autoantibodies, anti‐Ro52 autoantibodies in serum were measured and compared to MHC class II allele status. A clear association with pSS was detected for the DRB1*0301 and DRB3*0101 alleles, but these alleles were more closely associated with the presence of anti‐Ro52 autoantibodies than with pSS itself. Moreover, the DQA1*0501 and DQB1*0201 alleles were only associated with the presence of anti‐Ro52 autoantibodies. This study shows that the production of anti‐Ro52 autoantibodies in pSS is associated with the DRB1*0301, DRB3*0101, DQA1*0501 and DQB1*0201 alleles which are in strong linkage disequilibrium.

Serum Antibodies from Patients with Primary Sjögren’s Syndrome and Systemic Lupus Erythematosus Recognize Multiple Epitopes on the La(SS-B) Autoantigen Resembling Viral Protein Sequences

The aim of this study was to investigate the epitope recognition pattern of La(SS‐B) autoantibodies in sera from patients with Sjögren’s syndrome (SS) and systemic lupus erythematosus (SLE) using overlapping synthetic decapeptides on solidphase. Eighty different decapeptides with five amino acids overlap from the human La(SS‐B) autoantigen were synthesized on cellulose paper using F‐moc chemistry. Tests were performed with 14 SS and six SLE sera. The results showed that the immune response to the La(SS‐B) oligopeptides was restricted and unique for each individual with no particular pattern typical for each of the two diseases, apart from the fact that SLE sera gave positive reaction with fewer peptides. Regions within the N‐ and C‐termini harboured most of the positive sequences. The authors specifically addressed the possibility of a viral aetiology for disease development or autoantibody generation. In this context the most frequently recognized linear epitopes on the La(SS‐B)autoantigen showed sequence similarities with proteins from a range of ubiquitous human viruses, in particular from the herpes virus group. The La(SS‐B) autoantibodies may thus be generated through molecular mimicry.

DIFFERENTIAL IMMUNOLOGICAL ABERRATIONS IN PATIENTS WITH PRIMARY AND SECONDARY SJOGREN'S SYNDROME

The aim of the present study was to analyse possible differences in immunological features between patients with primary and secondary Sjögrens syndrome (SS). Ten patients with primary SS and 10 patients with secondary SS also suffering from rheumatoid arthritis, were identified according to established criteria for SS. Ten healthy, age‐matched women served as controls. The authors analysed the phenotypic characteristics of lymphocytes in peripheral blood as well as in focal inflammatory infiltrates of minor salivary gland biopsies. Functional analyses of T lymphocytes were performed after stimulation with mitogens and antigen. B cell activity was determined at the single cell level by spontaneous and mitogen induced immunoglobulin production. Serum levels of IL‐4, IL‐6 and IFN‐γ were also analysed. Patients with primary SS displayed a significantly higher degree of salivary gland inflammation and reduced salivary flow than did patients with secondary SS. Decreased in vitro T cell responses to antigen and mitogens were evident in both patient groups. The CD4/CD8 ratios in both peripheral blood and salivary gland lesions were significantly lower in primary SS compared with secondary SS patients. Polyclonal B cell activation, measured as the frequency of spontaneous immunoglobulin producing cells, was most prominent in primary SS, whereas a diminished response to poke‐weed mitogen (PWM), a T cell dependent B cell mitogen, was more pronounced in secondary SS. The results reveal certain immunological aberrations in the whole group of patients with SS. In addition, the authors demonstrated distinct differences in immune dysfunction between patients with primary and secondary SS, indicating that they may constitute separate entities.

Ro/SS-A- and La/SS-B-reactive B lymphocytes in peripheral blood of patients with Sjögren's syndrome

The aim of this study was to investigate the production of anti‐Ro/SS‐A and anti‐La/SS‐B antibodies in peripheral blood (PB) of patients with Sjögrens syndrome (SS). The ELISPOT method was performed to quantify the frequency of PB lymphocytes spontaneously secreting anti‐Ro/SS‐A and/or anti‐La/SS‐B antibodies. The total number of IgG‐, IgA‐ and IgM‐producing cells was also quantified. The recombinant Ro 52‐kD, Ro 60‐kD and La 48‐kD proteins were used as target antigens. Three of 18 SS patients had PB lymphocytes secreting IgG antibodies against the recombinant Ro 52‐kD protein. The same three patients had high serum titres of anti‐Ro 52‐kD antibodies. In addition, these patients were classified as having severe disease, and all three had focus scores of ≥ 8 in biopsies of the labial salivary glands (LSG). The correlation between the number of PB cells producing IgG antibodies against the recombinant Ro 52‐kD protein and the focus score was significant (P < 0.01). The results indicate that only SS patients with severe disease and high degree of local inflammation in LSG have B cells producing anti‐Ro/SS‐A antibodies in PB. Thus, most of the spontaneous autoantibody production must take place in other body compartments, e.g. in exocrineglands and probably also in the lymphoid organs and/or other mucosal sites.

Peripheral Blood in Sjögren's Syndrome Does Not Contain Increased Levels of T lymphocytes Reactive with the Recombinant Ro/SS-A 52 kD and La/SS-B 48 kD Autoantigens

Patients with Sjögrens syndrome (SS) frequently have anti-Ro/SS-A and anti-La/SS-B autoantibodies. The aim of this study was to investigate if these patients have peripheral blood lymphocytes (PBL) secreting IFN-gamma after short-term cultivation in the presence of Ro/SS-A and La/SS-B antigens. The frequency of PBL secreting IFN-gamma was examined in 12 SS patients and 11 healthy controls. The enzyme-linked immunospot (ELISPOT) assay was performed after 48 hours cultivation of PBL in the presence of recombinant Ro 52 kD protein or recombinant La 48 kD protein. The number of unstimulated IFN-gamma secreting cells in the SS patient group was not significantly different from that of the control group. Moreover, no increase in the number of IFN-gamma secreting cells after Ro/SS-A and La/SS-B stimulation was detected in the two groups. Thus, T cells reactive with the recombinant Ro 52 kD and La 48 kD proteins do not occur with any increased frequency in peripheral blood of SS patients.

Serum Markers in Rheumatoid Arthritis: A Longitudinal Study of Patients Undergoing Infliximab Treatment.

Objective. The aim of this study was to investigate the clinical effect and serum markers in a cohort of rheumatoid arthritis patients with moderate to high disease activity, participating in an open clinical phase IV study conducted in Norway between 2001 and 2003 receiving infliximab treatment. Method. A total of 39 patients were studied, with a mean age of 54 years and 12-year disease duration. The analyses were performed using serum from patients at four assessment time points: baseline and 3, 6, and 12 months after starting treatment with infliximab. A wide variety of clinical data was collected and disease activity of 28 joints and Simple Disease Activity Index were calculated. The joint erosion was determined by X-ray imaging and the Sharp/van der Heijde score was determined. Serum analysis included multiplex immunoassays for 12 cytokines, 5 matrix metalloproteases, and 2 VEGFs. Results. The majority of the RA patients in this study had initially moderate to high disease activity and the infliximab treatment reduced the disease activity significantly and also reduced any further joint destruction and improved disease status. Most of the serum levels of cytokines and metalloproteases remained unchanged during the course of the study, and we were unable to detect changes in TNF-α in serum. Serum levels of IL-6 and VEGF-A decreased significantly after initiation of infliximab treatment. Conclusion. The serum levels of IL-6 and VEGF-A may be promising disease markers as they vary with disease progression. The clinical significance of these findings is yet to be determined and has to be confirmed in future clinical trials before being applied in the clinics.

Calprotectin (S100A8/A9) should preferably be measured in EDTA-plasma; results from a longitudinal study of patients with rheumatoid arthritis

Abstract Calprotectin (S100A8/A9), a protein expressed in neutrophils and monocytes/macrophages in circulation and inflamed tissue, is associated with measures of disease activity in rheumatoid arthritis (RA) patients both when measured in ethylenediaminetetraacetic acid (EDTA)-plasma and in serum. We wanted to explore if EDTA-plasma or serum should be preferred for calprotectin as a marker of disease activity. Calprotectin was analysed in EDTA-plasma and serum by enzyme-linked immunosorbent assay (ELISA) at baseline in 141 RA patients, starting biologic disease-modifying anti-rheumatic drugs (bDMARDs), and after three months. Differences between plasma and serum levels of calprotectin were assessed by Wilcoxon signed rank test. Variability was assessed by quartile coefficient of dispersion. Spearman’s test explored correlations between calprotectin in plasma and serum and between calprotectin (plasma or serum) and clinical/ultrasound (US) measures of disease activity. Bland Altman plots were used for method comparisons. Conventional inflammatory markers were evaluated for comparison. Calprotectin had similar variability when measured in plasma and serum, but there was a significant difference in concentrations between plasma and serum (p < .001). The correlation coefficients at baseline between calprotectin measured in plasma/serum and measures of disease activity were rs = 0.62/0.46 for sum power Doppler score (PD), rs = 0.60/0.48 for assessor’s global visual analogue scale (VAS), rs = 0.59/0.43 for sum grey scale (GS) score and rs = 0.47/0.37 for swollen joint count of 32, all p < .001. Similar differences were found after three months. Calprotectin measured in plasma showed the strongest associations with assessments of disease activity, and EDTA-plasma should preferably be used when evaluating disease activity in RA patients.