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. 1999 Jan;115(1):203–207. doi: 10.1046/j.1365-2249.1999.00778.x

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

A-K Halse *, J B Harley *, U Kroneld †, R Jonsson *
PMCID: PMC1905201  PMID: 9933443

Abstract

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 Sjögren's 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.

Keywords: Sjögren's syndrome, labial salivary glands, peripheral blood, Ro/SS-A

INTRODUCTION

Sjögren's syndrome (SS) is a chronic autoimmune rheumatic disease characterized by severe dryness of the eyes and mouth, probably resulting from the lymphocytic infiltration of the lachrymal and salivary glands. In primary SS, patients have no associated connective tissue disease, while patients with secondary SS per definition also have another rheumatic disease, most commonly rheumatoid arthritis (RA) or systemic lupus erythematosus (SLE).

Hypergammaglobulinaemia and autoantibody production are seen in most patients with primary SS. The autoantibodies found in SS are directed against both organ-specific and non-organ-specific targets [1]. The organ-specific antibodies include antibodies to smooth muscle and microsomal thyroid. More common are the non-organ-specific antibodies including rheumatoid factor, anti-Ro/SS-A and anti-La/SS-B antibodies in addition to other antinuclear antibodies. The presence of anti-Ro/SS-A or anti-La/SS-B autoantibodies is one of the classification criteria suggested by the European Community Study Group on Diagnostic Criteria for SS [2]. Anti-Ro/SS-A antibodies can be detected in about 70% of SS patients, while anti-La/SS-B antibodies are found in about 60% of patients [3]. An association between these antibodies and certain subsets of the disease has been demonstrated [4,5].

The Ro/SS-A antigen has been identified as a ribonucleoprotein consisting of a 60-kD protein and one member of a group of related RNAs [6]. A 52-kD protein has also been described as a component of the Ro/SS-A antigenic particle [7]. The La/SS-B antigen consists of a 48-kD protein which binds to RNA polymerase III transcripts. Anti-La/SS-B reactivity usually coexists with anti-Ro/SS-A antibodies in sera from SS patients [8].

In this study the enzyme-linked immunospot (ELISPOT) assay [9] was performed to assess the presence of anti-Ro/SS-A-producing cells in labial salivary glands (LSG) and peripheral blood (PB) of SS patients. The anti-Ro/SS-A and anti-La/SS-B antibodies have in previous studies been demonstrated in saliva of SS patients [10,11], indicating a local production of these autoantibodies. Anti-Ro 52-kD antibody-producing cells have also been found in LSG, spleen and lymph nodes of MRL/lpr mice [12]. To our knowledge, however, no clear evidence for anti-Ro/SS-A antibody-producing cells in LSG or PB of SS patients has been presented. The aim of this study was therefore to assess whether these antibodies are produced by LSG B lymphocytes and/or by PB lymphocytes.

PATIENTS AND METHODS

Patients

LSG biopsies from seven female patients with clinically definite primary SS were obtained [13]. PB was obtained from two of these patients. The patients were attending the Department of Oral Diagnosis, Faculty of Odontology, University of Göteborg, Göteborg, Sweden. All of them were serologically positive for antibodies binding to bovine Ro 60 kD, and they had focus scores ranging between 7 and 12 after histological evaluation of LSG biopsies [14]. LSG biopsies and PB from two patients with non-specific arthralgia and one patient with fibromyalgia served as controls.

The study was approved by the Committee of Ethics at the University of Göteborg, and all procedures were performed after informed consent from the patients.

Bovine Ro 60 kD

Tissue extracts were prepared by homogenizing and extracting calf thymus gland in an equal amount (w/v) of PBS pH 7.2 with 2 mm dithiothreitol at 4°C. After centrifugation at 10 000 g for 1 h, the supernatant was fractionated using ammonium sulphate precipitation. The precipitate at 30% saturation was discarded, and the precipitate at 60% saturation was dissolved in PBS and dialysed against PBS for 48 h. The final protein concentration of this calf thymus extract was approx. 40 mg/ml.

The immunoadsorbent column used for purification of the Ro 60-kD antigen was prepared by coupling IgG from a patient who showed only anti-Ro/SS-A precipitins in immunodiffusion to cyanogen bromide (CNBr)-activated Sepharose 4B (Pharmacia Fine Chemicals, Piscataway, NJ) by the method of the manufacturer. We followed a procedure which has been described in detail elsewhere [15]. IgG was prepared by DEAE-cellulose chromatography, and dialysed against the coupling buffer (0.05 m NaCl, 0.1 m NaHCO3, pH 8.3). The protein concentration was adjusted to 30 mg/ml. CNBr-activated Sepharose (15 g) was washed with 1 l of 1.0 mm HCl solution and reacted with 5 ml of IgG solution on a rotating turntable for 2 h at room temperature. Unbound protein was removed by washing with coupling buffer and the residual active sites on the Sepharose beads were blocked by reaction with 1.0 m ethanolamine pH 8.0 for 1 h at room temperature.

Pooled eluates from the anti-Ro/SS-A immunoadsorbent column were further fractionated on a calibrated Bio-Gel A-0.5 m (BioRad Labs, Richmond, CA) column (1.5 × 165 cm). Fractions containing Ro/SS-A antigen activity detected by microcomplement fixation or ELISA were collected, concentrated and stored at −70°C.

Dispersal of LSG cells

The LSG biopsy was performed as described earlier [14]. Cryostat sections of LSG were examined under a microscope, and the degree of inflammation was determined using a focus scoring system [14].

Glandular tissue not needed for diagnostic examination was used for functional analysis and immediately immersed in ice-cold complete medium (RPMI 1640; Bio-Whittaker, Walkersville, MD) containing HEPES, l-glutamine, penicillin, gentamycin and streptomycin. At the laboratory the tissues were placed in sterile Petri plates and cut in a tissue-chopper (McIlwain Tissue Chopper; The Mickle Laboratory Engineering Co. Ltd, Gramshall, UK). The cells were washed in sterile PBS to remove blood and dissociated from the stromal tissue by use of the neutral protease enzyme Dispase (Boehringer Mannheim Biochemicals, Indianapolis, IN) as previously described [16]. The LSG tissues were incubated in a solution of Joklik's minimal essential medium (MEM) containing Dispase for 30 min at 37°C. The cell suspension was removed, mixed with incomplete medium (RPMI 1640) and centrifuged. This enzyme digestion process was repeated four times. The cells were washed and resuspended in complete medium containing 10% fetal calf serum (FCS). The mononuclear cells (MNC) were counted, the viability tested (> 90%), and the cell solution adjusted to the desired concentration. The method is described in detail elsewhere [17].

Preparation of MNC from PB

PB was collected in heparinized tubes and diluted with the same volume of PBS. The blood MNC were separated by density gradient centrifugation (Lymphoprep; Nycomed A/S, Oslo, Norway) [18,19]. The cells from the interphase were carefully collected and washed three times with PBS, and resuspended in complete medium (RPMI 1640) including l-glutamine, penicillin, gentamycin, streptomycin, fungizone and 5% heat-inactivated FCS. The MNC were counted and viability tested (> 90%), and the cell solution adjusted to the desired concentration.

ELISPOT assay

The ELISPOT assay was performed to detect single cells from LSG and PB secreting antibodies against the bovine Ro 60-kD protein [9]. The assay was performed using microtitre plates with 96 wells and nitrocellulose bottoms (Millititer-HA; Millipore Products Division, Bedford, MA). The plates were coated with affinity-purified bovine Ro 60-kD antigens. The proteins were dissolved in sterile PBS to a final concentration of 10 μg/ml, and 100 μl were added to each well overnight at 4°C. This antigen concentration was found to be optimal in preliminary experiments. In parallel, wells were also coated with 100-μl aliquots of diluted anti-human affinity-purified and isotype F(ab′)2 reagents specific for IgG (Jackson ImmunoResearch Labs, West Grove, PA) or IgA and IgM (Pel-Freez Biologicals, Rogers, AR) for enumeration of total IgG-, IgA- and IgM-producing cells in LSG and PB. Control wells were coated with FCS or PBS. The plates were washed with sterile PBS to remove unabsorbed antibodies, and 200 μl of RPMI 1640 were applied per well at 37°C for at least 30 min to block non-specific binding sites. Individual wells were filled with 100-μl aliquots containing 2.54–105 MNC in RPMI 1640 and 5% FCS. All cultures were performed in triplicates or more. The cells were incubated for 4 h at 37°C in a humid atmosphere containing 7% CO2. After incubation the plates were washed with PBS and PBS–Tween. Subsequently, 100 μl of catching antibody were added to each well. As catching antibodies we used alkaline phosphatase-conjugated goat anti-human IgG (Southern Biotechnology Associates, Birmingham, AL) or peroxidase-conjugated goat anti-human IgG, IgA or IgM (Sigma, St Louis, MO) diluted 1 : 500 in PBS–Tween. The plates were incubated overnight at 4°C. After washing with PBS the plates were enzymatically developed with 100 μl nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrate solution or AEC substrate solution (10 mg 3-amino-9-ethylcarbazole in 1 ml dimethyl formamide, diluted to 30 ml with 0.1 m citrate acetate buffer of pH 5, followed by filtration through a 0.45-μm filter and addition of 15 μl 30% H2O2). The reaction was stopped by washing with tap water. Enzyme activity was visualized on the nitrocellulose membrane as blue and red spots, respectively, which were counted in a stereomicroscope under ×40 magnification (Fig. 1). No spots appeared in control wells where FCS or PBS were used instead of capture antibody, or in coated wells subjected to medium without cells. The presented data are expressed as numbers of spots/105 MNC.

Fig. 1.

Fig. 1

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One well of a microtitre plate visualizing the spots on the nitrocellulose membrane. Each spot represents antibodies secreted by one B lymphocyte.

Statistical analysis

The Mann–Whitney test was used for statistical evaluation.

RESULTS

Number of B cells producing anti-bovine Ro 60-kD antibodies in LSG and PB from anti-Ro/SS-A-positive primary SS patients

To test the frequencies of LSG and PB lymphocytes producing antibodies against the bovine Ro 60 kD, the ELISPOT assay was performed, and PBS and FCS were used as control antigens.

All six patients investigated had LSG B cells producing anti-bovine Ro 60-kD antibodies of the IgG isotype (Table 1). PB from two of the patients was also tested for cells producing anti-bovine Ro 60-kD antibody of the IgG isotype, and in both patients such blood cells were found. The number of IgG anti-bovine Ro 60-kD antibody-producing cells was slightly higher in LSG than in blood. Due to the small size of the sample no statistical testing was performed.

Table 1.

Numbers of cells producing IgG, IgA and IgM and anti-bovine Ro 60 kD-specific antibodies of the three isotypes per 105 mononuclear cells from labial salivary glands (LSG) and peripheral blood (PB) of anti-Ro/SS-A-positive primary SS patients. Patients with arthralgia and fibromyalgia were used as controls.

graphic file with name cei0115-0203-t1.jpg

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One of six patients had LSG cells producing anti-bovine Ro 60-kD antibodies of the IgA isotype, whereas neither of two patients had PB cells producing such antibodies. One of five patients had LSG cells producing anti-bovine Ro 60-kD antibodies of the IgM isotype, while neither of two patients had PB cells producing such antibodies (Table 1).

Total number of immunoglobulin-producing cells in LSG and PB from patients with primary SS and controls

All seven primary SS patients investigated had LSG cells producing IgG, IgA and IgM at the mean levels of 1450, 950 and 500/105 MNC cells, respectively (Table 1). The number of IgG-producing cells was significantly higher than the number of IgM-producing cells (P < 0.02). The difference between the number of IgG-producing cells and IgA-producing cells, as well as the difference between the number of IgA- and IgM-producing cells, was not statistically significant (P > 0.05).

Two primary SS patients were also investigated for the number of total IgG-producing cells in blood. Both patients had such cells, but at a much lower level than in LSG. There was a seven-fold and 30-fold increase in IgG production, respectively, for the two patients, in LSG compared with blood. The same two primary SS patients were also investigated for PB cells secreting IgA and IgM. Neither of them had IgA-producing cells, whereas one patient had low levels of IgM-producing cells (Table 1).

Two patients with arthralgia and one patient with fibromyalgia all had LSG cells producing IgG, IgA and IgM. The level of immunoglobulin production in these patients was lower than in the primary SS patients, and the number of IgG-producing cells was slightly lower than the number of IgA- and IgM-producing cells. The two arthralgia patients had PB cells producing IgG at the same level as in the primary SS patients, while the fibromyalgia patient had no IgG-producing cells in blood (Table 1). Due to the low number of arthralgia and fibromyalgia patients, no statistical testing was performed between the groups.

Number of B cells producing anti-bovine Ro 60-kD antibodies related to the total number of immunoglobulin-producing cells in LSG and PB from anti-Ro/SS-A-positive primary SS patients

In two patients both LSG and PB were tested for anti-bovine Ro 60-kD antibody-producing cells of the IgG isotype and total numbers of IgG-producing cells, and the ratio between these two numbers was calculated. The first patient had 10/105 MNC producing anti-bovine Ro 60-kD antibodies of the IgG isotype and 262/105 MNC producing total IgG in LSG, which gave a ratio of 0.038. The same patient had 6.9/105 MNC producing anti-bovine Ro 60-kD antibodies of the IgG isotype and 8.3/105 MNC producing total IgG in PB, which gave a ratio of 0.83. The second patient had 20.5/105 MNC producing anti-bovine Ro 60-kD antibodies of the IgG isotype and 307/105 MNC producing total IgG in LSG, which gave a ratio of 0.067. This patient had 4.8/105 MNC producing anti-bovine Ro 60-kD antibodies of the IgG isotype and 44/105 MNC producing total IgG in PB, which gave a ratio of 0.11.

DISCUSSION

In this study we evaluated whether B cells specific for the Ro/SS-A antigen are present in LSG and/or PB of patients with primary SS. Total numbers of IgG-, IgA- and IgM-producing cells in LSG and PB were also measured. We used the ELISPOT assay, which permits quantification of secreted products at the single-cell level. This method has previously been widely used to evaluate the autoantibody production in PB and affected organs of different autoimmune diseases [20–25].

Interestingly, all six primary SS patients investigated had LSG cells producing anti-bovine Ro 60-kD antibodies of the IgG isotype, while only one patient had LSG cells producing such antibodies of the IgA isotype. This was unexpected, since IgA anti-Ro/SS-A antibodies previously have been found in saliva of a high number of SS patients [11]. These antibodies are supposed to be produced in LSG, but our results do not support this hypothesis. More probably IgA anti-Ro/SS-A antibodies are produced in other lymphoid organs, and later concentrated in saliva.

Two of the six SS patients were also tested for PB cells producing anti-bovine Ro 60-kD antibodies of the IgG isotype, and both had such antibodies. The total number of anti-bovine Ro 60-kD antibody-producing cells was somewhat higher in LSG than in blood. However, the number of anti-bovine Ro 60-kD antibody-producing cells compared with the total number of IgG-secreting cells was much higher in blood than in LSG. This finding is consistent with relative depletion of anti-bovine Ro 60-kD antibody-producing cells in LSG, and may have various potential explanations. For example, different methods were used for separation of LSG cells and PB cells, which may have influenced the relative number of antigen-specific lymphocytes. However, since only two SS patients were tested for both LSG and blood, no definite conclusions can be drawn.

Similar studies of local autoantibody production as presented in this paper have been performed in RA [20,21]. These investigators found anti-collagen type II antibody-producing cells in the synovial fluid of 50% of patients with active and destructive RA, but no such cells were found in PB. In addition, the IgG anti-collagen type II response occurred exclusively in patients who were positive for HLA-DR4 [20]. These findings do not support the hypothesis that anti-collagen type II antibody production is a secondary phenomenon to the cartilage destruction seen in RA, but rather indicate that the anti-collagen II antibody production is a T cell-dependent process. Thus, collagen may be a principal antigen in the induction and/or perpetuation of RA.

It is, however, not known if autoantibody production against the anti-Ro/SS-A and anti-La/SS-B antigens is a T cell-dependent process. Moreover, we have not been able to find higher levels of Ro/SS-A- and La/SS-B-reactive T cells in PB of SS patients than in healthy controls [26]. On the other hand, one study has demonstrated Ro/SS-A-, but not La/SS-B-reactive T cells in LSG of a limited number of SS patients. This supports the hypothesis that Ro/SS-A might function as an autoantigen recognized by autoreactive T cells and thereby trigger the progression of the exocrinopathy of SS [27].

While the mechanism by which anti-Ro/SS-A and anti-La/SS-B antibodies arise is not clearly understood, knowledge about the pathogenic role of these autoantibodies is increasing. There is now substantial evidence that antibodies to the Ro/SS-A and La/SS-B proteins directly participate in the pathogenesis of SS and other connective tissue disease manifestations [28–30]. Some studies have demonstrated anti-Ro/SS-A and anti-La/SS-B antibodies deposited in LSG of patients with SS [29,31], supporting the hypothesis that the production of these autoantibodies takes place in these tissues. To what extent these antibodies are participating in the local inflammation is not known.

In conclusion, we have demonstrated anti-bovine Ro 60 kD-secreting cells in LSG of patients with primary SS. All the investigated patients had B cells producing antibodies to this antigen in LSG tissue. Thus, our findings provide evidence for and support the previous reports suggesting a local production of autoantibodies in LSG of SS patients. Circulating B lymphocytes are normally in a resting state, and antibodies are thought to be produced mainly by non-circulating B cells. Nevertheless, we found PB lymphocytes secreting antibodies against the bovine Ro 60 kD in both the investigated SS patients. To evaluate to what extent anti-Ro/SS-A and/or anti-La/SS-B antibody-producing cells are present in PB of SS patients, a follow-up study has been performed including blood from a larger number of patients [32]. Future investigations should also include evaluation of anti-Ro/SS-A and anti-La/SS-B antibody-producing cells and search for Ro/SS-A- and La/SS-B-specific T cells in the lymphoid organs of SS patients. This might further elucidate the site of autoantibody production and the possibility that Ro/SS-A and La/SS-B proteins are genuine autoantigens in SS.

Acknowledgments

The authors wish to thank Maria Heyden for technical assistance and Dr Christopher Robinson for linguistic help. The study was supported by Aslaug Andersen Foundation for Rheumatological Research in Bergen, Inger R. Haldorsen Foundation, the Research Council of Norway grant no. 115563/320 and the European BIOMED concerted action BMH4-CT96-0595.

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