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Keywords:

  • autoimmunity;
  • B cells;
  • Ro/SSA;
  • salivary glands;
  • Sjögren's syndrome

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Primary Sjögren's syndrome (pSS) is characterized by the presence of autoantibodies against the ribonucleoprotein (RNP) particles Ro/SSA and La/SSB, and mononuclear cell infiltration of exocrine tissues, especially salivary and lachrymal glands. Low numbers of autoantigen-specific memory B cells and elevated levels of plasma cells have been detected previously in the peripheral blood (PB) of pSS patients compared to controls. As both Ro52 and Ro60-specific cells have been detected in the salivary glands (SG) of pSS patients, we aimed to characterize the SSA-specific B cell pattern in SG biopsies. A series of double immunohistochemical stainings were performed on paraffin-embedded tissue from 10 well-characterized pSS patients for each Ro52 and Ro60 along with CD19, CD5, CD20 or CD27, respectively. Ro52 and Ro60-specific cells detected in SG tissue were found to be CD19+ B cells located outside the CD19+/CD20+ B cell zones (BCZ) and also interstitially. These SSA-specific cells were also quantified. No SSA-specific cells were CD5+, indicating that they do not belong to the B-1 B cell subset. Furthermore, no SSA-specific cells were observed within the CD20+ BCZ. Hence, no SSA-specific memory B cells were detected in these individuals. Contrary to this, SSA-specific cells were found to be CD19+/CD27++, demonstrating that they are differentiating short or long-lived plasma cells. Taken together, our findings suggest that these lower levels of SSA-specific memory B cells in PB and absence of SSA-specific memory B cells in SG of pSS patients could result from activation of these cells into plasma cells at the site of inflammation.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Primary Sjögren's syndrome (pSS) is a systemic autoimmune disorder associated with humoral and cellular abnormalities, leading to manifestations and inflammation in exocrine glands, particularly the lachrymal and salivary glands (SG) [1, 2]. It is therefore characterized by sicca symptoms, including dry eyes and dry mouth [3]. Another distinguished feature of Sjögren's syndrome (SS) is the high production of autoantibodies against the intracellular proteins Ro/SSA and La/SSB, which can be detected in the peripheral blood (PB) of 60–90% of Sjögren's patients [4-6]. Furthermore, these autoantigen-specific cells have also been identified previously in the SG [7-9]. As a consequence, there is enhanced B cell differentiation and an increased level of antibody-secreting plasma cells in SS patients, a condition known as hypergammaglobulinaemia [10]. A result of this is the depressed percentage of circulating memory B cells [11-13]. Although it was believed previously that these memory B cells are, in turn, retained in the SG [14], recent findings have confirmed that there is indeed a low number of memory B cells in the SG of pSS patients alongside elevated levels of activated short- and long-lived plasma cells [15]. These differentiating plasma cells therefore constitute the greater portion of the total B cell infiltrates in the SG [16].

Generally, B cells are known to make up 20% of the infiltrating cell populations in exocrine glands [17, 18]. In some patients, these infiltrating cells can result in the formation of structures similar to those of organized secondary lymphoid tissue [19]. Indeed, 25% of pSS patients demonstrate ectopic germinal centres (GC) in their exocrine glands [20-22]. Hence, although it is not known whether B cell activation is a primary cause or a secondary effect in SS, B cells remain important in disease pathogenesis [23].

There are two main lineages of B cells, namely B-1 B cells that originate in the fetal liver and B-2 B cells that originate in the bone marrow [24]. B cells are classified in these two subpopulations according to their expression of CD5: a marker that is present on B-1 B cells yet absent on B-2 B cells [25]. In contrast to this, a general B cell marker expressed by all the B cell subsets is CD19 [26]. A further distinction of the B cell subpopulations could be attained by detection of the surface marker CD27, also known as the general memory B cell marker [27, 28]. However, this marker is also expressed by plasmablasts, plasma cells and long-lived plasma cells [22, 28, 29]. None the less, plasmablasts and plasma cells are CD20-negative, while naive and memory B cells both express CD20 [30-32]. This could explain why current therapeutic attempts in SS that relied on the elimination of CD20-positive cells, by administration of the anti-CD20 antibody Rituximab®, resulted in reduced numbers of B cells in the patients’ as-yet unchanged serum levels of autoantibodies, as the activated portion of the B cell populations remained untargeted [33-35].

Given that Ro/SSA-specific cells have been detected previously in the SG of SS patients through immunohistochemical (IHC) staining and the use of biotinylated Ro52 and Ro60 antigens [7-9], we were inspired to explore further the SSA-specific B cell pattern in the SG of pSS patients in the present study. Through a series of double IHC labelling of paraffin-embedded SG tissue sections with non-biotinylated Ro52 or Ro60 antigens, along with CD19, CD5, CD20 and CD27, respectively, we managed to account for the different subtypes of SSA-specific B cells present within the gland, in addition to their distribution and morphology. Furthermore, the memory B cell patterns in the PB and SG of these individuals have also been accounted for previously [15, 36]. We were therefore curious to see what significance this would have on the SSA-specific B cell pattern in these patients. Taken together, our findings could provide further insight into the pathogenesis of SS, in addition to evaluating the potential efficiency of the current therapeutic strategies applied.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Patients and controls

This study included lower labial minor SG biopsies obtained from 10 patients who were diagnosed with pSS; nine of these patients fulfilled the American–European consensus group criteria (AECC) for pSS [37]. In addition, four minor SG biopsies with normal morphology from subjects evaluated for SS, but not fulfilling the criteria (siccae controls), served as non-pSS tissue controls. All biopsies were performed between 1992 and 2011 at the Department of Otolaryngology/Head and Neck Surgery at Haukeland University Hospital in Bergen, Norway. These formalin-fixed, paraffin-embedded minor SG tissue sections were stained with haematoxylin and eosin (H&E) and evaluated by an oral pathologist to determine their focus score (FS). This is known as the number of mononuclear cell infiltrates with ≥50 mononuclear cells per 4 mm2. Given that focus scoring is a semi-quantitative method where FS values may differ depending on how deep in the gland the sections were taken, new H&E staining was performed for all 10 patients and their FS was re-evaluated [38, 39]. In this way potential discrepancies could be eliminated. The re-evaluated FS range was found to be from 1 to 3. Additionally, these H&E-stained sections were screened for the presence of GC-like structures.

Clinical data were obtained from the Department of Rheumatology, Haukeland University Hospital, which provided information collected during routine laboratory assessments. These include RF detection, anti-nuclear antibodies (ANA), anti-Ro/SSA and anti-La/SSB. Furthermore, PB and plasma attained in the year 2010 from these 10 patients have also been tested in a previous study [36], where the Ro/SSA- and La/SSB-specific memory B cell pattern and function in relation to the progression of the disease was characterized in these individuals. In addition, the number of SSA- and SSB-specific antibody secreting cells (ASC) was also accounted for, and a reassessment of the autoantibody production in these subjects was performed against each of the Ro52, Ro60 and La48 autoantigens. The characteristics of the pSS patients included in this study are shown in Table 1. All the studied subjects gave their informed consent, and the Committee of Ethics at the University of Bergen approved the study.

Table 1. Medical and experimental characteristics of the primary Sjögren's syndrome (pSS) patients included in the study.
Patient no.Focus score*Focus score (re-evaluated)GC +/– Number(2) of B cell zones/10 mm2 of SG tissueRo52+ cells/10 mm2 of SG tissueRo60+ cells/10 mm2 of SG tissueAnti-Ro52 secreting B cells/100000 PBMCs in PBAnti-Ro60 secreting B cells/100000 PBMCs in PBRo52 (μg/ml)§Ro60 (μg/ml)§%Ro60 of total IgG+ memory B cells in PB%Ro52 of total IgG+ memory B cells in PBNumber(2) memory B cells/ 10 mm2 of SG tissueANA**RF titre
  1. *Values are the number of focal infiltrates/4 mm2 area containing >50 mononuclear cells. New haematoxylin and eosin (H&E) staining was performed for all 10 patients and their FS was re-evaluated. Anti-SSA-secreting B cells measured by direct enzyme-linked immunospot (ELISPOT) previously and presented as spot-forming cells/100 000 PBMCs(1). §Autoantibody production was assessed by enzyme-linked immunoassay (ELISA) previously and presented in μg/ml for each Ro52 and Ro60(1). Autoantigen-specific memory B cells measured by ELISPOT previously and presented as a percentage of immunoglobulin (Ig)G+ memory B cells(1). **Values are attained by ELISA, range at 0–9·99. ANA: anti-nuclear antibodies; GC: germinal centre; NT: not tested; PB: peripheral blood; PBMC: peripheral blood mononuclear cells; RF: rheumatoid factor. (1)See Ref. [36]. (2)See Ref. [15].

pSS-13842+41491417100·000·0274+
pSS-14133646143955·8316·001117++
pSS-144200231041400·000·0350+
pSS-147115691835220·2900·0105+
pSS-14941217460000·000·0016++
pSS-1520003189753·2300·0110+
pSS-15803631766295·8116·3166+
pSS-16020069546600·000·0110++
pSS-163125138567900·000·00111+
pSS-1651192221373300·000·01012

Antigens

The following anti-human antigens were used in this study: Ro52 (0·23 μg/ml) (Cat. no. ATR 05-02; Arotec Diagnostics Limited, Wellington, New Zealand) and Ro60 (1·4 μg/ml) (Cat. no. ATR 02-02; Arotec Diagnostics Limited).

Primary antibodies

The following primary anti-human antibodies were used in this study at the indicated working dilution: mouse monoclonal Ro52 (1:5) (Cat. no. 57038; Progen, Heidelberg, Germany), mouse monoclonal Ro60 (1:200) (Cat. no. 57040; Progen), mouse monoclonal CD19 (1:50) (clone LE-CD19; Dako A/S, Carpinteria, CA, USA), mouse monoclonal CD5 (1:50) (clone 4C7; Dako A/S, USA), mouse monoclonal CD20 (1:3000) (clone L26; Dako A/S; Glostrup, Denmark) and mouse monoclonal CD27 (1:20) (clone 137B4; Nordic BioSite, Täby, Sweden).

Immunohistochemistry

Single-staining

A Leica serial microtome (Leica Instruments GmbH, Nussloch, Germany) was used to cut paraffin-embedded, formalin-fixed minor SG tissue (4–6 μM thick). The sections were placed on SuperFrost® Plus microscope slides and incubated overnight in a heat cabinet at 56°C. This was followed by deparaffinization and heat-induced epitope retrieval (HIER) with PT-Link (Dako, Denmark) for 20 min using Target Retrieval Solution [high pH (pH 9·0); Dako, Denmark]. Endogenous peroxidase activity was then blocked with 0·3% peroxidase (K4007; Dako, USA) for 10 min. Furthermore, the sections were incubated with antigen (Ro52 and Ro60), made up in antibody diluent (S0809; Dako, USA), for 60 min. This was followed by incubation with primary antibody [Ro52 monoclonal antibody (mAb) and Ro60 mAb)] for another 60 min and then horseradish peroxidase (HRP)-conjugated anti-mouse EnVision secondary antibody (K4007; Dako, USA) for 30 min. Thereafter, the sections were incubated for 10 min with diaminobenzidine (DAB), which was used as chromogen for development (K4007; Dako, USA). All incubations were performed at room temperature (RT), and Tris-buffered saline (TBS) containing 0·1% Tween (TBST) was used as washing buffer (pH 7·6) between every step for 10 min. Finally, the sections were counterstained with haematoxylin (S3301; Dako, Denmark) for 4 min, dehydrated using ethanol (70, 96 and 100%) and xylene, and mounted in Eukitt (O. Kindler GmbH & Co, Freiburg, Germany).

Double-staining

In order to study the general Ro52- and Ro60-specific B cell pattern in the SG, double-staining experiments were carried out with CD19 in combination with either Ro52 or Ro60. To identify CD19-positive B-1 B cells, CD5 was used in combination with Ro52 and Ro60, respectively. The memory B cell pattern was detected by double-staining of serial sections with either CD27 or CD20 in combination with Ro52 and Ro60, correspondingly. The sections were pretreated as described previously and incubated with Dual Endogenous Enzyme Block (K5361; Dako, Denmark) for 10 min in order to block endogenous peroxidase and alkaline phospatase (AP). The antigen was then added (Ro52 or Ro60) and the sections were incubated for 60 min at RT. The sections were then incubated with the first primary antibody (Ro52 mAb or Ro60 mAb) for another 60 min at RT. This was followed by the procedure described for single-staining. After the development of the antigen/first primary antibody with DAB, the sections were washed with water for 5 min, with TBST for 10 min, and then treated with Doublestain Block (K5361; Dako, Denmark) for 3 min. Thereafter, the sections were incubated with the second primary antibody (CD19, CD5, CD20, CD27) for 10 min at RT then placed at 4°C overnight, and again thawed for another 10 min at RT the following day. Binding of the second primary antibody was detected by the AP polymer and Permanent Red (PR, K5361; Dako, Denmark) incubation for 10 min. Similar to the single-staining method described previously, the sections were washed with TBST for 10 min between every step, and counterstained consecutively with haematoxylin, dehydrated and mounted in Eukitt. Filter paper was used for dehydrating the sections in order to avoid damaging the double-staining of the Ro52- and Ro60-specific cells with the use of alcohol and xylene. This might have resulted in high background staining, especially in instances where there was a great deal of saliva production in the gland.

Evaluation of staining

The minor SG sections were studied using a light microscope (Leica, DMLB, Leica Microsystems Wetzlar, Wetzlar, Germany) by three investigators. Both mononuclear cells in focal infiltrates and those located interstitially, i.e. in close proximity to the acinar or ductal epithelium, were analysed.

The single-positive Ro52- and Ro60-specific cells were counted with ×10 and ×20 magnification, using a grid. In addition, double-positive Ro52- and Ro60-specific memory B cells (CD20/CD27) were studied on serial sections using a ×10, ×40 or ×63 objective. Moreover, double-positive Ro52- and Ro60-specific B1 B cells (CD19/CD5) were also accounted for using a ×10, ×40 or ×63 objective. Cells were considered positive when 50% or more of the cell membrane was stained positively. All the glandular tissue from each patient was counted. The total area of the minor SG section was also measured for each of the patients. Together, this allowed the presentation of the data as number of Ro52- or Ro60-specific cells per 10 mm2 of SG tissue. Furthermore, the CD20-positive B cell zones (BCZ) and total number of double-positive memory B cells (CD20/CD27) were also accounted for in a previous study of these 10 pSS patients [15], as presented in Table 1.

Statistical analysis

Statistical significance was evaluated by Student's t-test and presented as the mean. Differences were considered significant when P ≤ 0·05. In addition, Pearson's correlation test was used to examine the association between the different parameters.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Study population

By relying upon the recently re-evaluated FS values, the 10 pSS patients included in this study were divided into four groups according to the degree of inflammation in their SG tissue (Fig. 1). One group consisted of patients with FS = 0 who exhibited little to no focal inflammation in their SG tissue, and three additional groups included patients with FS = 1, FS = 2 and FS = 3, respectively. SG tissue sections from four non-pSS subjects also served as the control group. These individuals had sicca symptoms, but had normal SG morphology.

figure

Figure 1. Haematoxylin and eosin (H&E) staining in salivary glands (SG) of primary Sjögren's syndrome (pSS) patients. H&E staining in a pSS patient with focus score (FS) = 1, another with FS = 2, a pSS patient with FS = 3 and a subject with normal gland (NG) histology where FS = 0. There is a general increase in mononuclear cell infiltration with increasing FS, while no focal infiltration was observed in the patient with NG histology.

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Studying the morphology of the different SG sections, one patient, pSS-138, was positive for GC-like structures (GC+). This individual had an FS of 2, which is consistent with what has been observed previously, where GC+ structures are more likely to occur in cases where the FS is ≥2 and there is increased focal inflammation and infiltrations [16, 19, 21]. However, this GC+ patient was negative for both ANA and autoantibodies. In contrast, eight of 10 patients in our study group were ANA-positive.

The total memory B cell number in the SG of these individuals was generally low, ranging from 0 to 17 cells per 10 mm2 of SG tissue, where 17 cells were observed in the FS = 3 group. No memory B cells were observed in the group with FS = 0. Also, a correlation has been found previously in these patients between the BCZ and the memory B cells [15]. Correspondingly, our FS = 0 group had no BCZ. In comparison to this, the percentage of immunoglobulin (Ig)G+ memory B cells that are specific for Ro52 and Ro60 in the PB of these individuals was also generally low in all 10 patients, ranging from 0 to 11%. None the less, the highest percentage values measured (6 and 11%) were for Ro52-specific IgG+ memory B cells, and observed consequently in the FS = 3 group. In addition, the number of anti-Ro52 and anti-Ro60-secreting B cells in PB was found to be generally low, where eight of the patients had fewer than 10 ASC per 100 000 PBMC. Also, five patients (representing all four FS groups) showed the common symptoms of pSS, i.e. is dry eyes and dry mouth.

Ro52- and Ro60-specific cells and CD19 expression in SG of pSS

In order to determine the total number of Ro52- and Ro60-specific cells in the SG of the pSS patients, single-staining with Ro52 and Ro60 antigens was carried out and the single-positive cells were counted using a grid at ×10 and ×20 magnification. Taking into consideration that our study group represented FS values of 0–3, we attempted to study the relation between the number of Ro52- and Ro60-specific cells to the FS. The mean number of Ro52-specific cells per 10 mm2 of SG tissue was found to be 41, 103, 144 and 25 for each FS = 0, FS = 1, FS = 2 and FS = 3, respectively. The average number of Ro60-specific cells was measured to be 82, 67, 99 and 80 per 10 mm2 of SG tissue for FS = 0, FS = 1, FS = 2 and FS = 3, in that order. In both instances, higher numbers of SSA-specific mononuclear cells were found in FS = 2 group, while the lowest number of cells was observed in the FS = 3 group for both Ro52 and Ro60. A high number of Ro52- and Ro60-specific cells was also detected in the FS = 0 group, even though no BCZ were identified in this group previously [15]. Importantly, SSA-specific cells were also detected in the non-pSS control group. Moreover, all 10 patients had Ro52- and Ro60-specific cells in their SG, even though SSA-specific serum autoantibodies could be detected in only four of these individuals (Table 1).

Because CD19 is a general B cell marker present on all the B cell subsets, double-staining for each of the Ro52 and Ro60 antigens with CD19 allowed identification of the general SSA-specific B cell pattern in the SG of these individuals. In addition, BCZ, detected with the use of CD20, were now verified using CD19 in the same subjects. In this way, localization of the Ro52- and Ro60-specific cells could also be studied in relation to the BCZ. Interestingly, both Ro52- and Ro60-specific cells observed in the SG biopsies of these 10 pSS patients were found to be CD19-positive. These SSA-specific cells were also generally observed interstitially and outside the BCZ (Fig. 2). No correlation was found between the number of BCZ and the number of Ro52- and Ro60-specific cells (data not shown).

figure

Figure 2. Ro52- and Ro60-specific cells and CD19 expression in salivary glands (SG) of primary Sjögren's syndrome (pSS) patients. Double-positive Ro52- and Ro60-specific cells (brown) that also express CD19 (red), a marker also used to identify the glandular B cell zones (BCZ) (red), in a pSS patient with focus score (FS) = 1.

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CD5 expression and B-1 B cell pattern for Ro52- and Ro60-specific cells in the SG of pSS patients

Having already established that these Ro52- and Ro60-specific cells are CD19-positive B cells, we wished to examine whether any of these cells belong to the B-1 B cell subset and could, in turn, give rise to natural antibodies. For these reasons, double-staining was carried out for each of the Ro52 and Ro60 antigens along with CD5, where tissue sections from human tonsils were used as a positive control. No Ro52- and Ro60-specific cells were found to be positive for CD5 (Fig. 3).

figure

Figure 3. CD5 expression and B-1 B cell pattern for Ro52- and Ro60-specific cells in the salivary glands (SG) of primary Sjögren's syndrome (pSS) patients. Double-staining identifying Ro52- and Ro60-specific cells (brown) and CD5-positive cells (red) in pSS patients. No SSA-specific cells that express CD5 are detected.

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CD20/CD27 double-positive memory B cell pattern and CD27-positive plasma cell pattern of Ro52- and Ro60-specific cells in SG of pSS patients

In order to identify Ro52- and Ro60-specfic memory B cells expressing CD27 and CD20, double-staining of serial sections from the SG biopsies was made using either Ro52 or Ro60 antigens along with CD20 and CD27, respectively. In addition, by staining the SG sections with CD20, we managed to identify the BCZ once again within the gland. Similar to the observation with CD19, all Ro52- and Ro60-specific B cells were observed outside the CD20-positive BCZ and interstitially, rendering them CD20-negative (Fig. 3).

Contrary to this, the double-staining of serial sections with either Ro52- or Ro60-antigens, along with CD27, showed that these SSA-specific cells were CD27-positive (Fig. 3).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

In this study we characterized the Ro52- and Ro60-specific B cell pattern in pSS through a series of double IHC staining of formalin-fixed, paraffin-embedded minor SG tissue from 10 well-defined pSS patients with varying FS (Fig. 1). Both Ro52- and Ro60-specific cells were detected in the SG of all 10 patients, only four of whom were positive for SSA-specific serum autoantibodies. Although local production of SSA-specific autoantibodies in the SG has been found previously to coincide with high levels of circulating autoantibodies in the sera of SS patients [8], our current results suggest that the presence of SSA-specific cells could perhaps also be independent of the systemic serum levels. Additionally, our FS = 2 group had the highest number of infiltrates, although a high number of SSA-specific cells was also observed in the FS = 0 group. Given that these SSA-specific cells were located sporadically within the SG and also interstitially, the degree of specific cells could, in turn, be independent of FS. The same pattern was also evident in our non-pSS control group, where all four individuals were positive for SSA-specific cells in their SG despite possessing normal gland morphology and negative serology. This could explain why no correlation was found between the number of SSA-specific infiltrates and increase in FS of our study population. Moreover, the Ro52 and Ro60 ASC in the PB of these same patients have also been accounted for in a previous study [36]. Both Ro52- and Ro60-specific ASC were present in the PB of all 10 patients, mainly in the FS = 3 group, despite possessing no serum autoantibodies at the time of admittance. This was consistent with what had been reported previously on how the presence of SSA-specific ASC in the PB of pSS patients also leads to detection in the SG [7]. None the less, the number of SSA-specific ASC in the PB of these 10 pSS patients was generally much lower than that of the SSA-specific infiltrates in the glands, suggesting that the SG is a homing site for these cells in pSS [16, 40]. However, the time-point at which the SG biopsies were taken in relation to disease progression for each subject might have an effect on the patterns studied. In addition to this, other factors such as chemokines and cytokines within the inflamed tissue may also influence these particular processes [41]. Therefore, based on the limited number of patients included in this study, only a trend could be indicated.

In order to determine whether these SSA-specific cells are indeed B cells, double-staining was performed for each of the Ro52 and Ro60 antigens with CD19, as CD19 is a B cell marker identified on all the B cell subsets [31, 42]. As expected, both Ro52- and Ro60-specific cells were found to be CD19-positive, confirming that they are indeed B cells (Fig. 2). In addition, the localization of these SSA-specific cells could be studied in relation to the BCZ, which were also identified in these individuals previously using CD20 [15]. Interestingly, both Ro52- and Ro60-specific cells were located interstitially and outside the BCZ, explaining why no correlation was observed between the number of BCZ and the number of SSA-specific cells.

Furthermore, to establish whether these CD19-positive SSA-specific cells detected in the SG of pSS patients belong to the B-1 B cell subset, double-staining was carried out with CD5 along with Ro52 and Ro60 antigens, respectively. No Ro52- and Ro60-specific cells were found to be positive for CD5 (Fig. 3). This indicates that SSA-specific cells are of the B-2 subset of B cells, in turn not giving rise to natural antibodies.

Moreover, double-staining of serial sections from the SG biopsies using either Ro52 or Ro60 antigens along with CD20 and CD27, respectively, revealed that these SSA-specific cells were CD20-negative, but were none the less CD27-positive (Fig. 4). As no Ro52- and Ro60-specific cells were CD20-positive, none of these SSA-specific cells could, in turn, be memory B cells. Consequently, no Ro52- and Ro60-specific memory B cells were detected in these 10 pSS patients. This is in tune with previous findings, where low levels of total CD20+/CD27+ memory B cells in the SG of these 10 subjects were accounted for [15]. Accordingly, low levels of Ro52- and Ro60-specific IgG+ memory B cells have also been observed previously in the PB of the same individuals [36]. Contrary to this, detection of CD27-positive Ro52- and Ro60-specific cells, which are also CD19-positive as explained above, indicated that these SSA-specific cells could either be plasmablasts, plasma cells or long-lived plasma cells. Because memory B cells have been found to migrate to the bone marrow and lymph nodes [43-45], a possibility for this lack of Ro52- and Ro60-specific CD20/CD27 double-positive memory B cells in the SG of our study group could be due to the SG not being the major homing site of memory B cells in the pathogenesis of pSS. However, it has also been established that long-lived plasma cells also tend to migrate to the bone marrow [16, 29, 46, 47]. Therefore, another possible explanation for these low numbers of memory B cells and elevated levels of plasma cells and long-lived plasma cells in the SG of pSS patients could be the result of activation of these memory B cells into plasma cells at the site of inflammation. This might also explain why current therapeutic interventions in SS that depend on administration of the anti-CD20 antibody Rituximab® have proved to be ineffective. This was also indicated in our study population, where 90% of the subjects were ANA-positive, in addition to the detection of autoantibodies in patients with high FS (Table 1). Bearing in mind that our results have shown Ro52- and Ro60-specific plasma cells and long-lived plasma cells to be the most abundant subtype of SSA-specific glandular B cells, and to be CD20-negative they would, in turn, not be affected by treatment with the anti-CD20 antibody Rituximab®. We therefore suggest that considering an additional mAb for therapy that targets the activated population of B cells could, perhaps, be a more effective therapeutic approach.

figure

Figure 4. CD20/CD27 double-positive memory B cell pattern of Ro52- and Ro60-specific cells in the salivary glands (SG) of primary Sjögren's syndrome (pSS) patients. (a) No double-positive cells expressing CD20 (red) and Ro52 (brown) are identified in a pSS patient with focus score (FS) = 2. Other naive B cells that are single-positive for CD20 and are infiltrating the B cell zones (BCZ) are also detected (red). Double-staining of a serial section from the same patient with Ro52-specific cells (brown) revealed that they are also positive for CD27 (red). The same structures in the left and right sections are marked with numbers 1 and 2. (b) No double-positive cells expressing Ro60 (brown) and CD20 (red) are identified in a pSS patient with FS = 2. Double-staining of a serial section from the same patient with Ro60-specific cells (brown) and CD27 showed that these autoantigen-specific cells also express CD27 (red). The same structures in the left and right sections are marked with numbers 1 and 2.

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In conclusion, we have shown that SSA-specific cells identified in the SG of pSS patients are indeed CD19-positive B cells that are CD5-negative, belonging in turn to the B-2 subset of B cells. Moreover, these individuals also display no SSA-specific memory B cells in their SG tissue, and a low number of total memory B cell infiltrations, in addition to decreased levels of circulating SSA-specific memory B cells. Contrary to this, elevated numbers of SSA-specific plasmablasts, plasma cells and long-lived plasma cells were also detected in the same individuals. Taken together, our findings suggest that the lack of SSA-specific memory B cells might be the result of the activation of these cells into CD20-negative antibody-secreting plasma cells and long-lived plasma cells at the site of inflammation, also rendering them resistant to Rituximab® treatment. Further studies need to be conducted on a murine model in order to gain a clearer understanding of memory B cell homing during the pathogenesis of pSS.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

We acknowledge with appreciation Professor Johan G. Brun from the Department of Rheumatology, Haukeland University Hospital for providing us with clinical information on the subjects and patients included in the study, Professor Anne Christine Johannessen at Section for Pathology, The Gade Institute, for the routine histological assessment of the salivary gland sections, and Kjerstin Jakobsen and Marianne Eidsheim at the Broegelmann Research Laboratory for excellent technical assistance. Finally, we would like to thank the Broegelmann Chair in Immunology Professor Roland Jonsson, the Broegelmann Research Laboratory for his scientific advice and guidance. This study was supported by the Faculty of Medicine and Dentistry at the University of Bergen, The Broegelmann Foundation and Western Norway Regional Health Authority.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References
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