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Levels of plasmacytoid dendritic cells and type-2 myeloid dendritic cells are reduced in peripheral blood of patients with primary Sjögren's syndrome
  1. Petra Vogelsang1,
  2. Johan G Brun2,3,
  3. Gunnvor Øijordsbakken4,
  4. Kathrine Skarstein4,
  5. Roland Jonsson1,2,
  6. Silke Appel1
  1. 1Broegelmann Research Laboratory, The Gade Institute, University of Bergen, Bergen, Norway
  2. 2Department of Rheumatology, Haukeland University Hospital, Bergen, Norway
  3. 3Institute of Medicine, Section for Rheumatology, University of Bergen, Bergen, Norway
  4. 4Section for Pathology, The Gade Institute, University of Bergen, Bergen, Norway
  1. Correspondence to Dr Silke Appel, Broegelmann Research Laboratory, The Gade Institute, University of Bergen, Laboratory building, 5th floor, N-5021 Bergen, Norway; silke.appel{at}


Objective Sjögren's syndrome (SS) is a lymphoproliferative autoimmune disease, characterised by dryness of the mouth and eyes. Dendritic cells (DC) are potent antigen-presenting cells crucial for initiating and maintaining primary immune responses. This study quantified interferon-producing plasmacytoid DC (pDC) and two myeloid DC subsets (mDC1 and mDC2) in peripheral blood (PB) from primary SS (pSS) patients and healthy controls.

Methods Blood samples from 31 pSS patients and 28 gender and age-matched healthy controls were analysed by flow cytometry using the Miltenyi Blood DC enumeration kit. The presence of pDC in salivary glands (SG) from pSS patients was analysed by immunohistochemistry.

Results Patients with pSS had significantly less pDC and mDC2 in PB compared with healthy controls. Moreover, pDC are present in SG from patients with pSS.

Conclusion Patients with pSS have alterations among DC populations in PB, and pDC are present in the SG, suggesting a potential role of these cells in SS.

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Sjögren's syndrome (SS) is a chronic inflammatory and lymphoproliferative autoimmune disease, characterised by progressive mononuclear cell infiltration within exocrine glands.1 Dryness of the mouth (xerostomia) and eyes (keratoconjunctivitis sicca) are the main organ-related clinical features affecting SS patients. A characteristic of SS is the presence of autoantibodies, such as anti-Ro/SSA and anti-La/SSB. Using the European–American consensus group criteria (AECC), the prevalence of SS among the population is estimated to be approximately 0.1–0.6%, with 90% of the affected patients being female.2 The aetiology of the disease is so far unknown, but it is assumed that a combination of several factors is involved in the occurrence of SS. As the cell infiltrates mainly consist of T cells and B cells,1 little attention has so far been directed to dendritic cells (DC).3

DC are key players in initiating an immune response and maintaining tolerance.4 They sample antigens from the environment and present them as peptide–MHC complexes to effector T cells in lymphoid organs. DC comprise a heterogeneous population of cells that is divided into two main subsets, myeloid DC (mDC) and plasmacytoid DC (pDC). The latter are also known as natural interferon-producing cells.5

Besides their immunogenic properties during infections of the body, DC also play a central role in preventing autoimmunity.6 During the steady state, DC induce and sustain peripheral tolerance by controlling regulatory T cells.7 Previously, it was shown that alterations among DC populations occur in autoimmune diseases such as systemic lupus erythematosus (SLE),8 rheumatoid arthritis (RA)9 and type 1 diabetes.10 The purpose of this study was to analyse the quantities of pDC, mDC1 and mDC2 in peripheral blood from patients with primary SS (pSS).

Materials and methods


All 31 pSS patients included in this study fulfilled the AECC for classification of SS2 and were recruited from the Department of Rheumatology at the Haukeland University Hospital in Bergen, Norway. The control group consisted of 28 gender and age-matched healthy controls who were recruited from the same geographical area as the patients. This study was approved by the Ethical Committee of the University in Bergen, Norway (242.06). All subjects studied gave their informed consent.

Laboratory assays

The analysis of anti-Ro/SSA, anti-La/SSB, antinuclear antibodies, serum immunoglobulin levels (IgG, IgA, IgM), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), Schirmer's test and salivary flow rate as well as medical records have been evaluated and obtained as part of the routine diagnostic evaluation for SS at the Department of Rheumatology, Haukeland University Hospital (table 1). Serum IFNα levels were determined as described by Reksten et al.11

Table 1

Clinical data on patients used in this study

Enumeration of DC subsets in peripheral blood

Blood was collected in heparin tubes and DC populations were stained with the Blood DC enumeration kit (Miltenyi Biotec, Bergisch Gladbach, Germany), according to the manufacturer's manual. The amount of leucocytes was determined using a CASY-Technology cell counter (Innovatis AG, Reutlingen, Germany). Cells were analysed on a BD FACSCanto I (BD Bioscience, San Jose, California, USA) flow cytometer and data analysis was performed using FlowJo software (Tree Star Inc, Ashland, Oregon, USA).


Frozen tissue sections of minor salivary gland (SG) biopsies from patients with pSS were stained with a monoclonal anti-human CD303 antibody (clone 104C12.08; Dendritics, Lyon, France) as described previously.12

Statistical analyses

Data are presented as the total cell count of each DC population per millilitre of blood or as a percentage of analysed leucocytes. Values are expressed as the median. For the statistical analysis of comparison between groups, a two-tailed Mann–Whitney U test was used. Correlation between parameters was calculated using the percentage of analysed leucocytes by Spearman's rank correlation for non-parametric data. All statistical analyses were performed using Prism (GraphPad Software Inc, La Jolla, California, USA).


Patients with pSS have reduced circulating pDC and mDC2

Patients with pSS have significantly reduced numbers of pDC and mDC2 in peripheral blood (PB) compared with healthy controls expressed either as median counts of DC per millilitre of blood (figure 1A; pDC: patients 1.3×104 vs controls 3.1×104; p=0.036; mDC2: patients 1.7×103 vs controls 3.1×103; p=0.0303) and the percentage of analysed leucocytes (figure 1B; pDC: patients 0.114 vs controls 0.275; p=0.0024; mDC2: patients 0.016 vs controls 0.0315; p=0.0025). There was no significant difference in the mDC1 population between patients and controls either as total cell count per millilitre of blood (patients 3.4×104 vs controls 3.7×104) or as the percentage of leucocytes (patients 0.349 vs controls 0.406).

Figure 1

Patients with primary Sjögren's syndrome (pSS) have significantly fewer plasmacytoid dendritic cells (pDC) and type-2 myeloid dendritic cells (mDC2) in peripheral blood (PB) compared with healthy controls. Results were acquired by using the blood DC enumeration kit on whole blood samples from pSS patients. (A) Absolute counts of DC subsets in PB from pSS patients and healthy controls. Patients (P, n=27) and healthy controls (C, n=18). (B) Percentage of DC subsets among leucocytes in PB from pSS patients and healthy controls. Patients (P, n=31) and healthy controls (C, n=28). (C) Correlation between levels of DC populations and Ro/SSA and La/SSB autoantibodies.

Correlation between DC populations and clinical parameters

In patients, we found a significant correlation between levels of pDC and mDC2 (p<0.0002) and between mDC1 and mDC2 (p<0.0001) but no correlation between levels of pDC and mDC1. In controls, we observed a correlation between pDC and mDC1 (p=0.0008), pDC and mDC2 (p<0.0002), mDC1 and mDC2 (p<0.0001).

We analysed the correlation between patients with Ro/SSA, La/SSB or Ro/SSA and La/SSB antibodies and levels of DC (figure 1C). Patients with only anti-Ro/SSA have significantly more pDC in PB than patients with anti-Ro/SSA and anti-La/SSB (p=0.0051).

No correlation between serum IFNα levels, focus score, germinal centres, CRP, ESR or duration of disease and the frequencies of DC populations in PB was found.

Frequency of pDC in minor SG of pSS patients

We aimed to correlate the amount of pDC in minor SG tissue and PB by choosing representative SG biopsies from patients with high, medium and low levels of pDC in PB and quantified pDC in SG tissue (figure 2). No obvious correlation between peripheral and tissue pDC could be observed.

Figure 2

Immunochemical staining for plasmacytoid dendritic cells (pDC) with an anti-BDCA-2 antibody in representative salivary glands from patients with primary Sjögren's syndrome. (A) Patient with high pDC levels in peripheral blood (PB). (B) Patient with low pDC levels in PB. (C) Patients with medium pDC levels in PB. (D) Overview over a lymphocytic infiltrate from a patient with medium pDC levels in PB revealing the localisation of the pDC in the periphery of the infiltrate. Magnification 200×.


Levels of the IFN-producing pDC and CD11c+ mDC have previously been described to be altered in patients with SLE, a disease closely related to SS,13 and elevated levels of IFNα in serum correlated to decreased numbers of pDC.14 In this study, we show that patients with pSS have decreased levels of pDC in PB, in confirmation of findings presented by Wildenberg et al.15 It is still controversially discussed whether SS leads to systemically increased IFNα levels as seems to be the case in SLE patients, because pSS patients usually do not show directly measurable increased IFNα levels in serum.16 Studies using an in-vitro reporter cell assay were able to show an increased IFNα activity using plasma from patients' samples.17 18 Here, we could not correlate DC numbers to serum IFNα levels because they were low in most of the samples examined. However, it would be interesting to analyse the IFN signature in different cell types and SG from patients used for this study and correlate it to pDC levels, because the upregulation of IFN-regulated genes in SG16 19 and monocytes15 from pSS patients has been shown previously.

pDC circulate with the PB through the body and migrate to inflamed tissues and secondary lymphoid organs. In patients with pSS, the presence of IFNα-containing cells in SG has been reported before.16 We did not observe an obvious correlation between peripheral and tissue pDC numbers, but the sample size of this study presents limitations to draw definite conclusions. We cannot rule out that time point of biopsies, onset of disease, duration of disease or other pathological events such as atrophy and fibrosis influence the detection of infiltrating pDC in the SG, making a correlation to pDC in PB difficult.

In contrast to pDC, not much is known about the function of mDC2. Interestingly, levels of mDC2 are not altered in type 1 diabetes or RA patients,9 10 suggesting that this DC population may play a distinct role in SS. Therefore, the function of mDC2 and their potential contribution to the development of SS has to be further elucidated. A problem to be solved is in general the very low frequency of mDC2 in PB, which is on average approximately 0.02% among leucocytes.

We observed a significant correlation between mDC1 and mDC2, pDC and mDC2, but no correlation between pDC and mDC1 in patients with pSS, although levels of all three DC populations correlate in healthy controls. This result is especially interesting considering the fact that mDC1 levels seem to be unaltered in patients and controls. We therefore conclude that an imbalance in DC levels plays a role in SS.

Patients with only anti-Ro/SSA have significantly more pDC in PB than patients with anti-Ro/SSA and anti-La/SSB, equalling amounts of patients without any Ro/SSA and La/SSB autoantibodies. The same pattern can be seen for mDC2 without statistical relevance, but not mDC1. We could not find a relation to clinical manifestations such as germinal centres, focus score and treatment or disease duration, but this might be due to limitations of our patient cohort.

The exact mechanism of how reduced DC levels influence the outcome of autoimmunity has not yet been revealed. Further studies on functional aspects of DC from patients with pSS have thus to be performed. Nevertheless, our study supports the finding that altered DC populations may play a crucial role in autoimmune diseases such as SS.


The authors would like to thank all patients and healthy controls for kindly donating blood for this study. They appreciate the help of Dagny Ann Sandnes, Marianne Eidsheim, Nina Harkestad and the laboratory staff at the Department of Rheumatology for taking the blood samples, and also wish to thank Ammon Peck for reading the manuscript.



  • Funding This study was supported by the Faculty of Medicine and Dentistry at the University of Bergen, the Strategic Research Program at Helse Bergen, the Western Norway Regional Health Authority, the Broegelmann Foundation, the Norwegian Cancer Society and the Bergen Research Foundation.

  • Competing interests None.

  • Ethics approval This study was conducted with the approval of the University of Bergen, Norway (approval number 242.06).

  • Patient consent Obtained.

  • Provenance and peer review Not commissioned; externally peer reviewed.