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Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 2006 Feb 13;103(8):2770–2775. doi: 10.1073/pnas.0510837103

Activation of IFN pathways and plasmacytoid dendritic cell recruitment in target organs of primary Sjögren’s syndrome

Jacques-Eric Gottenberg *,†, Nicolas Cagnard †,‡, Carlo Lucchesi †,‡, Franck Letourneur †, Sylvie Mistou †, Thierry Lazure §, Sebastien Jacques †, Nathalie Ba §, Marc Ittah *, Christine Lepajolec , Marc Labetoulle ∥, Marc Ardizzone †,**, Jean Sibilia **, Catherine Fournier †, Gilles Chiocchia †,††, Xavier Mariette †,††,‡‡
PMCID: PMC1413808  PMID: 16477017

Abstract

Gene expression analysis of target organs might help provide new insights into the pathogenesis of autoimmune diseases. We used global gene expression profiling of minor salivary glands to identify patterns of gene expression in patients with primary Sjögren’s syndrome (pSS), a common and prototypic systemic autoimmune disease. Gene expression analysis allowed for differentiating most patients with pSS from controls. The expression of 23 genes in the IFN pathways, including two Toll-like receptors (TLR8 and TLR9), was significantly different between patients and controls. Furthermore, the increased expression of IFN-inducible genes, BAFF and IFN-induced transmembrane protein 1, was also demonstrated in ocular epithelial cells by quantitative RT-PCR. In vitro activation showed that these genes were effectively modulated by IFNs in salivary gland epithelial cells, the target cells of autoimmunity in pSS. The activation of IFN pathways led us to investigate whether plasmacytoid dendritic cells were recruited in salivary glands. These IFN-producing cells were detected by immunohistochemistry in all patients with pSS, whereas none was observed in controls. In conclusion, our results support the pathogenic interaction between the innate and adaptive immune system in pSS. The persistence of the IFN signature might be related to a vicious circle, in which the environment interacts with genetic factors to drive the stimulation of salivary TLRs.

Keywords: autoimmune disease, innate immunity, salivary gland

Sjögren’s syndrome (SS) is a common systemic autoimmune disease that can be primary or occur in association with other autoimmune diseases. The prevalence of SS is second only to that of rheumatoid arthritis. This disease preferentially involves salivary and lachrymal glands, leading to the main symptoms of ocular and mouth dryness, but a wide range of other organs might be targeted (1). Moreover, primary SS (pSS) can be used as a prototypic disease for investigating the relation between autoimmunity and lymphoma, because 5% of patients with pSS develop B cell non-Hodgkin’s lymphoma (2, 3).

pSS is currently considered a complex disease involving environmental and genetic factors (4–6). However, the mechanisms driving the autoimmune process in pSS have not been unraveled, despite extensive studies. The absence of familial clustering of the disease (in contrast with that of other autoimmune diseases) precludes gene-mapping efforts. Until recently, no experimental model was particularly relevant concerning the pathogenesis of pSS. Thus, genome-wide gene expression profiling involving microarrays could be a good strategy to study the pathogenesis of this disease. This approach, which allows the simultaneous analysis of thousands of mRNA transcripts in a biologic sample, has been applied successfully in the investigation of various human malignant diseases (7). In the field of autoimmune diseases, most of the microarray studies used gene expression profiling of peripheral blood mononuclear cells (8, 9). Given the predominant organ specificity in pSS, we explored the gene expression of minor salivary gland tissue, a target organ of the disease, to provide insights into the pathogenesis of the disease.

Results

Salivary Gland Gene Expression Differentiates Most Patients with pSS from Controls.

Gene expression of minor salivary glands was compared between 7 patients with pSS and 7 controls, with 28 cDNA microarrays. Among the 7,261 genes analyzable, 424 genes showed significant differential expression (215 up-regulated, 209 down-regulated) in patients with pSS compared with controls (Table 3, which is published as supporting information on the PNAS web site). Among the 7,261 genes, 3,408 had no missing values in all 28 microarrays, and 120 of these showed significant differential expression between patients and controls (top 120 differentially expressed genes) (Table 4, which is published as supporting information on the PNAS web site).

Unsupervised clustering of all analyzable genes by use of treeview resulted in no particular grouping of the samples. Unsupervised hierarchical clustering was then applied to the data set of 120 genes to identify expression patterns, which provided a molecular signature allowing for differentiation of most patients with pSS from healthy controls (Fig. 1).

Fig. 1.

Fig. 1.

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Unsupervised hierarchical clustering of 120 top differentially expressed genes in patients with pSS. Expanded view of gene expression of selected differentially expressed genes in patients with pSS and controls. Expression level is shown by color: red, relatively high expression; green, low expression. The seven pSS samples clustered together and six of seven controls clustered together. Only one control sample (C10) was more closely associated with the pSS samples.

Differential Expression of Genes Involved in IFN Pathways in Salivary Glands of Patients with pSS.

Gene ontology analysis was used to organize the expression data of the 424 differentially expressed genes into their functional relationships on the basis of biological processes. Gene ontology terms of interest for autoimmunity included “immune response” for 10% of genes, “cellular defense response” for 12%, “response to external stimulus” for 7%, “cell death” for 5%, and “organogenesis” for 16% of genes. pathwayassist software revealed 111 genes to be involved in functional pathways. IFN-α and IFN-γ pathways involved 46 (10.8%) of these genes. Data from the literature confirmed 23 of these genes to be IFN-inducible (10). The expression of 21 of these genes was significantly up-regulated in pSS, with only two genes having a significantly down-regulated expression (Fig. 2 and Table 1). Most of these genes belonged both to IFN-I (IFN-α) and IFN-II (IFN-γ) pathways. These results led us to focus on the genes involved in IFN pathways in patients with pSS.

Fig. 2.

Fig. 2.

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Mean ratio of 424 differentially expressed genes in patients with pSS to those in controls. Mean pSS/controls ratio of the 424 differentially expressed genes in pSS. The 23 IFN-inducible genes are in red. Twenty-one of these genes showed significantly increased expression in pSS, and two showed significantly decreased expression.

Table 1.

Differential expression of genes involved in IFN pathways in patients with pSS

Gene name Gene symbol Unigen ID Gene group/function Mean ratio pSS patients/controls Pcorr P
Genes with increased expression in pSS
    IFN-induced transmembrane protein 1 (9–27) Hs.458414 IFITM1 IR 2.52 0.030 0.002
    CD6 antigen Hs.436949 CD6 IR 2.40 0.030 0.002
    NACHT. leucine-rich repeat and PYD containing 2 Hs.369279 NALP2 CD 2.32 0.030 0.002
    promyelocytic leukaemia Hs.89633 PML Protein ubiquitination 2.22 0.030 0.002
    INF-induced transmembrane protein 2 (1–8D) Hs.174195 IFITM2 IR 2.04 0.030 0.002
    MHC class I polypeptide-related sequence B Hs.211580 MICB IR 1.92 0.030 0.002
    IFN-induced transmembrane protein 3 (1–8U) Hs.374650 IFITM3 IR 1.88 0.030 0.002
    IL-4 receptor α-chain isoform a Hs.513457 IL4R IL4R 1.87 0.048 0.004
    Ets variant gene 5 (ets-related molecule) Hs.43697 ETV5 Nucleic acid binding 1.87 0.030 0.002
    Transporter 2. ATP-binding cassette Hs.502 TAP2 IR 1.76 0.030 0.002
    Spleen tyrosine kinase Hs.192182 SYK ISC 1.76 0.030 0.002
    β2-microglobulin Hs.48516 B2M IR 1.70 0.048 0.004
    Mitogen-activated protein kinase kinase kinase 1 Hs.508461 MAP3K1 Kinase activity 1.65 0.039 0.003
    TLR9 Hs.436439 TLR9 IR 1.48 0.048 0.004
    Diablo homolog Hs.169611 DIABLO CD 1.47 0.030 0.002
    EphA4 Hs.244624 EPHA4 ISC 1.37 0.030 0.002
    Collagen, type XVI. alpha 1 Hs.26208 COL16A1 Cell adhesion 1.35 0.030 0.002
    TLR8 Hs.272410 TLR8 IR 1.33 0.030 0.002
    Megakaryocyte-associated tyrosine kinase Hs.437808 MATK ISC 1.31 0.030 0.002
    PKC-δ Hs.155342 PRKCD CD/ISC 1.29 0.030 0.002
    Chemokine (C-C motif) ligand 20 Hs.75498 CCL20 IR 1.27 0.030 0.002
Genes with decreased expression in pSS
    Chemokine (C-C motif) ligand 18 Hs.412832 CCL18 IR 0.46 0.030 0.002
    Suppressor of cytokine signaling 3 Hs.527973 SOCS3 ISC 0.18 0.048 0.004
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Pcorr, corrected P value according to Benjamini and Hochberg’s correction; IR, immune response; CD, cell death; ISC, intracellular signaling cascade.

Two members of the Toll-like receptor (TLR) family (TLR8 and TLR9) showed significantly increased expression in patients with pSS. Likewise, the three members of the IFN-induced transmembrane protein family (IFITM1, IFITM2, and IFITM3) (11) showed significantly increased expression. Interestingly, the only member of the BAFF family present on the microarray, BCMA [one of the BAFF receptors (12)], showed significantly increased expression. Two genes, SOCS3 and CCL18, which play a role in the inhibition of inflammation, showed significantly decreased expression.

Quantitative RT-PCR Analysis of Genes Involved in IFN Pathways in Salivary Glands and in the Ocular Surface of Patients with pSS.

The expression of five genes (TLR8, TLR9, IFITM1, BAFF, and SOCS3) involved in IFN pathways was analyzed by quantitative RT-PCR. Genes with increased expression were TLR8; TLR9; IFITM1; BAFF, which was not printed on microarrays; and BCMA (Fig. 3A). SOCS3, an inhibitor of IFN-signaling pathways, showed significantly decreased expression, which confirmed the microarray data (Fig. 3A). Because ocular and mouth dryness might share common physiopathologic features, we investigated whether the expression of two IFN-inducible genes, IFITM1 and BAFF, was increased in conjunctival cells collected by impression cytology of the ocular surface. A significant 2.5-fold increase of IFITM1 and a 3-fold increase of BAFF were observed in conjunctival cells of patients with pSS compared with controls (Fig. 3B).

Fig. 3.

Fig. 3.

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Quantitative real-time RT-PCR analysis of expression of IFN-inducible genes in salivary glands and ocular epithelial cells. (A) Quantitative RT-PCR analysis of gene expression of IFN-inducible genes and BCMA in salivary glands. Gene expression of TLR8, TLR9, IFITM1, BAFF, and BCMA was significantly increased, and SOCS3 expression was significantly decreased in patients with pSS (n = 10) compared with controls (n = 10). (B) RT-PCR analysis of expression of IFN-inducible genes in ocular epithelial cells of patients with pSS (n = 5) and controls (n = 8). BAFF and IFITM1 expression were significantly increased in patients with pSS.

In Vitro IFN Induction of Gene Expression in Salivary Gland Epithelial Cells, Target Cells of Autoimmunity.

We determined the extent to which expression of IFN-inducible genes was indeed increased by IFN stimulation in the target organ of the disease. Because salivary gland epithelial cells (SGECs) are the main cell targets of the disease, we investigated whether IFITM1, a prototypic IFN-stimulated gene, could be regulated by IFN treatment in epithelial cells. SGECs showed strong increased expression of IFITM1 mRNA after stimulation with IFN-α and IFN-γ (98-fold and 32-fold compared with the baseline, P < 0.0001 and P = 0.01, respectively). Induction of IFITM-1 by IFN-α and IFN-γ was higher in SGECs cultured from patients with pSS than in controls, although not significantly (Fig. 4). In addition, BAFF was induced in epithelial cells after IFN-α/γ stimulation (data not shown).

Fig. 4.

Fig. 4.

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In vitro IFN induction of IFITM1 in salivary gland epithelial cells. Salivary gland epithelial cells were cultured in vitro after minor salivary gland biopsy of patients with pSS (n = 4) and controls (n = 6). IFITM-1 gene expression in epithelial cells was assessed by using quantitative RT-PCR at baseline and after IFN-α (2,400 unit/ml) or IFN-γ (5 ng/ml) stimulation during 2 days. Salivary gland epithelial cells showed a strong and significant increased expression of IFITM1 mRNA after stimulation with IFN-α and IFN-γ.

Presence of Plasmacytoid Dendritic Cells (pDCs) in Salivary Glands of Patients with pSS.

The increased expression of IFN-inducible genes in salivary glands and in conjunctival cells of the ocular surface, as well as increased gene expression of TLR9, an innate immunity receptor present in pDCs (13), led us to investigate the presence of pDCs, which secrete high amounts of IFN-α (14). pDCs, identified as CD123-positive cells, were present in all patients with pSS [mean number of pDCs/focus: 4 (range, 3–5)]. Similarly appearing plasmacytoid cells stained positively for BDCA2, a specific marker of pDCs (15), and TLR9 (Fig. 5). pDCs were not present in any controls.

Fig. 5.

Fig. 5.

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Identification of pDCs in the salivary glands of patients with pSS. Immunohistochemical study of salivary glands of patients with pSS (n = 5) and controls (n = 9). The results of one patient with pSS (A, C, and E) and one control (B, D, and F) are presented. pDCs, identified as CD123 positive cells (A), were present in all of the patients with pSS. Similarly appearing plasmacytoid cells stained positively for BDCA-2, a specific marker of pDCs (C), and TLR-9 (E). No pDCs were observed in controls.

Discussion

Our study of pSS as a prototypic autoimmune disease suggests the involvement of pDCs and IFN-mediated innate immunity in the pathogenesis of pSS.

Differential expression of 23 IFN-inducible genes led us to investigate the presence of a recently characterized subset of DCs, pDCs. pDCs are also called “natural IFN-producing cells” (16), because they are the most potent producers of IFN I, secreting up to 1,000-fold more IFN-α/β than other cell types in response to pathogen-associated molecular patterns or endogenous ligands. To our knowledge, the presence of pDCs in the salivary glands of patients with pSS had never been investigated in contrast with that of myeloid DCs. Myeloid DCs were reported to be present in the salivary glands of patients with pSS in large follicular inflammatory infiltrations (focus score ≥ 1) (17). By using immunohistochemistry we demonstrated that pDCs are present in the salivary glands of patients with pSS but absent in controls. Activation of pDCs through TLR receptors links innate to adaptive immunity, leading to increased secretion of type I IFN (IFN-α/β) and IL-12, which promotes IFN II (IFN-γ) secretion by T lymphocytes, NK cells and DCs (16). In addition, IFN-α and IL-6 secretion by pDCs results in plasma cell differentiation and antibody secretion. Last, activation of pDCs increases their capacities to present antigens, which remain lower than that of myeloid DCs. Thus, the presence of pDCs in the salivary glands of patients with pSS sheds light on the pathogenesis of the disease.

Why are pDCs present in salivary glands of patients with pSS? First, a defective homeostasis of this cell lineage can be hypothesized. Few data are available regarding the ontogeny of pDC lineage (18). ID3, an inhibitory transcription factor of the basic helix–loop–helix family, inhibits the development of pDCs (19). Interestingly, ID3−/− knockout mice were recently reported to develop an autoimmune disease closely mimicking pSS (20). The migration of pDCs could also be related to specific chemoattractants, promoting their homing into salivary glands in pSS and into various target organs in other autoimmune diseases, such as the skin [systemic lupus erythematosis (SLE) (21) and psoriasis (22)], synovial fluid [rheumatoid arthritis (23) and spondylarthropathy (24)], muscle [dermatomyositis (25)], and cerebral spinal fluid [multiple sclerosis (26)].

The first suspect for induction of IFN-α secretion by TLR stimulation of pDCs is viral infection (27). Interestingly, viruses such as Epstein–Barr, hepatitis C, retroviruses (human T-cell lymphotropic type I), or enteroviruses have been suggested to play a triggering role in the autoimmune epithelitis of pSS (28–31). However, no firm association between viral infections and pSS has been established. Viral components can be recognized by various TLRs, including TLR3, TLR7, TLR8, and TLR9 (27). We demonstrate that TLR8 and TLR9 have an increased expression in pSS, but the absence of overexpression of TLR3 and TLR7 does not rule out the involvement of these TLRs in the pathogenesis of the disease.

In addition to pathogen sensing, TLRs can also recognize self-antigens released from damaged host tissues. Thus, IgG bound to mammalian chromatin activates transgenic rheumatoid factor B cells after B cell antigen receptor recognition of the immune complexes and delivery of the DNA to TLR9 (32). Likewise, chromatin immune complexes stimulate myeloid DCs, pDCs, and DNA-reactive B cells by the coengagement of FCγ receptors and TLR9 (33, 34). Thus, TLR9 overexpression in pSS might contribute to B cell and pDC activation in this disease. Interestingly, TLR9-deficient lpr/lpr mice did not secrete DNA-specific antibodies but still produced RNA-reactive autoantibodies (35). It was recently demonstrated that self-RNA bound to autoantibodies actually leads to TLR7- and TLR8-driven stimulation of DCs and self-reactive B cells (36, 37). Interestingly, two-thirds of the patients with pSS have autoantibodies against SSA/SSB, which form immune complexes with double-stranded (Y RNA) and single-stranded RNA (38). In the present study, we demonstrate an increased expression of TLR8, which recognizes single-stranded RNA. Besides the potential activation of TLR8 by pathogens, single-stranded RNA bound by SSA might also stimulate TLR8 and explain the increased expression of IFN-α observed in pSS patients with anti-SSA/SSB antibodies (39). Thus, genetic factors, such as HLA class II or TGF-β gene polymorphisms, mainly involved in the predisposition to autoantibody production (4, 40–42), might interact with environmental factors to drive the stimulation of salivary TLRs, which results in the ongoing activation of IFN pathways. The respective contribution of genetic factors or environmental triggers in the activation of IFN pathways might be different in each individual patient. The persistent activation of the IFN pathways could also be due to a defective inhibitory feedback. Interestingly, SOCS3, an inhibitor of JAK-STAT, thus a potent physiological repressor of TLR- and IFN-signaling pathways (43), showed decreased expression in patients with pSS.

The specific role of IFN type I could not be differentiated from that of IFN type II on the basis of microarray data in pSS, as observed in SLE (8, 9). Due to a wide cross-talk of type I IFN pathways with type II IFN pathways, type I and II IFNs reciprocally affect each other’s production and signaling. However, IFN-α/β production is initiated at the early stages of the innate immune response and is likely to be the dominant factor (27). Thus, inhibition of IFN-α secretion by pDCs by using anti-BDCA2 antibodies completely inhibited the development of autoimmunity in a xenograft model of psoriasis (44). Interestingly, both IFN-α and IFN-γ induce BAFF expression by myeloid cells and salivary epithelial cells, which might represent one of the important pathogenic consequences of the activation of the IFN pathways in pSS. Indeed, BAFF overexpression in salivary glands of patients with pSS (45) might result in local activation and survival of autoreactive B and T cells. In addition, a higher expression of BCMA, one of BAFF receptors, was also observed in patients with pSS in our study and another recent microarray study (46).

pSS and SLE, which share some clinical and immunological features, now have two other pathogenic features in common: the presence of infiltrating pDCs in target organs (21) and activation of IFN pathways. The main difference between the two diseases might be related to the relative organ specificity of pSS. Accordingly, the serum IFN-α level was low or undetectable in patients with pSS (44) in contrast with those with SLE. Interestingly, our study with others suggest that, besides the fundamental role of IFN in lupus, the predominance of IFN-related genes could also be common in some organ-specific autoimmune diseases, including dermatomyositis (25), psoriasis (44), and now pSS, which have very distinct clinical phenotypes. This finding suggests that the physiopathological and clinical consequences of the activation of a common cytokine pathway essentially depend on resident cells of target organs.

This study also raises clinical and therapeutic perspectives. Regarding disease activity, assessing the level of expression of IFN-inducible genes might be beneficial in pSS, a disease for which no activity marker is currently available. We recently reported the association between increased serum β2-microglobulin and systemic features in pSS (47). Interestingly, in the present study, the gene expression of β2-microglobulin, which is also IFN-inducible, was significantly increased in patients with pSS. Thus, the gene expression of some IFN-inducible genes, creating an “IFN score,” might represent a relevant disease activity marker in pSS.

To date, therapy for pSS does not exist. The present results give insights into the lack of efficacy of anti-TNF-α therapy in pSS (48). Indeed, TNF-α-inducible genes were not clustered within the disease molecular signature of the present study. Moreover, because of the tight cross-regulation between TNF-α and IFN-I (49), anti-TNF therapy could lead to further enhancement of IFN pathways, already pathologically activated in pSS. Conversely, the data presented here provide a strong rationale for the development of new therapies to block IFN pathways or to inhibit the consequences of IFN production, such as BAFF secretion, in patients with pSS.

In conclusion, our results support the pathogenic interaction between the innate and adaptive immune system in pSS, a prototypic systemic autoimmune disease. The persistence of the IFN signature might be related to a vicious circle in which the environment interacts with genetic factors (HLA-DR-associated production of autoantibodies and subsequent formation of immune complexes with self-RNA) to drive the stimulation of salivary TLRs.

Materials and Methods

Patients.

Ten patients with pSS according to European-American consensus group criteria (50) and 10 control subjects participated in the study after their referral to two reference centers (the departments of rheumatology at the Bicêtre and Strasbourg Hospitals, France). Mean disease duration was 8 years. No features of other autoimmune diseases, including SLE or rheumatoid arthritis was observed in patients. Clinical and immunological features of patients are summarized in Table 2. The control subjects had subjective symptoms of oral or ocular dryness but met none of the objective criteria of SS (no autoantibodies and no lymphocytic infiltrates on minor salivary gland biopsy). Only women were included in both groups. The mean age of patients was 57 ± 17 years (range 40–80). The mean age of the controls was 56 ± 10 years (range 37–70). No subject had ever taken immunosuppressant medication. Informed consent was obtained from all study subjects, and the study was approved by the institutional review board of Bicêtre and Strasbourg Hospitals.

Table 2.

Clinical and biological features of patients with pSS

Patients Age, years Disease duration, years Chisholm and Mason’s score Anti-SSA antibody Anti-SSB antibody RF Anti-dsDNA antibody
    1 40 4 3 + + + −
    2 78 24 4 − − − −
    3 80 1 3 + − + −
    4 42 5 2 + + + −
    5 52 5 3 + − − −
    6 46 20 4 − − − −
    7 76 1 4 + + + −
    8 43 4 4 + − + −
    9 55 3 3 − − − −
10 60 2 3 + + + −
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RNA from minor salivary gland samples of patients 5, 9, and 10 did not reach a 28S/18S ribosomial RNA ratio between 1.5 and 1.7 and were used only for the quantitative RT-PCR analysis. RF, rheumatoid factor.

RNA Extraction and Amplification, Synthesis of Fluorescent cDNA from mRNA.

Minor labial salivary glands were dissected from the lower lip of subjects. Samples used for the microarray study were immediately placed in RNAlater (Qiagen, Courtaboeuf, France) for RNA extraction. Samples used for diagnostic focus scoring were fixed in formalin.

Tissue samples were homogenized with a Mixer Mill MM 300 (Qiagen), and total RNA was extracted with an RNEasy MicroKit (Qiagen) according to the manufacturer’s recommendations. The quality of RNA was measured with use of a 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA): 14 of the 20 samples (seven patients and seven controls) reached a 28S/18S ribosomal RNA ratio of 1.7 or greater and were kept for the following steps of the microarray procedure.

Histologically normal parotid tissue was obtained from four subjects without features of autoimmunity. These tissues were homogenized and pooled to constitute the reference RNA.

One microgram of RNA from the samples and the reference sample was amplified with use of a linear amplification kit (MessageAmp aRNA; Ambion, Huntingdon, U.K.) according to the manufacturer’s instructions. The quality and yield of cRNA was analyzed with use of an Agilent Technologies 2100 Bioanalyzer. A total of 250 ng of cRNA from each sample was used for cDNA synthesis. Fluorescent cDNA was synthesized by reverse transcription and indirect labeling and purified from the amplified cRNA with the use of the Fairplay Microarray Labeling kit (Stratagene). Ten microliters of cDNA was separated in two volumes of 5 μl, one labeled with Cy3 and the other with Cy5 (Amersham Pharmacia) and concentrated by drying under a vacuum in a rotary dessicator in a volume of 4 μl each.

Hybridization on cDNA Microarrays and Image Acquisition.

Gene expression analysis involved the use of cDNA microarrays (Commissariat à l’Energie Atomique, Paris) containing 10,752 human probes, including 1,560 controls, as reported in ref. 51. Twenty-eight microarrays were hybridized according to the manufacturer’s recommendations (Genopole, Evry, France) and as reported in Supporting Materials and Methods, which is published as supporting information on the PNAS web site. A dye-swap design was used for Cy3 and Cy5 labeling. For details on image acquisition, see Supporting Materials and Methods.

Statistical Analysis of Microarrays.

Spots flagged by Standard global locally weighed scatter plots with smoothing (lowess) and normalization (A_versus_M plot) were then analyzed by using genespring software (Agilent Technologies). Genes with >30% missing values in the patient or control groups were then removed. Analyses involved two sets of data comparing first the probes present in every slide (no missing values) and then the probes for which <30% of values were missing (and were estimated by the LSimpute adaptive method) (52). Statistical analysis involved use of bootstrap resampling with Benjamini and Hochberg’s correction.

Unsupervised clustering by sample and gene dimensions by hierarchical clustering was based on the nonparametric Spearman correlation distance and complete linkage-group aggregation technique. Results were validated by comparing the results of genespring (Agilent Technologies), cluster/treeview (53) and hclust of r. All of the microarray procedure was MIAME (Minimum Information About a Microarray Experiment) compliant. The microarray data were deposited in the ArrayExpress database (accession nos. E-MEXP-419 and A-MEXP-248).

Gene Ontology Analysis and Analysis of Pathways.

Gene ontology analysis involved use of fatigo web-based software (54). The pathwayassist software (Stratagene) was used to investigate the functional pathways represented by differentially expressed genes.

Quantitative RT-PCR.

The cDNA was amplified by quantitative real-time PCR by using the LightCycler system (Roche Diagnostics) according to the manufacturer’s instructions. For details on the conditions of PCRs and on sequences of PCR primers, see Supporting Materials and Methods.

Salivary Gland Epithelial Cell Culture.

After minor salivary gland biopsy, salivary gland epithelial cells were cultured in vitro as described in ref. 55. After 4 weeks of primary culture, IFITM-1 and BAFF gene expression in epithelial cells was assessed at baseline and after 48 h of stimulation with IFN-α or IFN-γ by real-time RT-PCR.

Conjunctival Impression Cytology of the Ocular Surface.

Imprints of the bulbar and palpebral conjunctiva were obtained by use of polyether sulfone filter (Supor Gelman Sciences, Ann Arbor, MI) in patients with pSS and controls. RNA was extracted with an RNEasy MicroKit (Qiagen). BAFF and IFITM-1 gene expression was analyzed by quantitative RT-PCR.

Immunohistochemistry.

Samples of salivary glands from patients with pSS and controls were immediately frozen and maintained at −80°C until use. Briefly, endogenous peroxydase activity was blocked with 3% hydrogen peroxide. Sections were incubated with mouse monoclonal antibodies against CD123 (IgG2a, clone 7G3; Becton Dickinson), BDCA2 (IgG1, clone AC144; Miltenyi Biotec, Paris), and TLR9 (IgG1, clone 26C93.2; Imgenex, San Diego). Sections were incubated with peroxydase-labeled polymer conjugated to streptavidin (EnVision+; DakoCytomation, Carpinteria, CA). To visualize the bound antibodies, slides were incubated in diaminiobenzidene substrate chromogen (DAKO). Tissue sections were counterstained with hematoxylin. In negative control sections, the primary antibodies were omitted, or an irrelevant antibody was applied at the same concentration as the primary antibody.

Supplementary Material

Supporting Information
pnas_0510837103_index.html (341.2KB, html)

Acknowledgments

We thank Professor Jean-Claude Brouet (Saint-Louis Hospital, Paris) for precious advice and Xavier Gidrol (Commissariat à l’Energie Atomique, Evry, France) for technical support.

Abbreviations

SS

Sjögren’s syndrome

pSS

primary SS

DC

dendritic cell

pDC

plasmacytoid DC

TLR

Toll-like receptor

SLE

systemic lupus erythematosis.

Footnotes

Conflict of interest statement: No conflicts declared.

Data deposition: The microarray data reported in this paper have been deposited in the ArrayExpress database (accession nos. E-MEXP-419 and A-MEXP-248).

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