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. 2014 Jul 24;177(3):618–629. doi: 10.1111/cei.12377

Dysregulation of the suppressor of cytokine signalling 3–signal transducer and activator of transcription-3 pathway in the aetiopathogenesis of Sjögren's syndrome

S R Vartoukian *, W M Tilakaratne †, N Seoudi *, M Bombardieri ‡, L Bergmeier *, A R Tappuni *,1, F Fortune *,1
PMCID: PMC4137846  PMID: 24827536

Abstract

The suppressor of cytokine signalling 3 (SOCS3) negatively regulates the Janus kinase (JAK)/signal transducer and activator of transcription-3 (STAT-3)/interleukin (IL)-17 pathway. The proinflammatory cytokine IL-17 is over-expressed in Sjögren's syndrome (SS) and is a key factor in its pathogenesis. We hypothesized that IL-17 over-expression in SS results from ineffective regulation by SOCS3. The expression of SOCS3 was analysed in peripheral blood mononuclear cells (PBMC) from SS cases, sicca controls (SC) and healthy controls (HC) and tissue samples from SS, SC and healthy salivary glands (HSG). PBMC and salivary gland tissue from SS and controls were dual-immunostained for SOCS3 and IL-17. IL-6-stimulated PBMC from SS and controls were evaluated for time-dependent STAT-3 activation and SOCS3 induction, and for IL-17 expression. Immunoblotting revealed greater levels of SOCS3 in PBMC from SS than SC (P = 0·017) or HC (P < 0·001). Similarly, the proportion of salivary-gland tissue cells staining for SOCS3 was significantly higher in SS than SC (P = 0·029) or HSG (P = 0·021). The cells in PBMC/salivary gland samples from controls predominantly expressed either SOCS3 or IL-17. However, there was a high frequency of SOCS3/IL-17 co-expression within cells of SS samples. IL-6-stimulation of PBMC from SS cases revealed prolonged activation of STAT-3 with reduced negative regulation by SOCS3, and enhanced expression of IL-17. This study showed that SOCS3 expression is up-regulated in SS. However, the absence in SS of the normal inverse relationship between SOCS3 and pSTAT-3/IL-17 indicates a functional disturbance in this signalling cascade. Consequently, a reduction in function, rather than a reduction in expression of SOCS3 accounts for the unregulated expression of IL-17 in SS, and may play a crucial role in aetiopathogenesis.

Keywords: IL-17, pathogenesis, Sjögren's syndrome, SOCS-3, STAT-3

Introduction

Sjögren's syndrome (SS) is a chronic autoimmune disorder which targets multiple exocrine glands and typically presents with sicca (dryness) of the oral, genital and ocular mucosa. Affected individuals may also exhibit clinical features associated with involvement of musculoskeletal, pulmonary, gastric, renal and nervous systems. Subjects manifesting signs and/or symptoms of dryness without systemic involvement, and not fulfilling the revised criteria of the American–European Consensus Group for the classification of SS [1], are by exclusion, usually classified under the term ‘sicca syndrome’.

In the aetiopathogenesis of SS, the proinflammatory cytokine interleukin (IL)-17 is the focus of interest as a potent inducer of inflammation and tissue damage [2–6]. IL-17 is over-expressed in salivary gland tissue [7–9], plasma [7] and saliva [10] from subjects with SS compared with controls. Furthermore, evidence from animal studies has demonstrated the induction of a SS-like phenotype by IL-17 [11,12] and the prevention of SS-like changes by reduction in IL-17 [12,13], suggesting a role for IL-17 in SS aetiology.

Suppressors of cytokine signalling (SOCS) are cytokine-inducible intracellular proteins which act as negative regulators of the Janus kinase/signal transducers and activators of transcription (JAK/STAT) signal transduction pathway, controlling the longevity and magnitude of cytokine signals and inhibiting the autocrine/paracrine effects of cytokines [14,15]. The binding of specific cytokines to their cognate receptors initiates a signalling cascade involving the phosphorylation of JAKs and the subsequent phosphorylation, dimerization and translocation to the nucleus of STATs, which induce the transcriptional up-regulation of target genes such as SOCS [16]. In turn, the induced SOCS proteins block cytokine signal transduction through the inhibition of STATs, JAKs or cytokine receptors, effectively forming a classic negative feedback loop [16].

The binding of the IL-6-family cytokines to the gp130 cell receptor triggers STAT-3 activation through phosphorylation of tyrosine 705 via JAK. pSTAT-3 induces the expression of SOCS3 [17,18], one of eight SOCS family proteins and a non-redundant feedback inhibitor of specific cytokines such as IL-6 [19]. Following induction by these cytokines via phosphorylated STAT-3 (pSTAT-3), SOCS3 binds to and inhibits the catalytic activity of JAKs via a non-competitive mechanism [20]. It contributes to the targeting of both JAK and cytokine receptor for proteosomal degradation [19]. It is also thought to have a direct physical interaction with STAT-3, which may be important in determining inhibitory efficacy [21]. In effect, SOCS3 inhibits downstream activation of STAT-3, regulating both quantity and type of STAT-3 signalling in a classical negative-feedback loop [14,22]. In so doing it attenuates the production of IL-17 from T helper type 17 (Th17) cells [17,19], a process that is dependent upon STAT-3 activation [23,24]. Several studies have demonstrated that SOCS3 expression correlates inversely with that of pSTAT-3 [25–27] and that of IL-17 [25,28–30].

In view of the association of SS with over-expression of IL-17, a proinflammatory cytokine that is normally negatively regulated by SOCS3, we hypothesized that SOCS3 plays a part in the ineffective regulation of IL-17 and thus contributes to the aetiopathogenesis of SS. In the absence of any previous investigation of SOCS3 expression in SS, the aim of this work was to investigate the local and systemic expression of SOCS3 (relative to pSTAT-3 and IL-17) in SS and controls, including an in -vitro exploration of the STAT-3/SOCS3 signal transduction pathway.

Materials and methods

Subjects and samples

Ethical approval was granted by St Thomas' Hospital Research Ethics Committee and written informed consent was obtained from study participants. Volunteers were recruited into three main cohorts, matched for age and gender: (i) primary Sjögren's syndrome (SS), subjects classified according to the revised criteria of the American–European Consensus Group [1] [presence of a minimum of four of the following six criteria: signs and symptoms of oral and ocular sicca, histopathological features of SS and positive serology (anti-Ro and/or anti-La autoantibodies), including one or both of the latter two criteria]; (ii) sicca controls (SC), equivalent to sicca syndrome, subjects with sicca signs/symptoms, not fulfilling the American–European criteria for SS [1], systemically well and unaffected by any known autoimmune disease; and (iii) healthy controls (HC), subjects without sicca, unaffected by autoimmune disease.

Peripheral blood was collected from 18 SS cases, 16 SC and 16 HC. Formalin-fixed paraffin-embedded minor salivary gland tissue was obtained from nine SS cases and eight SC controls. An additional cohort of four subjects who underwent radical neck dissection for suspected tumour metastases provided paraffin-embedded histologically healthy submandibular gland tissue (HSG).

Primary antibodies

Mouse anti-human SOCS3 (IgG2B clone 516919; R&D Systems, Minneapolis, MN, USA), mouse anti-human STAT-3 (IgG2a clone 124H6; Cell Signaling, Danvers, MA, USA), rabbit anti-human phospho-STAT-3 (Tyr705) [immunoglobulin (Ig)G clone EP2147Y; Millipore, Billerica, CA, USA] and rabbit anti-human IL-17 (H-132/sc-7927; Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies were validated by immunoblotting using control cell lysates. Resultant bands were of expected molecular weight: ∼30 kDa for SOCS3; ∼86 kDa and ∼79 kDa for the α and β isoforms of total STAT-3 and pSTAT-3; and ∼15 kDa or ∼30 kDa (homodimer) for IL-17.

Immunoblotting

Peripheral blood mononuclear cells (PBMC) were isolated from each blood sample by density-gradient centrifugation using Ficoll-Paque Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden). Cells were washed in phosphate-buffered saline (PBS) and suspended at a final concentration of 25 000 cells/μl in ice-cold lysis buffer containing 1% NP40, 1 mM dithiothreitol (DTT), 1 mM ethylenediamine tetraacetic acid (EDTA), 1 mM ethylene glycol tetraacetic acid (EGTA), 20 mM HEPES (pH 7·9), 420 mM NaCl, 1 mM NaV, 50 mM NaF, 1 mM phenylmethanesulphonyl fluoride (PMSF), 1 μg/ml aprotinin and 20% glycerol. After 30 min, lysates were sonicated for five pulses of 5 s and clarified at 12 000 g for 20 min (4°C). Duplicate 15 μg samples of protein extract from each subject were separated by sodium dodecyl phosphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to methanol-primed polyvinylidene difluoride (PVDF) membranes and examined for SOCS3, pSTAT-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression by indirect immunoblotting followed by enzyme chemiluminescence (ECL Prime Western Blotting Detection reagent; GE Healthcare) for signal detection. After stripping in 0·1 M glycine buffer (pH 2) for 5 min and retesting by enzyme chemiluminescence to confirm the absence of any detectable signal, membranes were reprobed for total STAT-3. Bands representing SOCS3 detection were semi-quantified by densitometry [ImageJ, National Institutes of Health (NIH), Bethesda, MD, USA] relative to GAPDH; those representing pSTAT-3 were normalized first relative to GAPDH, then relative to both GAPDH and total STAT-3. Use of an internal control on each gel allowed for normalization between gels.

Immunohistochemistry

Three-μm sections of salivary gland tissue were subject to indirect immunohistochemistry for SOCS3 and IL-17: antigen retrieval was performed by boiling in Tris-EDTA buffer (10 mM Trizma base, 1 mM EDTA, 0·05% Tween 20, pH 9) for 20 min, non-specific binding of antibody was blocked by incubation in buffer containing 10% normal goat serum and 1% bovine serum albumin (BSA) and endogenous peroxidase activity was quenched by incubating in 3% hydrogen peroxide (15 min). Ultimately, EnVision™+ Dual Link System-HRP (Dako, Glostrup, Denmark) was applied to the tissue and peroxidase activity was visualized under bright-field microscopy using a diaminobenzidine (DAB) chromogen against a Mayer's haematoxylin counterstain. Mean counts of SOCS3-expressing cells and mean total cell counts were determined by examining four photomicrographs from each of four subjects/cohort. The photomicrographs derived from randomly selected, ×400 magnification views under a Nikon Inverted Microscope ECLIPSE TE2000-S.

Dual-staining for SOCS3/IL-17 was performed on salivary gland tissue samples by indirect immunofluorescence. Goat anti-rabbit Alexa Fluor 488 (AF488) and goat anti-mouse Alexa Fluor 568 (AF568) fluorochrome-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, USA) targeted anti-IL-17 and anti-SOCS3 primary antibodies, respectively. Stained sections were visualized using a Zeiss LSM510 inverted confocal-laser-scanning system (Zeiss, Birmingham, UK) fitted with argon and helium–neon lasers. Checks were performed to ensure that the fluorescent emission was not due to ‘bleed through’ as a result of excitation by lower-wavelength emissions of AF488 fluorochrome. Frequency scatter-plots (generated in Image J, NIH) were used to analyse the degree of co-localization of SOCS3 and IL-17 staining in each sample.

The absence of non-specific binding by any of the antibodies was confirmed by subjecting additional tissue sections to immunohistochemistry as above, except that: (i) either primary, secondary or both antibodies were omitted; or (ii) primary antibody was substituted for either an isotype-matched control or a control serum (in lieu of monoclonal or polyclonal antibodies, respectively).

IL-6 stimulation of PBMC from SS, SC and HC: a time–course study evaluating STAT-3 activation and SOCS3 induction

Peripheral blood was collected from each of three SS cases, three SC and three HC, matched for age and gender. PBMC were isolated from the samples by density-gradient centrifugation using Ficoll-Paque Plus. Approximately 2 × 105 cells from each sample were applied to SuperFrost Plus microscope slides (VWR International, Radnor, PA, USA) using Shandon EZ Single Cytofunnels (Thermo Shandon, Runcorn, UK), fixed with ice-cold methanol for 10 min and dual-stained for SOCS3/IL-17 by indirect immunofluorescence (see method above). Following confirmation of > 97% cell viability, the remainder of each cell sample was suspended at a concentration of 2·5 × 106 cells/ml in complete medium: RPMI-1640 (Life Technologies, Glasgow, UK) containing 10% fetal calf serum (FCS), 2 mM L-glutamine, 50 U/ml penicillin and 50 U/ml streptomycin. Six-well plates were seeded with 10 × 106 cells/well and incubated at 37°C in 10% CO2. Baseline samples were harvested after 2 h of incubation. Wells were subsequently inoculated either with recombinant human IL-6 (Life Technologies) at a final concentration of 10 ng/ml, or with an equivalent amount of carrier medium (as ‘no-IL-6’ control). Serial samples were harvested from each well over 4 h from the point of ‘stimulation’. The samples were washed in cold PBS and cell lysates prepared as described in the ‘Immunoblotting’ section, except that cells were suspended in lysis buffer at a final concentration of 10 000 rather than 25 000 cells/μl. Fifteen μg of each protein extract was tested (in duplicate) by indirect immunoblotting using primary antibodies for SOCS3, pSTAT-3 and GAPDH, prior to reprobing for total STAT-3 (see above for methods). SOCS3 detection signal was semi-quantified by densitometry relative to GAPDH; the pSTAT-3 signal was normalized against total STAT-3 and GAPDH and all data were normalized between gels using an internal control.

IL-6 stimulation of PBMC from SS, SC and HC: a flow cytometry study evaluating IL-17 expression

Peripheral blood was collected from a total of 14 subjects (four SS cases, five SC and five HC). PBMC were isolated from the samples by density-gradient centrifugation over Ficoll-Hypaque. The isolated cells were cultured as described above and treated for 3·5 h with recombinant IL-6 at a final concentration of 20 ng/ml and at a cell density of 2 × 106 cells/treatment. Fifty ng/ml phorbol myristate acetate (PMA) and 1 μg/ml ionomycin acted as positive control. For the negative control, cells were subjected to carrier vehicle alone (acetic acid) at a concentration corresponding to that used in IL-6 treatment. A GolgiStop protein transport inhibitor (1 μl/ml monensin; BD Biosciences, San Jose, CA, USA) was added to the culture medium 1·5 h after the start of stimulation. Cells were washed with PBS and stained with extracellular stain [5 μl of allophycocyanin (APC) anti-human CD3/2 × 106 cells; BioLegend, San Diego, CA, USA], according to the manufacturer's instructions. Cell fixation and permeabilization was achieved by incubating the cells in the dark for 20 min with fixation/permeabilization solution (BioLegend). The permeabilized cells were then split into two and incubated either with 2 μl of phycoerythrin (PE) anti-human IL-17A/1 × 106 cells (BioLegend) or with the appropriate isotope control to exclude background staining (2 μl of PE mouse IgG1, K isotype control/1 × 106 cells) (BioLegend). In order to ensure accurate frequency measurements during flow cytometric acquisition, at least 200 000 cells were acquired. The frequency of IL-17-producing CD3+ cells was demonstrated as a percentage of the total number of CD3+ cells. The stimulation index for IL-6 treatment was calculated by dividing the frequency of IL-17 produced after treatment, by the frequency of IL-17 secretion for the equivalent negative control. The data were collected on a four-colour BD LSR II flow cytometer and analysed by high-performance BD fluorescence activated cell sorter (FACS)Diva software (BD Biosciences).

Statistical analysis

Statistical analysis was performed using spss version 18·0 for Windows with non-parametric tests (Mann–Whitney U-test, Kruskal–Wallis test and χ2 test with Yates's correction or Fisher's exact test, as indicated). Ordinal logistic regression (backward-model) was used to identify variables with a significant explanatory effect on the systemic expression of SOCS3. This test was applied initially to data from all three cohorts for the following variables: subject age, gender, cohort, unstimulated salivary flow rate and pSTAT-3 expression; the test was subsequently repeated with data from only SS and SC cohorts, and in that case also considered the prevalence of Ro/La autoantibodies as possible variables.

Results

Systemic expression of SOCS3 and pSTAT-3: PBMC

Table 1a compares the various clinical and laboratory parameters between the cohorts that were used in this study according to the recommendations of the American–European Consensus Group [1] to designate subjects to SS and, by exclusion, to SC categories. The SS and SC cohorts showed no significant difference in the prevalence of sicca symptoms, but there was a higher prevalence/severity in SS cases of sicca signs, histopathological and serological features of SS.

Table 1.

Demographic, clinical and laboratory data from cases and controls recruited for analysis of peripheral blood mononuclear cells (PBMC) (a) and salivary gland tissue (b)

(a) Subjects for PBMC analysis (b) Subjects for salivary gland tissue analysis
Characteristic SS (n = 18) SC (n = 16) HC (n = 16) Statistical signif. (P) SS (n = 9) SC (n = 8) HSG (n = 4) Statistical signif. (P)
Age: mean (s.d.) 53·69 (16·52) 56·38 (13·36) 46·63 (11·74) 0·107† (n.s.) 55·25 (13·84) 59·13 (16·13) 63·50 (13·63) 0·609† (n.s.)
Gender: male/female 1/17 0/16 3/13 0·132† (n.s.) 0/9 0/8 1/3 0·107† (n.s.)
Sicca symptoms
 Oral dryness 18/18 16/16 0 0·000† (s) 8/9 8/8 n.d. 1·000‡ (n.s.)
1·000‡ (n.s.)
 Ocular dryness 16/18 14/16 0 0·000† (s) 9/9 7/8 n.d. 0·471‡ (n.s.)
1·000‡ (n.s.)
 Time since onset (years): mean (s.d.) 11·13 (6·62) 9·80 (11·26) n.a. 0·206‡ (n.s.) 9·75 (8·02) 10·17 (6·24) n.d. 0·748‡ (n.s.)
Sicca signs
 Unstim. salivary flow rate (ml/min): mean (s.d.) 0·11 (0·22) 0·22 (0·19) 0·62 (0·34) 0·000† (s) 0·18 (0·26) 0·08 (0·08) n.d. 0·629‡ (n.s.)
0·011‡ (s)
 Positive Schirmer's test 17/18 4/16 n.d. 0·000‡ (s) 8/9 3/8 n.d. 0·050‡ (s)
Histopathology
 Labial gland biopsy
  Positive for SS 7/12 0 n.d. 0·017‡ (s) 9/9 0 n.d. 0·000‡ (s)
  Focus score: mean (s.d.) 2·44 (2·35) 2·00 (1·29)
  Negative for SS 5/12 7/7 0 8/8
Serology
 Autoantibodies
  ANA 16/18 8/16 n.d. 0·023‡ (s) 8/9 2/8 n.d. 0·015‡ (s)
  Ro (SSA) 14/18 0 n.d. 0·000‡ (s) 5/9 1/8 n.d. 0·131‡ (n.s.)
  La (SSB) 10/18 0 n.d. 0·000‡ (s) 4/9 1/8 n.d. 0·294‡ (n.s.)
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†

For difference between the three cohorts.

‡

For difference between SS and SC.

ANA = anti-nuclear antibodies; SSA = Sjögren's syndrome A; SSB = Sjögren's syndrome B; S = significant; SS = Sjögren's syndrome; SC = sicca controls; HC = healthy controls; HSG = healthy salivary gland; n.a. = not applicable; n.d. = no data; n.s. = not significant; s.d. = standard deviation.

Analysis of immunoblots revealed the level of SOCS3 expression in PBMC to be highest in SS, intermediate in SC and lowest in HC, with statistically significant differences between each pair of cohorts (Fig. 1a,b). Regression analyses applied to data from (i) all three cohorts and (ii) SS and SC only revealed that ‘cohort’ was the only explanatory variable for the level of SOCS3 expression in PBMC (P = 0·000 and P = 0·015, respectively).

Figure 1.

Figure 1

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Significantly greater expression of suppressor of cytokine signalling 3 (SOCS3) in Sjögren's syndrome (SS) cases than controls, both systemically in peripheral blood monuclear cells (PBMC) and locally in salivary gland tissue. (a–d) Relative expression of SOCS3, phosphorylated signal transducer and activator of transcription-3 (pSTAT-3) and STAT-3 was assessed in PBMC from SS (n = 18), sicca controls (SC) (n = 16) and healthy controls (HC) (n = 16) cohorts. (a) The immunoblot [with glyceraldehyde 3-phosphate dehydrogenase (GAPDH)] shows the results for three subjects from each cohort; (b,d) the bar charts represent mean normalized expression data derived from all samples (in duplicate) after densitometry analysis of immunoblots; (c) the bar chart shows the proportion of all subjects in each cohort with greater, equal or lesser relative expression of pSTAT-3 α than β. (e,f) SOCS3 expression in salivary gland tissue from SS (n = 9), SC (n = 8) and healthy salivary glands (HSG) (n = 4) was determined by immunohistochemistry. (e) The specimen photomicrographs show SOCS3 immunostaining [diaminobenzidene (DAB), brown] counterstained with haematoxylin (blue) (original magnification ×200); (f) the bar chart of mean counts is based on analysis of four ×400 magnification views for each of four subjects per cohort. The P-values in (b,d,f) were derived from statistical analysis with Mann–Whitney U- and Kruskal–Wallis tests (for two- and three-group comparisons, respectively), and the P-value in (c) with the χ2 test with Yates's correction.

The majority of subjects in each cohort showed greater expression of pSTAT-3α (86 kDa) than pSTAT-3β (79 kDa), with no statistically significant difference between the cohorts in the pattern of relative pSTAT-3α/β expression (P = 0·935) (Fig. 1c). Expression of pSTAT-3 was addressed as a combination of α and β isoforms. After normalization relative to GAPDH, pSTAT-3 expression in PBMC was proportionally greater in SS than in either SC or HC; however, the difference was statistically significant only between SS and HC (P = 0·004) (Fig. 1d). After taking into account a higher relative expression of total STAT-3 in SS than in controls, there was no significant difference in pSTAT-3 expression between any of the cohorts (Fig. 1d).

Local expression of SOCS3 and IL-17: salivary gland tissue

Table 1b represents data from subjects recruited into the SS, SC and HSG cohorts for analysis of salivary gland tissue. While labial gland tissue from SS cases showed histopathological features of SS (focal lymphocytic sialadenitis with one or more lymphocytic foci per 4 mm2 of glandular tissue), that of SC controls showed only non-specific sialadenitis.

SOCS3 was found to be variably expressed in salivary gland tissue: at acinar cells, ductal epithelial cells and foci of immune/inflammatory cells (Fig. 1e). The mean proportion of salivary gland cells staining for SOCS3 was significantly higher in SS [99·0%, standard deviation (s.d.) 0·9] than in either SC (52·3%, s.d. 35·2) or HSG (16·3%, s.d. 21·6) samples (P = 0·029 and P = 0·021, respectively). In contrast, the difference between the two control groups (SC and HSG) was statistically non-significant (P = 0·083) (Fig. 1f).

IL-17 was detected in cells of the immune/inflammatory infiltrate, ductal epithelium and acini in salivary gland tissue from subjects with SS, but was limited mainly to ductal epithelial cells in SC and HSG samples (as reported by Sakai et al. [31]). Of note, there were isolated large cells within the immune/inflammatory infiltrate found in SS and SC samples that showed particularly intense staining for IL-17. Further investigation by means of dual immunofluorescence using antibodies for IL-17 and CD68 (clone KP1) identified these prominent producers of IL-17 in inflamed minor salivary gland tissue as macrophages (data not shown).

Co-expression of SOCS3 and IL-17 in salivary glands

Salivary gland tissue from a subgroup of subjects (three from each of SS, SC and HSG cohorts) was examined for dual immunofluorescence with SOCS3 and IL-17 antibodies. Tissue samples from subjects within each cohort exhibited similar staining patterns and a representative of each cohort is shown in Fig. 2. The pattern of SOCS3/IL-17 staining differed between SS and control cohorts. In the salivary gland control tissues (SC and HSG), SOCS3 and IL-17 were expressed predominantly at discrete sites, with little evidence of co-local expression in the representative merged images or frequency scatter-plots: IL-17 was found primarily at epithelial cells lining large ducts and at macrophages, while SOCS3 stained most strongly at acini and lymphocytic infiltrates, where present. By contrast, there was a high frequency of SOCS3/IL-17 co-expression in salivary gland tissue from SS subjects, as shown in representative scatter-plots and confirmed by the presence of an almost universal yellow colour in merged images.

Figure 2.

Figure 2

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Contrasting pattern of suppressor of cytokine signalling 3 (SOCS3)/interleukin (IL)-17 expression in salivary gland tissue from Sjögren's syndrome (SS) cases and sicca controls (SC)/healthy salivary glands (HSG) controls: a high degree of SOCS3/IL-17 co-expression in SS, versus a predominantly discrete expression of SOCS3 and IL-17 in SC/HSG controls. Biopsy samples from three subjects per cohort (SS, SC and HSG) were stained with haematoxylin and eosin (H&E), and dual-immunostained for IL-17 (AlexaFluor488, green) and SOCS3 (AlexaFluor568, red). A single representative is shown for each cohort. The inset images are frequency scatter-plots (x-axis: red channel, y-axis: green channel).

In these SS tissues, IL-17 staining appeared relatively strong in the ductal epithelial cells, the cell infiltrates and the acini; all these sites also showed strong expression of SOCS3.

Co-expression of SOCS3 and IL-17 in PBMC

PBMC isolated from three SS cases, three SC and three HC were subject to both dual immunofluorescence for SOCS3/IL-17 and in-vitro cultivation for IL-6 stimulation (see below). Although, within each cohort, samples gave similar results, the pattern of staining differed between SS cases and SC/HC controls (Fig. 3a). Moreover, the results paralleled those reported earlier for dual-stained salivary gland tissue. Namely, cells within ‘control’ PBMC samples predominantly expressed either SOCS3 (shown in red) or IL-17 (green), resulting in the polarization of scatter in frequency scatter-plots, into two distinct regions abutting x- and y-axes. By contrast, SOCS3 and IL-17 were frequently co-expressed within cells of PBMC samples from SS, as evidenced in scatter-plots by a high frequency of combined red and green signals.

Figure 3.

Figure 3

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Co-expression of suppressor of cytokine signalling 3 (SOCS3) (red) and interleukin (IL)-17 (green) in peripheral blood mononuclear cells (PBMC) isolated from Sjögren's syndrome (SS) cases, sicca controls (SC) and healthy controls (HC) (a), and time-dependent activation of signal transducer and activator of transcription-3 (STAT-3) and induction of SOCS3 in cultured PBMC (from the same subjects) stimulated with 10 ng/ml IL-6 for up to 4 h (b–e). (a) SOCS3 and IL-17 are discretely expressed within PBMC from controls (SC and HC) but frequently co-expressed within cells from SS cases. Each of the three PBMC samples tested within each cohort gave similar results. Frequency scatter-plots are inset (x-axis: red channel, y-axis: green channel). (b–e) SOCS3 induction occurs early in SS; however, phosphorylated STAT-3 (pSTAT-3) detection is prolonged, there being no rapid decline in STAT-3 phosphorylation in concurrence with SOCS3 induction. (b,c) Baseline (BL) samples were collected after a 2-h pre-incubation phase and prior to IL-6 stimulation. Cell lysates were analysed by immunoblotting for SOCS3, pSTAT-3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), prior to reprobing for STAT-3. (b) The immunoblots shown are representative of one subject from each of the SS, SC and HC cohorts. (c) The bar charts represent mean normalized expression data after densitometry analysis of duplicate immunoblots and derive from three independent experiments for each cohort; pSTAT-3 data were normalized against total STAT-3 and GAPDH, SOCS3 was normalized against GAPDH, and all data were normalised between gels using an internal control. ‘SOCS3 induction time’ (defined as the time interval between maximal activation of STAT-3 upon IL-6 stimulation and maximal expression of SOCS3) is significantly shorter in stimulated PBMC from SS cases than from SC or HC. (e) ‘pSTAT-3 persistence time’ (defined as the time difference between maximal expression of SOCS3 after IL-6 stimulation and final detection of activated STAT-3 over the 4-h time–course) is significantly longer in SS cases than in SC or HC. (d,e) Analyses were based on normalized expression data from immunoblots; statistical significance testing for differences between the cohorts was performed using Mann–Whitney U-tests.

Time-dependent activation of STAT-3 and induction of SOCS3 in IL-6-stimulated PBMC from SS, SC and HC

‘No-IL-6’ control experiments showed minimal time-dependent fluctuation in the expression of pSTAT-3 and SOCS3 (data not shown), and protein expression was comparable to baseline in equivalent experiments using IL-6 stimulation.

In HC experiments, stimulation of PBMC with IL-6 activated STAT-3 and induced SOCS3 in the expected time-dependent fashion (Fig. 3b,c): in general, pSTAT-3 expression peaked within 30 min of exposure to IL-6. After a lag phase, SOCS3 protein expression increased, reaching a maximum approximately 1·5 h after stimulation. As indicated by mean data, this was followed by a rapid decline in STAT-3 phosphorylation, consistent with the regulatory role of SOCS3 on STAT-3 activation. pSTAT-3 detection dropped to baseline levels within 2 h of stimulation.

A similar trend was observed in SC experiments (Fig. 3b,c) with a lag between peak expression of pSTAT-3 and that of SOCS3, and a drop in pSTAT-3 detection to levels approaching those of baseline within, on average, 2 h of IL-6 stimulation and 30 min of peak SOCS3 expression.

The results derived from equivalent experiments on PBMC from SS cases (Fig. 3c) differed from those of control experiments in two main respects, as follows.

First, whereas STAT-3 activation in samples from SS followed the same time–course as that observed in HC (pSTAT-3 showing mean peak expression 30 min after IL-6 stimulation), SOCS3 induction was observed much earlier, both in relation to IL-6 exposure and to STAT-3 activation. Maximal SOCS3 expression occurred within 30 min (rather than within 1·5 h) of IL-6 stimulation, there being no lag phase between peak expression of pSTAT-3 and that of SOCS3. Moreover, SOCS3 induction was found to precede STAT-3 activation in one of the three SS cases tested. These results are summarized in Fig. 3d, which shows that mean ‘SOCS3 induction time’ (defined in the context of this work as the time interval between maximal activation of STAT-3 upon IL-6 stimulation and maximal expression of SOCS3) was statistically significantly shorter in SS cases than in SC (P = 0·046) or HC (P = 0·043).

Secondly, although there was a decline in the mean amount of pSTAT-3 expressed in SS samples over the course of the experiment, this decline was more gradual than that observed in HC samples, and strong induction of SOCS3 did not lead to a concurrent drop in STAT-3 phosphorylation to baseline levels (as was the case in experiments on SC and HC samples). Consequently, STAT-3 was activated for an extended time and still detectable above baseline levels at the conclusion of the experiment, 4 h after IL-6 stimulation. As shown in Fig. 3e, mean ‘pSTAT-3 persistence time’ (defined as the time difference between maximal expression of SOCS3 after IL-6 stimulation and final detection of activated STAT-3 over a 4-h time–course) was significantly longer in SS cases than in SC (P = 0·046) or HC (P = 0·041).

In summary, STAT-3 activation followed a normal time–course in SS samples; however, SOCS3 induction occurred early. Furthermore, SOCS3 had a reduced negative-regulatory effect on STAT-3 activation, resulting in prolonged detection of pSTAT-3 in samples from disease.

IL-17 expression in IL-6-stimulated PBMC from SS, SC and HC

Flow cytometric analysis evaluated IL-17 expression in IL-6-stimulated PBMC isolated from blood samples of four SS, five SC and five HC subjects. Following IL-6 stimulation there was (on average) a higher proportional increase in IL-17-expressing CD3+ cells within SS-derived samples, than in samples representing either of the control cohorts SC and HC (5·5 ± 3·5, 4·0 ± 2·8 and 3·2 ± 1·9, respectively, Fig. 4). There was no significant statistical difference between the three cohorts (P = 0·496) or between SS and HC (P = 0·387); however, the sample was small. Furthermore, the results may be influenced by the SS and SC patients but not HC using non-steroidal anti-inflammatory or hydroxychloroquine medication, both of which have been shown to reduce IL-17 production [32,33].

Figure 4.

Figure 4

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Higher stimulation index in Sjögren's syndrome (SS) cases than in controls for interleukin (IL)-17-expression in CD3+ cells. The frequency of IL-17-producing CD3+ cells was examined by flow cytometry after treatment of cultured peripheral blood monuclear cells (PBMC) for 3·5 h with 20 ng/ml IL-6 or with an equivalent concentration of carrier vehicle (negative control). The box-plots summarize the data derived from a minimum of four independent experiments for each cohort and represent the fold change in IL-17-expressing CD3+ cells upon stimulation. Statistical significance testing for the difference between the three cohorts was performed using the Kruskal–Wallis test.

Discussion

SOCS proteins are potent endogenous regulators of cytokine expression and are essential for stringent regulation of the immune system. SS is characterized by a disproportionately high expression of the proinflammatory cytokine IL-17 [7–10], which is a key player in disease aetiopathogenesis [2,11–13]. On the hypothesis that IL-17 over-expression in SS results from ineffective regulation by SOCS3, this study investigated SOCS3 expression in salivary gland tissue and peripheral blood from patients with SS (cases) and both SC and healthy people (controls).

In spite of some biological variation in the results, there was a statistically significant difference between the case and control cohorts in the expression of SOCS3, both local and systemic. Regression analysis confirmed the significance of ‘cohort’ (SS, SC or HC) as an explanatory variable for SOCS3 expression in PBMC.

The results revealed that the local and systemic expression of SOCS3 is up-regulated significantly in SS compared with both sicca (SC) and healthy (HC and HSG) controls. In line with the current work, human case–control studies investigating other autoimmune diseases (namely systemic lupus erythematosis, rheumatoid arthritis, Crohn's disease, autoimmune uveitis and psoriasis) have also demonstrated significant up-regulation of SOCS3 expression in disease [34–38].

There is some controversy surrounding the role of SOCS3 in the regulation of IL-17 production. While evidence indicates that SOCS3 is a negative regulator of STAT-3 activation [14,25,26], resulting in the reduction of its downstream target IL-17 [19,28–30,39–42], an isolated research paper [43] investigating human T cells has reported that SOCS3 expression promotes IL-17 production. It may be of relevance to note that the authors, being unable to down-regulate SOCS3 using short hairpin RNA, ultimately suppressed the gene with recombinant IL-7. The authors comment that exclusive inhibition of SOCS3 would be important, and the results need verification. In comparison, a recent study [41] also investigated human T cells. They silenced the SOCS3 gene with small interfering RNA and demonstrated suppression of IL-17 production mediated by SOCS3. On the basis that the majority of evidence points to the negative regulatory role of SOCS3 on IL-17 production, we had expected that SOCS3 would be down-regulated in SS, a disease associated with over-expression of proinflammatory IL-17. Our results, however, demonstrated that SOCS3 was up-regulated and we infer that SOCS3 may be dysfunctional in SS, unable to negatively regulate pSTAT-3 and unable to attenuate the IL-17 expression. This would help to explain the paradox that SOCS3 is up-regulated in SS and other autoimmune diseases, but appears to provide no protection in disease in humans (through modulation of inflammation), while forced expression of SOCS3 in animals offers protection against experimental arthritis [30,44].

Preliminary evidence for dysregulation of the SOCS3 inhibitory pathway in SS was provided by exploring the in-vivo inter-relationship between SOCS3 and IL-17 in salivary gland tissue and PBMC, and finding that it is atypical in SS. Cells within salivary gland tissue and PBMC from controls (SC, HSG, HC) showed in the main a discrete expression of SOCS3 and IL-17 (in accordance with the expected indirect inhibition of IL-17 by SOCS3), whereas analysis of corresponding cells/tissue from SS cases showed frequent co-expression of SOCS3 and IL-17. In the salivary gland and PBMC samples the observed staining patterns (as evaluated by frequency scatter-plots) were consistent for each cohort.

In-vitro examination of time-dependent changes in STAT-3 activation and SOCS3 induction in IL-6-stimulated PBMC confirmed the presence of a disturbance in SS in the SOCS3-STAT-3 negative-feedback loop. In PBMC from controls pSTAT-3 expression was short-lived, with levels declining rapidly in concurrence with SOCS3 induction, whereas in samples from SS cases detection of pSTAT-3 was significantly prolonged, with a slow decline in STAT-3 phosphorylation following SOCS3 induction. In conjunction with the observation that IL-6-induced activation of STAT-3 followed a similar time–course in SS as in HC samples, this suggests that there is normal activation of STAT-3 in SS with the subsequent failure of regulation of STAT-3 phosphorylation by SOCS3. This parallels the proposal by Ramos et al. [45] that the constitutive activation of STAT-3 in SS may be due to an abnormal inactivation of pSTAT-3 rather than an enhanced stimulation by upstream activators.

Although the kinetic profile for peak activation of STAT-3 was normal in SS, SOCS3 induction occurred early in relation to both IL-6 stimulation and STAT-3 activation. The time lapse between peak expression of pSTAT-3 and that of SOCS3 was negligible in SS. In one case the SOCS3 induction preceded STAT-3 activation. This implies that IL-6-mediated SOCS3 induction may occur independently of STAT-3 in disease. The observation that the normalized expression of pSTAT-3 in native PBMC was not significantly different in SS than in controls provides further evidence that SOCS3 over-expression in disease is not directly related to STAT-3. The detection of high levels of SOCS3 in SS may be due to transcriptional up-regulation through epigenetic activation of a SOCS3 promoter reporter construct, enhanced stability of mRNA or reduced protein degradation. The underlying molecular mechanisms require exploration.

SOCS3 had a less-than-normal attenuating effect on STAT-3 phosphorylation in SS samples, resulting in pSTAT-3 being detected for an abnormally long period. One would predict that as IL-17 is a downstream target of pSTAT-3, the production of IL-17 would be elevated even in the presence of SOCS3. Indeed, immunostaining revealed frequent co-expression of IL-17 and SOCS3 within individual cells from SS samples, a finding rarely seen in healthy PBMC or those from SC subjects. Furthermore, analysis of IL-17 expression in PBMC stimulated with IL-6 (in parallel with the experiment which evaluated STAT-3 activation and SOCS3 induction), suggested a higher stimulation index and increased IL-17 expression among CD3+ cells within SS-derived samples than in those from control subjects. Taken together, these findings reinforce the assumption that there is a functional disturbance in the SOCS3/STAT-3 negative-feedback loop in SS, which would explain the presence of high levels of IL-17 in disease, despite increased expression of SOCS3.

Recent evidence demonstrates that inhibitory efficacy of SOCS3 is dependent upon there being a close physical interaction between SOCS3 and both the JAK/gp130 receptor complex [20,46] and STAT-3 [21]. It suggests that a mutation in the binding/active sites of proteins involved in this signalling cascade [e.g. SOCS3 (kinase inhibitory region/KIR or Src homology 2/SH2 domains), JAK (GQM motif), STAT-3 or gp130 receptor] may be responsible for reduced regulatory efficacy of SOCS3 in SS. Forced expression of SOCS3 in animals has been shown to offer protection by preventing not only the induction, but also the progression, of experimental arthritis [30,44]. On the basis that SOCS3 is a non-redundant inhibitor of JAKs via a non-competitive mechanism, it is hoped that this work will lead ultimately to the development of new biological therapies for successful modulation of inflammation in SS. Potential therapeutic agents include modified peptides to emulate SOCS3 activity or JAK antagonists (jakinibs), the latter of which have recently shown great potential in treating rheumatic and other autoimmune diseases [47].

Clinically, the diagnosis of SS may be ambiguous, with confirmation resting at times on the presence of positive histopathological findings in minor salivary gland tissue. However, the value of such biopsies is limited by the potential of having false negative outcomes [48], making it sometimes difficult to differentiate those with SS from subjects with non-autoimmune sicca. The current work has revealed a notable difference between autoimmune SS cases and inflammatory SC controls (chronic sialadenitis) with respect to the co-expression of SOCS3 and IL-17 in salivary gland tissue. This distinguishing characteristic may help to differentiate between SS and chronic sialadenitis.

In conclusion, SS is associated with local and systemic up-regulation of SOCS3 expression. However, the inter-relationships between SOCS3 and pSTAT-3/IL-17 are atypical in SS, with reduced negative regulation by SOCS3 indicating a functional disturbance in this signal transduction pathway. A reduction in function (and not a reduction in expression level) of SOCS3 explains the ineffective regulation of proinflammatory IL-17, which is crucial to the aetiopathogenesis of SS. The results may help to establish a basis for more targeted diagnostic and therapeutic strategies for SS.

Acknowledgments

The authors would like to thank Professor K. Piper for histopathological diagnosis of clinical specimens.

Disclosure

The authors have no conflicts of interest to declare.

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