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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Arthritis Rheum. Author manuscript; available in PMC 2010 April 6.
Published in final edited form as:
PMCID: PMC2850059

Salivary Gland Tissue Expression of Interleukin-23 and Interleukin-17 in Sjögren’s Syndrome

Findings in Humans and Mice
Cuong Q. Nguyen, PhD, Min H. Hu, BS, Yi Li, MD, Carol Stewart, MS, DDS, and Ammon B. Peck, PhD



Recently, the Th1/Th2 paradigm has been expanded by the discovery of Th17 cells, a subset of CD4+ memory T cells characterized by their unique ability to secrete interleukin-17 (IL-17) family cytokines. Importantly, Th17 cells appear to be intimately involved in autoimmunity. We undertook the present study to investigate whether the Th17/IL-23 system is up-regulated in Sjögren’s syndrome (SS).


Sera, saliva, and salivary glands from C57BL/6.NOD-Aec1Aec2 mice (a model for primary SS), as well as sera, saliva, and salivary gland biopsy specimens obtained from patients with primary SS, were evaluated for IL-17 and IL-23 expression by immunohistochemistry, real-time polymerase chain reaction, and the Luminex system.


Immunohistochemical stainings of submandibular glands from C57BL/6.NOD-Aec1Aec2 mice and of salivary gland biopsy specimens from SS patients revealed strong positive staining for both IL-17 and IL-23 within lymphocytic foci and diffuse staining on epithelial tissues. Temporal expression of IL-17 and IL-23 in submandibular glands of C57BL/6.NOD-Aec1Aec2 mice correlated with expression of retinoic acid–related orphan receptor γt, the Th17 cell master control gene. While IL-17 could not be detected in saliva from 4–20-week-old C57BL/6.NOD-Aec1Aec2 mice, this cytokine was present in the blood of mice up to age 16 weeks. This contrasted with sera and saliva from SS patients, in which IL-17 and IL-6 were present at varying levels.


These results suggest that the Th17/IL-23 system is up-regulated in C57BL/6.NOD-Aec1Aec2 mice and SS patients at the time of disease. A correlation between up-regulated IL-17/IL-23 expression and specific clinical manifestations of SS has yet to be identified.

The various subsets of CD4+ Th cells regulating development of adaptive immunity possess the ability to differentiate into functionally distinct effector cells often defined by their cytokine profiles (1-3). Currently, Th cells are divided into 3 major subsets: Th1 cells (secreting interferon-γ [IFNγ]), Th2 cells (secreting interleukin-4 [IL-4], IL-5, and/or IL-13), and the recently defined Th17 cells (secreting IL-17 and/or IL-22) (4). The unique interactions and cross-talk between these 3 different CD4+ T cell lineages result in both the development and regulation of specialized immune responses against invading pathogens and other environmental antigens. However, regulation of the immune response can be circumvented, leading to states of hypersensitivity and autoimmunity resulting in tissue and organ pathology. Recently, much attention has focused on the relationship between innate responses and subsequent activation of specific adaptive immunity in an attempt to understand subsequent immune dysregulation. With the identification of the CD4+ Th17 cell population (5), which initially challenged the long-standing Th1/Th2 cell paradigm, several of these important relationships between innate and adaptive immunity are now being uncovered.

The IL-17–producing Th17 cells are developmentally and functionally divergent from the classic Th1 and Th2 cells (5,6). Upon stimulation by IL-6 and transforming growth factor β, a subset of naive CD4+ T cells can be activated to differentiate into Th17 cells (5). As mature memory cells, Th17 cell survival and maintenance appear to be dependent on IL-23, a heterodimeric cytokine comprising the p40 subunit expressed in IL-12 and a distinct p19 subunit (7). Activated Th17 cells secrete IL-17, a family of cytokines consisting of 6 members: IL-17A (IL-17), IL-17B, IL-17C, IL-17D, IL-17E (IL-25), and IL-17F (4). These are potent inflammatory cytokines that are actively involved in tissue inflammation by inducing expression of numerous proinflammatory cytokines and chemokines, such as IL-6, tumor necrosis factor (TNF), macrophage inflammatory protein 2, granulocyte colony-stimulating factor, CXCL1, CXCL2, CXCL5, matrix metalloproteinase 3 (MMP-3), and MMP-13, in other cell types (8). In addition, as part of the local inflammatory response, IL-17 is involved in the proliferation, maturation, and migration of neutrophils (9).

Recently, CD4+ Th17 cells have been shown to be tissue seeking and intimately involved in autoimmune diseases (e.g., Crohn’s disease [CD] [10,11], experimental autoimmune encephalomyelitis [EAE] [12], and collagen-induced arthritis [CIA] [13]) in which tissue destruction appears to result, in part, from the up-regulation of MMPs. In the present study, we asked whether the Th17/IL-23 system, involving both of the cytokines IL-23 and IL-17, is important in the development of Sjögren’s syndrome (SS), an autoimmune disease characterized primarily by destruction of acinar tissue within the salivary and lacrimal glands (14-23). Using an animal model of SS, we were able to investigate the temporal appearance of both IL-17– and IL-23–positive cells in the targeted glandular tissues, as well as the correlation between these cell populations and the presence of lymphocytic foci. In addition, we investigated whether similar results are present in specimens from SS patients.


Human subjects

Participants were selected from patients seen at the Center for Autoimmune Disorders at the University of Florida. Initial evaluation of these patients was performed by the University of Florida/Shands Department of Rheumatology. From a large cohort of patients who met the American–European Consensus Group criteria for primary SS (24), a subset of 21 patients was selected randomly to include those with whole unstimulated salivary flow rates below the SS criterion for hyposalivation (<0.1 ml/minute) and patients with flow rates above that level.

All patients underwent extensive serologic evaluations, which included tests for the presence of antinuclear antibodies (ANAs), anti-SSA/Ro, anti-SSB/La, anticentromere antibodies, anti–Scl-70, anti-Sm, anti-RNP, anti–double-stranded DNA, rheumatoid factor (RF), anticardiolipin antibodies, and lupus anticoagulant as well as levels of C3 and C4, level of C-reactive protein, thyroid profile, liver function screen, renal screen, complete blood cell count, iron profile, and erythrocyte sedimentation rate. In addition, all patients underwent an extensive medical examination. Following the initial evaluation by a rheumatologist, each patient was referred to the Oral Medicine Clinic for a review of his or her medical history, an oral examination, an unstimulated whole salivary flow rate, and a labial salivary gland biopsy. Specimens from 19 subjects not known to have SS were used as comparative controls. All procedures were reviewed and approved by the University of Florida Health Science Center Institutional Review Board.


Strains of mice used in this study were C57BL/6J and C57BL/6.NOD-Aec1Aec2. These mice were bred and maintained under specific pathogen–free conditions in the mouse facility of the Department of Pathology, Immunology and Laboratory Medicine’s within the Health Science Center at the University of Florida, Gainesville. The C57BL/6.NOD-Aec1Aec2 line, the development of which is described elsewhere (25), has been shown to develop a severe SS-like disease that mimics SS in humans (26). All mice received water and food ad libitum. Studies described herein were approved by the University of Florida Institutional Animal Care and Use Committee.


Unstimulated whole saliva was collected by the drooling method during the late afternoon, between 2:30 pm and 4:30 pm. Study participants were asked to refrain from oral hygiene procedures, smoking, eating, and drinking for at least 2 hours prior to the test session. They were seated comfortably in an upright position and instructed to allow their saliva to flow into a preweighed vessel for a period of 15 minutes. Sealed containers were then reweighed to determine the weight of saliva expectorated. The unstimulated salivary flow rate was determined by gravitation, using a scale accurate to 0.01 gm. On the assumption that 1 gm of saliva is equivalent to 1 ml, the measured volume was expressed as flow rate in ml/minute (27,28). The saliva samples were immediately frozen and stored at −80°C.

Labial salivary gland biopsy

Labial salivary gland biopsies were performed on SS patients in the Oral Medicine Clinic within 2 weeks of the initial Oral Medicine Clinic visit. A local anesthetic was injected into the lower lip followed by a small incision to the right or left of the lip midline. Five or 6 minor salivary gland lobules were carefully harvested and placed into formalin fixative. Resorbable sutures were placed. Standard paraffin preparations were prepared, sectioned at 5-μm thickness, and stained with hematoxylin and eosin (H&E). The slides were examined for the presence of lymphocytic infiltrates and/or foci by 3 board-certified oral and maxillofacial pathologists using standardized criteria. A “focus” was defined as an aggregate of ≥50 lymphocytes with a few plasma cells. The focus score was reported as the number of foci per 4 mm2 of tissue, up to a maximum of 12 foci (29,30).

Immunohistochemical staining for IL-17, IL-23, and B and T lymphocytes

Mouse submandibular glands, as well as biopsy specimens from the lips of patients with SS, were surgically removed and placed in 10% phosphate buffered formalin for 24 hours. Fixed tissues were embedded in paraffin and sectioned at 5-μm thickness. Paraffin-embedded slides were deparaffinized by immersion in xylene, followed by dehydration in ethanol. Antigen retrieval was done with 10 mM citrate buffer, pH 6.0. Following a 5-minute wash with Tris buffered saline–Tween, sections were incubated for 20 minutes at room temperature with blocking solution containing normal sera and avidin block (Vector, Burlingame, CA). Tissue sections were incubated overnight at 4°C with either anti–IL-17 or anti–IL-23, both at 1:100 dilution (Santa Cruz Biotechnology, Santa Cruz, CA). Isotype controls were done with rabbit IgG or goat IgG. The slides were washed for 5 minutes, followed by a 20-minute incubation with biotinylated goat anti-rabbit IgG or biotinylated rabbit anti-goat IgG (Vector). Following a 5-minute wash, slides were incubated for 30 minutes with horseradish peroxidase–conjugated avidin–biotin–peroxidase using the Vectastain ABC kit (Vector). The staining was developed by using diaminobenzidine substrate (Vector), and counterstaining was performed with hematoxylin. Similar procedures were followed for B and T cell staining. The SS labial salivary gland tissues were stained with anti-CD20 for B cells and anti-CD3 for T cells.

Determination of IL-17 levels in serum and saliva

Measurements of mouse or human IL-17 in serum and saliva samples were performed using the mouse IL-17 Bio-Plex Cytokine Assay (Bio-Rad, Hercules, CA) and the human IL-17 LINCOplex kit (Linco Research, St. Charles, MO). All procedures were performed according to the manufacturer’s instructions. Briefly, cytokine standard and samples were mixed in appropriate buffer and added to each well of a 96-well filter plate. Sonicated antibody-conjugated beads solution was added to each well and incubated in the dark at room temperature for 30 minutes. Following 3 washes with wash buffer, 25 μl of detection antibody was added to each well and incubated for 30 minutes at room temperature. Fluorescence intensity was determined by incubation with streptavidin–phycoerythrin solution. All readings were carried out using the Luminex system (Luminex, Austin, TX). Standard curves were generated from 3.1 to 10,200 pg/ml for IL-17 and from 3.2 to 10,400 pg/ml for IL-6. The lower cutoff level for detection by the software was 1 pg/ml.

RNA isolation and quantitative real-time polymerase chain reaction (PCR)

Total RNA was isolated from individual submandibular glands of C57BL/6.NOD-Aec1Aec2 and C57BL/6J mice using RNeasy Mini Kits (Qiagen, Valencia, CA). Complementary DNA (cDNA) was generated by reverse transcription using the Superscript II preamplification system (Invitrogen, Carlsbad, CA). All PCRs were performed at least twice using 3 replicates per sample on the iCycler iQ RT-PCR cycler (Bio-Rad) with SYBR Green I Master Mix from the same manufacturer. The reaction mixture contained 12.5 μl of iQ SYBR Green Supermix, 1 μg of cDNA, and 100 nm of each primer, and was brought to 25 μl with H2O. The thermal cycling conditions involved an initial denaturation step at 95°C for 3 minutes, followed by 50 cycles of 95°C for 30 seconds and 63°C for 30 seconds. Expression levels of each gene were quantified by measuring threshold cycle values using the 18S RNA gene as an endogenous control for normalization of target values. The final relative fold difference was expressed as n-fold differences in targeted gene expression relative to the C57BL/6 mice.

The sequences of the mouse primers were as follows: for IL-17, 5′-TCCACCGCAATGAAGACCCTGATA-3′ (forward) and 5′-ACAAACACGAAGCAGTTTGGGACC-3′ (reverse); for IL-17 receptor (IL-17R), 5′-AAGTTCGCCCAGTTCCTGATCACT-3′ (forward) and 5′-TGTGAGCTCTCTGACATGGTGCAT-3′ (reverse); for the IL-23 p19 subunit, 5′-AACAGATGCCCAGCCTGACTTCTA-3′ (forward) and 5′-AGGCCAACCGCTCGAGACTTTATT-3′ (reverse); for retinoic acid–related orphan receptor γt (RORγt), 5′-GCCTACAATGCCAACAACCACACA-3′ (forward) and 5′-ATTGATGAGAACCAGGGCCGTGTA-3′ (reverse); for T-bet, 5′-AGCCAGCCAAACAGAGAAGACTCA-3′ (forward) and 5′-AATGTGCACCCTTCAAACCCTTCC-3′ (reverse); for GATA-3, 5′-TCTCCAAGTGTGCGAAGAGTTCCT-3′ (forward) and 5′-AGATCTGTCGCTTTCGGGCTTCAT-3′ (reverse); and for 18S RNA, 5′-CTGCGGCTTAATTTGACTCAA-3′ (forward) and 5′-AACCAGACAAATCGCTCCA-3′ (reverse).

Enumeration of IL-17– and IL-23–positive cells in minor salivary glands of SS patients

Using immunohistochemistry, minor salivary gland biopsy specimens from 7 patients examined in this study appeared to give similar patterns of IL-17 and IL-23 staining. Using stained sections from SS patient 11 as a representative sample, 3 lymphocytic foci with different patterns of IL-17 and IL-23 staining were randomly selected for enumerating the number of positively and negatively stained cells. Cells showing positive staining were enumerated visually at higher magnification (projected on a screen) by 3 individuals blinded to the coded sections. The 3 values were compared to confirm similar counts.

Statistical analysis

All values presented are the mean ± SEM. Statistical differences were analyzed with the Student-Newman-Keuls test using GraphPad software (San Diego, CA). P values less than 0.05 were considered significant.


Detection of IL-17 and IL-23 in the submandibular glands of SS-susceptible C57BL/6.NOD-Aec1Aec2 mice at the time of disease onset

Although previous studies have identified the expression of multiple cytokines (e.g., IL-1β, IL-6, IL-10, TNFα, and IFNγ) in the submandibular glands of NOD/LtJ and NOD-derived congenic strains during development and onset of SS-like disease (31), little is known of the expression of IL-17 and IL-23. To determine whether these cytokines are part of the inflammatory response in the submandibular glands during the development of SS-like disease, histologic sections of submandibular glands obtained from C57BL/6.NOD-Aec1Aec2 mice at various ages ranging from 4 weeks to 26 weeks were stained with anti–IL-17 and anti–IL-23 antibodies.

As shown in Figure 1, neither IL-17 (produced by activated T cells) nor IL-23 (produced by activated antigen-presenting cells) was detected in the submandibular glands of 4- and 8-week-old C57BL/6.NOD-Aec1Aec2 mice (Figures 1A and B), 2 time points prior to lymphocytic infiltration into the submandibular glands. In contrast, both IL-17 and IL-23 were detected in the submandibular glands of 12- and 26-week-old C57BL/6.NOD-Aec1Aec2 mice (Figures 1C and D), 2 time points when lymphocytic foci are clearly visible. Thus, expression of these 2 cytokines correlated with the presence of lymphocytic foci. Interestingly, IL-17 and IL-23 were not observed in the submandibular glands of SS-nonsusceptible C57BL/6J mice even when leukocytic infiltrations were present (Figure 1E).

Figure 1
Presence of interleukin-17 (IL-17) and IL-23 in the submandibular glands of Sjögren’s syndrome–susceptible C57BL/6.NOD-Aec1Aec2 mice. Submandibular glands explanted from C57BL/6.NOD-Aec1Aec2 mice at ages 4, 8, 12, and 26 weeks ...

To confirm results of the immunohistochemical staining, we determined the temporal changes in gene expression for IL17, IL17R, and IL23 in the submandibular glands of 4-, 8-, 12-, 16-, and 20-week-old C57BL/6.NOD-Aec1Aec2 mice compared with age- and sex-matched C57BL/6J mice. Total RNA was converted to cDNA and used as template in real-time PCR. As shown in Figure 2A, the messenger RNA (mRNA) levels of IL17, IL17R, and IL23 in the submandibular glands of C57BL/6.NOD-Aec1Aec2 compared with C57BL/6J mice remained relatively similar from age 4 weeks to age 12 weeks. However, after age 12 weeks, a rapid up-regulation in the expression of IL17, IL17R, and IL23 was noted in C57BL/6.NOD-Aec1Aec2 mice, corresponding to the time when the immune attack on the glands is initiated. By age 20 weeks, expression levels of IL17 and IL23 returned to normal, while IL17R expression remained elevated.

Figure 2
Temporal expression of interleukin-17 (IL-17), IL-17 receptor (IL-17R), and IL-23 mRNA transcripts (A) and of T-bet, GATA-3, and retinoic acid–related orphan receptor γt (RORγt) mRNA transcripts (B) in the submandibular glands ...

Differential expression of transcription factors for CD4+ Th1, Th2, and Th17 cells in the submandibular glands of C57BL/6.NOD-Aec1Aec2 mice

A number of transcription factors have been identified that promote differentiation of CD4+ T cells into Th1, Th2, or Th17 cells. These include STAT-1, STAT-4, and T-bet for Th1 cells, STAT-6, GATA-3, and c-Maf for Th2 cells, and RORγt for Th17 cells. To identify transcription factor expression in the submandibular glands of C57BL/6.NOD-Aec1Aec2 mice compared with age- and sex-matched C57BL/6J mice between age 4 weeks and age 20 weeks, cDNA was used as template in real-time PCR with primers for T-bet, Gata3, and Rorγt. As shown in Figure 2B, T-bet expression was up-regulated nearly 3.5-fold in glandular tissue from 4-week-old C57BL/6.NOD-Aec1Aec2 mice but returned to normal levels by age 8 weeks. In contrast, expression of both Gata3 and Rorγt did not increase until after age 12 weeks, peaked at age ~16 weeks, and returned to normal by age 20 weeks. These data are highly indicative of the disease profile described for C57BL/6.NOD-Aec1Aec2 mice, in which IFNγ expression is essential early in the disease, but IL-4 expression is essential in the onset of clinical disease (32). The current data indicate that IL17 gene expression is coordinate with IL4 gene expression as well as with production of IgG1 isotypic autoantibodies postulated to effect SS-like disease.

Comparison of IL-17 levels in sera and saliva from C57BL/6.NOD-Aec1Aec2 mice

Although IL-17 is a potent inflammatory cytokine that is produced locally by activated CD4+ memory T cells, it remains a subject of speculation whether IL-17 is functionally more effective locally or systemically. To determine whether IL-17 is present in either the sera or saliva of SS-susceptible C57BL/6.NOD-Aec1Aec2 mice, sera and saliva were collected from mice at ages 4, 16, and 20 weeks. As shown in Figure 3, C57BL/6.NOD-Aec1Aec2 mice exhibited a high level of IL-17 at age 4 weeks, with decreasing levels thereafter. IL-17 was no longer detected in sera by age 20 weeks, a time when disease in the salivary glands is considered to be fully developed. In contrast, IL-17 was detected in saliva at extremely low levels at each of the 3 time points (data not shown). Interestingly, SS-nonsusceptible C57BL/6J mice showed similar patterns of IL-17 expression in serum (Figure 3) and saliva (results not shown) but at levels generally lower than those in the prediseased C57BL/6.NOD-Aec1Aec2 mice.

Figure 3
Temporal loss of detectable levels of interleukin-17 (IL-17) in sera from C57BL/6.NOD-Aec1Aec2 mice. Sera were prepared from C57BL/6J and C57BL/6.NOD-Aec1Aec2 mice at ages 4, 16, and 20 weeks (n = 4 per each time point and strain). IL-17 levels were determined ...

Detection of IL-17 and IL-23 in the minor salivary glands of SS patients

While the C57BL/6.NOD-Aec1Aec2 mouse has been advanced as an animal model that closely mimics human SS (26), it is not known whether similar expression of the Th17/IL-23 system occurs in SS patients. To determine whether IL-17 and/or IL-23 are expressed in the salivary glands of SS patients, 7 lip biopsy specimens that had been freshly explanted, fixed in buffered formalin, and embedded in paraffin were sectioned, and consecutive serial sections were stained with anti–IL-17 and anti–IL-23 antibodies. An additional consecutive section was stained with H&E to quantify the number of foci present in the gland sections.

Each biopsy specimen examined from the 7 SS patients exhibited similar staining patterns for IL-17 and IL-23, as shown in Figure 4 for 3 representative patient samples. The staining appeared localized to lymphocytic infiltrates and ductal cells, with less staining occurring in the acinar components. Close examination of individual lymphocytic foci revealed that 1) both IL-17 and IL-23 staining was diffuse, 2) IL-17 expression appeared stronger and more widely distributed than IL-23 expression, and 3) neither IL-17 nor IL-23 stained all mononuclear inflammatory cells associated with an individual focus. Enumeration of IL-17– and IL-23–positive cells within individual foci present in the same gland section indicated marked differences between the numbers of cells staining positive or negative for IL-17 and IL-23, as shown in Figure 5.

Figure 4
Cellular expression of interleukin-17 (IL-17) and IL-23 in the minor salivary glands of patients with Sjögren’s syndrome (SS), and presence of B and T lymphocytes determined by immunohistochemistry. Explanted minor salivary glands from ...
Figure 5
Enumeration of IL-17– and IL-23–positive/negative cells present in the minor salivary glands. Minor salivary glands from SS patient 11 were prepared and stained as described in Patients and Methods. The numbers of cells staining positively ...

Comparison of IL-17 and IL-6 levels in sera and saliva from SS patients with those in subjects without SS

Levels of IL-17 and IL-6 in both sera and saliva were determined using the Luminex system for 21 SS patients and 19 control subjects without SS, as described in Patients and Methods. Briefly, the mean levels of IL-17 in sera from SS patients did not differ significantly from those measured in sera from control subjects without SS, despite being slightly higher (Figure 6A). A similar relationship for IL-17 levels was also seen between saliva from SS patients and saliva from control subjects without SS (Figure 6A). In contrast, while the levels of IL-6 did not differ significantly between sera from SS patients and sera from control subjects without SS, the IL-6 levels in saliva from SS patients exhibited nearly a 3-fold increase over those in saliva from control subjects without SS (Figure 6B). This elevated level of IL-6 in SS patients may represent the driving force in promoting Th17 cells in the salivary glands (3335).

Figure 6
Levels of cytokines IL-17 and IL-6 in sera and saliva specimens from SS patients and control subjects without SS. Saliva and serum specimens collected from SS patients (n = 21) or from control subjects without SS (n = 19) were tested for levels of IL-17 ...

Lack of direct correlation between disease phenotype and IL-17 in SS patients

As presented above, lip biopsy specimens from SS patients exhibited strongly positive staining for both IL-17 and IL-23 that colocalized with visible infiltrating lymphocytes and/or lymphocytic foci. To identify possible correlations between IL-17/IL-23 staining and biomarkers of SS, the disease profiles of the SS patients were examined. Patients in whom primary SS was diagnosed exhibited normal clinical test results except for the presence of lymphocytic foci in minor salivary gland biopsy specimens, abnormal salivary flow rates, and/or the presence of ANAs, anti-SSA/Ro, anti-SSB/La, and RF. No patients were receiving corticosteroids at the time of the labial salivary gland biopsy. In addition, no patients were receiving methotrexate, which has been reported to affect IL-17R mRNA (35). Although a direct correlation with a specific pathologic marker or set of markers could not be identified, weak positive correlations were seen with RF in the serum or saliva, detectable IL-6 and IL-17 secreted in saliva, and hyposalivation (<0.1 ml/minute). While it has been reported that IL-17 may play a role in the inflammatory responses to Helicobacter pylori–infected gastric mucosa (36), none of the study participants was tested for H pylori (data not shown).


In the present study, we sought to determine whether the Th17/IL-23 system might be involved in the development of SS-like disease in C57BL/6.NOD-Aec1Aec2 mice and/or SS in humans. By taking advantage of the mouse model, it was possible to investigate whether there are temporal relationships between the Th17/IL-23 system, glandular inflammation, and clinical disease. Initial results indicate that both Th17 cells and IL-23–positive cells are present in the salivary glands, primarily located within the lymphocytic foci considered important and within prominent pathologic lesions associated with SS in both species. In contrast, and somewhat unexpectedly, lymphocytic foci occurring in older SS-nonsusceptible C57BL/6J mice (age >30 weeks) did not contain either Th17 cells or IL-23–positive cells, possibly indicating that these lymphocytic infiltrations are not pathologic lesions. Overall, these data therefore suggest, but do not prove, that these cell populations may play an important role in the development and/or onset of SS; however, there are no indications that either cell population is an actual effector of SS or that the levels of staining correspond to time of disease onset and/or severity. Nevertheless, the present study adds SS to the rapidly expanding list of autoimmune diseases (e.g., CD [10,11], EAE [12], multiple sclerosis [37], rheumatoid arthritis [RA] [38], psoriasis [39], and CIA [13]) in which the Th17/IL-23 system is now implicated.

While submandibular glands from diseased C57BL/6.NOD-Aec1Aec2 mice and lip salivary gland biopsy specimens from SS patients immunostained for IL-23 and IL-17 showed similar results, detection of these 2 cytokines in sera and saliva provided very different outcomes. In our mouse model, IL-17 could be detected only in sera prepared at early time points (i.e., up to age 16 weeks) when IL-17 could also be detected in sera from SS-nonsusceptible parental C57BL/6J mice, although in lesser quantities. In contrast, IL-17 was virtually undetectable in saliva at any of the time points examined (i.e., up to age 20 weeks). In the human specimens, IL-17 could be detected in 9 of the 21 serum samples from SS patients tested, a frequency similar to that in control subjects without SS (8 of 19). At the same time, IL-17 was detected in 11 of 21 saliva samples from SS patients, but in only 9 of 19 saliva samples from control subjects without SS. These data are consistent with those reported for RA patients, where IL-17 was not detected in serum samples but did appear in sera and synovial fluids from a subset of patients (33).

While the results of our current study involving cytokines seem inconsistent, they may actually represent the predicted changing profile associated with the natural progression of the SS disease state. For example, based on data obtained from the human samples, we would predict that as the disease in the C57BL/6.NOD-Aec1Aec2 mice progresses beyond the 20 weeks followed up in the current study, IL-17 should eventually be detected in the saliva of these mice as well. Conversely, based on our data obtained from the mouse studies, the level of IL-17 would be anticipated to be elevated in the sera of SS patients if examined at a time point prior to clinical disease.

In the present study, we found that at age 4 weeks, C57BL/6.NOD-Aec1Aec2 mice exhibited up-regulation of T-bet, the Th1 cell master control gene. This observation is consistent with previous studies by our group showing high levels of IFNγ production at this early age (40). Thus, our data are consistent with the concept that there is an early induction of a CD4+ Th1/Th17 pathway leading to systemic release of IL-17. Since IL-17 can induce an up-regulation of vascular cell adhesion molecule 1 that, in turn, makes vascular endothelium more adherent to intravascular lymphocytes, sites of inflammation would exhibit increased vascular adherence, permitting Th17 cells to gain access to the damaged tissues and to begin secreting cytokines that exacerbate the pathology (41). This cascade can include IL-6, which induces expression of intercellular adhesion molecule 1, which functions as a receptor for activated T cells expressing lymphocyte function–associated antigen 1, as well as Th2 cytokines that function to induce infiltrating B lymphocytes to produce autoreactive antibodies (42). Again, the coordinate up-regulation of Gata3, the Th2 cell master control gene, and of RORγt, the Th17 cell master control gene, is consistent with such a model.

Despite the apparent involvement of Th17 cells in the development and/or onset of SS, one cannot forget the importance of both the Th1 and Th2 pathways. In previous studies by our group, elimination of IFNγ in SS-susceptible mice ameliorated all pathologic and clinical signs of disease, while elimination of IL-4 prevented loss of secretory function (40,43). These observations indicate an essential role for both Th1 cell–and Th2 cell–associated cytokines. These data raise several important questions, including the question of when Th17 cells become involved in the autoimmune response and whether they act directly through secretion of inflammatory IL-17 family cytokines or by activating autoimmune T and B cells. If Th17 cells act in SS as reported in EAE, then IL-17 may not be required for initiation of SS, but it still represents a critical regulator of Th1 cells and their production of IFNγ (41), again consistent with our group’s data from the study of Ifng gene–knockout mice (40). In any event, the IL-23/CD4+ Th17/IL-17R system appears to bridge aspects of the innate and adaptive immune responses.

Finally, is it possible to utilize these new data concerning the Th17/IL-23 system to actually identify developing SS-like or clinical SS disease? In both our C57BL/6.NOD-Aec1Aec2 mouse model and a randomly selected patient population, we observed various overlaps in data with presumably nonsusceptible or normal, healthy controls (e.g., the presence of IL-17 in saliva from humans or the presence of IL-17 in sera from mice). Furthermore, no particular disease profile appears to correlate specifically with our Th17/IL-23 findings, although the number of patients (and animals) included in the present study is limited. SS is a very intricate and complex autoimmune disease in which significant alterations, both physiologic and immunologic, are disease phase dependent or disease stage associated. Future investigations need to take these issues into consideration in designing studies to examine the systemic and localized effects of the Th17/IL-23 system in SS.


Supported in part by the University of Florida’s Center for Orphaned Autoimmune Disorders. Dr. Nguyen’s work was supported by a Postdoctoral Fellowship through USPHS grant T32-DE-07200 from the NIH. Dr. Peck’s work was supported by USPHS grant DE-014344 from the NIH.


Dr. Peck holds stock or stock options in Ixion Biotechnology

AUTHOR CONTRIBUTIONS Dr. Nguyen had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Nguyen, Peck.

Acquisition of data. Nguyen, Hu, Li, Stewart.

Analysis and interpretation of data. Nguyen, Stewart, Peck.

Manuscript preparation. Nguyen, Stewart, Peck.

Statistical analysis. Nguyen, Hu, Stewart.


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