Abstract

The pathogenesis of Sjögren’s syndrome (SS) is poorly understood. To evaluate an autoimmunization-induced experimental SS model, we firstly observed the phenotype of lymphocyte infiltration in the enlarged submandibular gland (SG). Furthermore, significant activation of caspase-3 and a high ratio of Bax-to-Bcl-2 were detected, indicating the inflammatory apoptosis associated with developmental foci. Meanwhile, the dysregulated cytokines, such as tumor necrosis factor α, IL-1β and IL-6 mRNA expression, were found to be over-expressed. A progressive decrease of aquaporin 5 and its subcellular translocation from apical to basal membrane in SG was found to be associated with the abnormally expressed M3 muscarinic acetylcholine receptor. This pattern was found to be similar to that seen in human SS and possibly contributed to the saliva secretion deficiency. Thus, this autoimmunization-induced model recapitulates the key features of human SS and may have potential for studying the pathogenesis of human SS.

Introduction

Sjögren’s syndrome (SS) is a systemic autoimmune disease with connective tissue disorders (1) characterized by progressive destruction of salivary glands and lachrymal glands, eventually leading to the typical symptoms of dry mouth and dry eyes (2, 3). Although the etiology of SS is unknown, there is substantial evidence suggesting that immune-mediated inflammation may be the most important factor leading to the secretory dysfunction (4, 5).

The intense immune responses contribute to the alterations in cytokine network and histopathological lesion in exocrine glands (6, 7). In exocrine glands, pro-inflammatory cytokines, such as IL-1β and tumor necrosis factor α (TNF-α), along with other cytokines involved in T- and B-cell activation and auto-antibody production, such as IL-6, were found over-expressed (6, 8). Metalloproteinase 9 (MMP-9), the most efficient activator of precursor IL-1β (9), degrades the extracellular matrix (ECM) and further destroy the connective tissues (10). High activities of MMP-9 are found in patients with severe active SS (11). The later activation of tissue damage (such as apoptosis) leads to chronic inflammation and loss of physiological function (7, 12).

In SS patients, aquaporin 5 (AQP-5), a water-specific membrane channel protein, is found abnormally distributed, mostly in the basolateral acinar plasma membrane domain, instead of the apical membrane (13). The protein expression of AQP-5 is also involved in rapid transport of water across the cell membrane and used as a functional indicator of salivary flow rate (14). As identified in the salivary gland (15), AQP-5 is expressed in a variety of fluid-transporting epithelia, and the involvement of AQP-5 in saliva secretion has been clearly demonstrated in AQP-5-knockout mice that display a considerably reduced salivary volume and flow (16). A recent finding also demonstrated that AQP-5 could be inhibited by TNF-α in murine lung epithelial cells (17).

There is no effective treatment for patients with SS. Most treatments (anti-cholinergic medications, etc.) target the symptoms (18) with limitations (19). Given the complexity and heterogeneity of SS, the establishment of animal models which exemplify the key features seen in SS is therefore of value. Murine models to explore the pathogenesis of SS have been reported and reviewed in detail (3). Although the non-obese diabetic mouse model is commonly used for autoimmune diseases, the spontaneous development of diabetes, limited apoptosis-related molecules (20) and systemically lymphocytic infiltration in numerous other organs and tissues were considered as inadequacies of the model (3). The experimentally induced autoimmunized rat model was firstly established by White et al. (21) and was further evaluated using lymphocytic infiltration by Cutler et al. (22). However, this chronic SS model usually requires a repeated procedure for months or even longer (23). Thus, C57BL/6 mouse, an inbred strain highly susceptible to autoimmune induction in a shorter time period, was used as modification (21, 24). It was well described for the study of SS due to similarity in the pathological process (25). Our previous study showed glandular destruction with a decrease of AQP-5 (26) and imbalance of Th1/Th2 cytokines in this model was also reported by Wang et al. (27). However, the inflammatory apoptosis, cytokines disorder and the role of AQP-5 in the pathogenesis of salivary gland injury and dysfunction in this model are not known. Thus, the present study was designed to further evaluate this model.

Materials and methods

In vivo study

Mice. Female 8-week-old C57BL/6 mice of inbred strains, weighing 18–20 g, were obtained from Laboratory Animal Center of University of Hong Kong. Mice were bred and maintained at the Laboratory Animal Unit under specific pathogen-free conditions. The ambient temperature and humidity were maintained at 22°C and 50%, respectively. The lighting conditions were 12 h of light and 12 h of darkness. Mice were allowed to acclimatize for a minimum of 1 week.

Induction of SS model.

The SS model was induced by immunization with submandibular gland (SG) auto-antigen as previously described with some modifications (21, 27). Preparation of the SG auto-antigen was carried out on ice. Five C57BL/6 mice were sacrificed by using an overdose of pentobarbital. The bilateral SG from the mice were immediately removed under sterile conditions, dissected free from fat and connective tissues, weighed and centrifuged at 12 000 × g at 4°C for 20 s after homogenization in 2 ml of sterile saline solution per 100 mg SG. The sample was further centrifuged at 3000 × g for 15 min at 4°C. The supernatant was collected and protein concentration was determined by the bicinchoninic acid (BCA) assay (Sigma–Aldrich) (28) and then adjusted to 800 μg ml−1 in PBS and emulsified in an equal volume of CFA (Sigma–Aldrich) to a concentration of 400 μg ml−1. On day 0, each of the mice was injected subcutaneously with 0.1 ml of the emulsion. On day 14, the booster injection was carried out with the same dose of auto-antigen emulsified in Freund’s incomplete adjuvant (Sigma–Aldrich). Control mice were immunized with 0.1 ml of PBS per mouse. Body weight and water intake were measured every 2 days. The protocol for animal study had been approved by the Committee on the Use of Live Animals in Teaching and Research (CULATR) of the University of Hong Kong meeting the standards required by the UKCCCR guidelines (Project number CULATR-1727-08).

Serum and tissue collection.

On day 25, mice were sacrificed; blood was immediately collected and centrifuged at 3000 × g for 10 min to obtain the serum further analysis. After blood sampling, SG and spleen from each mouse were excized and weighted to calculate the SG index and spleen index. Both indexes were calculated as (organ weight/body weight) ×1000. The SG was then used for histological examination. The abundance of antibody to nuclear antigen (ANA) was detected by using mouse ANA ELISA kit (5210; Alpha Diagnostic International) according to the manufacturer’s specifications.

Immunohistochemistry and immunofluorescent staining.

SGs were weighed and fixed in a 4% PFA solution. Paraffin-embedded tissue sections (5 μm) were stained with hematoxylin and eosin (H&E) for determination of lymphocyte infiltration. Lymphocytes in H&E-stained SG sections were measured by the use of IPP software (ver6.0, Image-Pro Plus; Media Cybernetics, USA) and compared with control group sections. Diaminobenzidine staining was performed by using a peroxidase substrate kit following the instruction provided in the manual (Vector Vip SK-4600) (29).

For immunofluorescent staining, sections were cut and incubated with AQP-5 pAb (AB15858, 1:250; Millipore) or M3 muscarinic receptor (M3R) pAb (SC-9108, 1:50; Santa Cruz), followed by staining with goat anti-rabbit IgG-FITC (1:400; Millipore). Fluorescence microscopy employing a Zeiss fluorescent microscope was used for image capture. AxioVision digital imaging system was used to optimize signal-to-noise ratio.

Gelatin zymography and MMP-9 activity assay.

Gelatinase level in the serum samples of mice was measured by gelatin zymography, as previously described with slight modification (30). Diluted serum (all at a dilution of 1:10) or SG samples were incubated in loading buffer without β-mercaptoethanol for 30 min at room temperature and analyzed by electrophoresis on a 10% SDS–polyacrylamide gel containing 1 mg ml−1 gelatin (Sigma–Aldrich). MMP standard and protein marker were preloaded to indicate the molecular weight. After electrophoresis, the proteins were renatured by removing SDS from the gel using washing buffer containing 0.25% Triton X-100. This was followed by incubation at 37°C for 16 h in the developing buffer consisting of 50 mM Tris–HCl (pH 7.4), 0.15 M NaCl, 10 mM CaCl2, 2 mM ZnSO4, 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma–Aldrich), 0.005% Brij35 and 0.02% sodium azide. After the incubation, the gel was briefly rinsed in distilled water and stained with 0.25% Coomassie brilliant blue R250 for 1 h and then distained with 7% acetic acid. Gelatinase activity in the gel was visible as a clear area in the blue background, indicating an area where gelatin had been digested.

Preparation of nuclear extract.

Nuclear extract was prepared as previously described (31), and protein was quantitated using the BCA protein assay (Sigma–Aldrich). Briefly, the SG homogenates were left at 4°C in 10 ml buffer A [10 mM HEPES, pH 7.9, 10 mM KCl, 1.5 mM MgCl2, 1 mM dithiothreitol (DTT), 1 mM PMSF] for 15 min and then centrifuged at 18000 × g for 5 min. After removal of the supernatants (cytoplasmic extracts), the pelleted nuclei were washed with 30 ml buffer A and subsequently, cell pellets were re-suspended in 5 ml buffer B (20 mM HEPES, pH 7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 25% glycerol, 1 mM DTT, 0.5 mM PMSF) and incubated at 4°C for 30 min. The mixtures were centrifuged at 24 000 × g for 20 min, and the supernatants were used as nuclear extracts.

Western blot analysis.

Proteins were collected from mouse organs and serum samples as described previously (26). Protein samples were loaded on 12% SDS–polyacrylamide precast gels. All experiments were carried out in duplicate. After transfer to polyvinylidene fluoride membranes, they were incubated overnight with primary antibodies. Incubation with secondary antibody was performed in an identical manner. Bands were visualized by ECL Advanced Solution (GE Healthcare Life Sciences). The membranes were scanned and the density of each protein band was measured using Quantity One software (pdi, New York, NY, USA). β-Actin was determined as the loading control (SC-47778, 1:1000; Santa Cruz). Primary antibodies included rabbit anti-AQP-5 (AB15858, 1:1000; Millipore), rabbit anti-caspase-3 (9661S, 1:1000; Cell Signaling Technology), rabbit anti-NF-κB-p65 (sc-33039; Santa Cruz), rabbit anti-Bax(Δ21) (sc-6236; Santa Cruz), rabbit anti-Bcl-2(C21) (sc-783; Santa Cruz), rabbit anti-lamin B1 (ab16048, 1:1000; Abcam) and second antibodies anti-rabbit IgG-HRP (12-348, 1:1000; Millipore) and anti-mouse IgG-HRP (12-348, 1:1000; Millipore).

RT–PCR analysis.

RT–PCR experiments were carried out according to standard protocols. Total RNA in SG and serum samples were extracted with TRIZOL (Sigma–Aldrich) and the first-strand cDNA was synthesized using M-MuLV reverse transcriptase (Fermentas). Then cDNA was amplified by Green Master Mix (Promega) using the following primers:

β-actin: 5′-CCCCATTGAACATGGCATTG, 3′-ACGACCAGAGGCATACAGG

AQP-5: 5′-ATCTACTTCACCGGCTGTTCC, 3′-GTCAGCTCGATGGTCTTCTTC

TNF-α: 5′-ATCAGTTCTATGGCCCAGACCCT, 3′-TCACAGAGCAATGACTCCAAAGTA

IL-1β: 5′-TTGACGGACCCCAAAAGAT, 3′-GAAGCTGGATGCTCTCATCTG

IL-6:5′-GCTACCAAACTGGATATAATCAGGA, 3′-CCAGGTAGCTATGGTACTCCAGAA

The amplification program was set as follows: 2 min at 94°C, 30 s at 94°C, 30 s at 49°C and 50 s at 72°C. The cycle numbers were optimized for each primer pair and then were incubated for 10 min at 72°C. The resulting PCR products were analyzed by electrophoresis in 1% agarose gel containing Gel red and visualized by UV transillumination. The density and the width of each PCR product were measured using Quantity One software (pdi).

In vitro study on A-253 Cell line

Cell culture. A-253 cells from human salivary gland (HTB-41, American Type Culture Collection) were cultured in McCoy's 5A modified medium (Invitrogen) supplemented with 10% (v/v) fetal bovine serum (Invitrogen) and 1% (v/v) antibiotic solution (100 U ml–1 of penicillin and 100 mg ml−1 streptomycin) (Invitrogen) and incubated at 37°Cin an incubator in a humidified atmosphere of 5% CO2–95% air. Approximately 48 h before study, cells were seeded onto six-well tissue culture dishes at a density of 5 × 105 cells per well and were serum starved 12 h before study by replacing the serum-free medium.

Cell viability and nuclear staining with Hoechst 33258.

To determine the inducible apoptosis by the auto-antibodies in the model mice serum, we performed the cell viability test using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. A-253 cells were incubated with different concentrations of serum from control mice and model group for 1 h. Cells without any treatment was set as the negative control. Apoptotic nuclei were visualized with a fluorescent DNA-binding dye, Hoechst 33258, as previously described (26). Cells were then fixed with 4% PFA solution and stained with Hoechst 33258 (2 μg ml–1) for 10 min and observed under a fluorescence microscope (×20). Nuclear size reduction, chromatin condensation, intense fluorescence and nuclear fragmentation were considered as features of apoptosis.

AQP-5 inhibition by human TNF-α.

To support our findings that AQP-5 may be inhibited by the releasing cytokines, we performed AQP-5 inhibition in human salivary gland cells. A-253 cells were treated with human TNF-α as determined by a dose–response curve for the duration of the experiment and visualized by immunoblotting (Fig. 11). This experiment was replicated in its entirety at least twice with at least three independent wells per experimental group.

Data analysis and statistics.

Results are expressed as mean ± SD. Data were analyzed using an unpaired Student’s t-test to determine the difference between groups using SPSS 16.0 (*P < 0.05; **P < 0.01).

Results

SG and spleen enlargement in model mice

First, we observed the morphology change in the typical glands, SG and spleen in the autoimmunized mouse model. Figure 1 representatively showed the clear enlargement of SG and spleen of model mice compared with control mice. Quantitatively, the measurement of tissue index also demonstrated the significance increase in weight after the immunization.

Fig. 1.

Pathological change of autoimmunized mice. A swelling SGs and spleen in C57/BL6 mice of model group compared with control mice were observed. Increased tissue index of model mice quantified the enlargement in weight.

Fig. 1.

Pathological change of autoimmunized mice. A swelling SGs and spleen in C57/BL6 mice of model group compared with control mice were observed. Increased tissue index of model mice quantified the enlargement in weight.

Lymphocyte infiltration in SG of model mice

Furthermore, by examining the histopathological sections of the swelling SG, we found that mice immunized with the SG auto-antigen developed lymphocytic infiltration similar to that seen in human SS. The presence and degree of pathology in the form of lymphocytic infiltrates varied among the individual mice, but all clearly destructed, whereas control mice had little or no infiltration of lymphocytes in the salivary glands. As shown in Fig. 2(A) (a-d), the histopathological findings disclosed the time course of infiltrations in different degrees. On day 20, some of the mice in model group were found as previously defined as grade 3 foci (32) (>50 periductal lymphocytes and the presence of a germinal center-like structure) in the SS model mice (arrow), while no infiltrations were observed in the control mice immunized with PBS alone (Fig. 2A, e). Also, according to the scoring system, the mice were graded from 0 to 3, based on the presence of lymphocytes in each focus per 4 mm2 (Fig. 2B). Histological examination of the salivary gland and surrounding tissues did not reveal any bacterial or viral infections except for lymphocyte infiltration. The mean values of anti-ANA IgG were 12.66 μg ml−1 control group and 114.13 μg ml−1 model group according to the standard assay curve (Fig. 2C).

Fig. 2.

Development of autoimmunity of model mice. (A) H&E staining of SG. According to the time course, autoimmunization-induced mice developed severe lymphocytic infiltration and presented multiple foci from day 0–20 (a–d). The control mice immunized with PBS alone showed a grade 0 in (e). (B) The grading system showed high grade of model group but no infiltrations in the control. The average lymphocytes were counted by Image-Pro Plus. (C) The ANA levels were measured in control and model group. The mean values of control and model group were 12.66 and 114.13 μg ml−1, respectively. Pictures were taken with the original magnification of 10 × 10 lenses. Data were presented as mean ± SD of independent analyzed (n = 8) by independent t-test. **P < 0.01 versus control mice.

Fig. 2.

Development of autoimmunity of model mice. (A) H&E staining of SG. According to the time course, autoimmunization-induced mice developed severe lymphocytic infiltration and presented multiple foci from day 0–20 (a–d). The control mice immunized with PBS alone showed a grade 0 in (e). (B) The grading system showed high grade of model group but no infiltrations in the control. The average lymphocytes were counted by Image-Pro Plus. (C) The ANA levels were measured in control and model group. The mean values of control and model group were 12.66 and 114.13 μg ml−1, respectively. Pictures were taken with the original magnification of 10 × 10 lenses. Data were presented as mean ± SD of independent analyzed (n = 8) by independent t-test. **P < 0.01 versus control mice.

Increased water intake

Meanwhile, water intake of the mice during establishment of the model was measured. Mice in the SS model group exhibited increased drinking of water compared with the control group (Fig. 3).

Fig. 3.

Model mice with autoimmunization had a progressive increase of water intake compared with control mice. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. *P < 0.05 and **P < 0.01 versus control mice.

Fig. 3.

Model mice with autoimmunization had a progressive increase of water intake compared with control mice. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. *P < 0.05 and **P < 0.01 versus control mice.

Autoimmunization-induced apoptosis

Consequently, we evaluated the activation of apoptosis in response to the autoimmunization. Apoptosis is genetically regulated by the downstream intracellular Bcl-2 family of proteins and the caspase pathway activation (33). Therefore, we investigated Bax/Bcl-2 and caspase-3 as the two major execution programs downstream of the death signal (34). Analyzed by western blot, we found a high expression of the proapoptotic protein Bax but rarely the anti-apoptotic protein Bcl-2 in the SG of SS model mice (Fig. 4), showing the significant intracellular imbalance of apoptosis-regulated proteins. Furthermore, the amount of activated caspase-3 protein was observed to be ∼80-fold higher than control mice, strongly contributing to the developmental of autoimmunity in SS model mice.

Fig. 4.

The time course of Bax, Bcl-2 and caspase-3 activation (cleaved form) in SG was analyzed by western blot. Semi-quantification data were expressed as a fold of control group. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control mice. C: control; M: model.

Fig. 4.

The time course of Bax, Bcl-2 and caspase-3 activation (cleaved form) in SG was analyzed by western blot. Semi-quantification data were expressed as a fold of control group. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control mice. C: control; M: model.

Up-regulated mRNA expression of TNF-α, IL-1β and IL-6

There is extensive evidence for the role of TNF-α in the pathogenesis of SS (35, 36). In contrast to the minimum expression of TNF-α observed in control mice, the SS model mice showed a ∼2.5-fold higher mRNA level (Fig. 5). Furthermore, among the pro-inflammatory cytokines involved in the immune responses, IL-1β was reported to be a potent inducer of TNF-α and IL-6 (37, 38). The results showed, as assessed by RT–PCR, a ∼10-fold and ∼4-fold higher IL-1β mRNA expression in respectively the serum and SG of SS model mice compared with control mice (P < 0.001).

Fig. 5.

Cytokines network imbalance of autoimmunized model mice. mRNA expression of TNF-α, IL-1β and IL-6 was found significant increased in model mice. Semi-quantification data were expressed as a fold of control group. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control mice.

Fig. 5.

Cytokines network imbalance of autoimmunized model mice. mRNA expression of TNF-α, IL-1β and IL-6 was found significant increased in model mice. Semi-quantification data were expressed as a fold of control group. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control mice.

In view of the strong expression of TNF-α and IL-β which can both stimulate IL-6 (39) and IL-6 mRNA expression was subsequently observed to be ∼3.7-fold and ∼3-fold higher in serum and SG of model mice (P < 0.001, Fig. 2E).

Increased MMP-9 activity

The activity of MMP-9, a protease that activates precursor IL-1β in the extracellular environment, was also evaluated (40). By using gelatin zymography, a semi-quantitative analysis of the results revealed a 2.8-fold higher activity of MMP-9 in serum of model mice compared with control mice (P < 0.001) (Fig. 6).

Fig. 6.

MMP-9 activity was performed by gelatin zymography. Both in the serum and in the SG, the activity of MMP-9 was found significantly increased. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control mice. S: standard.

Fig. 6.

MMP-9 activity was performed by gelatin zymography. Both in the serum and in the SG, the activity of MMP-9 was found significantly increased. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control mice. S: standard.

Nuclear factor κB activation

Since the ability of TNF-α to induce such a wide variety of effects is likely due to its ability to activate multiple signal transduction pathways including nuclear factor κB (NF-κB) (41), we further verified the activation of NF-κB as its translocation from cytoplasm to nuclei. As shown in Fig. 7, we observed a significantly higher nuclear ratio of NF-κB and the increment of total expression in the SS model mice compared with control mice (normalized with lamin-B).

Fig. 7.

Activation of the NF-κB protein as its translocation form to nuclei in the SG of model mice (normalized with lamin-B) and semi-quantified as the nuclear ratio of NF-κB. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control mice.

Fig. 7.

Activation of the NF-κB protein as its translocation form to nuclei in the SG of model mice (normalized with lamin-B) and semi-quantified as the nuclear ratio of NF-κB. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control mice.

Abnormality of AQP-5 in SG of model mice

In a time course study of AQP-5 expression, we found a progressive reduction in SG of SS model mice compared with control mice (Fig. 8A and B, n = 8), suggesting the dysfunctional fluid transport. Furthermore, immunofluorescence labeling with a mouse AQP-5 antibody was performed on paraffin sections of mouse SG. In the SGs of control mice (Fig. 8C), AQP-5 labeling was present abundantly in the apical domains acinar cells (arrows) and close to the central lumen (arrow heads), which was consistent with the previous studies (13). In contrast, the SS model mice revealed AQP-5 expression primarily at the basal membrane of the acinar cells away from the lumen. Similar to the SS patients, the results indicate that translocation of AQP-5 may contribute to the saliva secretion deficiency.

Fig. 8.

Abnormality of AQP-5 expression in the model mice. (A) Time course of a progressive decrease of AQP-5 expression and semi-quantified as (B). (C) Representative AQP-5 localization in SG of C57/BL6 mice was performed by fluorescence immunohistochemistry staining. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. *P< 0.05 and **P < 0.01 versus control mice. Bars = 20 μm.

Fig. 8.

Abnormality of AQP-5 expression in the model mice. (A) Time course of a progressive decrease of AQP-5 expression and semi-quantified as (B). (C) Representative AQP-5 localization in SG of C57/BL6 mice was performed by fluorescence immunohistochemistry staining. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. *P< 0.05 and **P < 0.01 versus control mice. Bars = 20 μm.

Fig. 9.

Abnormality of M3R expression in model mice. (A) M3R protein in SG was found significantly increased of model mice. Compared with control mice (B and E), DAB staining (C) and immunofluoresence staining (G) of M3R represented a strong intensity and localized in the acini (arrow), intercalated and striated ducts (arrow heads) of SG of model mice. (D) and (F) were nuclear staining to (E) and (G), respectively. Data were presented as mean ± S.D. of three independent experiments analyzed by independent t-test. ** P <0.01 vs control mice. Bars=20 μm.

Fig. 9.

Abnormality of M3R expression in model mice. (A) M3R protein in SG was found significantly increased of model mice. Compared with control mice (B and E), DAB staining (C) and immunofluoresence staining (G) of M3R represented a strong intensity and localized in the acini (arrow), intercalated and striated ducts (arrow heads) of SG of model mice. (D) and (F) were nuclear staining to (E) and (G), respectively. Data were presented as mean ± S.D. of three independent experiments analyzed by independent t-test. ** P <0.01 vs control mice. Bars=20 μm.

Fig. 10.

A-253 cells apoptosis induced by serum of model mice. (A–C) Representative nuclear morphology visualized by a fluorescence microscope. (A) A-253 cells without any treatment were set as negative control. (B) 1:50 diluted serum of control mice treatment alone for 1 h. (C) 1:50 diluted serum of model mice treatment alone for 1 h. (D) Cell viability of A-253 treated by mice serum. NC, negative control; Ctrl, serum of control mice with 1:50 dilution. Diluted serum: serum of model mice was diluted as 30, 50, 80 and 100:1, respectively. Data were presented as a percentage of control (n = 6, mean ± SD; *P < 0.05 and **P < 0.01 versus control).

Fig. 10.

A-253 cells apoptosis induced by serum of model mice. (A–C) Representative nuclear morphology visualized by a fluorescence microscope. (A) A-253 cells without any treatment were set as negative control. (B) 1:50 diluted serum of control mice treatment alone for 1 h. (C) 1:50 diluted serum of model mice treatment alone for 1 h. (D) Cell viability of A-253 treated by mice serum. NC, negative control; Ctrl, serum of control mice with 1:50 dilution. Diluted serum: serum of model mice was diluted as 30, 50, 80 and 100:1, respectively. Data were presented as a percentage of control (n = 6, mean ± SD; *P < 0.05 and **P < 0.01 versus control).

M3R expression abnormality

The M3Rs are expressed on salivary and lachrymal glands, and thus, they should be key receptors involved in the production of saliva and tears after stimulation of acetylcholine (42). A compensatory increase of M3R expression in acini of SS patients was reported (43) and further established in vivo (44). This feature was usually observed in SS patients associated with autonomic dysfunction (45). Therefore, we further demonstrated this finding by immunoblotting, immunohistochemistry and immunofluoresence staining of M3R in SG (Fig. 9). Figure 9A clearly showed the up-regulation of M3R expression in the SG compared with the control mice. Furthermore, we examined the M3R expression in the histological sections (Fig. 9B–G). The M3R labeling was found localized in the acini (arrows), intercalated and striated ducts (arrow heads). As similarly seen in the patients of SS (43), the bright fluorescent M3R in Fig. 9G or Fig. 9C (dark region) revealed a significant increase compared with control mice (Fig. 9B and E). Nuclei were counterstained with the DNA dye Hoechst 33258 (Fig. 9D and F).

Serum of autoimmunized mice of SS model induced cell apoptosis and nuclear condensation

We also evaluated the ability of mouse serum to induce apoptosis in vitro as mentioned above. Nuclear morphology of A-253 cells stained with Hoechst 33258 showed smaller and brighter nuclei (Fig. 10C) than those in normal cells and cells treated with serum samples of control mice (Fig. 10A and B, respectively). Some of the cells were also fragmented. Cell viability determined by the MTT assay revealed abundant cell death in a dose-dependent manner induced by serum of SS model mice (Fig. 10D).

Fig. 11.

AQP-5 proteins were decreased in A-253 cells after the treatment with various concentrations of TNF-α. Representative western blot analysis of AQP-5 isolated from A-253 cells incubated in medium containing 50, 100, 200 and 500 U/ml human TNF-α for 8 h. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control untreated cells.

Fig. 11.

AQP-5 proteins were decreased in A-253 cells after the treatment with various concentrations of TNF-α. Representative western blot analysis of AQP-5 isolated from A-253 cells incubated in medium containing 50, 100, 200 and 500 U/ml human TNF-α for 8 h. Data were presented as mean ± SD of three independent experiments analyzed by independent t-test. **P < 0.01 versus control untreated cells.

TNF-α inhibited AQP-5 expression in A-253

We also directly assessed AQP-5 inhibition by increasing expressed TNF-α on human salivary gland A-253 cell line. By the analysis of western blot, significantly decreased AQP-5 protein levels were observed after treatment with TNF-α at 50, 100, 200 and 500 U ml–1 for 8 h (Fig. 11) in a dose-dependent manner. Consistently, AQP-5 protein was decreased maximally after treatment with 100 U ml–1 of TNF-α. Identical results were found in a previous study (17).

Discussion

Although SS was firstly described by Henrik–Sjögren >70 years ago (1933), the pathogenic mechanisms still remain unknown. In an attempt to characterize and diagnosis the patients of SS, intermittent unilateral or bilateral salivary gland enlargements were reported as the evidence obtained from those who assessed xerostomia (46–49). Subsequently, abnormalities reflecting oral exocrine involvement were detected (50); and this was further indicated as the histologic changes in salivary glands consisting of lymphoid infiltration in aggregates, dilatation or proliferation of ductal epithelium or glandular atrophy in most patients (51, 52). The diminished or absent glandular secretions and mucosal dryness, as the clinical feature were further investigated and AQP-5, were then reported to be decreased and abnormally distributed in the salivary gland and played the key role of saliva secretion deficiency (13, 53).

Even though animal models that fully mimic the human disease are lacking, it is extremely useful to explain individual aspects of pathology, particularly specific molecular pathways that contribute to disease development (23). In this study, we established the experimental SS mouse model and evaluated the immune disorder and the expression of AQP-5 of this model. Followed by the swelling gland of autoimmunized mice and the increased tissue index (Fig. 1), significant accumulation of lymphocytes infiltration was further found by examination of histological sections (Fig. 2A), which was consistent with our previous study (26). Meanwhile, autoimmunization of model mice produced high levels of serum ANA (Fig. 2C). There is growing evidence indicating that acinar and ductal epithelial cells of salivary gland undergo apoptosis, which has been proposed as a possible mechanism of glandular destruction in SS (2). Bcl-2 can inhibit the apoptosis based on the evidence that Bcl-2 transgenic mice have prolonged lymphocyte survival (54). It was further supported by the inhibition of activated Bax-regulated death signal (55), and the activation of caspase-3 (cleavage) appears to be indispensable for the apoptotic process (56). These two major executioners of apoptosis were therefore determined in the mouse model with time course (Fig. 4). This was also supported by our in vitro study of A-253 cells. Since several authors had demonstrated the increased apoptosis of salivary gland epithelium together with the presence of antinuclear auto-antibodies (especially Ro and La) (57), Sisto et al. (7, 58) demonstrated that auto-antibodies in the sera of patients with SS directly triggered the apoptotic process on A-253 cells. The possible mechanism was further investigated to be associated with the activation of Fcγ receptors (59). Herein, with high serum ANA levels detection, we also observed significant apoptosis of A-253 cells induced by diluted serum of model mice (Fig. 10).

Apoptotic cell death may be induced by the dysregulated cytokines network and reflected by the over-expression of pro-inflammatory cytokines (6). Although analysis of expression of Th1 and Th2 cytokine mRNA levels in SG has generated conflicting results (60), IL-6 mRNA is highly expressed in patients with SS (61). Furthermore, IL-1β and TNF-α are reported to be up-regulated and involved in the destruction of acinar structure in human salivary glands (37, 62, 63). In addition, Chrousos (39) stated the sequence of evens at the inflammatory site, that TNF-α and IL-1β stimulate each other, and both stimulate IL-6. The productions of pro-inflammatory cytokines involved in the autoimmune responses have also been proposed to induce SS (64). In this study, we showed the up-regulated mRNA expression of cytokines TNF-α, IL-1β and IL-6 in SG and serum of model mice in response to the autoimmunization (Fig. 5). As the most efficient protease that activates precursor IL-1β (9, 40), the up-regulated activity of MMP-9 was previously seen in sera and SG of SS patients but not MMP-2 (65, 66). This may result in the weakness of the tissue structure and allow the infiltration to contribute to the glandular destruction (67). We showed a significantly high activity of MMP-9 in both serum and SG of SS model mice but not in control mice (Fig. 6). Thus, the autoimmunization-induced apoptosis and the cytokines disorder were the first time evaluated in this model.

Associated with the dysfunction of SG in the time frame, the plasma membrane of cells in SG is a major barrier to water transport and aquaporins crucially regulate the membrane permeability to water. AQP-5 is highly expressed in SG (68) and important for saliva secretion (69). In response to the autoimmunization and the glandular destruction, we found a progressive reduction of AQP-5 protein expression in SG (Fig. 8A and B). A recent study of rabbit model of SS also revealed a significant reduction of AQP-5 in the lacrimal glands (70). This may lead to a speculation that the immune response to AQP-5 plays an important role in the secretory abnormality. Some traditional therapies such as TNF-α receptor inhibitor or infliximab were reported to ameliorate this abnormality (71, 72). Therefore, TNF-α may be responsible or at least in part to the AQP-5 reduction. This was also demonstrated by Towne et al. (17). To further support the reduced AQP-5 in SG that might be response to the increased TNF-α, we used human salivary gland cell line A-253 as the cell model by direct addition of human TNF-α (Fig. 11), suggesting the cytokines disorder may contribute to the dysfunctional saliva secretion. This may also partially responsible for the increased water intake of model mice (Fig. 3) and thus suggested the saliva secretion deficiency. However, due to the complexity of the relationships between the pro-inflammatory cytokines in SS, whether the AQP-5 inhibition induced by other cytokines is lack of investigation.

Moreover, the subcellular localization of AQP-5 was demonstrated to be of functional importance in the apical membrane by allowing water transfer between the lumen and the epithelial cells and rendering them water permeable (13, 73). Similar to the healthy person, our findings showed that fluorescence-labeled AQP-5 was localized and restricted to the apical plasma membranes in SG of control mice (Fig. 8C). In contrast, SS model mice exhibited AQP-5 expression primarily at the basal membrane. Based on ImageJ 1.41o software, the labeling index showing the decrease of AQP-5 in SS model mice consistent with western blot was semi-quantitatively evaluated (data not shown). The changes in salivary AQP-5 levels of expression and distribution were used as an indicator of salivary flow since this protein correlated with the saliva secretion and the effect of M3 muscarinic acetylcholine receptor (M3R) agonist (53). In general, the activation of M3R induced the translocation of AQP-5 to the apical plasma membrane in SG (74). However, M3R expression was usually found increase in the SS patients (43), resulted from the denervation or blockage of the receptor channel (75). Inhibition of neurotransmitter release may be caused by cytokines, whose release by lymphocytes and acinar cells in the exocrine glands is increased in SS patients (76). Dawson et al. (77), however, indicated that cytokines are unlikely to affect signal transduction directly in acinar cells. There was later evidence showing that the final alternative, block of the M3R, occurs in SS. Anti-M3R antibodies that act post-junctionally to bind and block parasympathetic neurotransmission were recently demonstrated (78). Therefore, traditional therapies targeting the M3R agonist such as cevimeline and pilocarpine can only afford temporary relief of the dryness symptoms but no improvement on long-term salivary function was shown (79). The immune response to M3R was emerging recently and considered to play a role in the progression of SS (80). This notion was further supported by Sumida et al. (81), that M3R auto-antigen could induce the autoimmune reactions in the SG. This M3R immunized mouse model disclosed the increased M3R reactive T cells, anti-M3R auto-antibodies, over-expressed M3R and reduced saliva volume, suggesting the importance of the pathogenesis of M3R in SS. Our findings showed the M3R expression was increased in model mice and localized in acinar structures (Fig. 9). Conversely, the up-regulated expression of M3R does not activate the translocation of AQP-5 to the apical membrane in the SG of model mice (Fig. 8), suggesting that the M3R was not activated and not fully functional. Instead of the activation of water channel in the acini, the over-expressed M3R and increased water intake suggested the long-term blockage during the autoimmunization. This was explained by Beroukas et al. (43,82) that the increased M3R was likely to be a compensatory effect in response to the M3R blockage in SS. Thus, our findings revealed the consistent pattern of AQP-5 and M3R that seen in the SS patients in this autoimmunization-induced mouse model, and the further study focusing on the anti-M3R auto-antibodies is necessary.

Conclusions

In summary, we describe herein developmental lymphocytic infiltration in the SGs by autoimmunization of female C57BL/6 mice. Further, the severe inflammation is accompanied by apoptosis and the dysregulation of pro-inflammatory cytokines. The observed up-regulated M3R, translocation of AQP-5 to basal membrane domain and decreased expression may contribute to the increased water intake. These results lend credence to the contention that the autoimmunization model recapitulated the key features of human SS. This model may have potential for studying the pathogenesis and screening therapeutic drugs of human SS.

Funding

University of Hong Kong Project No. (201011159206 and 201007160006)

We thank Mr Song for assistance in examination of tissue pathology and Dr Shaw, Dr Ng and Dr Sze for providing reagents.

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