Results

Caspase-11 gene expression is upregulated in the exocrine glands of SjS-susceptible mice

To confirm our earlier results⁴ obtained with microarray analyses showing an increased gene expression of caspase-11 in the SMX of 8-week-old C57BL/6.NOD-Aec1Aec2 mice carrying two NOD-derived genetic intervals (Figure 1a), the levels of caspase-11 mRNA in both the SMX of 8- and 12-week-old NOD/LtJ, C57BL/6.NOD-Aec1Aec2 and C57BL/6J mice were determined by semiquantitative reverse transcription-PCR. As presented in Figure 1b, caspase-11 expression was increased 4.6-fold (P<0.01) in the SMX of C57BL/6.NOD-Aec1Aec2 mice compared with C57BL/6 mice at 8 weeks of age, decreasing slightly by 12 weeks of age (bar graphs). Interestingly, caspase-11 gene expression was also increased 1.7-fold (P<0.05) and 1.8-fold (P<0.05) in the lacrimal glands of both C57BL/6.NOD-Aec1Aec2 and NOD/LtJ mice at 8 weeks of age, and remained elevated at 12 weeks of age (data not presented).

Figure 1.

Increased caspase-11 expression in the SMX of the SjS-prone C57BL/6.NOD-Aec1Aec2 mouse before lymphocytic infiltration. (a) Two NOD-derived genetic intervals, namely autoimmune exocrine loci 1 and 2 on chromosomes 3 and 1, respectively, in the disease-prone C57BL/6.NOD-Aec1Aec2 mouse are depicted. (b) Elevated caspase-11, its major transcription factors STAT-1, and Nfkb1 in the salivary glands of C57BL/6.NOD-Aec1Aec2 were confirmed by semiquantitative RT-PCR. (c) Caspase-11 in the SMX was stained with FITC-labeled anti-mouse caspase-11 antibody in the C57BL/6.NOD-Aec1Aec2 at 8 weeks. Arrows indicate positive staining for caspase-11. Magnification, 20. (d) Double staining of caspase-11 with anti-Cd11c antibody (dendritic cells) and anti-F4/80 antibody (macrophages) revealed that cells positive for caspase-11 were also positive for both cell types. Arrows indicate double-stained cells, which are shown in yellow. Magnifications, 10 and 40. Aec, autoimmune exocrine loci; RT-PCR, reverse transcription-polymerase chain reaction; SjS, Sjögren's syndrome; SMX, submandibular glands; STAT, signal transducers and activators of transcription.

Full figure and legend (318K)

To determine whether either of the known transcription factor STAT-1 and NF-B for caspase-11 is concomitantly upregulated with caspase-11, their gene expressions were measured (Figure 1b). STAT1 gene expression proved to be upregulated in the SMX of both NOD/LtJ and C57BL/6.NOD-Aec1Aec2 mice (2.3-fold, P<0.01) at 8 weeks of age. Similarly, expression of Nfkb1 (p50) was upregulated in the SMX of C57BL/6.NOD-Aec1Aec2 mice. In contrast, Nfkb2 (p52) was slightly downregulated in the SMX of both C57BL/6.NOD-Aec1Aec2 and NOD/LtJ mice at 8 weeks of age. By 12 weeks of age, Nfkb1 was downregulated in the SMX, whereas Nfkb2 remained at low levels.

Caspase-11 expression is detected in macrophage and dendritic cells

Caspase-11 protein expression was confirmed by immunohistochemistry on the SMX of 8-week-old C57BL/6.NOD-Aec1Aec2 mice (Figure 1c). A slide of human breast cancer tissue was used as a positive control for anti-caspase-11 antibody activity (inset). To localize caspase-11 protein expression in the salivary glands, fluorescein isothiocyanate (FITC-) or phycoerytherin (PE-) (or Texas Red-) labeled antibodies were used for caspase-11 and other cell-type markers. Double-staining caspase-11 expressing cells with a dendritic cell (CD11c) or a macrophage (F4/80) cell marker revealed that caspase-11 was expressed by both cells, showing positively stained cells in yellow when the images were merged (Figure 1d). Caspase-11-positive cells surrounded acinar and/or ductal cell units as indicated by white arrows in Figure 1d.

STAT-1 but not NF-B activity is concomitantly upregulated with caspase-11 in the salivary glands of SjS-susceptible mice

Elevated STAT1 and Nf-b1 gene expression in the SMX of 8-week-old C57BL/6.NOD-Aec1Aec2 mice raised the question, which transcription factor induces caspase-11 gene expression, leading us to perform electrophoretic gel mobility shift assays (EMSAs). Nuclear extracts were prepared from pooled SMX freshly explanted from 8-week-old female C57BL/6.NOD-Aec1Aec2 mice, incubated with biotin-labeled oligonucleotides specific for the DNA binding site of either STAT-1 or NF-B, and separated by PAGE. As shown in Figures 2a and b, NF-B showed no statistically significant increase in activity, whereas STAT-1 activity (upregulated >20%) was found in the SMX of 8-week-old NOD/LtJ and C57BL/6.NOD-Aec1Aec2 mice, when compared with age- and sex-matched C57BL/6 mice. Furthermore, a doubling of the amount of nuclear extract for the NF-B assay only slightly increased visualization. In the presence of unlabeled (or cold) oligonucleotide probe, binding was decreased indicating binding specificity. Unexpectedly, the binding activities of STAT-1 and NF-B in nuclear extracts prepared from C57BL/6.NOD-Aec1Aec2 lacrimal glands were consistently below detection levels by EMSA, supporting the dichotomy observed in the underlying pathology and timing of SjS-like disease progression within the SMX versus lacrimal glands of NOD/LtJ and C57BL/6.NOD-Aec1Aec2 mice.

Figure 2.

Concomitant increase in STAT-1 activity in the SMX of C57BL/6.NOD-Aec1Aec2 at 8 weeks. (a) Increased STAT-1 activity was detected by EMSA in the glands isolated at 8 weeks. Inhibition of binding is shown with a cold probe (unlabelled probe) incubation. The right panel indicates bar graphs generated by densitometer analyses on EMSA results for NF-B. (b) Absence of STAT-1 and NF-B activity in the lacrimal gland (LAC) at 8 weeks was detected whereas elevated STAT-1 was shown in the SMX of C57BL/6.NOD-Aec1Aec2 and NOD/LtJ mouse. Increased amount of nuclear extract (ten micrograms) isolated from 0.5 g of pooled glands (n=5–7 mice) was used per lane to enhance binding activity for NF- B. The right panel indicates bar graphs generated by densitometer analyses on EMSA results for STAT-1. The experiments were carried out three times per molecule of interest. ^*P<0.05 in comparison with C57BL/6. EMSA, electrophoretic gel mobility shift assay; NF-B, nuclear factor-B; SMX, submandibular glands; STAT, signal transducers and activators of transcription.

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Caspase-11 activates caspase-1 but not caspase-3

To better define the consequences of elevated caspase-11 in the SMX of an SjS mouse model during the preclinical phase of disease when apoptosis of acinar tissue is prevalent, we analyzed the activity of caspase-1 and caspase-3. As shown in Figure 3a, caspase-1, but not caspase-3, activity was upregulated in the SMX of 8-week-old C57BL/6.NOD-Aec1Aec2 mice compared with C57BL/6 mice, although the baseline activities for caspase-3 were higher overall. To confirm activation of the caspase-1 pathway, we examined whether IL-1 and/or IL-18, two factors whose secretion is strongly regulated by the activation of caspase-1,⁶ were also upregulated. Reverse transcription-PCR analyses indicated highly elevated IL-1 and IL-18 in the SMX of 8-week-old C57BL/6.NOD-Aec1Aec2 mice (Figure 3b). Interestingly, activation of caspase-1 in the local target tissue (that is, the salivary glands) of SjS corresponded with upregulated IL-18 cytokine expression in the saliva but not in sera before disease onset (Figure 3c). Gradual increase in IL-18 production in saliva as well as sera was apparent at 12 weeks, which is the time when lymphocytes start infiltrating into the SMX.

Figure 3.

Activation of caspase-1-mediated pathway in C57BL/6.NOD-Aec1Aec2 before disease onset. (a) Caspase-1 activity was upregulated significantly in the SMX of C57BL/6.NOD-Aec1Aec2 at 8 weeks. Experiments were performed in triplicate. ^**P<0.01 in comparison with C57BL/6. (b) Genes downstream of caspase-1 such as IL-1 and IL-18 in the C57BL/6.NOD-Aec1Aec2 mouse at 8 weeks were analyzed by RT-PCR. The C57BL/6.NOD-Aec1Aec2 mice were positive for these genes whereas the SMX from other mouse strains showed either negative or weak expression (n=5–7 female mice). -actin was used as a control for normalization. (c) IL-18 protein expression was elevated in the saliva from C57BL/6.NOD-Aec1Aec2 at 8 weeks by ELISA. Pooled saliva and sera were used for ELISA (n=5 female mice). ^*P<0.05 and ^**P<0.01 in comparison with the age-matched C57BL/6 mouse. IL, interleukin; RT-PCR, reverse transcription-polymerase chain reaction; SMX, submandibular glands; ELISA, enzyme-linked immunosorbent assay.

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Apoptosis is more prevalent in the SMX of SjS-prone mice before disease onset in comparison with disease-free mice

To identify whether activation of caspase-11 with subsequent activation of the caspase-1 pathway is associated with apoptotic events in the exocrine glands of SjS-like disease-susceptible mice, apoptotic cell death in the SMX of 8-week-old NOD/LtJ and C57BL/6.NOD-Aec1Aec2 mice was examined. Histological sections of salivary glands were prepared and transferase-mediated dUTP-biotin nick end labeling (TUNEL) stained (Figures 4A and B). Visualization using a fluorescent microscope fitted with a red filter for Cy3 revealed that both NOD/LtJ and C57BL/6.NOD-Aec1Aec2 mice showed more abundant apoptotic cell death than C57BL/6 control mice (Figure 4B). Nuclease-treated slides (PC in Figure 4A) were served as a positive control.

Figure 4.

Increased epithelial cell death in the glands of disease-prone mice at 8 weeks and lack of direct colocalization of caspase-11 with TUNEL-positive cells. (A) TUNEL staining was performed on the prediseased salivary glands; upper panel at 10 and lower panel at 40 magnifications. (B) Percentages of TUNEL-positive cells are shown as a bar graph. For each mouse, three slides were evaluated for TUNEL-positive cells, which were counted using a cell counter. (C) Caspase-3-positive cells (yellow arrows in b) were colocalized with TUNEL-positive cells (red arrows). White arrows indicate caspase-11-positive cell. Magnification, 40. NC, negative control; PC, positive control treated with nuclease; TUNEL, transferase-mediated dUTP-biotin nick end labeling.

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Caspase-11 is not detected in TUNEL-positive acinar or ductal cells

Experiments were carried out to investigate whether caspase-11 plays a direct role in increased apoptotic acinar cell death in the salivary glands before disease onset. Freshly prepared sections of SMX from 8-week-old C57BL/6.NOD-Aec1Aec2 mice, first treated to identify TUNEL-positive cells, were counterstained with FITC-conjugated anti-mouse caspase-11 antibody. As presented in Figure 4C, caspase-11 (white arrow) was present in the cells located outside or between the acinar or ductal areas, whereas TUNEL-positive cells (red arrows) were acinar or ductal cells. Although these dying cells were colocalized with caspase-3 (Figure 4C-a, yellow arrow) showing positivity inside the cytoplasm, our finding indicates that not all TUNEL-positive cells were positive for caspase-3.

Caspase-1 in conjunction with IFN- is essential to increased apoptotic cell death of human salivary gland epithelial cells

Lack of colocalization between caspase-11- and TUNEL-positive cells led us to hypothesize that caspase-11 functions in a noncell autonomous manner, rather than directly killing the cells, by activating caspase-1 and a subsequent pro-inflammatory cytokine release into the microenvironment, resulting in increase in apoptotic cell death of salivary epithelial cells. This hypothesis was tested using a human salivary gland (HSG) cell line⁹ cocultured with a THP-1 human monocyte cell line, which was stimulated with LPS for the induction of IL-1 and IL-18 in the presence and absence of IFN-, in an attempt to duplicate observations in the in vivo environment. Our earlier data from an SjS mouse model lacking IFN-² showed the absence of disease and predisease phenotype, clearly indicating that IFN- is critical not only for the onset of the disease, but also for the predisease stage. In addition, in the NOD mouse, IFN- was upregulated by two-fold in the SMX before disease onset.² Our current data indicate that increased apoptosis of HSG cells occurred only in the presence of LPS-stimulated THP-1 cells when IFN- was present (Figures 5a and b, P<0.01). The increased rate of apoptotic cell death was reversed to a normal level when caspase-1 transcription in THP-1 cells was downregulated by siRNA (Figure 5). The normalized caspase-1 knockdown efficiency was greater than 70%, as shown in Figure 5c (P<0.01).

Figure 5.

Inhibition of apoptotic cell death of HSG cells by caspase-1 knockdown in THP-1 cells. (a) HSG cells were cultured in the absence or presence of LPS- and/or IFN--stimulated THP-1 cells. After removing culture media or stimulated THP-1 cells from the culture, apoptotic HSG cells were analyzed by TUNEL assays. For the caspase-1 inhibition study, siRNA to caspase-1 was transfected into THP-1 cells before stimulation with LPS and IFN- and cocultured with HSG cells. (b) Percent TUNEL-positive cells were presented as a bar graph. The experiment was repeated three times for reproducibility (^**P<0.01). (c) Knockdown efficiency of caspase-1 was compared with a housekeeping protein golgin-97 by western blotting and depicted as a bar graph. The experiment was repeated twice and caspase-1 protein level was normalized to golgin-97. HSG, human salivary gland; IFN, interferon; LPS, lipopolysaccharide; TUNEL, transferase-mediated dUTP-biotin nick end labeling.

Full figure and legend (193K)

Discussion

Recent identification of caspase-11 as one of the differentially expressed genes at 8 weeks of age in SjS-prone C57BL/6.NOD-Aec1Aec2 mice, together with an abnormal glandular homeostasis in prediseased NOD mice, led us to hypothesize the importance of intrinsic properties of target tissues (such as availability of antigen(s) and changed activity of antigen presenting cells by pro-inflammatory cytokines) for the breakdown of peripheral tolerance and the activation of autoreactive immune cells.^{1, 3, 4, 10, 11} Our efforts at understanding initial molecular events triggering onset of SjS identified three important findings pertaining to a caspase-11-mediated pathway in the salivary glands. First, caspase-11 expressed primarily in macrophages/dendritic cells is upregulated in the SMX before disease onset and is apparently associated with the enhanced transcriptional activity of STAT-1. Second, these events seem to lead to secretion of pro-inflammatory cytokine production from the local tissue through caspase-1 activation, as indicated by elevated IL-18 levels in saliva, capable of inducing increased epithelial cell death rather than cell-autonomous killing of epithelial cells. Third, caspase-1 in macrophages/dendritic cells and IFN- in the salivary gland microenvironment play a critical role in the death of residential epithelial cells, shown both in vivo and in vitro analyses. This scheme is illustrated in Figure 6.

Figure 6.

Schematic representation of the current working hypothesis. Inductive viral profile results in alterations in the target tissue through the activation of IFN-STAT and a subsequent induction of caspase-11. Upregulated caspase-1 activity produces mature IL-1 and IL-18 from macrophages and dendritic cells, which may play a role in the activation of caspase-3 or induction of caspase-3-independent apoptotic factors in neighboring acinar and/or ductal cells. Elevated pro-inflammatory cytokines in the SMX enhances IFN- production by epithelial cells, resulting in further activation of macrophages. Areas where further investigation is needed for confirmation are depicted with dotted lines (that is, questions as to whether caspase-11 is a constituent of the NALP inflammasome or why IFN- and consequently STAT-1 are upregulated in the SMX before disease onset). The assembled proteins (the inflammasome) in response to signal recognition by LRR of the NALP leads to the activation of caspase-1, depicted in the bracket. Our current findings are depicted in bold arrows. ASC, apoptosis–associated speck-like protein containing a caspase-activating recruitment domain; DC, dendritic cells; IFN, interferon; IL, interleukin; LRR, leucine-rich repeats; NALP, NACHT-, LRR- and PYD-containing proteins; SMX, submandibular glands; STAT-1, signal transducers and activators of transcription.

Full figure and legend (184K)

Recently, we postulated that a potential signal for induction of STAT-1 transcriptional activity may come from latent/recurrent viral infection in the salivary glands, especially reactivation of endogenous virus in case of mice housed under specific pathogen-free (SPF) conditions, thus promoting interferon induction as an anti-viral defense mechanism by epithelial cells or natural killer cells in the glands. Increased IFN-, known to be present in exocrine glands of NOD-derived mice,² can enhance the activity of macrophages and/or dendritic cells in the tissues, resulting in the production of caspase-11. This is supported by the fact that caspase-11 is known to be induced only when signals from IFN- or LPS through the activation of STAT-1 or NF-B transcription factors are synthesized.

A study on experimental autoimmune encephalomyelitis indicates that caspase-11 is highly expressed in both oligodendrocytes and infiltrating cells and colocalized with activated caspase-3, suggesting that a pathway involving caspase-11 and caspase-3 is important in the execution of oligodendrocyte death in experimental autoimmune encephalomyelitis lesions.⁵ However, the role of caspase-11 found in the SMX of SjS-prone C57BL/6.NOD-Aec1Aec2 mice differed from that in experimental autoimmune encephalomyelitis models in that caspase-11 in our model was neither produced by dying cells nor colocalized with caspase-3. In our SjS model, caspase-11 is apparently correlated with caspase-1 activity. Furthermore, abundant TUNEL-positive cells in the SMX of C57BL/6.NOD-Aec1Aec2 mice, together with decreased caspase-3 activity and the fact that not all TUNEL-positive cells were positive for caspase-3, suggest that acinar cell apoptosis induced by pro-inflammatory cytokines in the microenvironment may involve a caspase-3-independent pathway.

During early disease pathogenesis of SjS, the roles of IFN- seem to be indispensable based on our earlier study² as well as our current study. IFN- is required but not sufficient for the induction of increased cell death in the target tissue of SjS. In addition, our earlier studies² indicate that in the absence of IFN-, mice exhibited neither secretory dysfunction nor alterations in predisease markers, strongly conclusive of our current findings. The requirement for caspase-1 and IFN- in enhanced cell death, proven by in vitro coculture study with siRNA targeting caspase-1 in THP-1 cells, may originate from the fact that the IFN- is essential for caspase-1 activation, which cleaves pro-IL-1 and IL-18 to produce mature forms of IL-1 and IL-18 for its release.¹² Therefore, synergistic effects between cytokines induced and cleaved by the activation of caspase-11 and caspase-1, and IFN-/STAT-1 are essential for driving chronic inflammatory conditions in the targeted glands even before disease onset.

Increased expression of IL-1 can cause the activation of the signal cascade leading to the activation of several transcription factors involved in inflammatory responses.¹³ IL-18, originally described as an IFN--inducing factor, is a potent inflammatory stimulant produced by macrophages and dendritic cells and is known to enhance antigen-specific clonal expansion of IFN--producing T cells,¹⁴ suggesting that IL-18 may impact T-cell immunity in both nonlymphoid and lymphoid tissues by bridging the innate and adaptive arms of the immune system through IFN- during the early stage of SjS. A study also indicates that IL-18 produced by Kupffer cells stimulates T_H1 and natural killer cell cytotoxic activity by increasing their production of FasL (CD 95), which ultimately induces apoptosis in Fas-bearing hepatocytes, causing liver injury.¹⁵ In a similar manner, IL-18 produced by phagocytic cells may upregulate FasL on natural killer cells and Fas on epithelial cells in the SMX through caspase-1 activation, resulting in subsequent apoptotic processes in the epithelial cells. Interestingly, IL-18 and IL-1 have been reported to be upregulated in both sera and the SMX of SjS patients.^{16, 17} Considering the results of our current study, one might speculate that IL-1 and IL-18 are upregulated in the target tissue of SjS patients as well, starting at the early disease stage.

Recent studies indicate that a set of caspases (that is, human caspase-1, caspase-4 and caspase-5, along with murine caspase-11 and caspase-12), considered as 'inflammatory caspases', are involved in the proteolytic maturation of inflammatory cytokines.^{18, 19} Recent studies have identified a complex of proteins, referred to as the 'inflammasome', functions in innate immunity by regulating inflammatory caspase-1 activation.^{20, 21, 22} Proteins that make up the inflammasomes are members of the NACHT-, LRR- and PYD-containing proteins (NALP) family of proteins, although information on its exact expression or binding partners is relatively scarce.²³ The inflammasomes are hypothesized to act as an early sensor detecting danger signals because the stimulation of the inflammasome triggers a series of internal reactions that ultimately activates caspase-1, which subsequently produces mature IL-1 and IL-18 for regulating immune cells. Important questions that we are currently investigating include whether caspase-11 activates caspase-1 as a part of the inflammasomes, whether increased epithelial cell death in the target tissues confers target tissue specificity in autoimmune SjS and whether abnormal regulation of the inflammasome translates to SjS in humans. Our current results seem to point to the potential existence of abnormal regulation of the inflammasome in the SMX of NOD-derived SjS-like disease-susceptible mice (Figure 6).

In summary, this study shows that caspase-11 plays a critical role in the susceptibility of mice to SjS-like disease by upregulating caspase-1-mediated pathway, which is vital for apoptotic cell death of the neighboring epithelial cells. In addition, the presence of IFN- in the environment is essential for caspase-1-induced cell death of salivary epithelial cells. The repeated occurrence of pro-inflammatory cytokine secretion and apoptotic cell death following reactivation of latent or persistent viral infection may lead to chronic inflammatory conditions in the salivary glands in SjS. STAT-1 activation rather than NF-B activation in the SMX of disease-prone mice seems to be responsible for caspase-11 induction. Overall, our observations underscore the potentially critical roles of myeloid cell populations and of intracellular pattern recognition through the inflammasomes activating caspase-1 in the early pathogenesis of SjS. Crossing knockouts onto the SjS-prone mouse strain will confirm critical roles of inflammatory caspases and their therapeutic values in delaying disease onset and progression of SjS.

Methods

Animals

C57BL/6J, C57BL/6.NOD-Aec1Aec2 and NOD/LtJ were bred and maintained under SPF conditions within the Animal Care Services at the University of Florida, Gainesville. The animals were maintained on a 12 h light--dark schedule and provided water and food ad libitum. For this study, female mice were killed at either 8 or 12 weeks of age. Both breeding and use of these animals were approved by the University of Florida IACUC. The mice were killed using American Veterinary Medical Association approved procedures.

Expression profiles of caspase-11 and related molecules by reverse transcription-PCR

Total RNA was prepared from freshly isolated SMX using the RNeasy Mini Kit (Qiagen, Valencia, CA, USA). Semiquantitative PCRs were carried out using 1 g of cDNA as template. Following an initial denaturation at 94 °C for 4 min, each PCR was carried out for 34 cycles consisting of 94 °C for 30 s, optimal annealing temperature for 30 s and 72 °C for 1 min. PCR products were analyzed by electrophoresis using 2% agarose gels. PCR band intensities were compared with -actin using the Flourchem Imaging densitometer system (Alpha Innotech Corporation; San Leandro, CA, USA). The primer sequences are as follows:

-actin-forward: 5'-CCTGACCCTAAGGCCAACCG-3' (398 bp; 57 °C);

-actin-reverse: 5'-GCTCATAGCTCTTCTCCAGGG-3';

STAT1-forward: 5'-TCCCGTACAGATGTCCATGA-3' (84 bp; 57 °C);

STAT1-reverse: 5'-GCCTGATTAAATCTTTGGGCA-3';

Caspase-11-forward: 5'-ATGGCCGTACACGAAAGGCTCTTA-3' (376 bp; 57 °C);

Caspase-11-reverse: 5'-GCCTGCACAATGATGACTTTGGGT-3';

Nfkb1-forward: 5'-TGAAGCAGCTGACAGAAGACACGA-3' (350 bp; 56 °C);

Nfkb1-reverse: 5'-TTCATCTATGTGCTGCCTCGTGGA-3';

Nfkb2-forward: 5'-AGTTGACTGTGGAGCTGAAGTGGA-3' (336 bp; 61 °C);

Nfkb2-reverse: 5'-TGGCCTCGGAAGTTTCTTTGGGTA-3';

IL-1-forward: 5'-CTCCATGAGCTTTGTACAAGG-3' (245 bp; 55 °C);

IL-1-reverse: 5'-TGCTGATGTACCAGTTGGGG-3';

IL-18-forward: 5'-ACTGTACAACCGCAGTAATACGG-3' (434 bp; 55 °C);

IL-18-reverse: 5'-AGTGAACATTACAGATTTATCCC-3'.

Analysis of STAT-1 and NF-B transcriptional activity by electrophoretic mobility shift assay

The SMX and lacrimal glands from SjS-prone C57BL/6.NOD-Aec1Aec2, NOD/LtJ and disease-resistant C57BL/6 mice at 8 weeks were analyzed by EMSA. Pooled glands (0.5 g) from each strain were used to obtain 5–10 g l^-1 concentration of nuclear extract following the instructions in the Nuclear Extraction Kit (Panomics, Inc., Redwood City, CA, USA). Nuclear extracts were incubated with 2.0 l of 5 binding buffer, 1.0 l of poly d (I-C) (1 g l^-1), 1.0 l of biotin-labeled STAT-1 or NF-B probe (10 ng l^-1) and 5.0 l of distilled water at 15–20 °C for 30 min. For negative controls, an unlabeled cold STAT-1 or NF-B probe was added. Samples (5–10 g per lane) were run on a 6.0% polyacrylamide gel at 4 °C using 120 V. After electrophoresis, the samples were transferred onto Pall Biodyne B membranes (Pall Corporation, Ann Arbor, MI, USA) by electroblotting (300 mA). The membrane was baked for 1 h at 85 °C in a dry oven for immobilization. Blocking, incubating with streptavidin-HRP conjugate, washing, developing with hydrogen peroxide and luminol and membrane exposures on Hyperfilm ECL for 30 s were performed following the manufacturer's instruction for the EMSA kit (Panomics).

Caspase activity assay

The SMX from disease-prone C57BL/6.NOD-Aec1Aec2, NOD/LtJ and disease-resistant C57BL/6 mice were analyzed at 8 weeks. From each strain, 1–1½ glands were used to acquire 3–7 g l^-1 concentration of gland lysate following manufacturer's instructions from BioVision (Mountain View, CA, USA). Gland lysates (50 g per well) were placed into a 96-well flat bottom plate and incubated in the dark with 50 l of 2 reaction buffer (containing 10 mM dithiothreitol) and 5.0 l of either 1 mM YVAD-AFC (7-amino-4-trifluoromethyl coumarin) substrate (50 M final concentration) for caspase-1 or DEVD-AFC for caspase-3 followed by incubation at 37 °C for 90 min. Negative controls consisted of the same reactions in the absence of gland lysate. After incubation, the samples were read in a microplate fluorometer equipped with a 400-nm excitation filter and 505-nm emission filter. Experiments were performed in triplicate.

Measurement of apoptotic cells by TUNEL staining and colocalization with caspases

The SMX and lacrimal glands were freshly explanted and fixed in 10% neutral-buffered formalin for additional processing. After deparaffinization, slides were placed for antigen retrieval in 0.1 M citrate buffer, pH 6.0 (Biogenex, San Ramon, CA, USA), and microwaved (350 W) for 6 min. Slides were washed twice in phosphate-buffered saline and the tissues were stained following the instructions provided in the in situ Cell Detection Kit, TMR Red (Roche Applied Science, Indianapolis, IN, USA). Slides were analyzed under a fluorescent microscope using an excitation wavelength in the range of 520–560 nm (maximum 580 nm, red) (Carl Zeiss Inc., Thornwood, NY, USA).

For colocalization with caspase-11, separate sets of TUNEL-stained slides were treated first with blocking buffer (Dako, Fort Collins, CO, USA), stained for 1 h with rabbit anti-mouse caspase-11 antibody (Calbiochem, San Diego, CA, USA) diluted 1:50, and then developed for 45 min using a FITC-conjugated donkey anti-rabbit IgG antibody (Molecular Probes, OR, USA) diluted 1:1000. As this antibody for murine caspase-11 is cross-reactive with human caspase-4, slides with human breast cancer were used as positive controls following the manufacturer's guideline. For colocalization with caspase-3, TUNEL-stained slides were stained with rabbit anti-mouse caspase-3 antibody (Abcam, Cambridge, MA, USA) at 1:50 dilution for 1 h, and then, incubated with FITC-conjugated donkey anti-rabbit IgG antibody (Molecular Probes) at 1:1000 dilution for 45 min. Stained sections were mounted with 4',6-diamidino-2-phenylindole mounting medium (Vector, Burlingame, CA, USA) and observed at 20 and 40 magnifications. Slides were analyzed under a fluorescence microscope (Carl Zeiss) using an excitation wavelength in the range of 520–560 nm (maximum 580 nm, red) for TUNEL-positive cells and 488 nm range for caspase-11- or caspase-3-positive cells.

Identification of caspase-11 expressing cells

Slides of freshly explanted SMX from female C57BL/6.NOD-Aec1Aec2 mice at 8 weeks of age were prepared for immunostaining as described above. Antigen retrieval was performed by incubating the slides in Trilogy (Cell Marque, Austin, TX, USA) for 30 min at 95 °C. One set of slides was incubated first with rabbit anti-mouse caspase-11 antibody (Calbiochem), followed by FITC-conjugated donkey anti-rabbit IgG antibody, as described above. This set of slides was then counterstained with PE-conjugated hamster anti-mouse CD11c antibody (BD Biosciences, San Jose, CA, USA) at 1:100 dilution for 45 min. A second set of slides was first incubated with a solution containing both rabbit anti-mouse caspase-11 antibody and rat anti-mouse F4/80 antibody (Serotec, Raleigh, NC, USA) at 1:50 dilution for 1 h, then a solution containing both FITC-conjugated donkey anti-rabbit IgG antibody (Molecular Probes) at 1:1000 dilution and AffiniPure Texas Red conjugated rabbit anti-rat IgG antibody (Jackson ImmunoResearch, West Grove, PA, USA) at 1:100 dilution and incubated for 45 min. Stained sections were mounted with 4',6-diamidino-2-phenylindole mounting medium (Vector) and observed at 20 and 40 magnifications.

Measurement of mouse IL-18 by enzyme-linked immunosorbent assay

Mouse sera and saliva from C57BL/6 and C57BL/6.NOD-Aec1Aec2 strains (n=5) at 8 and 12 weeks of age were tested for mouse IL-18 with a sandwich ELISA kit (MBL International, Woburn, MA, USA), following the manufacturer's instructions.

Detection of apoptotic HSG cells cocultured with THP-1 cells

THP-1 human monocytes obtained from American Type Culture Collection (Manassas, VA, USA) were cultured in RPMI 1640 medium with supplements. HSG cells were seeded at 5 10⁵ cells per well in 6-well plates containing glass coverslips and cultured in complete media. The next day, HSG culture media were removed from the cells and 5 10⁵ THP-1 cells in 1 ml THP-1 growth media containing 2 g ml^-1 LPS (Sigma, St Louis, MO, USA) and 10 ng ml^-1 IFN- (BD Biosciences) were added. The THP cells were incubated for 48 h at 37 °C before removal. HSG cells on coverslips were fixed in 4% paraformaldehyde for 1 h and permeabilized with 0.1% Triton X-100 in 0.1% sodium citrate buffer for 2 min on ice. To detect apoptotic cells, the In Situ Cell Death Detection Kit, TMR red (Roche Applied Science) was used according to the manufacturer's protocol. Fluorescence images were taken with Zeiss Axiovert 200 M microscope and a Zeiss AxioCam MRm camera using the 10 0.75 NA objectives. Color images were assessed using Adobe Photoshop version 7. Cells were counted using Cell-Profiler image analysis software²⁴ to detect 4',6-diamidino-2-phenylindole staining, and TUNEL-positive cells were counted using a cell counter.

Caspase-1 siRNA transfection

siRNA targeting caspase-1 was transfected into THP-1 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to manufacturer's instructions. The siRNA used in this study was purchased from Ambion (Austin, TX, USA) and dissolved in nuclease-free water, and the resulting 20 M stock was stored at -80 °C before use. The sense and antisense strands are as follows: 5'-GGUUCGAUUUUCAUUUGAGtt-3' and 5'-CUCAAAUGAAAAUCGAACCtt-3'. THP-1 cells transfected with siRNA targeting caspase-1 were incubated for 48 h, washed once in growth media, and then cocultured with HSG cells as described above.

Verification of caspase-1 knockdown by western blotting

THP-1 cells transfected with siRNA targeting caspase-1 were lysed 48 h after transfection, and cell extracts were loaded onto a 10% sodium dodecyl sulfate-PAGE gel and transferred to nitrocellulose. The following antibodies and dilutions were used: rabbit anti-caspase-1 antibodies at 1:50 (Abcam) and rabbit anti-golgin-97 antibodies at 1:200.²⁵ Secondary goat anti-rabbit antibodies conjugated to horse radish peroxidase were used at 1:10 000 dilutions (Southern Biotech, Birmingham, AL, USA). Densitometric analysis of the developed film was performed using Image J software. Caspase-1 protein levels were normalized to golgin-97 to determine knockdown efficiency.

Statistical analyses

Statistical significances were determined using the Student's t-test. P<0.05 values were considered to be significant.

Conflict of interest

The authors state no conflict of interest.

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Acknowledgements

This study was supported by NIH grants U24 DE016509 (SC), DE016705 (SC), and DE015152 (ABP). We acknowledge Miss Reshma Patel for her assistance with immunofluorescent staining for caspase-11 and caspase-3.