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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Arthritis Rheum. Author manuscript; available in PMC Aug 5, 2009.
Published in final edited form as:
PMCID: PMC2721328
NIHMSID: NIHMS130450

Inflammatory Stimuli Accelerate Sjögren’s Syndrome-like Disease in NZB/WF1 Mice

Umesh S. Deshmukh, PhD, Yukiko Ohyama, DDS, Harini Bagavant, MBBS, PhD, Xiaoti Guo, DDS, Felicia Gaskin, PhD, and Man Shu Fu, MD, PhD

Abstract

Objective

To determine whether induction of systemic inflammation would accelerate Sjögren’s syndrome (SS) development in genetically susceptible mice.

Methods

Female NZB/WF1 mice were treated either with Freund’s incomplete adjuvant (IFA) or PBS at monthly intervals. Salivary gland function was monitored by measuring pilocarpine induced saliva volume. Mice were sacrificed at different time points and investigated for sialoadenitis and salivary gland infiltrating cells. Sera were analyzed for autoantibodies to salivary gland antigens, nuclear antigens and Ro60.

Results

While IFA treated mice had significantly decreased salivary secretion 7 weeks after the initial treatment, PBS treated controls did not develop it until 17 weeks. At 7 week time point, the severity of sialoadenitis and the number of T and B cells infiltrating the salivary glands was not different between the two groups. However, at this time point the IFA treated mice showed significantly higher frequency of B220+, CD11clo, Ly6C+, mPDCA-1+, dendritic cells in the salivary glands. While levels of autoantibodies between the two groups were not different at early time point, by late time point IFA treated mice had higher levels.

Conclusion

Our data suggest that generalized inflammatory stimuli can accelerate the development of SS-like disease in NZB/WF1 mice. The glandular dysfunction at earlier time points did not correlate with the severity of sialoadenitis or levels of autoantibodies. Instead, it was associated with increased frequency of plasmacytoid dendritic cells in the gland. Thus, glandular dysfunction in SS can develop prior to the generation of a robust adaptive autoimmune response.

Introduction

Sjögren’s syndrome (SS) is a chronic autoimmune disorder mainly affecting the salivary and lacrimal glands. The disease is characterized by infiltration of mononuclear cells into the glands leading to the formation of multiple lymphoid foci and glandular destruction (1). Autoantibodies to Ro60, a ubiquitously present ribonucleoprotein, are often found in the sera of patients with SS. However, their contribution towards the pathogenesis of the disease is not understood. Also not clear are the events initiating lymphocytic infiltration into the exocrine glands. However, it is widely believed that interaction of environmental and genetic factors is responsible. This study was designed to test the hypothesis that a sustained non-specific inflammatory stimulus in genetically susceptible individuals would lead to the acceleration of SS-like disease. To test this hypothesis we decided to employ the NZB/WF1 mice as a genetically susceptible mouse strain and IFA as an inflammatory stimulus. The NZB/WF1 mouse is one of the earliest described models for spontaneous SS and lupus (2). Although effects of exogenous triggers such as pristane (3) and IFA plus dying cells (4) on acceleration of lupus-like disease have been demonstrated, effects on SS-like disease are not known. This study demonstrates that IFA treatment accelerates SS-like disease in the NZB/W F1 mice.

Materials and Methods

Mice

All experiments involving mice were performed according to the protocol approved by the Animal Care and Use Committee of the University of Virginia. NZB/WF1 female mice were purchased from the Jackson Laboratories (Bar Harbor, ME) and maintained under specific pathogen-free conditions. At 8–10 weeks of age, mice were injected subcutaneously with 150µl of 1:1 emulsion of IFA in PBS at 2 sites (footpad and tail base). Three additional injections of IFA were administered by intraperitoneal route at monthly intervals.

Salivary gland function

Salivary gland function was assessed by measuring the amount of saliva produced following pilocarpine stimulation (1µg/g body weight) by the method described by Lin et al (5).

Histopathology

Formalin fixed salivary glands were embedded in paraffin and 5µm sections were stained with Hematoxylin and Eosin. Slides were read in a blinded fashion using coded samples. Severity of sialoadenitis scored on a scale of 1–4 as described previously (6).

Flow cytometry

Mice were perfused with saline solution and submandibular salivary glands harvested. Single cell suspensions of salivary glands were prepared, suspended in PBS with 1% BSA and 0.1% sodium azide and stained for flow cytometry analysis using standard procedures. Plasmacytoid dendritic cells were identified by three color staining using anti-B220-cychrome, anti-Ly6C (HK1.4)-FITC (Abcam) and anti-mPDCA-1-PE (Miltenyi Biotech) antibodies (7). Appropriate matched isotype antibodies were used as negative controls. Greater than 105 events were acquired per sample. Data was analyzed using Flow Jo software.

Autoantibody detection

Reactivity to salivary gland antigens was determined by western blotting using a mouse salivary gland extract as substrate using a method standardized for detection of kidney antigen reactive antibodies (8). The presence of antibodies to Ro60 was determined by ELISA using recombinant mouse Ro60 as described previously (9).

Statistical analysis

Statistical significance of variance was determined by the unpaired Students t-test or non parametric Mann Whitney test using Graphpad Prism software.

Results

IFA treatment accelerates salivary gland dysfunction in the NZB/WF1 mice

To determine the functional consequences of IFA treatment on salivary gland function, mice were monitored for pilocarpine induced salivation. Figure 1A shows that IFA treated mice had a significantly lower saliva volume than the PBS treated group at 7, 11 and 14 weeks after treatment. In the IFA treated group, a significant drop in saliva volume over the initial volume was seen by 7 wks after treatment (15–17wks of age, p=0.0005). In the PBS treated group a significant drop over the initial saliva volume was only observed by 17 wks after first treatment (25–27 wks of age, p<0.0001), which is indicative of spontaneous disease development in the NZB/WF1 mice. These data clearly demonstrate that IFA treatment accelerated salivary gland dysfunction in the NZB/WF1 mice.

Figure 1
Glandular dysfunction in NZB/W F1 mice treated with IFA does not correlate with the severity of sialoadenitis

Salivary gland dysfunction and severity of sialoadenitis

To study lymphocytic infiltration in the salivary glands, 5 mice per group treated either with IFA or PBS were sacrificed at different time points and sections of salivary glands graded for severity of sialoadenitis (figure 1B). These time points were based on preliminary experiments performed in independent cohorts of mice (4–5 per group). At 7 weeks after treatment, only 1 out of 5 mice in each group had mild inflammation in the submandibular glands. Parotid and sublingual glands did not show any inflammation. However, at this time point IFA treated group had developed glandular dysfunction. By 11 weeks after the initial treatment while 4/5 mice in IFA treated group showed clearly defined lymphoid foci, only 2/5 mice in the PBS treated group showed evidence for a much milder inflammation. The mean severity of sialoadenitis was significantly higher (p=0.0317) in the IFA treated mice by 15 weeks after treatment with 100% incidence (4/4 mice) than the PBS treated group with 40% incidence (2/5 mice). These results demonstrate that while IFA treatment had significantly accelerated sialoadenitis in the NZB/WF1 mice, at early time points the severity of sialoadenitis did not correlate with glandular dysfunction.

Autoantibody levels and glandular dysfunction in IFA treated NZB/WF1 mice

To determine whether the differences seen in salivary gland dysfunction between the IFA and PBS treated groups was associated with autoantibodies; sera were analyzed for autoantibodies to salivary gland antigens, SSA/Ro60 and anti-nuclear antibodies. Reactivity to salivary gland antigens was determined by western blotting using a salivary gland extract (Figure 2A). The pattern or intensity of reactivity with salivary gland antigens was not different at the pretreatment time point or 9–10 weeks after the initial treatment in both groups of mice. However, the intensity of autoantibody reactivity was much higher at 16–17 weeks after treatment in the IFA group. In IFA treated mice some bands observed at 9–10 weeks were not visible at 16–17 weeks. The variability in autoantibody patterns is often observed in lupus-prone mice and the reasons are not known. Similar results were obtained in an additional cohort of mice. Similar to our study, Bondanza et al (4) did not detect autoantibodies (ANA, nucleosome, dsDNA, β2GPI) in IFA treated NZB/W F1 mice at 16 weeks of age (10 wk after treatment).

Figure 2
Analysis of autoantibodies to salivary gland antigens by western blot (top panel) and Ro60 by ELISA (bottom panel)

To determine the contribution of anti-Ro60 antibody in the pathogenesis of the disease, reactivity to Ro60 was analyzed at different time points (Figure 2B). Antibody reactivity to Ro60 increased significantly in both groups of mice by 9–10 weeks after treatment when compared with the initial time point. However, the differences in reactivity to Ro60 between the PBS and IFA treated groups were not statistically significant. However, by 16–17 weeks the IFA treated mice had significantly higher (p=0.007) levels of anti-Ro60 antibodies. Similar trend was observed in anti nuclear antibody titers determined using NIH3T3 cells as substrate (data not shown) Antibodies to cholinergic muscarinic receptors are shown to have pathogenic consequences in SS. The western blot analysis employing whole salivary gland extract (that includes membrane proteins) indirectly suggests that these antibodies might not be critical in this mouse model at early time point. In summary these data suggest that autoantibodies do not seem to play a major role in the initiation of glandular dysfunction.

Increased numbers of plasmacytoid dendritic cells are seen in salivary glands of IFA treated mice

Lymphocytic foci were undetectable by immunohistochemistry in PBS or IFA treated mice at 6–7wks after treatment (or 16–17 weeks of age). This trend is similar to the kinetics of sialoadenitis in untreated NZB/W F1 mice reported by Jonsson et al (2). However, in that study immunohistochemical analysis showed presence of T and B cells in submandibular glands. Thus, to investigate the potential changes in numbers of infiltrating immune cells, not organized into inflammatory foci, single cell suspensions of submandibular salivary glands were studied for presence of T cells, B cells, macrophages and dendritic cells by flow cytometry. At 6wks, there were no significant differences in the frequency of T cells, B cells or macrophages between PBS and IFA treated groups (data not shown). However, the percentage of CD11c+ dendritic cells (% of the total salivary gland cells) in IFA treated mice (0.038±0.006%) were significantly increased (p=0.0079) than the PBS treated mice (0.021±0.0006%). Further analysis showed a significant increase in the frequency of the CD11clo, B220+, Ly6C+, and mPDCA-1+ plasmacytoid dendritic cell population (7) in salivary glands of IFA treated mice over the PBS treated mice (Figure 3). This increase was not a general occurrence, as analysis of spleen cells from the same mice showed a significantly decreased (p<0.008) percentage of pDC in spleens of IFA group (0.72±0.12 %) than in the spleens of PBS group (1.41±0.02%). However, the reasons for this decrease are not known.

Figure 3
Frequency of plasmacytoid dendritic cells is increased in submandibular salivary glands of NZB/W F1 mice treated with IFA (top panel) compared to PBS treated mice (bottom panel)

Discussion

To address the role of systemic inflammation in the development of SS, we repeatedly treated NZB/WF1 mice with IFA as an inflammatory agent. Our study demonstrates that IFA treatment accelerated the development of SS-like disease in the NZB/WF1 mice. The major observations from our study are that in initial stages of the disease 1) glandular dysfunction did not correlate with the severity of lymphocytic infiltration/foci in the salivary gland. 2) Autoantibodies to salivary gland antigens or Ro60 may not play a major role in the induction of glandular dysfunction. Instead, we observed increased numbers of plasmacytoid dendritic cells in the salivary glands of IFA treated mice. Plasmacytoid dendritic cells are major producers of type I interferons and have been shown to accumulate in the salivary glands of patients and NOD mice (10). However their role in the pathogenesis the disease is difficult to assess. Microarray gene expression analyses of salivary gland biopsies from patients have demonstrated upregulation of several IFN-inducible genes which is suggestive of localized effects of type I interferon production (11, 12).

Although the NZB/WF1 mouse model for SS has been one of the earliest described models, studies for salivary gland function have not been carried out. The disparity between the severity of sialoadenitis and salivary gland dysfunction observed in our model has also been reported for lacrimal gland function in this mouse strain (13). Similarly in the NOD mouse, Jonsson et al have reported that at later stages of the disease the changes in saliva volume correlated more with the changes in cytokine profiles than with the degree of lymphocytic infiltration (14). Intraperitoneal injection of IFA has been shown to induce formation of granulomas, adhesions, and upregulation of inflammatory cytokines such as IL-6, IL-12 and TFN-[alpha](15). Based on our study we would like to propose that sustained inflammatory stimuli in genetically susceptible individuals might alter the chemokine production by salivary glands resulting in lymphocytic infiltration. Thus, glandular dysfunction seen at earlier time points might be a cumulative effect of inflammatory cytokines as well as localized type I interferon production in the salivary glands. Direct experimental evidence for the role of pDCs in salivary gland dysfunction is being currently sought.

In our experimental model we did not see differences in the levels of autoantibodies to salivary gland-antigens between the IFA and PBS treated groups at 5–6 and 9–10 weeks after treatment. At this time point the glandular dysfunction was already evident in the IFA treated mice. These data suggest that autoantibodies might not have a primary role in the induction of disease. However, at later stages of the disease (16–17 weeks after treatment) higher autoantibody levels were observed in the IFA treated mice. Also the level of T and B cell infiltration and MHC II expression in the salivary gland was much higher in the IFA treated mice (data not shown). This demonstrates an enhanced systemic and localized adaptive autoimmune response in the mice that might be critical for the progression of the disease. The observations from our experimental model of accelerated SS-like disease are highly relevant towards understanding the mechanisms for pathogenesis of SS. In genetically susceptible individuals, repeated exposure to environmental stimuli, such as infectious microorganisms can lead to aberrant activation of innate immune system that might contribute towards the development of glandular dysfunction and disturbed glandular homeostasis. Ensuing adaptive autoimmune responses and their interaction with the innate immune responses would be responsible for sustaining the chronic nature of the disease.

Acknowledgments

Grant support; This work was supported in part by grants from the National Institutes of Health: K01AR051391 (USD); K01DK063065 and R01DK069769 (HB); and R01AR047988, R01DE001254 and P50AR45222 (SMF).

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