Novel animal models for Sjögren's syndrome: expression and transfer of salivary gland dysfunction from regulatory T cell-deficient mice

Abstract

IL-2 knockout (KO), IL-2Ralpha KO and scurfy mice lack the CD4+CD25+ regulatory T (Treg) cells and develop severe inflammation in multiple organs, although organs affected vary among these strains. We asked if salivary and lacrimal glands, the main organs affected in Sjögren's syndrome, are targeted in these strains. Severe lymphocyte and neutrophil infiltration in the salivary and lacrimal glands and a decrease in salivary secretory function were observed in IL-2 KO and IL-2Ralpha KO mice, but not in scurfy mice. Interestingly, transfer of lymph node cells from scurfy mice to RAG-1 KO recipients rapidly and effectively induced inflammation and loss of function in the salivary glands. Furthermore, we observed that daily LPS feeding in scurfy mice also induced inflammation in the salivary glands. Our study demonstrates several novel models for Sjögren's syndrome, including an adoptive transfer model that shows that scurfy mice have dormant salivary gland-specific autoreactive lymphocytes that can be activated by certain environmental factors, such as those present in RAG-1 KO mice.

Fig. 1

Two of the three Treg cell deficient mouse strains display inflammation in the salivary and lacrimal glands. Three strains of mice that lack CD4⁺CD25⁺ Treg cells were compared for inflammation in their colons (A-D), lungs (E-H), lacrimal glands (I-L) and salivary glands (M-P). B6 mice were used as control. The B6, IL-2Rα KO and IL-2 KO mice were 8-10 weeks old, while scurfy mice were 3-4 weeks old. At least 4 mice were examined from each strain. The figure is composed of representative H&E stained sections of the indicated tissues from each strain. Representative acini and ducts are labeled with Ac and Dt respectively.

Fig. 2

Flow cytometric analysis of infiltrating cells in the salivary glands. Single cell suspension of salivary gland was prepared from B6, IL-2Rα KO, and IL-2 KO mice, as described in “Materials and Methods.” Total viable cell number was determined. Cells were co-stained with anti-CD8 and anti-CD4 mAb (top panels), or anti-NK1.1 and anti-Gr-1 mAb (lower panels). To facilitate analysis, the latter staining was gated on CD3⁻ cells.

Fig. 3

Salivary gland function was impaired in the IL-2 and IL-2Rα KO mice. We examined salivary gland function in 8-10 week old mice, as described in the “Materials and Methods” section. Each circle represents an individual mouse and the average is indicated by the bar. The volume of saliva secreted (A) and the lag time for saliva secretion (B) were impaired in both IL-2Rα KO and IL-2 KO mice, as compared to age- and sex-matched B6 mice (p< 0.05 in all cases).

Fig. 4

Lymph node cells from scurfy mice could transfer salivary gland dysfunction to RAG-1 KO mice. (A) Total LN cells from scurfy or age-matched B6 mice were adoptively transferred into 6-week old male RAG-1 KO mice, as described in “Methods.” Salivary gland function was determined three weeks later. The scurfy LN cells inhibited saliva secretion (left) and prolonged the lag time for saliva secretion (right). The circles represent individual mice and the bars represent the means (p<0.05 in all cases). (B) On histological examination, there was a remarkable infiltration of cells in the salivary (panel A, C) and lacrimal glands (panel E) of RAG-1 KO mice that received LN cells from scurfy mice, as compared to RAG-1 KO mice that received cells from B6 donors (panels B, D and F). Representative acini and ducts are labeled with Ac and Dt respectively.

Fig. 5

Transfer of scurfy LN cells induces inflammation in neonatal RAG-1 KO recipients. We transferred 5×10⁶ LN cells from 3-week old scurfy mice into 6-day old RAG-1 KO mice. Unlike the scurfy donors (left panels), the RAG-1 KO recipients developed inflammation in their salivary glands. Four mice were examined in each group. The data are representative H&E stained sections from two independent experiments.

Fig. 6

Feeding LPS triggers inflammation in the salivary glands of scurfy mice. We fed neonatal scurfy mice with LPS (10 μg/per day) from day 6 to day 21 after birth. The H&E stained sections of salivary glands, examined at the end of the experiment, displayed a significant degree of inflammation, as compared with the untreated control.