IL-4-STAT6 signal transduction-dependent induction of the clinical phase of Sjögren's syndrome-like disease of the nonobese diabetic mouse

Abstract

NOD.B10-H2(b) and NOD/LtJ mice manifest, respectively, many features of primary and secondary Sjögren's syndrome (SjS), an autoimmune disease affecting primarily the salivary and lacrimal glands leading to xerostomia (dry mouth) and xerophthalmia (dry eyes). B lymphocytes play a central role in the onset of SjS with clinical manifestations dependent on the appearance of autoantibodies reactive to multiple components of acinar cells. Previous studies with NOD.IL4(-/-) and NOD.B10-H2(b).IL4(-/-) mice suggest that the Th2 cytokine, IL-4, plays a vital role in the development and onset of SjS-like disease in the NOD mouse model. To investigate the molecular mechanisms by which IL-4 controls SjS development, a Stat6 gene knockout mouse, NOD.B10-H2(b).C-Stat6(-/-), was constructed and its disease profile was defined and compared with that of NOD.B10-H2(b).C-Stat6(+/+) mice. As the NOD.B10-H2(b).C-Stat6(-/-) mice aged from 4 to 24 wk, they exhibited leukocyte infiltration of the exocrine glands, produced anti-nuclear autoantibodies, and showed loss and gain of saliva-associated proteolytic enzymes, similar to NOD.B10-H2(b).C-Stat6(+/+) mice. In contrast, NOD.B10-H2(b).C-Stat6(-/-) mice failed to develop glandular dysfunction, maintaining normal saliva flow rates. NOD.B10-H2(b).C-Stat6(-/-) mice were found to lack IgG1 isotype-specific anti-muscarinic acetylcholine type-3 receptor autoantibodies. Furthermore, the IgG fractions from NOD.B10-H2(b).C-Stat6(-/-) sera were unable to induce glandular dysfunction when injected into naive recipient C57BL/6 mice. NOD.B10-H2(b).C-Stat6(-/-) mice, like NOD.B10-H2(b).IL4(-/-) mice, are unable to synthesize IgG1 Abs, an observation that correlates with an inability to develop end-stage clinical SjS-like disease. These data imply a requirement for the IL-4/STAT6-pathway for onset of the clinical phase of SjS-like disease in the NOD mouse model.

FIGURE 1

Expression of MHC class II (I-A^b) and CD23 on B cells following IL-4 stimulation in vitro. Purified B lymphocytes from NOD.B10-H2^b.C-Stat6^+/+ and NOD.B10-H2^b.C-Stat6^−/− mice were cultured in medium and stimulated for 48 h with 20 ng/ml recombinant mIL-4. After 48 h, cells were harvested and analyzed for MHC class II (I-A^b) and CD23 expression using flow cytometry. A, MHC class II (I-A^b) expression. B, CD23 expression. Gray, NOD.B10-H2^b.C-Stat6^+/+ mice. Black, NOD.B10-H2^b.C-Stat6^−/− mice.

FIGURE 2

Detection of proteolytic activity against PSP in saliva. Saliva, collected from individual mice, was assayed for proteolytic activity on a 16-mer oligopeptide containing the NLNL enzymatic site for a disease-activated serine kinase. Using reverse-phase HPLC, the synthetic 16-mer oligopeptide eludes at 13.9 min (A). Saliva from BALB/c mice was incapable of enzymatically digesting the oligopeptide (B). Saliva from NOD/LtJ (C), NOD.B10-H2^b (D), NOD.B10-H2^b.C-Stat6^+/+(E), and NOD.B10-H2^b.C-Stat6^−/− (F) mice all showed full digestion of the oligopeptide with the two degradation products of the oligopeptide eluting at 9.2 and 12.8 min, respectively.

FIGURE 3

Histological examination of the exocrine glands. Submandibular glands removed from groups of mice at ages ranging from 20 to 28 wk were fixed in 10% formalin. Each gland was serially sectioned (5-µm thickness) and histology performed on two sections cut 50 µm apart. Each section was stained with Mayer’s H&E dye (A, C, and E) or immunofluorescent Abs for determining the distribution of B (anti-B220, red) and T (anti-CD3, green) cells in lymphocytic foci (B, D, and F). The sections were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (blue). The numbers of focus were counted across whole histological sections of glands from 20-wk-old female NOD.B10-H2^b mice (n = 8; A and B), 28-wk-old female NOD.B10-H2^b.C-Stat6^+/+ mice (n = 18; C and D), and 25-wk-old female NOD.B10-H2^b.C-Stat6^−/− mice (n = 23; E and F). All H&E sections and immunofluorescent staining was examined at ×200 magnification. The number of foci for the two sections was averaged for comparisons.

FIGURE 4

Detection of ANAs. Serum samples obtained from groups of mice at various ages were pooled, diluted 1/40, and incubated with HEp-2-fixed substrate slides for 30 min at room temperature in a humidified chamber. The substrate slides were then incubated 30 min at room temperature with different isotype-specific secondary FITC-conjugated goat anti-mouse Abs diluted 1/50, then viewed using a Zeiss Axiovert 200 M fluorescent microscope at ×200 magnification. Sera—pooled from two mice selected from each experimental group of NOD.B10-H2^b.C-Stat6^+/+ and NOD.B10-H2^b.C-Stat6^−/− mice—were used, and the experiment was repeated two additional times.

FIGURE 5

Examination of stimulated salivary flow rates. Groups of 4-wk-old (n = 7) or 24-wk-old (n = 12) female NOD.B10-H2^b.C-Stat6^+/+ and NOD.B10-H2^b.C-Stat6^+/− mice, and 4-wk-old (n = 5) or 24-wk-old (n = 8) NOD.B10-H2^b.C-Stat6^−/− mice were injected with isoproterenol/pilocarpine to stimulate saliva secretion. Saliva was collected from each mouse for 10 min starting 1 min after injection of the secretagogue. The volume of each sample was measured. Statistical analysis was performed by Student Newman-Keuls test using GraphPad InStat software.

FIGURE 6

Detection of M3R isotypic autoantibodies by immunofluorescence. Sera collected from NOD.B10-H2^b.C-Stat6^+/+ and NOD.B10-H2^b.C-Stat6^−/− mice were incubated at a dilution of 1/50 with newly cloned M3R-transfected Flp-In CHO cells (see Ref. and Ref. 19) for 1 h in a humidified chamber at room temperature. The cells were washed five times (5 min each wash) with PBS, then incubated 30 min at room temperature with FITC-conjugated secondary Abs specific for IgM (A and B), IgG (C and D), IgG2b (E and F), or IgG1 (G and H) isotypes diluted 1/100 (Serotec). Cells were again washed and visualized using a Zeiss Axiovert 200 M microscope at ×100 magnification. Images represent an exposure time of 25 ms.

FIGURE 7

Salivary flow rates of C57BL/6 mice after injections of IgG isolated from NOD.B10-H2^b.C-Stat6^+/+ or NOD.B10-H2^b.C-Stat6^−/− mice. Temporal changes in salivary flow rates of male C57BL/6 mice injected with whole IgG collected from individual 6-wk-old NOD.B10-H2^b.C-Stat6^+/+(n = 4F) or NOD.B10-H2^b.C-Stat6^−/− (n = 5M and 3F) mice (A) and 16-wk-old NOD.B10-H2^b.C-Stat6^+/+(n = 5F) and NOD.B10-H2^b.C-Stat6^−/− (n = 2M and 3F) mice (B). Mice were injected i.p. with 500 µg of fractionated IgG. All values are expressed as mean salivary flow rates ± SEM. Statistical analysis was performed by two-tailed, paired Student t test using GraphPad InStat software (*, p < 0.05).