Materials
The plasmid (pGEX-4T-2–α-fodrin) was a generous gift from Professor Hayashi of the Tokushima University School of Dentistry in Japan.
Large-scale bacterial expression and fusion protein purification
A 5 ml saturated culture of pGEX-4T-2–α-fodrin-transformed bacteria grown in Luria-Bertani(LB) medium supplemented with ampicillin (100 μg/ml) was used to inoculate 200 ml LB medium. This culture was allowed to reach the log phase (A600 = 0.6) before induction with Isopropyl β-D-1-Thiogalactopyranoside (IPTG) (1 mM final). The induction was carried out for 4 hours at 37°C (300 rpm), and then the bacteria were harvested by centrifugation (1,200 × g for 10 min, 4°C). The resulting pellets were resuspended in 24 ml ice-cold 10 mM Tris (pH 7.5), 10% glycerol, 10 mM dithiothreitol. Bacteria were lysed by sonication, and bacterial inclusion bodies were collected by centrifugation (40,000 × g for 10 min, 4°C). The sample was passed over a 5 ml glutathione-Sepharose affinity column equilibrated with PBS, and the fusion protein was eluted with 10 ml elution buffer (5 mM glutathione, 50 mM Tris, pH 8.0) collected in 2 ml fractions. The final yield of purified α-fodrin was 2 mg.
Mice
Female NOD mice (4 weeks old) were purchased from the Laboratory Animal Resource Center of the Peking University Medical Science Center and were acclimatized to the center for 1 week before starting the experimental protocol. Animals were maintained in a specific pathogen free environment, and were fed standard rodent chow and water ad libitum. The study was approved by the Animal Care and Use Committee of the Peking University People's Hospital.
Treatment protocol
Four groups, each consisting of eight NOD mice, were used. Groups I and II were immunized every other day with 1 μg and 10 μg.
Glutathione transferase–α-fodrin fusion protein, respectively. Group III was immunized with 10 μg Glutathione transferase every other day, and Group IV was immunized with 10 μl PBS every other day. Immunization was given by nasal administration. Preimmune bleeds were collected from all mice. Postimmune bleeds were collected every 2 weeks. Mice were killed at 17 weeks of age by cervical dislocation.
Peptides
The sequence of the type 3 muscarinic acetylcholine receptor second loop polypeptide (KRTVPPGECFIQFLSE) is a known M3R epitope based on previous studies [5]. This linear peptide was synthesized using solid-phase techniques on an Applied Biosystems Peptide Synthesizer (APEX396, AAPPPEC, Kentucky, USA) at SBS Gene Technology Company (Shanghai, China). The peptide was purified by reversed-phase high-performance liquid chromatography to a purity >90%.
Quantitation of anti-type 3 muscarinic acetylcholine receptor polypeptide antibody by ELISA
A solid-phase immunoassay for the type 3 muscarinic acetylcholine receptor polypeptide (M3RP) was performed as previously described. Briefly, 96-well ELISA plates (Costarvinyl, Cambridge, MA, USA) were coated with 100 μl of 10 μg/ml M3RP in 0.5 M carbonate buffer, pH 9.6. Coating was performed at 4°C overnight, followed by blocking with 100 μl of 5% milk in PBS (pH 7.4) containing 0.1% BSA. After washing with PBS–Tween 20, the wells were then loaded with 100 μl mouse serum (1:100 dilution) and were incubated at room temperature for 2 hours, at which point the wells were washed five times with PBS–Tween 20. Then 100 μl goat anti-mouse IgG conjugated to peroxidase (1:4,000; Zhongshan Technology Company, Beijing, China) was added to each well and incubated for 1 hour at room temperature. After washing five times with PBS–Tween 20, the bound antibodies were detected with the substrate, O-phenylenediamine. The reaction was stopped by addition of 100 μl of 2.5 M sulfuric acid to each well. Plates were read at a wavelength 492 nm (absorbance, OD492 nm) with an ELISA plate reader (BIO-RAD Model 550; MICROPLATE READER, Hercules California, USA). Each serum sample was assayed in duplicate. The results were expressed as optical density units ± standard deviation. An optical density value greater than two standard deviations from the mean optical density value of the control sera was considered positive.
Autoantibody quantitation
The IgG class autoantibodies against SSA, SSB, rheumatoid factor (Euroimmun, Lübeck, Germany) and anti-α-fodrin antibody (AESKI, Wendelsheim, Germany) were measured by ELISA. Antinuclear antibodies were measured by immunofluorescence according to the manufacturer's instructions (Euroimmun).
Preparation of lymphocytes and flow cytometry
Lymphocyte suspensions were prepared from spleens and lymph nodes. For flow cytometric analysis, 1 × 106 cells were incubated with fluorescein isothiocyanate-labeled anti-CD4 and activated protein C-labeled anti-CD25 (BD Biosciences, Franklin Lakes, NJ USA). For intracellular staining of Foxp3, lymphocytes were first surface-labeled (CD4 and CD25), were then fixed and permeabilized using the Cytofix/Cytoperm kit (BD Biosciences), and finally were stained with anti-Foxp3-PE (BD Biosciences). Cells were analyzed by flow cytometry (BECTONDICKINSON, FACSort, Franklin Lakes, NJ USA).
Analysis of cytokine production
Samples of blood were collected at the end of the experiment. Serum levels of IFNγ and IL-10 were measured with a commercial ELISA kit (R&D, Minneapolis, Mn. USA) according to the recommendations of the manufacturer.
Histological and immunohistochemical analysis
After the mice were killed, the submandibular and sublingual salivary glands were surgically removed, fixed in 10% buffered formalin, and embedded in paraffin. Four-micrometer sections were prepared. H&E staining was performed to determine the degree of inflammation. Infiltrates appear as periductal and perivascular foci within the glandular architecture of the salivary glands. Focal scores classified as 1 or above, which consist of clusters of >50 lymphocytes in a 4 mm2 area, are considered abnormal. Immunostaining was performed by the avidin–biotin complex method, using anti-α-fodrin polyclonal antibody (Cell Signaling, Danvers, Boston, USA).
Measurement of the water volume intake of mice
We fed and watered the mice carefully. Each bottle of water was measured to 200 ml on the first weekday, and the remaining water volume at the end of the week was measured to calculate the volume of water drunk by the mice. Food was given to the mice separate from the water. Mice were separated and the volume of water was measured every week. The volumes of the groups were then compared.
Statistical analysis
Experimental findings were presented using the Mann–Whitney U test (two-tailed, independent). Analyses were carried out using SPSS 13.0 software (SPSS® Base 13.0, Wacker Drive, Chicago, USA). The statistical significance level was set at P = 0.05.