Table 1.
Demographics and clinical data for patients.
Figure 1.
Increased membrane expression of GFP-tagged-recombinant human aquaporin 5
(rhAQP5) following carbachol stimulation in HSG cells. (A) M3R expression was detected in HSG cells (red color) using polyclonal (a, b) or monoclonal (c) antibody by immunocytochemistry. Stained cells were observed at ×200 (a) and ×400 (b, c) magnifications. (B) hAQP5 protein expressions were examined by western blotting in total HSG cell lysates purified from non- or rhAQP5 vector-transfected HSG cells (a) and in HSG cell membrane fractions purified from transfected cells with or without CCh treatment (b) in duplicates #1 and #2. (C) To confirm the hAQP5 trafficking in HSG cells, control GFP (a) or rhAQP5 (c) vector transfected HSG cells were treated with carbachol (CCh, 100 μM) (a and c) or no stimulation (b). Images were taken under microscope with a ×400 magnification using a Zeiss Axiovert 200M microscope and obtained with AxioVs40 software (Ver. 4.7.1.0, Zeiss).
Figure 2.
Inhibition of hAQP5 trafficking in rhAQP5 vector-transfected HSG cells in the presence of 4-DAMP.
(A) rhAQP5 vector-transfected HSG cells were stimulated with CCh (100 μM) in the presence or absence of 4-DAMP (10 μM), prenzepine (10 μM), and mixture of 4-DAMP and prenzepine (M3R or M1R specific antagonist, respectively). (B) To monitor hAQP5 trafficking, original GFP pictures in (A) were converted into ‘Fire’ 3D LUT (lookup tables) setting in ImageJ software and only GFP signal above the fluorescent signal threshold (≥80, red or yellow color in converted picture), was captured in (B) as positive GFP signal. Yellow arrows indicate the positive GFP signal on the membrane and green arrows indicates signal in the cytoplasm in HSG cells. Cells were observed at ×200 magnification using a Zeiss Axiovert 200M microscope and images were obtained with AxioVs40 software (Ver. 4.7.1.0, Zeiss).
Figure 3.
Presence of anti-M3R autoantibodies in SjS plasma detected by immunocytochemistry.
(A) HSG cells were pre-incubated with (a) healthy control (HC) plasma, (b, e) Sjögren's syndrome (SjS) plasma, (c) pre-absorbed SjS plasma, or (d) IgG depleted SjS plasma for 24 hours. Endogenous expressions of M3R (red color) on HSG cells were detected by using poly- (a–d) or mono-(e) clonal antibody. Immunofluorescence staining of HSG cells following pre-incubation with SjS plasma indicated co-localization [yellow or orange color in merged picture (b) and (e)] of human IgG (green color) and M3R (red color) on HSG cells. This specific binding was confirmed by pre-absorbed (c), IgG depleted plasma (d), or monoclonal antibody specific for M3R (e). Stained cells were observed at a ×200 magnification. (pM3R* Ab: polyclonal anti-hM3R antibody; mM3R Ab: monoclonal anti-hM3R antibody). (B) HSG cells were incubated with purified IgG from five pooled-SjS plasma samples and polyclonal anti-M3R antibody followed by Alexa Fluor 488 and 568-labeled secondary antibody incubation, respectively. Yellow (b, c, d, e, and h) or white arrows (a, f, and g) in the image picture indicate co-localization or adjacent staining of IgG and M3R, respectively. poly α-M3R ab, polyclonal antibody; mono α-M3R ab, monoclonal antibody; Green arrows, IgG staining; red arrows, M3R staining; white arrows, M3R and IgG adjacent staining; yellow arrows, co-localization of IgG and M3R. Cells were observed at ×400 magnification using a Zeiss Axiovert 200M microscope and images were obtained with AxioVs40 software (Ver. 4.7.1.0, Zeiss).
Figure 4.
Inhibition of AQP5 trafficking to HSG cell plasma membrane by SjS plasma.
(A) rhAQP5 vector transfected HSG cells were pre-incubated with five different healthy control (HC, (a)), five SjS plasma (b), or mono-clonal anti-M3R antibody (c) for 24 hours (only three representative pictures are shown). rhAQP5 GFP signal trafficking in HSG cells after CCh treatment were acquired with a spinning disk confocal microscope with a 63X oil-immersion objective, using a cascade-cooled EMCCD camera. HSG cells were observed at 30-second intervals for 15 minutes with 1/250 second fixed exposure time. Representative still images were captured after 10 minutes and rhAQP5 signals were converted and analyzed by ImageJ software (1.46a, open source software, http://rsb.info.nih.gov/ij/). Pictures were converted into ‘Fire’ 3D LUT (lookup tables) setting in ImageJ software and only GFP signal above the fluorescent signal threshold (≥80, red or yellow color in converted picture), was considered for quantitative analysis (% of positive signal on the membrane) in (B) to compare rhAQP5 trafficking among CCh treated groups. Each column represents the mean ± SEM of 5, p value less than 0.05 was considered significantly different by two-tailed unpaired Student's t-test, **p<0.01.
Figure 5.
Reduced CCh-induced intracellular calcium releases by SjS IgG.
(A) Immunoprecipitation (IP)-Western blot analysis using an extract of M3R-3xHA vector transfected HSG cells and pooled plasma samples from five HC or SjS plasma demonstrated that IgG in SjS plasma immunoprecipitated HA-tagged M3R (75 kDa), which was recognized by mouse monoclonal anti-HA antibodies. (B) HSG cells were pre-incubated with media only (negative control) or IgG from HC (10 μg/ml), SjS (10 μg/ml) or monoclonal anti M3R antibody (10 μg/ml) for 24 hours and treated with ionomycin (100 μM) or carbachol (100 μM). Data are based on three independent experiments (n = 5). Each column represents the mean ± SEM, p value less than 0.05 was considered significantly different by one-way ANOVA test.
Figure 6.
Inhibition of CCh-induced hAQP5-GFP transfected HSG cell shrinkage by SjS plasma.
(A) hAQP5-GFP transfected HSG cells were pre-incubated with HC or SjS plasma for 24 hours and stimulated with CCh. Representative still images were captured before and after 30 minutes of CCh treatment. (B) Cell volume changes were quantified by measuring the areas within the cell membrane before and after CCh stimulation and normalized by the area before stimulation. Cross section of the cell at center of z-axis was considered for the analysis. Data are based on three independent experiments and all cells under a microscopic view were counted for calculation each time. Each column represents the mean ± SEM, p value less than 0.05 was considered significantly different by two-tailed unpaired Student's t-test, *p<0.05.