Participants and specimens
Serum samples were collected from six patients with active PV (Table 1) prior to treatment and three healthy volunteers. PV was diagnosed based on clinical features, histopathology, and immunofluorescence findings, and all the patients were in the active stage of the disease. The serum from the healthy volunteers was used as a control. The inclusion criteria of patients with PV were as follows: (i) met the diagnostic criteria for PV; (ii) patients did not use immunosuppressants, glucocorticoids, or antibiotics within the last 30 days; (iii) patients did not have an autoimmune disease except for PV; (iv) patients did not have significant vital organ dysfunction. Exclusion criteria were as follows: (i) patients that were pregnant; (ii) pemphigus induced by drugs; (iii) patients with malignant tumors. Informed consent was obtained from all patients enrolled in the study. The serum from all six patients was mixed to avoid bias of an individual sample [16]. The mixed serum was prepared by heating at 56°C for 30 min to inactivate complement. An anti-Dsg antibody was tested by enzyme-linked immunosorbent assay (ELISA) assay. The anti-Dsg antibody concentration in the mixed serum was 169 U/mL for Dsg1 and 117 U/mL for Dsg3. Anti-Dsg antibody titers were negative in the mixed serum from the three healthy volunteers (Dsg1: 1 U/mL; Dsg3: 1 U/mL, 2 females and 1 males, 26–29 years old). This study and all relevant experiments were reviewed and approved by the Guangzhou Institute of Dermatology Research Ethics Committee (NO. 201802, Guangzhou, Guangdong Province, China).
Antibodies and reagents
The primary antibodies included mouse anti-Dsg3 antibody (Abcam, Cambridge, UK), rabbit anti-p38 MAPK, rabbit anti-phospho-p38 MAPK (Thr180/Tyr182, Cell signaling Technology, Danvers, MA), mouse anti-HSP27, rabbit anti-phospho-HSP27 (Ser82, Cell signaling Technology), and rabbit anti-GAPDH (Affinity Biosciences, OH, USA). The secondary antibodies included a goat anti-mouse antibody, goat anti-rabbit antibody (Abclonal, Wuhan, China), and Cy3-conjugated goat anti-mouse (Abclonal) antibody.
Other regents included FK506 (Med Chem Express, Shanghai, China), clobetasol propionate (CP) (Macklin, Shanghai, China) and a p38 MAPK inhibitor, SB203580 (Med Chem Express). AK23 (BML, Tokyo, Japan), which is a pathogenic monoclonal antibody targeting Dsg3, was utilized for the incubation steps in the cell culture model at a concentration of 1 μg/ml and was used as a positive control,
Cell culture and study design
The human keratinocyte cell line HaCaT (MissBio Co., Ltd. Guangzhou, China) was used to establish an in vitro model of PV according to a previous study[33, 34]. Cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin/streptomycin and 5% fetal bovine serum (FBS) at 37°C in a humidified atmosphere containing 5% CO2.
Four experimental groups were designated in the study: (ⅰ) normal control group (Con), which refers to the group that only received DMEM with 5% FBS medium; (ⅱ) normal healthy sera group (NH), refers to the group with the presence of 5% healthy sera in DMEM medium; (ⅲ) PV sera group (PV), refers to the group with the presence of 5% PV sera in DMEM medium; and (ⅳ) AK23 group, refers to 1 μg/mL AK23 in 5% FBS DMEM medium. The HaCaT cells were sub-cultured until reaching 60–70% confluency, and exposed to different conditions as mentioned above for another 24 h. For FK506 studies, cells were incubated in 5% PV sera or AK23 with 100 nM FK506 and/or 1 μM CP.
Dispase-based Keratinocytes Dissociation Assay
The dispase-based keratinocyte dissociation assay is currently the main tool for the analysis of antibody-induced acantholysis in in vitro models of PV[35]. This assay was performed as previously described[31, 36, 37]. Briefly, HaCaT cells were seeded in 24-well plates and grown to 60–70% confluency and exposed to different conditions as mentioned above. Cell monolayers were washed with pre-warmed Hank’s Balanced Salt Solution (HBSS, Solarbio, Beijing, China) and subsequently released from the well-bottom by incubation with 2.4 U/ml Dispase II (Solarbio, Beijing, China) dissolved in HBSS for 30 min at 37°C. Next, the dispase solution was aspirated with a pipette, and then HBSS was added into each well. Each condition was treated for 30 min with thiazolyl blue tetrazolium bromide (namely, methylthiazol tetrazolium, MTT) (Sigma-Aldrich, Taufkirchen, Germany) at a final concentration of 10 µM to better visualize the monolayer sheets. Mechanical stress was applied to the monolayers of each group by pipetting the monolayers 10 times with a 1-mL electric pipette (Eppendorf, Wesselingberzdorf, Germany) for the production of cell fragments. The numbers of cell fragments were counted using a binocular microscope (CKX41, Olympus, Tokyo Japan). Each experiment was conducted three times.
Western blotting assay
HaCaT cells were grown in 6-well plates for 18–24 h until the cultures reached 70% to 80% confluency, and then the medium was replaced 5% FBS medium for Con group, 5% healthy sera in DMEM medium for NH group, 5% PV sera in DMEM medium for PV sera group and 1 μg/mL AK23 in 5% FBS DMEM medium for AK23 group. For FK506 studies, cells were incubated in 5% PV sera or AK23 with 100 nM FK506 and/or 1 μM CP. After which, total cell lysates were prepared from HaCaT cells by extracting the proteins in RIPA buffer (Beyotime, Shanghai, China) with protease and phosphatase inhibitors. Proteins (20 μg) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene fluoride (PVDF) membranes (Merck Millipore, Darmstadt, Germany). The PVDF membranes were blocked for 2 hours with 5% non-fat milk in 0.1% Tris-buffered saline with Tween (TBST) and immunoblotted with primary antibodies (GAPDH 1:10000, Dsg3 1:1000, p38MAPK 1:1000, p-p38MAPK 1:1000, HSP27 1:1000, and p-HSP27 1:1000) at 4°C for another 12–16 h. For phosphoproteins, the PVDF membranes were blocked with 5% bovine serum albumin (BSA) in 0.1% TBST.
Immunofluorescence assay
The cells were grown directly on glass coverslips for 18–24 h in a 24-well plate and then continuously for 24 h after replacing the medium with or without supplements as described above. The cells were fixed in a 4% paraformaldehyde solution for 15 min, washed three times with phosphate buffer saline (PBS), incubated 10 min with 0.1% Triton X-100 (in PBS), and further washed three times with PBS. Subsequently, the cells were blocked with 5% BSA for 1 h at 25°C. The samples were then incubated with an anti-Dsg3 antibody (1:250 dilution) at 4°C for 12–16 h, followed by several rinses with PBS. A Cy3-conjugated goat anti-mouse antibody was added as the secondary antibody (1:500 dilution), and the samples were incubated for 1 h at 25°C. A fluorescence microscope (BX63, Olympus) was used for image acquisition.
Statistical analysis
Western blots were visualized and analyzed using Image J (NIH, Maryland, USA, http:imagej.nih.gov/ij). Statistical significance was assessed using an one-way ANOVA followed by Student-Newman-Keuls correction using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA) for comparison of multiple groups. Differences were deemed significant when the calculated p value was < 0.05. The data are expressed as the mean ± SE.