PV IgG associates with lipid raft markers in vivo and is trafficked to endosomes

Previous studies have determined that desmosome disassembly and endocytosis occur in a lipid raft-dependent manner (Delva et al., 2008; Stahley et al., 2014). In vivo, patient IgG (hIgG) colocalized with raft markers CD59 and caveolin-1 both at cell borders and in vesicular-like puncta (Figure 2). A number of studies using cell culture models have found that the PV IgG-Dsg complex is internalized from the plasma membrane via lipid raft-mediated endocytosis, resulting in degradation of Dsg3 and reduced plasma membrane levels of the adhesive protein (Calkins et al., 2006; Cirillo et al., 2007; Delva et al., 2008; Jolly et al., 2010; Kitajima, 2014; Mao et al., 2014; Mao et al., 2011; Saito et al., 2012; Sato et al., 2000; Schulze et al., 2012). To determine if PV IgG is internalized to endocytic compartments in patient tissue, biopsy sections were stained for hIgG and the early endosomal marker EEA-1. Nearly every cell, particularly near sites of blister formation, displayed multiple puncta at or near the cell periphery containing hIgG and EEA1 (Figure 3a). Interestingly, steady state Dsg3 levels were decreased in PV patient tissue compared to normal human control tissue (Figure 3b, S5), further suggesting that PV IgG induces Dsg3 endocytosis and degradation in vivo.

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Figure 2

PV patient IgG colocalizes with lipid raft markers in vivo

(a) Colocalization of PV patient IgG (hIgG) with lipid raft markers CD59 (top panel) and caveolin-1 (middle panel), and non-raft marker clathrin (bottom panel) in biopsies from PV patient tissue. Colocalization is observed both at cell borders and in vesicular-like structures (asterisks). Note lack of clathrin colocalization in vesicular puncta. Images from P5. Scale bar, 5 μm. (b) Quantification of hIgG colocalization (Mander's coefficient) with membrane markers at cell-cell borders. Means ± SEM, *p < 0.05; n = at least 54 cell borders from biopsies derived from 3 different patients. (c) Quantification of vesicular hIgG colocalization (Mander's coefficient) with membrane markers. Means ± SEM; *p < 0.05; n = 30 vesicles analyzed from three patient biopsies.

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Figure 3

PV patient IgG is internalized in vivo and Dsg3 levels are reduced

(a) PV IgG (hIgG) colocalizes with early endosomal marker EEA1. Numbered enlargements highlight vesicular colocalization. Images from P5. Scale bar, 2 μm. (b) Quantification of Dsg3 protein levels relative to adherens junction protein p120 in normal and PV patient tissue. Image J was used to analyze wide-field microscopy images of tissue. Following background subtraction, Dsg3 intensity was normalized to p120 levels using Image J. Means ± SEM; *p < 0.05; Data were collected from 3 NH and 5 PV patient biopsies. Average fluorescence intensity measurements were derived from analysis of 6-10 images from each biopsy.

Desmosomes are smaller and split in PV patients

Ultrastructural studies of desmosome morphology in PV patients and mouse models have suggested that desmosomes either split at the adhesive interface or are reduced in size (Shimizu et al., 2004; van der Wier et al., 2014). We utilized SIM imaging to assess desmosome morphology in patient tissue and directly compared these changes to cultured keratinocytes exposed to patient IgG. We took advantage of information from previous immuno-gold EM studies defining the position of various desmosomal proteins (North et al., 1999) and the sub-diffraction limit resolution of SIM to identify bona fide desmosomes in tissues and cells by immunofluorescence localization. Using this approach, antibodies directed against the carboxyl terminal domain of DP result in a “railroad track” pattern of fluorescence that defines the localization of the inner desmosomal plaque, whereas antibodies against the Dsg extracellular domain identify the adhesive core (Figure 4a-c).

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Figure 4

Desmosomes in PV patients are smaller and split

(a) Desmosome schematic depicting staining with N-terminal Dsg3 antibody (hIgG in patient tissue) and a C-terminal desmoplakin (DP) antibody. (b) SIM resolves the plaque to plaque distance and desmosomes appear as regions of parallel DP staining, or ‘railroad’ tracks, along a cell border (1) or a sandwich of DP-Dsg3-DP staining (2). Split desmosomes appear as regions of green-red staining (3). (c) In vivo examples of (b). Scale bar, 0.3 μm. (d-f) Normal human (NH) and PV patient biopsies stained as detailed above and imaged by SIM. Railroad tracks were used to identify and measure desmosome size. (d) DP staining. Arrows indicating intercellular space highlight smaller desmosomes. Images from N3 and P1. Scale bar, 0.5 μm. (e) Many split (or half) desmosomes with IgG staining (asterisks) are observed adjacent to the blister space. Small desmosomes (arrowheads) were also observed. DP (green), hIgG (red). Images from P5. Scale bar, 0.5 μm. (f) Quantification of desmosome size. Means ± SEM; * p < 0.05 comparing PV-whole or PV-split to NH; measurements were obtained from 6-163 desmosomes per patient; data from three NH controls and six PV patients.

This pattern of DP railroad track staining was used to identify and measure desmosome size in human tissue. Desmosomes in NH samples averaged 0.43 microns in size, while desmosomes in PV patients were significantly smaller, averaging 0.35 microns (Figure 4d,f). This observation is consistent with recent electron microscopy studies of PV patients (van der Wier et al., 2014) and analysis of desmosomes in cultured keratinocytes treated with PV IgG (Saito et al., 2012; Tucker et al., 2014). Interestingly, SIM revealed three types of fluorescence staining patterns in patient tissue (Figure 4b-c, e). First, when DP railroad track staining was intact, we often observed hIgG deposition in the desmosome core (red) that was bracketed by DP staining on opposing keratinocyte membranes (green). Second, a few instances of DP railroad track staining devoid of hIgG deposition in the desmosomal core were also observed, possibly indicating Dsg-depleted desmosomes (Figure S6). Third, we observed numerous split desmosomes in which hIgG was adjacent to only one “rail” of DP (Figure 4e, asterisks), indicating desmosome splitting. The presence of split desmosomes in patients and mouse models of PV has been attributed to pathogenic antibodies physically interfering with, or sterically hindering, desmosomal cadherin adhesive interactions (Amagai et al., 1992; Sharma et al., 2007; Stahley and Kowalczyk, 2015; Tsunoda et al., 2003). If steric hindrance were the sole mechanism for loss of adhesion, we would predict that desmosomes would be split but remain otherwise unchanged morphologically. Interestingly, the majority of split desmosomes in PV patient tissue were also found to be smaller than intact desmosomes in NH tissue (Figure 4f). The observation that desmosomes were often found to be smaller and occasionally depleted of Dsg supports a pathomechanism model for PV that includes altered desmosome assembly or disassembly dynamics.

Mechanical stress causes desmosome splitting

Our analysis of patient tissue using SIM revealed striking similarities in desmosomal protein organization and trafficking in cultured keratinocyte models and PV patient tissue. However, split desmosomes have not been reported in cell culture models of disease, possibly because unlike patient tissue, cultured keratinocytes are not exposed to mechanical stress. To test the hypothesis that mechanical stress causes desmosome splitting, cultured keratinocyte monolayers were subjected to a dispase cell fragmentation assay (Ishii et al., 2005) and then processed for immunofluorescence analysis by SIM. Similar to normal human tissue, the keratinocyte cell sheet exposed to NH IgG remained adhesive and numerous intact desmosomes were observed (Figure 5). As expected, keratinocytes exposed to PV IgG dissociated upon exposure to mechanical stress. SIM analysis of the free edge of the fragmented cell sheet exposed to PV IgG revealed extensive desmosome splitting (Figure 5) that was strikingly similar to that observed at the blister edge in PV patient biopsies (Figure 4e). These results indicate that the application of mechanical stress causes desmosomes that are weakened by PV IgG to split at the adhesive interface.

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Figure 5

Mechanical stress causes desmosome splitting in cultured keratinocytes exposed to PV IgG

Keratinocyte monolayers exposed to either normal human (NH) or PV IgG were subjected to mechanical stress by repeated pipetting and then processed for SIM. The keratinocyte sheet exposed to NH IgG display intact desmosomes at cell borders indicated by the green-red-green ‘rail-road’ track staining pattern (top panel). The free edge of the fragmented sheet exposed to PV IgG exhibits multiple split desmosomes (bottom panel) indicated by only a green-red staining. Scale bar, 0.5 μm.

Antibodies

The following antibodies were used in this study: mouse anti-Dsg3 antibody AK15 (Tsunoda et al., 2003) was a kind gift from Dr. Masayuki Amagai (Keio University, Tokyo); rabbit anti-desmoplakin antibody NW6 was a kind gift from Dr. Kathleen Green (Northwestern University); mouse anti-Dsg1 antibody P124 (Progen Biotechnik GmbH, Heidelberg); mouse anti-desmoplakin I/II antibody (Fitzgerald, Acton, MA); rabbit anti-γ-catenin (plakoglobin, H-80) and rabbit anti-p120 antibodies (Santa Cruz Biotechnology, Santa Cruz, CA); mouse anti-E-cadherin (HECD-1, Abcam, Cambridge, MA); mouse anti-CD59-FITC conjugated antibody (Invitrogen, Grand Island, NY); rabbit anti-caveolin-1 antibody (BD Biosciences, San Jose, CA); rabbit anti-early endosomal antigen-1 antibody (EEA1) (Thermo Scientific, Waltham, MA). Secondary antibodies conjugated to Alexa Fluors were purchased from Invitrogen. PV sera (used in Figure 5) was a generous gift from Dr. M. Amagai. PV patient sera used in all other Figures were obtained from patients seen at Emory University, Department of Dermatology. IgG was purified from PV sera according to the manufacturer's protocol using Melon Gel IgG Purification Resins and Kits (Thermo Fisher Scientific, Rockford, IL).

Human tissue biopsy processing

Perilesional biopsies (mucosa lip or skin) from six mucocutaneous PV patients seen at the Emory Clinic Dermatology Department were collected and stored at −80°C. 5 μm sections from the biopsies were mounted onto glass slides and processed for immunostaining as described below.

Cells and culture conditions

Primary human keratinocytes (HKs, passage 2 or 4) were isolated as previously described (Calkins et al., 2006) and cultured in KBM-Gold basal medium (100 μM calcium) supplemented with KGM-Gold Single-Quot Kit (Lonza, Walkersville, MD). For Figure 1, HKs were cultured to 70% confluence on glass coverslips and switched to 550 μM calcium 16-18 hrs to induce junction assembly. HKs were exposed to NH IgG or IgG from PV patients for 6 hrs at 37°C, processed for wide-field immunofluorescence and then analyzed for clustering as described below. For the dispase assay in Figure 5, HKs were cultured to 100% confluence in 4-well tissue culture plates and switched to 50 μM calcium to prevent any junction assembly for 16-18 hrs prior to switching to 550 μM calcium for 3 hrs to allow for junction assembly. HKs were exposed to NH or PV IgG for 3hrs at 37°C and processed for a dispase fragmentation assay followed by SIM, as described below.

Immunofluorescence

Patient tissue slices were allowed to come to room temperature and immunostained with primary and secondary antibodies for 1 hr each at room temperature with triple PBS⁺ washes between antibody incubations. HKs in Figure 1 were fixed in methanol and processed for immunofluorescence. Primary antibodies described above and patient IgG present in tissues was detected with Alexa Fluor-conjugated secondary antibodies. Widefield fluorescence microscopy was performed as previously described (Stahley et al., 2014). Super-resolution structured illumination microscopy (SIM) was performed using the N-SIM system equipped with a 100x/1.49 NA oil immersion objective and 488- and 561-nm solid-state lasers. 3D SIM images were captured with an EM charge-coupled device camera (DU-897, Andor Technology, UK) and reconstructed using NIS-Elements software with the N-SIM module (version 3.22, Nikon, Melville, NY). Colocalization analysis via Mander's coefficient was performed using ImageJ Fiji and plugin JACoP (Bolte and Cordelieres, 2006).

Clustering analysis

Protein clustering was measured as previously described (Saito et al., 2012). Briefly, lines were drawn along cell borders to measure fluorescence intensity using ImageJ/Fiji (NIH, Bethesda, MD). A custom designed MATLAB program identified all local maxima (peaks) with an intensity at least half the maximum intensity. The clustering index is defined as 1 over the number of peaks per 5 μm distance.

Dispase-based fragmentation assay with immunofluorescence

Following PV IgG treatment, cells were subjected to a dispase fragmentation assay as previously described (Saito et al., 2012). In parallel, cell sheets/fragments were fixed in paraformaldehyde, permeablized in Triton X-100 and incubated with primary and secondary antibodies in the tissue culture wells. Gentle washes with PBS⁺ were carried out between Triton and antibody incubations. Cell sheets/fragments were then mounted in ProLong Gold (Molecular Probes, Eugene, OR) and then imaged by SIM.

We acknowledge members of the Kowalczyk laboratory for their insightful advice and discussions, and especially Ms. Susan Summers for keratinocyte isolations. We greatly appreciate the assistance of Ms. Bridget Bradley (Emory Dermatology) and Sue Manos (Emory Pathology) in obtaining and processing pemphigus patient samples. This work was conducted using funding and instrumentation made available through the Integrated Cellular Imaging Core (ICI) of Emory University. This work was supported by NIH R01AR048266 to APK and R21AR066920 to ALM. SNS was supported by NIH T32GM008367. MFW was supported by NIH UL1TR000454.