Characterization of bead-to-cell junctions.

For characterization of bead-to-cell junctions by immunofluorescence, we used Dsg1-coated latex-sulfate beads rather than Dynabeads because the latter displayed strong autofluorescence whereas the former are nonfluorescent. After settlement on the surface of HaCaT cells for 30 minutes, most beads had recruited endogenous Dsg1, Dsg3, and plakoglobin to the bead-to-cell junctions (Figure ​(Figure4,4, A–D), as shown by halo-like accumulation of immunofluorescence specific for these proteins in the immediate vicinity of the beads (arrows in Figure ​Figure4,4, B and D). Beads coated with Fc fragments only (used as controls) did not induce formation of such bead-to-cell contacts (Figure ​(Figure4,4, E and F). With respect to staining for Dsg1, it has to be mentioned that the Dsg1 antibody used for staining is directed against the extracellular domain of Dsg1 and thus cannot discriminate between cellular Dsg1 and recombinant Dsg1-Fc bound to the bead surface. However, as seen in Figure ​Figure4B,4B, the amount of Dsg1-Fc bound to the bead surface was in most experiments too low to create any significant immunosignal. Beads not inducing immunopositive bead-to-cell contacts were in the range of roughly 10–30%, which is close to the value of loosely attached beads determined by laser tweezer experiments (20%). To further characterize the structure of bead-to-cell contacts, monolayers were processed for transmission and scanning electron microscopy. During the rather harsh conditions of fixation, dehydration, and embedding/critical point drying, only the tightly (specifically) bound beads remained attached to the cell surface. Cell-to-bead junctions were found at the tip of finger-like processes between the cell surface and adhering microbeads (arrow in Figure ​Figure5).5). These processes closely mimicked the desmosome-bearing processes between neighboring cells (arrowhead). Phagocytosis of beads was not observed under these conditions and also did not occur during the subsequent incubation period with PF-IgGs (30–60 minutes).

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Figure 4

Localization of Dsg1, Dsg3, and plakoglobin at cell-to-bead contacts. Dsg1-coated beads (A–D) and Fc-coated beads (E and F) were immunostained following settlement on HaCaT cells for 30 minutes. Immunostaining for Dsg1 (B) showed localization of Dsg1 at the cell surface underneath most Dsg1-coated beads, which are visualized in A by corresponding phase contrast microscopic image. Note halo-like accumulation of cellular Dsg1 and plakoglobin around the bead attachment sites (arrows in B and D). Similarly, Dsg3 (C) was detected at cell-bead contacts. In contrast, no immunoreactivity for Dsg1 was found underneath Fc-coated beads visualized in a corresponding phase contrast figure (E and F). Scale bar: 10 μm for all panels (n = 5).

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Figure 5

Characterization of cell-to-bead contacts induced by Dsg1-coated beads. Scanning electron microscopy (A) and transmission electron microscopy (B) were used to characterize contacts between HaCaT cells and Dsg1-coated beads formed after 30 minutes of settlement. Finger-like processes were present underneath beads with direct contact to HaCaT cells (arrows). These processes were similar to the desmosome-bearing processes found at contact sites between neighboring cells (arrowhead). Scale bars: 2.5 μm (A), 1.25 μm (A, inset); 1.0 μm (B).

PF-IgGs reduced adhesion of Dsg1-coated microbeads to the surface of HaCaT cells.

The effect of PF-IgG fractions on Dsg1 binding was studied by laser tweezer experiments. For this purpose, Dsg1-coated beads were allowed to settle on the cell surface for 30 minutes before their binding was tested by separating forces of the laser beam. Under these control conditions, about 80% of Dsg1-coated beads were tightly bound to the cell surface. Incubation periods longer than 30 minutes did not change the overall outcome of the assay. Therefore, all studies were performed with the 30-minute protocol. When EGTA (5 mM, 30 minutes) was added to the culture medium, the number of bound beads dropped to 27% ± 1% of control values (Figure ​(Figure6),6), documenting that Dsg1-mediated adhesion is strongly Ca²⁺-dependent (n = 6). To demonstrate that bead binding was specifically mediated by Dsg1 and to rule out the possibility that loss of bead binding following incubation with EGTA was caused by secondary effects, we performed experiments with the mouse monoclonal antibody directed against the extracellular domain of Dsg1. At antibody concentrations of 1:50, the immunolabeling signal of dot blots of recombinant Dsg1 displayed intensities similar to the signal obtained with PF-IgGs (Figure ​(Figure1).1). Anti-Dsg1 reduced the number of bound Dsg1-coated beads to HaCaT cells to 27% ± 7% of control values and thus to the same extent as EGTA. In the presence of control IgGs (35 μg/ml), the number of beads bound to the cell surface was unaffected (n = 6). However, when HaCaT cells with surface-bound beads were postincubated with PF-IgG fractions (PF1- and PF2-IgGs, 35 μg/ml, 30 minutes), the number of bound beads dropped significantly to 51% ± 2% and 28% ± 4% of control values, respectively (n = 6). When PF2-IgGs were depleted of Dsg1 autoantibodies by immunoabsorption with Dsg1-coated beads (see above), the PF2-IgG–induced loss of bead binding was completely abolished (Figure ​(Figure6),6), indicating that Dsg1-specific antibodies from the PF-IgG fraction were responsible for these effects. This conclusion was confirmed by control experiments using VE-cadherin–coated beads for immunoabsorption. No significant reduction of PF2-IgG–induced loss of Dsg1-mediated binding was observed under these conditions (42% ± 4% of control values, n = 5 each).

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Figure 6

Effect of PF-IgGs on binding of Dsg1-coated beads to HaCaT cells. Beads were allowed to settle on the surface of HaCaT cells for 30 minutes (control). Number of bound beads was reduced by simultaneous incubation of EGTA (5 mM, 30 minutes). Incubation of monolayers with attached beads for an additional 30 minutes with IgG fractions from 2 patients (PF1- and PF2-IgG, 35 μg/ml each) as well as with a monoclonal antibody (1:50) directed against the extracellular domain of human Dsg1 significantly reduced the number of bound beads (label in white box and white bars). Immunoabsorption using Dsg1-coated beads but not control absorption using VE-cadherin–labeled beads completely abolished the effect of PF-IgGs on bead adhesion. Preincubation of HaCaT cells with PF-IgGs prior to bead settlement also reduced bead binding (label in gray box and gray bars) whereas preincubation of beads with PF-IgGs did not inhibit bead binding. In contrast, the monoclonal Dsg1 antibody reduced bead binding also when applied for preincubation with beads, indicating a different mechanism underlying the reduction of Dsg1 adhesion (n = 6 for each condition).

To investigate whether PF-IgGs inhibit Dsg1-mediated adhesion directly or act indirectly by complex cellular responses, we performed preincubation studies in which either HaCaT cells or Dsg1-coated beads were preincubated with PF-IgGs before beads were allowed to settle on the cell surface. When HaCaT cells (35 μg/ml) were preincubated for 30 minutes with PF1-IgGs before beads were applied after removal of PF-IgG–containing culture medium, binding of Dsg1-beads was reduced to 33% ± 8% of control levels and thus was not significantly different from experiments in which PF-IgGs were applied subsequently to binding of Dsg1-coated beads (n = 5). However, when instead Dsg1-coated beads were preincubated with PF1-IgGs (35 μg/ml, 30 minutes) in vitro before settlement on HaCaT cells, 93% ± 3% of beads compared with controls bound to the cell surface, indicating that PF-IgGs did not inhibit Dsg1-mediated binding to HaCaT cells (n = 5). In contrast, the monoclonal anti-Dsg1 reduced bead binding to 29% ± 2% when cells were preincubated with the antibody and also reduced the number of bound beads to 26% ± 1% when Dsg1-coated beads were preincubated with the antibody, indicating that the mechanisms by which PF-IgGs and the monoclonal antibody affected Dsg1-mediated adhesion are profoundly different. Moreover, these experiments demonstrate that PF-IgG treatment of cultured keratinocytes reduced Dsg1-mediated adhesion not by steric hindrance of Dsg1 transinteraction but by involved secondary cellular mechanisms.

PF-IgGs induced dissociation of keratinocytes and loss of Dsg1.

In our study, incubation with PF-IgGs induced formation of numerous intercellular gaps in monolayers of HaCaT cells. Dissociation of keratinocytes in response to incubation with PF-IgGs was paralleled by profound loss of Dsg1 immunoreactivity. Whereas Dsg3 was still present in cellular processes spanning intercellular gaps, Dsg1 staining had largely disappeared in cell groups exhibiting gaps. These data are in line with previous studies that demonstrated that PV antibodies induced Dsg3 degradation (10, 11). Moreover, recent studies reported that PV-IgGs both reduced gene expression and increased phosphorylation of Dsg3 and other desmosomal plaque components. Expression of cytoskeletal proteins like cytokeratins was also found to be reduced following treatment with PV-IgGs (30). In this context, we believe our results are the first to demonstrate that PF-IgGs cause dissociation of cultured keratinocytes within a time course similar to that described previously for antibodies from PV patients (12). Also in favor of signaling events, other studies demonstrated that PV-IgGs caused activation of protein kinase C and required the expression of plakoglobin for cell dissociation (7, 12, 25). Moreover, our experiments indicate that inhibition of Dsg1 binding by steric hindrance is not sufficient to induce keratinocytes to detach. The monoclonal Dsg1 antibody reduced binding of Dsg1-coated beads as well as transinteraction of single Dsg1 molecules (AFM experiments) but was not sufficient to cause dissociation of keratinocytes. If cell dissociation in vitro reflects the skin blistering activity of PF-IgG, these results indicate that reduction of Dsg1-mediated adhesion by steric hindrance may not be sufficient for the pathogenesis of PF.

Cytochemistry.

HaCaT cells were grown on cover slips to confluence as described above (7 days) and incubated with PF-IgGs or control IgGs for 24 hours at 37°C. After incubation with PF-IgG, culture medium was removed and monolayers fixed for 10 minutes at room temperature (RT) with 2% formaldehyde (freshly prepared from paraformaldehyde) in PBS. Afterwards, monolayers were treated with 0.1% Triton X-100 in PBS for 5 minutes at RT. After rinsing with PBS at RT, HaCaT cells were preincubated for 30 minutes with 10% normal goat serum (NGS) and 1% BSA at RT and incubated for 16 hours at 4°C with mouse monoclonal antibodies directed against the ectodomain of human Dsg1 (clone p124; Progen Industries Ltd.) or Dsg3 (Zytomed) (dilution 1:100 in PBS each). After several rinses with PBS (3 times for 5 minutes each time), monolayers were incubated for 60 minutes at RT with Cy3-labeled goat anti-mouse IgGs (Dianova). For visualization of F-actin, Alexa-phalloidin (MoBiTec; diluted 1:60 in PBS) was used (incubation for 1 hour at RT). Cells incubated with antibodies or Alexa-phalloidin were rinsed with PBS (3 times for 5 minutes each time). Cover slips were mounted on glass slides with 60% glycerol in PBS containing 1.5% n-propyl gallate (Serva) as antifading compound. For visualization of Dsg1, Dsg3, and plakoglobin in association with bound Dsg1-coated beads, Dynabeads could not be used because of strong autofluorescence. Therefore, immunolocalization studies were performed with latex sulphate beads (Interfacial Dynamics Corp.). Bead solution (125 μl; 5 μl packed beads) was washed twice in 2-morpholino-ethane sulfonic acid (MES) (25 mM, pH 6.0) buffer by centrifugation and resuspension at 3000 g for 10 minutes. Beads were resuspended in 500 μl MES including 2.5 μg Dsg1-Fc and incubated overnight at RT under permanent slow overhead rotation. After centrifugation at 3000 g for 10 minutes, beads were washed 3 times in 1 ml PBS containing 0.1% albumine. Beads were stored for up to 10 days under permanent overhead rotation at 4°C. After incubation of HaCaT with Dsg1-coated microbeads for 30 minutes, culture medium was removed and monolayers were treated as described above. Monolayers were examined using an LSM 510 (Zeiss). Images were processed using Adobe Photoshop 7.0 software.

Purification of PF-IgG.

Sera from 2 PF patients whose diagnosis was confirmed clinically, histologically, and serologically and from a volunteer without any skin disease (control) were used for the present study. Subjects provided informed consent to the Department of Dermatology, University of Lübeck for the research studies. IgG fractions were purified by affinity chromatography using protein A agarose (Oncogene), eluted by citrate buffer (25 mM, pH 2.4), and immediately dialyzed against HBSS. The purity of isolated IgGs was checked by Coomassie blue staining of 7.5% SDS-PAGE. Activities against Dsg1 of IgGs purified from PF/control sera and purchased mouse monoclonal antibody directed against Dsg1 were adapted by dot blot immunodetection analysis of recombinant Dsg1-Fc (0.1–1 μg per dot) transferred to nitrocellulose using mouse monoclonal Dsg1 antibody followed by HRP-labeled goat anti-mouse IgGs (Dianova) as secondary antibody and the enhanced chemiluminescence technique (Amersham Biosciences) or biotinylated PF-IgGs and biotinylated control IgGs followed by HRP-labeled streptavidin (Dianova). For biotinylation, 300 μg of PF-IgGs was incubated twice for 2 hours at RT with 20 μg/μl N-hydroxy-succimide–biotin (NHS-Biotin) (Pierce Biotechnology Inc.), and afterwards reaction was stopped using saturated glycin in PBS followed by dialysis against PBS overnight at 4°C. Dot blotting of HaCaT cells was performed as indicated above using the monoclonal antibodies directed against Dsg1 and Dsg3, respectively.

Preparation of Fab fragments.

PF-IgGs were dialyzed overnight against buffer A (0.1 M Tris, pH 8.0, 2 mM EDTA) at 4°C. Papain (Sigma-Aldrich) was incubated in buffer A containing dithiothreitol (DTE) for 15 minutes at 37°C for activation and added at 1% final concentration to PF-IgG fractions for a 2-hour incubation at 37°C. Afterwards, reaction was stopped by incubation for 1 hour at 37°C in 2 mM iodacetamid (35). Preparations of Fab fragments were analyzed by Coomassie blue staining of 7.5% SDS-PAGE and by Western blotting using HRP-labeled goat anti-FC IgGs or goat anti-Fab IgGs (Dianova; 1:600 each) followed by HRP-labeled rabbit anti-goat IgGs as outlined above. Capability of Fab fragments to bind to recombinant Dsg1 was demonstrated by dot blot analysis.

Recombinant Dsg1-Fc.

The transfer vector pEVmodPFIg, containing the sequence of the entire extracellular domain (EC 1–5) of Dsg1 and the constant region of human IgG 1 (Fc), was a kind gift from Masayuki Amagai (Keio University School of Medicine, Tokyo, Japan) (4). For expression in eucaryotic cells, the Dsg1-Fc sequence was excised from pEVmodPFIg by digestion with BglII and KpnI and ligated with similarly cut pEGFP-N3 mammalian expression vector (BD Biosciences — Clontech). Dsg1-Fc and VE-cadherin were expressed by stably transfected CHO cells and purified from culture supernatants by affinity chromatography using protein A agarose (Oncogene). The protein was eluted by citrate buffer (25 mM, pH 2.4) and immediately dialyzed against HBSS for 16 hours at 4°C and stored in aliquots at –80°C. Protein content was determined using the Bradford method, and purity was checked by Coomassie blue staining of 10% SDS-PAGE. Western blotting and immunodetection were performed as described above using mouse monoclonal antibody against Dsg1 as well as goat anti-human IgGs directed against the Fc-portion of the fusion protein. Both antibodies detected the recombinant Dsg1-Fc fusion protein as a single band migrating slightly above the calculated molecular weight (not shown).

Coating of polystyrene beads.

After vortexing, 10 μl solution of protein A–coated superparamagnetic polystyrene microbeads (Dynabeads, diameter 2.8 μm; Dynal) containing 2 × 10⁹ beads/ml were washed 3 times using 100 μl of buffer A (100 mM sodium phosphate buffer, pH 8.1). Washing was performed by immobilization of beads for 1 minute in a magnetic tube holder (MPC-E-1; Dynal Biotech) and reuptake in the corresponding buffer. Washed beads were suspended in 100 μl of 100 mM sodium phosphate buffer, pH 8.1, in HBSS containing 10 μg of Dsg1-Fc or of the Fc part of human IgGs (Dianova) and allowed to react for 16 hours at 4°C under permanent slow overhead rotation to avoid aggregation. After washing 3 times for 5 minutes each time in 100 μl of buffer A and 3 times for 5 minutes each time in buffer B (100 mM sodium borate, pH 9.0), beads were incubated for 45 minutes at RT in 100 μl buffer B containing 0.54 mg dimethyl pimelimidate dihydrochloride (DMP) (Pierce Biotechnology Inc.) to covalently cross-link protein A and bound Fc parts. After washing 2 times for 5 minutes each time in buffer C (100 μl 0.2 M ethanolamine, pH 8.0), beads were incubated in buffer C for 2 hours at RT. Finally, beads were washed 3 times for 5 minutes each time in HBSS and stored in HBSS at 4°C for up to 8 days under permanent slow overhead rotation to avoid aggregation of beads. The concentration of beads in these stocks was about 1.6 × 10⁸ beads/ml.

Immunoabsorption experiments.

PF-IgGs were added to 1 ml of DMEM (35 μg/ml final concentration) and incubated overnight at 4°C with Dynabeads coated with 15 μg recombinant Dsg1-Fc or VE-cadherin–Fc as described above. To saturate potential free protein A binding sites, beads were postincubated with 200 μl of human control IgGs (0.3 μg/ml) for 1 hour at RT. Following immunoabsorption, beads were separated using a magnetic tube holder, and DMEM containing the IgG fractions was applied to HaCaT monolayers.

Laser tweezer.

The setup was used as described previously (17). The home-built laser tweezer setup consisted of an Nd:Yag laser (1064 nm), the beam of which was expanded to fill the back aperture at a high NA-objective (100 × 1.3 oil; Zeiss), coupled through the epi-illumination port of an Axiovert 135 microscope (Zeiss) and reflected to the objective by a dicroic mirror (FT 510; Zeiss). Through all experiments, the laser intensity was 42 mW in the focal plane. Coated beads (10 μl of stock solution) were suspended in 200 μl of culture medium and allowed to interact with HaCaT monolayers for 30 minutes at 37°C before measuring the number of bound beads (control values). Beads were considered tightly bound when resisting laser displacement at the 42-mW setting. For every condition, 100 beads were counted. Afterwards, control and PF-IgG fractions (35 μg/ml) were applied for 30 minutes, and the number of bound beads was counted again. Percentage of beads resisting laser displacement under various experimental conditions was normalized to control values. For preincubation experiments, either HaCaT cells or Dsg1-coated beads were incubated with PF-IgGs (35 μg/ml, 30 minutes) and rinsed twice in HBSS before beads were allowed to settle on HaCaT cells for 30 minutes. Negative controls were performed using Dsg1-coated beads incubated on the surface of HaCaT cells in the presence of 5 mM EGTA (Roth) or in the presence of monoclonal mouse IgG antibody (1:50 in HBSS) directed against Dsg-1 (clone p124; Progen Industries Ltd.).

Scanning electron microscopy.

HaCaT monolayers with adhering Dsg1-coated Dynabeads were fixed overnight with 6.5% glutaraldehyde in HBSS. After dehydration with graded acetone series, critical point drying, and sputtercoating with palladium-gold (CPD 030, BAL-TEC), cells were examined with a JSM-840 scanning electron microscope (JEOL).

Transmission electron microscopy.

HaCaT monolayers with adhering Dsg1-coated Dynabeads were fixed in 2.5% glutaraldehyde containing 0.01% ruthenium red in 0.1 M sodium cacodylate, pH 7.35, for 1 hour at 4°C. After rinsing in 0.1 M cacodylate (3 times for 5 minutes each time), cells were postfixed in 2% OSO₄ in 0.1 M cacodylate for 1 hour at RT. Cells were rinsed again in cacodylate and dehydrated in ascending concentrations of methanol (25%, 50%, 70%, 80%, 95% [twice], 100% [twice]; 10 minutes each time). Following incubation in 70% methanol, monolayers were placed for 1 hour in saturated uranyl acetate in 70% methanol for 1 hour in the dark. After dehydration, cells were equilibrated in propylene oxide (2 times for 10 minutes each time) and embedded in Epon 812, semithin sections (1 μM) were stained with toluidine blue, and ultrathin sections were contrasted with uranyl acetate and lead citrate and examined with a LEO AB 912 electron microscope.

Atomic force microscopy measurements.

Dsg1-Dsg1 interactions were characterized by force-distance measurements of Dsg1 coupled via flexible linkers to the tip and substrate of a Bioscope AFM driven by a Nanoscope III controller (Digital Instruments Inc.). Dsg1 was linked covalently to the Si3N4 tip of the cantilever (Park Scientific Ltd.) and freshly cleaved mica plates (Wacker) using PEG spacers containing an amino-reactive cross-linker group (NHS ester) at 1 end and a thiol-reactive group (2-[pyridyldithio] propionate) at the other, as previously described in detail (21). The NHS group served to link PEG to free amino acid groups at both the Si3N4 tip and the SiOH plate introduced by treatment of the tip and plate with 2-aminoethanol HCl (Sigma-Aldrich). Binding events were measured in HBSS by force-distance cycles at amplitudes of 300 nm and at frequencies ranging from 1–2 Hz. Force-distance cycles were performed either at constant lateral positions or with lateral shifts of 1 nm/s. Force-distance cycles were analyzed as described previously in detail (15, 16).

We are grateful to Masayuki Amagai (Keio University School of Medicine, Tokyo, Japan) for supplying us with the Dsg1-Fc construct and helpful discussion and to P. Boukamp (German Cancer Research Center (DKFZ), Heidelberg, Germany) for supplying the HaCaT cell line. We thank Gabriele Königer and Siglinde Schenk for excellent technical assistance. These studies were supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 487, TP B5).