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. Author manuscript; available in PMC: 2009 Oct 1.
Published in final edited form as: J Invest Dermatol. 2008 Nov 27;129(4):908–918. doi: 10.1038/jid.2008.339

Disruption of desmosome assembly by monovalent human pemphigus vulgaris monoclonal antibodies

Xuming Mao 1, Eun Jung Choi 1, Aimee S Payne 1
PMCID: PMC2743719  NIHMSID: NIHMS124306  PMID: 19037235

Abstract

The intercellular interactions of the desmosomal cadherins, desmoglein and desmocollin, are required for epidermal cell adhesion. Pemphigus vulgaris (PV) is a potentially fatal autoimmune blistering disease characterized by autoantibodies against desmoglein (Dsg) 3. During calcium-induced desmosome assembly, treatment of primary human keratinocytes with pathogenic monovalent anti-Dsg3 mAbs produced from a PV patient causes a decrease of Dsg3 and desmoplakin but not desmocollin (Dsc) 3 in the Triton-insoluble fraction of cell lysates within 2 hours. Immunofluorescence and antibody ELISA studies suggest that pathogenic mAbs cause internalization of cell surface Dsg3 but not Dsc3 via early endosomes. Electron microscopy demonstrated a lack of well-formed desmosomes in keratinocytes treated with pathogenic compared to nonpathogenic mAbs. In contrast, pathogenic mAbs caused late depletion of Dsg3 from preformed desmosomes at 24 hours, with effects on multiple desmosomal proteins including Dsc3 and plakoglobin. Together, these studies indicate that pathogenic PV mAbs specifically cause internalization of newly synthesized Dsg3 during desmosome assembly, correlating with their pathogenic activity. Monovalent human PV anti-Dsg mAbs reproduce the effects of polyclonal PV IgG on Dsg3 and will facilitate future studies to further dissect the cellular mechanisms for the loss of cell adhesion in pemphigus.

Keywords: autoimmunity, dermatology, skin

Introduction

Pemphigus vulgaris (PV) is a potentially life-threatening autoimmune blistering disorder of the skin and mucous membranes characterized by serum autoantibodies against the desmosomal cadherins, desmoglein (Dsg) 3 and Dsg1 (Stanley and Amagai, 2006). The pathognomonic histologic feature of PV is suprabasilar acantholysis, leading to the classic “tombstone” appearance of basal keratinocytes that have lost both lateral and apical intercellular adhesion. The serum anti-Dsg autoantibody profile varies depending on the disease type (Amagai et al., 1999; Ding et al., 1997). Patients with mucosal dominant PV typically demonstrate anti-Dsg3 autoantibodies only, while patients with mucocutaneous PV have circulating autoantibodies against both Dsg3 and Dsg1. Individuals with anti-Dsg1 autoantibodies only develop blistering in the superficial layer of the skin in the related disease, pemphigus foliaceus.

Desmogleins are members of the cadherin protein superfamily, which includes the classical cadherins as well as the desmosomal cadherins, Dsg and desmocollin (Dsc) (Getsios et al., 2004). Four Dsg isoforms and three Dsc isoforms have been identified in humans, with each Dsc isoform demonstrating “a” and “b” splice variants (Kljuic et al., 2003; Whittock and Bower, 2003; Smith et al., 2004). Dsg3 and Dsc3 are the primary desmosomal cadherins in the basal layers of the epidermis, the site of PV pathology (Mahoney et al., 1999; North et al., 1996). The extracellular domain of Dsg3 can mediate weak homophilic adhesion (Amagai et al., 1994), but heterophilic adhesion between Dsg and Dsc is thought to form the basis of robust intercellular desmosome adhesion (Chitaev and Troyanovsky, 1997; Syed et al., 2002; Marcozzi et al., 1998). Intracellularly, both Dsg and Dsc “a” cytoplasmic domains bind plakoglobin, which links the desmosomal cadherins to the keratin intermediate filament network through the cytoplasmic plaque protein desmoplakin. These keratin-linked multimolecular structures are responsible for the characteristic electron microscopic appearance of desmosomes (North et al., 1999).

Epitope mapping studies have demonstrated that pathogenic PV antibodies tend to bind calcium-sensitive conformational epitopes at the amino terminus of Dsgs (Futei et al., 2000; Sekiguchi et al., 2001). This antigenic region is predicted to form the trans-adhesive interface between cells based on homology to the crystal structure of the classic cadherins (He et al., 2003). These and other studies have led to the “steric hindrance” hypothesis of disease, in which pathogenic antibodies directly interfere with desmosomal cadherin intercellular interactions. As desmogleins have been shown to internalize in the absence of intercellular contact (Demlehner et al., 1995), the steric hindrance model closely ties into mechanisms of desmosome assembly and disassembly (Kitajima, 2002). Previous studies have shown varying effects of PV IgG on desmosome assembly and disassembly, with results depending on cell confluence and the type of cell lines used (Calkins et al., 2006; Aoyama and Kitajima, 1999; Kitajima et al., 1987; Sato et al., 2000; Cirillo et al., 2006). There is increasing evidence that modulators of diverse signaling pathways can inhibit PV IgG-induced acantholysis, including p38 MAPK (Berkowitz et al., 2006), rho family GTPases (Waschke et al., 2006), protein kinase C (Sanchez-Carpintero et al., 2004), and c-myc (Williamson et al., 2006), among others (reviewed previously (Sharma et al., 2007; Muller et al., 2008)). Additionally, PV autoantibodies have been reported to target alternative antigens, including cholinergic receptors and E-cadherin (Nguyen et al., 2000b; Nguyen et al., 2000a; Evangelista et al., 2008).

Part of the difficulty in studying pathogenic mechanisms in PV is the lack of well-defined, reproducible anti-Dsg autoantibody among laboratories, as serum autoantibody profiles vary among patients and even within the same patient over time, and the use of clinical procedures such as plasmapheresis to obtain large volumes of patient sera is declining. To address this issue, we and others have cloned human pathogenic and nonpathogenic anti-Dsg3 monoclonal antibodies (mAbs) from patients using phage display and heterohybridoma production, respectively (Payne et al., 2005; Bhol and Ahmed, 2002; Yeh et al., 2006). Additionally, pathogenic and nonpathogenic anti-Dsg3 mAbs have been isolated from an active immune mouse model of PV, in which splenocytes from Dsg3-deficient mice are passively transferred to Rag2-deficient mice expressing Dsg3 (Amagai et al., 2000). In a recent study, anti-Dsg3 mAbs from the active immune mouse model of PV did not demonstrate the same extent of Dsg3 depletion from desmosomes as PV IgG, and the residual levels of Dsg3 did not correlate with the adhesive strength of treated cells (Yamamoto et al., 2007). Here, we have investigated the effects of monovalent human PV anti-Dsg mAbs on Dsg3 incorporation into the desmosome, separately examining desmosome assembly and disassembly pathways in primary human epidermal keratinocytes (PHEK). The current study uses well characterized and reproducible sources of pathogenic PV anti-Dsg mAbs (Payne et al., 2005), with nonpathogenic PV anti-Dsg mAbs as controls, to ensure that our findings are not due to polyclonal effects of IgG or whole sera on alternative cellular targets. Additionally, we have examined Dsg3 in comparison to its binding partner Dsc3. The use of primary human keratinocytes as a model system allows for correlation of subcellular fractionation and immunofluorescence localization studies with antibody pathogenicity. We provide evidence that monovalent PV anti-Dsg mAbs preferentially deplete Dsg3 but not Dsc3 from desmosomes during desmosome assembly, correlating with their pathogenic activity.

Results

Pathogenic PV mAbs preferentially deplete Dsg3 but not Dsc3 from the desmosome during calcium-induced desmosome assembly

Calcium induction is a commonly used method for studying desmosome assembly (Watt et al., 1984). Prior studies have shown that in low (<0.1 mM) calcium, Dsgs are synthesized but are rapidly endocytosed and degraded in the absence of calcium or intercellular contact (Demlehner et al., 1995). In high calcium media, the half-life of Dsg increases from approximately 4 to 24 hours, concomitant with the increased partitioning of Dsg into the Triton X-100 insoluble fraction of cells (Pasdar and Nelson, 1989; Williamson et al., 2006). In order to characterize the localization of desmosomal cadherins in PHEK during calcium-induced desmosome assembly, we first examined the immunofluorescence localization of Dsg3 and Dsc3 in PHEK grown in basal media (containing 0.07 mM calcium) and after the switch to media containing 1.2 mM calcium (Figure 1). Under low calcium conditions (time “0”), both Dsg3 and the cytoplasmic plaque protein desmoplakin are localized in an endoplasmic reticular or diffuse cytoplasmic pattern, with no apparent cell surface staining. Within two hours of calcium induction, cell surface staining of both Dsg3 as well as the cytoplasmic plaque protein desmoplakin is apparent, with the majority of Dsg3 being promoted to the cell membrane within 4–6 hours after calcium induction. Interestingly, Dsc3 demonstrates focal cell surface staining even under low calcium conditions, together with a diffuse cytoplasmic pattern). Similar to Dsg3, the majority of Dsc3 localizes to the cell membrane in a punctate pattern within 4 hours after calcium induction. Therefore, to study the effects of PV mAbs on desmosome assembly, we treated PHEK with PV mAbs at the time of calcium induction, when the majority of Dsg molecules are translocating to the cell surface. In order to study desmosome disassembly, we first induced desmosomes in PHEK with 0.4 mM to 1.2 mM calcium media for 12–20 hours, at which time the majority of Dsg3 is desmosomal (as evidenced by co-localization with desmoplakin), and then treated with PV mAbs.

Figure 1. Localization of desmosomal cadherins during calcium-induced desmosome assembly in primary human epidermal keratinocytes.

Figure 1

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PHEK seeded on glass coverslips in basal DK-SFM media (0.07 mM calcium) were shifted to DK-SFM media containing 1.2 mM calcium for the indicated amount of time. Cells were processed for single or double-label immunofluorescence using primary antibodies specific for Dsg3 (H-145), desmoplakin, or Dsc3 (U114). Cell nuclei were stained with DAPI. Scale bar, 10 microns.

The pathogenicity of the monovalent PV mAbs has previously been characterized (Payne et al., 2005; Payne et al., 2007). Supplementary Table 1 indicates the formal mAb name previously published, a shortened designation used in this study based on antibody pathogenicity (P=pathogenic, NP=nonpathogenic), and the cadherin specificity of the mAb, including its deduced amino acid epitope if known. All PV mAbs recognize Dsg3. None of the PV mAbs cross-react with Dsc3 or E-cadherin. Although some mAbs cross-react with Dsg1, Dsg1 protein is minimally induced in the first 24 hours after the calcium switch (Supplementary Figure 1).

We first sought to determine the effects of pathogenic and nonpathogenic PV mAbs on desmosomal cadherins during calcium-induced desmosome assembly. Confluent PHEK cultures were treated with a pathogenic PV mAb (P1), nonpathogenic PV mAb (NP1), negative control recombinant mAb (Neg), or PBS at the time of calcium induction. Since desmosomes are known to partition into the Triton X-100 insoluble fraction of cell lysates, at specified time points cells were processed to obtain the Triton X-100 soluble and insoluble fractions, followed by immunoblot analysis to determine protein levels of Dsg3 and Dsc3. Consistent with our immunofluorescence data (Figure 1), calcium induces a marked increase of Dsg3 in the Triton X-100 insoluble fraction over 4 hours (Figure 2A, upper panel, compare controls at time 0, 2, and 4 hours). The pathogenic PV mAb (P1) causes a reduction of Dsg3 in the Triton X-100 insoluble fraction within 2 hours of antibody treatment, compared to nonpathogenic NP1 mAb, Neg control mAb, and vehicle controls. This effect is more significant 4 hours after antibody treatment. The Triton X-100 soluble fraction demonstrates a transient increase of Dsg3 at 2 hours in response to P1 mAb, followed by an almost complete depletion at 4 hours after antibody treatment. Mild decreases in plakoglobin are observed in both the soluble and insoluble fractions. Desmoplakin is similarly reduced in the Triton X-100 insoluble fraction 2 and 4 hours after treatment with P1 mAb (Figure 2B). In contrast, Dsc3 was not significantly affected in the Triton X-100 soluble or insoluble fraction by PV mAbs.

Figure 2. Pathogenic PV mAb causes loss of Dsg3 and desmoplakin but not Dsc3 from the Triton X-100 insoluble fraction of keratinocyte lysates during calcium-induced desmosome assembly.

Figure 2

Figure 2

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PHEK were treated with a pathogenic PV mAb (P1), nonpathogenic PV mAb (NP1), negative control mAb (Neg), or PBS for the indicated amount of time during calcium-induced desmosome assembly. Equal amounts of protein from the Triton X-100 soluble and insoluble fractions were analyzed by SDS-PAGE, followed by immunoblotting for (A) Dsg3 (5G11) and plakoglobin, or (B) Dsc3 (U114) and desmoplakin, with keratin and beta-tubulin as loading controls.

To determine whether pathogenic PV mAbs affect desmosomal cadherins in pre-assembled desmosomes, confluent PHEK were first cultured in 0.4 mM calcium for 16 hours to induce desmosomes, followed by treatment with PV mAbs. Pathogenic P1 mAb but not NP1 mAb significantly reduces the protein level of Dsg3 in both the Triton X-100 soluble and insoluble fractions after 24 hours, although no significant changes are observed 2 and 8 hours after mAb treatment (Figure 3). Interestingly, Dsc3 and to a lesser extent plakoglobin also decreased in the Triton X-100 insoluble fraction in response to pathogenic P1 mAb after 24 hours.

Figure 3. Pathogenic PV mAb causes delayed depletion of both Dsg3 and Dsc3 from keratinocytes demonstrating preformed desmosomes.

Figure 3

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PHEK were first incubated with 0.4 mM calcium media overnight to stimulate desmosome assembly, followed by treatment with a pathogenic (P1) or nonpathogenic (NP1) PV mAb for the indicated amount of time. Equal amounts of protein from the Triton X-100 insoluble fractions were analyzed by SDS-PAGE, followed by immunoblotting for Dsg3 (5G11), Dsc3 (U114), or plakoglobin, with keratin as a loading control.

Taken together, these data demonstrate that pathogenic PV mAb causes an early and specific depletion of Dsg3 from the Triton X-100 insoluble fraction of keratinocytes during desmosome assembly, while pathogenic effects on Dsg3 in preformed desmosomes are delayed and associated with depletion of multiple desmosomal molecules.

Pathogenic PV mAbs induce internalization of cell surface Dsg3 but not Dsc3

To further investigate the mechanism by which PV mAbs deplete Dsg3 in keratinocytes, we examined the immunofluorescence localization of desmosomal cadherins in cells treated with PV mAbs during desmosome assembly. Consistent with our subcellular fractionation studies, cell surface Dsg3 and desmoplakin are reduced in PHEK treated with pathogenic P1 mAb modestly at 2 hours and more significantly 4 hours after antibody treatment, concomitant with an increase in cytoplasmic vesicular staining of Dsg3 (Figure 4A). In both NP1 and P1-treated cells, linear streaks of Dsg3 staining were observed at the 2 hour time point, which may represent recruitment of Dsg3 to nascent points of cell-cell contact during desmosome assembly. In contrast, Dsc3 cell surface localization is not significantly affected by pathogenic P1 mAb after 4 hours of antibody treatment (Figure 4B).

Figure 4. Pathogenic PV mAb causes loss of cell surface Dsg3 and desmoplakin but not Dsc3 during desmosome assembly.

Figure 4

Figure 4

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PHEK were treated with pathogenic (P1) or nonpathogenic (NP1) PV mAb during calcium-induced desmosome assembly. At the specified time points, cells were processed for single or double-label immunofluorescence using primary antibodies against (A) Dsg3 (5G11), desmoplakin, or (B) both Dsg3 (5G11) and Dsc3 (H-50) after 4 hours of mAb treatment. Scale bar, 10 microns.

Antibody-induced internalization of Dsg3 correlates with PV mAb pathogenicity

Because phage display isolated multiple PV anti-desmoglein mAbs, we were interested in characterizing whether the effects observed for Dsg3 could be reproduced by other pathogenic and nonpathogenic PV mAbs. During calcium-induced desmosome assembly, both pathogenic (P1 and P2) PV mAbs, but not two nonpathogenic (NP1 and NP2) PV mAbs, cause a decrease of Dsg3 in both the Triton X-100 soluble and insoluble fraction of cell lysates after 4 hours of antibody treatment (Figure 5A). Similarly, both P1 and P2, but not NP1 and NP2, PV mAbs cause a loss of cell surface Dsg3 in PHEK after 4 hours of antibody treatment (Figure 5B). Because prior studies have shown that PV IgG is internalized together with Dsg3 (Calkins et al., 2006), we examined whether pathogenic PV mAbs are more rapidly depleted from the keratinocyte culture media than nonpathogenic PV mAbs. PV mAbs were incubated with PHEK during calcium-induced desmosome assembly, and aliquots of culture supernatant were removed at the specified time points for Dsg3 ELISA. Nonpathogenic PV mAbs demonstrated a mild decrease in keratinocyte culture supernatants over 48 hours. However, P1 mAb was rapidly depleted from keratinocyte culture supernatants, with moderate effects on P2 mAb (Figure 5C).

Figure 5. Antibody-induced depletion of Dsg3 correlates with PV mAb pathogenicity.

Figure 5

Figure 5

Figure 5

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PHEK were treated with pathogenic or nonpathogenic PV antibodies for the indicated amount of time during calcium-induced desmosome assembly. (A) Immunoblot analysis of Dsg3 (5G11) in the Triton X-100 soluble and insoluble fractions of keratinocytes treated with different pathogenic (P1 and P2) and nonpathogenic (NP1 and NP2) PV mAbs. (B) Immunofluorescence analysis of cells treated with pathogenic (P1 and P2) and nonpathogenic (NP1 and NP2) PV mAbs. Cells were stained for Dsg3 (5G11, green), and nuclei were stained with DAPI (blue). Scale bar, 10 microns. (C) The relative concentrations of PV mAbs in PHEK culture medium were monitored for 0–48 hrs and measured by Dsg3 ELISA. Data points represent mean values of four independent experiments.

As these data suggested that cell surface Dsg3 may be internalized in response to pathogenic PV mAbs, we investigated whether the cytoplasmic vesicular pattern of Dsg3 colocalized with the early endosomal marker EEA-1. PHEK were cultured with pathogenic P1 mAb in 1.2 mM calcium for one hour, followed by double-label immunofluorescence for the early endosomal marker EEA-1 and the pathogenic P1 mAb, which would be bound only to Dsg3 that had reached the cell surface and would not label newly synthesized outgoing Dsg3. Punctate intercellular localization of Dsg3 is observed after one hour of calcium induction (Figure 6, red arrows), representing antibody-bound cell surface Dsg3. Significant co-localization of EEA-1 and P1 mAb, but not NP1 mAb, is observed beneath the plasma membrane (Figure 6, yellow arrows). EEA-1 positive vesicles lacking Dsg3 are present, prominent in NP1-treated cells (Figure 6, green arrows).

Figure 6. Antibody-bound Dsg3 is internalized from the cell surface into early endosomes.

Figure 6

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PHEK were treated with P1 or NP1 PV mAb during calcium-induced desmosome assembly. After 1 hour, cells were processed for double-label immunofluorescence using Alexa 594-conjugated primary antibodies against the HA tag of the pathogenic PV mAb (shown in red) and Alexa 488 secondary antibodies against primary antibodies binding the early endosomal marker EEA-1 (shown in green). Red arrows, antibody-bound cell surface Dsg3. Yellow arrows, co-localization of internalized P1 mAb with EEA-1. Green arrows, EEA-1 positive/PV mAb negative early endosomes. Scale bar, 10 microns.

Taken together, these data suggest that pathogenic PV mAbs bind newly synthesized Dsg3, preventing desmosome assembly through internalization of Dsg3 into an early endosomal compartment.

Interference with Dsg3 incorporation into the desmosome inhibits calcium-induced desmosome assembly

As our immunofluorescence as well as biochemical data demonstrated that Dsc3 was still localized to the cell surface and the Triton X-100 insoluble fraction of cell lysates despite depletion of Dsg3 by pathogenic PV mAbs during desmosome assembly, we were interested in determining whether Dsc3 alone could nucleate desmosomes through homophilic extracellular interactions. Since desmosomes are well defined at the ultrastructural level by their characteristic appearance, we induced PHEK with P1 and NP1 mAbs in 1.2 mM calcium media for one hour, followed by processing of cells for electron microscopy. In NP1-treated cells after one hour of calcium induction, multiple desmosomes were observed by electron microscopy, demonstrating typical apposing electron dense plaques attached to keratin intermediate filaments (Figure 7A–7B). In one out of 44 NP1 frames, an apparent half-desmosome was observed, although sectioning artifact of an intact desmosome cannot be excluded (Figure 7C). In contrast, most P1-treated PHEK demonstrated no apparent desmosomes despite multiple areas of cell-cell contact (Figure 7D). In some P1-treated cells, desmosome-like structures lacking well defined electron dense plaques were observed (Figure 7E–7F). 44–49 random intercellular frames of P1 and NP1-treated cells were captured by an independent electron microscopy researcher, and the number of desmosomes or desmosome-like structures (in the case of P1-treated cells) were tallied. NP1-treated cells demonstrate an average of 11.1 desmosomes per 10,000 square microns, and P1-treated cells demonstrate an average of 1.8 desmosome-like structures per 10,000 square microns, with no desmosomes demonstrating electron dense plaques. No half desmosomes were observed in P1-treated cells. These data indicate that interference with Dsg3 incorporation into the desmosome by pathogenic PV mAbs disrupts overall desmosome assembly.

Figure 7. Interference with Dsg3 incorporation into the desmosome prevents calcium-induced desmosome assembly.

Figure 7

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PHEK were treated with a pathogenic (P1) or nonpathogenic (NP1) PV mAb during calcium-induced desmosome assembly. After 1 hour cells were processed for electron microscopy. (A) Long arrows indicate desmosomes displaying typical electron dense plaques and keratin intermediate filament insertion. Scale bar, 2 microns. (B) Higher magnification view of desmosomes in control NP1-treated keratinocytes. Scale bar, 1 micron. (C) One half-desmosome was observed in control NP1-treated keratinocytes. Scale bar, 0.5 microns. (D) In P1-treated cells, multiple points of cell-cell contact without desmosome formation were observed. Scale bar, 2 microns. (E) In rare P1-treated cells, desmosome-like structures were observed lacking well-defined electron dense plaques (short arrows). Scale bar, 2 microns. (F) Higher magnification view of desmosome-like structures in P1-treated cells lacking electron dense plaques. Scale bar, 0.5 microns.

Discussion

The major novel finding of the current study is that monovalent human PV anti-Dsg mAbs preferentially disrupt desmosome assembly in human keratinocytes, correlating with their pathogenic activity (Figures 5 and 7). We have demonstrated that pathogenic PV mAbs cause an early depletion of Dsg3 and desmoplakin but not Dsc3 in the Triton X-100 insoluble fraction of keratinocyte lysates during calcium-induced desmosome assembly (Figure 2), concomitant with a loss of cell surface Dsg3 and desmoplakin by immunofluorescence analysis (Figure 4). Pathogenic PV mAbs likely affect newly synthesized (non-desmosomal) Dsg3, as they directly bind cell surface Dsg3 shortly after calcium induction with subsequent internalization (Figure 6). These events result in prevention of desmosome assembly in cells treated with pathogenic PV mAbs by electron microscopy (Figure 7). In contrast, pathogenic PV mAb does not demonstrate significant effects on Dsg3 in pre-assembled desmosomes until 24 hours after antibody treatment, even in lower (0.4 mM) calcium media (Figure 3). This depletion of Dsg3 is paralleled by similar decreases in Dsc3 and plakoglobin, suggesting that the delayed effects of PV mAbs on preformed desmosomes are not due to specific induction of Dsg3 disassembly from desmosomes. Instead, consistent with our electron microscopy findings, prevention of Dsg3 incorporation into the desmosome likely leads to destabilization of the overall desmosomal structure, with subsequent turnover of multiple desmosomal molecules.

Triton insolubility is a classic biochemical feature of desmosomes. However, recent studies have suggested that non-desmosomal desmogleins may partition into lipid rafts, which share the biochemical property of Triton X-100 insolubility (Delva et al., 2008; Nava et al., 2007). Immunofluorescence allows for analysis of the overall effects of pathogenic antibodies on subcellular localization but is suboptimal for defining desmosomal localization, as the level of resolution is often not sufficient to distinguish desmosomes from non-desmosomal plasma membrane. The most well-defined experimental definition of desmosomes is by electron microscopy, with the classic appearance of the electron dense cytoplasmic plaques and intercellular midline. Immunoelectron microscopy requires analysis of hundreds of sections, making this approach logistically difficult for evaluating the overall subcellular trafficking of proteins, especially those of low cellular abundance. Given these limitations, the subcellular localization, immunofluorescence, and electron microscopy data presented are internally consistent, indicating that pathogenic PV mAbs impair assembly of Dsg3 but not Dsc3 into desmosomes.

A previous study examining anti-Dsg3 mAbs isolated from the active immune mouse model of PV showed relatively modest effects of individual pathogenic mAbs on depletion of Dsg3 from the Triton X-100 insoluble fraction of cells, although multiple mAbs in combination showed stronger effects (Yamamoto et al., 2007), consistent with previous reports on the synergistic pathogenicity of various anti-Dsg3 mAb combinations (Kawasaki et al., 2006). It is unclear whether this lack of correlation is secondary to fine differences in anti-Dsg mAb specificity across species, which we have noted when using our human PV mAbs in neonatal mouse passive transfer studies (Payne et al., 2005). In our study, the immunofluorescence and biochemical findings with human-derived PV mAbs correlate highly with PV mAb pathogenicity in primary human keratinocytes. Although the PV mAbs described here are monovalent single chain variable fragment (scFv) antibodies, initial studies using the identical variable fragments cloned into a bivalent IgG molecule have demonstrated similar results by immunofluorescence and immunoblotting (data not shown).

In all pathogenic models for PV IgG, including cultured keratinocyte dispase assay, ex vivo human skin injection, and passive transfer to neonatal mice, loss of cell adhesion is observed within 1–6 hours after antibody treatment ((Payne et al., 2005) and data not shown). Interestingly, the cultured keratinocyte dispase assay is performed under conditions of desmosome assembly, treating cells with PV mAbs at the time of calcium induction. When desmosomes are induced prior to PV IgG exposure, we and others have found that 16–24 hour incubations with PV IgG are required for optimal pathogenicity (Ishii et al., 2005; Calkins et al., 2006). Both of these time frames correspond with the time to depletion of Dsg3 in the Triton X-100 insoluble fraction of cells under the conditions studied, suggesting that ultimately desmosomal rather than non-desmosomal depletion of Dsg3 is the critical pathogenic event in PV.

Prior studies in the field have drawn varying conclusions regarding a primary role of PV IgG in desmosome assembly versus disassembly. One early study suggested that PV IgG does not inhibit calcium-induced desmosome formation in DJM-1 squamous carcinoma cells (Kitajima et al., 1987). However, this study only examined cytoplasmic keratin organization, and other desmoglein isoforms such as Dsg1 or Dsg2 could have nucleated functional desmosomes within these cells. In agreement with our findings, subsequent immunoelectron microscopy experiments suggested that PV IgG binds cell surface Dsg3 prior to desmosome incorporation, leading to its endocytosis (Sato et al., 2000). Immunofluorescence and subcellular fractionation experiments from different laboratories have demonstrated that PV IgG most rapidly affects detergent-soluble cell surface Dsg3, although effects on detergent-insoluble Dsg3 were not observed until 24 hours or later (Aoyama and Kitajima, 1999; Calkins et al., 2006). Interestingly, many studies have found a stronger effect of PV IgG on subconfluent cells or after wounding, when desmosomal assembly may be the primary pathway of Dsg trafficking. Collectively these data appear to lend more support to a pathogenic role of PV mAbs in desmosome assembly as opposed to disassembly. Our data using human anti-Dsg PV mAbs indicate that monovalent PV mAbs reproduce the pathologic effects of bivalent PV IgG, providing evidence that the anti-Dsg serum antibodies are responsible for the desmosomal disruption previously observed with PV IgG.

One of the strongest arguments for a role of PV IgG in desmosome disassembly derives from immunoelectron microscopy studies in the active immune mouse model of PV (Shimizu et al., 2004). Post-embedding labeling of oral mucosal biopsies demonstrated in vivo mouse IgG labeling of whole and split desmosomes without keratin retraction. The paucity of cytoplasmic or nondesmosomal labeling suggested that anti-Dsg antibodies primarily bind desmosomal Dsg3, causing disassembly through direct interference with intercellular desmosomal cadherin interaction. However, these studies could not distinguish pathogenic from nonpathogenic antibody labeling of desmosomal Dsg, and other immunoelectron microscopy studies have shown that split desmosomes observed after PV IgG treatment of cultured keratinocytes demonstrate a reduced Dsg3 to Dsc3 ratio, indicating that partial depletion of Dsg3 from desmosomes during assembly is sufficient to cause loss of functional desmosomal adhesion (Shu et al., 2005). Ultimately, studies on the in vivo half life of cell surface Dsg3 will be required to confirm whether the pathogenic effects of PV mAbs on Dsg3 during desmosome assembly can account for the time course of blister formation in various PV pathogenic models.

One interesting observation is that Dsc3, but not Dsg3 or desmoplakin, demonstrates focal cell surface staining in basal (0.07 mM) calcium media (Figure 1), indicating a potential non-desmosomal role for cell surface Dsc3. Prior studies have shown that Dsc can localize to the lateral plasma membrane of Madin-Darby canine kidney cells together with E-cadherin in low calcium media, and that Dsc is the first desmosomal cadherin to localize to the cell surface after the switch to high calcium media (Burdett and Sullivan, 2002). Further studies have demonstrated that the Dsc cytoplasmic domain can interact with beta-catenin and plakoglobin, independent of its desmosomal adhesive function (Hanakawa et al., 2000), suggesting that Dsc may provide the link for adherens junction-dependent desmosome assembly. A desmosome-independent biological role for Dsc is further supported by the d6.5 embryonic lethality of Dsc-deficient mice, prior to the appearance of desmosomes (Den et al., 2006). Interestingly, the desmoplakin-null mouse also exhibits d6.5 embryonic lethality (Gallicano et al., 1998). An epidermal-specific deletion of desmoplakin formed partially adhesive desmosomes, although they were functionally compromised (Vasioukhin et al., 2001). A non-desmosomal function for Dsc3 and desmoplakin could contribute to the cell surface staining of Dsc3 and desmoplakin observed after pathogenic PV mAb treatment (Figure 4). Alternatively or in addition, the poorly formed sites of keratin intermediate filament insertion observed in Figure 7E–F could reflect desmoplakin-bound Dsc3, which would correlate with Dsc3 and desmoplakin retention in the Triton X-100 insoluble fraction (Figure 2B). Future immunoelectron microscopy studies would be required to substantiate this hypothesis.

It is interesting to note the emerging parallels between desmosomal and classical cadherin-based adhesion. Similar to our current findings with Dsg-blocking antibodies, early studies using E-cadherin blocking antibodies demonstrated a marked loss of cell surface E-cadherin during calcium-induced junction assembly, with more modest effects on preformed adherens junctions (Gumbiner et al., 1988). The regulation of cadherin adhesiveness is a complex process that can be modulated through multiple mechanisms including junction assembly, disassembly, and more rapid alterations in intermolecular affinities (Gumbiner, 2000). Interestingly, numerous signaling pathways regulating adherens junction assembly have been implicated in PV pathogenesis, including protein kinase C and rho family GTPases, among others (Sharma et al., 2007; Muller et al., 2008; Waschke, 2008). It is not surprising, therefore, that multiple chemical inhibitors have been demonstrated to inhibit acantholysis by PV IgG, because perturbations that generally increase intercellular adhesion (by adherens junctions or desmosomes) may be expected to ameliorate the effects of anti-Dsg autoantibodies. Future experiments may determine whether chemical inhibitors of acantholysis are specific to Dsg3 and desmosomes, or due to modulation of other adhesion pathways. Ultimately, these studies may broaden our understanding of cross-talk among epidermal adhesive junctions and lead to novel strategies for treating autoimmune blistering diseases.

Materials and Methods

Antibodies

The production and characterization of single chain variable fragment (scFv) PV mAbs used in these studies have been previously described (Payne et al., 2005; Payne et al., 2007), including negative control scFv mAbs produced in the same bacterial recombinant system (E1M2, a human anti–red blood cell antibody, and AM3-13).

Primary antibodies: mouse monoclonal anti-Dsg3 (5G11, Invitrogen), rabbit polyclonal anti-Dsg3 (H-145, Abcam), mouse anti-desmoplakin (BioDesign International), mouse monoclonal anti-Dsc3 (Meridian Life Science Inc.), rabbit polyclonal anti-Dsc3 (H-50, Santa Cruz Biotechnology Inc.), mouse monoclonal anti-keratin (Zymed), mouse monoclonal anti-plakoglobin (11E4, anti-γ catenin, Chemicon), mouse anti-EEA-1 (BD Biosciences), rat monoclonal anti-HA (3F10, Roche).

Secondary antibodies or reagents: HRP-conjugated donkey anti-mouse IgG (Jackson ImmunoResearch Lab, Inc.); HRP-conjugated rat anti-HA (3F10, Roche); Alexa 488-conjugated donkey anti-human IgG, Alexa 488-conjugated goat anti-mouse and rabbit IgG, Alexa 594-conjugated goat anti-mouse, rat, or rabbit IgG (Molecular Probes, Invitrogen); Alexa 594-conjugated anti-HA and Alexa Fluor 633 phalloidin (Molecular Probes, Invitrogen).

Cell Culture

Primary human epidermal keratinocytes (PHEK) isolated from neonatal foreskin were expanded and stored frozen for later use by the University of Pennsylvania Dermatology core facility. Cells from passages 4–5 were cultured in basal defined keratinocyte–serum-free media (DK-SFM) (Invitrogen) containing 0.07 mM CaCl2 supplemented with penicillin/streptomycin. Cells were maintained in a humidified incubator with 5% CO2 at 37°C. For experiments on desmosome assembly, cultures were exposed to PV mAbs (40–100 μg/mL) in DK-SFM supplemented with 0.4–1.2 mM CaCl2 for up to 24 hours. For experiments on desmosome disassembly, cells were incubated with DK-SFM supplemented with 0.4–1.2 mM CaCl2 for 16–24 hours prior to exposure to PV mAbs.

Cell fractionation and immunoblot

PHEK were cultured to 90–100% confluence in DK-SFM with 5% CO2 at 37°C before experiments. After treatments, cells were chilled on ice and washed with ice cold PBS containing calcium and magnesium (DPBS, Mediatech Inc). For analysis of Triton X-100-soluble or -insoluble fractions, cells were initially lysed in buffer containing 1% Triton X-100, 10 mM Tris-HCl (pH 7.5), 140mM NaCl, 5mM EDTA, 2mM EGTA, with protease inhibitors (P8340, Sigma) for 15 minutes on ice, followed by centrifugation at 16,000×g for 20 minutes at 4°C. The resulting supernatant was collected as the cytosol/membrane (nondesmosomal) fraction. The Triton X-100-insoluble pellets (containing desmosomal fractions) were solubilized in Laemmli sample buffer containing 5% beta-mercaptoethanol. Total protein was determined by protein assay (RC-DC, BioRad), and approximately 25 μg of each sample fraction was separated by SDS-PAGE. The gels were transferred to nitrocellulose, and membranes were incubated with mouse anti-Dsg3 mAb 5G11 (Invitrogen), mouse monoclonal anti-desmocollin 3 (U114, Meridian Life Science Inc.), mouse monoclonal anti-keratin (Zymed), or mouse monoclonal anti-plakoglobin (11E4 anti-γ catenin, Chemicon) dilutedin PBS/5% milk. Blots were washed with PBS containing 0.1% Tween-20 and then incubated with either HRP-conjugated goat anti-mouse (Bio-Rad) or donkey anti-mouse (Jackson ImmunoResearch Labs, Inc.) secondary antibodies diluted in PBS/5% milk. Blots were developed using ECL Plus reagent (Amersham Biosciences).

Immunofluorescence microscopy studies

PHEK were seeded on coverslips pre-incubated with collagen solution. For assembly experiments, cells were treated with PV mAbs in DK-SFM containing 1.2 mM calcium for the indicated amount of time, and then fixed in PBS containing 4% paraformaldehyde (freshly diluted from a 20% paraformaldehyde solution, Electron Microscopy Sciences) for 20 minutes at 4 °C, or with ice cold methanol or methanol/acetone (1:1) for 15 minutes at 4 °C. For cells fixed with 4% paraformaldehyde, a 10 minute incubation with PBS containing 0.2% Triton X-100 was used for cell permeabilization. Cells were blocked in PBS containing 2% BSA for 30 minutes at room temperature, followed by incubation with primary antibodies in blocking solution for 1 hour at room temperature. After three washes in PBS, cells were incubated with appropriately conjugated secondary antibodies diluted 1:200 in blocking solution for 30–60 minutes at room temperature. Nuclear staining was performed with DAPI (Sigma) or with ProLong Gold antifade reagent with DAPI (Molecular Probes, Invitrogen). Immunofluorescence was visualized with an Olympus BX61 microscope. Images were acquired using Slidebook 4.2 software (Olympus) and a Hamamatsu Orca ER camera, using consistent time exposures among samples from each experiment. Nearest neighbor deconvolution was performed on the acquired images.

Antibody ELISA

PHEK were incubated with 100μg/mL pathogenic PV mAbs (P1 and P2) or nonpathogenic antibodies (NP1 and NP2) in DK-SFM supplemented to 0.4 mM calcium. 10 μl of culture medium was removed at time point 0, 2, 4, 6, 8, 12, 24, and 48 hours. The samples were immediately diluted in Dsg3 ELISA sample diluent (MBL International Corp.) for a starting OD450 of approximately 0.5–1.0, stored at 4°C until all samples had been collected, then incubated on Dsg3 ELISA plates according to the manufacturer’s directions. After washing, plates were developed with HRP-conjugated anti-HA antibody(3F10, Roche) and tetramethylbenzidine substrate. Absorbance was read at 450 nm.

Electron microscopy

Confluent PHEK were seeded onto collagen coated Permanox removable chamber coverglass (LabTek) and grown to confluence in DK-SFM, followed by a one hour incubation with PV mAbs in DK-SFM supplemented to 1.2 mM CaCl2. Cells were prepared for transmission electron microscopy analysis according to previously published methods (Allen et al., 1996; Dyer et al., 2006). Briefly, at the end of antibody incubation, the cells were washed in PBS and fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M sodium cacodylate buffer for 1 hour. The cells were post-fixed with 1% osmium tetroxide and dehydrated with ethanol. After infiltration, selected areas of the slide were selected for flat embedding with pre-made blank epoxy blocks and polymerized at 68°C for 48 hours. One surface semithin section was cut and stained with toluidine blue to screen the cell population, and ultrathin sections were cut parallel to the culture surface. After stain with uranyl acetate and lead citrate, the sections were examined with a FEI Tecnai-T12 transmission electron microcope operated at 80kv. To evaluate intercellular contact, random digital images (X1100) were taken from multiple mesh holes with a Gatan 2K digital camera using image acquisition software (DigitalMicrograph) from the same vendor.

Supplementary Material

Supplementary data file

Acknowledgments

We wish to thank John Stanley and John Seykora for helpful discussions, Chris Marshall for assistance with maintenance and plating of primary human epidermal keratinocyte cultures, and Qian-Chun Yu for the acquisition of electron microscopy images. This work was supported by NIH/NIAMS K08 AR053505 and a University Foundation Research award to ASP.

Abbreviations list

Dsc

desmocollin

Dsg

desmoglein

EEA

early endosomal antigen

NP

nonpathogenic

P

pathogenic

PHEK

primary human epidermal keratinocytes

PV

pemphigus vulgaris

Footnotes

Conflict of Interest

The authors state no conflict of interest.

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Supplementary Materials

Supplementary data file
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