Search

Background: Herpes simplex virus (HSV) infection in the skin causes small grouped vesicles characterized by acantholytic cells and multinucleated giant cells (MGCs). Although viral factors have been studied as fusion proteins, little is known how the differentiation status of keratinocytes is involved in the formation of MGCs by HSV-1 infection., Objective: As the human epidermis is composed of several layers of keratinocytes that undergo terminal differentiation, we aimed to elucidate whether the differentiation status of keratinocytes affects viral entry, propagation, cell-to-cell transmission of HSV-1, and MGC formation., Methods: HaCaT cells and normal human epidermal keratinocytes were cultured in either low- or high-Ca 2+ medium. After HSV-1 infection, cellular morphology, viral propagation, and expression of cytoskeletal and intercellular adhesion molecules were examined sequentially. Viral entry, replication, and expression of HSV receptors were analyzed. Cell-to-cell transmission and fusion after HSV-1 infection was evaluated using the Cell Tracker™ Red CMTPX dye system., Results: Keratinocytes in high-Ca 2+ medium formed MGCs, but those in low-Ca 2+ medium formed single nuclear round cells in response to HSV-1 infection. HSV-1 entered the keratinocytes more effectively in low-Ca 2+ than in high-Ca 2+ medium, although transcripts of HSV receptors were comparable in both conditions. HSV-1 could replicate more efficiently in high-Ca 2+ than in low-Ca 2+ medium. A cell-to-cell fusion assay showed that HSV-1-infected and adjacent-uninfected keratinocytes were involved in MGCs in high-Ca 2+ but not in low-Ca 2+ medium., Conclusion: Differentiated keratinocytes promote MGC formation by cell-to-cell fusion with resolution of cell membrane and cell-to-cell transmission of HSV-1 from infected keratinocytes to neighboring uninfected keratinocytes., (Copyright © 2018 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All rights reserved.)

The receptor for advanced glycation end products (RAGE) is involved in the pathogenesis of many inflammatory, degenerative, and hyperproliferative diseases, including cancer. Previously, we revealed mechanisms of downstream signaling from ligand-activated RAGE, which recruits TIRAP/MyD88. Here, we showed that DNAX-activating protein 10 (DAP10), a transmembrane adaptor protein, also binds to RAGE. By artificial oligomerization of RAGE alone or RAGE-DAP10, we found that RAGE-DAP10 heterodimer formation resulted in a marked enhancement of Akt activation, whereas homomultimeric interaction of RAGE led to activation of caspase 8. Normal human epidermal keratinocytes exposed to S100A8/A9, a ligand for RAGE, at a nanomolar concentration mimicked the pro-survival response of RAGE-DAP10 interaction, although at a micromolar concentration, the cells mimicked the pro-apoptotic response of RAGE-RAGE. In transformed epithelial cell lines, A431 and HaCaT, in which endogenous DAP10 was overexpressed, and S100A8/A9, even at a micromolar concentration, led to cell growth and survival due to RAGE-DAP10 interaction. Functional blocking of DAP10 in the cell lines abrogated the Akt phosphorylation from S100A8/A9-activated RAGE, eventually leading to an increase in apoptosis. Finally, S100A8/A9, RAGE, and DAP10 were overexpressed in the psoriatic epidermis. Our findings indicate that the functional interaction between RAGE and DAP10 coordinately regulates S100A8/A9-mediated survival and/or apoptotic response of keratinocytes., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

Here we show that keratinocytes in psoriatic lesional skin express increased Toll-like receptor (TLR) 9 that similarly localizes with elevated expression of the cathelicidin antimicrobial peptide LL-37. In culture, normal human keratinocytes exposed to LL-37 increased TLR9 expression. Furthermore, when keratinocytes were exposed to LL-37 and subsequently treated with TLR9 ligands, such as CpG or genomic DNA, they greatly increased production of type I IFNs. This response mimicked observations in the epidermis of psoriatic lesional skin as keratinocytes in psoriatic lesions produce greater amounts of IFN-β than normal skin lacking LL-37. The mechanism for induction of type I IFNs in keratinocytes was dependent on TLR9 expression but not on a DNA-LL-37 complex. These findings suggest that keratinocytes recognize and respond to DNA and can actively participate in contributing to the immunological environment that characterizes psoriasis.

The corneodesmosomes in the stratum corneum are critical for the maintenance of stratum corneum integrity. To evaluate the normal and diseased keratinization states in the epidermis, we studied the distribution of desmoglein 1 (DSG1), a major component of corneodesmosomes, in samples of the stratum corneum obtained by tape stripping, a non-invasive method. Samples were collected from lesional skin of four patients with psoriasis and three with lichen planus, and from non-lesional skin of three volunteers. Upper stratum corneum cells were obtained by tape stripping and skin biopsies were obtained from adjacent sites. Tape-stripped samples were examined by immunofluorescence microscopy using anti-DSG1 monoclonal antibody, in combination with histopathology of skin biopsies. In normal human stratum corneum, which shows basket-woven orthokeratosis, DSG1-containing fluorescent dots were distributed on the lateral cell-cell contact areas of plasma membrane, but not on the dorsal/ventral plasma membrane, and formed a well-ordered hexagonal network structure. In psoriatic stratum corneum, fluorescent dots were distributed throughout the cell membrane at ventral aspects of corneocytes as well as at the lateral cell-cell contacts. In lichen planus, fluorescent dots were distributed homogeneously and/or heterogeneously on the ventral surface in some cells. Adjacent cells lacked DSG1 at the lateral cell-cell contacts, but were instead separated by distinctive black-gap lines. These results suggest that the intercellular adhesion by DSG1 may depend on the lateral plasma membrane in normal human stratum corneum, on the dorsal/ventral plasma membrane in lichen planus, and on both lateral and dorsal/ventral plasma membranes in psoriatic stratum corneum. Tape stripping and DSG1 immunofluorescence visualizes adhesion features of corneocytes and has considerable potential for evaluation of abnormal keratinization and the process of healing in response to treatment., (© 2010 Japanese Dermatological Association.)

Epidermal cells adhere to the basement membrane zone through cell-matrix junctions termed hemidesmosomes. During wound healing, hemidesmosomes are disassembled to allow keratinocytes to move over wound sites. Such movement is mediated by both hemidesmosome protein complexes (HPCs) and focal contacts (FCs). In this study, we analyzed the interaction between HPCs and FCs in live HaCat cells expressing yellow fluorescent protein (YFP)-tagged beta4 integrin and cyan fluorescent protein (CFP)-tagged alpha-actinin as markers of HPCs and FCs, respectively. In HaCat cells migrating to repopulate wounds, FC proteins cluster rapidly in the direction of the wound. HPC assembly then follows and the newly formed HPCs occupy sites vacated by the disassembled FCs. HPC dynamics are dramatically reduced, and HaCat cells cease migration upon treatment with reagents that affect FC integrity/function. Upon treatment with reagents that destabilize HPCs, the dynamics of FCs in HaCat cells at the edges of wounds are enhanced, although FC assembly is irregular and the migration of the cells is aberrant. We also show that the complex interaction between hemidesmosomes and FCs in keratinocytes is myosin dependent and requires energy. In summary, we suggest that HPCs and FCs dynamics are tightly co-regulated in keratinocytes undergoing migration during wound healing.

Although desmocollins (Dscs) and desmogleins (Dsgs) are known to be bound to each other to form desmosomes, neither their interactions nor regulations that occur in human keratinocytes grown in low and high Ca2+medium has been determined. In this study, we investigated whether Dsc3 interacts with Dsg3 in a cell line of human squamous cell carcinoma keratinocytes (DJM-1) grown in low (0.05 mm) or high (1.27 mm) Ca2+ medium. Anti-Dsc3 monoclonal antibody did not co-immunoprecipitate Dsg3 nor plakoglobin with Dsc3 in low Ca2+ culture, whereas it co-immunoprecipitated plakoglobin already at 10 min and Dsg3 at 60 min after Ca2+ -switch in association with Dsc3 phosphorylation at serine residues. These results suggest that both the binding of Dsc3 to plakoglobin and Dsc3 phosphorylation are involved in Dsc3 binding to Dsg3 during Ca2+ -induced desmosome assembly.

We have shown that binding of bullous pemphigoid (BP)-patient IgG (BP-IgG) causes the internalization of BP180 from the cell membrane. This study examined whether BP-IgG treatment can deplete cultured keratinocytes of BP180, how it affects cellular levels of alpha6 and beta4 integrins (by western blot analysis using monoclonal antibodies to these antigens), and whether it reduces adhesion of cells to the culture dish (by a vibration detachment assay). All BP-IgG or BP sera with high values of BP180-ELISA from 18 BP patients before and after oral corticosteroid treatment showed dramatically decreased BP180 in cells after 6 hours of BP-IgG stimulation, whereas alpha6 and beta4 integrin levels were not decreased. Even IgG from patients in whom oral corticosteroid had suppressed active blistering could deplete cells of BP180, as long as sera retained a high value of BP180-ELISA. On the other hand, reduction of cell BP180 content increased detachment of cells from the dish. These results suggest that BP-IgG reduces hemidesmosomal BP180 content, weakening the adhesion of hemidesmosomes to the lamina densa. In the presence of BP180 deficiency, inflammation generated by BP180 immune-complex formation might then tear the weakened lamina lucida, and this could lead to generation of the BP-specific split at the lamina lucida.

We recently showed that p120 catenin (p120ctn), which is an armadillo family protein member that binds to E-cadherin (E-cad), is also localized to desmosomes by directly or indirectly binding to desmogleins (Dsg). We examined whether p120ctn is associated with Dsg1 and Dsg3, as compared with E-cad and plakoglobin (PG), in keratinocytes grown in high or low Ca2+, using a human squamous cell carcinoma cell line, DJM-1 cells. The cell lysate of DJM-1 cells grown in high- or low-Ca2+ media was immunoprecipitated with anti-Dsg1/2 and Dsg3 antibodies, and we examined whether p120ctn is associated with Dsg1 and Dsg3. Then, we observed the co-localization between Dsg3 and p120ctn in cells grown in high- or low-Ca2+ medium on double-staining immunofluorescence microscopy using anti-p120ctn and anti-Dsg3 antibodies. Immunoprecipitates with anti-Dsg1/2 and Dsg3 antibodies in cells grown in high-Ca2+ medium contained p120ctn. In contrast, in low-Ca2+ medium, p120ctn was co-immunoprecipitated with neither Dsg1 nor Dsg3, but was co-immunoprecipitated with E-cad in cells grown in both high- and low-Ca2+ media. Dsg3 was associated with PG in cells grown in both low- and high-Ca2+ media. On immunofluorescence microscopy, p120ctn and Dsg3 were independently observed in cells grown in low-Ca2+ medium; p120ctn, but not Dsg3, was observed in a linear pattern at the cell-cell boundary. However, they were co-localized at cell-cell contacts in cells grown in high-Ca2+ medium. Thus, these proteins are not co-localized in low Ca2+ medium. These results suggest that p120ctn plays an important role in Ca2+-induced desmosome formation.

Background: Although pemphigus vulgaris (PV)-IgG has been shown to activate urokinase plasminogen activator (uPA) in cultured keratinocytes, activation of uPA is thought to have no primary role in PV-acantholysis, because PV-IgG is still pathogenic in uPA- and tissue-PA-knockout mice., Objective: To determine if PV-IgG-induced uPA activation is due to specific antibody against Dsg3, we examined whether or not pathogenic monoclonal anti-Dsg3 antibody can activate uPA, because PV-IgG is thought to contain antibodies against unknown antigens besides Dsg3., Methods: We stimulated cultured normal human and DJM-1 keratinocytes with monoclonal anti-Dsg3 IgG1 antibodies (pathogenic AK23, AK19 and nonpathogenic AK18, AK20), negative control monoclonal mouse IgG1 and positive control PV-IgG. Cells were treated with IgGs over a time course of 24h, and uPA-protein content and activity in the culture medium were determined by enzyme-linked immunosorbent assay (ELISA) and chromogenic assay, respectively., Results: The uPA-protein content in samples treated with or without pathogenic, nonpathogenic, control monoclonal mouse IgG1s and PV-IgGs increased continuously up to 24h, with no differences between samples, suggesting a spontaneous secretion. In contrast, uPA activity in the culture medium of cells treated with PV-IgG increased dramatically, whereas that of cells treated with all AK-IgGs and control monoclonal mouse IgG1 did not increase at all., Conclusion: These results suggest that PV-IgG-dependent uPA activation is not related to anti-Dsg3 antibody activity, which is an essential factor in PV-IgG acantholysis, and that it may be due to other antigens than Dsg3 or unknown factors contained in PV-IgG fraction.

Pemphigus vulgaris is an autoimmune blistering disease caused by antibodies against desmoglein (Dsg) 3. We previously reported that pemphigus vulgaris (PV)-IgG caused the formation of Dsg3-depleted desmosomes in normal human cultured keratinocytes and DJM-1, a human squamous cell carcinoma cell line. In the present study, we injected PV-IgG and normal human IgG into neonatal mice and examined the quantities of Dsg3 in the mouse skin. We showed that injection of PV-IgG into neonatal mice caused suprabasal blister formation and approximately 30% reduction of Dsg3 in mouse epidermal keratinocytes, compared to mice injected with normal human IgG. In addition, we showed that the quantity of Dsg3 in the skin of patients with PV did decrease, as compared to that in healthy volunteers. Our data suggests the reduction of Dsg3 might be relevant to blister formation. These results also suggest that even a partial depletion of Dsg3 may contribute to blistering in PV patients.

The Rho GTPase and Fyn tyrosine kinase have been implicated previously in positive control of keratinocyte cell-cell adhesion. Here, we show that Rho and Fyn operate along the same signaling pathway. Endogenous Rho activity increases in differentiating keratinocytes and is required for both Fyn kinase activation and increased tyrosine phosphorylation of beta- and gamma-catenin, which is associated with the establishment of keratinocyte cell-cell adhesion. Conversely, expression of constitutive active Rho is sufficient to promote cell-cell adhesion through a tyrosine kinase- and Fyn-dependent mechanism, trigger Fyn kinase activation, and induce tyrosine phosphorylation of beta- and gamma-catenin and p120ctn. The positive effects of activated Rho on cell-cell adhesion are not induced by an activated Rho mutant with defective binding to the serine/threonine PRK2/PKN kinases. Endogenous PRK2 kinase activity increases with keratinocyte differentiation, and, like activated Rho, increased PRK2 activity promotes keratinocyte cell-cell adhesion and induces tyrosine phosphorylation of beta- and gamma-catenin and Fyn kinase activation. Thus, these findings reveal a novel role of Fyn as a downstream mediator of Rho in control of keratinocyte cell-cell adhesion and implicate the PRK2 kinase, a direct Rho effector, as a link between Rho and Fyn activation.

In order to elucidate the function of c-Src in keratinocytes, we studied the intracellular distribution of its active and inactive form in cultured normal human keratinocyte, using anti-c-Src monoclonal antibody clone 28, which recognizes the active form of c-Src (dephosphorylated at COOH-terminal residue Tyr 530), and monoclonal antibody clone 327 which recognizes both active and inactive forms. Since c-Src has been suggested to be involved in the control of cell adhesion in other cells, we produced a dynamic condition of cell migration by cutting culture cell colonies into squares to form a mesh pattern with a blade (culture wound model). Before cutting, the active form was expressed in cells located only at the periphery of colonies or isolated migrating cells, and was associated with microtubules. Wounding the colony generated a dramatic and rapid activation of c-Src in a few rows of cells along the cut edges, which were made even at the middle of colony, resulting in the association of the active form with microtubules. This increase of the active form was also detected by immunoblotting of cell extracts. These reactions were inhibited by 1 mM sodium orthovanadate, a protein-tyrosine phosphatase inhibitor. ST 638, a potent Src family tyrosine kinase inhibitor, inhibited the migration of keratinocytes in the culture wound healing model. These results suggest that wounding the culture causes activation of c-Src in keratinocytes, and thus activated c-Src may play a role in the function of microtubules during cell migration, especially at an early stage of wound healing.

To determine the assembly pathway of desmoglein 3 (Dsg3) into desmosomes and the subsequent effects of pemphigus vulgaris immunoglobulin G (PV-IgG) on such, we employed a time-lapsed labeling for FITC/Rhodamine (Rod) double-stained immunofluorescence and 5-nm/10-nm gold double-stained immunoelectron microscopy by using PV-IgG, which was confirmed to react specifically Dsg3. Cells from a human squamous cell carcinoma cell line (DJM-1) were first treated briefly with PV-IgG (3 min), then incubated in either anti-human IgG-FITC or 5-nm gold antibody-containing medium (5 min), followed by a 60-minute chase in normal medium without antibodies. The same cells were reincubated with PV-IgG medium for 3 minutes, followed by either anti-human IgG-Rod or 10-nm gold antibodies for 5 minutes. Using this method, FITC and 5-nm gold particles show the fate of Dsg3-PV-IgG complexes during the following 60-minute chase. IgG-Rod or 10-nm gold particles, which are bound during the last 5 minutes of the chase, show Dsg3 molecules newly expressed on the cell surface during the 60-minute-chase period. Initially, Dsg3 formed two types of small clusters on the nondesmosomal plasma membrane, ie, either half-desmosome-like clusters with keratin intermediate filament (KIF) attachment or simple clusters without KIF attachment. The PV-IgG binding to Dsg3 caused the internalization of the simple clusters into endosomes, but not the half-desmosome-like clusters. After the 60-minute-chase period, both types of cell surface Dsg3 clusters were labeled with only 10-nm gold, suggesting that new Dsg3 molecules were being delivered to the cell surface. Desmosomes were labeled with both 5-nm gold and 10-nm gold, whereas the half-desmosome-like clusters were labeled with only 10-nm gold, suggesting that the desmosomes themselves were not split. These results suggest that Dsg3 first forms simple clusters, followed by KIF-attachment, and then becomes integrated into desmosomes, and that PV-IgG-induced internalization of the nondesmosomal simple clusters of Dsg3 may represent the primary effects of PV-IgG on keratinocytes.

We have investigated transmembrane signaling for the regulation of desmosomes and hemidesmosomes, using a human squamous cell carcinoma cell line (DJM-1) and normal human keratinocytes. This review discusses the involvement of protein kinase C (PKC) in regulation of these junctions, and signaling pathways involved in cell-cell detachment induced by pemphigus vulgaris (PV) IgG in a culture system. Cells grown in low-Ca++ conditions, which lack desmosomes, rapidly form desmosomes upon a low-normal Ca++-shift in association with PKC-activation and, in turn, PKC-activation by 12-O-tetradecanoylphorbol-13-acetate (TPA) induces desmosome formation even in low-Ca++ conditions. TPA induces serine-phosphorylation of the 180 kDa-bullous pemphigoid antigen (BPAG2), generating 190 kDa-phosphorylated BPAG2, and dissociates BPAG2 from hemidesmosomes. TPA-treatment also causes secretion of urokinase-type plasminogen activator (uPA) and expression of its receptor (uPAR), which activates plasminogen to plasmin and may digest extracellular domains of desmosomes and hemidesmosomes. These results suggest that PKC may play a role in activation of desmosome turnover and dysfunction of hemidesmossomes, and thus a role in up-migration of keratinocytes. Binding of PV-IgG to Dsg3 induces activation of diverse isoenzymes of PKC, linked to uPA secretion and uPAR expression. Furthermore, PV-IgG binding alone induces the serine-phosphorylation of Dsg 3, associated with its dissociation from plakoglobin and its deletion from desmosomes. This PV-IgG-induced Dsg 3-phosphorylation and Dsg 3-deletion from desmosomes may impair desmosome formation, whereas PV-IgG-induced PKC signaling mediates the uPA secretion and uPAR expression leading to digestion of preexisting desmosomes from the outside of the cell. These two different PV-IgG-activated signaling pathways may play a key role in acantholysis in PV.

Pemphigus vulgaris (PV) is an autoimmune blistering skin disease, which is characterized by autoantibodies to a specific desmosomal constituent, i.e. desmoglein 3 (Dsg3). In this study, we analyzed phosphorylation of desmosomal proteins and their molecular interactions after PV-IgG binding to Dsg3 using DJM-1 cells, a squamous cell carcinoma cell line, and normal human keratinocytes. Cells were metabolically labeled with 32P inorganic phosphate, followed by stimulation with the IgG fractions from five PV patients or normal individuals for 20 min. Phosphorylation of specific desmosomal components and their molecular interactions were studied in immunoprecipitates using PV-IgG and anti-plakoglobin (PG) antibodies. PV-IgG binding alone induced the phosphorylation of Dsg3 at serine residues. Although Dsg3 and PG were coprecipitated by PV-IgG-immunoprecipitation when treated with normal IgG, PG was not coprecipitated with Dsg3 when stimulated with PV-IgG, suggesting that PV-IgG binding to Dsg3 caused the dissociation of Dsg3 from PG. These results demonstrate that the binding of pathogenic PV autoantibodies to the cell surface antigen Dsg3, which is an adhesion molecule categorized into desmosomal cadherins, caused particular phosphorylation of Dsg3 and its dissociation from PG.

Ganglioside IV3 (NeuAc)2, II3 (NeuAc)2-GgOse4Cer (GQ1b), which induces terminal differentiation in keratinocytes, was previously found to enhance the mass content of inositol 1,4,5-trisphosphate and intracellular calcium concentration ([Ca++]i), peaking at 30 seconds. In the present study, the biphasic accumulation of 1,2 diacylglycerol, i.e., the first transient and the second sustained phase, was observed in cultured human keratinocytes stimulated by GQ1b. On the other hand, II3 NeuAc-LacCer (GM3), which inhibits keratinocyte proliferation without inducing differentiation, did not cause diacylglycerol formation. Phosphatidylethanol, produced by transphosphatidylation and a potential marker for phospholipase D activity, was produced by the exposure to GQ1b in the presence of ethanol. The second sustained phase of diacylglycerol was repressed by ethanol, indicating that the diacylglycerol-formation pathway via phospholipase D followed by phosphatidic acid phosphohydrolase would in part account for the second diacylglycerol phase. Furthermore, this second phase of GQ1b-induced diacylglycerol generation was reduced by pretreatment with propranolol, an inhibitor of phosphatidic acid phosphohydrolase. In addition, the levels of [3H]choline, a direct metabolite of the phospholipase D pathway, were elevated within 1 min after GQ1b addition and then sustained for at least 20 min. Taken together, the results suggest that the phospholipase D pathway may contribute to the second phase of diacylglycerol formation, which might be involved in differentiation.

Bradykinin (BK) is one of the key mediators of inflammation and a weak mitogen. We have previously demonstrated that BK induced the generation of inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) which caused Ca2+ mobilization in human keratinocytes. In this study, BK-induced Ca2+ responses were examined in primary cultured human keratinocytes by video imaging fluorescence microscopy using fura-2. Intracellular calcium concentration ([Ca2+]i) level increased to a peak within 30 s after BK addition and decreased gradually to the basal level. The existence of the broad shoulder in the [Ca2+]i profile was suggested to be due to the Ca2+ influx from the external medium, because this disappeared in the presence of 0.5 mM EGTA. Pretreatment with phorbol-12-myristate-13-acetate (PMA), a protein kinase C (PKC) activator, significantly resulted in reduction of the descending shoulder of BK-induced increase in [Ca2+]i. A 20-min pretreatment with PKC inhibitors, H-7 or staurosporine, reversed the decrease by PMA in the shoulder of BK-induced Ca2+ response. Furthermore, the BK-induced [45Ca] uptake was inhibited by EGTA and PMA. Ins(1,4,5)P3 generation induced by BK peaked at 20 s and returned to the basal level at 60 s. There were no significant differences in Ins(1,4,5)P3 levels at 20 and 60 s among the cells exposed to BK alone, BK with PMA pretreatment (20 min) and BK with PMA+H-7 pretreatment. These results suggest that the BK-induced Ca2+ influx, which was shown as shoulder, may be negatively modulated by PKC in primary cultured human keratinocytes.

The article focuses on a study related to novel IFIH1 mutation in the pincer domain underlies the clinical features of both Aicardi–Goutières and Singleton–Merten syndromes in a Japanese girl. It mentions Immunohistochemical stainings6 revealed that p-STAT3 was strongly expressed in the nuclei of keratinocytes in the patient. it also mentions heterozygous missense mutation in the pincer domain of IFIH1 leading to overlapping clinical phenotypes of type I IFN unregulated signaling.

Background Pemphigus vulgaris (PV) is characterized by autoantibodies against desmoglein (Dsg) 3 or both Dsg1 and Dsg3, i.e. desmosomal adhesion molecules. Objectives We examined whether or not PV IgG binding to Dsg3 directly impairs the adhesion of desmosomes. Methods For immunofluorescence microscopy, keratinocytes were first incubated with PV IgG for 30 min in low Ca2+ medium, in which no desmosomes were formed, and then for 1 h in high Ca2+ medium to generate desmosomes. For immunoelectron microscopy, after a 30-min incubation with PV IgG in low Ca2+ medium, cells were incubated with antihuman IgG with 5-nm gold particles for 5 min; after washing, cells were further incubated in high Ca2+ medium for 1 h. For tracing of PV IgG/Dsg3 immune complexes formed in the desmosomal core domain, cells were first incubated with PV IgG for 5 min to allow PV IgG to bind the desmosomal core domain and were further incubated with PV IgG-free medium for different times. Results Immunofluorescence microscopy revealed that PV IgG bound in a random-punctate pattern on the cell surface in low Ca2+ medium was translocated to the cell–cell contacts forming a dotted-linear distribution, suggesting desmosome generation even in the presence of PV IgG. Immunoelectron microscopy revealed that half-desmosome-like structures decorated with gold particles in low Ca2+ keratinocytes coupled to form desmosomes and gold particles were sandwiched in the desmosomal core domain after Ca2+ switch, even though their surfaces were covered with PV IgG/antihuman IgG 5-nm gold particles. In the tracing experiments, although PV IgG demonstrated a dotted-linear distribution along the cell–cell contacts colocalized with desmoplakin (DPK) after a 30-min tracing, it disappeared from cell–cell contacts after a 5-h tracing, leaving DPK and desmocollin 3. Conclusions These results suggest that the PV IgG/Dsg3 immune complexes are excluded from the desmosomal core domain rather than directly splitting the desmosome. [ABSTRACT FROM AUTHOR]