In Vitro Keratinocyte Dissociation Assay for Evaluation of the Pathogenicity of Anti- Desmoglein 3 IgG Autoantibodies in Pemphigus Vulgaris Ken Ishii,

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In Vitro Keratinocyte Dissociation Assay for Evaluation of the Pathogenicity of Anti- Desmoglein 3 IgG Autoantibodies in Pemphigus Vulgaris Ken Ishii, Reiko Harada, Itsuro Matsuo, Yuji Shirakata, Koji Hashimoto, Masayuki Amagai Journal of Investigative Dermatology Volume 124, Issue 5, Pages 939-946 (May 2005) DOI: 10.1111/j.0022-202X.2005.23714.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Expression pattern of desmoglein (Dsg) isoforms in cultured human epidermal keratinocytes. Normal human epidermal keratinocytes (NHK) were cultured to confluence in low-calcium medium and switched into 1.2 mM calcium-containing medium at 4, 9, or 24 h before harvest. In the exfoliative toxin A (ETA) lane, NHK were incubated with 0.5 μg per mL of ETA for 2 h after being cultured in high-calcium medium for 24 h. KU8 (squamous carcinoma cell line) and A431 (vulvar carcinoma cells) were also included. Total cell lysates were separated by 7.5% SDS-PAGE and subjected to immunoblot with antibodies against Dsg1 (982), Dsg2 (6D8), and Dsg3 (5H10). Note that NHK express Dsg3 and Dsg1 predominantly whereas KU8, and A431 cells express Dsg2 mainly. Journal of Investigative Dermatology 2005 124, 939-946DOI: (10.1111/j.0022-202X.2005.23714.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Treatment with combination of a pathogenic AK23 anti-desmoglein (Dsg) 3 monoclonal antibody and exfoliative toxin A (ETA)-dissociated cell sheets of normal human epidermal keratinocytes (NHK). Monolayers of NHK were treated with either AK23 monoclonal anti-Dsg3 antibody (1 μg per mL) overnight (B), ETA (1 μg per mL) for 2 h (C), or a combination of both reagents (D). Dissociation assay was performed as described in the Material and Methods section. Note that cell sheets were minimally dissociated when treated with only AK23 or ETA, whereas the cell sheets broke into numerous small fragments when treated with both AK23 and ETA. Numbers indicate a mean of numbers of fragments counted by image pro software using three captured images. Journal of Investigative Dermatology 2005 124, 939-946DOI: (10.1111/j.0022-202X.2005.23714.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Dissociation assay to detect the pathogenic strength of anti-desmoglein (Dsg) 3 antibodies in disruption of intercellular adhesion. (A) Monolayers of normal human epidermal keratinocytes were treated with various monoclonal anti-Dsg3 antibodies. After 2 h incubation with 0.5 μg per mL of exfoliative toxin A, monolayers were separated from culture dishes by incubation with dispase. Subsequently, they were subjected to mechanical stress by pipetting using 1 mL of pipetman five times. AK7 does not have cross-reactivity with human Dsg3 and was used as a negative control. AK20 has reactivity against human Dsg3, but failed to induce apparent blisters by passive transfer to neonatal mice. AK23 has reactivity against human Dsg3 and has a pathogenic activity shown by passive transfer to neonatal mice. (B) Quantification was obtained by counting the number of fragments of cell sheets as described in the Material and Methods section. (C) The cell sheets were incubated with AK23 at concentrations of 0.1, 0.5, 1, and 5 μg per mL. Each condition was in duplicate. Bars represent standard deviations. Journal of Investigative Dermatology 2005 124, 939-946DOI: (10.1111/j.0022-202X.2005.23714.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Dissociation assay to detect pathogenic activity in disruption of intercellular adhesion of pemphigus vulgaris (PV) sera. Ten sera patients with PV in active stage (five sera that have only anti-desmoglein (Dsg) 3 autoantibody (open circle) and five sera that have both Dsg3 and Dsg1 autoantibodies (filled dot)) and 11 sera from normal healthy controls were examined by the dissociation assay treated with and without exfoliative toxin A (ETA). A dashed line indicates a cut-off value, defined as an average plus 2 SD of normal control sera. Bars indicate averages of each group of samples. The dissociation scores of PV sera were significantly higher than that of normal controls (Mann–Whitney U test, p=0.003). In the assay without treatment of ETA, the dissociation scores for PV sera were negative except two samples, which contain Dsg1 in addition to Dsg3 autoantibodies. Journal of Investigative Dermatology 2005 124, 939-946DOI: (10.1111/j.0022-202X.2005.23714.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Dissociation scores correlate well with the clinical disease severity in pemphigus vulgaris (PV). Two mucocutaneous-type PV (Pt. 1 and 2) and four mucosal dominant-type PV (Pt. 3–6) were studied using in vitro dissociation assay and desmoglein (Dsg) 3 ELISA. Serum samples were taken when patients were in an active stage and in remission. Ratio of values to those in an active stage was shown for Dsg3 ELISA and in vivo dissociation assay. “A” and “R” represent an active stage and remission, respectively. Journal of Investigative Dermatology 2005 124, 939-946DOI: (10.1111/j.0022-202X.2005.23714.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Detection of pathogenic heterogeneity of anti-desmoglein (Dsg) 3 autoantibodies in pemphigus vulgaris (PV) sera. Serum samples from six PV patients that have similar reactivity against Dsg3 were examined by dissociation assay (approximately 100 index in Dsg3 ELISA). Three of these samples were collected when the patients were in active stage. Three samples were collected when the patients were in remission. Journal of Investigative Dermatology 2005 124, 939-946DOI: (10.1111/j.0022-202X.2005.23714.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions