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-55- Bullons(2) 9/4 Room-C
PHOSPHORYLATION AND DISSOCIATION OF DESMOGLEIN 1, 3 AND plakoglobin induced by pemphigus igg.
Yumi Aoyama1, Koji M Owada2, Yasuo Kitajima1.
1Department of Dermatology, Gifu Univ. School of Med, Gifu, 2Inst. of Mol. and Cell. Biol. for Pharmaceut. Sci., Kyoto Pharmaceut. University Kyoto, Japan.
We previously have demonstrated that pemphigus vulgaris (PV)-IgG activates phospholipase C/Ca2+/ C kinase signaling pathways leading to cell-cell detachment. In this study, we have analyzed phosphorylation of desmosomal proteins and their mutual interactions after PV-IgG has bound to the antigens on the cell surface in DJM-1 cells. 32Pi-labeled cells were stimulated with IgG from 6 PV patients and normal individuals. Phosphorylation of Dsg1, 3 and PG were detected only when stimulated with PV-IgG. Serine phosphorylation was confirmed in Dsg3. Phosphorylation of Dsg1 and 3 were partially inhibited with staurosporine. TPA failed to phosphorylate Dsg1 and 3. PV-IgG-induced phosphorylated Dsg was dissociated from PG, while normal-IgG treated nonphosphorylated Dsg3 was associated with PG. These results suggest that PV-IgG causes¡¡phosphorylation and dissociation of Dsg3 and PG in cultured keratinocytes, which may involved in signaling pathways leading to acantholysis.
-56- Bullons(2) 9/4 Room-C
IMMUNOREACTIVITY OF PEMPHIGUS SERA AGAINST THE CALCIUM BINDING SITES ON THE PEMPHIGUS VULGARIS ANTIGEN.
Tsutomu Muramatsu, Toshihiko Shirai.
Department of Dermatology,Nara Medical University,Nara,Japan.
GST fusion proteins (FPs) were generated to five regions of the pemphigus vulgaris (PV) antigen. FP1, FP2 and FP3 were FPs containing the extracellular calcium binding sites. FP4 and FP5 were FPs encoding the non-calcium binding sites of the extracellular domain and intracellular domain, respectively. With the immunoblot analysis, we examined the sera from the patients with PV (n=20), pemphigus foliaceus (PF, n=12), paraneoplastic pemphigus (PNP, n=2) and bullous pemphigoid (BP, n=2), and 4 normal (NM) sera. Fourteen out of 20 PV sera reacted with these FPs. In contrast, 2 PF sera reacted only with FP1,and PNP, BP and NM sera showed no positive reaction.
-57- Bullons(2) 9/4 Room-C
LOW EXPRESSION OF DESMOGLEIN1 AND HIGH EXPRESSION OF DESMOGLEIN3 IN ORAL MUCOUS MEMBRANE.
Yuji Shirakata1, Masayuki Amagai2, Yasushi Hanakawa1, Takeji Nishikawa2, Koji Hashimoto1.
1Department of Dermatology, Ehime University School of Medicine, Ehime, 2Department of Dermatology, Keio University School of Medicine, Tokyo, Japan.
Oral lesions are seen in most cases of pemphigus vulgaris (PV), whereas they are only rarely seen in pemphigus foliaceus (PF). However, both PV antigen (desmoglein 3 or Dsg3) and PF antigen (Dsg1) are thought to be expressed because both PV and PF sera showed positive immunofluorescence staining on oral mucosa. To explain this apparent paradox, we examined expression level of both types of Dsg in human skin, oral mucous membrane, and esophagus by immunofluorescence staining and immunoblotting. For immunofluorescence staining we used Dsg isotype-specific antibodies produced by immunoadsorbing PV sera by either recombinant Dsg1 or Dsg3 baculoprotein. Staining intensity of Dsg3 was much higher than that of Dsg1 in both oral mucous membrane and esophagus. We then compared their expression level by immunoblotting between skin and mucous membrane. The total amount of desmosomal protein per well was adjusted by the staining intensity of desmoplakin, a cytoplasmic plaque protein of desmosomes, as an internal control. In the skin, expression level of Dsg1 was much higher than that of Dsg3. In contrast, in oral mucous membrane Dsg3 expression was much higher than Dsg1. These data indicate that the difference of oral mucosa involvement between PV and PF should be explained by the difference of expression level of Dsg1 and Dsg3.
-58- Bullons(2) 9/4 Room-C
ASSOCIATION BETWEEN CLINICAL PICTURE AND PRODUCTION OF ANTI-DESMOGLEIN 1 ANTIBODY IN PEMPHIGUS VULGARIS.
Ken Ishii1, Masayuki Amagai1, Russell P. Hall3, Atsushi Takayanagi2, Shinobu Gamou2, Nobuyoshi Shimizu2, Takeji Nishikawa1.
1Department of Dermatology, 2Department of Molecular Biology, Keio University School of Medicine, Tokyo, Japan. 3Duke University Medical Center, Durham, USA.
We developed antigen-specific ELISAs with baculovirus expressed Desmoglein (Dsg) to detect autoantibodies of sera from patients with pemphigus. More than half of sera from pemphigus vulgaris (PV) patients were positive for Dsg1 in addition to Dsg3. The purpose was to determine the correlation between clinical picture and anti-Dsg1 antibody (Ab) titer in PV. First, to exclude a possibility that anti-Dsg1 reactivity in PV sera is due to cross-reactivity by anti-Dsg3 Ab, inhibition ELISA was tested for 9 PV sera showing positive reactivity in both Dsg3 and Dsg1 ELISA. The reactivity against Dsg1 was inhibited by pre-adsorption with rDsg1 but not with rDsg3. Conversely, the reactivity against Dsg3 was inhibited by pre-adsorption with rDsg3 but not with rDsg1. Thus, PV have specific Ab against Dsg1 in addition to anti-Dsg3 Ab. To examine the correlation between clinical picture and production of anti-Dsg Ab, clinical picture of PV was divided into two groups: oral dominant type (15 cases) as oral lesion with minimal skin involvement and skin dominant type (12 cases) as significant skin lesion with oral lesion. Although anti-Dsg3 Ab levels were not significantly different between the two groups, anti-Dsg1 Ab levels were significantly higher in skin dominant type. Considering that anti-Dsg1 Abs in PV are not pathogenic, production of anti-Dsg1 Abs in PV may represent an epiphenomenon.
-59- Bullons(2) 9/4 Room-C
ANALYSIS OF HLA CLASSII GENES IN JAPANESE PATIENTS WITH PEMPHIGUS.
Ikuko Higashimine, Takashi Iida, Takaya Fukumoto, Yukio Yamashina, Sachiko Miyagawa, Toshihiko Shirai.
Department of Dermatology,Nara Medical University, Nara, Japan.
To find evidence of potential HLA class II allele associations with pemphigus in Japanese patients who have a relatively homogeneous ethnic background, we genotyped HLA-DRB1,DQA1,DQB1 and DPB1 alleles in 16 patients with pemphigus ( 9 patients with PV and 7 patients with PF ) by PCR-PFLP.¡¡ All 9 PV patients and 5 of 7 PF patients carried one or two alleles of HLA-DRB1¡ö04 ( 0403,0406 ) and HLA-DRB1¡ö14 ( 1401,1405,1406 ) subtypes. Sequence analysis of these DRB1¡ö04 and DRB1¡ö14 alleles revealed the amino acid homology of phenylalanine at position 26 and valine at position 86 with the DRB1¡ö0402 allele which reportedly conferrs a strong susceptibility to PV in Ashkenazi Jews.¡¡Our findings , together with previous HLA studies on PV patients of different ethnic groups, suggest that HLA-DRB1¡ö04 and DRB1¡ö14 alleles are commonly associated with pemphigus across racial barriers.
-60- Bullons(2) 9/4 Room-C
ENVOPLAKIN IS ONE OF THE PARANEO-PLASTIC PEMPHIGUS ANTIGEN COMPLEX.
Chie Kiyokawa1, Tadashi Karashima1, Osamu Mori1, Takashi Hashimoto1, Takeji Nishikawa2, Grant J. Anhalt3, Kathleen J. Green4, Fiona M. Watt5.
1Department of dermatology, Kurume University School of Medicine, Fukuoka, 2Department of dermatology, Keio University school of Medicine,Tokyo,Japan. 3Johns Hopkins University, Baltimore, 4Northwestern University, Chicago, USA. 5Imperial Cancer Reserch F
Envoplakin is a component of cornified cell envelop with a protein structure resembling desmoplakin and BP230. Envoplakin is thought to play a role in association between cornified cell envelop and desmosomes. Paraneoplastic pemphigus sera react with protein complex of 250kD, 230kD, 210kD, 190kD,170kD. However, we previously reported that the 210kD may not be composed of only desmoplakin II. In this study, careful observation of immunoprecipitation results indicated that the 210kD protein is a doublet of two closely related proteins. Furtermore, by immunoblotting using antibodies specific to both desmoplakin and envoplakin, we found the 210kD protein is a doublet of two proteins, and larger and smaller proteins are corresponding to desmoplakin II and envoplakin, respecitively. These results indicate that renvoplakin is one of the paraneoplastic pemphigus antigen complex.