059 Analysis of HLA class II genes in Japanese patients with pemphigus

JSID

112

058

055 PHOSPHORYLATION AND DISSOCIATION OF DESMOGLEIN 1.3 AND PLAKOGLOBIN INDUCED BY PEMPHIGUS IgG. Y. Aoyama’, KM. Owadaz, and Y. Kitajimal. ‘Department of Dermatology, Gifu Univ. School of Med.. Gifu, *Inst. of Mol. and Cell. Biol. for Pharmaceut. Sci., Kyoto

Phammceut

Univ.,

Kyoto, Jamn.

We previously have demonstrated that pemphigus vulgaris (PV)-IgG activates phospholipase C/Caz+/ C kinase signaling pathways leading to cell-cell detachment. In this study, we have analyzed phosphorylation of dcsmosompl proteins and their mutual interactions after PV-IgG has bound to the antigens on the cell surface in DJM-1 cells. 3*Pi-labeled cells were stimulated with IgG from 6 PV patients and normal individuals. Phosphorylation of Dsgl, 3 and PG were detected only when stimulated Serine phosphorylation was confirmed in Dsg3. with PV-IgG. Phosphorylation of Dsgl and 3 were partially inhibited with staurosporine. TPA failed to phosphorylate Dsgl 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 keratinccytes. which may involved in signaling pathways leading to acantholysis.

CALCIUM

VULGARIS

OF PEMPHIGUS BINDING

ANTIGEN.

SITES

SERA AGAINST

ONTHE

T.Muramatsu.

PEMPHIGUS

TShirai.

Department

of

Nara Medical University,Nara,Japan.

Dermatology,

GST fusion proteins (FPs) were generated to five regions of the pcmphigus

vulgaris

containing

the extracellular

(PV)

antigen.

FPs encoding the non-calcium and intracellular

FPl, FP2 and FP3 were FPs

calcium

we examined the sera from

bullous pamphigoid

binding sites.

FP4 and FP.5 were

binding sites of the extracellular

domain, rapectively.

foliaceus (PF, n=12),

With the immunoblot

the patients with PV (n=20),

paraneoplsstic

domain

analysis,

pemphigus

pemphigus (PNP, n=2) and

(BP, n=2), and 4 normal (NM)

sera. Fourteen

out

of 20 PV sera reacted with thcsc FPs. In contrast, 2 PF sera reacted only

ASSOCIATION BB’IWBBN CLINICAL PICTURE AND PRODUCTION OP ANTIDESMOOLEIN I ANTIBODY IN PBMPHIGUS VULGARIS &.J&@&j&aa&.

Departmenu of ‘Dermatology and 2Molecular Biology, Keio Universily School of Medicine, Tokyo, 3Duke University Medical Ccntcr, Durham, USA. We developedandgen-specific ELISAs with bscubvirus expressed Desmoglcin (Dsg) to detecLautoantibodiesof sers from patients with pemphigus. Mcfe than hslf of sera from pemphigus vulgaris (PV) patients were positive for Dsgl in sddilion to Dsg3, The purpose was LOdccumine the correlation betweenclinical pictun andanti-Dsgl andbody (Ab) liter in PV. First. to exclude s possibility that anti-Dsgl ructivily in PV sers is due to cross-reactivity by anti-Dsg3 Ab. inhibilion ELISA wss (esled for 9 PV sers showing positive rcacdvity in both Dsg3 and Dsgl ELISA. nK reactivity against Dagl wss inhibiled by pro-sdsorpdon with rDsgl but not with rDsg3. Converacly, ths reacdvily against Dsg3 was inhibited by padsorption with rDsg3 but not with rDsgl. Thus. PV have specific Ab agsinst Dsgl in addition 10and-Dsg3 Ab. To examine the cmreladon between clinical picture and pmducdon of anti-Dsg Ab. clinical picture of PV wss divided into two gmups: oral dominant type (IS cases) ss oral lesion with minimal skin involvement and skin dominant type (12 csses) as significant skin lesion with oral lesion. Although anti-Dsg3 Ab levels were na significandy different between the lwo groups. and-Dsgl Ab levels were signiticandy higbzr in skin dominant type. Considting thsl rind-Dsgl Abs in PV sm not pathogenic, production of snti-Dsgl Abs in PV may represent an epiphenomenon.

059

056 IMMUNORBACTIVITY THE

Abstracts

with FPl,and

PNP,BP and NM sera showed no positive reaction.

057 LOW EXPRESSIONOFDESMOCLElN1ANDHIG~EXPRESSlONOFDESMOGLEIN3 IN ORAL MUCOUS MEMBRANE. Y Shirakata . M. Ama&. Y. Hanakawa’. T. Nishikawa’. K. H& ‘Department of Dermatology, Ehime University School of Medicine, fhime. ‘Department of Dermatology, Keio University School of Medicine, Tokyo, Japan. Oral lesions are seen in must cases uf pemphiyus vulgaris (PV), whereas they are only rarely seen in pemphigus foliaceus (I’F). Iiowever, both PV antigen (desmoylein 3 or Dsg3) and PF antiSen (Dsgl) are thought m be expressed because both PV and PF sera showed positive immunofluorescence staining on oral ~IYCOEP. 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 Dsu isotvoe-soecific antibodies produced by immunoadsorbiig PV sera by e&r re&nbinant Dsgl or Dsg3 baculoprotein. Staining intensity of Dsg3 was much higher than thar of Dsgl in both oral mucus 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. 111the skin, expression level of Dsgl was much higher than that of Dsg3. In contrast, in oral mucous membrane Dsg3 expression was much higher than Dsgl. These dam indicate that the difference of oral mucosa involvement between PV and PFshould be explained by the difference of expression level of Dsgl and Dsg3.

ANALYSIS OF HLA CLASS II GENES IN JAPANESE PATIENTS WITH PEMPHIGUS. I . Higasbimine ‘, T. Iida I, T. Fukumoto I, Y. Yamaahina ‘, S. hfiyagawa ‘, T. Shirai ‘. 1 Department of Dermatology, Nara Medical University, Nara Japan. To find evidence of potential HLA class II allele aesociations with pemphigua in Japanese patients who have a relatively homogeneous ethnic background, we genetyped HLA-DRBl,DQAl,DQBl and DPBl alleles in 16 patients with pemphigus ( 9 patienta with PV and 7 patients with PF ) by PCR-PM&‘. AI1 9 PV patients and 6 of 7 PF patients carried one or two alleles of HLA-DRBI’M ( 0403.0466 ) and HLA-DRB1.14 ( 1401,1406,1406 ) subtypes. Sequence analysis of these DRB1’04 and DRB1*14 alleles revealed the amino acid homology of phenylalanine at position 26 and valine at position 66 with the DRBl* 0402 allele which reportedly conferrs a strong susceptibility to PV in Asbkenati Jews. Our finding8 , together with previous HLA studies on PV patients of different ethnic groupa, auggeet that HLA-DRB1.04 and DRB1’14 alleles are commonly associated with pemphigue across racial barriers.

060 ENVOPLAKIN IS ONE OF THE PARANBOPLASTIC PEMPHIGIJS ANIlGBN COMPLBX. C.I.IuImT.3 Jgid&wa*a3.KJ.Grrco4.*.

IDept. of Dennstol. Kurum

Univ. School of Medicine. Fukuoks, Jspsn, *tkpt. of Dcnnstol. Keio Univ. School of Medicine, Tokyo, Japsn. 3Johns Hopkins Universily, Bsltimon. USA, 4Nndhwcstm Univ. Chicago. USA, %mperisl CmkzerRcs. Fund, London. UK Eavoplskin is s mmponcnt of amiticd cell envelop with oi protein structure nssmbling desmplskin snd BPUO. Em+skin is thought to plsy I mlc in associslion behveenmmitied cell envelop md dwmoaoms. Psrsneoplsstic pemphigus sem rud with protein complex of *SOLD, 23OkD. ZlOkD, 191&D, 17OkD. However, we previously qmted lbsl the 21OkD msy not be amposed of only dunplskin II. In Ibis study, amlid obstrvstion of immuwprecipitstion results indicstedthst the 2lOkD protein is I do&et of two closely &ted pmtcins. Furhlermon, by immunobloaiag using u&bodies specitic 10both desmophkin md envoplskin, we fovod the 210kD pmtein is I doublet of hvo proteins, andluger 4 smsller pmteins uc conespoDdingto desmplskio II lad envoplr kin, rrspecilivcly. Thsse resuks indicatethll envoplskin is mu of the pamne@utic pempbigus snligen complex.