HLA Class II Polymorphism Contributes to Specify Desmoglein Derived Peptides in Pemphigus Vulgaris and Pemphigus Foliaceus
Descrição do Produto
doi:10.1006/jaut.2000.0388, available online at http://www.idealibrary.com on
Journal of Autoimmunity (2000) 15, 67–73
HLA Class II Polymorphism Contributes to Specify Desmoglein Derived Peptides in Pemphigus Vulgaris and Pemphigus Foliaceus Pascale Loiseau1, Laurence Lecleach2, Catherine Prost3, Virginia Lepage1, Marc Busson1, Sylvie Bastuji-Garin4, Jean-Claude Roujeau2 and Dominique Charron1 1
Service d’immunologie et d’histocompatibilite´, Hoˆpital Saint-Louis, Paris, France 2 Service de dermatologie, Hoˆpital Henri Mondor, Cre´teil, France 3 Service de dermatologie, Hoˆpital Saint-Louis, Paris, France 4 Service de Sante´ Publique, Hoˆpital Henri Mondor, Creteil, France
Received 2 February 2000 Accepted 10 May 2000 Key words: Human leucocyte antigen, desmoglein, peptide, pemphigus
Susceptibility to Pemphigus, an autoimmune disease of the skin, has been previously linked to DRB1*0402, 1401/04 and DQB1*0503 in pemphigus vulgaris (PV), to DRB1*0102, 0404, 1402/06 in endemic pemphigus foliaceus in Brazil and to DRB1*04 in Italian patients suffering from pemphigus foliaceus (PF). The disease is caused by autoantibodies against desmoglein (Dsg1 in PF, Dsg3 in PV). Molecular typing of 57 French patients suffering from PV (37) and from PF (20) confirmed previous results concerning PV and showed that DRB1*0102 and 0404 are susceptible molecules to PF in France. We have analysed the characteristics of the ‘pockets’ of the susceptibilityassociated molecules to PV and PF and we showed that (i) in PV, two kinds of Dsg3 derived peptides may be presented by HLA-DR according to HLA polymorphism (DRB1*0402 or DRB1*14/0406), (ii) the same Dsg1 peptides may be presented by DRB1*0102, DQB1*0404 or DRB1*14 in PF, (iii) the DRB1*14/0406 PV-related molecules may be able to present Dsg1 and Dsg3 peptides thereby providing an explanation for the cases of PV with combined responses to Dsg1 and to Dsg3 which are typified by a muco-cutaneous clinical phenotype. © 2000 Academic Press
Introduction
adhesion with resultant blister formation. Two forms of Pemphigus exist: Pemphigus vulgaris (PV) and Pemphigus folliaceus (PF) which are distinguishable by clinical (mucous and cutaneous blisters in PV, exclusively cutaneous lesions in PF) and histological aspects (suprabasal acantholysis in PV, more superficial acantholysis in PF). The autoantibodies recognize epitopes of the desmoglein 3 (Dsg3) or desmoglein 1 (Dsg1) molecule in PV and PF respectively. Endemic and non-endemic PF have been described with a predominance of endemic PF in Brazil and in NorthAfrica (Tunisia). A strong association with MHC molecules has already been shown in PV (DRB1*0402 and DQB1*0503) [2, 3, 4, 5, 6] and in endemic PF (DRB1*0102, 04040, 1402 or 1406) [7, 8, 9]. The HLA susceptibility to PV has not been determined in France, and previous studies (with the exception of a recent study of Italian patients) [10] concerning nonendemic PF were carried out on small patient groups and were therefore inconclusive. In this study, we report the HLA-DRB1 and DQB1 alleles distribution in 57 French patients suffering from PV (37) and from non-endemic PF (20) and we show the contribution of the MHC susceptible molecules to the specificity of the desmoglein derived peptides.
Human autoimmune diseases have a striking genetic association with particular alleles of major histocompatibility complex (MHC) class I or II genes. The MHC class II molecules are highly polymorphic membrane glycoproteins [1] which bind peptide fragments of proteins and display them for recognition by CD4+ T cells. The structural characterization of MHC-peptide complexes and the identification of allele-specific peptide-binding motifs have redefined the field of autoimmunity. Based on this knowledge, the structural basis of MHC-linked susceptibility to autoimmune diseases can be reassessed according to the presentation of (self) peptides specific for particular disease-associated MHC molecules and the specificity for the target organ of the immune attack. Pemphigus is a chronic acantholytic blistering disease mediated by autoantibodies that bind to keratinocyte cell adhesion molecules (desmogleins) causing a loss of cell Correspondence to: Dr P. Loiseau, Service d’immunologie et d’histocompatibilite´, Hoˆpital Saint-Louis, 1, Avenue Claude Vellefaux, 75010 Paris, France. Fax: +33 0142494889. 67 0896–8411/00/050067+07 $35.00/0
© 2000 Academic Press
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Table 1. Increased allelic HLA-DRB1 frequencies and DQB1 among French patients with pemphigus vulgaris (PV) and pemphigus foliaceus (PF)
HLA Class II genes
DRB1* 0402 1401 1404 0102 0404 DQB1* 0302 0503 02
Allelic frequency
Allelic frequency Control (n=106)
PV patients (n=37)
P value
RR
PF patients (n=20)
P value
RR
0.005 0.052 0.005 0.005 0.037
0.310 0.135 0.094 0.027 0.067
5.4 10 −16 0.01 4.2 10 −5 0.10 (NS) 0.27 (NS)
91.6 3.1 17.2 4.9 1.9
0.050 0.050 0 0.100 0.175
1 (NS) 0.95 (NS) 1 (NS) 6.2 10 −5 0.018
9.5 1.12 1.7 19.1 4.1
0.100 0.066 0.257
0.414 0.257 0.07
9.7 10 −7 5.3 10 −5 7 10 −4
6.9 5.3 0.16
0.315 0.052 0.105
5.3 10 −5 0.7 (NS) 0.12 (NS)
6.9 0.9 0.36
RR=Relative Risk. NS=Non-significant.
Materials and Methods Patients Fifty-seven patients from France were included in a prospective study between December 1993 and June 1997. Among the 57 patients, 37 (21 females and 16 males mean age 50.5±14) suffered from PV, and 20 (11 females and nine males, mean age 51±16) from PF. The ethnic origin of the patients was diverse including Caucasian, Asian and North-African patients. The diagnosis of pemphigus was based on clinical features, standard histology and a direct immunofluorescence study of skin biopsy specimens and an indirect immunofluorescence study on serum samples.
Controls A hundred and six healthy controls from France were enrolled in this study.
HLA typing DRB1 and DQB1 medium resolution typings were performed using the PCR-SSO reverse dot blot kits from Innogenetics (InnoLipa DRB key and InnoLipa DQB kits respectively) (Zwijndrecht, Belgium). DRB1 allelic determination was performed using a local PCR-SSP/RFLP technique [11].
Statistical analysis Analysis was done with HLASTAT software. The estimation of gene frequencies was direct as there
were no recessive genes. Comparisons for phenotypes were made between groups allele by allele, and the P value was corrected (pc) multiplying by the number of tested alleles at each locus considered. Odds ratios were also calculated for each allele.
Results DRB1*0402, DRB1*1401/04, DQB1*0302 and DQB1*0503 alleles are associated with susceptibility to PV The frequency of the association of different DRB1* alleles with PV is reported in Table 1. A strong positive association was found in French PV patients with the DRB1*0402, two different DR14 (1401, 1404), DQB1*0302 and DQB1*0503 alleles (Table 1). Thirtythree out of 37 PV patients in France were either DRB1*0402 or DRB1*1401/04 or both (P=10 −16). Only 13 of 106 controls had this status. The increased frequency of DRB1*0402 corroborates previous studies in Ashkenazi Jewish patients [2] and in non-Jewish Caucasians [3, 4, 5, 6, 10]. The DRB1*1401 or 1404 alleles were found associated with PV in non-Jewish patients of European ancestry (Italy, Sardinia, Spain, Boston area) [3, 4, 5, 6] and in Asian populations (India, Pakistan, Japan) [12, 13, 14, 15, 16, 17]. All the DRB1*04 PV patients were DQB1*0302 and al the DRB1*14 were DQB1*0503 therefore suggesting association of two presumed haplotypes with susceptibility to PV in France: DRB1*0402-DQB1*0302 and DRB1*1401 or 1404-DQB1*0503. An important and significant decrease (P=0.0007) in DQB1*02 frequency was observed in French PV patients, confirming previous studies from India and Italy [6, 10, 13]. The DQB1*02 decrease was associated
HLA class II in Pemphigus vulgaris and foliaceus
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Table 2. Polymorphic residues of DR beta 1 chain forming the pockets of the DR molecules (A) associated with PV in French and Japanese patients (B) associated with endemic and non-endemic PF AA position DRB1* A. Pemphigus vulgaris 0402 0406 1401 1404 1405 1406 B. Pemphigus foliaceus 0404 0102 1402 1406
P4
P1 86
13
70
71
74
V V V V V V
H H S G S S
D Q R R R Q
E R R R R R
V V G V
H F S S
Q Q Q Q
R R R R
P7
P9
78
P6 11
28
30
47
67
71
9
37
57
60
A E E E E A
Y Y Y Y Y Y
V V S S S S
D D D D D E
Y Y Y Y Y Y
Y Y Y Y Y Y
I L L L L L
E R R R R R
E E E E E E
Y S F F F N
D D A A D D
Y Y H H Y Y
A A A A
Y Y Y Y
V L S S
D E E E
Y C Y Y
Y Y Y Y
L L L L
R R R R
E W E E
Y S N N
D D D D
Y Y Y Y
The amino acid residues are designated according to the letter code. D, E negatively charges AA, are in bold. K, R positively charged AA, are underlined.
with a decreased frequency of both DRB1*03 (P=0.008) and DRB1*07 (P=0.006) alleles.
DRB1*0102 and DRB1*0404 alleles are associated with susceptibility to PF Three allelic frequencies were significantly increased in French PF patients: DRB1*0404 (0.175 v. 0.037 P=0.018 Relative risk (RR)=4.1), DRB1*0102 (0.1 v. 0.005 P=6.2 10-5 RR=19.1) and DQB1*0302 (0.315 v. 0.1 P=5.3 10-5 RR=6.9) (Table 1). All of the DRB1*0404 PF patients were DQB1*0302 leading to the definition of a presumed haplotype of susceptibility to PF: DRB1*0404-DQB1*0302. The alleles associated with PF in France (DRB1*0404 and 0102), encode the same DR57-78 sequence. This epitope is strongly associated with PF since 17/40 alleles in PF patients shared the epitope against 31/212 in controls (P=0.00001). The other alleles of susceptibility to endemic PF in Brazil (DRB1*1402 and 1406) [9] encode molecules sharing the same DR57-78 epitope.
Classification of the DR PV and PF associated molecules according to the pockets of their peptide binding groove Table 2A shows the polymorphic residues of the DR1 chain forming the pockets of the DR molecules associated with PV in French and Japanese patients. The pockets of the DRB1*0402 molecule are quite different from DRB1*1401/04 ones, especially in the P4 pocket where the two negatively charged amino acids (AA) of the DRB1*0402 molecule at position DR 70 and 71 are replaced by two positively charged AA in the
DR14 molecules (Table 2A). The DRB1*1401 and 1404 molecules which are also associated with PV have the same pockets except for one difference (S or G at position DR13). In Japanese PV patients, the DRB1*04 restriction element is not the DRB1*0402 but the DRB1*0406 molecule. These molecules share in common the same P1, P6 and P9 pockets but differ by the P4 and P7 ones (Table 2A). The P4 and P7 pockets of DRB1*0406 are positively charged (arginine at position 71) and are therefore unlikely to bind a positively charged peptide such as DRB1*0402 molecule. In PV Japanese patients, additional DRB1*14 alleles were observed: DRB1*1405, 1406 [15, 16, 17]. The DRB1*1401, 05, 06 and 0406 alleles share the same P1 and P7 pockets and also a functionally identical P4 pocket (Table 2A). Indeed, it has been suggested that substitution of R for Q at position DR70 would be conservative and that the neutral hydrophillic residue (Q) can be considered to be equivalent to the only slightly larger and positively charged residue, R. So, there are great similarities of the pockets of the DR14 and DRB1*0406 PV related molecules. In conclusion, the DR molecules associated with PV can be separated into two different groups (DRB1*0402 and DRB1*14/0406) according to the characteristics of their peptide binding groove pockets. Table 2B reports the DR1 polymorphic AA forming the pockets of the DRB1*0404, 0102 and 1402/06 molecules shared by PF patients in France (nonendemic PF) and in Brazil (endemic PF) [7, 8, 9]. DR molecules associated with PF are very similar according to the characteristics of their peptide binding pockets: they all have the same P1 pocket, the same positively charged P4 pocket, the same P7 pocket with a negatively charged aa (E or D) at position DR28 and a positively charged aa (R) at position DR71, and a similar P9 pocket.
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Table 3. Motifs of peptides bound to DRB1*0101, 0402, 0404, molecules and the proposed motif of peptides able to bind to DRB1*0102 and to both DRB1*0102 and 0404 molecules DRB1*
P1
P4
0402
VLMIF
0101
VLMIF WY
0404
VLMIF
0102
VLMIF
0102 and 0404
VLMIF
KR no DE LAIV MNQ no HKR ALIV WDEFY no KR ALIV MNQ no HKR ALIV DE no HKR
DR pocket P6 STNQK
P7
P9
HKN PQR
AGH NQS ALV INFY
ADHIL MNPST
ALV QGSTK
AGSTP STNQR AGSTP
ALV INFY
ST no charged AA
ALV no DE
The AA residues are shown using the letter code.
Discussion PF and PV are autoimmune diseases mediated by autoantibodies that bind to the keratinocyte cell adhesion molecule (Dsg1 and 3 respectively) causing loss of cell adhesion. The Dsg show considerable homology (about 80%) in the extracellular domains EC1, 2 and 3, less homology (about 50%) in the extracellular domain EC4 and in the intracellular portion, whereas no homology has been observed in the juxtamembranar EC5 domain. The disease can be reproduced in neonatal mice by passive transfer of patients’ IgG indicating that humoral immunity plays an important role in the pathogenesis. Several studies have demonstrated that T cell clones derived from PF and PV patients were stimulated by Dsg1 and Dsg1 3 peptides, that CD4+ T cells are the major T cell population responding to Dsg3 and that Dsg3-specific T cells secrete Th2 cytokines capable of inducing antibody production in B lymphocytes [18, 19, 20]. Recognition by T cells of Dsg epitopes is therefore critical for the initiation and perpetuation of a Dsg-specific T cell response providing help to the B cells that produce Dsg-specific auto-antibodies. The interaction between the TCR (T Cell Receptor) and the MHC-peptide complex is the first step in triggering T cell help. In the skin, keratinocytes and Langherans cells may be involved in the presentation of the self-peptide since they express HLA class II molecules either constitutively (Langherans cells) or after exposure to interferon-alpha for example, following local inflammation or UV exposure (keratinocytes). Pemphigus vulgaris A strong positive association of French PV patients with DRB1*0402, DRB1*1401, 1404, DQB1*0302 and DQB1*0503 alleles.
Since the allele frequency of DRB1*0402 is low (
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