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Abstract The three most common canine autoimmune blistering skin diseases (AISBD), bullous pemphigoid
(BP), mucous membrane pemphigoid (MMP) and epidermolysis bullosa acquisita (EBA) have recently been
separated based on clinical, histological and immunological grounds. The objectives of this study were to determine
the isotype profiles of circulating autoantibodies in these dermatoses. Serum was collected from 5 dogs with BP,
15 with MMP and 11 with EBA. All sera were tested using an indirect immunofluorescence method using saltsplit canine gingiva as substrate. Anti-basement membrane IgG autoantibodies were detected in all patients.
Among the IgG autoantibodies, IgG1 and IgG4 were encountered most frequently, while IgG2 and IgG3 were
uncovered in some dogs. IgE autoantibodies were detected more often than IgA or IgM autoantibodies in any
of the three entities. The predominance of IgG1, IgG4 and IgE autoantibody isotypes in dogs with AISBD is
very similar to the situation found in humans with the homologous diseases.
Keywords: animal model, autoantibody, autoimmunity, epidermal basement membrane, epidermolysis bullosa
acquisita, immunoglobulin subclass, isotype, pemphigoid.
INTRODUCTION
The denomination autoimmune subepidermal blistering dermatoses (AISBD) refers to a group of skin
diseases associated with the presence of autoantibodies
directed against components of the basement membrane zone (BMZ). In humans and dogs, bullous pemphigoid (BP), mucous membrane pemphigoid (MMP;
previously called cicatricial pemphigoid) and epidermolysis bullosa acquisita (EBA) are the conditions that
are most frequently encountered within this group.
At this time, the classification of human AISBD is
based upon the clinical features, histological findings,
and on the characterization of antigens targeted by
circulating autoantibodies. Based on different clinical
and immunological features, BP,1,2 EBA3 and MMP
have been individualized as separate entities.4
In recent years, various studies have investigated the
isotype profile of circulating autoantibodies in human
individuals affected with these three conditions. These
observations have led to the demonstration of IgG1,
IgG4, IgE and IgA autoantibodies in sera of human
beings affected with BP.57 Similarly, IgG1, IgG4 and
IgA autoantibodies have been uncovered in the serum
of MMP and EBA patients.69 These studies often have
24
C. Favrot et al.
Table 1. Criteria for diagnosing canine
AISBD
BP
MMP
EBA
Acquired disease
Erosive and/or vesicular lesions
Distribution*
Subepidermal vesicles
Predominant Inflammation
Main Antigen Targeted
yes
yes
SKIN/mm
yes
eosinophils neutrophils
collagen XVII
yes
yes
MM/skin
yes
cell poor
collagen XVII
yes
yes
SKIN/
yes
neutrophils
collagen VII
BP, bullous pemphigoid; EBA, epidermolysis bullosa acquisita; Eos, eosinophils; MM,
mucous membrane; MMP, mucous membrane pemphigoid; Neuts, neutrophils.
*Upper case indicates major site of lesions; lower case indictaes minor site of lesions.
Table 2. Immunoreagents
Specificity
Identification
Dilution
Source
Canine IgG
(Fc-specific)
Canine IgA
(Fc-specific)
Canine IgM
(Fc-specific)
Canine IgE
Canine IgG1
Canine IgG2
Canine IgG3
Canine IgG4
Mouse IgG
(whole molecule)
A40 118F
1:50
A40 104F4
1:50
A40 116F11
1:50
5.91
B6 (SN)
E5 (SN)
A3G6 (SN)
A5 (SN)
F-0257
1:1000
1:10
1:10
1:10
1:10
1:40
SN: supernatant.
25
Subject
IgG
IgG1
IgG2
IgG3
IgG4
IgA
IgM
IgE
BP 1
BP 2
BP 3
BP 4
BP 5
Total #
Total percentage
1:100
1:50
1:100
1:4000
1:100
5
100
1:100
1:25
1:50
1:500
1:25
5
100
BDL
BDL
BDL
BDL
BDL
0
0
1:25
BDL
BDL
BDL
1:10
2
40
1:10
BDL
BDL
1:10
1:10
3
60
BDL
BDL
BDL
BDL
BDL
0
0
1:50
BDL
BDL
1:10
1:10
3
60
1:10
BDL
1:10
BDL
1:10
3
60
RESULTS
Isotypes of circulating autoantibodies in canine BP
The sera from all five dogs with BP contained circulating IgG basement membrane-specific autoantibodies
that were detectable by our indirect IF method at 1:10
dilution (Table 3; Figs 13). The titres of autoantibodies varied from 1:50 to 1:4000 (median: 1:100). The IgG
autoantibodies belonged predominantly to the IgG1
(5/5 dogs; median titre: 1:50) and IgG4 (3/5 dogs;
median titre: 1:10) isotypes. IgG2 antibodies were not
detected at 1:10 dilution. Only two dogs exhibited low
titres of IgG3 autoantibodies.
Also, low levels of circulating basement membrane
autoantibodies of IgM and IgE isotypes were detected
in 3/5 and 3/5 cases, respectively. Autoantibodies of
IgA class were not observed with this indirect IF
technique. All isotypes of canine BP autoantibodies
bound to antigenic epitopes situated on the epithelial
side of salt-split gingival lip.
Isotypes of circulating autoantibodies in canine MMP
Detectable circulating basement membrane-specific IgG
autoantibodies were found in all 15 (100%) dogs with
MMP at 1:10 dilution (Table 4; Figs 1, 2 and 4). The
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C. Favrot et al.
Figure 3. Indirect IF staining profile in canine BP. Serum from a dog with BP (case BP4) was tested by indirect IF, at 1:10 dilution, for detection
of basement membrane-specific autoantibodies. High fluorescent detection of IgG and IgG1, low detection of IgG4, and weak detection of IgM
autoantibodies that bound to the epithelial side of the splits (arrowheads) (bar = 90 m).
Subject
IgG
IgG1
IgG2
IgG3
IgG4
IgA
IgM
IgE
MMP 1
MMP 2
MMP 3
MMP 4
MMP 5
MMP 6
MMP 7
MMP 8
MMP 9
MMP 10
MMP 11
MMP 12
MMP 13
MMP 14
MMP 15
Total #
Total percentage
1:100
1:50
1:10
1:100
1:50
1:100
1:1000
1:50
1:25
1:100
1:50
1:50
1:250
1:100
1:250
15
100
1:50
1:50
BDL
1:100
1:50
1:100
1:500
1:50
1:25
1:100
1:50
1:25
1:100
1:100
1:100
14
93
BDL
BDL
1:10
BDL
BDL
BDL
BDL
1:10
BDL
BDL
BDL
BDL
BDL
BDL
BDL
2
13
BDL
BDL
1:10
BDL
BDL
1:50
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
1:10
3
20
BDL
1:10
BDL
1:10
1:10
1:10
BDL
BDL
BDL
BDL
BDL
1:10
BDL
1:10
BDL
6
40
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
1:10
1:25
BDL
BDL
BDL
BDL
BDL
2
13
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
0
0
1:10
BDL
1:10
1:10
1:50
BDL
1:10
BDL
BDL
BDL
1:10
BDL
1:10
1:10
BDL
8
53
Figure 4. Indirect IF staining profile in canine MMP. Serum from a dog with MMP (case MMP5) was tested by indirect IF, at 1:10 dilution, for
detection of basement membrane-specific autoantibodies. IgG, IgG1, IgG4 and IgE autoantibodies bound to the epithelial side of the splits
(arrowheads) (bar = 90 m).
median IgG autoantibody titre was 1:100. The predominant IgG subclasses of autoantibodies were IgG1
and IgG4, which were found in 14 (93%) and 6 (40%)
of 15 dogs, respectively. Median titres of IgG1 and IgG4
autoantibodies were 1:75 and 1:10, respectively. IgG2
and IgG3 autoantibodies were detected in rare subjects.
Basement membrane targeting autoantibodies of
27
Subject
IgG
IgG1
IgG2
IgG3
IgG4
IgA
IgM
IgE
EBA 1
EBA 2
EBA 3
EBA 4
EBA 5
EBA 6
EBA 7
EBA 8
EBA 9
EBA 10
EBA 11
Total #
Total percentage
1:100
1:500
1:1000
1:500
1:25
1:10
1:250
1:250
1:2000
1:100
1:250
11
100
1:100
1:500
1:1000
1:500
1:25
BDL
1:250
1:250
1:2000
1:100
1:50
10
91
BDL
1:10
BDL
1:25
1:25
1:10
BDL
1:50
1:25
BDL
BDL
6
55
BDL
BDL
1:50
BDL
BDL
BDL
BDL
1:50
1:50
BDL
BDL
3
27
1:10
1:50
1:250
1:25
BDL
BDL
BDL
1:25
1:500
1:25
BDL
7
64
BDL
1:10
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
1:10
2
18
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
BDL
1:25
BDL
1
9
BDL
BDL
BDL
1:25
BDL
BDL
BDL
BDL
1:25
1:10
BDL
3
27
Figure 5. Indirect IF staining profile in canine EBA. Serum from a dog with EBA (case EBA2) was tested by indirect IF, at 1:10 dilution, for
detection of basement membrane-specific autoantibodies. High fluorescent detection of IgG and IgG1, medium fluorescent detection of IgG4
and weak fluorescent detection of IgG2 and IgA autoantibodies on the dermal side of the clefts (arrowheads) (bar = 90 m).
DISCUSSION
The aim of this study was to determine the isotype
profile of circulating autoantibodies in dogs affected
with the most common AISBD, and to assess whether
these autoantibodies exhibited a particular class and
subclass distribution between diseases. As is the case for
human patients with the same affections, we confirmed
that most basement membrane-specific serum autoan-
28
C. Favrot et al.
ACKNOWLEDGEMENTS
This study was financed by funds of a competitive
research grant awarded by the European Society of
Veterinary Dermatology (ESVD). The authors are
indebted to Professor MJ Day for the gift of the
monoclonal antibodies specific for the canine IgG
subclasses.
17.
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34. Amo, Y., Ohkawa, T., Tatsuta, M. et al. Clinical significance of enzyme-linked immunosorbent assay for the
detection of circulating anti-BP180 autoantibodies in
patients with bullous pemphigoid. Journal of Dermatological Science 2001; 26: 1418.
Rsum Il a t rcemment possible de diffrencier les trois plus frquentes dermatoses bulleuses souspidermiques (pemphigoide bulleuse [BP], pemphigode des muqueuses [MMP] et pidermolyse bulleuse acquise
[EBA]) sur la base de critres cliniques, histologiques et immunologiques. Les buts de cette tude taient de dterminer les profils isotypiques des autoanticorps circulants dans ces maladies. Le srum de 5 chiens BP, 15 chiens
MMP et 11 chiens EBA a t collect et test en utilisant une mthode dimmunofluorescence indirecte avec
comme substrat une gencive canine clive par le sel. Des autoanticorps IgG anti-membrane basale ont t dtects
chez tous les patients. Parmi ces IgG, les IgG1 et IgG4 taient rencontres le plus souvent, alors que les IgG2 et
IgG3 taient absentes chez certains chiens. Des autoanticorps IgE taient plus frquemment rencontrs que des
IgA ou des IgM pour les trois maladies. La prdominance dIgG1, IgG4 et IgE chez les chiens prsentant une
dermatose auto-immune bulleuse sous-pidermique est trs semblable la situation rencontre chez les patients
humains souffrant de maladies quivalentes.
Resumen Las tres enfermedades ampollosas y autoinmunes de la piel ms frecuentes en la especie canina, penfigoide bulloso (PB), penfigoide de las membranas mucosas (PMM) y epidermlisis bullosa adquirida (EBA),
se han separado recientemente en trminos clnicos, histolgicos e immunolgicos. Los objetivos del estudio
fueron determinar el perfil de isotipos de autoanticuerpos circulantes en estas dermatosis. Se tomaron muestras
serolgicas de cinco perros con PB, 15 con PMM y 11 con EBA. Todos los sueros fueron analizados mediante
un mtodo indirecto de immunofluorescencia utilizando separacin por NaCl (salt-split) y enca canina como
substrato. Se detectaron autoanticuerpos IgG antimembrana basal en todos los pacientes. De los autoanticuerpos IgG, los isotipos IgG1 e IgG4, se hallaron con ms frecuencia, mientras que los IgG2 e IgG3 fueron detectados slo en algunos perros. Autoanticuerpos IgE fueron detectados ms a menudo que los autoanticuerpos IgA
o IgM en las tres entidades. El predominio de los isotipos de autoanticuerpos IgG1, IgG4 e IgE en perros con
enfermedades ampollosas autoinmunes de la piel es muy similar a la situacin encontrada en humana con
enfermedades homlogas.
Zusammenfassung Die drei hufigsten autoimmunen Hauterkrankungen mit Blasenbildung beim Hund, bullses Pemphigoid [BP], Schleimhautpemphigoid [SP] und Epidermolysis bullosa acquisita [EBA] sind krzlich
aus klinischen, histologischen und immunologischen Grnden separiert worden. Das Ziel dieser Studie war, das
Isotypenprofil von zirkulierenden Antikrpern bei diesen Erkrankungen zu erstellen. Serum wurde von fnf
Hunden mit BP, 15 mit SP und 11 mit EBA gewonnen. Alle Seren wurden mittels einer indirekten Immunfluoreszenzmethode und mit Salz getrennter Hundemundschleimhaut (Gingiva) als Substrat getestet. AntiBasismembran IgG Autoantikrper wurden bei allen Patienten festgestellt. Unter den IgG Autoantikrpern wurden
IgG1 und IgG4 am hufigsten angetroffen, whrend IgG2 und IgG3 bei einigen Hunden gefunden wurden. IgE
Autoantikrper wurden bei den drei Syndromen fter als IgA oder IgM Autoantikrper festgestellt. Die Prdominanz von IgG1, IgG4 und IgE Autoantikrper Isotypen bei Hunden mit mit Blasenbildung einhergehenden
autoimmunen Hauterkrankungen hnelt der Situation beim Menschen mit entsprechenden Erkrankungen.