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Veterinary Dermatology 2003, 14, 23 30

Blackwell Science, Ltd

Isotype determination of circulating autoantibodies in canine


autoimmune subepidermal blistering dermatoses
CLAUDE FAVROT*, STANLEY M. DUNSTON, MANON PARADIS* and
THIERRY OLIVRY
*Universit de Montral, Facult de Mdecine Vtrinaire, Saint-Hyacinthe, Qubec, Canada Department of
Clinical Sciences, College of Veterinary Medicine, NC State University, 4700 Hillsborough Street, Raleigh,
NC 27606, USA
(Received 22 May 2002; accepted 31 August 2002)

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

Correspondence: Thierry Olivry. Fax: +1-919-513-6336; E-mail:


Thierry_Olivry@ncsu.edu
Present address: Clinic for Small Animal Medicine, University of
Zrich, Switzerland.
2003 European Society of Veterinary Dermatology

established correlations between autoantibody isotype


profiles and duration or severity of the affections, or
sometimes with treatment outcome and eventual
remissions.1012
Canine BP was described for the first time in 1976.13
Since then, numerous cases have been reported in
the literature with the generic denomination of BP
(reviewed in Olivry & Chan17). These published cases
have exhibited different clinical features, histological
characteristics and prognoses; thus it is likely that these
dogs suffered from different clinicopathological entities.
Since 1995, an attempt was made to differentiate canine
AISBD based on the identification of targeted autoantigens. These efforts have led to the individualization
of canine BP,14 EBA15 and MMP as separate entities.16
At this time, there are no studies investigating the
profile of circulating autoantibodies in canine patients
affected with AISBD. Thus, there is no information on
the presence (or absence) of circulating IgA, IgE and
IgM in these sera, nor does one has any knowledge about
the variability of IgG subclasses in these conditions.
The goals of this study were to determine whether
there were differences, among the three principal
canine AISBD, in the isotypes of circulating autoantibodies detected by means of an indirect immunofluorescence (IF) method. We confirm that, as is the case in
human individuals affected with such entities, circulating autoantibodies belong predominantly to IgG1,
IgG4 and IgE isotypes, and that there are only minor
noticeable differences in autoantibody isotype profiles
between BP, MMP and EBA in dogs.
23

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

Bethyl Laboratories (Montgomery, TX)

A40 104F4

1:50

Bethyl Laboratories (Montgomery, TX)

A40 116F11

1:50

Bethyl Laboratories (Montgomery, TX)

5.91
B6 (SN)
E5 (SN)
A3G6 (SN)
A5 (SN)
F-0257

1:1000
1:10
1:10
1:10
1:10
1:40

B. Hammerberg, NC State University


M.J. Day, University of Bristol
M.J. Day, University of Bristol
M.J. Day, University of Bristol
M.J. Day, University of Bristol
Sigma Immunochemicals, St Louis, MO

SN: supernatant.

MATERIALS AND METHODS


Study subjects
Serum samples were obtained from 31 dogs diagnosed
with one of the three principal AISBD. In each patient
(5 affected with BP, 15 with MMP and 11 with EBA),
the diagnosis was made on clinical and histological
features and determination of targeted autoantigens
(Table 1).17,18 Also, a direct IF assay had revealed the
presence of autoantibodies and/or complement deposited linearly at the dermalepidermal junction in each
study subject (data not shown). There were no age
(range 1.515 years) or sex predilection in the study
subjects. Similarly, it was not possible to demonstrate
any breed predilection although German Shepherd Dogs
and Great Danes appeared over-represented in the
MMP and EBA groups, respectively. Only 1 of 31 dogs
had been treated previously with immunosuppression.
The short duration of this treatment (3 days) was felt to
be of no influence on autoantibody serum levels.
Epithelial substrates
Two frozen sections (5 m thickness) of salt-split
canine gingival lip, processed as described previously,15
were used for each experiment. These two sections were
obtained from different dogs to prevent variability in
IF detection of autoantibodies as described in our
recent study.19 All skin biopsy specimens were snapfrozen in liquid nitrogen after OCT compound embedding (Tissue-Tek, Sakura Finatek, Torrance, CA) and
were stored at 70 C until indirect IF was performed.15
The salt-split technique results in a clefting of the
dermalepidermal junction in the middle of the lamina
lucida and enhances the exposition of basement

membrane antigens.15,20 Moreover, this technique allows


the differentiation of canine BP from EBA, the former
being associated with autoantibody binding to the
epidermal side of artificial clefts, the latter to the dermal
side.14,15
Salt-split canine gingival lip was selected as preferential substrate after results from preliminary studies
had confirmed its usefulness in enhancing detection of
circulating autoantibodies in dogs with AISBD.19
Indirect immunofluorescence assay
The indirect IF assay was performed as described
previously.15 Briefly, 5 m cryosections of two salt-split
canine gingival lips were deposited onto glass slides, air
dried for 10 min, fixed in acetone for 10 min, rinsed in
phosphate-buffered saline (PBS) and then overlaid in
1% normal goat serum solution for 20 min for blocking
of nonspecific antibody binding. Sections were then
incubated for 1 h with the patients sera in a moist
chamber.
Following several rinses with PBS, skin sections were
then incubated for 30 min (anti-Ig G, IgA and IgM) or
60 min (anti-IgE, IgG subsets) at room temperature
with a panel of secondary antibodies (Table 2).
After incubation with the fluorescein-conjugated antiIgG, -IgA and -IgM polyclonal antibodies, sections
were rinsed in PBS, mounted with Vectashield-DAPI
(Vector, Burlingame, CA) and finally cover-slipped.
Following overlay with monoclonal antibodies specific
for canine IgE and IgG subsets, sections were incubated 30 min with antimouse-IgG-fluorescein, then
processed as described above.
In all indirect IF studies, negative controls consisted
of two slides in which patient serum was replaced by

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 23 30

Autoantibody isotypes in blistering dermatoses


Table 3. Isotypes of circulating autoantibodies
in canine bullous pemphigoid

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

BDL: below detection limit of 1:10 serum screening dilution.

Figure 1. Immunoglobulin class autoantibody


titers in dog with autoimmune subepidermal
blistering dermatoses. Sera from dogs with
BP, MMP and EBA were tested by indirect
IF on salt-split canine gingival lip sections. Each
data point corresponds to the fluorescence
extinction titre of basement membrane specific
autoantibodies of the four immunoglobulin
classes. Horizontal line: detection limit (1:10).

Figure 2. IgG subclass autoantibody titers


in dog with autoimmune subepidermal
blistering dermatoses. Sera from dogs with
BP, MMP and EBA were tested by indirect
IF on salt-split canine gingival lip sections. Each
data point corresponds to the fluorescence
extinction titre of basement membrane specific
autoantibodies of the four IgG subclasses.
Horizontal line: detection limit (1:10).

PBS (one slide) or normal dog serum (one slide). Of


note is that low titres (1:10) of antibasement membrane
IgG autoantibodies are detected only rarely using this
technique in normal dogs or dogs with nonblistering
skin diseases (< 10% of cases) (Favrot & Olivry, unpublished data). As positive controls, we used the serum
of a dog with pemphigus vulgaris, for which previous
studies had demonstrated the presence of circulating
IgG, IgG1, IgG2, IgG3 and IgG4 antikeratinocyte
autoantibodies. Also, we stained normal canine spleen
with all secondary antibodies as described above. The
use of this substrate permitted the visualization of plasma
cells producing all isotypes of immunoglobulins.
Interpretation of indirect immunofluorescence results
All sections were observed with an epifluorescence
microscope. The pattern of specific fluorescence
observed in the various canine AIBSD has been
described elsewhere.1416,19 Disease-specific fluorescence pattern was recorded on salt-split gingival lip
sections (e.g. epidermal aspect for BP and MMP; on
dermal side for EBA) as present or absent at a 1:10
screening serum dilution. When observed at 1:10,
additional step-wise serum dilutions were tested, and
the final fluorescence extinction titre was tabulated.

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

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 2330

26

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

Table 4. Isotypes of circulating autoantibodies


in canine mucous membrane pemphigoid

BDL: below detection limit of 1:10 serum screening dilution.

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

IgA, IgM and IgE classes were detected in 2 (13%), 0


(0%) and 8 (53%) of 15 dogs, respectively. The median
autoantibody titres detected with this method were
lower than 1:50 for both IgA and IgE.
As for dogs with BP, all isotypes of canine MMP
autoantibodies bound to antigenic epitopes situated
on the epidermal side of salt-split gingival lip.

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 23 30

Autoantibody isotypes in blistering dermatoses


Table 5. Isotypes of circulating autoantibodies
in canine epidermolysis bullosa acquisita

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

BDL: below detection limit of 1:10 serum screening dilution.

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).

Isotypes of circulating autoantibodies in canine EBA


Circulating autoantibodies of IgG class were found
in all 11 dogs with EBA at 1:10 dilution (Table 5;
Figs 1, 2 and 5). The median IgG autoantibody titre
was 1:250. The predominant IgG subclasses of autoantibodies were IgG1 and IgG4, which were found in 10
(median titre: 1:250) and 7 (median titre: 1:25) of 11
dogs, respectively. Low titres of autoantibodies of IgG2
and IgG3 subclasses were detected in six (median titre:
1:25) and three subjects (median titre: 1:50), respectively.
Basement membrane autoantibodies of IgA, IgM and
IgE classes were detected in two, one and three dogs,
respectively. The median IgE autoantibody titre was 1:25.
In contrast to dogs with BP and MMP, autoantibodies found in all dogs with EBA bound to the dermal
side of salt-split gingival lip.

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-

tibodies belong to the IgG class and IgG1 and IgG4


subclasses. In most cases, the titre of IgG1 autoantibodies was higher than that of the IgG4 ones. Also, we
uncovered occasionally circulating autoantibodies of
IgA and IgE classes, and IgG2 or IgG3 subclasses. The
similarity in clinical and pathological lesions, in immunological findings as well as the similitude of isotypes
among species with the same diseases provides additional evidence for the relevance of canine AISBD to
study the pathogenesis and treatment of their human
counterparts.
In this study, we elected to employ an indirect IF
assay to look for circulating autoantibodies. This technique has been considered for a long time as one of the
best methods for detecting circulating antibasement
membrane autoantibodies in human patients with
AISBD. However, it has been shown that the sensitivity
of indirect IF tests can be greatly influenced by the
selection of epithelial substrates.21 Moreover, Woodley
et al. demonstrated that the use of salt-split skin, separated following an incubation in 1 sodium chloride,
allowed better exposition of basement membrane antigens and, subsequently, led to an increased sensitivity
of indirect IF reactions.22
In veterinary dermatology, even though it has been
accepted as dogma that the indirect IF method is not
useful to permit detection of circulating autoantibodies

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 2330

28

C. Favrot et al.

in dogs with autoimmune blistering diseases23 recent


data contradict this opinion. Indeed, it was recently
demonstrated that the reliability of indirect IF techniques for the diagnosis of such canine diseases also
depended upon the substrates utilized.19,20,24 Lately, we
documented that the use of intact and salt-split canine
gingival lip proved rewarding in enhancing detection of
autoantibodies in dogs with AISBD.19
In this study, we established that the serum of all
dogs with AISBD contained circulating autoantibodies of IgG class, thus confirming earlier observations.14 16,25 This high rate of positive detection also
validates the relevance of the substrate and technique
employed herein. These results are comparable with
those of recent studies of human patients with AISBD
for which indirect IF percentages of detection are
reported up to100% of cases. However, such successful
rates were sometimes achieved via testing of undiluted
or even concentrated sera.26,27 Also, one of these
studies stressed that tests were more rewarding when
carried out on sera collected on untreated patients
presented during an active episode of the disease.26 In
general, most sera employed in our study had been
collected during an active phase of their disease, but
sometimes these dogs had been treated with various
anti-inflammatory medications prior to venepuncture.
According to most reports, the serum of humans
with BP always contains basement membrane-specific
IgG, but IgA and IgE are also frequently encountered.6
In fact, human BP is considered a unique autoimmune
blistering disease because of the common detection of
high levels of circulating IgE autoantibodies.28 The
majority of BP IgG autoantibodies belong to IgG1
and IgG4 subclasses, uncovered in 100 and 78% of
tested human sera, respectively.7
In comparison, our study, albeit performed on only
five canine BP sera, established that basement membrane targeting autoantibodies of IgG class were
always detected, and that IgM and IgE autoantibodies
could also be found. Similarly to the results reported in
human patients with BP, canine IgG autoantibodies
are predominantly of IgG1 and IgG4 isotypes.
In humans affected with MMP or EBA, circulating
autoantibody isotype profiles appear very similar, the
principal distinction between these two diseases residing in the antigen targeted by autoantibodies.6,8
Human patients with EBA and MMP most commonly
exhibit basement membrane-targeting serum autoantibodies of IgG class.3,8,29,30 Occasionally, however, IgA
autoantibodies are also found.9 In contrast to individuals with BP, IgE autoantibodies are not usually
detected. In most studies, IgG autoantibodies belong
to IgG1 and IgG4 subclasses.8,31,32 More recently, a
more heterogeneous isotype distribution has been
found, with autoantibodies of all four IgG subclasses
discovered almost equally.33
In this study using canine sera, the antibody profiles
overall were similar between MMP and EBA, the only
differences between the entities being the higher rate of
detection of antibasement membrane IgE autoanti-

bodies in dogs with MMP and IgG2 in patients with


EBA. In contrast to the results reported in humans,
basement membrane-specific IgE autoantibodies do
not seem to be restricted to canine patients with BP. On
the contrary, low titres of these autoantibodies have
been uncovered in a large proportion of canine patients
with MMP (53%) and in some with EBA (27%). The
results of our study, albeit being limited to sera from 11
dogs with EBA, are in agreement with those reported
in the study by Gandhi et al.,33 as we detected autoantibodies of all classes and subclasses. Sera from dogs
with MMP also exhibited a wide diversity of autoantibodies of most classes, except IgM, and subclasses.
Numerous studies have been carried out to determine
the relationship between disease severity and autoantibody titres in human patients with AISBD. For example, increasing titres of IgG4 autoantibodies seem to be
associated with clinical remission of BP.10 In another
report, IgE autoantibody levels seem to decrease during successful treatment.12 Remarkably, human BP IgE
autoantibodies target mainly the 230 kDa antigen
BPAG1, and antibodies against this protein are
believed to not be pathogenic.28 When tested by ELISA
using the NC16A segment of collagen XVII, serum
IgG autoantibody levels correlate with the evolution of
BP in human patients.34 Similarly, Setterfield et al. also
demonstrated that IgG and IgA autoantibody serum
levels correlated with the activity of human MMP.11 In
contrast, when IgG subclass levels were analysed, a
relationship between IgG1 and IgG4 titres and EBA
disease activity was not discovered.27 At this time,
serial titres of basement membrane autoantibodies
have not been performed on serum collected from dogs
with AISBD, and it is not known whether antibody
titres, or isotype variations could be seen with treatmentinduced remission.
Our results suggest that circulating autoantibodies
can be found in dogs with AISBD using canine saltsplit gingival lip substrates. When present, basement
membrane autoantibodies are usually of IgG class, and
less frequently of IgA, IgM and IgE isotypes. IgG
autoantibodies belong usually to IgG1 or IgG4 subclasses. Future studies will be performed to determine
whether the autoantibody profiles vary during clinical
evolution, whether the appearance/disappearance of
a specific isotype is predictor of positive/negative
outcome, and whether specific disease variants are
associated with unique autoantibody profiles, thus
suggesting new avenues of research into the pathogenesis of these diseases.

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.

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 23 30

Autoantibody isotypes in blistering dermatoses


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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.

2003 European Society of Veterinary Dermatology, Veterinary Dermatology, 14, 23 30

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