Molecular diagnosis of autoimmune skin diseases

Rom J Morphol Embryol 2014, 55(3 Suppl):1019–1033
RJME
REVIEW
Romanian Journal of
Morphology & Embryology
http://www.rjme.ro/
Molecular diagnosis of autoimmune skin diseases
ROXANA CHIOREAN1), MICHAEL MAHLER2), CASSIAN SITARU1,3)
1)
Department of Dermatology, University of Freiburg, Germany
2)
INOVA Diagnostics, Inc., San Diego, CA, USA
3)
BIOSS Centre for Biological Signalling Studies, Albert-Ludwigs University, Freiburg, Germany
Abstract
A wide range of skin diseases are associated with autoimmune responses against skin-specific or ubiquitous antigens. In many of these
diseases, including autoimmune blistering disorders, collagenoses and vasculitides, extensive clinical and experimental evidence shows that
autoreactive T-cells and/or autoantibodies play a major pathogenic role, allowing their classification as autoimmune diseases. The presence of
tissue-bound and circulating autoantibodies does not only bear relevance for disease pathogenesis, but also allows developing robust
diagnostic tools and molecular therapeutic approaches. Thus, various immunofluorescence methods, as well as molecular immunoassays,
including enzyme-linked immunosorbent assay and immunoblotting, belong to the modern diagnostic algorithms for these disorders. This
review article describes the immunopathological features of autoimmune skin diseases and the molecular assays currently available for their
diagnosis and monitoring.
Keywords: autoimmunity, molecular diagnosis, blistering skin diseases, collagen diseases.
 Introduction
During the last decades, extensive research has focused
on the pathogenesis and diagnosis of autoimmune skin
diseases, which show a steadily increasing incidence
and prevalence [1]. Criteria to classify a condition as an
autoimmune disease were initially modeled on the
Koch’s postulates by Ernest Witebsky in the 1950s and
further refined by Rose and Bona in 1993 [2, 3]. They
include circumstantial evidence from clinical and laboratory
findings (e.g., the presence of antibody deposition in the
affected tissue), indirect evidence, based on the reproduction of the disease by immunizing animals with the
putative autoantigen and a direct proof, provided by
disease induction through the passive transfer of autoantibodies and/or autoreactive T-cells [3].
The presence of tissue-bound and circulating autoantibodies does not only bear relevance for pathogenesis,
but also allows developing robust diagnostic tools and
molecular therapeutic approaches. Various molecular
assays show increasing importance for the diagnosis and
monitoring of a series of autoimmune skin diseases [4].
While in some diseases, including autoimmune blistering
disorders, collagenoses and vasculitides, the detection of
autoantibodies is highly important for diagnosis, in others,
such as psoriasis, vitiligo or alopecia areata, the diagnosis
is primarily made on clinical grounds [5–10].
In this article, we present an overview on the use of
molecular immunoassays for the diagnosis and monitoring
of autoimmune skin diseases.
 Overview of diagnostic procedures in
autoimmune skin diseases
As with other diseases, a thorough patient history and
a skillful clinical investigation provide a strong index of
suspicion for an autoimmune skin disease and guide the
ISSN (print) 1220–0522
further diagnostic work-up. While the histopathological
examination of a lesional skin biopsy is an important
diagnostic tool, further imaging and laboratory investigations may be needed to support the diagnosis.
Direct immunofluorescence (IF) microscopy
Importantly, classifying a disease as autoimmune
requires detection of tissue-bound and/or circulating
autoantibodies. Deposits of immunoreactants (typically
immunoglobulins and complement components) in the
perilesional skin are detected by direct immunofluorescence
(IF) microscopy, which remains the gold standard for
the diagnosis of autoimmune bullous diseases and is an
important part of the diagnostic work-up in cutaneous
lupus and vasculitides [5, 11]. Thus, by direct IF microscopy
deposition of different immunoreactants in the skin of
the patients is detected using fluorochrome-labeled antibodies (e.g., specific for human IgG, IgA, IgM and C3)
(Figure 1). For the direct IF, the site of the biopsy relative
to the primary lesion (i.e., lesional or perilesional) should
be selected depending on the suspected disease (Table 1).
Table 1 – Optimal site of biopsy for direct immunofluorescence microscopy
Suspected diagnosis
Site of biopsy
Autoimmune bullous Perilesional erythematous skin of
fresh bullae.
skin diseases
Lesional skin.
Lichen planus
Active skin lesions, older than 60 days
Collagenoses
(exposed/not exposed to sun).
Recent lesions, <24 h.
Vasculitides
While a strict perilesional biopsy is required when
autoimmune skin blistering diseases are suspected, in
order to avoid false negative results, for other diseases,
including vasculitides and connective tissue disease, the
biopsy should be performed from the lesion [11].
ISSN (on-line) 2066–8279
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Figure 1 – Detection of tissue-bound and serum autoantibodies in autoimmune skin diseases. Direct immunofluorescence (IF) and histopathological examination can be performed on a skin biopsy sample. IIF, ELISA,
immunoblotting and immunoprecipitation can be performed using a serum sample. Direct IF microscopy detects
presence of antigen deposits in a skin biopsy, using a fluorochrome-labeled antibody that binds to immunoreactant
structures from patient’s skin. Indirect immunofluorescence (IIF) microscopy demonstrates presence of circulating
autoantibodies. Autoantibodies from patient’s serum bind to the antigenic structures of the substrate (monkey
esophagus, rat bladder). A second incubation, with a fluorochrome-labeled antibody, specific for the first antibody
takes place after. ELISA method quantifies the serum level of specific autoantibodies. Plate wells are coated with an
antigen and incubated with patient’s serum. Binding of specific autoantibodies to the antigenic protein takes place.
Subsequently, wells are incubated with a second antibody, conjugated with an enzyme. A reaction takes place, leading
to a change of color, which is directly proportional with the concentration of autoantibodies from patient’s serum.
Indirect IF microscopy
By indirect IF microscopy, autoantibodies in patient’s
sera may be detected. As shown in the Figure 1, this
method involves two incubation steps. First, autoantibodies
from patient’s sera are incubated with a substrate containing the putative autoantigen(s). The choice of the
substrate mainly depends on the disease suspected and
on the antigen expression levels in different tissues (e.g.,
HEp2 cells, rat liver, granulocytes, monkey esophagus,
or salt-split skin). Subsequently, in a second incubation
step, the sections are incubated with a fluorochromelabeled antibody specific for human immunoglobulins
classes [11].
To characterize the molecular specificity of circulating
autoantibodies several immunoassays, including ELISA,
immunoblotting and immunoprecipiation may be used.
The enzyme-linked immunosorbent assay (ELISA) is used
to quantify the level of antigen-specific autoantibodies in
autoimmune skin diseases. For this purpose, microtiter
plate wells are coated with the antigen and incubated with
patient’s serum. Binding of specific autoantibodies to the
antigenic protein is detected by a subsequent incubation
with a secondary antibody specific for human immunoglobulins (e.g., IgG, IgM, IgA or IgE) conjugated with an
enzyme. Finally, a chromogenic substrate is added and the
ensuing color reaction is measured by spectrophotometry
(Figure 1).
Immunoblotting
Immunoblotting (also referred to as Western blotting)
may be also used to identify the molecular target of
autoantibodies in autoimmune skin diseases. After separation by SDS-PAGE, the substrate (i.e., recombinant
proteins or tissue extracts) are transferred to a nitrocellulose membrane and then incubated with patients’
sera. Subsequently, the membrane is incubated with an
enzyme-conjugated secondary antibody and the protein
bands are visualized by a color or chemiluminescent
reaction [11].
Immunoprecipitation
Immunoprecipitation is a technique used to precipitate
the autoantigen(s) in solution. An extract of radioactively
or biotin-labeled keratinocyte whole cell extracts or
supernatant is mixed with patient’s serum or purified
IgG. The formed immune complexes containing the
autoantigen are precipitated using beads coated with
protein A/G. The precipitates are separated by SDSPAGE, visualized by protein staining and/or may be
subjected to immunoblotting, using antibodies specific
for the putative autoantigen [5, 11].
 Autoimmune skin blistering diseases
Detection of autoantibodies, as well as characterization
of their microscopic binding pattern and of their molecular
specificity are obligate requirements for diagnosing an
autoimmune blistering disease.
The main autoantigens involved in the pathogenesis
of autoimmune blistering diseases are depicted in Figure 2.
A diagnostic algorithm for autoimmune blistering skin
diseases is shown in Figure 3 and detailed below.
Molecular diagnosis of autoimmune skin diseases
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Figure 2 – Localization of autoantigen in autoimmune dermatoses. In pemphigus diseases, autoantigens are represented
by cell junction molecules of keratinocytes. In pemphigoid diseases, the autoantigens mainly belong to hemidesmosomal
anchoring complex at the dermo-epidermal junction. In epidermolysis bullosa acquisita, the main autoantigen is
collagen VII. Adapted from [4].
Pemphigus diseases
Pemphigus vulgaris
Pemphigus vulgaris is an autoimmune blistering disease,
characterized by intraepidermal acatholytic cleavage and
presence of autoantibodies against cell junction molecules
of keratinocytes [12]. While several autoantigens have been
incriminated as targets of autoantibodies in pemphigus,
desmoglein 3 and desmoglein 1 were extensively characterized as autoantigens and are widely used in the routine
diagnosis [13–17]. Direct IF microscopy demonstrates
presence of intraepidermal deposits of IgG and/or C3,
in a “cobblestone” or “fishnet” intercellular pattern [18].
Indirect IF microscopy is usually performed on monkey
esophagus and shows binding of autoreactive IgG from
patient’s sera to epithelia with an intercellular pattern
[11, 18]. ELISA typically demonstrates the presence of
autoreactive IgG against desmoglein 3. In addition, autoantibodies against desmoglein 1 may be also detected [5].
Pemphigus foliaceus
Pemphigus foliaceus represents a superficial variant of
pemphigus, characterized by skin blistering with lack of
mucosal involvement, the presence of subcorneal
cleavage and of autoantibodies against desmoglein 1
[5]. The main antigen is represented by desmoglein 1
[14]. Direct and indirect IF findings are similar to those
in pemphigus vulgaris. Commercially available ELISA
kits may be used to detect the presence of autoreactive
IgG against desmoglein 1 [17, 19]. Key laboratory
diagnostic features for pemphigus vulgaris and foliaceus
are shown in Table 2.
Table 2 – Key laboratory diagnostic features in
pemphigus
Investigation
Direct IF microscopy
Finding
Intraepidermal deposits of IgG and/or
C3, in a “cobblestone” or “fishnet”
intercellular binding pattern.
Indirect IF microscopy
Intercellular binding pattern of
(e.g., monkey
autoantibodies (IgG) to epithelial cells.
esophagus)
Autoantibodies (IgG) against
Immunoassay (ELISA)
desmoglein 3 and desmoglein 1.
Importantly, the profile of autoantibody molecular
specificity correlates well with the clinical form of
pemphigus [20]. In about 80% of pemphigus patients,
detection of autoantibodies against desmoglein 3 alone
correlates with a mucosal form of pemphigus vulgaris.
Detection of autoantibodies to both desmoglein 3 and 1
is generally seen in patients with the mucocutaneous
form of pemphigus vulgaris, while the exclusive
reactivity to desmoglein 1 is documented in patients
with pemphigus foliaceus [20, 21].
Figure 3 – Algorithm of diagnosis for autoimmune blistering skin diseases.
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Molecular diagnosis of autoimmune skin diseases
Paraneoplastic pemphigus
Paraneoplastic pemphigus is an autoimmune multiorgan syndrome characterized by a pathogenic autoimmune response to several epithelial proteins occurring
in the setting of neoplasia [22, 23]. The autoantigens of
paraneoplastic pemphigus include desmoglein 1 and 3,
the bullous pemphigoid antigen of 230 kDa (BP230),
periplakin, evoplakin, desmoplakin and the alpha-2macroglobulin-like-1 [22, 24]. Direct IF microscopy shows
an association between IgG and/or C3 deposits in the
epidermis, with an intercellular pattern and also along
the dermal–epidermal junction, with a linear pattern.
Indirect IF microscopy demonstrates binding of autoreactive IgG from patient’s sera to epithelial cells on
monkey esophagus, in an intercellular pattern and might
also show linear deposits in the basement membrane
zone. A more specific analysis may be performed on
transitional epithelia (e.g., rat bladder), a substrate rich in
plakins, which facilitates detection of binding autoreactive
IgG [5, 25]. The methods used for the characterization
of the molecular specificity of autoantibodies in paraneoplastic pemphigus include immunoprecipitation using
radioactively-labeled keratinocytes, ELISA and immunoblotting [5]. By ELISA or immunoblotting, specific autoantibodies may be detected using recombinant antigens
(desmoglein 1 and 3, BP230, periplakin, evoplakin) or
epidermal extracts (Table 3).
Table 3 – Key laboratory diagnostic features in paraneoplastic pemphigus
Investigation
Finding
IgG and/or C3 deposits in the epidermis
Direct IF
with an intercellular binding pattern and
microscopy
linear at the dermal–epidermal junction.
Binding of autoantibodies (IgG) in an
Indirect IF
microscopy (e.g., intercellular pattern and linear deposits
monkey esophagus) along the basement membrane.
Immunoassay
Evoplakin, periplakin, plectin, desmoglein
(ELISA, immuno1 and 3, desmocollin 1 and 3, BP230,
blotting, immunoalpha-2-macroglobulin-like-1.
precipitation)
Immunoprecipitation was the gold diagnostic standard,
but, due to the costs and required time, it remained reserved
to a couple of specialized laboratories worldwide [5, 26].
IgA pemphigus
IgA pemphigus is a pemphigus variant characterized
by the presence of autoreactive IgA against antigens on
the surface of keratinocytes [27]. Two types of disease
are distinguished, including the intraepidermal neutrophilic
IgA dermatosis and the subcorneal pustular dermatosis.
In the subcorneal pustular dermatosis subtype, the main
autoantigen is represented by desmocollins 1–3 [5, 27].
While autoantibodies against desmoglein 1 and 3 may be
found in a few patients with intraepidermal neutrophilic
IgA dermatosis, the major autoantigen of this pemphigus
variant is still elusive [28–30]. Direct IF microscopy
demonstrates the presence of IgA deposits in the epidermis,
with an intercellular pattern. By indirect IF microscopy
on monkey esophagus, binding of autoreactive IgA from
patient’s sera to epithelial cells with an intercellular
pattern may be documented. To characterize the molecular
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specificity of autoreactive IgA, an indirect IF microscopy
assay using desmocollin-transfected COS-7 cells was
developed [5, 27].
Pemphigoid diseases
Bullous pemphigoid. Pemphigoid gestationis
Bullous pemphigoid is a blistering disease mostly
affecting elderly, characterized by subepidermal cleavage
and presence of autoantibodies against hemidesmosomal
antigens [5, 11]. Pemphigoid gestationis is a subepidermal
blistering disease associated with pregnancy or gestational
trophoblastic diseases and presence of autoantibodies
against hemidesmosomal proteins [31–33]. The major
targets of autoantibodies in patients with bullous pemphigoid and pemphigoid gestationis are the hemidesmosomal components collagen XVII/BP180 and BP230
[34, 35]. Direct IF microscopy demonstrates presence of
linear deposits of IgG and C3 at the dermo-epidermal
junction [5, 11]. Indirect IF microscopy on salt-split skin
demonstrates the presence of IgG and C3 antibodies that
bind to the epidermal site of the artificial cleavage [36].
By ELISA or immunoblotting, autoantibodies against
BP180 and BP230 may be detected. Key laboratory
diagnostic features for bullous pemphigoid and pemphigus
gestationis are shown in Table 4.
Table 4 – Key laboratory diagnostic features of bullous
pemphigoid and pemphigoid gestationis
Investigation
Direct IF
microscopy
Indirect IF
microscopy
(on salt-split skin)
Immunoassay
(e.g., ELISA or
immunoblotting)
Findings
Linear IgG and/or C3 deposits along the
dermal–epidermal junction.
Binding of autoantibodies (IgG) to the
epidermal side of the artificial cleavage.
Autoantibodies (IgG) targeted against
BP180 and/or BP230.
Commercially available ELISA systems allow for
easy quantitative detection of autoantibodies against the
immunodominant region of BP180 and BP230 and are
widely used as diagnostic and monitoring tools in pemphigoid diseases [37, 38].
Mucous membrane pemphigoid
Mucous membrane pemphigoid is a subepithelial
autoimmune blistering disease, primarily involving the
mucosae [39, 40]. The main autoantigens are localized
at the dermal–epidermal junction and include laminin
332 [41], α6β4 integrin [42] and BP180 [42, 43]. Direct
IF microscopy reveals linear deposits of IgG, IgA and
C3 antibodies at the dermal–epidermal junction. Patients
with mucous membrane pemphigoid often show low
serum autoantibody reactivity; binding of autoreactive
IgG or IgA on the dermal or epidermal side may not be
detected in up to 50% of the cases by indirect IF performed on human salt-split skin [5, 11]. Immunoblotting
performed on extracellular matrix of keratinocytes and
immunoprecipation of cultured keratinocytes are two
highly sensitive methods used for anti-laminin 332 antibody detection [44]. Key laboratory diagnostic features
for mucous membrane pemphigoid are shown in Table 5.
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Table 5 – Key laboratory diagnostic features in mucous
membrane pemphigoid
Investigation
Direct IF
microscopy
Indirect IF
microscopy
(on salt-split skin)
Immunoassay
(e.g., ELISA or
immunoblotting)
Finding
Linear deposits of IgG, A and/or C3 along
the dermal–epidermal junction.
Binding of autoantibodies (IgG/IgA) to the
epidermal/dermal side of the artificial
cleavage.
Autoantibodies (IgG/A) against collagen
XVII/BP180, laminin 332, α6β4 integrin.
Linear IgA disease
Table 6 – Key laboratory diagnostic features in linear
IgA disease
Direct IF
microscopy
Indirect IF
microscopy
(on salt-split skin)
Immunoassay
(e.g., ELISA or
immunoblotting)
Investigation
Direct IF
microscopy
Indirect IF
microscopy
(on salt-split skin)
Immunoassay
(e.g., ELISA or
immunoblotting)
Finding
Linear IgG and C3 deposits along the
dermal–epidermal (DEJ) junction.
Binding of autoantibodies to the dermal side
of the artificial cleavage.
Autoantibodies (IgG) targeted against
laminin γ1.
Epidermolysis bullosa acquisita
Linear IgA disease is a subepidermal blistering disease
showing linear IgA deposits along the epidermal basement
membrane [45]. The main antigens are the 97 kDa protein
(LABD97) and 120 kDa (LAD-1), which represent proteolytic cleavage products of BP180 protein [46, 47]. In
some patients, the main antigen is represented by
collagen VII. For this subtype of LAD, the name of
IgA-mediated epidermolysis bullosa acquisita has been
proposed [46]. Direct IF microscopy shows linear IgA
deposits at the DEJ. Indirect IF microscopy on saltsplited skin demonstrates presence of autoreactive IgA
binding to the epidermal or dermal side. Presence of
specific autoreactive IgA is evidenced by ELISA and
immunoblotting. Immunoblotting using recombinant
BP180 ectodomain or supernatant of cultured keratinocytes
demonstrates the presence of autoreactive IgA targeting
the shed ectodomain of BP180 [47]. ELISA using recombinant BP180 or collagen VII can be performed, in order
to measure the titer of autoreactive IgA [48]. Key laboratory diagnostic features for linear IgA disease are shown
in Table 6.
Investigation
Table 7 – Key laboratory diagnostic features of antip200 pemphigoid
Finding
Linear IgA deposits along the dermal–
epidermal junction.
Autoantibodies (IgA) binding to the
epidermal side of the artificial cleavage.
Autoantibodies (IgA) specific the shed
ectodomain of collagen XVII/BP-180 (LAD-1).
Anti-p200 pemphigoid
Anti-p200 pemphigoid is an autoimmune blistering
disease, characterized by subepidermal blisters and autoantibodies targeting a 200 kDa protein (p200) [49]. The
main antigen is represented by a dermal p200 protein,
which has been recently identified as the laminin γ1 chain
[50]. Direct IF microscopy demonstrates linear deposits
of IgG and C3 at the dermal–epidermal junction. Indirect
IF microscopy shows autoreactive IgG binding to the
dermal side. Immunoblotting on dermal extracts demonstrates presence of specific antibodies against the 200 kDa
protein [49, 51]. ELISA reveals presence of specific
autoantibodies against laminin γ1 [52]. Key laboratory
diagnostic features for anti-p200 pemphigoid are shown
in Table 7.
Epidermolysis bullosa acquisita is a chronic autoimmune blistering disease, characterized by subepidermal
cleavage and presence of autoantibodies against collagen
VII [5, 11, 53]. Key laboratory diagnostic features of
epidermolysis bullosa acquisita are summarized in Table 8.
Table 8 – Key laboratory diagnostic features in epidermolysis bullosa acquisita
Investigation
Direct IF
microscopy
Indirect IF
microscopy
(on salt-split skin)
Immunoassay
(e.g., ELISA or
immunoblotting)
Finding
Linear IgG and/or C3 deposits along the
DEJ.
Binding of autoantibodies at the dermal
side of the DEJ.
Autoantibodies (IgG and/or IgA) targeted
against collagen VII.
Direct IF microscopy shows linear IgG and C3 at the
dermal–epidermal junction. Indirect immunofluorescence
on human salt-split skin demonstrates binding of autoreactive IgG or IgA to the dermal side. Immunoblotting
using dermal recombinant proteins or dermal extract
reveals presence of autoantibodies targeted against different
epitopes of collagen VII. Levels of collagen VII-specific
autoantibodies may be measured by ELISA, using
various recombinant forms of the autoantigen [54–57].
Dermatitis herpetiformis
Dermatitis herpetiformis is a chronic subepidermal
autoimmune blistering disease, associated with a glutensensitive enteropathy and characterized by typical clinical
and immunopathological findings [58, 59]. Direct IF
microscopy demonstrates the presence of granular deposits
along the dermal–epidermal junction, with an accentuation
at the dermal papillae. Indirect IF microscopy on monkey
esophagus reveals autoreactive IgA binding to endomysium. ELISA demonstrates presence of autoreactive
IgA targeting the epidermal and tissue transglutaminase
[60–62]. Key diagnostic features of dermatitis herpetiformis are shown in Table 9.
Table 9 – Key laboratory diagnostic features in dermatitis herpetiformis
Investigation
Finding
Direct IF microscopy Granular IgA deposits along the DEJ.
Indirect IF microscopy
IgA anti-endomysial autoantibodies.
(e.g., monkey esophagus)
Autoantibodies (IgA) against tissue/
Immunoassay
epidermal transglutaminase.
(e.g., ELISA)
Connective tissue diseases
Anti-nuclear antibodies (ANA) represent a serological
Molecular diagnosis of autoimmune skin diseases
hallmark for a group of systemic autoimmune diseases,
including systemic lupus erythematosus (SLE), systemic
sclerosis, idiopathic inflammatory myopathies (IIM),
Sjögren’s syndrome and overlap syndromes [63, 64]. In
addition, ANA may test positive in a series of additional
autoimmune diseases, including autoimmune hepatitis,
primary billiary cirrhosis and rheumatoid arthritis [65–
67]. However, ANA usually in low titers and generally
showing specific binding patterns may be found also in
10–20% of the general population [68–70]. The majority
of healthy individuals with positive ANA test have antibodies to dense fine speckled antigen. Using a specific
assay for anti-DFS70 antibodies, it was demonstrated that
these antibodies can be used to differentiate apparently
healthy individuals from patients with connective tissue
disease [71]. While, by definition, ANA comprise autoantibodies against components of the nucleus and mitotic
apparatus, in practice, autoantibodies targeting further
cellular components, including cytoplasmic organelles,
cytoskeleton and cell membrane, which may be detected
on the same substrate, are also reported [72]. Coining a
more appropriate term for designating autoantibodies
against ubiquitous cellular components with diagnostic
relevance is a still unmet need in the field of autoimmune
diagnostics. Although several methods for detection of
ANA antibodies have been developed, indirect IF assay
on HEp-2 cells (a cell line of laryngeal carcinoma cells)
is considered the gold standard [73]. Table 10 summarizes
the major autoantibody binding patterns on HEp-2 cells
and their clinical correlations.
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In addition to their diagnostic significance, ANA may
help to predict the course of the autoimmune disease.
Thus, similar to other autoimmune diseases such as autoimmune diabetes mellitus, occurrence of ANA may
precede with months or years the onset of clinical SLE.
Furthermore, the appearance of autoantibodies in patients
with SLE tends to follow a predictable course, with a
progressive accumulation of specific autoantibodies before
the onset of SLE, while patients are still asymptomatic
[74].
Lupus erythematosus
Lupus erythematosus encompasses a spectrum of
clinical conditions, ranging from cutaneous disease to
systemic manifestations [75]. Dermatologists distinguish
several forms of cutaneous lupus, including erythematosus
chronic cutaneous lupus erythematosus (CCLE), subacute
lupus eythematosus (SCLE) and acute lupus erythematosus
(ACLE; Figure 4) [76].
Table 10 – Immunofluorescent autoantibody binding
patterns on HEp2 cells in systemic autoimmune
diseases
HEp-2 pattern
Nuclear
homogenous
Autoantigen
Clinical association
dsDNA
SLE
SLE, drug-induced
lupus
Histones
Nuclear rim
(membranous)
with metaphase
staining
dsDNA
Nuclear matrix
Heterogenous
ribonuclear proteins
(hnRNP)
Coarse
speckled
Sm, U1,2,4,6-snRNP
Fine speckled
SSA/Ro, SSB/La, Mi-2,
RNA-polymerase II
SLE
SLE
Scleroderma
Mixed connective
tissue disease
SLE
Systemic sclerosis
SLE
Systemic sclerosis
Dermatomyositis
Nucleolar
staining
Pleomorphic
speckled
Centriole
staining
Midbody
Centromere
staining
DFS70
Clumpy nucleolar
SSc, SLE
PCNA
SLE
Centriole/centrosome
proteins
Scleroderma
Mitotic spindle agent
(MSA2)
Centromere proteins
(CENP)
DFS70/LEDGF
CREST
Scleroderma
CREST syndrome
Low likelihood of
systemic
autoimmune
rheumatic disease
Figure 4 – Main subtypes of lupus erythematosus.
ACLE: Acute cutaneous lupus erythematosus; DLE:
Discoid cutaneous lupus erythematosus; CCLE: Chronic
cutaneous lupus erythematosus; SCLE: Subacute
cutaneous lupus erythematosus; SLE: Systemic lupus
erythematosus. Modified from Walling & Sontheimer,
Am J Clin Dermatol, 2009 [76].
CCLE is defined by the presence of chronic, localized,
circumscribed, hyperkeratotic and erythematous plaques,
which often demonstrate healing with scars [77, 78].
Cutaneous discoid lupus erythematosus (CDLE) represents
the most common subtype of CCLE [76]. The diagnosis
of CCLE and CDLE is strongly suggested by the typical
clinical appearance. Histopathological examination confirms the diagnosis and correlates well with the clinical
picture [79]. While not strictly required for diagnosis,
the direct IF microscopy may strengthen the suspected
diagnosis, by documenting the presence of broad deposits
of immunoglobulins and complement at the dermal–
epidermal junction (the lupus band), more often found
in the sun-exposed areas [18].
SCLE may be seen as an intermediate form of lupus
in patients having extensive cutaneous involvement and
some signs of systemic involvement, but without meeting
enough criteria to be classified as SLE [76]. The main
clinical features are photosensitivity and presence of
skin lesions that heal without scarring [76, 80]. Histopathological examination confirms the diagnosis. Limited
systemic involvement is often present. Up to 60–80% of
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the patients show positivity of ANA, more often specific
for Ro/SS-A antigens. ACLE includes two forms: localized
ACLE and generalized ACLE. While localized ACLE
presents as a “butterfly malar rash”, generalized ACLE
might present as a photosensitive maculopapular rash,
often associated with systemic involvement [76].
Diagnosis of cutaneous lupus erythematosus is based
on clinical, histo- and immunopathological findings [81].
The histology of the diseased skin of LE patients
shows some typical characteristics, including vacuolar
degeneration of the basal layer with thickened subepidermal basement membrane, as well as perivascular
and periadnexal lymphocytic infiltrates, which may help
to establish the diagnosis [76, 82]. The lupus band test is
defined by the presence of linear deposits of IgG, A, M
and/or C3 at the dermal–epidermal junction [18, 83].
Cytoid bodies and deposits in the keratinocyte nuclei
may also be present [18]. Altogether, the lupus band
may be documented in about 70–80% of patients with
SLE, when sun-exposed non-lesional skin is tested and
in about 55% cases, when sun-protected non-lesional
skin is studied. In patients with CLE, the lupus band test
of non-lesional skin is usually negative, although the
lesional skin may frequently show immune deposits at
the DEJ [84].
Systemic lupus erythematosus
Systemic lupus erythematosus (SLE) represents a multiorgan disease associated with systemic autoimmunity [6].
The diagnosis is guided by criteria developed for clinical
studies by the American College of Rheumatology (ACR)
[85, 86], which propose that the presence of four out of
the 11 listed criteria establish the SLE diagnosis (Table 11).
Table 11 – ACR criteria for diagnosis of SLE
1. Malar rash.
2. Discoid rash.
3. Photosensitivity.
4. Oral ulcers (usually painless).
5. Nonerosive arthritis (involving two or more peripheral joints).
6. Pleuritis or pericarditis.
7. Renal disorder [(1) Persistent proteinuria >0.5 g/day or more
than 3+ OR (2) Cellular casts – red cell, hemoglobin, tubular,
granular, mixed].
8. Neurological disorders (seizures or psychosis, in the absence
of other offending causes).
9. Hematological disorder [(1) Hemolytic anemia with
3
reticulocytosis OR (2) Leukopenia <4000/mm on ≥2
3
occasions OR (3) Lymphopenia <1500/mm on ≥2 occasions)
3
OR (4) Thrombocytopenia <100.000/mm in the absence of
offending drugs].
10. Immunological disorder: Anti-DNA positive autoantibodies
OR Anti-Sm positive autoantibodies OR Positive finding of
antiphospholipid autoantibodies [(1) Abnormal serum level
of IgM or IgG anticardiolipin antibodies OR (2) A positive
test result for lupus anticoagulant OR (3) A false positive
result for at least six months confirmed by immobilization of
Treponema pallidum or FTA absorption test].
11. An abnormal titer of ANA (antinuclear autoantibodies),
at any time point, in the absence of drugs.
Two of these criteria include the detection of autoantibodies, namely the presence of ANA and autoantibodies specific to certain antigens, which show a high
association with SLE. The main types of autoantibodies
detected in SCLE and SLE are shown in Table 12.
Table 12 – Main targets of autoantibodies in lupus
erythematosus
Autoantibodies against Clinical association
dsDNA
Sm
Ribosomal P
SS-A/Ro-60
Ro-52/TRIM21
SS-B/La
Nucleosomes
SLE
SCLE
SLE
SLE
SLE
SCLE
SLE
SLE
SCLE
SLE
Positivity
[percentage]
40–90%
40%
10–30%
10–30%
40–60%
60–90%
50–80%
20–30%
40%
60–90%
High titers of ANA and/or the presence of autoantibodies specific to dsDNA, Sm, nucleosomes, PCNA
are highly suggestive of SLE [64, 87–89]. Presence of
an antiphospholipid syndrome should be ruled out by
performing the lupus anticoagulant and testing for autoantibodies specific for cardiolipin and β2-glycoprotein I
[90, 91].
Monoclonal gammopathies, which tend to occur more
frequently in SLE patients [92], should be investigated
by measuring serum concentrations of IgG, IgA and
IgM, as well as by performing a serum electrophoresis
and/or immunofixation in serum and urine.
Scleroderma
Scleroderma represents an autoimmune disease, with
cutaneous and systemic involvement, characterized by
the presence of an inflammatory reaction, followed by
thickening and tightening of the dermis and damage
of internal organs. Scleroderma may present as skinlocalized or systemic disease.
Localized scleroderma
Localized scleroderma (morphea) is a term encompassing a spectrum of sclerotic autoimmune diseases
that primarily affect the skin, but also might involve
underlying structures such as the fat, fascia, muscle, and
bones [93, 94]. While the direct IF microscopy is usually
negative, occasionally it may reveal the presence of
granular IgM deposits along basement membrane zone
[18]. Although ANA are sometimes positive, they are
not specific for the localized scleroderma [93].
Systemic sclerosis
Systemic sclerosis (SSc) is a clinically heterogeneous
disease, characterized by dysfunction of fibroblasts
(determining increased deposition of extracellular matrix),
small vessel vasculopathy and production of autoantibodies [95]. Systemic sclerosis may present as a limited
cutaneous systemic sclerosis (acrosclerosis-sclerodactyly)
and diffuse cutaneous systemic sclerosis. CREST syndrome
is considered a limited form. The ACR/EULAR criteria for
classification of systemic sclerosis are shown in Table 13.
Diagnostic criteria include the presence of specific
autoantibodies. ANA are present in approximately 90%
of patients with systemic sclerosis. While autoantibodies
with several molecular specificities such as anti-centromere,
anti-Scl-70 and anti-RNA Pol III antibodies are highly
suggestive for systemic sclerosis, autoimmunity against a
Molecular diagnosis of autoimmune skin diseases
range of further antigens may associated with scleroderma
and overlap syndromes (Table 14) [96–101].
Autoantibodies against
PM-Scl75
Table 13 – The American College of Rheumatology/
European League Against Rheumatism criteria for
the classification of systemic sclerosis
Item/Sub-items
Weight/
Score
Skin thickening of the fingers of both hands
extending proximal to the metacarpophalangeal
joints (sufficient criterion)
Skin thickening of the fingers (only count to the
higher score)
▪ Puffy fingers
▪ Sclerodactyly of the fingers distal to the
metacarpophalangeal joints, but proximal to the
proximal interphalangeal joints
Fingertip lesions (only count to the higher score)
▪ Digital tip ulcers
▪ Fingertip pitting scars
Teleangiectasia
Abnormal nailfold capillaries
Pulmonary arterial hypertension and/or interstitial
lung disease (maximum score is 2)
▪ Pulmonary arterial hypertension
▪ Interstitial lung disease
Raynaud’s phenomenon
Systemic sclerosis-related autoantibodies
▪ Anticentromere
▪ Anti-topoisomerase I
▪ Anti-RNA polymerase III
9
2
4
2
3
2
2
Clinical associations
Specific antibodies
Centromere (CENPA;
CENPB)
Scl-70
RNA-polymerase III
Th/To-RNP
Fibrillarin
Associated antibodies
U3-RNP
B23
Ku
U1-RNP
Ro-52/TRIM-21
PDGFR
NOR90
Clinical associations
Dematomyositis/polymyositis
Younger patients
Active disease
Joint contractures
Increased myopathy
Decreased GI involvement
Arthritis
Myositis
Joint contractures
Limited cutaneous systemic
sclerosis
Association with SLE
Limited clinical utility
Limited clinical utility
Immunoblotting and ELISA kits for detection of
autoantibodies associated with systemic sclerosis are
commercially available and broadly used in clinical
practice.
Idiopathic inflammatory myopathies
2
2
3
3
Table 14 – Autoantibodies in systemic sclerosis
Autoantibodies against
PM-Scl100
1027
Limited cutaneous systemic
sclerosis
CREST syndrome
Digital ischemia
Risk of HTAP
Esophageal involvement
Diffuse systemic sclerosis
Pulmonary fibrosis
Cardiac involvement
Neoplasms
Diffuse systemic sclerosis
Renal crisis
Severe cutaneous involvement
Limited cutaneous systemic
sclerosis
Pulmonary involvement
Kidney involvement
Gut involvement
Severe prognosis
Limited cutaneous systemic
sclerosis
Diffuse systemic sclerosis
Cardiac involvement
Skeletal muscle involvement
Limited cutaneous systemic
sclerosis
Diffuse systemic sclerosis
Pulmonary involvement
HTAP
Idiopathic inflammatory myopathies (IIM), including
dermato- (DM) and polymyositis (PM) are chronic autoimmune diseases, which involve primarily the muscles and
the skin, may be associated with neoplasia and may
demonstrate systemic involvement [102, 103]. Diagnosis
is guided by criteria published by Bohan and Peter
(Table 15).
Table 15 – Bohan and Peter criteria for diagnosis of
dermatomyositis
1. Symmetric proximal muscle weakness.
2. Muscle biopsy.
3. Increased level of serum skeletal muscle enzymes.
4. Characteristic pattern on electromyography.
5. Typical cutaneous findings.
Dermatomyositis may be associated with interstitial
lung disease (ILD), in this case often labeled as antisynthetase syndrome (ASS), a condition characterized
by myositis, interstitial lung disease, fever, arthritis,
mechanic’s hands and Raynaud phenomenon [104].
Although direct IF microscopy occasionally reveals the
presence of C5-9 deposits in small blood vessels or
along the basement membrane zone, this feature is not
characteristic for dermatomyositis [18].
Autoantibodies with different molecular specificities
are involved in the pathogenesis of idiopathic inflammatory myopathies and associated disorders, several being
specific for myositis, while others being also found in
other systemic autoimmune diseases (Table 16).
Table 16 – Autoantibodies in idiopathic inflammatory
myopathies
Myositis-specific antibodies
Mi-2
SRP
Jo-1, PL-7, PL-12, EJ, OJ, KS,
YRS, Zo
Clinical associations
Characteristic skin
involvement
Photosensitivity
Good prognosis
Aggressive necrotizing
myositis
Low therapy response
Antisynthetase syndrome
Roxana Chiorean et al.
1028
Myositis-specific antibodies
CADM-140/MDA-5
MJ/NXP-2
P155/140 (TIF1-γ)
SAE
HMGCR
Myositis-associated antibodies
Ro-52/TRIM-21
Ku
PM/Scl
Clinical associations
Amyopathic dermatomyositis
Rapidly progressive
interstitial lung disease
Juvenile dermatomyositis
Calcinosis
Good response at therapy
Association with malignancy
Severe skin disease
Mild myopathy
Necrotizing myopathy
Statin-associated myopathy
Clinical associations
Association with Jo-1
positivity
Interstitial lung disease
Photosensitivity
Joint involvement
Overlap
dermatomyositis/SLE
Overlap systemic
sclerosis/dermatomyositis
Myositis-specific antibodies are highly selective,
being found predominantly in patients with idiopathic
inflammatory myopathies. They target nuclear and cytoplasmic components involved in gene transcription,
protein synthesis and translocation [105–107]. Detection
of autoantibodies to one or more of the eight aminoacyltransfer RNA synthetases – the most well recognized of
which is anti-histidyl (Jo-1) – is necessary to establish
the diagnosis of an antisynthetase syndrome [104].
Myositis-associated antibodies are found also in patients
with overlap syndromes and other connective tissue
diseases [107]. Other autoantibodies in idiopathic inflammatory myopathies are directed to the signal recognition
particle (SRP), 3-hydroxy-3-methylglutaryl coenzyme
(HMGCR), chromodomain helicase DNA binding protein 4
(Mi-2), SAE/small ubiquitin-related modifier (SUMO-1),
MJ/nuclear matrix protein 2 (NXP2), melanoma differentiation-associated gene 5 (MDA5)/clinically amyopathic
dermatomyositis p140 (CADM-140), transcription intermediary factor (TIF-1) gamma (p155/140) [106]. Immunoblotting/ELISA kits for detection of these autoantibodies
are commercially available and are routinely used by
physicians in clinical practice.
Vasculitides
Vasculitis is defined as an inflammation of blood
vessels, characterized by destruction of the vessel wall
[7]. According to the Chapel Hill Consensus, vasculitides
have been classified in three main subtypes, depending
on the vessel’s type involved, including large-, mediumand small-vessel vasculitis (SVV). Large-vessel vasculitides include Takayasu disease and giant cell temporal
arteritis. Medium-sized vessel vasculitides encompass
Kawasaki disease and polyarteritis nodosa. Small-vessel
vasculitides include Churg–Strauss syndrome, (Wegener’s)
granulomatosis with polyangitis, microscopic polyangiitis,
cutaneous leukocytoclastic angiitis, essential cryoglobulinemic vasculitis and Henoch–Schönlein purpura [108].
A subgroup of small-vessel vasculitides are associated
with autoreactive IgG against the cytoplasm of neutrophils
(ANCA) [109]. Erythema elevatum et diutinum is increasingly regarded as a vasculitis variant associated with
ANCA of the IgA class [110, 111].
Cutaneous vasculitis present with protean manifestations, including urticaria, infiltrative erythema, petechiae,
purpura, purpuric papules, hemorrhagic vesicles and bullae,
nodules, livedo racemosa, deep (punched out) ulcers and
digital gangrene. Guided by clinical data, histopathological
information coupled with direct IF microscopy and detection of ANCA greatly facilitate a precise and accurate
diagnosis of localized and systemic vasculitis syndromes
[7, 18, 109, 112]. A diagnostic algorithm in primary
cutaneous vasculitides is shown in Figure 5 [113].
Figure 5 – Diagnostic algorithm in primary cutaneous vasculitides. Modified from Kawakami, J Dermatol, 2010 [113].
Molecular diagnosis of autoimmune skin diseases
These varied morphologies are a direct reflection of
size of the vessels and extent of the vascular bed affected,
ranging from a vasculitis affecting few superficial, small
vessels in petechial eruptions, to extensive pan-dermal
small vessel vasculitis in hemorrhagic bullae, to muscular
vessel vasculitis in lower extremity nodules with livedo
racemosa [112]. Skin biopsy, extending to subcutis and
taken from the earliest, most symptomatic, reddish or
purpuric lesion is crucial for obtaining a high-yielding
diagnostic sample. Based on histology, vasculitis can
be classified on the size of vessels affected and the
dominant immune cell mediating the inflammation (e.g.,
neutrophilic, granulomatous, lymphocytic, or eosinophilic)
[112]. Disruption of small vessels by inflammatory cells,
deposition of fibrin within the lumen and/or vessel wall
coupled with nuclear debris allows for the confident
recognition of small vessel, mostly neutrophilic vasculitis
(also known as leukocytoclastic vasculitis). Direct IF
microscopy typically reveals IgG, IgA and C3 deposits
in the walls of dermal blood vessels [18, 112].
Measurement of ANCA is a rationale extension of
the diagnostic work-up. While p-ANCA mainly target the
myeloperoxidase and are often found in patients with
Churg–Strauss syndrome or microscopic polyangiitis,
c-ANCA often target the proteinase 3 (PR3) and are
characteristic for granulomatosis with polyangiitis [109].
Cryoglobulins are immunoglobulins that precipitate
in vitro, at temperatures less than 370C and produce organ
damage by increased blood hyperviscosity and immunemediated mechanisms. Three types of cryoglobulinemia
are recognized, according to the clonality and type of
immunoglobulins. Type I consists of monoclonal immunoglobulin, generally either IgM or IgG. Type II cryoglobulins are a mixture of monoclonal IgM and polyclonal
IgG. The IgM component of type II cryoglobulins has
rheumatoid factor activity (i.e., these immunoglobulins
bind to the Fc portion of IgG). Type III cryoglobulins are
a mixture of polyclonal IgM and IgG [114]. Detection
of cryoglobulins and/or cryofibrinogen is a mandatory
diagnostic step when suspecting a cryoglobulinemic
vasculitis [115, 116]. Cryoglobulinemia and cryofibrinogenemia may be primary (essential) or secondary to
other underlying disorders, such as carcinoma, infection,
vasculitis, collagen disease, or associated with cryoglobulinemia. Therefore, in this case, the presence of an
underlying cause, such as hepatitis B, C, SLE, Sjögren’s
syndrome or neoplasia should be ruled out.
 Laboratory diagnosis in further autoimmune skin diseases
The autoimmune nature of further chronic skin
diseases, including psoriasis, vitiligo, and alopecia areata
is supported by extensive clinical and experimental
evidence. Psoriasis is a T-cell-mediated inflammatory
skin disorder, multifactorial in its etiology [8, 117]. It
affects about 2% of the worldwide population. There is
a wide range of clinical presentations of the disease, but
the most common variety is the chronic plaque psoriasis.
The evolution of a psoriatic lesion entails a complex
interplay between environmental and genetic factors,
which sets the scene for a cascade of events that activate
1029
dendritic cells and T-cells [8, 118]. Alopecia areata appears
to be T-cell-dependent autoimmune disease with typical
clinical manifestation [10, 119]. Vitiligo is a skin disease
that causes patchy depigmentation of the epidermis and
afflicts approximately 0.5% of the population, without
preference for race or gender [120]. Its pathogenesis
appears to involve both autoantibodies and T-cells, which
may induce tissue damage by several mechanisms [121–
123]. The diagnosis of these dermatoses is primarily made
on clinical grounds and the laboratory work-up plays an
ancillary function. However, inflammation, liver and
kidney parameters may be used to monitor the response
to treatment and the side effects of the systemic antiinflammatory therapies.
In further inflammatory dermatoses, including chronic
ulcerative stomatitis, lichen planus and erythema multiforme, an autoimmune ethiopathogenesis is suspected,
but not yet proven by clinical and experimental data. In
lichen planus, in addition to histopathology, the direct IF
microscopy is a useful diagnostic tool, which may show
the presence of cytoid bodies and shaggy fibrinogen
deposition at the dermal–epidermal junction [18, 124,
125].
Chronic ulcerative stomatitis manifests more frequently
in Caucasian middle-aged women with painful, exacerbating and remitting oral erosions, and ulcerations. The
histological features are non-specific, with a chronic
inflammatory infiltrate, often appearing similar to oral
lichen planus. Diagnosis of chronic ulcerative stomatitis
requires surgical biopsy with immunofluorescence microscopic examination. Immunofluorescence studies show
circulating and tissue-bound autoantibodies to a protein,
DeltaNp63alpha, which is a normal component of stratified
epithelia [126, 127].
 Perspectives and conclusion
The future development of state-of-the-art diagnostic
tests for autoimmune diseases is directly linked to sustained and extensive fundamental and translational research.
A detailed definition of pathogenic human autoantibodies
would allow the development of quantitative tests, which
would ideally reflect disease activity in patients [5, 11].
A further important avenue for improvement is related
to the use of multiplex technologies, which allow multiple
antibodies to be analyzed simultaneously, contributing to
diagnosis and patient stratification in the field of autoimmune diseases. The need for standardization may be
addressed by robotic preparation of slides and development of a computer-aided IF pattern analysis system
[128, 129]. Advanced systems are currently being developed, that provide fully automated readings of IF images
and software algorithms for the mathematical description
of the IF patterns. These tests, using collections of up to
hundreds of antigens are amenable to full automation
and high-throughput screening for autoantibodies and
should greatly reduce the costs involved [5].
Detection of tissue-bound and circulating autoantibodies plays an essential role in the diagnosis of autoimmune skin diseases. The target antigens of these autoantibodies have been extensively characterized over the
past decades. Detection of tissue-bound and serum auto-
1030
Roxana Chiorean et al.
antibodies and characterization of their molecular specificity allows for a rapid and accurate diagnosis of autoimmune skin diseases. Various IF methods, as well as
molecular immunoassays, including ELISA and immunoblotting, became an essential part of the modern diagnostic
algorithms for these disorders. Fundamental and translational research combining pathogenetic studies, bioinformatic analyses, automation and high throughput
approaches will greatly facilitate the development of a
next generation of improved diagnostic tools [5, 11].
Acknowledgments
The work of the authors is supported by grants from
the Deutsche Forschungsgemeinschaft SI-1281/5-1 (CS),
from the European Community’s Seventh Framework
Programme [FP7–2007–2013] under grant agreement
No. HEALTH–F4–2011–282095 (CS).
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Corresponding author
Cassian Sitaru, MD, PhD, Department of Dermatology, University of Freiburg, Hauptstrasse 7, D–79104 Freiburg,
Germany; Phone +49–(0)761–27067690, Fax +49–(0)761–27068290, e-mail: [email protected], cassian.
[email protected]
Received: December 10, 2013
Accepted: November 5, 2014