Rom J Morphol Embryol 2014, 55(3 Suppl):1019–1033 R J M E EVIEW Romanian Journal of R 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, diseases.

 Introduction further diagnostic work-up. While the histopathological examination of a lesional skin biopsy is an important During the last decades, extensive research has focused diagnostic tool, further imaging and laboratory investi- on the pathogenesis and diagnosis of autoimmune skin gations may be needed to support the diagnosis. diseases, which show a steadily increasing incidence and prevalence [1]. Criteria to classify a condition as an Direct immunofluorescence (IF) microscopy were initially modeled on the Importantly, classifying a disease as autoimmune Koch’s postulates by Ernest Witebsky in the 1950s and requires detection of tissue-bound and/or circulating further refined by Rose and Bona in 1993 [2, 3]. They autoantibodies. Deposits of immunoreactants (typically include circumstantial evidence from clinical and laboratory findings (e.g., the presence of antibody deposition in the immunoglobulins and complement components) in the affected tissue), indirect evidence, based on the repro- perilesional skin are detected by direct immunofluorescence duction of the disease by immunizing animals with the (IF) microscopy, which remains the gold standard for putative autoantigen and a direct proof, provided by the diagnosis of autoimmune bullous diseases and is an disease induction through the passive transfer of auto- important part of the diagnostic work-up in cutaneous antibodies and/or autoreactive T-cells [3]. and vasculitides [5, 11]. Thus, by direct IF microscopy The presence of tissue-bound and circulating auto- deposition of different immunoreactants in the skin of antibodies does not only bear relevance for pathogenesis, the patients is detected using fluorochrome-labeled anti- but also allows developing robust diagnostic tools and bodies (e.g., specific for human IgG, IgA, IgM and C3) molecular therapeutic approaches. Various molecular (Figure 1). For the direct IF, the site of the biopsy relative assays show increasing importance for the diagnosis and to the primary lesion (i.e., lesional or perilesional) should monitoring of a series of autoimmune skin diseases [4]. be selected depending on the suspected disease (Table 1). While in some diseases, including autoimmune blistering Table 1 – Optimal site of biopsy for direct immuno- disorders, collagenoses and vasculitides, the detection of fluorescence microscopy autoantibodies is highly important for diagnosis, in others, Suspected diagnosis Site of biopsy such as psoriasis, vitiligo or alopecia areata, the diagnosis Autoimmune bullous Perilesional erythematous skin of is primarily made on clinical grounds [5–10]. skin diseases fresh bullae. In this article, we present an overview on the use of Lichen planus Lesional skin. molecular immunoassays for the diagnosis and monitoring Active skin lesions, older than 60 days Collagenoses of autoimmune skin diseases. (exposed/not exposed to sun). Vasculitides Recent lesions, <24 h.  Overview of diagnostic procedures in While a strict perilesional biopsy is required when autoimmune skin diseases autoimmune skin blistering diseases are suspected, in As with other diseases, a thorough patient history and order to avoid false negative results, for other diseases, a skillful clinical investigation provide a strong index of including vasculitides and connective tissue disease, the suspicion for an autoimmune skin disease and guide the biopsy should be performed from the lesion [11].

ISSN (print) 1220–0522 ISSN (on-line) 2066–8279 1020 Roxana Chiorean et al.

Figure 1 – Detection of tissue-bound and serum autoantibodies in autoimmune skin diseases. Direct immuno- fluorescence (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 autoantibodies in autoimmune skin diseases. After sepa- ration by SDS-PAGE, the substrate (i.e., recombinant By indirect IF microscopy, autoantibodies in patient’s sera may be detected. As shown in the Figure 1, this proteins or tissue extracts) are transferred to a nitro- method involves two incubation steps. First, autoantibodies cellulose membrane and then incubated with patients’ from patient’s sera are incubated with a substrate con- sera. Subsequently, the membrane is incubated with an taining the putative autoantigen(s). The choice of the enzyme-conjugated secondary antibody and the protein substrate mainly depends on the disease suspected and bands are visualized by a color or chemiluminescent on the antigen expression levels in different tissues (e.g., reaction [11]. HEp2 cells, rat liver, granulocytes, monkey esophagus, Immunoprecipitation or salt-split skin). Subsequently, in a second incubation step, the sections are incubated with a fluorochrome- Immunoprecipitation is a technique used to precipitate labeled antibody specific for human immunoglobulins the autoantigen(s) in solution. An extract of radioactively classes [11]. or biotin-labeled keratinocyte whole cell extracts or To characterize the molecular specificity of circulating supernatant is mixed with patient’s serum or purified autoantibodies several immunoassays, including ELISA, IgG. The formed immune complexes containing the immunoblotting and immunoprecipiation may be used. autoantigen are precipitated using beads coated with The enzyme-linked immunosorbent assay (ELISA) is used protein A/G. The precipitates are separated by SDS- to quantify the level of antigen-specific autoantibodies in PAGE, visualized by protein staining and/or may be autoimmune skin diseases. For this purpose, microtiter subjected to immunoblotting, using antibodies specific plate wells are coated with the antigen and incubated with for the putative autoantigen [5, 11]. patient’s serum. Binding of specific autoantibodies to the antigenic protein is detected by a subsequent incubation  Autoimmune skin blistering diseases with a secondary antibody specific for human immuno- globulins (e.g., IgG, IgM, IgA or IgE) conjugated with an Detection of autoantibodies, as well as characterization enzyme. Finally, a chromogenic substrate is added and the of their microscopic binding pattern and of their molecular ensuing color reaction is measured by spectrophotometry specificity are obligate requirements for diagnosing an (Figure 1). autoimmune blistering disease. The main autoantigens involved in the pathogenesis Immunoblotting of autoimmune blistering diseases are depicted in Figure 2. Immunoblotting (also referred to as Western blotting) A diagnostic algorithm for autoimmune blistering skin may be also used to identify the molecular target of diseases is shown in Figure 3 and detailed below.

Molecular diagnosis of autoimmune skin diseases 1021

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 [14]. Direct and indirect IF findings are similar to those in pemphigus vulgaris. Commercially available ELISA Pemphigus vulgaris kits may be used to detect the presence of autoreactive Pemphigus vulgaris is an autoimmune blistering disease, IgG against desmoglein 1 [17, 19]. Key laboratory characterized by intraepidermal acatholytic cleavage and diagnostic features for pemphigus vulgaris and foliaceus presence of autoantibodies against cell junction molecules are shown in Table 2. of keratinocytes [12]. While several autoantigens have been Table 2 – Key laboratory diagnostic features in incriminated as targets of autoantibodies in pemphigus, pemphigus desmoglein 3 and desmoglein 1 were extensively charact- Investigation Finding erized as autoantigens and are widely used in the routine Intraepidermal deposits of IgG and/or diagnosis [13–17]. Direct IF microscopy demonstrates Direct IF microscopy C3, in a “cobblestone” or “fishnet” presence of intraepidermal deposits of IgG and/or C3, intercellular binding pattern. Indirect IF microscopy in a “cobblestone” or “fishnet” intercellular pattern [18]. Intercellular binding pattern of (e.g., monkey autoantibodies (IgG) to epithelial cells. Indirect IF microscopy is usually performed on monkey esophagus) esophagus and shows binding of autoreactive IgG from Autoantibodies (IgG) against Immunoassay (ELISA) patient’s sera to epithelia with an intercellular pattern desmoglein 3 and desmoglein 1. [11, 18]. ELISA typically demonstrates the presence of Importantly, the profile of autoantibody molecular autoreactive IgG against desmoglein 3. In addition, auto- specificity correlates well with the clinical form of antibodies against desmoglein 1 may be also detected [5]. pemphigus [20]. In about 80% of pemphigus patients, Pemphigus foliaceus detection of autoantibodies against desmoglein 3 alone correlates with a mucosal form of pemphigus vulgaris. Pemphigus foliaceus represents a superficial variant of Detection of autoantibodies to both desmoglein 3 and 1 pemphigus, characterized by skin blistering with lack of is generally seen in patients with the mucocutaneous mucosal involvement, the presence of subcorneal form of pemphigus vulgaris, while the exclusive cleavage and of autoantibodies against desmoglein 1 reactivity to desmoglein 1 is documented in patients [5]. The main antigen is represented by desmoglein 1 with pemphigus foliaceus [20, 21].

1022 Roxana Chiorean et al.

une blistering skin diseases. une blistering skin Algorithm of diagnosis for autoimm of diagnosis Algorithm Figure 3 – Figure 3 –

Molecular diagnosis of autoimmune skin diseases 1023

Paraneoplastic pemphigus specificity of autoreactive IgA, an indirect IF microscopy Paraneoplastic pemphigus is an autoimmune multi- assay using desmocollin-transfected COS-7 cells was organ syndrome characterized by a pathogenic auto- developed [5, 27]. immune response to several epithelial proteins occurring Pemphigoid diseases in the setting of neoplasia [22, 23]. The autoantigens of paraneoplastic pemphigus include desmoglein 1 and 3, . Pemphigoid gestationis the bullous pemphigoid antigen of 230 kDa (BP230), Bullous pemphigoid is a blistering disease mostly periplakin, evoplakin, desmoplakin and the alpha-2- affecting elderly, characterized by subepidermal cleavage macroglobulin-like-1 [22, 24]. Direct IF microscopy shows and presence of autoantibodies against hemidesmosomal an association between IgG and/or C3 deposits in the antigens [5, 11]. Pemphigoid gestationis is a subepidermal epidermis, with an intercellular pattern and also along blistering disease associated with pregnancy or gestational the dermal–epidermal junction, with a linear pattern. trophoblastic diseases and presence of autoantibodies Indirect IF microscopy demonstrates binding of auto- against hemidesmosomal proteins [31–33]. The major reactive IgG from patient’s sera to epithelial cells on targets of autoantibodies in patients with bullous pem- monkey esophagus, in an intercellular pattern and might phigoid and pemphigoid gestationis are the hemides- also show linear deposits in the basement membrane mosomal components collagen XVII/BP180 and BP230 zone. A more specific analysis may be performed on [34, 35]. Direct IF microscopy demonstrates presence of transitional epithelia (e.g., rat bladder), a substrate rich in linear deposits of IgG and C3 at the dermo-epidermal plakins, which facilitates detection of binding autoreactive junction [5, 11]. Indirect IF microscopy on salt-split skin IgG [5, 25]. The methods used for the characterization demonstrates the presence of IgG and C3 antibodies that of the molecular specificity of autoantibodies in para- bind to the epidermal site of the artificial cleavage [36]. neoplastic pemphigus include immunoprecipitation using By ELISA or immunoblotting, autoantibodies against radioactively-labeled keratinocytes, ELISA and immuno- BP180 and BP230 may be detected. Key laboratory blotting [5]. By ELISA or immunoblotting, specific auto- diagnostic features for bullous pemphigoid and pemphigus antibodies may be detected using recombinant antigens gestationis are shown in Table 4. (desmoglein 1 and 3, BP230, periplakin, evoplakin) or Table 4 – Key laboratory diagnostic features of bullous epidermal extracts (Table 3). pemphigoid and pemphigoid gestationis Table 3 – Key laboratory diagnostic features in para- Investigation Findings neoplastic pemphigus Direct IF Linear IgG and/or C3 deposits along the Investigation Finding microscopy dermal–epidermal junction. IgG and/or C3 deposits in the epidermis Indirect IF Direct IF Binding of autoantibodies (IgG) to the with an intercellular binding pattern and microscopy microscopy epidermal side of the artificial cleavage. linear at the dermal–epidermal junction. (on salt-split skin) Binding of autoantibodies (IgG) in an Immunoassay Indirect IF Autoantibodies (IgG) targeted against (e.g., ELISA or microscopy (e.g., intercellular pattern and linear deposits BP180 and/or BP230. monkey esophagus) along the basement membrane. immunoblotting) Immunoassay Evoplakin, periplakin, plectin, desmoglein (ELISA, immuno- Commercially available ELISA systems allow for 1 and 3, desmocollin 1 and 3, BP230, blotting, immuno- alpha-2-macroglobulin-like-1. easy quantitative detection of autoantibodies against the precipitation) immunodominant region of BP180 and BP230 and are Immunoprecipitation was the gold diagnostic standard, widely used as diagnostic and monitoring tools in pem- but, due to the costs and required time, it remained reserved phigoid diseases [37, 38]. to a couple of specialized laboratories worldwide [5, 26]. Mucous membrane pemphigoid IgA pemphigus Mucous membrane pemphigoid is a subepithelial IgA pemphigus is a pemphigus variant characterized autoimmune blistering disease, primarily involving the by the presence of autoreactive IgA against antigens on mucosae [39, 40]. The main autoantigens are localized the surface of keratinocytes [27]. Two types of disease at the dermal–epidermal junction and include are distinguished, including the intraepidermal neutrophilic 332 [41], α6β4 integrin [42] and BP180 [42, 43]. Direct IgA dermatosis and the subcorneal pustular dermatosis. IF microscopy reveals linear deposits of IgG, IgA and In the subcorneal pustular dermatosis subtype, the main C3 antibodies at the dermal–epidermal junction. Patients autoantigen is represented by desmocollins 1–3 [5, 27]. with mucous membrane pemphigoid often show low While autoantibodies against desmoglein 1 and 3 may be serum autoantibody reactivity; binding of autoreactive found in a few patients with intraepidermal neutrophilic IgG or IgA on the dermal or epidermal side may not be IgA dermatosis, the major autoantigen of this pemphigus detected in up to 50% of the cases by indirect IF perfor- variant is still elusive [28–30]. Direct IF microscopy med on human salt-split skin [5, 11]. Immunoblotting demonstrates the presence of IgA deposits in the epidermis, performed on extracellular matrix of keratinocytes and with an intercellular pattern. By indirect IF microscopy immunoprecipation of cultured keratinocytes are two on monkey esophagus, binding of autoreactive IgA from highly sensitive methods used for anti-laminin 332 anti- patient’s sera to epithelial cells with an intercellular body detection [44]. Key laboratory diagnostic features pattern may be documented. To characterize the molecular for mucous membrane pemphigoid are shown in Table 5.

1024 Roxana Chiorean et al. Table 5 – Key laboratory diagnostic features in mucous Table 7 – Key laboratory diagnostic features of anti- membrane pemphigoid p200 pemphigoid

Investigation Finding Investigation Finding Direct IF Linear deposits of IgG, A and/or C3 along Direct IF Linear IgG and C3 deposits along the microscopy the dermal–epidermal junction. microscopy dermal–epidermal (DEJ) junction. Indirect IF Binding of autoantibodies (IgG/IgA) to the Indirect IF Binding of autoantibodies to the dermal side microscopy epidermal/dermal side of the artificial microscopy of the artificial cleavage. (on salt-split skin) cleavage. (on salt-split skin) Immunoassay Immunoassay Autoantibodies (IgG/A) against collagen Autoantibodies (IgG) targeted against (e.g., ELISA or (e.g., ELISA or XVII/BP180, laminin 332, α6β4 integrin. laminin γ1. immunoblotting) immunoblotting)

Linear IgA disease Epidermolysis bullosa acquisita Linear IgA disease is a subepidermal blistering disease Epidermolysis bullosa acquisita is a chronic auto- showing linear IgA deposits along the epidermal basement immune blistering disease, characterized by subepidermal membrane [45]. The main antigens are the 97 kDa protein cleavage and presence of autoantibodies against collagen (LABD97) and 120 kDa (LAD-1), which represent proteo- VII [5, 11, 53]. Key laboratory diagnostic features of lytic cleavage products of BP180 protein [46, 47]. In epidermolysis bullosa acquisita are summarized in Table 8. some patients, the main antigen is represented by Table 8 – Key laboratory diagnostic features in epider- collagen VII. For this subtype of LAD, the name of molysis bullosa acquisita IgA-mediated epidermolysis bullosa acquisita has been Investigation Finding proposed [46]. Direct IF microscopy shows linear IgA Direct IF Linear IgG and/or C3 deposits along the deposits at the DEJ. Indirect IF microscopy on salt- microscopy DEJ. Indirect IF Binding of autoantibodies at the dermal splited skin demonstrates presence of autoreactive IgA microscopy side of the DEJ. binding to the epidermal or dermal side. Presence of (on salt-split skin) Immunoassay specific autoreactive IgA is evidenced by ELISA and Autoantibodies (IgG and/or IgA) targeted (e.g., ELISA or immunoblotting. Immunoblotting using recombinant against collagen VII. immunoblotting) BP180 ectodomain or supernatant of cultured keratinocytes demonstrates the presence of autoreactive IgA targeting Direct IF microscopy shows linear IgG and C3 at the the shed ectodomain of BP180 [47]. ELISA using recom- dermal–epidermal junction. Indirect immunofluorescence binant BP180 or collagen VII can be performed, in order on human salt-split skin demonstrates binding of auto- to measure the titer of autoreactive IgA [48]. Key labora- reactive IgG or IgA to the dermal side. Immunoblotting tory diagnostic features for linear IgA disease are shown using dermal recombinant proteins or dermal extract in Table 6. reveals presence of autoantibodies targeted against different epitopes of collagen VII. Levels of collagen VII-specific Table 6 – Key laboratory diagnostic features in linear IgA disease autoantibodies may be measured by ELISA, using various recombinant forms of the autoantigen [54–57]. Investigation Finding Dermatitis herpetiformis Direct IF Linear IgA deposits along the dermal– microscopy epidermal junction. Dermatitis herpetiformis is a chronic subepidermal Indirect IF Autoantibodies (IgA) binding to the autoimmune blistering disease, associated with a gluten- microscopy epidermal side of the artificial cleavage. (on salt-split skin) sensitive enteropathy and characterized by typical clinical Immunoassay and immunopathological findings [58, 59]. Direct IF Autoantibodies (IgA) specific the shed (e.g., ELISA or ectodomain of collagen XVII/BP-180 (LAD-1). microscopy demonstrates the presence of granular deposits immunoblotting) along the dermal–epidermal junction, with an accentuation at the dermal papillae. Indirect IF microscopy on monkey Anti-p200 pemphigoid esophagus reveals autoreactive IgA binding to endo- Anti-p200 pemphigoid is an autoimmune blistering mysium. ELISA demonstrates presence of autoreactive disease, characterized by subepidermal blisters and auto- IgA targeting the epidermal and tissue transglutaminase antibodies targeting a 200 kDa protein (p200) [49]. The [60–62]. Key diagnostic features of dermatitis herpeti- main antigen is represented by a dermal p200 protein, formis are shown in Table 9. which has been recently identified as the laminin γ1 chain Table 9 – Key laboratory diagnostic features in derma- [50]. Direct IF microscopy demonstrates linear deposits titis herpetiformis of IgG and C3 at the dermal–epidermal junction. Indirect Investigation Finding IF microscopy shows autoreactive IgG binding to the Direct IF microscopy Granular IgA deposits along the DEJ. Indirect IF microscopy dermal side. Immunoblotting on dermal extracts demon- IgA anti-endomysial autoantibodies. (e.g., monkey esophagus) strates presence of specific antibodies against the 200 kDa Immunoassay Autoantibodies (IgA) against tissue/ protein [49, 51]. ELISA reveals presence of specific (e.g., ELISA) epidermal transglutaminase. autoantibodies against laminin γ1 [52]. Key laboratory diagnostic features for anti-p200 pemphigoid are shown Connective tissue diseases in Table 7. Anti-nuclear antibodies (ANA) represent a serological

Molecular diagnosis of autoimmune skin diseases 1025 hallmark for a group of systemic autoimmune diseases, In addition to their diagnostic significance, ANA may including systemic lupus erythematosus (SLE), systemic help to predict the course of the autoimmune disease. sclerosis, idiopathic inflammatory (IIM), Thus, similar to other autoimmune diseases such as auto- Sjögren’s syndrome and overlap syndromes [63, 64]. In immune diabetes mellitus, occurrence of ANA may addition, ANA may test positive in a series of additional precede with months or years the onset of clinical SLE. autoimmune diseases, including autoimmune hepatitis, Furthermore, the appearance of autoantibodies in patients primary billiary cirrhosis and [65– with SLE tends to follow a predictable course, with a 67]. However, ANA usually in low titers and generally progressive accumulation of specific autoantibodies before showing specific binding patterns may be found also in the onset of SLE, while patients are still asymptomatic 10–20% of the general population [68–70]. The majority [74]. of healthy individuals with positive ANA test have anti- bodies to dense fine speckled antigen. Using a specific Lupus erythematosus assay for anti-DFS70 antibodies, it was demonstrated that Lupus erythematosus encompasses a spectrum of these antibodies can be used to differentiate apparently clinical conditions, ranging from cutaneous disease to healthy individuals from patients with connective tissue systemic manifestations [75]. Dermatologists distinguish disease [71]. While, by definition, ANA comprise auto- several forms of cutaneous lupus, including erythematosus antibodies against components of the nucleus and mitotic chronic cutaneous lupus erythematosus (CCLE), subacute apparatus, in practice, autoantibodies targeting further lupus eythematosus (SCLE) and acute lupus erythematosus cellular components, including cytoplasmic organelles, (ACLE; Figure 4) [76]. 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. Table 10 – Immunofluorescent autoantibody binding patterns on HEp2 cells in systemic autoimmune diseases HEp-2 pattern Autoantigen Clinical association Figure 4 – Main subtypes of lupus erythematosus. dsDNA SLE ACLE: Acute cutaneous lupus erythematosus; DLE: Nuclear Discoid cutaneous lupus erythematosus; CCLE: Chronic SLE, drug-induced homogenous Histones lupus cutaneous lupus erythematosus; SCLE: Subacute Nuclear rim cutaneous lupus erythematosus; SLE: Systemic lupus (membranous) erythematosus. Modified from Walling & Sontheimer, dsDNA SLE with metaphase Am J Clin Dermatol, 2009 [76]. staining SLE CCLE is defined by the presence of chronic, localized, Heterogenous circumscribed, hyperkeratotic and erythematous plaques, Scleroderma Nuclear matrix ribonuclear proteins which often demonstrate healing with scars [77, 78]. (hnRNP) Mixed connective tissue disease Cutaneous discoid lupus erythematosus (CDLE) represents the most common subtype of CCLE [76]. The diagnosis Coarse SLE Sm, U1,2,4,6-snRNP of CCLE and CDLE is strongly suggested by the typical speckled Systemic sclerosis clinical appearance. Histopathological examination con- SLE SSA/Ro, SSB/La, Mi-2, firms the diagnosis and correlates well with the clinical Fine speckled Systemic sclerosis RNA-polymerase II picture [79]. While not strictly required for diagnosis, Dermatomyositis the direct IF microscopy may strengthen the suspected Nucleolar Clumpy nucleolar SSc, SLE diagnosis, by documenting the presence of broad deposits staining of immunoglobulins and complement at the dermal– Pleomorphic PCNA SLE speckled epidermal junction (the lupus band), more often found in the sun-exposed areas [18]. Centriole Centriole/centrosome Scleroderma SCLE may be seen as an intermediate form of lupus staining proteins CREST in patients having extensive cutaneous involvement and Mitotic spindle agent Midbody Scleroderma (MSA2) some signs of systemic involvement, but without meeting Centromere Centromere proteins enough criteria to be classified as SLE [76]. The main CREST syndrome staining (CENP) clinical features are photosensitivity and presence of Low likelihood of skin lesions that heal without scarring [76, 80]. Histo- systemic DFS70 DFS70/LEDGF autoimmune pathological examination confirms the diagnosis. Limited rheumatic disease systemic involvement is often present. Up to 60–80% of

1026 Roxana Chiorean et al. the patients show positivity of ANA, more often specific Table 12 – Main targets of autoantibodies in lupus for Ro/SS-A antigens. ACLE includes two forms: localized erythematosus Positivity ACLE and generalized ACLE. While localized ACLE Autoantibodies against Clinical association presents as a “butterfly malar rash”, generalized ACLE [percentage] SLE 40–90% might present as a photosensitive maculopapular rash, dsDNA often associated with systemic involvement [76]. SCLE 40% Diagnosis of cutaneous lupus erythematosus is based Sm SLE 10–30% on clinical, histo- and immunopathological findings [81]. Ribosomal P SLE 10–30% SLE 40–60% The histology of the diseased skin of LE patients SS-A/Ro-60 shows some typical characteristics, including vacuolar SCLE 60–90% degeneration of the basal layer with thickened sub- Ro-52/TRIM21 SLE 50–80% SLE 20–30% epidermal basement membrane, as well as perivascular SS-B/La and periadnexal lymphocytic infiltrates, which may help SCLE 40% to establish the diagnosis [76, 82]. The lupus band test is Nucleosomes SLE 60–90% defined by the presence of linear deposits of IgG, A, M High titers of ANA and/or the presence of auto- and/or C3 at the dermal–epidermal junction [18, 83]. antibodies specific to dsDNA, Sm, nucleosomes, PCNA Cytoid bodies and deposits in the keratinocyte nuclei are highly suggestive of SLE [64, 87–89]. Presence of may also be present [18]. Altogether, the lupus band an antiphospholipid syndrome should be ruled out by may be documented in about 70–80% of patients with performing the lupus anticoagulant and testing for auto- SLE, when sun-exposed non-lesional skin is tested and antibodies specific for cardiolipin and β2-glycoprotein I in about 55% cases, when sun-protected non-lesional [90, 91]. skin is studied. In patients with CLE, the lupus band test Monoclonal gammopathies, which tend to occur more of non-lesional skin is usually negative, although the frequently in SLE patients [92], should be investigated lesional skin may frequently show immune deposits at by measuring serum concentrations of IgG, IgA and the DEJ [84]. IgM, as well as by performing a serum electrophoresis Systemic lupus erythematosus and/or immunofixation in serum and urine. Systemic lupus erythematosus (SLE) represents a multi- Scleroderma organ disease associated with systemic autoimmunity [6]. Scleroderma represents an autoimmune disease, with The diagnosis is guided by criteria developed for clinical cutaneous and systemic involvement, characterized by studies by the American College of (ACR) the presence of an inflammatory reaction, followed by [85, 86], which propose that the presence of four out of thickening and tightening of the dermis and damage the 11 listed criteria establish the SLE diagnosis (Table 11). of internal organs. Scleroderma may present as skin- Table 11 – ACR criteria for diagnosis of SLE localized or systemic disease. 1. Malar rash. Localized scleroderma 2. Discoid rash. Localized scleroderma (morphea) is a term encom- 3. Photosensitivity. passing a spectrum of sclerotic autoimmune diseases 4. Oral ulcers (usually painless). that primarily affect the skin, but also might involve 5. Nonerosive arthritis (involving two or more peripheral joints). underlying structures such as the fat, fascia, muscle, and 6. Pleuritis or pericarditis. bones [93, 94]. While the direct IF microscopy is usually 7. Renal disorder [(1) Persistent proteinuria >0.5 g/day or more than 3+ OR (2) Cellular casts – red cell, hemoglobin, tubular, negative, occasionally it may reveal the presence of granular, mixed]. granular IgM deposits along basement membrane zone 8. Neurological disorders (seizures or psychosis, in the absence [18]. Although ANA are sometimes positive, they are of other offending causes). not specific for the localized scleroderma [93]. 9. Hematological disorder [(1) Hemolytic anemia with reticulocytosis OR (2) Leukopenia <4000/mm3 on ≥2 Systemic sclerosis occasions OR (3) Lymphopenia <1500/mm3 on ≥2 occasions) OR (4) Thrombocytopenia <100.000/mm3 in the absence of Systemic sclerosis (SSc) is a clinically heterogeneous offending drugs]. disease, characterized by dysfunction of fibroblasts 10. Immunological disorder: Anti-DNA positive autoantibodies (determining increased deposition of extracellular matrix), OR Anti-Sm positive autoantibodies OR Positive finding of small vessel vasculopathy and production of autoanti- antiphospholipid autoantibodies [(1) Abnormal serum level of IgM or IgG anticardiolipin antibodies OR (2) A positive bodies [95]. Systemic sclerosis may present as a limited test result for lupus anticoagulant OR (3) A false positive cutaneous systemic sclerosis (acrosclerosis-sclerodactyly) result for at least six months confirmed by immobilization of and diffuse cutaneous systemic sclerosis. CREST syndrome Treponema pallidum or FTA absorption test]. is considered a limited form. The ACR/EULAR criteria for 11. An abnormal titer of ANA (antinuclear autoantibodies), at any time point, in the absence of drugs. classification of systemic sclerosis are shown in Table 13. Diagnostic criteria include the presence of specific Two of these criteria include the detection of auto- autoantibodies. ANA are present in approximately 90% antibodies, namely the presence of ANA and auto- of patients with systemic sclerosis. While autoantibodies antibodies specific to certain antigens, which show a high with several molecular specificities such as anti-centromere, association with SLE. The main types of autoantibodies anti-Scl-70 and anti-RNA Pol III antibodies are highly detected in SCLE and SLE are shown in Table 12. suggestive for systemic sclerosis, autoimmunity against a

Molecular diagnosis of autoimmune skin diseases 1027 range of further antigens may associated with scleroderma Autoantibodies against Clinical associations and overlap syndromes (Table 14) [96–101]. PM-Scl75 Dematomyositis/polymyositis Table 13 – The American College of Rheumatology/ Younger patients European League Against Rheumatism criteria for Active disease the classification of systemic sclerosis Joint contractures Weight/ Increased Item/Sub-items PM-Scl100 Score Decreased GI involvement Skin thickening of the fingers of both hands Arthritis extending proximal to the metacarpophalangeal 9 joints (sufficient criterion) Ku Myositis Skin thickening of the fingers (only count to the Joint contractures higher score) Limited cutaneous systemic U1-RNP ▪ Puffy fingers 2 sclerosis ▪ Sclerodactyly of the fingers distal to the Ro-52/TRIM-21 Association with SLE metacarpophalangeal joints, but proximal to the 4 PDGFR Limited clinical utility proximal interphalangeal joints NOR90 Limited clinical utility Fingertip lesions (only count to the higher score) ▪ Digital tip ulcers 2 Immunoblotting and ELISA kits for detection of ▪ Fingertip pitting scars 3 autoantibodies associated with systemic sclerosis are Teleangiectasia 2 commercially available and broadly used in clinical Abnormal nailfold capillaries 2 practice. Pulmonary arterial hypertension and/or interstitial lung disease (maximum score is 2) Idiopathic inflammatory myopathies ▪ Pulmonary arterial hypertension 2 Idiopathic inflammatory myopathies (IIM), including ▪ Interstitial lung disease 2 dermato- (DM) and polymyositis (PM) are chronic auto- Raynaud’s phenomenon 3 immune diseases, which involve primarily the muscles and Systemic sclerosis-related autoantibodies 3 the skin, may be associated with neoplasia and may ▪ Anticentromere demonstrate systemic involvement [102, 103]. Diagnosis ▪ Anti-topoisomerase I is guided by criteria published by Bohan and Peter ▪ Anti-RNA polymerase III (Table 15). Table 15 – Bohan and Peter criteria for diagnosis of Table 14 – Autoantibodies in systemic sclerosis dermatomyositis Autoantibodies against Clinical associations 1. Symmetric proximal muscle weakness. Specific antibodies 2. Muscle biopsy. Limited cutaneous systemic 3. Increased level of serum skeletal muscle enzymes. sclerosis 4. Characteristic pattern on electromyography. Centromere (CENPA; CREST syndrome 5. Typical cutaneous findings. CENPB) Digital ischemia Risk of HTAP Dermatomyositis may be associated with interstitial Esophageal involvement lung disease (ILD), in this case often labeled as anti- Diffuse systemic sclerosis synthetase syndrome (ASS), a condition characterized Pulmonary fibrosis Scl-70 by myositis, interstitial lung disease, fever, arthritis, Cardiac involvement mechanic’s hands and Raynaud phenomenon [104]. Neoplasms Although direct IF microscopy occasionally reveals the Diffuse systemic sclerosis presence of C5-9 deposits in small blood vessels or RNA-polymerase III Renal crisis along the basement membrane zone, this feature is not Severe cutaneous involvement characteristic for dermatomyositis [18]. Limited cutaneous systemic Autoantibodies with different molecular specificities sclerosis are involved in the pathogenesis of idiopathic inflam- Th/To-RNP Pulmonary involvement matory myopathies and associated disorders, several being Kidney involvement specific for myositis, while others being also found in Gut involvement other systemic autoimmune diseases (Table 16). Fibrillarin Severe prognosis Table 16 – Autoantibodies in idiopathic inflammatory Associated antibodies myopathies Limited cutaneous systemic sclerosis Myositis-specific antibodies Clinical associations Diffuse systemic sclerosis Characteristic skin U3-RNP involvement Cardiac involvement Mi-2 Photosensitivity Skeletal muscle involvement Good prognosis Limited cutaneous systemic Aggressive necrotizing sclerosis SRP myositis Diffuse systemic sclerosis B23 Low therapy response Pulmonary involvement Jo-1, PL-7, PL-12, EJ, OJ, KS, Antisynthetase syndrome HTAP YRS, Zo

1028 Roxana Chiorean et al.

Myositis-specific antibodies Clinical associations MJ/nuclear matrix protein 2 (NXP2), melanoma differ- Amyopathic dermatomyositis entiation-associated gene 5 (MDA5)/clinically amyopathic CADM-140/MDA-5 Rapidly progressive dermatomyositis p140 (CADM-140), transcription inter- interstitial lung disease mediary factor (TIF-1) gamma (p155/140) [106]. Immuno- Juvenile dermatomyositis blotting/ELISA kits for detection of these autoantibodies MJ/NXP-2 Calcinosis are commercially available and are routinely used by Good response at therapy physicians in clinical practice. P155/140 (TIF1-γ) Association with malignancy Severe skin disease Vasculitides SAE Mild myopathy Vasculitis is defined as an inflammation of blood Necrotizing myopathy HMGCR vessels, characterized by destruction of the vessel wall Statin-associated myopathy [7]. According to the Chapel Hill Consensus, vasculitides Myositis-associated antibodies Clinical associations have been classified in three main subtypes, depending Association with Jo-1 on the vessel’s type involved, including large-, medium- positivity and small-vessel vasculitis (SVV). Large-vessel vasculi- Ro-52/TRIM-21 Interstitial lung disease tides include Takayasu disease and giant cell temporal Photosensitivity arteritis. Medium-sized vessel vasculitides encompass Joint involvement Kawasaki disease and polyarteritis nodosa. Small-vessel Overlap Ku vasculitides include Churg–Strauss syndrome, (Wegener’s) dermatomyositis/SLE Overlap systemic granulomatosis with polyangitis, microscopic polyangiitis, PM/Scl sclerosis/dermatomyositis cutaneous leukocytoclastic angiitis, essential cryoglobu- linemic vasculitis and Henoch–Schönlein purpura [108]. Myositis-specific antibodies are highly selective, A subgroup of small-vessel vasculitides are associated being found predominantly in patients with idiopathic with autoreactive IgG against the cytoplasm of neutrophils inflammatory myopathies. They target nuclear and cyto- (ANCA) [109]. Erythema elevatum et diutinum is increa- plasmic components involved in gene transcription, singly regarded as a vasculitis variant associated with protein synthesis and translocation [105–107]. Detection ANCA of the IgA class [110, 111]. of autoantibodies to one or more of the eight aminoacyl- Cutaneous vasculitis present with protean manifest- transfer RNA synthetases – the most well recognized of ations, including urticaria, infiltrative erythema, petechiae, which is anti-histidyl (Jo-1) – is necessary to establish purpura, purpuric papules, hemorrhagic vesicles and bullae, the diagnosis of an antisynthetase syndrome [104]. nodules, livedo racemosa, deep (punched out) ulcers and Myositis-associated antibodies are found also in patients digital gangrene. Guided by clinical data, histopathological with overlap syndromes and other connective tissue information coupled with direct IF microscopy and detect- diseases [107]. Other autoantibodies in idiopathic inflam- ion of ANCA greatly facilitate a precise and accurate matory myopathies are directed to the signal recognition diagnosis of localized and systemic vasculitis syndromes particle (SRP), 3-hydroxy-3-methylglutaryl coenzyme [7, 18, 109, 112]. A diagnostic algorithm in primary (HMGCR), chromodomain helicase DNA binding protein 4 cutaneous vasculitides is shown in Figure 5 [113]. (Mi-2), SAE/small ubiquitin-related modifier (SUMO-1),

Figure 5 – Diagnostic algorithm in primary cutaneous vasculitides. Modified from Kawakami, J Dermatol, 2010 [113].

Molecular diagnosis of autoimmune skin diseases 1029

These varied morphologies are a direct reflection of dendritic cells and T-cells [8, 118]. Alopecia areata appears size of the vessels and extent of the vascular bed affected, to be T-cell-dependent autoimmune disease with typical ranging from a vasculitis affecting few superficial, small clinical manifestation [10, 119]. Vitiligo is a skin disease vessels in petechial eruptions, to extensive pan-dermal that causes patchy depigmentation of the epidermis and small vessel vasculitis in hemorrhagic bullae, to muscular afflicts approximately 0.5% of the population, without vessel vasculitis in lower extremity nodules with livedo preference for race or gender [120]. Its pathogenesis racemosa [112]. Skin biopsy, extending to subcutis and appears to involve both autoantibodies and T-cells, which taken from the earliest, most symptomatic, reddish or may induce tissue damage by several mechanisms [121– purpuric lesion is crucial for obtaining a high-yielding 123]. The diagnosis of these dermatoses is primarily made diagnostic sample. Based on histology, vasculitis can on clinical grounds and the laboratory work-up plays an be classified on the size of vessels affected and the ancillary function. However, inflammation, liver and dominant immune cell mediating the inflammation (e.g., kidney parameters may be used to monitor the response neutrophilic, granulomatous, lymphocytic, or eosinophilic) to treatment and the side effects of the systemic anti- [112]. Disruption of small vessels by inflammatory cells, inflammatory therapies. deposition of fibrin within the lumen and/or vessel wall In further inflammatory dermatoses, including chronic coupled with nuclear debris allows for the confident ulcerative stomatitis, lichen planus and erythema multi- recognition of small vessel, mostly neutrophilic vasculitis forme, an autoimmune ethiopathogenesis is suspected, (also known as leukocytoclastic vasculitis). Direct IF but not yet proven by clinical and experimental data. In microscopy typically reveals IgG, IgA and C3 deposits lichen planus, in addition to histopathology, the direct IF in the walls of dermal blood vessels [18, 112]. microscopy is a useful diagnostic tool, which may show Measurement of ANCA is a rationale extension of the presence of cytoid bodies and shaggy fibrinogen the diagnostic work-up. While p-ANCA mainly target the deposition at the dermal–epidermal junction [18, 124, myeloperoxidase and are often found in patients with 125]. Churg–Strauss syndrome or microscopic polyangiitis, Chronic ulcerative stomatitis manifests more frequently c-ANCA often target the proteinase 3 (PR3) and are in Caucasian middle-aged women with painful, exacer- characteristic for granulomatosis with polyangiitis [109]. bating and remitting oral erosions, and ulcerations. The Cryoglobulins are immunoglobulins that precipitate histological features are non-specific, with a chronic in vitro, at temperatures less than 370C and produce organ inflammatory infiltrate, often appearing similar to oral damage by increased blood hyperviscosity and immune- lichen planus. Diagnosis of chronic ulcerative stomatitis mediated mechanisms. Three types of cryoglobulinemia requires surgical biopsy with immunofluorescence micro- are recognized, according to the clonality and type of scopic examination. Immunofluorescence studies show immunoglobulins. Type I consists of monoclonal immuno- circulating and tissue-bound autoantibodies to a protein, globulin, generally either IgM or IgG. Type II cryoglo- DeltaNp63alpha, which is a normal component of stratified bulins are a mixture of monoclonal IgM and polyclonal epithelia [126, 127]. IgG. The IgM component of type II cryoglobulins has rheumatoid factor activity (i.e., these immunoglobulins  Perspectives and conclusion bind to the Fc portion of IgG). Type III cryoglobulins are a mixture of polyclonal IgM and IgG [114]. Detection The future development of state-of-the-art diagnostic of cryoglobulins and/or cryofibrinogen is a mandatory tests for autoimmune diseases is directly linked to sus- diagnostic step when suspecting a cryoglobulinemic tained and extensive fundamental and translational research. vasculitis [115, 116]. Cryoglobulinemia and cryofibri- A detailed definition of pathogenic human autoantibodies nogenemia may be primary (essential) or secondary to would allow the development of quantitative tests, which other underlying disorders, such as carcinoma, infection, would ideally reflect disease activity in patients [5, 11]. vasculitis, collagen disease, or associated with cryoglo- A further important avenue for improvement is related bulinemia. Therefore, in this case, the presence of an to the use of multiplex technologies, which allow multiple underlying cause, such as hepatitis B, C, SLE, Sjögren’s antibodies to be analyzed simultaneously, contributing to syndrome or neoplasia should be ruled out. diagnosis and patient stratification in the field of auto- immune diseases. The need for standardization may be addressed by robotic preparation of slides and develop-  Laboratory diagnosis in further auto- ment of a computer-aided IF pattern analysis system immune skin diseases [128, 129]. Advanced systems are currently being deve- The autoimmune nature of further chronic skin loped, that provide fully automated readings of IF images diseases, including psoriasis, vitiligo, and alopecia areata and software algorithms for the mathematical description is supported by extensive clinical and experimental of the IF patterns. These tests, using collections of up to evidence. Psoriasis is a T-cell-mediated inflammatory hundreds of antigens are amenable to full automation skin disorder, multifactorial in its etiology [8, 117]. It and high-throughput screening for autoantibodies and affects about 2% of the worldwide population. There is should greatly reduce the costs involved [5]. a wide range of clinical presentations of the disease, but Detection of tissue-bound and circulating autoanti- the most common variety is the chronic plaque psoriasis. bodies plays an essential role in the diagnosis of auto- The evolution of a psoriatic lesion entails a complex immune skin diseases. The target antigens of these auto- interplay between environmental and genetic factors, antibodies have been extensively characterized over the which sets the scene for a cascade of events that activate past decades. Detection of tissue-bound and serum auto-

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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