44 Autoantibodies in Atopic Eczema
N. Mothes, I. Mittermann, K. Aichberger, P. Valent, R. Valenta
44.1
Introduction
Atopic eczema (AE) is a chronic inflammatory skin dis- ease with a biphasic course consisting of an acute inflammatory Th2-dominated phase and a chronic phase, reminiscent of a delayed-type immune reaction, with the appearance of Th1-like immune responses.
AE is thus a rather unusual manifestation of IgE- mediated allergies because the immediate reaction caused by the cross-linking of effector cell (i.e., mast cells, basophils) -bound IgE that leads to the release of inflammatory mediators within a few minutes (e.g., histamine and leukotrienes) does not dominate the disease. Skin lesions in AE are characterized by the influx of T cells and thus resemble eczematous features [1 – 3]. It was demonstrated very early that AE patients exhibit much stronger lymphoproliferative responses in response to allergens than patients suffering from other manifestations of atopy (e.g., allergic rhinocon- junctivitis, asthma) [4]. The important contribution of T cells to the pathogenesis of AE has now been well established [1, 5, 6].
AE affects 10 % – 20 % of children and 1 % – 3 % of adults. The majority of AE cases belong to the extrinsic form, which is due to IgE recognition of a broad variety of allergens. Patients suffering from extrinsic forms of AE are typically sensitized against a broad variety of allergens and frequently exhibit elevated levels of total serum IgE antibodies directed against allergens [7 – 10]. However, also intrinsic forms of AE have been described that resemble the typical clinical criteria of AE but are not characterized by the production of IgE antibodies with specificity for defined allergens [7].
For most forms of respiratory allergy (e.g., rhino- conjunctivitis, allergic asthma) and gastrointestinal allergy, IgE-sensitization to certain allergens and dis-
ease symptoms are highly related. In AE, it is possible that patients mount IgE antibodies against a variety of environmental allergens but disease exacerbations often lack an obvious association between contact to allergens and clinical symptoms [7].
Several explanations for this phenomenon can be considered. First, it is possible that the disease-elicit- ing allergens are not known and hence the association cannot be demonstrated. In this context, it should be mentioned that a broad variety of unusual allergens have been discovered to be related to AE (e.g., bacte- ria, yeast) [1, 11, 12]. Second, it may be considered that different pathogenetic mechanisms are operative in AE vs other forms of allergy that are more difficult to reveal by diagnostic tests. For example, skin prick testing with allergens is useful for most forms of aller- gy, whereas atopy patch testing, a test principle simi- lar to epicutaneous testing used for delayed-type hypersensitivity, has been found suitable for AE diag- nosis [13].
Third, allergens and allergen-derived peptides may
reach the skin also via endogenous routes. In this con-
text, it has been shown that AE patients contain aller-
gen-specific T cells that home to the skin, and anti-
gen-presenting cells in AE may undergo extensive
trafficking [14, 15]. Thus even allergens and allergen-
derived peptides taken up via the gastrointestinal
tract may reach the skin via various mechanisms [7,
16 – 18]. This book chapter is dedicated to summariz-
ing recent findings pointing to the possibility that also
IgE recognition of autoantigens and other autoim-
mune phenomena may be involved in the pathogene-
sis of AE [19].
44.2
Similarities and Cross-Reactivities Between Environmental Allergens and Human Proteins:
The Concept of IgE Autoimmunity is Reborn
Already in the 1920s, several investigators reported that human skin dander could trigger immediate hypersen- sitivity reactions and it has been demonstrated by cuta- neous testing and RAST technology that atopic patients form IgE antibodies not only against environmental allergens, but also against human proteins [20 – 25].
However, with the discovery and characterization of several extremely potent environmental allergens, the concept that IgE autoreactivity could play a pathogenet- ic role in atopy fell into oblivion. It was reborn when the cDNA coding for a birch pollen allergen was isolated
Table 44.1. List of autoantigens in atopic eczema patients. The antigenic structures, their molecular weight (kDa), their function and references are displayed and grouped in target structures of IgE antibodies and IgG antibodies in atopic eczema (AE)
Environmental allergens with similarity to human proteins
MW (kDa)
Origin Function Refer-
ences
Profilin 12 – 17 All eukaryotic cells Actin-binding protein partici-
pating in the phosphoinositide pathway and a signal transduction
[26]
Albumin (dog, cat) 60 – 70 Mammalian serum proteins [29]
Calcium-binding allergens (plants, fish)
8 – 23 Binding and transport of calcium [30]
Cytochrome allergens [31]
MnSOD Manganese superoxide dismutase [34]
P2-protein
⎫
⎬
⎭
Most eukaryotic cells
Ribosomal phosphoprotein type 2 [33]
Target structures of IgE autoantibodies in AE
Hom s 1 (SART 1) 73.4 Mammalian cells: skin, lung, colon, liver
Recognized by cytotoxic T cells of cancer patients
[39, 40]
Hom s 2 ( [ -NAC) 23.2 Most eukaryotic cells: skeletal muscle, liver
Sequence-specific sorting and trans- location of intracellular proteins
[41 – 43, 71]
Hom s 3 (BCL7B) 20.1 Mammalian cells: gall bladder, skeletal muscle, placenta, liver, ocular ciliary body
Putative oncogene [41, 44]
Hom s 4 (CALC) 54 Eukaryotic cells: skin, brain, lung, breast, liver, heart
Calcium-binding protein [41, 45]
Hom s 5 (KER)
⎫
⎬
⎭
Atopy related autoantigens (ara)
42.6 Mammalian cells: epithelial tissues
Formation of intermediate filaments
[41; 46]
Target structures of IgG autoantibodies in AE
IgE antibodies ⎫
⎬ ⎭
Mammalian cells
⎫
⎬
⎭
Cell nucleus
Most euka- ryotic cells
[56 – 64]
Nuclear proteins [66, 67]
DFS-70 70 Transcription coactivator p75, lens
epithelium-derived growth factor (LEDGF)
[68]
p80-coilin
⎫
⎬
⎭ Nuclear proteins
80 RNA processing and cellular
trafficking [69, 70]
and the corresponding allergen was identified as profi- lin, an ubiquitous actin-binding protein found in dif- ferent pollen, fungi and even in humans [26 – 28].
Recombinant profilin from birch reacted with IgE anti- bodies from sensitized allergic patients, induced spe- cific basophil activation and profilin-sensitized indi- viduals were found who, due to cross-reactivity with birch profilin, mounted IgE autoantibodies to human profilin [26]. The molecular nature of several other environmental allergens with similarity and/or cross- reactivity to human proteins was revealed (Table 44.1).
These allergens included dog and cat albumin [29], cal-
cium-binding allergens from plants and fish (reviewed
in [30]) and the cytochrome family of plant allergens
[31], manganese superoxide dismutase (MnSOD) and a
ribosomal P2-protein (P2-protein) from Aspergillus
418 44 Autoantibodies in Atopic Eczema
S KIN G A S T R OINT E S T INA L
T R A C T
E YE S R E S P IR A T OR Y
T R A C T
R eleas e of A utoallergens
IgE
FcHRI mas t c ell
IFN-J A P C
E c zema, P ruritus T is s ue damage
T cell epitope MHC
TCR
FcHRII FcHRI
T c ells T
E xogenous A llergens
Inflammation
A utos ens itization, Inflammation fumigatus. The latter two showed cross-reactivity with
human proteins and induced immediate-type and T cell-mediated autoimmune reactions [32, 33]. Humoral and cell-mediated autoreactivity to the corresponding human proteins in vitro and in vivo were demonstrat- ed. For MnSOD, inhibition studies showed that patients’ IgE antibodies recognized common epitopes between the enzymes from humans and other species, including Drosophila enzyme. Rare exposure to MnSOD of the latter species suggested molecular mim- icry as the mechanism for cross-reactivity [34].
Studies on the three-dimensional structure of aller- gens revealed that despite low or lacking sequence identity between environmental allergens and human proteins (e.g., profilin, timothy grass pollen allergen, Phl p 2), environmental allergens can mimic the struc- ture of human proteins [35, 36].
The identification of highly cross-reactive allergens that even cross-reacted with endogenous antigens gave rise to the rediscovery of IgE autoreactivity, but the pathogenetic relevance of this finding remained unclear. Furthermore, IgE recognition of cross-reac- tive autoantigens was not associated with certain forms/manifestations of atopy, extent of disease severi- ty, or other clinically important findings.
44.3
The Discovery that the Occurrence of IgE Autoantibodies Is Frequently Associated with Atopic Eczema
In order to investigate whether IgE autoreactivity is associated with certain manifestations of atopy or oth- er immunologically mediated diseases, the approach of testing sera from various patient groups and healthy non-allergic individuals for IgE reactivity to nitrocellu- lose-blotted human proteins from various cell types was taken [37]. The latter study revealed several impor- tant aspects. First, it was found that IgE autoreactivity can be found in a high percentage of AE patients but not in healthy persons, in patients suffering from mild forms of allergy and not in patients suffering from oth- er immunologically-mediated disorders (Fig. 44.2). IgE autoreactivity was directed against a large variety of human proteins expressed in various cell types and no major autoantigens could be identified. The specificity of IgE autoreactivity was demonstrated by inhibition studies by testing for IgG autoreactivity, and IgE allore-
Fig. 44.1. Pathomechanisms of autoallergy in AE patients. In sensitized atopic individuals, contact with exogenous allergens (orange asterisks) activates effector cells (e.g., mast cells) for immediate-type reactions and Th2 cell activation, which then may lead to tissue damage and the release of autoantigens (blue asterisks). These autoallergens can autosensitize and then cross-link IgE antibodies on mast cells or be presented to T cells
activity was excluded [37]. Although no major IgE-
reactive autoantigens, which are recognized by the
majority of AE patients, could be identified, a logical
next step was to attempt to characterize some of the
IgE-reactive autoantigens in detail.
44.4
Identification of IgE-Reactive Autoantigens by Molecular Cloning
For the identification and characterization of IgE-reac- tive autoantigens, an approach was taken that had been applied for the characterization of environmental aller- gens before (reviewed in [38]). Expression cDNA libraries prepared from the mRNA of human tissues were screened with serum IgE from AE patients to search for cDNAs coding for IgE-reactive autoantigens.
Five IgE-reactive autoantigens have been described by screening of human cDNA libraries and were named according to the allergen nomenclature as Hom s (Homo sapiens) antigens (Table 44.1).
Hom s 1 was the most frequently detected autoanti- gen recognized by serum IgE from patients suffering from AE. The DNA sequence of Hom s 1 codes for a 72.3-kDa protein highly expressed in the skin and to a lesser extent in other target organs of atopy (e.g., lung, gastrointestinal tract) [39]. Hom s 1 shares almost complete sequence identity with SART1, an antigen recognized by cytotoxic T cells of patients suffering from squamous esophageal cancer [40]. Using a rabbit anti-serum against purified Hom s 1, this autoantigen was detected within the epidermis, especially in the cytoplasm of suprabasal keratinocytes and in fibro- blasts and endothelial cells within the dermis [39].
Hom s 2 showed sequence identity with the human [ -chain of the nascent polypeptide-associated complex (NAC), a protein required for signal sequence-specific sorting and translocation of intracellular proteins [41 – 43]. The cDNA sequence codes for a protein with 23.2-kDa and by sequence analysis was found to be expressed in histogenetically unrelated tissues (Table 44.1). Hom s 2-homologous proteins were found in mice, insects, plants (Arabidopsis thaliana), yeast, and protozoa [42]. Using the purified and recombinant E.
coli-expressed Hom s 2 in circular dichroism experi- ments, it was demonstrated that [ -NAC represented a folded protein with mixed [ - and q -sheet conforma- tion and exhibited remarkable stability as well as refol- ding capacity [42].
Hom s 3, a protein that is expressed in skeletal mus- cle, gall bladder, placenta, liver, and ocular ciliary body is a 20.1-kDa protein called BCL7B. It possibly is an oncogene which occurs in many different tissues of the human body [41, 44].
Another recently identified autoantigen recognized
by IgE antibodies of AE patients was termed Hom s 4. It belongs to a new subfamily of calcium-binding pro- teins, which like Hom s 2, was found to induce T cell autoreactivity [42]. The complete Hom s 4 cDNA codes for a 54-kDa basic protein containing two calcium- binding domains. Using Hom s 4-specific antibodies Aichberger et al. demonstrated that the protein is strongly expressed within epidermal keratinocytes and dermal endothelial cells [45]. Moreover, it was shown that the recombinant Hom s 4 exhibited IgE cross-reac- tivity with exogenous calcium-binding allergens from plants and fish.
Hom s 5 is human cytokeratin type II (Table 44.1), a component of the mammalian cytoskeleton taking part in the formation of intermediate filaments in epithelial tissues [41, 46].
For some cloned IgE-reactive autoantigens, e.g., Hom s 2 sequence similarities with exogenous antigens present in yeast, plants, animals, and bacteria were found [42], whereas for other autoallergens no proteins with similarity to exogenous allergens have been found yet.
The unusual finding that most of the characterized IgE-reactive autoantigens represented intracellular proteins was confirmed by biochemical studies dem- onstrating that these autoantigens can be detected in the nuclear > microsomal > mitochondrial > cyto- plasmic fraction of A431 cells as well as in a variety of human tissue specimens (brain, bone, intestine, liver, lung, muscle, skin, uterus) and effector cells of atopy (basophils, mast cells, T cells) [47]. The biochemical studies thus confirmed the broad expression of IgE- reactive autoantigens in a variety of cell types and tis- sues. Skin-specific expression thus does not seem to be the reason why these autoantigens are recognized by IgE antibodies from AE patients. The biochemical studies on IgE-reactive autoantigens point to another feature of IgE-reactive autoantigens: it appears that these autoantigens are “better” recognized by IgE when they are altered by denaturation or other modifi- cations, indicating that “slightly altered self ” could be the target for IgE autoantibodies.
As stated above, molecular and histochemical char- acterization of autoallergens as well as fractionation experiments showed that most of them represented intracellular proteins [41, 47]. The fact that autoaller- gens represent mainly intracellular proteins raises the question of how these autoallergens are released to induce inflammation in AE patients.
420 44 Autoantibodies in Atopic Eczema
44.5
How Intracellular Antigens Can Contribute to the Pathogenesis of Atopic Eczema
Obviously, intracellular antigens are released whenever cells die, either due to necrosis or apoptosis. In fact, it has been demonstrated that keratinocytes in AE lesions undergo apoptosis and it is therefore possible that intracellular proteins, including autoantigens, are released by this mechanism [48]. Keratinocyte damage may occur via immunological mechanisms in AE patients but may be simply caused by allergic inflam- mation induced by exogenous allergens, by scratching and superinfections (Fig. 44.1). That IgE-reactive auto- antigens are indeed released into the circulation and can be detected complexed to IgE antibodies in AE patients has been demonstrated [39, 41]. Using anti- BCL7B antibodies, IgE-specific BCL7B immune com- plexes isolated with Sepharose-coupled anti-human IgE antibodies were detected by immunoblotting [41].
Likewise, Hom s 1 IgE immune complexes were detect- ed [39]. Circulating IgE autoallergen immune com- plexes may reach target organs of atopy and, when bound via Fc 5 -receptors on effector and inducer cells of atopy and may induce degranulation, mediator release or activation of T cells. For exogenous allergens, it has in fact been demonstrated that presentation via the low-affinity receptor (Fc 5 RII/CD23) as well as via the high-affinity receptor (Fc 5 RI) leads to enhanced T cell activation [49, 50]. The clinical relevance of the lat- ter findings has, however, not yet been established and it is unclear whether this mechanism is valid also for autoantigens. The description of IgE-dependent hista- mine-releasing factors in sera of atopic individuals [51, 52] has prompted the investigation of the possibility that these factors may be IgE–autoallergen immune complexes that can activate mast cells or basophils.
Sera from patients containing such histamine-releas- ing factors were described as IgE
+sera by their ability to induce basophil degranulation without the addition of allergens [51, 52]. However, a recent study conduct- ed to establish a possible link between IgE autoreactivi- ty and IgE-dependent histamine release factors has indicated that the two phenomena are rather distinct from each other [53].
44.6
IgE Autoreactivity as a Possible Marker for Chronic Inflammation and Tissue Damage in Atopic Eczema
While the pathogenetic relevance of IgE autoreactivity has not been proven, it has been demonstrated that the intensity of IgE autoreactivity is associated with dis- ease severity and exacerbation [41]. In another study, it was found that IgE autoreactivity to nitrocellulose blotted human proteins increased when skin symp- toms occurred in response to contact with environ- mental allergens [54]. Furthermore, it was shown that IgE antibody reactivities to autoallergens decreased treatment of skin manifestations with cyclosporin A [55].
The latter studies indicate that IgE autoreactivity is a useful parameter to monitor chronic inflammation and tissue damage in AE patients.
44.7
IgG Autoantibodies in Atopic Eczema Patients
While IgE autoreactivity is strongly associated with AE and disease severity of AE, the importance of IgG auto- reactivity in AE is less clear. Some investigations showed the presence of anti-IgE antibodies in the sera of AE patients [56 – 58] but also in normal individuals [59, 60]. Because of the difficulty of purifying anti-IgE antibodies, which seem to exist in the form of immune complexes with IgE in serum only in very small quanti- ties, controversial results were obtained and the ques- tion of biological relevance still remains unproven [61 – 64]. Other IgG autoantibodies comprising antinu- clear antibodies (ANAs), well known in systemic rheu- matic diseases (e.g., SLE, Sjögren syndrome, scleroder- ma) [65] were recently reported to be present in some patients with AE [66, 67]. However, no association between positive ANA levels and a high amount of total IgE, disease duration, or the presence of respiratory atopy could be found.
Other authors described a typical, dense, fine-
speckled ANA pattern on Hep-2 cells of AE patients
who also recognized a 70-kDa protein on immuno-
blots. The antigen thus was termed dense fine-speckled
70 (DSF 70) [68]. The authors reported that 9 % – 30 %
of AE patients from different populations had anti-
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 N 220
97 66 45 30
20 14
kDa
