Serum IgE Autoantibodies Target Keratinocytes in Patients with Atopic Dermatitis

Previous studies have shown that sera of patients with severe atopic dermatitis (AD) contain IgE specific for self-proteins, supporting the hypothesis of autoreactivity as a pathogenic factor in AD. In this study, we screened a large panel of AD patients (n=192) by western blotting (WB) for IgE reactivity not only against the human epithelial cell line A431 but also against primary keratinocytes (KCs). To investigate autoantigenic cell structures in detail, normal human skin and primary KCs were incubated with sera from both WB-reactive patients and, for control purposes, healthy individuals, and analyzed by immunohistology, confocal laser microscopy, and flow cytometry. Our analysis revealed that 28% of AD patients, but not healthy individuals, display serum IgE autoreactivity by WB analysis. The individual IgE reaction patterns of the sera pointed to the existence of unique as well as common specificities against epidermal or A431-derived proteins. Immunostainings identified cytoplasmic and, occasionally, also cell membrane-associated moieties as targets for autoreactive IgE antibodies. Interestingly, in certain autoreactive patients, the surface-staining pattern was accentuated at cellular contact sites. We conclude that IgE autoreactivity is common, particularly among severe AD patients, and that non-transformed primary cells are needed for characterization of the entire spectrum of IgE-defined autoantigens. Previous studies have shown that sera of patients with severe atopic dermatitis (AD) contain IgE specific for self-proteins, supporting the hypothesis of autoreactivity as a pathogenic factor in AD. In this study, we screened a large panel of AD patients (n=192) by western blotting (WB) for IgE reactivity not only against the human epithelial cell line A431 but also against primary keratinocytes (KCs). To investigate autoantigenic cell structures in detail, normal human skin and primary KCs were incubated with sera from both WB-reactive patients and, for control purposes, healthy individuals, and analyzed by immunohistology, confocal laser microscopy, and flow cytometry. Our analysis revealed that 28% of AD patients, but not healthy individuals, display serum IgE autoreactivity by WB analysis. The individual IgE reaction patterns of the sera pointed to the existence of unique as well as common specificities against epidermal or A431-derived proteins. Immunostainings identified cytoplasmic and, occasionally, also cell membrane-associated moieties as targets for autoreactive IgE antibodies. Interestingly, in certain autoreactive patients, the surface-staining pattern was accentuated at cellular contact sites. We conclude that IgE autoreactivity is common, particularly among severe AD patients, and that non-transformed primary cells are needed for characterization of the entire spectrum of IgE-defined autoantigens. epithelial tumor cell line antibody atopic dermatitis eczema area and severity index horseradish peroxidase Investigators' Global Assessment Score immunohistology keratinocyte western blotting Atopic dermatitis (AD) is a chronic relapsing inflammatory skin disease occurring in 5–20% of children and 1–3% of adults. Allergen studies indicate that approximately 80% of AD patients have elevated levels of total and exoallergen-specific IgE, which is directly associated with the severity of disease (Tan et al., 1996Tan B.B. Weald D. Strickland I. Friedmann P.S. Double-blind controlled trial of effect of housedust-mite allergen avoidance on atopic dermatitis.Lancet. 1996; 347: 15-18Abstract Full Text PDF PubMed Scopus (390) Google Scholar). Moreover, there exists a small proportion of patients with the typical signs of AD in which sensitization to a broad panel of exoallergens is not detectable by in vitro or in vivo tests (Schmid-Grendelmeier et al., 2001Schmid-Grendelmeier P. Simon D. Simon H.U. Akdis C.A. Wuthrich B. Epidemiology, clinical features, and immunology of the “intrinsic” (non-IgE-mediated) type of atopic dermatitis (constitutional dermatitis).Allergy. 2001; 56: 841-849Crossref PubMed Scopus (240) Google Scholar). This finding supports the involvement of either a non-immunological or an autoreactive pathomechanism in the perpetuation of the clinical manifestations. In fact, in the past decade autoreactive IgE antibodies against self-proteins have been identified in AD (Valenta et al., 1996Valenta R. Maurer D. Steiner R. Seiberler S. Sperr W.R. Valent P. et al.Immunoglobulin E response to human proteins in atopic patients.J Invest Dermatol. 1996; 107: 203-208Crossref PubMed Scopus (119) Google Scholar; Natter et al., 1998Natter S. Seiberler S. Hufnagl P. Binder B.R. Hirschl A.M. Ring J. et al.Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.FASEB J. 1998; 12: 1559-1569Crossref PubMed Scopus (110) Google Scholar; Fluckiger et al., 2002Fluckiger S. Scapozza L. Mayer C. Blaser K. Folkers G. Crameri R. Immunological and structural analysis of IgE-mediated cross-reactivity between manganese superoxide dismutases.Int Arch Allergy Immunol. 2002; 128: 292-303Crossref PubMed Scopus (48) Google Scholar). Studies concerning endogenous antigens as triggers in AD have shown that sera from atopic patients, particularly those from patients with severe AD, contain IgE antibodies against human proteins expressed in histogenetically different cell types, most importantly in epithelial cells (Valenta et al., 1996Valenta R. Maurer D. Steiner R. Seiberler S. Sperr W.R. Valent P. et al.Immunoglobulin E response to human proteins in atopic patients.J Invest Dermatol. 1996; 107: 203-208Crossref PubMed Scopus (119) Google Scholar; Natter et al., 1998Natter S. Seiberler S. Hufnagl P. Binder B.R. Hirschl A.M. Ring J. et al.Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.FASEB J. 1998; 12: 1559-1569Crossref PubMed Scopus (110) Google Scholar; Seiberler et al., 1999aSeiberler S. Bugajska-Schretter A. Hufnagl P. Binder B.R. Stockl J. Spitzauer S. et al.Characterization of IgE-reactive autoantigens in atopic dermatitis. 1. Subcellular distribution and tissue-specific expression.Int Arch Allergy Immunol. 1999; 120: 108-116Crossref PubMed Scopus (24) Google Scholar, Seiberler et al., 1999bSeiberler S. Natter S. Hufnagl P. Binder B.R. Valenta R. Characterization of IgE-reactive autoantigens in atopic dermatitis. 2. A pilot study on IgE versus IgG subclass response and seasonal variation of IgE autoreactivity.Int Arch Allergy Immunol. 1999; 120: 117-125Crossref PubMed Scopus (34) Google Scholar). Some of the IgE-defined epithelial autoantigens (for example, Hom s 1–5) have been identified and characterized at the molecular level (Mossabeb et al., 2002Mossabeb R. Seiberler S. Mittermann I. Reininger R. Spitzauer S. Natter S. et al.Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.J Invest Dermatol. 2002; 119: 820-829Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). These autoantigens are intracellular, mostly cytoplasmic proteins (Natter et al., 1998Natter S. Seiberler S. Hufnagl P. Binder B.R. Hirschl A.M. Ring J. et al.Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.FASEB J. 1998; 12: 1559-1569Crossref PubMed Scopus (110) Google Scholar), suggesting that their release from damaged tissue would trigger responses mediated by IgE or T cells (Valenta et al., 1998Valenta R. Natter S. Seiberler S. Wichlas S. Maurer D. Hess M. et al.Molecular characterization of an autoallergen, Hom s 1, identified by serum IgE from atopic dermatitis patients.J Invest Dermatol. 1998; 111: 1178-1183Crossref PubMed Scopus (131) Google Scholar) in AD patients. Recombinant human autoallergens characterized so far result from immunoscreenings of an epithelial carcinoma cell line (A431) (Seiberler et al., 1999aSeiberler S. Bugajska-Schretter A. Hufnagl P. Binder B.R. Stockl J. Spitzauer S. et al.Characterization of IgE-reactive autoantigens in atopic dermatitis. 1. Subcellular distribution and tissue-specific expression.Int Arch Allergy Immunol. 1999; 120: 108-116Crossref PubMed Scopus (24) Google Scholar, Seiberler et al., 1999bSeiberler S. Natter S. Hufnagl P. Binder B.R. Valenta R. Characterization of IgE-reactive autoantigens in atopic dermatitis. 2. A pilot study on IgE versus IgG subclass response and seasonal variation of IgE autoreactivity.Int Arch Allergy Immunol. 1999; 120: 117-125Crossref PubMed Scopus (34) Google Scholar), which may not perfectly reflect the physiological repertoire of proteins expressed in the epidermis. Skin-derived autoantigens potentially relevant for the disease could thus have been overlooked. Therefore, we wanted to analyze the range of IgE autoreactivities against primary epidermal cell-derived proteins and to compare them with those against A431-derived proteins. Furthermore, we wished to discover against which cell type, subcellular structure, or non-cellular formations of the skin the IgE-autoantibodies were directed. By doing so, we sought to build a basis for future investigations of their molecular nature. In a first set of experiments, we analyzed the prevalence of epithelial cell-reactive serum IgE autoantibodies in a large study population of AD patients. A total of 192 patient sera were screened by western blotting (WB) with protein extracts derived from the epithelial cell line A431 and with epidermal extracts from the skin of one healthy donor. A serum was considered “autoreactive” if the IgE contained in it recognized at least one band in one of the protein extracts used. Healthy individuals completely lacked serum IgE reactivity against the above-mentioned protein extracts, whereas approximately one-fourth (28%, n=54) of all AD patients exhibited IgE reactivity against both epithelial extracts (Figure 1). IgE autoreactivity against proteins derived from normal dermis was absent in all tested sera (data not shown), probably due to the cellular paucity of this tissue. IgE autoreactivity against primary epidermal extracts was irrespective of the healthy skin donor, indicating ubiquitous occurrence of autoantigens in healthy human skin. (Figure S1). Download .jpg (.03 MB) Help with files Figure S1WB with serum of one autoreactive AD patient and 5 different epidermal protein extracts: Protein extracts of 5 different ex-vivo human epidermal preparations (lane 1-5), one extract of cultured primary human fore-skin KC (lane 6), one extract of the cell line A431 (lane 8) and buffer control (lane 7) were separated by SDS-PAGE-electrophoresis, blotted onto nitrocellulose-membranes, cut into strips, blocked and incubated with the autoreactive serum of AD patient V (non surface-reactive) or with healthy control serum I; serum IgE-reactivity was detected using HRP-conjugated mouse anti-human IgE mAb. A representative example out of three autoreactive sera is shown. Each autoreactive serum recognized identical numbers and molecular weights in the different protein extracts. Next, we set out to identify common clinical features of autoreactive AD patients. Statistical analysis of the clinical data revealed that autoreactive AD patients exhibit significantly (P<0.001) higher total serum IgE levels when compared with non-autoreactive AD patients. Despite this significant correlation between IgE autoreactivity and high total serum IgE levels (>1,000 IU ml−1), there also existed several AD patients with elevated serum IgE levels (<1,000 IU ml−1) who did not show autoreactivity against epithelial proteins. These patients provide an internal control and indicate that IgE autoreactivity does not occur as a non-specific phenomenon due to high IgE levels. Disease activity, as determined by the Investigators' Global Assessment (IGA) Score and the Eczema Area and Severity Index (EASI), was also significantly (P<0.001) higher in autoreactive patients than in non-autoreactive ones (Figure 2). These data are in agreement with earlier observations showing that AD patients with serum IgE autoreactivity form a distinct subpopulation of patients with severe disease and considerably higher total serum IgE (Natter et al., 1998Natter S. Seiberler S. Hufnagl P. Binder B.R. Hirschl A.M. Ring J. et al.Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.FASEB J. 1998; 12: 1559-1569Crossref PubMed Scopus (110) Google Scholar). In contrast to this observation, we were not able to find a significant correlation within the group of autoreactive AD patients between the intensity of the WB reaction or a special recognized moiety on the one hand and the EASI and IGA on the other (data not shown), indicating that IgE autoreactivity is an important but not the only relevant factor in this complex skin disease. Interestingly, all of the IgE-autoreactive patients met the criteria of the extrinsic-type AD (that is, increased serum IgE and/or sensitization to environmental allergens; Wollenberg et al., 2000Wollenberg A. Kraft S. Oppel T. Bieber T. Atopic dermatitis: pathogenetic mechanisms.Clin Exp Dermatol. 2000; 25: 530-534Crossref PubMed Scopus (66) Google Scholar), whereas in the group of the non-autoreactive AD patients 17% had a characteristic intrinsic-type AD (Schmid-Grendelmeier et al., 2001Schmid-Grendelmeier P. Simon D. Simon H.U. Akdis C.A. Wuthrich B. Epidemiology, clinical features, and immunology of the “intrinsic” (non-IgE-mediated) type of atopic dermatitis (constitutional dermatitis).Allergy. 2001; 56: 841-849Crossref PubMed Scopus (240) Google Scholar; Novak and Bieber, 2003Novak N. Bieber T. Allergic and nonallergic forms of atopic diseases.J Allergy Clin Immunol. 2003; 112: 252-262Abstract Full Text Full Text PDF PubMed Scopus (339) Google Scholar). To investigate which protein extract is best suited for the detection of autoreactivity in AD patients, we compared reactivity against A431 with that against proteins extracted from freshly isolated keratinocytes (KCs) by WB. Of the 54 patients with IgE autoantibodies, 45 (=83%) recognized primary epidermis-derived proteins and 42 (=78%) recognized A431-derived proteins. Although most AD sera (61%) reacted with antigens in both the extracts (Table 1), 12 AD patients (=22%) reacted exclusively with extracts from epidermal tissue and nine (=17%) exclusively with A431 extracts. These data indicate that potentially autoreactive patients could have been overlooked if the screening was solely performed with protein extracts derived from the cell line A431 and that a maximum detection can be achieved by a combination of both protein extracts. There was no difference in the clinical scores and the IgE levels within the autoreactive AD patients detected by the two distinct antigenic sources (Table 1).Table 1Statistical analysis of the autoreactive AD patientsAutoreactivity against% of autoreactive AD patients (n=54)Number of tested persons (n)Intrinsic type of AD (n)Mean serum IgE (IU ml−1) (SD)Mean IGA (SD)Mean EASI (SD)A431 and epidermis613301,788* (±463.6)3.4* (±1.2)22.2* (±17.2)A431 alone17901,551* (±775.2)3.5* (±1.4)25.9* (±16.3)Epidermis alone221201,265* (±710.2)3.6* (±1.0)28.9* (±9.5)Non-autoreactive013823681 (±835.2)2.4 (±1.2)10.0 (±10.6)AD, atopic dermatitis; EASI, eczema area and severity index; IGA, Investigators' Global Assessment score.*No statistically significant differences between the three subgroups of AD patients. Open table in a new tab AD, atopic dermatitis; EASI, eczema area and severity index; IGA, Investigators' Global Assessment score. *No statistically significant differences between the three subgroups of AD patients. A detailed analysis of the band pattern in the western blots revealed that the IgE-autoreactive moieties in epidermal extracts differed in their molecular weights from those identified in A431 extracts by the very same sera. When analyzing interindividual serum reactivity against cell line A431, we could not detect prevalently recognized moieties among autoreactive AD patients. In contrast, when using primary epidermal extracts as an antigenic source, we were able to identify a common band of approximately 30 kDa in 60% of all autoreactive AD patients (Figure 1). Next, we sought to determine which cells of the skin express IgE-defined autoantigens. For this purpose, frozen sections of normal human skin were incubated with the sera of 15 autoreactive AD patients or five healthy controls. Serum IgE that bound to the sections was detected with an anti-IgE antibody. We found that six of 15 patients, but none of the healthy individuals, showed detectable IgE reactivity against normal human epidermis. Figure 3 shows representative examples of such reaction patterns. Similar to the results obtained by WB, we detected an interindividually heterogeneous IgE-binding pattern. For example, serum IgE of AD patient I showed predominantly nuclear labeling, whereas that from patient II was preferentially bound to membrane-associated structures. In total, two of the 15 AD patients had clearly detectable serum IgE reactivity against the cell periphery of KCs. Next, primary undifferentiated KCs were incubated with sera of the six AD patients, which had shown epidermal IgE binding by immunohistology (IH), and sera of two healthy controls. Using immunofluorescence microscopy, we were able to confirm the results derived from IH and showed that serum IgE binding is most pronounced at sites of cellular contact in the two patients with IgE reactivity against membrane structures (Figure 3). This indicates that junctional structures may be targets for autoreactive IgE of certain patients. Anti-IgE immunostainings of KCs incubated with sera from healthy persons gave no detectable signal. To determine whether the serum IgE of our patients exhibiting peripheral staining by immunofluorescence binds to extracellular or intracellular membrane structures of KCs, we performed FACS analysis with viable KCs that were non-enzymatically detached and probed with autoreactive AD serum. Surface-bound IgE was detected with mouse anti-human IgE mAb and Alexa488-labeled goat anti-mouse IgG antibody (Ab). Using this method, we found serum IgE binding to detached viable primary KCs in three of 15 autoreactive AD patients (Figure S2). This binding of the autoreactive IgE in the three AD sera was almost completely prevented when the KCs were trypsin-treated before incubation with patient sera (data not shown), demonstrating that freely accessible surface proteins are the targets of autoreactive serum IgE. Surface IgE binding was limited to epithelial cells, with a significant preferential binding to primary KCs, and could not be detected on other tumor or primary cell types (Table S1), suggesting the recognition of KC-specific autoantigens. Download .jpg (.04 MB) Help with files Figure S23 out of 15 AD patients revealed serum IgE binding to the surface of vital KC: KC were non-enzymatically detached (Cell Dissociation Buffer™ enzyme-free, Invitrogen), washed, blocked in 1% PBS/BSA + 1% goat serum and incubated on ice for 1h with patient serum (1:5). Surface-bound IgE was detected with the mouse anti-hu-IgE mAb B3102E8 (Southern Biotech) and a goat anti-mouse-IgG-A488 Ab (Invitrogen). To gate out the dead cells, PI (Invitrogen, final conc.: 1μM) was added. Samples were analyzed using a FACS-Scan™ flow cytometer (Becton Dickinson).Only viable, PI negative cells are shown. Positive surface stainings are depicted in colors: AD patient II is shown in red, AD patient III in green and AD patient IV in yellow. Download .doc (.12 MB) Help with doc files Table S1epithelial cells In a next step, we also performed immunostainings with autoreactive sera on primary KCs cultured on slides (Figure 4). By this method, we detected membrane labeling of the viable cells in five of 15 autoreactive AD patients. Compared with FACS analysis, two additional, but rather weak, KC surface–reactive AD patients were identified by immunofluorescence, possibly due to the higher sensitivity of the latter method. Although serum IgE reactivity against human proteins had been reported to occur in AD sera some 10 years ago (Valenta et al., 1996Valenta R. Maurer D. Steiner R. Seiberler S. Sperr W.R. Valent P. et al.Immunoglobulin E response to human proteins in atopic patients.J Invest Dermatol. 1996; 107: 203-208Crossref PubMed Scopus (119) Google Scholar), our knowledge about the significance of this phenomenon is still incomplete. The recombinant human autoallergens characterized so far (Hom s 1–5) result from immunoscreenings of the epithelial carcinoma cell line A431 with sera of autoreactive AD patients (Natter et al., 1998Natter S. Seiberler S. Hufnagl P. Binder B.R. Hirschl A.M. Ring J. et al.Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.FASEB J. 1998; 12: 1559-1569Crossref PubMed Scopus (110) Google Scholar). Results obtained previously show that IgE reactivity of AD sera against these structures is a rather infrequent event (Natter et al., 1998Natter S. Seiberler S. Hufnagl P. Binder B.R. Hirschl A.M. Ring J. et al.Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.FASEB J. 1998; 12: 1559-1569Crossref PubMed Scopus (110) Google Scholar; Valenta et al., 1998Valenta R. Natter S. Seiberler S. Wichlas S. Maurer D. Hess M. et al.Molecular characterization of an autoallergen, Hom s 1, identified by serum IgE from atopic dermatitis patients.J Invest Dermatol. 1998; 111: 1178-1183Crossref PubMed Scopus (131) Google Scholar; Mossabeb et al., 2002Mossabeb R. Seiberler S. Mittermann I. Reininger R. Spitzauer S. Natter S. et al.Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.J Invest Dermatol. 2002; 119: 820-829Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar; Aichberger et al., 2005Aichberger K.J. Mittermann I. Reininger R. Seiberler S. Swoboda I. Spitzauer S. et al.Hom s 4, an IgE-reactive autoantigen belonging to a new subfamily of calcium-binding proteins, can induce Th cell type 1-mediated autoreactivity.J Immunol. 2005; 175: 1286-1294Crossref PubMed Scopus (65) Google Scholar). As the phenomenon of serum IgE autoreactivity occurs in approximately a quarter of our study population, Hom s 1–5 are likely to represent only a small fraction of the target antigens recognized by autoreactive IgE, suggesting that important autoreactive targets still await identification. To address this issue, we decided to use primary epidermal cell extracts as a screening tool for IgE autoreactivity and to compare the results with the previously used extracts derived from A431. Our screening revealed that a considerable portion of AD patients (28%) exhibit serum IgE autoreactivity against epidermal extracts and extracts from the epithelial cell line A431. This autoreactivity in AD patients was significantly correlated with the severity of the disease, defined by the amount of total serum IgE and the clinical scores on the EASI and IGA. This relationship suggests that IgE autoantibodies are the result of repetitive and severe injury of the skin. Thus, it is conceivable that environmental exoallergens initiate the acute phase of the allergic cutaneous disease (Valenta et al., 2000Valenta R. Seiberler S. Natter S. Mahler V. Mossabeb R. Ring J. et al.Autoallergy: a pathogenetic factor in atopic dermatitis?.J Allergy Clin Immunol. 2000; 105: 432-437Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) and thereby destruction of KCs and release of autoantigens into the circulation. IgE autoimmunity may consequently contribute to chronicity of the disease even in the absence of environmental allergens. This hypothesis is supported by longitudinal studies showing that IgE responses to autoallergens occasionally increase during periods of disease exacerbation, and by the discovery that some environmental allergens share structural and functional similarities with human proteins (Crameri et al., 1996Crameri R. Faith A. Hemmann S. Jaussi R. Ismail C. Menz G. et al.Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus.J Exp Med. 1996; 184: 265-270Crossref PubMed Scopus (172) Google Scholar; Meyer et al., 1999Meyer C. Appenzeller U. Seelbach H. Achatz G. Oberkofler H. Breitenbach M. et al.Humoral and cell-mediated autoimmune reactions to human acidic ribosomal P2 protein in individuals sensitized to Aspergillus fumigatus.J Exp Med. 1999; 189: 1507-1512Crossref PubMed Scopus (98) Google Scholar; Kinaciyan et al., 2002Kinaciyan T. Natter S. Kraft D. Stingl G. Valenta R. IgE autoantibodies monitored in a patient with atopic dermatitis under cyclosporin A treatment reflect tissue damage.J Allergy Clin Immunol. 2002; 109: 717-719Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Interestingly, all the autoreactive AD patients were of the extrinsic type (total serum IgE >100 IU ml−1 and/or sensitization against common allergens). Three autoreactive patients displayed elevated total serum IgE but no sensitization against common allergens. These patients may be sensitized to exoallergens not tested by us, or, alternatively, the elevated total serum IgE may result from a “pure” reactivity against autoallergens. The latter explanation is in accordance with a report of two intrinsic-type AD patients with IgE autoimmunity against an exoallergen-crossreactive human protein (Schmid-Grendelmeier et al., 2005Schmid-Grendelmeier P. Fluckiger S. Disch R. Trautmann A. Wuthrich B. Blaser K. et al.IgE-mediated and T cell-mediated autoimmunity against manganese superoxide dismutase in atopic dermatitis.J Allergy Clin Immunol. 2005; 115: 1068-1075Abstract Full Text Full Text PDF PubMed Scopus (184) Google Scholar). An explanation for the significant correlation of autoreactivity and severity of the disease is that elevated autoreactive serum IgE levels themselves account for the worsening of disease. IgE immune complexes could target effector cells like mast cells and basophils (Valenta et al., 1996Valenta R. Maurer D. Steiner R. Seiberler S. Sperr W.R. Valent P. et al.Immunoglobulin E response to human proteins in atopic patients.J Invest Dermatol. 1996; 107: 203-208Crossref PubMed Scopus (119) Google Scholar; Natter et al., 1998Natter S. Seiberler S. Hufnagl P. Binder B.R. Hirschl A.M. Ring J. et al.Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.FASEB J. 1998; 12: 1559-1569Crossref PubMed Scopus (110) Google Scholar), initiating the release of preformed mediators (that is, histamine, proteoglycans, tryptase, chymase), the production of cytokines (that is, IL-4, IL-5, IL-13), and, consequently, an allergic tissue response (Irani et al., 1989Irani A.M. Sampson H.A. Schwartz L.B. Mast cells in atopic dermatitis.Allergy. 1989; 44: 31-34Crossref PubMed Scopus (62) Google Scholar). Support for this idea comes from the finding that purified recombinant IgE autoantigens caused histamine release from basophils and induced immediate type-I skin reactions (Valenta et al., 1991Valenta R. Duchene M. Pettenburger K. Sillaber C. Valent P. Bettelheim P. et al.Identification of profilin as a novel pollen allergen; IgE autoreactivity in sensitized individuals.Science. 1991; 253: 557-560Crossref PubMed Scopus (597) Google Scholar, Valenta et al., 1996Valenta R. Maurer D. Steiner R. Seiberler S. Sperr W.R. Valent P. et al.Immunoglobulin E response to human proteins in atopic patients.J Invest Dermatol. 1996; 107: 203-208Crossref PubMed Scopus (119) Google Scholar; Crameri et al., 1996Crameri R. Faith A. Hemmann S. Jaussi R. Ismail C. Menz G. et al.Humoral and cell-mediated autoimmunity in allergy to Aspergillus fumigatus.J Exp Med. 1996; 184: 265-270Crossref PubMed Scopus (172) Google Scholar; Natter et al., 1998Natter S. Seiberler S. Hufnagl P. Binder B.R. Hirschl A.M. Ring J. et al.Isolation of cDNA clones coding for IgE autoantigens with serum IgE from atopic dermatitis patients.FASEB J. 1998; 12: 1559-1569Crossref PubMed Scopus (110) Google Scholar; Appenzeller et al., 1999Appenzeller U. Meyer C. Menz G. Blaser K. Crameri R. IgE-mediated reactions to autoantigens in allergic diseases.Int Arch Allergy Immunol. 1999; 118: 193-196Crossref PubMed Scopus (56) Google Scholar). When we compared the moieties recognized in epidermal extracts and extracts derived from A431, we found incoherent band patterns in most autoreactive patients, indicating the presence of distinct autoantigens in the two protein extracts. Based on these results, we propose that extracts of freshly isolated skin cells reflect the in vivo antigen situation better than the A431 tumor cell extracts, and are thus indispensable for screening for autoreactive AD patients. In addition, a substantial proportion of autoreactive AD patients showed serum IgE reactivity selectively against proteins derived from epidermal and not A431 extracts, suggesting that autoreactive AD patients might have been overlooked when the screening was performed solely with cell lin