Autoimmune phenomena were observed in the development of both cellular and humoral responses. In fact, there may be significant overlap between the autoreactive and protective antibodies since polyreactive antibodies represent a substantial part of the normal repertoire. Antibodies against specific self-antigens are typically associated with systemic or organ-specific autoimmune diseases, but may be also found in neoplastic diseases and even in healthy subjects. EBA is a prototypical organ-specific autoimmune disease affecting the skin and the mucous membranes associated with autoantibodies against collagen VII [1
]. The blister-inducing potential of collagen VII-specific autoantibodies has been shown in different ex vivo
and animal models [13
]. Autoimmunity against collagen VII has been described in IBD, which may be clinically associated with EBA [2
]. Interestingly, collagen VII-specific autoantibodies are present in IBD patients, which do not show blistering skin disease [2
]. The aim of the present study, was to analyze collagen VII-specific autoantibodies, including IgG subclasses in large groups of healthy blood donors or patients with autoimmune blistering diseases.
The rapid and accurate routine diagnosis of most autoimmune diseases relies on the detection of autoantibodies with known molecular specificity. Several immunoassays have been developed so far for the detection of autoantibodies against collagen VII (Table ) [22
]. To further improve the detection of collagen VII-specific autoantibodies, in the present study we have generated a chimeric antigen substrate containing virtually all autoepitopes, which have been reported in previous epitope mapping studies in patients.
Sensitivity and specificity of ELISA systems for the detection of autoantibodies against collagen VII
Wet lab epitope mapping studies and our present in silico
analysis have shown that the epitopes targeted by autoantibodies are localized within the NC1, NC2 and most probably the hinge region of the antigen [19
]. Therefore, for measuring autoantibodies against collagen VII we have generated a chimeric protein containing all putative epitope-bearing regions of collagen VII, including its NC1 and NC2 domains and the hinge fragment. The chimeric protein, which was produced in a human cell line to ensure optimal posttranslational modifications, shows an increased yield and is more stable compared with the full-length collagen while containing all the epitopes/regions in one copy per molecule. Thus, its use as a substrate does not require preadsorption against bacterial proteins and ensures strict equimolar concentration of the NC1, NC2 and hinge regions.
Interestingly, our in silico analysis predicted the existence of both linear and conformational epitopes. Since immunoblotting is not quantitative and uses denatured antigen, to measure autoantibodies against both linear and conformational epitopes on collagen VII, we have used ELISA.
Possible isolated reactivity against hinge was reported so far only in 3 children with EBA [21
] and was apparently present in our relatively large cohort of adult patients only in one patient. This resulted in a slightly lower specificity of the ELISA using the fused NC1 and NC2 domains when compared with the form containing NC1, hinge and NC2 in the present study. The percentage of EBA patients with autoantibodies targeting only epitopes outside its NC1 and NC2 domains is unknown. However, since EBA was generally previously diagnosed by reactivity with the NC1 domain of collagen VII, patients showing only reactivity against the hinge region may have been largely excluded from our study. Therefore, the immunossay described here allows characterizing the prevalence of autoantibodies against the hinge region of collagen VII and should increase the yield of EBA patients, which will test positive by ELISA. In addition, as suggested by previous studies [21
], the reactivity against the hinge region of collagen VII may be associated with inflammatory rather than mechanobullous blistering disease. The new recombinant proteins generated in this study will help clarifying this aspect in larger cohorts of patients with disease associated with autoimmunity against collagen VII.
Low levels of collagen VII-specific autoantibodies were detected in a number of patients with other autoimmune blistering diseases and healthy subjects. In patients with autoimmune and inflammatory diseases the presence of autoantibodies against collagen VII may be the result of an epitope spreading process. Our findings are in line with the observation that healthy blood donors and patients without skin blisters show pemphigoid autoantibodies [36
]. The pathogenic significance of collagen VII-specific autoantibodies in other autoimmune blistering diseases and in healthy subjects is still unclear. As shown in our present study, these autoantibodies do not show binding to the dermal-epidermal junction by indirect IF microscopy suggesting that they do not bind in vivo
. In addition, our previous in vivo
studies documented that the mere presence of tissue-bound autoantibodies in experimental EBA does not result in skin disease [11
Autoantibodies with different molecular specificity are present in healthy individuals and in patients with various diseases (e.g., ANAs prevalence is about 3-15%). We have found collagen VII-specific autoantibodies in 4% and 18% of patients with CD and UC, respectively. Our present results in CD patients are in line with our previous study showing collagen VII-specific autoantibodies by immunoblotting in 5.8% of CD and 5.8% of UC patients, in contrast to over 60% of CD patients initially reported [23
]. Interestingly, we measured collagen VII-specific autoantibodies in a higher percentage of UC patients compared with 5.8% and 12.9%, that were reported in the previous studies [39
]. The reason for this discrepancy is not known and future studies in larger number of IBD patients should help defining the prevalence of collagen VII-specific autoantibodies in these patients. There is apparently no correlation of collagen VII-specific autoantibodies with inflammation markers in patients with CD and UC. While several hypotheses have been advanced, the induction of autoimmune response against collagen VII and the pathogenic significance of specific autoantibodies in inflammatory bowel disease is still elusive [2
As with other autoantibody-induced diseases, ELISA levels of collagen VII-specific autoantibodies likely correlate with the disease severity [13
]. It is therefore expected that measuring the autoantibody levels, will help predicting short-term clinical evolution in EBA patients and guide therapeutic decisions. A more general prognostic value of the levels of EBA autoantibodies for the long-term disease course, including the resistance to treatment, has not yet been addressed. Addressing this question, which requires a more wider approach in prospective clinical study, should be addressed in the future using the tools generated this study.
Our present results strongly suggest the existence of non-pathogenic autoimmunity against collagen VII in patients and healthy individuals. Why autoantibodies specific to collagen VII and XVII do not induce tissue damage in all individuals and conditions is not known. Inadvertent autoimmune responses may be uncoupled from disease by various mechanisms, including cryptic B cell autoepitopes, typically seen in Goodpasture syndrome [39
], anatomic, cellular and molecular barriers that avert either tissue deposition of immune complexes [42
] or the engagement of inflammatory effectors by tissue-bound antibodies [44
]. In this context, the IgG subclass is a major determinant of autoantibody pathogenicity. Similar to a previously published report, our IgG subclass analysis revealed a dominant IgG4 response in patients with collagen VII-specific autoantibodies [45
]. However, while the other subclasses of autoantibodies were less represented we could not document a strict restriction to IgG1 and IgG4 [45
]. The mechanisms of tissue damage in EBA may be inflammatory, requiring the activation of complement and leukocytes by bound autoantibodies, or non-inflammatory just involving the binding of autoantibodies independent of their Fc portions [13
]. Our own results and data from the literature show that in addition to IgG1, which shows good Fcγ-dependent complement- and leukocyte-activating capacity, non-complement-fixing IgG4 autoantibodies contribute to tissue damage by activating the leukocytes, albeit with a reduced efficiency compared with IgG1 [46
]. In addition, IgG4 could induce tissue damage just by binding to collagen VII in an Fc-independent manner [13
]. While experimental data to support this hypothesis are still scarce, our present findings suggest that IgG4 may unfold its pathogenic potential in this way.