Paraneoplastic pemphigus sera react strongly with multiple epitopes on the various regions of envoplakin and periplakin, except for the c-terminal homologous domain of periplakin

Paraneoplastic pemphigus sera react with multiple plakin family proteins, among which only envoplakin and periplakin are constantly detected by immunoblotting using normal human epidermal extracts. Using bacterial expression vectors containing polymerase chain reaction-amplified cDNA, we have prepared variously truncated recombinant glutathione-S-transferase-fusion proteins of envoplakin and periplakin, which presented N-terminal, central and C-terminal domains of each protein, as well as the so-called C-terminal homologous domain of envoplakin and the junctional regions of these domains. By immunoblotting using these 11 recombinant proteins, we demonstrated that most of the 26 paraneoplastic pemphigus sera reacted very strongly with multiple recombinant proteins of envoplakin and periplakin, except for the C-terminal homologous domain of periplakin. We also examined the reactivity with these recombinant proteins of other blistering diseases, including pemphigus vulgaris, pemphigus foliaceus, and bullous pemphigoid, and found that a few nonparaneoplastic pemphigus sera showed a weak reactivity with some of the recombinant proteins. Interestingly, some sera showed relatively strong reactivity with the C-terminal homologous domain of periplakin to which paraneoplastic pemphigus sera reacted less frequently. These results indicate that, although nonparaneoplastic pemphigus sera occasionally show a weak reactivity with envoplakin and periplakin, the pathogenicity and the mechanism of antibody production in these cases may be different from those in paraneoplastic pemphigus.     

Characterization of bullous pemphigoid antibodies by use of recombinant bullous pemphigoid antigen proteins

The seroreactivity of patients with bullous pemphigoid (BP) to recombinant proteins representing sequences in the carboxyl domain of the murine 230-kD BP antigen (BPA) was determined. Sera from 133 patients with BP, 20 patients with pemphigus, and 21 normal subjects were examined by Western blotting by using two recombinant proteins: RP120 (MW = 120 kD), representing the C-terminal half of the 230-kD BPA, and RP60 (MW = 60 kD), representing the C-terminal quarter. These RP120 and RP60 were recognized by 84% and 61%, respectively, of the BP sera that reacted with the 230-kD BPA in epidermal extract, and not by any of pemphigus or normal sera. Furthermore, these RP120 and RP60 were not recognized by any BP sera that reacted only with the 170-kD BPA, which is known to be another major BPA. These findings indicate that one or more of the major antigenic regions localizes in the carboxyl-half domain of the 230-kD BPA, and also suggest that the 230-kD BPA may be distinct from the 170-kD BPA.     

Autoantibodies in lichen planus pemphigoides react with a novel epitope within the C-terminal NC16A domain of BP180.

Lichen planus pemphigoides is an autoimmune subepidermal blistering disease. The finding of immunoglobulin G antibodies directed against the basement membrane zone differentiates it from bullous lichen planus. The aim of this study was to identify the target antigen of lichen planus pemphigoides autoantibodies. Sera from lichen planus pemphigoides patients (n = 4) stained the epidermal side of NaCl-split human skin in a pattern indistinguishable from that produced by bullous pemphigoid sera. In bullous pemphigoid, the autoimmune response is directed against BP180, a hemidesmosomal transmembrane collagenous glycoprotein. We previously demonstrated that bullous pemphigoid sera predominantly react with a set of four epitopes (MCW-0 through MCW-3) clustered within a 45 amino acid stretch of the major noncollagenous extracellular domain (NC16A) of BP180. By immunoblotting and enzyme-linked immunosorbent assay, lichen planus pemphigoides sera were also strongly reactive with recombinant bullous pemphigoid 180 NC16A. The lichen planus pemphigoides epitopes were further mapped using a series of overlapping recombinant segments of the NC16A domain. All lichen planus pemphigoides sera reacted with amino acids 46-59 of domain NC16A, a protein segment that was previously shown to be unreactive with bullous pemphigoid sera. Two lichen planus pemphigoides sera, in addition, reacted with the immunodominant antigenic region associated with bullous pemphigoid. In conclusion, there are now five bullous diseases that are associated with an autoimmune response to BP180: bullous pemphigoid; pemphigoid/herpes gestationis; cicatricial pemphigoid; linear immunoglobulin A disease; and lichen planus pemphigoides. In addition, we have identified a novel epitope within the BP180 NC16A domain, designated MCW-4, that appears to be uniquely recognized by sera from patients with lichen planus pemphigoides.     

The major cicatricial pemphigoid antigen is a 180-kD protein that shows immunologic cross-reactivities with the bullous pemphigoid antigen.

Recent studies have shown that sera from patients with cicatricial pemphigoid (CP) contained autoantibodies against epidermal antigens of molecular weight 230 kD and/or 180 kD by immunoblotting, similar to those recognized by bullous pemphigoid (BP) sera. Previous immunoprecipitation studies have shown that BP sera only precipitated the 230-kD antigen. To characterize the CP antigen(s) we tested 10 CP sera, 10 BP sera, and four controls by both immunoprecipitation of radiolabeled cells and immunoblotting of epidermal extracts. For immunoprecipitation, we used 0.5% NP-40 extracts of both normal human keratinocytes and Pam cells. All CP sera precipitated a 180-kD protein that co-migrated with the BP180 antigen precipitated by some individual BP sera. Two of these CP sera also faintly bound a 230-kD protein of similar molecular weight as the major BP230 antigen. CP and BP sera with an immunoblotting pattern of 180 kD immunoprecipitated a co-migrating 180-kD protein. CP sera reacting by immunoblotting with the 230-kD antigen precipitated the 180-kD and/or the 230-kD antigen. In contrast, BP sera reacting with the 230-kD antigen only precipitated this antigen. In further experiments, labeled 0.5% NP-40 extracts from Pam cells were first preabsorbed with a reference BP serum and then immunoprecipitated with CP sera. Under these conditions, CP sera that immunoprecipitated both 180-kD and 230-kD proteins with the standard procedure no longer precipitated these proteins. Our results suggest that a 180-kD protein is the major CP target-antigen that demonstrated immunologic cross-reactivities with the BP180 and the BP230 antigens.     

Autoantibody formation against a 190-kDa antigen of the desmosomal plaque in pemphigus foliaceus

Summary We report changes in the antigen recognition pattern of sera from two pemphigus foliaceus patients with a long-term follow-up. The patients' sera were analysed by immunoblotting using different antigenic sources: cultured human keratinocytes, bovine tongue epithelium and a recombinant protein corresponding to the C-terminal end of the 230-kDa bullous pemphigoid antigen. While initial serum samples reacted exclusively with the 160-kDa desmoglein 1, the later sera reacted both with desmoglein 1 and a 190-kDa antigen immunolocalized to the desmosomal plaque, previously demonstrated to be recognized by sera of some patients with paraneoplastic pemphigus. lgG subclass analysis further showed that antidesmoglein 1 antibodies were of lgGl and/or IgG4 subclasses, while anti-190-kDa antibodies were lgG3. The patients were free of malignancy.     

Comparative study of autoantigens for various bullous skin diseases by immunoblotting using different dermo-epidermal separation techniques

We investigaged the reactivity of pemphigus vulgaris (PV), pemphigus vegetans, pemphigus foliaceus (Pf), Brazilian Pf, bullous pemphigoid (BP), and epidermolysis bullosa acquisita (EBA) sera with an immunoblot analysis using human epidermal and dermal extracts as a source of antigen. To obtain epidermal and dermal extracts three different dermo-epidermal separation methods were used: namely, ethylenediaminetetraacetic acid (EDTA) separation, heat separation, and dispase separation. All the 15PV and the seven pemphigus vegetans sera demonstrated a 130-kDa PV antigen in epidermal extracts obtained by all the three methods. Furthermore, three PV sera also showed a 160-kDa Pf antigen, desmoglein. Ten of 14 Pf sera and six of 15 Brazilian Pf sera reacted with desmoglein in the same pattern in all the three epidermal extracts. Fifteen of the 22 BP sera showed reactivity with 230-kDa BP antigen in the same pattern in all the three epidermal extracts, whereas 14 BP sera delected the 180-kDa BP antigen in extracts of EDTA-and heat-separated epidermis but not in dispase-separated epidermal extract. Dermal extracts were obtained by EDTA- and heat-separated dermis, and all six EBA sera labelled a 290-kDa EBA antigen in both samples. These results suggest that heat-separated skin is as useful as EDTA-separated skin for detecting various autoantigens, but heat separation is preferable because the preparation time is shorter.     

Human placental amnion is a novel substrate for detecting autoantibodies in autoimmune bullous diseases by immunoblotting

BACKGROUND: Identification of antigens by immunoblotting techniques, using epidermal and dermal extracts, is regarded as essential for making a definitive diagnosis in autoimmune bullous diseases (AIBDs). These procedures involve epidermal-dermal separation for subsequent protein extraction, which may result in partial loss of some antigenic polypeptides and changes in the conformational epitopes targeted by autoantibodies in AIBDs. It may therefore be necessary to use different substrates for consistent results. Objectives To evaluate the usefulness of human placental amnion extract as a substrate for immunoblotting in the diagnosis of AIBDs. METHODS: We checked the structural components of the desmosomes and basement membrane zone (BMZ) of amnion by electron microscopy. Using immunofluorescence and immunoblotting techniques, we tested the amnion immunoreactivity with antibodies to desmosomal and BMZ proteins, and with sera from 76 patients with AIBDs including pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid (BP), pemphigoid gestationis, linear IgA bullous dermatosis, epidermolysis bullosa acquisita, paraneoplastic pemphigus and mucous membrane pemphigoid. RESULTS: The desmosomes and BMZ of the amnion tissue were ultrastructurally similar to those in skin. Antigen mapping confirmed that amnion contains all the proteins that were recognized by a panel of monoclonal and polyclonal antibodies. Immunoblotting showed that the antibodies clearly detected bands corresponding to desmogleins 1 and 3, desmocollins 1 and 2, desmoplakins 1 and 2, three subunits (alpha3, beta3 and gamma2) of laminin 5, BP antigens 1 and 2, the 97-kDa LAD antigen and type VII collagen. In addition, most of the patient sera (82%) reacted exclusively with their respective antigens. CONCLUSIONS: Harvesting proteins from amnion does not require epidermal-dermal separation, and a sufficient yield of desmosomal and hemidesmosomal proteins can be obtained. Therefore, amnion may be a more reliable source of substrate than skin samples for immunoblot analysis of AIBDs.     

Detection of autoantibodies against bullous pemphigoid and pemphigus antigens by an enzyme-linked immunosorbent assay using the bacterial recombinant proteins

Abstract To develop a new method to evaluate autoantibodies in various autoimmune bullous skin diseases, we examined reactivity of bullous pemphigoid (BP) and pemphigus vulgaris (PV) patients' sera with partial bacterial fusion proteins of the 230 kD BP antigen (BPAG1) and PV antigen, respectively, by an enzyme-linked immunosorbent assay (ELISA), and compared the results with those of immunoblotting. We used two fusion proteins derived from the mouse BPAG1 and a fusion protein derived from the amino-terminus (EC 1-2) of PV antigen. For both BP and PV sera, the ELISA scores were well correlated with the reactivities on immunoblot assays. The present study indicates that ELISA using recombinant antigen proteins in various autoimmune bullous skin diseases will be a new useful technique to detect the autoantibodies. However, development of recombinant proteins with the entire molecule and correct conformation will be necessary to establish a perfect ELISA system in the future.     

Childhood bullous pemphigoid associated with IgA antibodies against BP180 or BP230 antigens

Linear IgA bullous dermatosis (LABD) comprises a heterogeneous group of subepidermal blistering disorders characterized by in situ bound IgA antibodies in epidermal basement membrane. We report three children presenting clinical and immunopathological features characteristic of LABD. By immunoblotting, the three patients' sera contained IgA antibodies that reacted against the bullous pemphigoid (BP) antigen 180 and or BP230, molecular markers for BP. In addition, IgG antibodies directed against the ectodomain of BP180 were detected by an enzyme-linked immunosorbent assay using a eukaryotic recombinant form of BP180. Consistent with recent studies suggesting that the LABD antigen 1, the predominant autoantigen of LABD, is either a proteolytic product of BP180 or an isoform of the BP180 gene, our findings indicate that a subset of children with features of LABD have a distinct form of BP associated with an IgA response.     

Immunoreactivity of bullous pemphigoid (BP) autoantibodies against the NC16A and C-terminal domains of the 180 kDa BP antigen (BP180): immunoblot analysis and enzyme-linked immunosorbent assay using BP180 recombinant proteins

The 180 kDa bullous pemphigoid (BP) antigen (BP180) is known to be recognized by sera from patients with BP, herpes gestationis (HG) and cicatricial pemphigoid (CP). A series of previous studies using BP180 recombinant proteins has shown that most sera from patients with BP and HG react with the NC16A domain of BP180, an extracellular non-collagenous region just adjacent to the plasma membrane. In contrast, the C-terminal region of BP180 has been reported to be one of the epitopes of CP. In the present study, we examined the immunoreactivity of 110 BP sera against the NC16A and C-terminal domains of BP180 using immunoblot analysis and enzyme-linked immunosorbent assay (ELISA). Immunoblot analysis revealed that 100 (91%) and 26 (23.5%) of the 110 BP sera recognized the NC16A and C-terminal domains, respectively. The results of the ELISA were correlated with those of immunoblotting. There were no specific or significant clinical features such as severe involvement of mucous membranes and scarring in BP patients whose sera reacted with the C-terminal region. These findings suggest that some BP sera react with the C-terminal region of BP180 without any association with the characteristic clinical features of CP.     

A case of bullous pemphigoid with antidesmoplakin autoantibodies

Paraneoplastic pemphigus, a recently identified disease entity, is associated with autoantibodies against a variety of epidermal proteins including desmoplakins I and II, and the 230-kDa bullous pemphigoid (BP) antigen. We report an 84-year-old Japanese man who had typical clinical and histopathological features of BP, but in whom indirect immunofluorescence, using normal human skin as the substrate, revealed concomitant serum antibasement membrane zone and antikeratinocyte cell surface autoantibodies. His serum showed reactivity similar to that which is seen with antidesmoplakin monoclonal antibody on immunofluorescence of cardiac muscle and urinary bladder. With immunoblotting, using various antigen sources, the patient's serum reacted with desmoplakins I and II. and with both the 230- and 180-kDa BP antigens. Immunogold electron microscopy also indicated the presence of antidesmoplakin antibodies. Although the significance of the antidesmoplakin antibodies in this patient is unknown, the findings in this case may provide an insight into understanding the occurrence of antidesmoplakin antibodies in paraneoplastic pemphigus.     

Molecular heterogeneity of the bullous pemphigoid antigen as detected by immunoblotting

Sera from 18 patients with bullous pemphigoid (BP) and 20 controls (normal volunteers and patients with pemphigus) were tested against extracts of human epidermis by immunoblotting techniques. BP sera from individual patients showed different patterns of reactivity with the same epidermal extract, and each pattern consisted of one or more bands. A total f three bands of 240, 200, and 180 KD reacted with BP sera, and none of three bands was detected in the control sera. The 240 KD and 180 KD bands reacted strongly with some sera and were most frequently observed (44% and 50%, respectively). The 200 KD band was less frequently observed (22%). Eleven percent of the BP sera did not react to any bands. These results indicate the presence of several antigens reactive with autoantibodies in the sera of BP patients, rather than a single molecule.     

Major antigenic epitopes of bullous pemphigoid 230 kDa antigen map within the C-terminal end of the protein. Evidence using a 55 kDa recombinant protein

In order to obtain greater insight into the nature of B-cell epitopes in bullous pemphigoid (BP), we generated a BP recombinant protein of 55 kDa M_r (rBP 55) from a cDNA sequence encoding for the carboxyterminal region of the 230 kDa BP antigen. Serum IgG from guinea-pigs immunized with rBP 55 stained the basement membrane zone of normal human skin and immunoprecipitated the rBP 55 protein, and also the 230 kDa BP antigen recovered from extracts of cultured keratinocytes, thus confirming that the rBP 55 amino acid sequence is present in native BP antigen. The reactivity of sera from 60 patients with BP was analysed using an immunoblot assay on epidermal protein extracts and on the rBP 55 protein. Forty of the 60 BP sera (66%) contained autoantibodies to the 230 kDa polypeptide in an epidermal extract, and 37 of these 40 sera (92%) recognized the rBP 55 protein. In contrast, no reactivity against rBP 55 was detected with 20 BP sera devoid of autoantibodies against the 230 kDa antigen. Likewise, sera from patients with autoimmune blistering skin disorders other than BP (epidermolysis bullosa acquisita or pemphigus vulgaris), and control sera, were unreactive to rBP 55. These results clearly demonstrate the immunogenicity and antigenicity of the C-terminal end of the 230 kDa BP antigen. They confirm that this 555 amino acid segment, corresponding to rBP 55, contains major epitopes which can bind BP patients' autoantibodies, and suggest that the rBP 55 protein could be useful for further characterization of these B-cell epitopes.     

A 230-kD basic protein is the major bullous pemphigoid antigen

Recent studies have demonstrated that bullous pemphigoid (BP) antigen, in extracts of cultured human keratinocytes and normal human epidermis, is a 230-kD polypeptide. However, other recent studies have suggested that there is heterogeneity of BP antigen at the molecular level. To demonstrate that this 230-kD polypeptide is the major BP antigen and to further characterize it, we tested multiple BP sera by both immunoprecipitation of extracts of radiolabeled cultured human keratinocytes and immunoblotting of extracts of normal human epidermis. Thirty-six of 37 BP sera immunoprecipitated the 230-kD polypeptide, and 11 of 13 BP sera identified the 230-kD polypeptide by immunoblotting. Eighty-six disease and normal control sera did not bind the 230-kD BP antigen. Immunoprecipitation was a more sensitive assay than was immunoblotting to detect the 230-kD antigen; BP sera that were weak or negative by immunoblotting were strongly positive by immunoprecipitation. To demonstrate that this 230-kD polypeptide shared epitopes, defined by BP sera, with the epidermal basement membrane zone, we showed that BP IgG affinity purified on this polypeptide bound the epidermal basement membrane zone by immunofluorescence. To further characterize the BP antigen and to compare the antigen synthesized by cultured human keratinocytes to the antigen extracted from epidermis, we analyzed the 230-kD BP antigen by two-dimensional gel electrophoresis. BP antigen immunoprecipitated from culture extracts or BP antigen identified by immunoblots of epidermal extracts was a single spot on two-dimensional gels, with a pI of about 8. These data indicate that, although other polypeptides are sometimes identified (e.g., a 166 kD band by immunoprecipitation or a 180 kD band on immunoblots), the major BP antigen identified in cultured human keratinocytes is similar to the antigen found in normal human epidermis and is a basic 230-kD polypeptide.     

Specific detection of anti-cell surface antibodies in herpes gestationis sera

Abstract We examined 42 herpes gestationis sera with immunofluorescence of normal human skin sections, and found that anti-keratinocyte cell surface antibodies were detected specifically in 10 herpes gestationis sera. The diagnosis of these herpes gestationis cases was confirmed by detecting antibodies against the 180 kD bullous pemphigoid antigen with immunoblotting of its fusion protein. The results of immunoadsorption assay using baculoproteins of both pemphigus vulgaris and pemphigus foliaceus antigens indicated that the herpes gestalionis sera did not recognize common pemphigus antigens. Immunoblotting of human epidermal extracts and immunofluorescence of various tissues also suggested that the sera did not recognize any other desmosomal components or paraneoplastic pemphigus antigens. The significance of this reactivity is unclear. However, because no control bullous pemphigoid sera showed this reactivity, it may suggest a different pathophysiology between herpes gestationis and bullous pemphigoid.     

Comparative study of autoantigen profile between Colombian and Brazilian types of endemic pemphigus foliaceus by various biochemical and molecular biological techniques

BACKGROUND: Besides Brazilian endemic pemphigus foliaceus (EPF), we have described another focus of EPF in Colombia. Our previous study suggested that Colombian EPF seemed to react various plakin family proteins, such as envoplakin, periplakin and BP230. OBJECTIVE: To further characterize the Colombian EPF and study the difference from Brazilian EPF, we examined the antigen profile of the two types of EPF. METHODS AND RESULTS: Immunoblotting using normal human epidermal extracts revealed that 38% Colombian EPF sera and 25% Brazilian EPF sera showed IgG antibodies reactive with desmoglein (Dsg) 1, pemphigus foliaceus antigen. The sera of both types of EPF showed protein bands co-migrating with plakin family proteins, particularly periplakin. Immunoblotting analyses using recombinant proteins of various domains of envoplakin, periplakin and BP230 revealed that a considerable number of Colombian EPF sera reacted with recombinant proteins of periplakin, while only few Brazilian sera reacted with some of the recombinant proteins of any plakins. Enzyme-linked immunosorbent assay (ELISA) for Dsg1 and Dsg3 showed that Dsg1 was reacted by almost all sera of both types of EPF. However, unexpectedly, while none of Colombian EPF sera reacted with Dsg3, about half of Brazilian EPF sera reacted with Dsg3. CONCLUSION: These results suggested that the Colombian EPF is basically similar to Brazilian EPF in terms that major antigen is Dsg1, but there were some different antigen profiles between the two types of EPF.     

The presence of anti–basement membrane zone antibodies in the sera of patients with non–bullous lupus erythematosus

We performed indirect immunofluorescence (IF) studies using 1 mol/1 sodium chloride split skin to determine whether or not a positive IF is specific to patients with bullous lupus erythematosus (LE). We examined the sera from 21 patients with systemic LE (SLE), three of which were obtained from two SLE patients and one subacute cutaneous LE (SCLE) patient with bullous eruptions. As a comparison, we also studied the sera from patients with discoid LE (DLE,n= 7). SCLE (n= 1), systemic sclerosis (SSc, n= 20), bullous pemphigoid (n= 2) and normal individuals (n= 10). Sera from 16 SLE, four DLE and two SSc revealed a linear deposition of IgG isotype antibody at the epidermal side and/or the dermal side on indirect IF of split skin. The sera from three patients with bullous eruption and from 12 patients of SLE, SCLE, DLE without bullous eruption or SSc were further analysed by immunoblotting using five defined antigens, i.e, dermal extract, epidermal extract, three fusion proteins of 230 kDa bullous pemphigoid antigen (BPAG), 180 kDa BPAG, and human epidermolysis bullosa acquisita (EBA) antigen. Two SLE sera as well as one of the SCLE and the DLE serum reacted with 230 kDa BPAG in epidermal extract, and one of the SCLE and the DLE serum also reacted with the fusion protein of 180 kDa BPAG, No serum reacted with the dermal extract or the fusion protein of 230 kDa BPAG or EBA antigen. There was no consistent correlation between split–skin IF results and immunoblotting results. These results may suggest that even non–bullous LE patients often have autoantibodies to the basement membrane zone antigens, most of which are less pathogenic. Although we rarely examine the sera from non–bullous LE patients, we should keep this phenomenon in mind to avoid overestimating the results of split–skin test and immunoblotting.     

IgG autoantibodies from a lichen planus pemphigoides patient recognize the NC16A domain of the bullous pemphigoid antigen 180

Lichen planus pemphigoides (LPP) most likely encompasses a heterogeneous group of subepidermal autoimmune blistering disorders occurring in association with lichen planus. We describe the case of a 49-year-old patient with features characteristic of LPP. Direct immunofluorescence microscopy studies demonstrated linear deposits of C3 along the cutaneous basement membrane, while circulating IgG autoantibodies directed against the epidermal side of skin separated by 1 M NaCl were detected. The patient's serum contained IgG autoantibodies immunoblotting a recombinant form of bullous pemphigoid antigen 180 (BP180), but not the COOH-terminus of BP230. By using deletion mutants, it was found that IgG reactivity was restricted to the NC16A domain of BP180, the region harboring the major antigenic sites targeted by IgG autoantibodies from patients with the bullous pemphigoid group of disorders. Our findings provide support to the idea that a subset of patients with LPP have a distinct form of bullous pemphigoid associated with lichen planus.     

Studies of cicatricial pemphigoid autoantibodies using direct immunoelectron microscopy and immunoblot analysis

We studied 11 consecutive patients with classical cicatricial pemphigoid (CP) using direct immunoelectron microscopy (IEM) and Western immunoblotting analysis. Direct IEM performed in the skin or gingival mucosa revealed in all 11 CP patients that immunoglobulins and complement deposits were usually thick and discontinuous along the dermoepidermal junction, mostly localized on the lamina densa and occasionally in the lamina lucida. By direct IEM, the ultrastructural aspect in CP differs from the pattern observed in bullous pemphigoid (BP) and from that of chronic epidermolysis bullosa acquisita (EBA). Nine CP patients were studied by Western immunoblotting and, of these nine, only two had detectable anti-basement membrane zone (BMZ) antibodies by indirect immunofluorescence on salt-split skin. By immunoblotting performed on protein extracts of heat-separated epidermis, eight out of the nine CP sera specifically reacted with two protein bands of approximately 230-240 kD and 180 kD, similar to those recognized by BP sera in co-migration experiments. By immunoblotting on skin BMZ extracts, none of these nine CP sera recognized the 290-kD major polypeptide of EBA antigen. Taken together, these results suggest that, in CP, the target-antigen, as identified on immunoblots, is similar to BP antigen, but with an abnormal expression within the dermoepidermal junction of patients, which may in part explain the scarring course of the disease.     

IgG autoantibodies from bullous pemphigoid patients recognize multiple antigenic reactive sites located predominantly within the B and C subdomains of the COOH-terminus of BP230

Bullous pemphigoid is a subepidermal bullous disorder characterized by an autoantibody response against the bullous pemphigoid antigen 230 (BP230) and the bullous pemphigoid antigen 180 (BP180), a cytoplasmic component and a transmembrane component, respectively, of hemidesmosomes. Although immunodominant sequences within the extracellular domain of BP180 have been identified, characterization of the antigenic sites on BP230 is still incomplete. To identify autoantibody-reactive sites on BP230 and to examine whether the targeted regions are contained within functionally important domains, recombinant fragments encompassing almost the entire BP230 were used to assess the reactivity of 25 bullous pemphigoid sera by immunoblotting. Our results demonstrate that (i) the region bearing the B and C subdomains of the COOH-terminus of BP230 contains immunodominant sequences recognized by the majority of bullous pemphigoid sera; (ii) additional autoantibody- reactive sites are present over extended regions of the NH2-terminal half of BP230 without evidence for antigenic cross-reactivity between the NH2- and COOH-termini of BP230; and, finally, (iii) autoantibodies reacting with the BP230 tail predominantly belong to the IgG4 and IgG1 subclasses, suggesting that both autoreactive TH2 and autoreactive TH1 cells regulate the autoantibody response to immunodominant sequences of BP230. As the COOH- terminus of BP230 mediates the attachment of keratin intermediate filaments to the hemidesmosomal plaque, whereas its NH2-terminus contains sequences important for its interaction with other constituents of hemidesmosomes, autoantibodies to BP230 might precipitate subepidermal blister formation and perpetuate the disease not only by eliciting an inflammatory reaction but also by interfering with the function of BP230 and thus the stability of hemidesmosomes.