Localized Bullous Pemphigoid: Report of a Case with an Immunofluorescence and Electron Microscopical Studies on the Lesional Distribution of 180-KD Bullous Pemphigoid Antigen, β4 Integrin,and Type VII Collagen

A 67-year-old woman with a left-sided hemiplegia had localized bullous pemphigoid demonstrating typical clinical lesions on the left pretibial skin and the radial-side skin of the right forearm. The histology showed a subepidermal blister with extensive hyperkeratosis, hypergranulosis, and acanthosis. Direct immunofluorescence revealed distinct linear deposits of IgG and C3 at the dermo-epidermal junction in the perilesional skin and in the roof of the blisters, but few deposits in nonlesional skin. Electron microscopy revealed separation in the lamina lucida. Indirect immunofluorescence of type VII collagen showed its localization in the blister floor. The distribution of the 180-KD bullous pemphigoid antigen (BPA) and β4 integrin, hemidesmosomal transmembrane proteins, were studied in the lesional skin by indirect immunofluorescence. Both 180-KD BPA and β4 integrin were localized in the blister roof. By immunoelectron microscopy, β4 integrin was detected in small groups on the cell surface facing the blister cavity. Since the epitope of the monoclonal antibody to 180-KD BPA used here is known to be localized at a distance of 20 to 50 nm from the membrane surface and this epitope retained in the blister roof, it appears that the blister was produced in the deep lamina lucida. The lesions were cleared with topical 0.05% clobetasole propionate ointment.     

Expression of the alpha 6 beta 4 integrin in lesional skin differentiates bullous pemphigoid (BP) from epidermolysis bullosa acquisita (EBA).

The integrin alpha 6 beta 4 complex is a protein of the membrane of basal keratinocytes, localized at the surface of cells in contact with the basement membrane zone in normal skin. The expression of alpha 6 beta 4 was investigated in several autoimmune blistering skin diseases including bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA), bullous systemic lupus erythematosus (BSLE), and pemphigus vulgaris (PV) by an indirect immunofluorescence technique. In lesional bullous skin of BP, alpha 6 beta 4 expression was either absent, or in some cases represented an unusual irregular patchy staining. In contrast, in lesional bullous skin from EBA, BSLE, and PV, alpha 6 beta 4 expression was comparable to that observed in normal skin, i.e., a linear staining of the BMZ. Thus, analysis of the alpha 6 beta 4 integrin reactivity on lesional skin, in conjunction with the typical localization of collagen IV, allows a rapid and accurate distinction between BP and EBA.     

Re-epithelialization rate and protein expression in the suction-induced wound model: comparison between intact blisters, open wounds and calcipotriol-pretreated open wounds

We have investigated re-epithelialization following induction of suction blisters in humans in intact blisters, open wounds, i.e. blister roofs removed immediately after blister induction, and calcipotriol-pretreated open wounds. Intact blisters simulate blister healing in bullous disease, while open wounds simulate re-epithelialization during wound healing. Re-epithelialization was clearly faster in open wounds than in intact blisters, and was not affected by calcipotriol pretreatment. Bullous pemphigoid antigen 2 (BP180), bullous pemphigoid antigen 1 (BP230), plectin/hemidesmosomal 1 protein (HD1), laminin 5, laminin α5, laminin β1, type VII collagen, tenascin-C, β4, αvβ5, α5 and α9 integrins were studied in intact blisters and open wounds by immunohistochemistry. Hemidesmosomal plaque proteins BP230 and plectin/HD1, which connect the keratin cytoskeleton to the hemidesmosome, appeared earlier at the leading edge in intact blisters than in open wounds. Band-like immunostaining in the basement membrane for laminin 5, α5 and β1 chains was continuous in blister bases, but partially discontinuous in open wound bases. The other antigens studied showed similar expression in intact blisters and open wounds. BP180, BP230, plectin/HD1, β4 integrin, laminin 5 and tenascin-C expression were further studied in calcipotriol-pretreated open wounds. Calcipotriol did not affect the expression of these antigens. The immunohistochemical results suggest that the keratin cytoskeleton is linked to the basal plasma membrane of migrating basal cells via BP230 and plectin/HD1 earlier in the more slowly re-epithelializing blisters than in open wounds. An intact laminin sheath may inhibit keratinocyte migration in intact blisters.     

Case with Brunsting–Perry‐like localized subepidermal blister formations and immunoglobulin G antibodies against unidentified basement membrane zone antigen

Brunsting–Perry type bullous pemphigoid is defined by the blister formation limited to the head and neck, and autoantibodies to type VII collagen are detected in several cases. However, the pathomechanisms and autoantigens in this condition remain unknown. We report a 20‐year‐old female patient with a more than 2‐year history of recurrent tense blisters localized on the face with no distinct atrophic scar formation. The patient had neither extensive sun exposure nor a history suggestive of contact dermatitis. Oral betamethasone was effective on the skin lesions. Histopathology revealed subepidermal blister formation with dermal infiltrates of neutrophils. Although direct and indirect immunofluorescence tests detected immunoglobulin G antibodies to the basement membrane zone (BMZ), no known dermal or epidermal autoantigens were detected in immunoblot analyses. Therefore, this case may be a rare variant of Brunsting–Perry type localized bullous pemphigoid with autoantibodies to an undetermined BMZ antigen.     

Anti-p200 pemphigoid: a novel autoimmune subepidermal blistering disease

Anti-p200 pemphigoid is a recently defined autoimmune subepidermal blistering disease characterized by circulating and tissue-bound autoantibodies to a 200-kDa protein (p200) of the dermal-epidermal junction (DEJ). This DEJ constituent is thought to be important for adhesion of basal keratinocytes to the underlying dermis. While the exact identity of p200 remains unknown, it has been demonstrated to be immunologically and biochemically distinct from all major autoantigens of the DEJ, including bullous pemphigoid antigens 180 and 230, laminin 1, 5 and 6, alpha6beta4 integrin, and type VII collagen. Clinically, most reported cases present with tense blisters as well as urticarial papules and plaques, closely resembling bullous pemphigoid. Histopathological examination of lesional skin biopsies shows subepidermal split formation and superficial inflammatory infiltrate typically dominated by neutrophils. Immunopathologically, linear deposits of immunoglobulin (Ig)G and C3 are detected along the DEJ by direct immunofluorescence microscopy of perilesional skin. Indirect immunofluorescence microscopy of patients' sera on NaCl-split human skin demonstrates circulating IgG autoantibodies labeling the dermal side of the split. By immunoblotting, these autoantibodies recognize a 200-kDa protein of human dermis. Biochemical characterization of the p200 molecule revealed a noncollagenous N-glycosylated acidic protein with an isoelectric point of approximately 5.5. We present an overview of the pathogenesis, clinical features, diagnosis and treatment of this new disease entity.     

Linear IgA dermatosis with IgA and IgG autoantibodies to the 180 kDa bullous pemphigoid antigen (BP180): evidence for a distinct subtype

BACKGROUND: Autoantibodies in linear immunoglobulin A (IgA) disease (LAD) are reported to be of IgA class and directed against a 97-120 kDa epidermal antigen. METHODS: We report a 39-year-old woman with clinical features of LAD and with circulating IgA and IgG autoantibodies to the 180 kDa bullous pemphigoid antigen (BP180). RESULTS: Histopathology of lesional skin revealed a subepidermal blister with mixed inflammatory cell infiltrate. Direct immunofluorescence of perilesional skin showed linear deposits of IgA along the dermal-epidermal junction. The antigen specificity of the patient's circulating antibodies was determined by Western blotting and enzyme-linked immunoabsorbent assay (ELISA) using various antigen sources, including cultured human keratinocytes, dermal protein lysates, and purified laminin-5, as well as proteins corresponding to BP180, the 230 kDa bullous pemphigoid antigen (BP230), laminin-5 subunits, and collagen IV alpha1-alpha6 chains. IgA and IgG antibodies in the patient's serum were directed against BP180, and no IgA or IgG reactivity was found against the other skin antigens. CONCLUSIONS: These data provide evidence for the presence of a subtype of LAD with dual IgA and IgG autoimmune response to BP180.     

Cicatricial pemphigoid with circulating autoantibodies to beta4 integrin, bullous pemphigoid 180 and bullous pemphigoid 230

Cicatricial pemphigoid is a heterogeneous group of autoimmune subepidermal blistering diseases associated most commonly with autoantibodies to bullous pemphigoid (BP)180 and less frequently with those to laminin 5 or type VII collagen. In addition, a few cases have been described with autoantibodies to the beta4 subunit of alpha6beta4 integrin. We describe a patient with extensive disease of ocular, oral, pharyngeal, laryngeal and genital mucous membranes that healed with scarring of conjunctivae. IgG autoantibodies bound to the dermal-epidermal junction on direct immunofluorescence (IF) microscopy and to the epidermal side of 1 mol L(-1) NaCl-split skin on indirect IF microscopy. Our patient's circulating IgG recognized a 205-kDa protein in extracts of 293T cells transfected with the beta4 subunit of alpha6beta4 integrin and in the cell extract of DJM-1 cells. Our patient's IgG and IgA autoantibodies also reacted with full-length BP180 derived from epidermal extracts and the ectodomain of BP180 (LAD-1) derived from culture supernatant of keratinocytes. In addition, a weak IgG reaction with BP230 was noted. The disease rapidly responded to dexamethasone-cyclophosphamide pulse therapy, and immunoblot reactivity to both beta4 integrin and BP180 decreased according to disease activity.     

Alpha-6 (CD 49f) integrin expression in genetic and acquired bullous skin diseases

Bullous pemphigoid (BP) antigen and alpha 6 integrin are hemidesmosome-associated glycoproteins of basal keratinocytes. In this work, the immunoreactivity of antibodies to BP and to alpha 6 in salt- or dispase-split human skin, and in 46 biopsy specimens of various genetic and autoimmune bullous dermatoses taken from various body sites, was studied by double-labeling immunofluorescence. In all specimens, both antigens localized at the same side of the blisters observed, i.e. the roof of the bulla in cases with a junctional or dermolytic cleavage, or the floor of the blister in those with intraepidermal cleavage. Immunostaining for alpha 6 was strong and present in all specimens studied, whereas the one obtained with the BP serum was absent from some specimens. These results show that the BP antigen and the alpha 6 integrin colocalize at the level of cleavage in bullous diseases; however, the more consistent and reproducible reactivity obtained with the anti-alpha 6 antibody suggests that this should be preferentially used in the immunohistochemical investigation of bullous dermatoses.     

Defining target antigens in linear IgA disease using skin from subjects with inherited epidermolysis bullosa as a substrate for indirect immunofluorescence microscopy

Identification of target antigens in immunobullous disorders usually involves laborious techniques such as immunoblotting and immunoprecipitation, which do not always provide conclusive data. This is particularly true of linear IgA disease (LAD) in which the target antigen has often proved difficult to identify. As an alternative means of antigen identification in five adult patients with LAD, we performed indirect immunofluorescence (IIF) microscopy on a panel of skin samples taken from subjects with different forms of inherited epidermolysis bullosa (EB). Skin samples were selected that showed a complete absence of immunostaining for a specific basement membrane zone (BMZ) molecule (type VII collagen, laminin 5 or the 180-kDa bullous pemphigoid antigen BP180). In each case, the underlying genetic mutations had been defined and shown to consist of premature termination codons on both alleles of the particular gene, resulting in total ablation of the encoded protein. Two epidermal-binding LAD sera showed BMZ fluorescence on all substrates except BP180-deficient skin, suggesting that the target antigen was BP180, or a closely related molecule. In contrast, two dermal-binding LAD sera were positive on all substrates except the type VII collagen-deficient skin, suggesting that the target antigen was likely to be type VII collagen. One LAD serum sample, which showed combined dermal and epidermal fluorescence on normal salt-split skin, was also positive on all substrates tested, suggesting a target antigen other than type VII collagen, laminin 5 or BP180. The study confirms that LAD is a heterogeneous disorder and illustrates that IIF using a panel of skin samples which lack specific BMZ molecules, taken from subjects with inherited EB, is a relatively simple and useful tool to help identify target antigens in immunobullous disorders.     

A case of linear IgA disease presenting initially with IgG immune deposits

We describe a case of linear IgA bullous disease initially presenting with histopathologic and immunofluorescent findings consistent with bullous pemphigoid. Initial immunofluorescent studies demonstrated a predominance of linear IgG at the basement membrane zone (BMZ) of perilesional skin and a low titer circulating IgG anti-BMZ antibody. Repeat studies 3 years later revealed a predominance of linear IgA immune deposits at the BMZ and no circulating anti-BMZ antibody. Dapsone therapy was initiated at this time with a good therapeutic response noted. Suction blister studies, immunoelectron microscopy, split skin immunofluorescent studies and Western immune blot were performed and provided indirect evidence that the BMZ antigen in this case is distinct from the bullous pemphigoid antigen component of the BMZ.     

Anti‐p200‐Pemphigoid – Eine neue blasenbildende Autoimmundermatose

Anti‐p200 pemphigoid is an autoimmune skin disease characterized by tense blisters, subepidermal split formation, and mainly neutrophilic inflammatory infiltration of the dermal‐epidermal junction (DEJ). Direct immunofluorescence microscopy of perilesional skin biopsies demonstrates linear deposits of IgG and C3 along the DEJ, while by indirect immunofluorescence microscopy on NaCl‐split human skin, patients' IgG labels the dermal side. The antigenic target of the autoantibodies is a 200 kD protein (p200) of the lower lamina lucida that can be detected in human dermal extracts by immunoblotting. While p200 is thought to be important for cell‐matrix adhesion, its exact identity is unknown. To date, the p200 autoantigen has been demonstrated to be distinct from bullous pemphigoid antigens 180 und 230, laminin 1, 5, and 6, α6β4 integrin, and type VII collagen. Biochemical characterization of the p200 molecule revealed a noncollagenous N‐glycosylated acidic protein with an isoelectric point of approximately 5.5. We provide an overview on pathogenesis, clinical features, diagnosis, and treatment of this unique autoimmune dermatosis.     

An immunohistochemical study of the distribution of plasminogen and plasminogen activators in bullous pemphigoid

Abnormalities of the cutaneous plasminogen/plasminogen activator system have been associated with acantholytic disorders, psoriasis, keratinocytes in culture, and epidermis in healing wounds. The present study was undertaken to investigate the possible role of the plasmin/plasminogen protease system in lesion development in bullous pemphigoid (BP). Using polyclonal antibodies and a fluorescent technique, the immunohistochemical distribution of plasmin/plasminogen, fibrinogen and the plasminogen activators, urokinase (uPA) and tissue plasminogen activator (tPA), were studied in lesional and non-fesional skin from nine BP patients, one with linear IgA disease (LAD) and one with pemphigoid gestationis (PG). The distribution of the proteases was compared with that in normal skin (n=4) and in suction blisters (n=2). In normal skin, fibrinogen, tPA and uPA were absent from the epidermis and plasminogen was confined to the basal layer. Uninvolved BP skin was identical to controls. Focal areas of suprabasal plasminogen expression in the region of a blister was seen in 3/9 BP lesions and in 1/2 suction blisters. In 6/9 BP lesions and both uninvolved and lesional LAD and PG skin were identical to controls, and no suprabasal expression of plasminogen was present. These findings suggest that suprabasal plasminogen expression is unlikely to play a fundamental role in the pathogenesis of blister formation in BP as enhanced expression was not present in every case and the finding was not specific to BP, also occurring in a suction blister. Enhanced plasminogen expression rather may be a reflection of the processes of tissue repair.     

Acquired skin disease of hemidesmosomes.

The hemidesmosome is a membrane-associated supramolecular dermal epidermal complex linking the cytoskeleton of the basal keratinocyte to structures within the papillary dermis. Different components of this complex have been identified as autoantigens in autoimmune bullous skin diseases. Some of the autoantigens have been characterized at the molecular level. Little is known, however, about the factors that initiate the production of autoantibodies. By histopathology, acquired skin diseases of hemidesmosomes show subepidermal blisters and by direct immunofluorescence, linear deposits of IgG, C3 or IgA at the dermal epidermal junction. Bullous pemphigoid (BP) is the most common acquired disease of hemidesmosomes. Two proteins, BP180 and BP230, have been identified as primary targets of autoantibodies in BP. In addition, pemphigoid/herpes gestationis, lichen planus pemphigoides, cicatricial pemphigoid and linear IgA disease are characterized by an immune response to BP180. Laminin 5 is another well-characterized anchoring filament-lamina densa component of hemidesmosomes. Patients with autoantibodies to laminin 5 show the clinical phenotype of cicatricial pemphigoid. Other acquired skin diseases of the hemidesmosomes reveal autoantibodies to a plectin-like protein, the beta4 subunit of alpha6beta4 integrin, uncein and a not yet characterized 168 kDa protein. Recently, diseases with autoantibodies to 105 and 200 kDa proteins of the lower lamina lucida have been reported. The association of these autoantigens with hemidesmosomes still needs to be demonstrated. Finally, anchoring fibrils associate with the dermal epidermal anchoring complex. The major structural component of anchoring fibrils is type VII collagen, the autoantigen of epidermolysis bullosa acquisita.     

The localization of the bullous pemphigoid and cicatricial pemphigoid antigens: direct and indirect immunofluorescence of suction blisters

The location of in vivo bound immunoreactants was studied in 37 patients with subepidermal blistering diseases by direct immunofluorescence (IMF) on suction blisters taken from uninvolved forearm skin. The patients studied included 18 with bullous pemphigoid (BP), nine with cicatricial pemphigoid (CP), three with acquired epidermolysis bullosa (EBA) and 7 hybrid cases. The patterns of IMF in the suction blisters were: BP, epidermal 1, dermal 1, combined 4, negative 12; CP, epidermal 1, dermal 2, negative 6; EBA, dermal 2, negative 1; and 'hybrid' patients, epidermal 3, negative 4. The different patterns of suction blister staining could not be correlated with the clinical features of the patients in respect of mucous membrane involvement, scars or milia or a history of skin fragility. Both BP and CP are probably heterogeneous in respect of their antigen specificity, and the two diseases cannot reliably be distinguished by the patterns of direct IMF on suction blisters. In addition, some individual patients with BP have more than one target antigen as indicated by a combined pattern of suction blister fluorescence. The lack of correlation between the pattern of suction blister fluorescence and the clinical features suggests that factors other than antigen specificity determine the clinical expression of subepidermal blistering diseases.     

Basement membrane changes in lichen planopilaris

Background  Lichen planopilaris (LPP) is an inflammatory disease that affects the scalp and tends to produce cicatricial alopecia. The inflammatory process frequently results in the disruption of the basal cell of the external root sheath of the hair follicle.
Objectives  To investigate the alterations in the basement membrane zone (BMZ) in LPP by immunohistochemistry.
Methods  Skin biopsies from six patients with LPP plus six normal controls were studied by immunohistochemistry with antibodies to the following BMZ components: cytokeratin 5, cytokeratin 14, BP230 (bullous pemphigoid), BP180, plectin, laminin 5, collagen IV and collagen VII.
Results  The localization and staining of the hemidesmosome, laminin and collagen components were strikingly different in the inflamed follicular epithelium when compared to the uninvolved follicles or interfollicular epithelium in active LPP lesions. The hemidesmosome-associated complexes were weakly expressed and discontinuous in involved hair follicles. The expression of laminin-5, type IV collagen and type VII collagen was disrupted and not linear along the BMZ with finger-like projections of the staining protruding into the dermis. The expression of the intermediate filaments was normal.
Conclusion  These alterations in the BMZ in LPP may explain the abnormal healing at follicular level which leads to irreversible hair loss and scarring in this condition.     

Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes

This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence of serum and exogenous growth factors. The expression of various BM components was analyzed on the protein and mRNA level. Irrespective of the presence or absence of fibroblasts, keratin 14, hemidesmosomal proteins plectin, BP230 and BP180, and integrins alpha1beta1, alpha2beta1, alpha3beta1, and alpha6beta4 were expressed but laminin 1 was absent. Only in the presence of fibroblasts or of various growth factors, laminin 5 and laminin 10/11, nidogen, uncein, type IV and type VII collagen were decorating the dermal/epidermal junction. These findings indicate that the attachment of basal keratinocytes to the dermal matrix is most likely mediated by integrins alpha1beta1 and alpha2beta1, and not by laminins that bind to integrin alpha6beta4 and that the epithelial-mesenchymal cross-talk plays an important role in synthesis and deposition of various BM components.     

Immunofluorescent analysis of the basement membrane zone in lichen planus suggests destruction of the lamina lucida in bullous lesions

Lichen planus is an inflammatory dermatosis which is characterized histologically by an intense lymphocytic infiltrate at the dermal epidermal junction. This frequently results in disruption of the basement membrane zone, occasionally causing clinical blisters. In order to better understand the specific portion of the basement membrane zone which is disrupted by the lymphocytic infiltrate, we examined 7 cases of lichen planus with antibodies directed against anchoring filaments (GB3), the bullous pemphigoid antigen, anchoring fibrils (type VII collagen) and type IV collagen. In lesions without separation at the BMZ, all antibodies were strongly expressed, as in normal skin. In lesions with early separation, there was a focal decrease in GB3 staining, but types VII and IV collagen labelled normally. In lesions resulting in blisters, GB3 staining was essentially absent, and anti-types IV and VII collagen remained, but stained in a disrupted, less discrete pattern. The bullous pemphigoid antigen showed only slight deviation from the normal staining pattern. These findings suggest that the basement membrane zone in lichen planus is disrupted in the lamina lucida region. The lamina densa and sub-lamina clensa zones remain intact even in bullous lesions of lichen planus.     

The interleukin-2 receptor in lesions and serum of bullous pemphigoid

Summary The interleukin-2 receptor (IL-2R) is mainly expressed on activated T cells. Depending on its rate of synthesis, a portion is released from the cell surface as soluble IL-2R (sIL-2R). Since the role of mononuclear cells in the pathology of bullous pemphigoid (BP) is not well understood, we determined the sIL-2R in both blister fluid and serum of 15 BP patients with generalized disease before initiating systemic treatment. In addition, we obtained both lesional and perilesional skin biopsies and examined the mononuclear infiltrate with a panel of monoclonal antibodies. In BP blisters, sIL-2R levels were significantly increased (2070±350 U/ml), (±SEM) compared with serum samples taken at the time of blister puncture (1340±290 U/ml). In six patients with blisters due to second-degree burns or friction and in five suction blister volunteers, sIL-2R levels were normal in both blisters and serum. In BP, elevated serum levels decreased to normal during therapy, correlating with disease activity. The immunohistology showed that 30% of mononuclear cells in the dermal infiltrate of lesional skin expressed the IL-2R, whereas only 15% were positive in perilesional skin. IL-2R-positive cells are the most likely source of the shed receptor in BP blisters. Our results indicate the presence of activated T cells in lesions and peripheral blood of BP and thus underline the importance of cell-mediated immune mechanisms in the pathology of this disease.Presented in part at the Clinically Orientated Symposium on Bullous Disorders of the European Society for Dermatological Research, London, January, 1991     

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.     

Increased expression of eotaxin and its specific receptor CCR3 in bullous pemphigoid.

Several skin infiltrating inflammatory cells, such as eosinophils, neutrophils and activated T lymphocytes, are involved in bullous pemphigoid (BP) blister formation. The presence of CD4+ T cells able to produce IL-4 and IL-5 suggests Th2 involvement in the disease. The role of eotaxin in the recruitment of eosinophils into inflammatory sites has been recently described and the specific eotaxin receptor, CCR3, has been documented to be expressed on eosinophils, basophils, and Th2 cells. In this study, we analyzed by immunohistochemistry the expression of both eotaxin and CCR3 in lesional skin from patients with active BP (n = 10) and control subjects affected with pemphigus vulgaris (PV) (n = 3); furthermore eotaxin concentration in BP sera and blister fluids was also evaluated by enzyme-linked immunosorbent assay (ELISA), in comparison to sera from PV and normal donors (n = 10) and to suction blisters from 3 healthy volunteers. A strong immunostaining for eotaxin and CCR3 in BP skin specimens in lesional and, to a lesser extent, in perilesional skin was observed. CCR3 expression was documented on both eosinophils and T cells infiltrating skin lesions. Eotaxin serum levels were significantly higher in BP patients when compared to healthy donors (p = 0.003) and PV patients (p = 0.01). The highest eotaxin concentration was detected in BP blister fluids, in respect to both corresponding BP sera and blister fluids from normal donors (p = 0.003). These results account for the role of eotaxin in the recruitment of activated cells at inflammatory sites during BP and the expression of CCR3 on infiltrating T lymphocytes further supports the involvement of Th2 cells in the pathogenesis of BP.