IgG antibodies against immunodominant C-terminal epitopes of BP230 do not induce skin blistering in mice

Bullous pemphigoid, the most common autoimmune blistering disease in Western Europe and the USA is characterized by the presence of circulating and tissue-bound autoantibodies against the hemidesmosomal proteins BP230 and BP180/collagen XVII. After binding to their target antigens at the basement membrane of the dermal–epidermal junction these autoantibodies are thought to trigger an inflammatory cascade comprising complement- and granulocyte-dependent reactions that result in tissue damage. Whereas the role of anti-BP180 antibodies has been extensively characterized, few and conflicting data is available on the contribution of anti-BP230 antibodies to bullous pemphigoid pathogenesis. Therefore, we addressed in the present study the role of autoantibodies to BP230 in experimental bullous pemphigoid. Rabbit polyclonal antibodies generated against epitopes of the C-terminal fragment of murine BP230 bound to the basement membrane and activated the complement system ex vivo. Affinity-purified antibodies were subsequently subcutaneously transferred into neonatal and adult BALB/c mice. In vivo, we observed a dose-dependent binding of transferred antibodies in the murine skin; however, there was no complement activation and these mice showed no clinical or histological signs of inflammatory disease, in contrast to mice receiving anti-BP180 antibodies. We further conducted ex vivo experiments and demonstrated that rabbit IgG anti-BP230-specific antibodies, in contrast to antibodies from bullous pemphigoid patients or rabbit IgG anti-BP180 antibodies used as positive controls, did not activate human granulocytes to induce dermal–epidermal separation in skin cryosections. Our present findings demonstrate that antibodies against BP230 are non-pathogenic in experimental models of bullous pemphigoid and suggest that proper activation of the complement and granulocytes represent prerequisites for conferring bullous pemphigoid autoantibodies their tissue destructive potential.     

A Korean case of anti-p200 pemphigoid

Anti-p200 pemphigoid is a newly defined autoimmune subepidermal blistering disease, which is characterized by the presence of IgG autoantibodies to the dermal side of 1 M NaCl split skin as well as by the reactivity of these antibodies to a novel 200-kDa antigen on immunoblot analysis of a dermal extract. We describe a 49-year-old Korean male who presented with a bullous eruption on the whole body, which clinically resembled bullous pemphigoid or epidermolysis bullosa acquisita. A histopathological examination of a lesional skin biopsy specimen showed an area of dermal-epidermal separation and mixed dermal inflammatory infiltrates consisting of lymphocytes, neutrophils, and eosinophils. Direct immunofluorescence showed a linear deposition of IgG and C3 along the basement membrane zone. Indirect immunofluorescence demonstrated circulating IgG autoantibodies directed against the dermal side of the 1 M NaCl split skin. Immunoblot analysis of dermal extracts revealed the patient's sera recognized the 200-kDa antigen. This is the first Korean case of an anti-p200 pemphigoid who showed good response to the treatment with systemic corticosteroids and dapsone.     

IgE-mediated mechanisms in bullous pemphigoid and other autoimmune bullous diseases

Autoimmune bullous diseases (AIBDs) are characterized by autoantibodies against structural proteins of the dermal–epidermal junction (in pemphigoid diseases) and the epidermal/ epithelial desmosomes (in pemphigus diseases). By far, the most common AIBD is bullous pemphigoid, which is immunopathologically characterized by autoantibodies against BP180 (type XVII collagen) and BP230. IgG and, to a lesser extent, IgA autoantibodies are the major autoantibody isotypes in these disorders. IgE autoantibodies are increasingly reported in particular in bullous pemphigoid. The development of specific and sensitive anti-BP180 IgE ELISA systems, the report of two experimental murine models employing IgE autoantibodies against BP180, and the successful treatment of bullous pemphigoid with the anti-IgE antibody omalizumab have raised interest in the role of IgE autoantibodies and the modulation of their production in AIBDs. Here, the relevance of IgE autoantibodies in the diagnosis, pathophysiology, and treatment decisions of AIBDs, with a focus on bullous pemphigoid, is reviewed.     

Primary Sézary syndrome commonly shows low-grade cytologic atypia and an absence of epidermotropism

The dermatopathologic findings in cases of Sézary syndrome (SS) that arise in patients without a previous diagnosis of mycosis fungoides have not been well characterized. We evaluated the histologic findings in skin biopsy specimens from 31 patients with such primary SS and correlated them with clinical and hematologic parameters at the time of biopsy. The most characteristic histologic finding was the presence of a dermal perivascular lymphoid infiltrate, usually with mild to moderate cytologic atypia and variable numbers of eosinophils; epidermotropism was absent or minimal in 19 cases (61%). Reactive epidermal changes such as spongiosis, parakeratosis, and acanthosis also were present frequently (27 [87%], 17 [55%], 19 [61%] cases, respectively). The number of eosinophils present in skin biopsy specimens correlated with the level of peripheral blood lymphocytosis. In erythrodermic patients or patients with persistent xerosis and pruritus, it is important to carefully evaluate the degree of lymphocyte atypia in the dermal perivascular infiltrate and correlate with blood flow cytometric findings to diagnose primary SS. Many cases will lack the epidermotropism usually seen in mycosis fungoides.     

Crescentic glomerulonephritis and subepidermal blisters with autoantibodies to alpha5 and alpha6 chains of type IV collagen

We describe a novel autoimmune disease characterized by severe subepidermal bullous eruption and crescentic glomerulonephritis with autoantibodies directed against the noncollagenous domain of the alpha5 and alpha6 chains of type IV collagen. Biopsy of perilesional skin revealed a subepidermal blister with marked polymorphonuclear infiltrate with linear deposits of IgA and C3. Light microscopy of a kidney biopsy specimen revealed a crescentic glomerulonephritis, and immunofluorescence microscopy showed linear basement membrane staining for IgA (3+), C3 (1+), and IgG (1+). No electron-dense deposits were observed by transmission electron microscopy. The patient's autoantibodies reacted with normal human skin and kidney: IgA (3+) and IgG (1+) antibodies stained the basement membrane zones of skin, renal glomerulus, and some tubules. The identity of the target antigen was determined by immunochemical analyses of candidate antigens using the patient's autoantibodies. The patient's IgA and IgG autoantibodies reacted with a 185- to 190-kDa antigen from a human dermal extract that was distinguished from the other dermal or epidermal antigens, including the 145- to 290-kDa (type VII collagen) epidermolysis bullosa acquisita antigen, the 165- to 200-kDa alpha3 laminin mucous membrane cicatricial pemphigoid antigen, and the 230-kDa and the 180-kDa bullous pemphigoid antigens. Patient's IgA and IgG autoantibodies further reacted with the alpha5(IV) and weakly with the alpha6(IV) chains of type IV collagen by Western blot and ELISA. This report expands the repertoire of bullous skin disorders and provides an explanation for the association of anti-type IV collagen autoantibodies and glomerulonephritis with subepidermal blisters.     

Association of KM genotype with bullous pemphigoid

Association of kappa light chain immunoglobulin allotypes with bullous pemphigoid was examined in 101 Caucasian patients. Km alleles were determined by polymerase chain reaction amplification followed by restriction enzyme digestion. The frequency of Km(3)/Km(1,2)kappa light-chain genotype was found to be significantly associated with the disease, while that of the Km(3)homozygous genotype was significantly higher in patients with both anti-BPAG1 and anti-BPAG2 autoantibodies.     

An Extract from Cultured Human Keratinocytes that Contains the Major Autoantigens Related to Autoimmune Bullous Skin Diseases

Autoantibodies characteristic of autoimmune bullous skin diseases (AIBDs) can be detected by immunoblotting on epidermal, dermal, or bovine muzzle extracts. However, none of those substrates contain all the autoantigens involved in AIBDs, and the diagnosis requires the use of various substrates. Human keratinocytes were cultured under such conditions that they expressed the major autoantigens associated with AIBDs. Forty-two sera with antiepidermal antibodies were immunoblotted on the keratinocyte extract. Bands corresponding to desmoglein III, desmoglein I, BPAg2, BPAg1, and type VII collagen were found in 38 sera. Desmoplakins I and II were revealed by specific monoclonal antibodies. A review of the patients' charts showed a perfect correlation between the blots and the diagnoses of pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, cicatricial pemphigoid, and epidermolysis bullosa acquisita. Four sera revealing no band typical of AIBD were from patients with no autoimmune skin disease. Therefore, a single extract of keratinocytes can be used for the differential diagnosis of AIBDs.     

Deposition of eosinophil granule proteins precedes blister formation in bullous pemphigoid. Comparison with neutrophil and mast cell granule proteins.

Eosinophils, neutrophils, and mast cells have all been implicated in the pathogenesis of bullous pemphigoid (BP). To comparatively characterize the involvement of these cells in BP, 10 lesional skin biopsy specimens were identified retrospectively and studied for tissue localization of eosinophil, neutrophil, and mast cell granule proteins. Subsequently, multiple skin biopsies of lesions in various developmental stages were obtained from 3 patients with untreated BP. Involved and uninvolved skin specimens were also obtained from 2 patients. Using indirect immunofluorescence, retrospectively identified lesions showed eosinophils and extracellular granule protein deposition prominently in areas of blistering. Evolving lesions showed eosinophil granule protein deposition in all stages but was most marked in early erythematous and prebullous (urticarial) lesions and was minimal in uninvolved skin. Vascular cell adhesion molecule-1, E-selectin, and P-selectin were detected on vessels and very late activation antigen-4 was detected on mononuclear cells and eosinophils by immunoperoxidase staining of lesions. Eosinophil granule proteins were increased in the peripheral blood, urine, and blister fluid. Blister fluids caused increased eosinophil survival that was inhibited by antibodies to interleukin-5 and interleukin-3. Although neutrophil and mast cell infiltration was observed, extracellular granule protein deposition from these cells was minimal except in two specimens. These results demonstrate that eosinophils infiltrate and deposit granule proteins early in the development of BP lesions, that eosinophil-activating cytokines are present in blister fluid, and that eosinophil-selective adhesion molecules are present. These studies strongly support a role for eosinophils in blister formation in BP.     

Vessel associated adhesion molecules in normal skin and acute graft-versus-host disease.

Immunohistological staining of skin from normal donors and bone marrow transplant recipients was undertaken using antibodies to two vessel associated adhesion molecules, endothelial leucocyte adhesion molecule-1 (ELAM-1). In normal skin ELAM-1 staining was restricted to a variable but generally small number of endothelial cells which were significantly increased in graft-versus-host disease (GvHD), but only when the fully developed histological picture of epidermal basal damage and leucocytic infiltration was present. All other biopsy specimens from marrow recipients taken before or after transplantation were similar to those of normal controls even in the presence of a clinical rash consistent with early GvHD. Although VCAM-1 positivity was seen on a few endothelial cells in normal skin, staining was mainly observed on dermal dendritic cells surrounding blood vessels and adnexal structures. In specimens with histological evidence of GvHD, positive perivascular dendritic cells were increased and were accompanied by the appearance of large numbers of similar cells dispersed throughout the upper dermis. Biopsy specimens from marrow recipients before and after transplantation resembled those from normal donors except for the presence of a rash after transplantation when some specimens, which lacked the leucocytic infiltrate diagnostic of GvHD, showed an increase in VCAM-1 positive cells, particularly in the upper dermis. The identification of these cells may therefore be useful in diagnosing early GvHD.     

Diagnosis and classification of pemphigus and bullous pemphigoid

Pemphigus and bullous pemphigoid represent the two major groups of autoimmune blistering diseases. Pemphigus has three major variants: pemphigus vulgaris, pemphigus foliaceus and paraneoplastic pemphigus and is characterized by autoantibodies directed against the cell surface of keratinocytes, producing acantholysis that in turn leads to intraepithelial blisters in the skin and/or mucous membranes. In bullous pemphigoid, the autoantibodies are present at the dermo-epidermal junction and attack the hemidesmosomes, causing subepidermal blister formation. The classification of the major variants of both the pemphigus group and bullous pemphigoid can be based on the combination of clinical, histopathological and immunopathological criteria. Many tools are available for the diagnosis of these entities including biopsy, direct and indirect immunofluorescence, immunoprecipitation, immunoblotting and ELISA. However, currently there are no generally accepted criteria for the diagnosis of these disorders. The present review provides a proposal for diagnostic criteria.     

Pathogenesis of bullous pemphigoid

Bullous pemphigoid, the most common autoimmune blistering disease, is induced by autoantibodies against type XVII collagen. Passive transfer of IgG or IgE antibodies against type XVII collagen into animals has revealed not only the pathogenicity of these antibodies but also the subsequent immune responses, including complement activation, mast cell degranulation, and infiltration of neutrophils and/or eosinophils. In vitro studies on ectodomain shedding of type XVII collagen have also provided basic knowledge on the development of bullous pemphigoid. The pathogenic role of autoreactive CD4+ T lymphocytes in the development of the pathogenic autoantibodies to type XVII collagen should also be noted.     

Morphology of atopic eczema

The routine examination of skin biopsy specimens embedded in paraffin and strained with hematoxylin-eosin has failed to allow differentiation of atopic eczema from other types of eczematous dermatitis. The use of 1-μm plastic-embedded sections permits the recognition of in filtrating cell types and blood vessel alterations, thus allowing a refined method to examine cutaneous lesions and permit better definition of cutaneous structures than can be achieved in routinely-processed specimens.
Acute vesicular lesions exhibited marked epidermal intercellular edema (spongiosis) and a dermal inflammatory infiltrate of lymphocytes, and activated lymphocytes with normal numbers of mast cells that exhibited various degrees of hypogranulation. Only rare eosinophils, neutrophils, and basophils were noted. Venular alterations included endothelial cell hypertrophy without necrosis. In lichenified plaques there was epidermal hyperplasia with a dermal inflammatory infiltrate that included increased numbers of fully granulated mast cells and increased numbers of lymphocytes and monocyte-macrophages. Alterations of venules included marked endothelial cell hypertrophy and basement membrane thickening. Cutaneous nerves exhibited demyelination and fibrosis.
Also, increased numbers of Langerhans' cells have been noted in the epidermis of chronic lesions. Despite the absence of eosinophils, major basic protein has been demonstrated in the dermis by direct immunofluorescence techniques. Studies of lymphocyte subsets have shown increased numbers of CD4 + T lymphocytes.     

Indirect cutaneous immunofluorescence : II Clinical significance

Sera of 532 patients with bullous diseases, connective tissue diseases and malignancies were tested for pemphigus epidermal intercellular fluorescence (ICF) and for the bullous pemphigoid ;tubular' band by the indirect fluorescent antibody technique. Human normal skin cryostat sections were used.The band and ICF were seen primarily only in bullous pemphigoid and pemphigus respectively, Some indirect band and ICF-negative patients demonstrated positive direct results in involved skin. suggesting that direct tests should be performed in indirect negative patients clinically thought to have pemphigus or bullous pemphigoid.No close correlation was found between disease activity and positive or negative indirect tests in bullous pemphigoid and pemphigus. Steroids did not interfere with positive results of this diagnostically extremely valuable test.     

Detection and characterization of anti-envoplakin linker autoantibodies in paraneoplastic pemphigus using specific bead-based assay

Paraneoplastic pemphigus (PNP) is characterized by an autoantibody response directed against desmosomal antigens, desmogleins and plakin's proteins, i.e., periplakin and envoplakin. Notably, PNP antibodies were shown to recognize major epitopes located in the linker subdomain of human envoplakin and thus, may constitute a diagnostic marker of PNP. In this study, a recombinant envoplakin-linker subdomain (rENV-L) was produced and used to develop a specific bead-based assay to determine the prevalence and titers of anti-rENV-L antibodies in patients with different types of autoimmune bullous skin diseases. Sera from 33 PNP, 41 pemphigus vulgaris, 46 pemphigus foliaceus, 24 bullous pemphigoid and 74 normal subjects were analyzed by rENV-L bead-based assay: 23/33 (69.7%) PNP, 2/24 (8.3%) bullous pemphigoid and 4/74 (5.4%) healthy control sera showed IgG reactivity against rENV-L. For PNP, the sensitivity and specificity of the assay were 69.7% and 94.6%, respectively. Among the PNP sera reacting with envoplakin and periplakin by immunoblot analysis, 80.8% contained anti-rENV-L antibodies. Finally, we showed that an anti-rENV-L mAb that recognizes both envoplakin and periplakin gave the same fluorescence pattern on rat bladder sections than PNP sera. Thus, anti-rENV-L antibodies constitute a useful diagnostic marker of PNP and our bead-based assay, notably combined with other epidermal autoantigens, a useful tool to diagnose PNP.     

Pemphigus and pemphigoid antibody production by pokeweed mitogen-stimulated peripheral blood mononuclear cell cultures in vitro

Peripheral blood mononuclear cells from pemphigus vulgaris and bullous pemphigoid patients produced antiepithelial antibodies in a pokeweed mitogen-stimulated in vitro cell culture system. Antiintercellular substance (ICS) antibodies were detected in supernatants of cell cultures from four of four pemphigus vulgaris patients by indirect immunofluorescence. Similarly, antibasement membrane (BM) antibodies were detected in supernatants of cell cultures from three of four bullous pemphigoid patients. No anti-ICS or anti-BM antibodies were detected in supernatants of cell cultures of six normal controls, although antinuclear antibodies were detected occasionally. This pokeweed mitogen-stimulated in vitro cell culture system is suggested as a model to study the role of defects in immunoregulation in the pathogenesis of pemphigus vulgaris and bullous pemphigoid.     

Eosinophils in skin lesions of erythema multiforme

To investigate the controversy regarding the presence of eosinophils in skin lesions of erythema multiforme, we undertook a retrospective clinicopathologic study of 19 recent cases that fulfilled clinical and histopathologic criteria for the disease. At least a few eosinophils were observed in 13 of 19 cases, and in four cases there were more than three per high-power field, qualifying as "tissue eosinophilia." Immunofluorescence studies in three cases with eosinophils failed to show the linear basement membrane zone fluorescence characteristic of bullous pemphigoid. Giemsa stains revealed that mast cells were present in lesions both with and without eosinophils. The only clinical features that distinguished patients with tissue eosinophilia from those without were an older age of incidence and a longer duration of disease prior to biopsy. Drugs were implicated as a causative factor in some patients both with and without eosinophils, but all four patients with tissue eosinophilia were believed to have drug-induced disease. We conclude that eosinophils do occur in skin lesions of erythema multiforme and are occasionally numerous.     

Immunoreactivity for LN2 and LN3 distinguishes small cell carcinomas from non-small cell carcinomas in the lung

Immunoreactivity to LN2 and LN3, monoclonal antibodies that recognize components of the class II major histocompatibility complex, was assessed in 72 cases of non-small cell lung carcinoma (NSCLC) (32 biopsy specimens, 40 resection specimens) and 64 cases of small cell carcinoma (56 biopsy specimens, 8 resections) of the lung. All cases were reviewed independently by three pathologists for histological classification. Only 1 of the 64 small cell carcinomas showed immunoreactivity for LN2, and none of the 64 cases showed reactivity for LN3. Among the non-small cell carcinomas, 25 of 48 cases were positive for LN2 and 43 of 71 were positive for LN3; the sensitivity was greater for adenocarcinoma (78.5%) than for squamous cell carcinoma (37%). A combined sensitivity of 64.7% was observed when the results of LN2 and LN3 were combined, and this sensitivity was not significantly diminished in the biopsy subset of cases (59.4%). Differentiation within histological subtypes of NSCLC (ie, well, moderate, or poorly differentiated) did not alter test sensitivity. In conclusion, LN2 and LN3, used alone or in combination, appear highly specific for non-small cell carcinoma and moderately sensitive in both biopsy and resection specimens; therefore, these antibodies may be diagnostically useful in distinguishing small cell from non-small cell carcinoma of the lung.     

Lymphotoxin detected in the blister fluid of bullous pemphigoid patients

The role of lymphocytes in the pathogenesis of bullous pemphigoid was examined by assaying the blister fluid obtained from bullous pemphigoid patients for the presence of the lymphokine, lymphotoxin. Blister fluids from six bullous pemphigoid were assayed on L-929 target cells for the presence of cytolytic molecules in the standard lymphotoxin assay. Three of six blister fluids obtained from bullous pemphigoid patients and one linear IgA bullous dermatosis patient contained significant levels of cytolytic activity. Control blister fluids from suction blisters, herpes, pemphigus, and toxic epidermal necrolysis patients did not contain cytolytic activity. Serum from five bullous pemphigoid patients also had no cytolytic activity. Neutralization studies using rabbit anti--lymphotoxin demonstrated that 54 to 88% of the cytolytic activity found in bullous pemphigoid blister fluid was due to -lymphotoxin. These results indicate that lymphotoxin is locally released in the skin of bullous pemphigoid lesions and is detectable in blister fluids.     

Evaluation of Recombinant Antigen-Based Assays for Diagnosis of Bullous Autoimmune Diseases

The diagnosis of autoimmune bullous diseases is based on clinical observation and on the presence of autoantibodies directed to molecules involved in the adhesion systems of the skin. Immunofluorescence assays are the currently accepted method for detection of autoantibodies; such assays depend greatly on the skill of operators and are difficult to standardize. Recombinant desmoglein-1 (Dsg1), Dsg3, and BP180 peptides, the main autoantigens in pemphigus or bullous pemphigoid, have been used to develop new quantitative enzyme immunoassays (EIA) for the detection of specific antibodies. The present study was undertaken to evaluate the sensitivity and specificity of these immunoassays and to determine the correlation between the results and the clinical aspects of diseases. Serum samples from patients with pemphigus vulgaris, pemphigus foliaceus, bullous pemphigoid, or mucous membrane pemphigoid, from healthy individuals, and from patients with unrelated autoimmune conditions were tested. Anti-desmoglein reactivity was detected in all the patients with pemphigus and in none of the controls. Patients with the more benign form of cutaneous disease had anti-Dsg1 antibodies, while patients with deeper cutaneous lesions or with mucosal involvement had anti-Dsg3 reactivity also, or exclusively. The BP180-based assay was positive for 66.6% of patients with bullous pemphigoid and for none of the patients with mucous membrane pemphigoid, and no reactivity was detected in the control sera. In conclusion, the anti-Dsg1 and anti-Dsg3 assays are useful in the diagnosis of pemphigus and provide information on the clinical phenotype of the disease. However, the sensitivity of EIA for detection of autoantibodies in bullous pemphigoid should be improved by the use of additional antigens or epitopes.