Bullous Disorders

Bullous Disorders

Diagnostic Value of Indirect Immunofluorescence on Sodium Chloride- Split Skin in Differential Diagnosis of Subepidermal Autoimmune Bul-lous Dermatoses

Introduction.—Serum samples from a group of patients with subepidermal autoimmune bullous dermatoses (SABD) were tested by immunofluorescence (IF) on sodium chloride-split skin (SSS) to determine the diagnostic value of this technique in differentiating the pemphigoid group of SABD from epidermolysis bullosa acquisita (EBA). Previous studies suggest that the use of SSS as a substrate for indirect IF testing of serum is more sensitive than indirect IF on intact skin.

Methods.—Serum samples were obtained from 142 patients, 98 with bullous pemphigoid (BP), 23 with cicatricial pemphigoid (CP), 10 with EBA, and 1 with anti-type IV collagen bullous dermatosis. The samples were analyzed using IF on SSS and immunoblot assay on epidermal and dermal extracts, a recombinant protein corresponding to the C-terminal end of the 230-kd BP antigen, and purified laminin-5. Binding sites of serum to the epidermal and/or dermal sides of SSS were correlated with their antigen specificities.

Bullous DisordersResults.—Three types of staining were observed: a linear fluorescence along the epidermal side (116 serum samples), the dermal side (19 samples), or a combined pattern with an equivalent staining on both sides of the SSS (7 samples). All 10 PG samples and most BP (93/98) and CP (13/23) samples contained autoantibodies reactive to basement membrane zone antigens localized to the epidermal side of the SSS. Dermal staining was seen in 100% of EBA, 22% of CP, and 3% of BP samples. Combined staining was present in 22% of CP and 2% of BP samples. A good correlation was found between the epidermal staining pattern and the results obtained from immunoblot assay. Of the 116 samples with an epidermal pattern on SSS, 105 defined a BP antigen and none reacted with the EBA antigen or laminin-5 polypeptides. Five samples with epidermal staining from patients with CP reacted with a 168—kd mucosal antigen.

Conclusion.—Indirect IF on SSS may be useful as a screening test in the serologic diagnosis of SABD and may be the only test required when the serum labels on the epidermal side. When dermal staining occurs, however the differential diagnosis between the pemphigoid group of bullous dermatoses and EBA may require an immunoblot assay.

This study evaluated sera from 142 patients with bullous pemphigoid, cicatricial pemphigoid, pemphigoid gestationis, and epidermolysis bullosa acquisita. The authors confirm the long known sensitivity of saline split skin in the evaluation of patients with bullous pemphigoid. More importantly, they describe a heterogeneous population in which immunofluorescence is positive along the dermal portion of saline split skin. These patients may have antibodies against either type VII collagen (the epidermolysis bullosa acquisita antigen), laminin-5, type XVII collagen or type IV collagen. In this circumstance, immunoblotting is necessary to more accurately categorize these individuals.

Sensitivity of Indirect Immunofluorescence, Substrate Specificity, and Immunoblotting in the Diagnosis of Pemphigus

Introduction.—Bullous diseases are diagnosed on the basis of clinical, histologic, and immunologic findings. Several immunologic tests are used to confirm diagnosis of pemphigus and to differentiate between its superficial and deep forms. Sera from 59 patients with pemphigus were tested to compare the sensitivity of the most commonly used assays to detect pemphigus antibodies: indirect immunofluorescence (HF) and immunoblotting (IB).

Methods.—Fifty-two sera were obtained from 41 patients with pemphigus vulgaris (PV) and 22 from 18 patients with pemphigus foliaceus (PF). Control sera from 8 healthy individuals and 19 patients with melanoma were also tested by IIF and IB. The IIF studies were conducted with 2 substrates: monkey and guinea pig esophagus.

Results.—Fifty-four sera (86%) from the patients with pemphigus were positive with IIF testing for intercellular antibodies against monkey or guinea pig esophagus at a titer greater than 1:20. The average end-point titer was 206. All control sera proved negative. Sensitivity of the assay was 87% in patients with PV and 86% in patients with PF. The IB assay, however, was far less sensitive in PF than in PV (positive in 45% vs. 83% of sera, respectively). In general, antibodies in PV reacted more strongly against monkey esophagus and those in PF reacted more strongly against guinea pig esophagus. When intercellular antibodies reacted equally strongly to both substrates, the chances of a patient having one or the other condition were similar. There was good concordance between the results of IIF and IB assays for PV, but not for PF, antibodies.

Discussion.—Immunofluorescence proved to be a more sensitive test for the detection of circulating pemphigus antibodies than immunoblotting, particularly in patients with PF. The use of both tests can increase the likelihood of detecting these antibodies. Monkey esophagus is a good substrate for the detection of PV antibodies, whereas guinea pig esophagus is better for the detection of PF antibodies.

This study evaluates the sensitivity of different immunologic tests in the diagnosis of pemphigus vulgaris and pemphigus foliaceus. The authors conclude that indirect immunofluorescence using monkey or guinea pig esophagus is more sensitive than immunoblotting for the detection of cir-culating antibodies in pemphigus vulgaris and pemphigus foliaceus. They also demonstrate that guinea pig esophageal substrate more strongly reacts with pemphigus foliaceus serum, whereas monkey esophagus substrate more strongly recognized pemphigus vulgaris serum.

Development and Characterization of Desmoglein-3 Specific T Cells From Patients With Pemphigus Vulgaris

Purpose.—Patients with the autoimmune skin disease pemphigus vulgaris (PV) have intraepithelial blisters in the skin and mucous membranes. They also show anti-desmoglein-3 (Dsg3) autoantibodies bound to the surface of keratinocytes from the skin lesions, as well as circulating autoantibodies in the serum. Humoral immunity is thought to play an important pathogenetic role in PV, as evidenced by the finding that passive transfer of IgG from affected patients reproduces the disease in newborn mice. However, the role of T lymphocytes is unclear. The response of T lymphocytes from patients with PV to Dsg3 fusion proteins was investigated, along with the properties of Dsg3-specific T cells.

Methods and Findings.—Proliferation of T cells from PV patients was specifically induced by 3 immunoreactive segments of the Dsg3 ectodomain. T lymphocytes from 14 PV patients were studied. In all cases but 1, the T lymphocytes responded to at least 1 of the Dsg3 peptides, whereas control T lymphocytes did not respond. The Dsg3 peptides stimulated a mainly CD4-positive T cell population. Further studies were performed to develop Dsg3-specific T cell lines and clones, which expressed a CD4-positive memory T cell phenotype and secreted a Th2-like cytokine profile in response to stimulation. Only an anti-HLA-DR-anti- body inhibited proliferation of Dsg3-specific T cell clones in response to recombinant Dsg3—anti-HLA-DQ or -DP antibodies did not.

Conclusions.—This study demonstrates a polyclonal T cell response against Dsg3 in PV. The T-cell response is just part of the complex range of PV reactivity to self antigens. The findings will be of value in clarifying the cellular immune abnormalities that lead to the production of pathogenic IgG antibodies in PV.

The authors attempt to unravel the pathogenesis of pemphigus vulgaris, and demonstrate that desmoglein-3 specifically induces a proliferation of T cells in affected patients. They thus provided evidence that the T cell response to desmoglein-3 is polyclonal and, therefore, plays not only a permissive role in the expression of this disease, but may actually promote the generation of autoantibodies.

Lack of Mucosal Involvement in Pemphigus Foliaceus May Be Due to Low Expression of Desmoglein 1

Purpose.—In patients with pemphigus, autoantibodies induce loss of keratinocyte cell adhesion, thus leading to blister formation. The 2 major subtypes are pemphigus vulgaris (PV) and pemphigus foliaceus (PF), the autoimmune targets of which are desmoglein (Dsg)3 and Dsg1, respectively. In both forms, oral mucosa demonstrates positive immunofluores¬cence staining; however, oral mucosal lesions are observed mainly in PV. Expression of Dsg3 and Dsgl in the squamous mucosal epithelium and epidermis was assessed in an attempt to explain the difference in mucosal involvement between PV and PF.

Methods.—Immunofluorescence staining was performed using Dsg iso- type-specific antibodies produced by immunoadsorption of PV sera with recombinant Dsg1 or Dsg3 baculoprotein. Expression of Dsgl and Dsg3 in epidermal and oral mucosal extracts was compared by immunoblotting. In these studies, the total amount of desmosomal proteins applied was adjusted to produce the same degree of staining intensity for desmoplakin, a cytoplasmic plaque protein of desmosomes.

Results.—In the immunofluorescence studies of the skin, Dsg3-specific antibodies stained keratinocyte cell surfaces mainly in the basal and immediate suprabasal epidermal layers. With Dsg1-specific antibodies, staining was most intense in the superficial epidermis. There was greater staining intensity for Dsg1 than Dsg3. In the oral mucosa, anti-Dsg3-specific antibodies stained throughout the epithelium, most intensely in the upper two thirds. Although Dsg1-specific antibodies also stained throughout the epithelium, the staining was not as intense as for Dsg3. Staining for Dsg3 was stronger than for Dsg1 in other mucosal epithelia as well. In immunoblotting studies of mucosal extract, the Dsgl band was weaker than the Dsg3 band. In the epidermal extract, the opposite was true.

Conclusions.—These findings indicate that the distribution of Dsg1 and Dsg3 expression is similar throughout the squamous mucosal epithelia. However, expression of Dsg1 is much less intense than that of Dsg3. Thus anti-Dsg1 autoantibodies in PF may block the activity of Dsg1 in the mucosal epithelia. However, Dsg3 within the same desmosomes may be adequate for cell-cell adhesion. This could account for the lack of oral mucosal lesions in most patients with PF.

In skin, the blisters that occur in PF. form within the granular zone as a result of autoantibodies directed against Dsg1, which is the predominant desmoglein subtype present within the upper portion of the epidermis. Desmoglein 3 on the other hand, which mediates PV, is a predominant subtype within the lower portion of the spinous layer. Shirakata et al. have demonstrated, however, that within mucosal tissue there is no stratification of the expression of these 2 Dsg subtypes. That is, both Dsg3 and Dsg1 are present throughout the mucosal epithelium, and Dsg3 is uniformly the predominant subtype. These authors, therefore, propose that the absence of mucosal involvement in PF is caused by the absence of preferential expression of Dsg1 within any layer of mucosal epithelium.

Intercellular IgA Vesiculopustular Dermatosis: An Additional Case and a Review of the Literature

Introduction.—Recent studies report 28 cases demonstrating clinical features of vesiculopustular eruptions with intercellular IgA deposits in the epidermis. Although described under different names, these cases are now recognized as a distinct clinical entity. The patient reported here illustrates a typical case of an intraepidermal neutrophilic IgA dermatosis (IEN) type of intercellular IgA vesiculopustular dermatosis (IAVPD).

Case Report.—Man, 79, was seen for skin lesions localized on the trunk, groin, and nape of the neck. Lesions varied from 1 to 4 cm in size, extended peripherally with vesiculation, and exhibited a flower-like configuration. The oral mucosa was not involved and Nikolsky’s sign was negative. The patient’s serum IgA level was elevated to 516 mg/dL. Intercellular deposition of IgA was revealed by direct immunofluorescence of the perilesional skin. Histologic examinations revealed intraepidermal bullae containing many neu¬trophils and a few eosinophils. Treatment with an oral corticosteroid was not effective, but the lesions responded dramatically to dapsone (75 mg/day). The patient has been free of eruptions for 18 months and requires no continuing medication.

Conclusion.—Intercellular IgA deposits in the epidermis and atypical clinical features characterize IAVPD. Eighteen previous cases were of the subcorneal pustular dermatosis (SPD) type and 12 were of the IEN type. Both tend to localize to the trunk and spare the oral mucosa. Tables included in the article compare SPD and IEN types by clinical appearance, location of IgA deposits in the epidermis, and effective treatment.

The authors present a new case of IAVPD, a relatively rare entity but an important clinical subset of the bullous dermatoses. The authors provide an extensive review of the literature, including subcorneal variants as well as intraepidermal variants. Reviewed are the reactivities of those variants that recognize desmocollin I and II (115-kD and 105-kD, respectively) as well as evidence demonstrating that these autoantibodies are distinct from those seen in pemphigus vulgaris/foliaceus. This paper emphasizes the need to perform immunofluorescence in cases of these bullous diseases as this particular subset of patients may preferentially respond to dapsone.

A Major Role for Neutrophils in Experimental Bullous Pemphigoid

Background.—The inflammatory subepidermal blistering disease bullous pemphigoid (BP) has been linked to an IgG -autoimmune response to BP 180, a hemidesmosomal protein. Experiments in a passive transfer mouse model have produced a blistering skin disease similar to BP in response to antibodies to the murine BP180 ectodomain. Subsequent studics suggested that inflammatory cells may play an immunopathogenic role in this condition. The role of neutrophil involvement in the pathogenesis of BP was studied in this disease model.

Methods and Findings.—BALB/c mice were treated with neutrophil- specific antibodies to deplete them of circulating neutrophils. In these animals, anti-mBP180 IgG did not produce the BP-like blistering skin disease. The neutrophil-depleted mice showed no signs of inflammatory infiltration or blistering, although they did show deposition of IgG and complement at the dermal-epidermal junction.

Further experiments were performed in C5-deficient mice, which resist the disease-producing activity of anti-mBP180 IgG. In these animals, in- tradermal administration of interleukin-8 or C5a, both neutrophil chemoattractants, conferred susceptibility to anti-mBP180 IgG. BALB/c mice were given intraperitoneal injections of interleukin-8, producing peritoneal sequestration of neutrophils. This treatment blocked neutrophil infiltration of the skin after anti-mBP190, thus preventing development of the BP-like disease.

Conclusions.—These experiments provide initial evidence that neutro-phils are involved in the pathogenesis of BP. In the mouse model studied, neutrophils are recruited to the skin through a C5-dependent pathway, where they play a key role in subepidermal blistering. The findings may provide not only useful pathogenic insights but also potentially useful approaches to treatment.

This elegantly done study establishes the key role of neutrophils in the blistering that occurs in BP and demonstrates the essential function of C5a in recruiting neutrophils to the target site. Other studies by the same and other authors have found that subepidermal blistering in this experimental model is mediated by molecular interaction between Fc of anti-BP180 IgG and Fc receptors of neutrophils, and is dependent upon neutrophil elastase activity. These findings may lead to new approaches to the treatment of BP.

Pemphigoid Presenting as Atypical Excoriated Prurigo: Regarding 11 cases

Purpose.—It is sometimes unrecognized that simple excoriated prurigo or an atypical pruriginous eruption can be an early sign of bullous pemphigoid. Eleven patients with excoriated prurigo or pruriginous eruption, in which the diagnosis was made only by direct immunofluorescence, were reported.

Patients.—The patients were 8 men and 3 women (age range, 52-99). All had simple prurigo or prurigo with other nonspecific lesions. All patients had constant itching that did not respond to topical therapy but was severe enough to interfere with the patients’ sleep and general welfare. The histologic findings did not suggest bullous pemphigoid. However, the correct diagnosis was made by direct immunofluorescence studies. A series of biopsy specimens from cases that were clinically diagnosed as prurigo but were negative on direct immunofluorescence testing was studied as well.

Findings.—The histologic findings in the 11 immunofluorescence-positive and 7 immunofluorescence-negative cases were similar. The histologic diagnosis in the immunofluorescence-negative cases was excoriated prurigo in 2 cases, excoriated eczema in 2, suspected bullous pemphigoid in 2, and eosinophilic spongiosis in 1. In the immunofluorescence-positive group, the histologic diagnoses were prurigo in 5 cases, eczema in 2, nonspecific in 2, toxidermia in 1, and lichenification in 1. In most specimens, eosinophils were a rare finding. However, they were present to some extent in prurigo associated with urticarial lesions or with a bulla or fissure in the epidermis or at the dermal-epidermal junction.

Conclusion.—Histologic examination cannot distinguish between simple prurigo and bullous pemphigoid. The diagnosis of bullous pemphigoid cannot be made based on the presence of subepidermal fissures and eosinophils. Direct immunofluorescence is the only reliable way to distinguish bullous pemphigoid from prurigo.

Depending on the degree of cutaneous inflammation, bullous pemphigoid can be an extremely pruritic dermatosis. Urticarial plaques are often seen, occasionally in the absence of bullae. Gengoux and Lachapelle report 11 patients seen with simple prurigo or prurigo associated with other nonspecific lesions. Pruritus, often of unknown etiology, is a common symptom in the elderly, and the authors argue that bullous pemphigoid should be considered in the investigation of a possible causative factor. As the histology in these patients was nonspecific, the diagnosis could only be made by direct immunofluorescence testing.

T-Cell Receptor Vβ Expression Is Restricted in Dermatitis Herpetiformis Skin

Introduction.—Patients with dermatitis herpetiformis (DH) have skin lesions with a significantly increased number of CD3+ T-cells compared with uninvolved skin. The role of these cells in the pathogenesis of DH skin lesions is unknown. A finding of restricted T-cell receptor (TCR) Vβ gene expression, demonstrated in psoriasis, sarcoidosis, and other disorders, could indicate that recognition of a specific antigen(s) or superantigen is important in the pathogenesis of DH skin lesions. This hypothesis was examined in a study of skin biopsies from patients with DH.

Methods.—The skin biopsies were from 10 patients ranging in age from 27 to 71 years. Two 4-mm punch biopsies, 1 from the blister and 1 from uninvolved skin, were taken 7 days after discontinuation of treatment with dapsone and/or sulphonamides. The expression of 11 T-cell receptor Vβ families in biopsy material was examined by immunoperoxidase staining and compared with peripheral blood lymphocytes (PBLs).

Results.—Compared with uninvolved skin, the upper dermis of involved skin exhibited a significantly increased number of CD3-positive lymphocytes (mean 17 vs. 89 per high-power field, respectively). No CD3-positive lymphocytes were found in the epidermis. Six of the 10 patients had significant over-representation of Vβ2, 8 had significant over-representation of Vβ5.2/5.3, and 7 had significant over-representation of Vβ5.3. Over-representation was considered significant if the number of T-cells expressing the Vβ receptor in the skin was at least double the number present in the PBLs.

Conclusion.—Over-representation of specific TCR Vβ subsets (Vβ2, Vβ5.2/5.3, Vβ5.3) was found in DH lesional skin compared with blood. There was a particularly large number of T-cells expressing Vβ2 (in 1 patient, 50% of infiltrating T-cells expressed Vβ2). These findings implicate the involvement of an antigen or superantigen in the pathogenesis of DH skin lesions.

The authors categorize the overexpression of specific T-cell receptor Vβ families 2, 5.2/5.3 and 5.3 in patients with dermatitis herpetiformis. Their work suggests the possibility that recognition of a superantigen is involved in the pathogenesis of this disorder.