Assessment of skin basement membrane zone antibodies in the urine of patients with acquired subepidermal immunobullous diseases

BACKGROUND: In bullous pemphigoid (BP), cicatricial pemphigoid (CP) and linear IgA disease (LAD), autoantibodies to the basement membrane zone (BMZ) are found in skin and mucosa, blood and blister fluid. OBJECTIVES: To assess whether BMZ antibodies might also be detected in urine. METHODS: Urine and serum samples from 62 patients (32 with BP, 17 with CP and 13 with LAD) were analysed for antibody isotypes and subclasses by indirect immunofluorescence, and urine and serum samples from 40 patients (25 with BP, eight with CP and seven with LAD) were screened for target antigens using immunoblotting. RESULTS: Fourteen of 32 patients with BP had detectable levels of IgG BMZ autoantibodies in their urine, and all 32 had positive sera. Of these 14 BP patients, 13 had epidermal-binding serum autoantibodies at a titre > 1 : 160, and one had dermal-binding serum antibodies at a titre of 1 : 40. BMZ autoantibodies were not detected in the urine of the CP or LAD patients, but the corresponding sera were of low titre or negative. IgG subclasses (IgG1-4) were less frequently detected in urine than in serum. IgG4 was the predominant subgroup found (10 urine samples and all 14 sera), followed by IgG1 (two urine samples and 12 sera); IgG2 was detected in a single urine sample and three sera, and IgG3 was not detected. Eight of 25 BP and one of eight CP urine samples were positive on immunoblotting, and bound BP230 and/or BP180 with IgA and/or IgG autoantibodies. IgA autoantibodies were not detected in the urine of the seven LAD patients. The corresponding sera were often more positive, with 21 of 25 BP, five of eight CP and six of seven LAD sera immunoblotting the major BP antigens. CONCLUSIONS: The detection of IgG autoantibodies from urine samples using indirect immunofluorescence correlated with a high titre of IgG autoantibodies in the serum. IgG and IgA autoantibodies in the urine were detected by immunoblotting, although less frequently than in serum. The finding of BMZ antibodies in the urine of many BP patients may have clinical relevance, and may have a restricted application in the diagnosis of immunobullous disease.     

Leukocytoclastic vasculitis in association with linear epidermal basement membrane zone immunoglobulin deposition: Linear vasculitis

Cutaneous leukocytoclastic vasculitis (LCV) has a distinctive clinical and light microscopic presentation; however, the etiologic basis of LCV is varied. Most cases are attributable to immune complex deposition within a vessel wall and represent an Arthus type III immune complex reaction. The prototypic immunoreactant profile is characterized by granular deposits of components of complement activation in concert with immunoglobulin within the cutaneous vasculature. We encountered nine patients with vasculitic and/or vesiculobullous clinical presentations exhibiting an LCV in association with an immunoreactant profile characterized by homogeneous linear deposits of immunoglobulin along the dermal epidermal junction in a fashion resembling an autoimmune vesiculobullous disease.Among the clinical presentations were palpable purpura, urticarial vasculitis, and vesiculobullous eruptions with supervening purpura. Two patients with Crohn disease presented with classic palpable purpura with biopsy-proven LCV, and direct immunofluorescence (DIF) studies demonstrated linear immunoglobulin G (IgG) with floor localization on the salt-split skin assay. Four patients with systemic lupus erythematosus (SLE) showed purpuric vesiculobullous lesions, with evidence of a neutrophilic interface dermatitis and LCV in three of the four. The remaining patient had urticarial nonbullous lesions showing small-vessel vasculitiswith a neutrophilic interface dermatitis. In all of the patients with SLE, DIF studies showed linear immunoglobulin deposits within the basement membrane zone (BMZ). These constellation of findings clinically, light microscopically, and by immunofluorescence were those of a vasculitic presentation of bullous systemic lupus erythematosus. Two patients had linear IgA disease, which was drug induced in one and paraneoplastic in the other, and the dominant morphology on biopsy in both cases was an LCV. One patient microscopically demonstrated drug-associated and eosinophilic enriched LCV with DIF studies showing striking linear deposits of IgG suggestive of bullous pemphigoid, which was consistent with a vasculitic presentation of drug-induced bullous pemphigoid. In all cases, typical granular vascular immunoglobulin and complement deposition compatible with immune complex mediated vasculitis was observed. It is likely that local immune complexes derived from BMZ antigen bound to antibody are pathogenically relevant. We propose the designation of linear vasculitis for this unique scenario of LCV and linear immunoglobulin epidermal BMZ staining, which in some cases represents a vasculitic presentation of conventional autoimmune vesiculobullous disease.     

Presence and interpretation of vascular immune deposits in human skin: the value of direct immunofluorescence.

Direct immunofluorescence investigation of the skin is an easy and valuable technique to establish the diagnosis immune complex vasculitis. Vascular immune deposits can be found in 60-80% of all cases. Absence of vascular immune deposits, however, does not exclude vasculitis per se, since the dynamics of the vasculitic process limit their presence in time. Knowledge of these dynamics is indispensable for both the clinician and the interpreter. Several practical options are discussed that may increase sensitivity. The specificity of vascular immune deposits has become a complex matter. Different immunoglobulin classes have different specificity, indicating that specificity also depends on the relative incidence of individual immunoglobulin classes. Some of these relative incidences seem to have changed over the years. Furthermore, several non-vasculitic diseases and conditions have now been described, that may show fluorescent pictures similar to vasculitis and thus decrease specificity.     

The appearance of four basement membrane zone antigens in developing human fetal skin

In order to study the ontogeny of various structural and antigenic components of the basement membrane zone of human skin, we have examined skin specimens from 20 aborted fetuses ranging in gestational ages from 6 to 25 weeks, utilizing light microscopy, transmission electron microscopy, and indirect immunofluorescence with antibodies to bullous pemphigoid antigen, laminin, type IV collagen, and to the antigen defined by KF-1 monoclonal antibody. Both laminin and type IV collagen were detectable as early as 6 weeks of gestational age. In contrast, bullous pemphigoid antigen and the antigen defined by KF-1 antibody were not detectable before 10 weeks and 16 weeks, respectively. The appearance of bullous pemphigoid antigen correlated with stratification of the epidermis and the formation of hemidesmosomes and anchoring fibrils at the basement membrane zone. KF-1 antigen is first expressed when the epidermis is further stratified, hemidesmosomes and anchoring fibrils are present in greater numbers and with increased frequency at the dermal-epidermal junction, and hair follicles have begun to bud downward from the basal layer of the epidermis. Our findings suggest an orderly sequence to the appearance of these basement membrane zone components within human skin.     

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.     

Circulating anti-skin basement membrane zone antibodies in a patient with Goodpasture''s syndrome

We report the case of a patient with atypical bullous pemphigoid and haemoptysis in whom circulating IgG skin basement membrane zone antibodies were demonstrated. Six years later she developed acute renal failure due to Goodpasture's syndrome. This is the first case to raise the possibility of a link between epidermal and glomerular basement membrane antibodies.     

Immunofluorescence on split skin for the detection and differentiation of basement membrane zone autoantibodies.

The autoimmune subepidermal bullous diseases are characterized by autoantibodies to the basement membrane zone of stratified squamous epithelium. Recent studies have shown that the antibodies have characteristic ultrastructural and antigenic binding properties and that differentiating between those properties can be useful in distinguishing one disease from another. Immunofluorescence microscopy is widely used to detect basement membrane zone autoantibodies. The test has traditionally used tissue substrates with an intact basement membrane zone. Those substrates are limited because autoantibody binding cannot always be detected and because autoantibodies with different ultrastructural and antigenic binding properties cannot be distinguished from each other. Normal human skin that has been separated through the basement membrane zone (i.e., split skin) has recently been used as a substrate for detecting and characterizing basement membrane zone autoantibodies by immunofluorescence. Studies indicate that split skin is a more sensitive substrate than intact skin for detecting the antibodies and that antibodies with different ultrastructural binding sites can often be differentiated from one another on split skin. Those studies suggest split skin is the substrate of choice for the routine immunofluorescence evaluation of autoimmune subepidermal bullous diseases.     

A prospective immunofluorescence study of immune deposits in the skin of primary Sj?gren's syndrome

There are conflicting opinions concerning the epidermal immunofluorescence pattern in primary Sj?gren's syndrome. In a prospective study of 12 patients we found a characteristic pattern of epidermal nuclear/cytoplasmic IgG deposits in 8 (67%). This pattern was associated with the presence of antibodies against SSA/Ro and SSB/La in the serum and was also found in 2 out of 5 LE patients with monospecific antibodies against SSA/Ro. There is a resemblance to the pattern of dust-like particles described in the diseased skin of patients with subacute cutaneous LE. In one patient with primary Sj?gren's syndrome, IgG deposits were confined to epidermal cell nuclei (in vivo ANA). Instead of antibodies against SSA/Ro or SSB/La, this particular patient had nRNP-antibodies. From this study, we conclude that the epidermal IgG deposits in primary Sj?gren's syndrome may represent antibody binding to the sites within epidermal cells where the respective antigens are located.     

The basement membrane and its significance in congenital and acquired diseases of the skin

Basal membranes are an ubiquitous component of all human organs and fulfil a large variety of functions. They separate epithelial from mesenchymal tissue and control the passage of substances, of inflammatory as well as tumor cells. They form the extracellular cytoskeleton, regulate growth processes, and play an important part in wound healing. Ultrastructurally, the lamina densa can be distinguished from the lamina lucida. Anchoring fibrils connect the basal membrane of the dermoepidermal junction with the underlying dermis, while anchoring plates connect it with the epidermal cells. During the last few years, various components of the basal membrane have been biochemically analyzed. Different proteins were characterized, and their corresponding morphological structure could be identified. The growing knowledge regarding the structure and composition of the dermo-epidermal junction has led to a better understanding of many diseases involving this structure, in particular inborn and acquired bullous diseases, but also that of the role of the basal membrane in metastasis and tumor invasions. The characterization of antigens in bullous diseases with circulating antibodies has led to exact diagnostic criteria concerning the often overlapping disorders and allows, e.g., the differentiation between bullous pemphigoid and epidermolysis bullosa acquisita.     

The basement membrane zone in lichen sclerosus: an immunohistochemical study

The alteration in expression of basement membrane zone (BMZ) components in lichen sclerosus was investigated by immunohistochemical staining of skin biopsies from seven patients with histologically confirmed disease compared with controls. Monoclonal antibodies and polyclonal sera directed against proteins of the hemidesmosomes, anchoring fibrils, lamina lucida, lamina densa and BMZ collagens were used. Characteristic histological appearances at the dermo-epidermal junction were reflected in widespread alterations in antigen expression in the epidermal basement membrane and the papillary dermis. Expression of the proteins which constitute the structural scaffold (collagen IV and VII) were increased in lichen sclerosus. Expression of hemidesmosomal proteins which mediate adhesion and cell to matrix interaction (α6/β4 and hullous pemphigoid antigen) and expression of anchoring filament components were markedly reduced, suggesting that the epidermal cells are exposed to selective damage.     

Blister fluid for the diagnosis of subepidermal immunobullous diseases: a comparative study of basement membrane zone autoantibodies detected in blister fluid and serum

The subepidermal immunobullous diseases bullous pemphigoid (BP), cicatricial pemphigoid (CP), pemphigoid gestationis (PG) and linear IgA disease (LAD) are characterized by circulating and in vivo deposition of antibodies to antigens in the cutaneous basement membrane zone (BMZ). Indirect immunofluorescence (IMF) of serum is a routine diagnostic test to detect circulating BMZ antibodies in these diseases. We have compared the titres of IgG and IgA and their subclasses, also of IgM and IgE BMZ antibodies in serum and aspirated blister fluid in 35 adult patients with subepidermal immunobullous diseases: BP ( n  = 30), PG ( n  = 2), CP ( n  = 1), and LAD ( n  = 2), by indirect IMF on intact and salt-split skin. The antibody titre in blister fluid was the same or one dilution less than serum in most cases and there was no significant difference between these results ( P  > 0.05). IgG1 and IgG4 were the predominant subclasses in both blister fluid and serum in BP. Indirect IMF of serum and blister fluid was also carried out on trypsinized epidermal cells in a subgroup of patients with BP ( n  = 19). Typical polar fluorescence was obtained in all 14 cases which had positive indirect IMF on intact and split skin. Our findings demonstrate that blister fluid can be used as an alternative to serum for indirect IMF in subepidermal immunobullous diseases. This avoids the need for venesection and has a practical application in children and those with poor venous access.