Direct and indirect immunofluorescence (IF) plays a role in the evaluation of immunobullous diseases and their mimics, and in the investigation of vascular injury syndromes and autoimmune connective tissue disease (CTD). IF mapping may be an important adjunct in the assessment of congenital epidermolysis bullosa syndromes and in Alport disease, in which antibodies are directed at certain components of the basement membrane zone to assay for their deficiency. In many cases of immunobullous and autoimmune CTDs, correlation with direct IF results is useful and often decisive in lesional evaluation and thus in patient management.
- •
Direct immunofluorescence is a key adjunct in the investigation and diagnosis of a variety of dermatoses.
- •
In particular, the evaluation of immunobullous and connective tissue disease and vasculitis is often incomplete without direct immunofluorescence testing.
- •
Immunofluorescence using knockout models can be used to map antibody specificities in some immunobullous disorders.
- •
The incubation of patient serum with human umbilical cord endothelia in indirect immunofluorescence assays using a linking antibody has a role in predicting the severity of disease and the distribution of organ system involvement in certain systemic connective tissue disease states.
Introduction
The 2 principal techniques used in immunofluorescence (IF) testing are direct IF (DIF) and indirect IF (IIF). DIF testing uses fluorescein-conjugated antibodies monospecific for immunoglobulin (Ig) G, IgM, and IgA and complement fractions including C3, C3d, C4d, and C1q directly overlaid on frozen sections of patient tissue. IIF by definition uses a linking antibody, and that term should be used irrespective of the test substrate, be it patient lesional tissue or nonhuman esophagus, skin, or bladder. However, in common terminology, IIF refers to the application of patient serum in concert with fluorescein-conjugated anti-IgG and a mucosal substrate such as guinea pig or monkey esophagus or rat bladder. The 3 main disease categories in which DIF may be a critical diagnostic adjunct are vesiculobullous disorders, vasculitis, and autoimmune connective tissue disease (CTD) ( Box 1 ). The light microscopy of several other non–immune-based disorders can mimic certain of the conditions mentioned earlier, but most of these non–immune-based blistering diseases have nonspecific or negative DIF and IIF profiles ( Table 1 ). This article focuses on the various conditions associated with diagnostic IF profiles and their pathophysiologic basis. The dominant focus is on the immunobullous diseases, bullous pemphigoid (BP), epidermolysis bullosa acquisita (EBA), cicatricial pemphigoid, P200 pemphigoid, linear IgA disease, dermatitis herpetiformis (DH), and bullous systemic lupus erythematosus (SLE). In addition, IF application in the evaluation of CTDs including lupus erythematosus, mixed connective tissue disease (MCTD), dermatomyositis (DM), and sclerodermatomyositis is discussed. Its value in the approach to cutaneous vasculitis is considered. The use of the antiendothelial cell antibody assay is also discussed, which may be used in concert with routine microscopy and DIF in evaluating for certain CTDs associated with microvascular injury, as exemplified by DM and MCTD. Immunofluorescent studies have also been used to elucidate the presence or absence of certain basement membrane zone (BMZ) components in geno dermatoses such as congenital epidermolysis bullosa and Alport syndrome. Consideration is also given to nondiagnostic DIF profiles that may be encountered in skin biopsy material.
Autoimmune vesiculobullous disorders:
Antibodies directed to components of the epidermal basement membrane zone
Bullous and cicatricial pemphigoid
Herpes gestationis
P200 pemphigoid
Linear IgA disease
Bullous systemic lupus erythematosus
Epidermolysis bullosa acquisita
Antibodies to intercellular components of the epidermis
Pemphigus foliaceus
Pemphigus vulgaris
IgA pemphigus
Drug-induced pemphigus
Paraneoplastic pemphigus
Diseases associated with an immune complex–based pattern of IF
Dermatitis herpetiformis
IgA vasculitis
Mixed cryoglobulinemic vasculitis
Urticarial vasculitis
Lupus erythematosus (specifically in the context of the positive lupus band test)
Diseases associated with antibodies to endothelium with C5b-9 as the critical effector mechanism of vascular injury
Lupus erythematosus associated with anti-Ro antibodies
Dermatomyositis
Antiphospholipid antibody syndrome
Bullous disorders unrelated to an autoimmune-based cause
Porphyria
Pseudoporphyria
Bullosa diabeticorum
Collagen vascular disease
Lupus erythematosus
Mixed connective tissue disease
Dermatomyositis
Sclerodermatomyositis
Vasculopathic disorders with distinctive immunofluorescent profiles
IgA vasculitis/Henoch-Schönlein purpura
Mixed cryoglobulinemic vasculitis
Antineutrophil cytoplasmic antibody–positive vasculitic syndromes
Hypocomplementemic urticarial vasculitis
Antiphospholipid antibody syndrome
| Disorder with Negative/Nonspecific IF | Differential Excluded by Virtue of Negative IF |
|---|---|
| Subcorneal pustulosis | IgA pemphigus |
| Hailey-Hailey disease | Pemphigus |
| Bullous impetigo | Pemphigus |
| Darier disease | Pemphigus |
| Grover disease | Pemphigus |
| Acantholytic pityriasis rubra pilaris | Pemphigus |
| Bullous insect bite reaction | Bullous pemphigoid |
| Bullous drug reaction | Bullous pemphigoid |
| Lichen planopilaris | Discoid lupus erythematosus |
| Drug-induced lichenoid photodermatitis | Subacute cutaneous lupus erythematosus |
| Non–IgA-associated vasculitis | IgA vasculitis/Henoch-Schönlein purpura |
Autoimmune conditions associated with antibodies to components of the BMZ
Normal Constituents of the BMZ
The BMZ contains antigens that are the targets of antibodies in autoimmune bullous disorders ( Table 2 ). The uppermost zone is the dermal pole of the basal cell plasma membrane, which contains hemidesmosomes that in turn have an intracellular and extracellular portion. The BMZ proper comprises the lamina lucida or lamina rara, which is traversed by anchoring filaments. The lamina densa surmounts the fibrous network of the sub-BMZ proper; this sub-basal lamina fibrillar network contains anchoring fibrils that are contiguous with the lamina densa and insert in the dermis.
| Component | Location in DEJ |
|---|---|
| Type IV collagen | Lamina densa |
| Laminin | Lamina lucida |
| Heparan sulfate proteoglycan | In clusters on either side of the lamina densa |
| BP antigen | Basal cell plasma membrane |
| EBA antigen | Lamina densa-sublamina densa |
BP antigens
There are 2 BP antigens. One has an intracellular location and is associated with hemidesmosomes; it is designated BP Ag1 (230 kDa). Analysis of its amino acid sequence has revealed that it belongs to the same protein family as the desmosomal plaque protein desmoplakin; this homology accounts for the only known molecular relationship between hemidesmosomes and desmosomes. The genes that encode BPAg1 and desmoplakin are located on chromosome 6. The C-terminal domains are responsible for binding the cytokeratin filaments and are likely the major antigenic epitope. This protein is of major importance for tonofilament binding.
The other BP antigen has both intracellular and extracellular components and is designated BPAg2 (180 kDa). The gene that encodes this antigen is found on chromosome 10q. It is a transmembrane protein with an extracellular C terminus. Although the intracellular portion is basic and is part of the hemidesmosomal plaque, the longer extracellular portion contains collagenlike domains. Because it has intracellular protein features but also manifests collagen components, it is designated as collagen XVII. The extramembranous domain NC16a is the dominant antigenic epitope site for BP and herpes gestationis. The BP180 ectodomain exhibits an extended conformation spanning the lamina lucida and projecting into the lamina densa. The terminal portion of the BP180 ectodomain is in close apposition to the lamina densa and is the autoreactive site for cicatricial pemphigoid.
Antibodies to BPAg1 and BPAg2 are involved in the pathogenesis of BP, with 70% of patients manifesting circulating antibodies against BP230 and 55% having antibodies to BP180. The serum level of anti-BPAg1 autoantibodies inversely correlates with prognosis. For BP and herpes gestationis, IgG4 is the predominant IgG anti-BPAg2 subclass. Patients with BP have IgG anti-BP180, which reduces hemidesmosomal BP180, weakening hemidesmosomal attachment to the lamina densa. BP180 immune complex formation induces inflammation and may damage the lamina lucida and explain the BP split at the level of the lamina lucida. High titers of both IgG and IgE anti-NC16a in patients’ sera indicate a more severe disease course. Enzyme-linked immunosorbent assay (ELISA) for both IgG and IgE anti-NC16a compared with IgG alone gives better diagnostic and prognostic information. High titer of BP180 antibodies correlates with high risk of relapse after cessation of therapy in patients with BP.
Deficiency of BPAg2 and a reduced amount of the corresponding messenger RNA has recently been implicated in generalized atrophic benign epidermolysis bullosa, a form of nonlethal junctional epidermolysis bullosa clinically characterized by generalized blistering after birth, atrophic healing, and alopecia with onset in childhood.
Laminin 5
Laminin 5 is localized in the lamina lucida where it may contribute to the formation of anchoring filaments. It is a high-molecular-weight glycoprotein complex of epithelial basement membranes that is identical to the autoantigen epiligrin and has also been referred to as BM 600, nicein, and kalinin. Autoantibodies directed against laminin 5 are relevant for a certain subtypes of cicatricial pemphigoid referred to as antiepiligrin cicatricial pemphigoid. Mutations in the laminin 5 chain can also be observed in certain forms of junctional epidermolysis bullosa.
Uncein
Uncein is an anchoring filament component that consists of 3 polypeptide chains (100, 135, and 165 kDa) and is related to, but distinct from, laminin 5. Antibodies to uncein have recently been described in a disease that has overlap features of cicatricial pemphigoid and EBA.
Ladinin
A component of anchoring filaments, ladinin (LAD-1), with a molecular weight of 97 kDa, is a novel secretory protein that has basic properties as a result of enrichment of the N-terminal end with basic amino acids. Immunoelectron microscopy localizes ladinin to the lamina lucida beneath the hemidesmosomes. Ladinin seems to be the autoantigen in most cases of linear IgA disease and in chronic bullous disease of childhood.
EBA antigen
Localized within and subjacent to the lamina densa, the EBA antigen constitutes a fibronectin heterodimer that represents the noncollagenous domain of type VII collagen (145 kDa and 290 kDa). The antibody in bullous SLE manifests similar antigen specificity. Mainly synthesized and secreted by keratinocytes, type VII collagen is found in basement membranes beneath the stratified squamous epithelia of the skin, lip, buccal mucosa, upper esophagus, trachea, vagina, and amnion of mammals; it is not found in birds, reptiles, or fish. It is the constituent molecule of the anchoring fibrils that attach the basement membrane to the underlying dermis.
Salt-Split Skin Assay
Incubating normal skin with a 1-M solution of sodium chloride separates the epidermis from the dermis, whereby the upper lamina lucida and the hemidesmosomes including the BP antigens remain adherent to the epidermis. The other BMZ components, including lower lamina lucida antigenic components such as laminin 5, the lamina densa, and anchoring fibrils, remain with the dermal side after separation. This technique enables more precise localization of immunoreactants relative to the lamina lucida, increasing the specificity of DIF. There are certain distinctive vesiculobullous disorders showing localization of immune reactants to the roof of the saline-induced split, namely BP, herpes gestationis, cicatricial pemphigoid (excluding the antiepiligrin variant), and certain cases of linear IgA disease in which the antigenic specificity is to BP antigens. Those diseases showing floor localization include P200 pemphigoid, antiepiligrin pemphigoid, EBA, bullous SLE, and a subset of patients with linear IgA disease.
BP
BP occurs most commonly in the elderly and is associated with intense pruritus, blisters, and frequent truncal localization. Exogenous triggers such as drug therapy or trauma are implicated in some cases. BP is attributable to antibodies to BPAg1 or BPAg2, those components of the BMZ that localize above the salt-split skin zone. BPAg2 is a transmembrane protein, although the antigenic target eliciting BP is the extracellular noncollagenous zone. The less common antigenic epitope, BPAg1, localizes to the hemidesmosomes.
Light microscopy
The biopsy shows an eosinophil-rich subepidermal blistering dermatosis. Early phases of the disease may manifest an eosinophilic interface dermatitis with incipient or no cleft formation or, in some cases, urticarial changes ( Fig. 1 A, B). An elderly individual presenting with an intensely pruritic eruption may represent a prebullous lesion of BP.
