Cristina Has1 and Yinghong He1
Research Techniques Made Simple: Immunofluorescence Antigen Mapping in Epidermolysis Bullosa
Journal of Investigative Dermatology (2016) 136, e65-e71
INTRODUCTION
Inherited epidermolysis bullosa (EB) is a group of genetic diseases that is defined by fragility of the skin and mucous membranes.
EB is characterized by a broad spectrum of molecular defects and clinical severity, with more than 30 subtypes described so far (Fine et al., 2014).
EB is the consequence of mutations in genes coding for proteins involved in adhesion of epidermal keratinocytes to each other or to the underlying dermis (Figure 1).
Development of monoclonal antibodies enabled identification of dysfunctional proteins by immunofluorescence antigen mapping (IFM).
Sequencing of gene panels or whole-exome sequencing may allow direct discovery of the underlying genetic defect. Importantly,
IFM provides complementary information to mutation analysis, allowing clinicians and researchers to understand the consequences of the genetic defect on a protein and tissue level.
MOLECULAR BASIS OF EPIDERMAL AND DERMAL-EPIDERMAL ADHESION
The main adhesive structures in the skin examined with IFM are supramolecular protein complexes including desmosomes, hemidesmosomes, the basement membrane, and anchoring fibrils (Figure 1). Desmosomes are major intercellular junctions that provide a high degree of resistance to mechanical forces through stable molecular interactions between desmosomal plaques composed of desmoplakin, plakoglobin, plakophilins, and keratin intermediate filaments. The desmosomal cadherins, desmogleins (1e3), and desmocollins (1e3) insert with one end into the desmosomal plaque and accomplish intercellular connections through interactions between their extracellular domains. In basal keratinocytes facing the basement membrane, keratin intermediate filaments consisting of keratin-5 and -14 heterodimers insert into the inner plaques of the hemidesmosomes containing bullous pemphigoid antigen 1 (BPAG1e, BP230) and plectin, which then interact with the transmembrane proteins, integrin-b4 and collagen XVII (BPAG2, BP180). On the extracellular side, integrin-a6b4 and collagen XVII bind to laminin-332, which is inserted into the lamina densa, a tight molecular network of collagen IV and proteoglycans. Here, collagen VII is attached from the dermal side, in the form of anchoring fibrils, which ensure stable adhesion between the basement membrane and the underlying dermis
HOW IS IFM PERFORMED?
Biopsy technique and tissue processing The choice of the biopsy site is critical for maximizing the quality of IFM results. The skin sample should be taken from skin around a recent blister (less than 12 hours). In older blisters, inflammation and tissue regeneration may induce artifacts. If no fresh blister is present, the skin should be rubbed with an eraser to induce new blister formation, and the biopsy sample should be taken several minutes later. A 3e4-mm punch biopsy should be performed at the rubbed area; if more detailed RNA studies and extended IFM panels are desired, at least a 4-mm punch is recommended. To preserve the proteins and epitopes, the biopsy sample should either be immersed in Michel’s transport medium or normal saline or be directly snap frozen. Recently, ex vivo blister induction was proposed as more sensitive than in vivo blister induction (Mozafari et al., 2014). To avoid artificial cleavage of the skin and degradation of proteins, shipment of samples to the laboratory to perform IFM should not exceed 1e3 days.
Antigen-antibody interaction and visualisation
Monospecific antibodies at experimentally determined dilutions are directly applied to a 5-mm tissue section on a glass slide and incubated for 2 hours or overnight (Figure 2). Usually no blocking or permeabilization step is necessary. The choice of the primary antibodies depends on the available clinical data and on the complexity of the question. A minimal panel of antibodies against collagen types IV, VII, XVII and laminin-b3 chain can be used. For detailed analyses, extended panels can be applied, including antibodies against any protein involved in rare EB subtypes. Four washing steps of 5 minutes each are carried out to remove excess primary antibody. For dilution of antibodies and washing, Tris- or phosphate-buffered saline can be used. A secondary antibody against the IgG of the host species of the primary antibody, conjugated with a fluorescent compound, is then applied to the skin section for 1 hour.
Figure 2. Schematic diagram of the immunofluorescence antigen mapping technique. The indicated antigen-specific primary antibodies are incubated on the tissue substrate. The fluorescence-conjugated secondary antibodies bind to the unlabelled primary antibodies and fluorescence is visualized by microscopy
The most commonly used fluorescent compounds are fluorescein isothiocyanate (excitation and emission at 495 and 519 nm, respectively) and Alexa Fluor (e.g., 488 cyan-green, excitation and emission at 495 and 519 nm, respectively; Invitrogen, Karlsruhe, Germany), the latter having greater photostability and higher fluorescence intensity. After washing, the stained specimen is mounted with a coverslip using a fluorescence mounting medium (e.g., Dako, Hamburg, Germany). Because fluorochromes are prone to photobleaching, rapid but careful observation with fluorescence microscopy is required.
Controls
As a positive control against which to compare a patient’s samples, a normal skin sample should be stained in parallel using the same reagents. As a negative control, secondary antibodies without primary antibodies should be applied.
INTERPRETATION OF THE RESULTS
The interpretation of the results is described in detail in Table 1. In brief, the position of the cleavage plane relative to collagen IV, a marker of the basement membrane that is not affected in EB, indicates a junctional (collagen IV at blister floor) or a dermal (collagen IV at blister roof) blister. In EB simplex, the cleavage mainly occurs within the basal epidermal layer; the fluorescent signals for keratin, plectin, bullous pemphigoid antigen-1 (BPAG1), collagen XVII, or integrin-a6b4 appear at the floor of the blister. In Kindler syndrome, the layer of skin cleavage is variable: intraepidermal, junctional, or dermal. In addition, the intensity of the immunoreactivity as compared with normal skin reflects the relative protein expression in the skin of the patient and has prognostic value. Assessment of the immunofluorescence staining intensity can be done by the observer using a subjective scoring method or by using appropriate software (e.g., ImageJ, available from the National Institutes of Health at http://imagej.nih.gov/ij/).
COMPARISON BETWEEN IFM AND OTHER METHODS
A comparative study between transmission electron microscopy and IFM indicated the superiority of IFM in the diagnosis of EB (Yiasemides et al., 2006). Nevertheless, in particular situations transmission electron microscopy may deliver important morphologic details (e.g., the discovery of a particular subtype of EB simplex because of exophilin-5 gene mutations) (McGrath et al., 2012). IFM
IDENTIFICATION OF NEW EB SUBTYPES AND OF MOLECULAR DISEASE MECHANISMS BY IFM
Over the past three decades, IFM significantly contributed to scientific progress in understanding EB, including the identification of new genetic mutations, the illumination of mechanisms underlying a variety of mutations, and the discovery of revertant mosaicism. In the last example, IFM provided initial proof of collagen XVII expression in normal-appearing revertant areas (Jonkman et al., 1997). Integrin-a3 deficiency in interstitial lung disease, nephrotic syndrome, and EB was elucidated based on IFM findings. In addition, in severely ill patients without clinical evidence for cutaneous fragility and unclassified integrin-a3 variants, IFM analysis of the skin allowed clarification of the molecular basis of the disease (Figure 3) (He et al., 2016). In a late-onset subtype of junctional EB associated with a distinct collagen XVII missense mutation, IFM studies shed light on the disease mechanisms (Figure 4) (Has et al., 2014). Finally, assessment of the relative protein amount by IFM is an important technique to evaluate the efficacy of therapeutic interventions (e.g., cell, protein, or gene therapy).
Immunofluorescence antigen mapping in epidermolysis bullosa Cristina Has and Yinghong He
Clinical and molecular heterogeneity of EB. Upper panel: Residual superficial erosions after blistering on the heel of a boy with acral peeling syndrome (APSS) and transglutaminase 5 mutations, p.[G113C];[L214CfsX15]. Grouped blisters and crusts on the foot of a girl with EB simplex (EBS) caused by the keratin 14 mutation, p.R125C. (c) The right hand of a woman with junctional EB (JEB) and collagen XVII mutations, p.[M1T];[R1226X], showing blisters, erosions, crusts, hypopigmentation and nail loss. Feet of a boy with dystrophic EB (DEB) (c.[425A>G];[3276G>A]) demonstrate crusts, extensive scarring, webbing of the toes, and nail loss. The left hand of a young man with Kindler syndrome (KS) homozygous for the frame shift mutation p.[D153RfsX3];[D153RfsX3], demonstrates pronounced skin atrophy, incipient webbing of the finders and nail dystrophy. Lower panel: In the left panel immunofluorescence staining of normal human skin with antibodies against desmoplakin (red) and collagen IV (green) is shown. Nuclei are in blue. The levels of skin cleavage which correspond to the main EB types and subtypes and the defective proteins are indicated on the right side of the figure.
IFM with the skin sections from a healthy control and a patient with ILNEB with an intronic unclassified variant.
Immunofluorescence staining of the skin of a healthy control and the patient are shown by confocal microscopy. Integrin a3, integrin a6, laminin a3 and collagen VII appear in green.
IFM with the skin sections from a healthy control and three patients with junctional EB and COL17A1 mutations.
Immunofluorescence staining was performed with the following domain specific antibodies against collagen XVII: Endo2, NC16A and HK139, and skin sections from a healthy control, and three patients (P1-3).
MULTIPLE CHOICE QUESTIONS For each question, more than one answer may be correct.
1. Which protein used as a marker in IFM can be altered in dystrophic EB?
Collagen type IV Collagen type XVII Collagen type VII Laminin-332 E. None of the above
Collagen type IV Collagen type XVII Collagen type VII
Laminin-332
E. None of the above
It may indicate the skin layer where cleavage occurs.It may indicate the mutated gene and dysfunctional protein in EB.It may indicate the presence of revertant mosaicism in the skin of an EB patient.The specimen may contain areas with artificial cleavage.It always yields specific and clear results.
Near a recent blister (less than 12 hours) An erosive area The skin should be rubbed with an eraser to induce new blister formation, and a biopsy of that site should be taken several minutes later Palms or soles Any blister will indicate the layer where skin cleavage occurs.
Near a recent blister (less than 12 hours) An erosive area The skin should be rubbed with an eraser to induce new blister formation, and a biopsy of that site should be taken several minutes later
Palms or soles Any blister will indicate the layer where skin cleavage occurs.
In a junctional blister, collagen IV stains at the blister floorImmunoreactivity for collagen VII is alteredImmunoreactivity for collagen XVII may be alteredCollagen VII stains at the blister roofThe level of cleavage can be assessed by hematoxylin and eosin staining
A minimal panel of antibodies can be employedFor an extended IFM, antibody costs are significantly high.Primary antibodies are always conjugated with a fluorescent compound.A negative control (secondary antibodies without primary antibodies) should always be run.An extended panel of primary antibodies can be used depending on the complexity of the question.