Cristina Has1 and Yinghong He1

 Cristina Has1 and Yinghong He1

Research Techniques Made Simple: Immunofluorescence Antigen Mapping in Epidermolysis Bullosa

Journal of Investigative Dermatology (2016) 136, e65-e71

 

INTRODUCTION

  Figure 1. Morphologic and ultrastructural features of human skin and level of skin cleavage in EB. Transmission electron microscopy pictures depicting desmosomes (upper panel) and the basement membrane zone (lower panel) are shown. The disease-relevant molecular components of desmosomes (upper panel) and the basement membrane zone (lower panel) are depicted schematically. (Transmission electron microscopy courtesy of Dr. Ingrid Hausser, Heidelberg, Germany). Classically, EB was divided into three main types according to where skin cleavage occurs: in the basal epidermal layer, in the lamina lucida, or below the lamina densa of the basement membrane. Subsequently, new genes were discovered, and currently the EB spectrum includes Kindler syndrome and disorders exhibiting epidermal fragility (modified from Has et al., 2013, with permission from Elsevier and Dr. Ingrid Hausser). BPAG1e, bullous pemphigoid antigen-1e; DEB, dystrophic epidermolysis bullosa; EBS, epidermolysis simplex bullosa; JEB, junctional epidermolysis bullosa.

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 Michelís 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 patientís 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/).

 
  Figure 3. Immunofluorescence antigen mapping performed on skin sections from a healthy control (Co) and a patient with interstitial lung disease, nephrotic syndrome, and EB with an intronic unclassified variant. Confocal microscopy was used for visualization. Integrin-a3, integrin-a6, laminin-a3, and collagen VII appear in green. The positions of the blisters are depicted by a cross, and nuclei appear in blue. Scale bars ľ 100mm (reprinted from He et al., 2016, with permission from Elsevier).
  Figure 4. Immunofluorescence antigen mapping performed on skin sections from a healthy control (Co) and three patients with junctional epidermolysis bullosa and COL17A1 mutations (confocal microscopy). Immunofluorescence staining was performed with the following domain-specific antibodies against collagen XVII: Endo2, NC16A, and Hk139. Patients 1 (P1) and 2 (P2) had the mutation p.R1303Q, and patient 3 (P3) had loss-of-function mutations leading to absence of collagen XVII. Note apicolateral staining in the basal keratinocytes with the antibody Endo2 in P1 and P2, similar to the control skin. Immunostaining with HK139 and NC16A, which recognize the ectodomain of collagen XVII, showed a broad, irregular distribution below the level of the basement membrane in intact skin of P1 and P2 and presence of the signal at both roof and base of a blister in P3. Immunofluorescence staining with an antibody against the laminin-g2 chain (clone GB3) shows broad, irregular distribution below the level of the basement membrane in the intact skin of P1. Laminin- g 2 immunostaining is present at both roof and floor of the blister in P2, and only at the blister floor in P3. Crosses indicate blisters, and nuclei are stained with DAPI. Scale bars ľ 50 mm (modified from Has et al., 2014, with permission from Elsevier).

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.

  What immunofluorescence mapping (IFM) does?
  HOW IS IFM PERFORMED? Schematic diagram of the IFM technique
The indicated antigen-specific primary antibodies are incubated to the tissue substrate. The secondary antibodies bind to the unlabelled primary antibodies. Visualization is achieved through the host species specific secondary antibodies, which are fluorescence conjugated.
  Morphologic and ultrastructural features of human skin and level of skin cleavage in EB. TEM pictures depicting desmosomes (upper panel) and the basement membrane zone (lower panel) are shown. The disease-relevant molecular components of desmosomes (upper panel) and the basement membrane zone (lower panel) are depicted schematically. (TEM courtesy of Dr. Ingrid Hausser, Heidelberg, Germany). Classically, EB was divided into three main types according to where skin cleavage occurs: in the basal epidermal layer, in the lamina lucida, or below the lamina densa of the basement membrane. Subsequently, new genes were discovered and currently, the EB spectrum includes the Kindler syndrome and disorders exhibiting epidermal fragility.
(Modified after Chapter 147 by Has C., Bruckner-Tuderman L. and Uitto J., in Emery and Rimoin's Principles and Practice of Medical Genetics, Edited by:David L. Rimoin, Reed E. Pyeritz and Bruce Korf ISBN: 978-0-12-383834-6)
  Primary antibodies for IFM - minimal panel
  Primary antibodies for IFM - extended panel
  Interpretation of the results of IFM (1)
  Interpretation of the results of IFM (2)
  IFM reveals the molecular consequences of an ITGA3 intronic unclassified variant in a patient with ILNEB

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 domain specific antibodies  in junctional EB with COL17A1 mutations

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 

2-Which of the following is not true regarding IFM?

    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.

3-Which is the best biopsy site if EB is suspected and use of IFM is requested?

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.

4-Which of the following statements is true in junctional EB?

In a junctional blister, collagen IV stains at the blister floor
Immunoreactivity for collagen VII is altered
Immunoreactivity for collagen XVII may be altered
Collagen VII stains at the blister roof
The level of cleavage can be assessed by hematoxylin and eosin staining

5-Which of the following is wrong regarding the primary antibodies used in IFM?

A minimal panel of antibodies can be employed
For 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.