Granulocyte-dependent Autoantibody-induced Skin Blistering

Summary

In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human epidermolysis bullosa acquisita (EBA)¹.

Abstract

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to Witebsky's postulates, one of the criteria in diagnosing a disease as autoimmune is the reproduction of the disease in experimental animals by the passive transfer of autoantibodies. For epidermolysis bullosa acquisita (EBA), a prototypic organ-specific autoimmune disease of skin and mucous membranes, several experimental models were recently established. In the animal model described in our present work, purified IgG antibodies against a stretch of 200 amino acids (aa 757-967) of collagen VII are injected repeatedly into mice reproducing the blistering phenotype as well as the histo- and immunopathological features characteristic to human EBA ¹. Full-blown widespread disease is usually seen 5-6 days after the first injection and the extent of the disease correlates with the dose of the administered collagen VII-specific IgG. The tissue damage (blister formation) in the experimental EBA is depending on the recruitment and activation of granulocytes by tissue-bound autoantibodies ^2,-4. Therefore, this model allows for the dissection of the granulocyte-dependent inflammatory pathway involved in the autoimmune tissue damage, as the model reproduces only the T cell-independent phase of the efferent autoimmune response. Furthermore, its value is underlined by a number of studies demonstrating the blister-inducing potential of autoantibodies in vivo and investigating the mechanism of the blister formation in EBA ^1,3,-6. Finally, this model will greatly facilitate the development of new anti-inflammatory therapies in autoantibody-induced diseases. Overall, the passive transfer animal model of EBA is an accessible and instructive disease model and will help researchers to analyze not only EBA pathogenesis but to answer fundamental biologically and clinically essential autoimmunity questions.

Protocol

1. Preparation of Pathogenic Antibody

Note: Purification and concentration of the antibodies should be performed on the same day, as antibodies are not to be stored in 0.1 M glycine pH 2.5-3 (elution buffer) over night (ON).

Affinity purification of IgG: use 25 ml of immune rabbit serum:

1. Thaw the rabbit serum ON at 4 °C, mix it 1:1 with 1xPBS (running buffer) and centrifuge it at 1260 x g for 10 min at 20 °C. Optionally, a filtering step with filter paper might be included after centrifugation in case the serum is fatty.
2. Meanwhile, wash the protein G matrix filled column with 10 bed volumes (bed volume=matrix volume) of 1XPBS (running buffer).
3. Apply the diluted and filtered serum to the column and incubate 1 hr on the rocking platform at room temperature (RT).
4. Collect the flow-through (FT) in a 50 ml Flacon tube. Do not discard it till the concentration and titer of the purified IgG is known.
5. Wash the matrix with 5 bed volumes of 1XPBS and in the meantime prepare 50 ml Falcon tubes for collecting the eluted IgG fraction. Pipette 1 ml pH neutralizing buffer: 1 M TRIS pH 10 in each tube (otherwise the proportion of 1:20 TRIS buffer relative to the amount of eluate to be collected).
6. Apply 100-150 ml elution buffer: 0.1 M glycine pH 2.5-3 to the column and collect 50 ml fraction(s). Flip the tube 2-3x, measure the pH and add neutralizing buffer until you've reached the pH 7.2-7.4.
7. Collect few drops of IgG eluate and using the elution buffer as etalon measure the OD at 280 nm. If the OD is <0.1 stop collecting.
8. Regenerate the protein G matrix and store it following the manufacturer's instructions.

Concentration by ultrafiltration with Amicon tubes:

1. Wash the Amicon Ultra (15 ml /30KDa) ultrafiltration tubes with 1XPBS by centrifuging 15-20 min at 3,220 x g at 4 °C. Discard the FT from the Amicon.
2. Fill the Amicons with 15 ml of eluted IgG fraction and centrifuge for 25-30 min at 3,220 x g and 4 °C; centrifugation time always depends on the protein content of the eluate.
3. Discard FT and repeat ultrafiltration till you have no eluted fraction left.
4. Wash the concentrated IgG extensively, twice, with 1XPBS 25-30 min by centrifugation.
5. Collect the "sticky", yellowish antibody solution from the Amicon filter in a final volume of 1,500-2,000 μl of 1XPBS.
6. Measure concentration of IgG preparation using a spectrophotometer or NanoDrop:
   Conc. (mg/ml) = A(280 nm) x dilution factor /1,4
7. Wash and store the Amicons following the manufacturer's instructions.

2. Injection of Collagen VII-specific IgG into Mice

Before starting the actual experimental procedure, make sure that the experimental protocol is written and all materials are prepared for the experiment.

1. Prepare the data sheets for the disease scoring, ELISAs, immunofluorescence (IF) analysis and myeloperoxidase (MPO) assay ⁴.
2. Label the tubes for blood and organs, cryomolds as well as histology processing and embedding cassettes.
3. The antibody solutions should be prepared fresh or thawed from stocks and characterized with regard to their reactivity (titer by IF microscopy and/or ELISA). The sterile filtered antibody solution should be diluted in PBS in a way that the required dose for injection varies between 250-1000 μl. Make sure that you have enough IgG to perform the entire experiment.
4. Prepare fresh anticoagulant: heparin 20 U/ml and 8.7 mg/ml ketamine/ 1.3 mg/ml xylazine mixture for anesthesia. When sacrificing the mice have labeled tubes with 3.7% formaldehyde prepared.
5. Sterilize surgical instruments (scalpel, scissors, fine point and curved forcipes) and prepare syringes (insulin syringes, 1 and 2 ml syringes), needles, digital camera and extra memory card.

Note: Perform all procedures on ketamine/xylazine or isoflurane narcotized mice. Isoflurane is the preferred anesthesia option for the daily skin checks, as mice recover quickly. However, when taking pictures, 87 mg/kg ketamine and 13 mg/kg xylazine is usually injected subcutaneously. For euthanasia the ketamine/xylazine dosage is increased to 130 mg/kg ketamine and 20 mg/kg xylazine.

1. Check the already marked animals for their general health condition, skin and fur appearance, weigh them and measure their ear thickness (always stick to the same ear).
2. Register observed values and changes in the clinical evaluation sheet.
3. Collect 20-30 μl (2-3 drops) blood from the tail vein in a syringe containing the same volume of anticoagulant.
4. Disinfect the fur and skin using 80% ethanol.
5. Inject the antibody solution subcutaneously into the back using an insulin syringe. (For certain sites (e.g., the ears) only small volumes (10-50 μl) are feasible to be injected.)

Injections should be repeated every second day, 4 times. Balb/c and C57BL6 mice of a body weight of approximately 20 g start to develop the disease 3-4 days after the first administration of 400-500 up to 750 μg/g body weight/injection autoantibodies.

When finishing the experiment:

1. Put the mice under deep narcosis by injecting 130 mg/kg and 20 mg/kg ketamine/xylazine subcutaneously, exsanguinate them from the posterior vena cava and cut the heart out.
2. Collect organs/tissue samples such as skin: tail, ear, blood and esophagus and put them in previously labeled and PBS or 3.7% formaldehyde filled tubes.

When returning to the laboratory:

1. Centrifuge the blood at 1,200 x g, 10 min at RT to separate plasma, transfer the plasma to properly labeled tubes and store them accordingly.
2. Embed the skin biopsy in Optimal Cut Temperature medium using cryomolds, properly label, and store them at -80 °C.
3. Place the tissue samples meant for histology in the labeled embedding molds and keep them in 3.7% formaldehyde till paraffin embedding.

3. Clinical Evaluation of Disease Severity

Mice should be examined daily for lesions of skin and mucosa and the findings should be recorded using appropriate forms. The criteria for early euthanasia is skin lesions affecting more that 20% of the body surface and a weight loss of 5-10% of the total body weight in three consecutive days, counting for a disease score of 5 (Table 1).

1. Place the mouse on its belly. Start with the head area, namely with the right ear and check for blisters, erosions, crust on the inner as well as on the outer side. Continue the same way with the left ear.
2. Right and left eyes are controlled for signs of erythema, alopecia, erosions and/or crust.
3. Forehead and snout area are brushed through next, continuing with the ventral part of the snout, lip and the mucous membrane of the mouth.
4. Examine the right front leg starting from the paw, moving upwards; switch to the left front leg. Same procedure with the right and left hind legs.
5. Brush through the fur on the neck and back area with a curved fine point forceps.
6. Turn the mouse on its back and check the ventral part as well as the ventral sides of the limbs the same way as previously.
7. Check the tail.
8. Take pictures of relevant body parts where the mice develop disease. Attention must be paid to the quality and quantity of the pictures.
9. Calculate the clinical disease scores.

4. Analysis of Skin and Plasma Samples

1. Prepare, store, and/or process all collected tissues according to the plan.
2. Cut frozen tissue sections for IF detection of rabbit antibodies and mouse complement system components.
3. Analyze the different protein and enzyme content (MPO assay) of frozen tissue and/or organ extracts.
4. Section the paraffin embedded tissue and stain with H&E to visualize the extent of tissue damage.
5. Analyze the plasma by ELISA, immunoblot.

Results

The passive transfer of antigen specific antibodies results in a full blown disease in mice, resembling at clinical, histological and immunopathological levels the human EBA. Blisters, crusts, erosions and alopecia develop on the ears, snout, paws, legs, back and around the eyes of the mice. The first clinical signs of the disease will most probably appear on the ears and/or head area. Deposition of specific rabbit IgG, and mouse complement C3 is detected by direct IF at the dermal epidermal junction in cryosections of p...

Discussion

The passive transfer of autoantibodies into experimental animals is a major approach for demonstrating their pathogenicity ^{7, -12}. Animal models obtained through this method, in addition to being the indirect evidence for autoimmunity ¹³, allow the investigation of the efferent phase of the pathogenic mechanism. The passive transfer model of antibody-induced granulocyte-dependent skin blistering of epidermolysis bullosa acquisita (EBA) was used to dissect the mechanisms of tissue damage in autoimmun...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
recombinant antigen			The plasmids encoding for the recombinant forms of murine collagen VII are available from the corresponding author.
immune rabbit serum	www.eurogentec.com		We had New Zealand White rabbits immunized with 200 μg of antigen, 3 times at 2 week intervals. For this purpose we have used the services of Eurogentec S.A., Belgium.
Protein G agarose	Roche Applied Science	11243233001
Balb/c mice	Charles River Laboratories
OCT compound, Tissue-Tek	Sakura Finetek	4583	OCT, optimal cutting temperature
Cryomold standard	Sakura Finetek	4557	25 mm × 20 mm × 5 mm
Cryomold intermediate	Sakura Finetek	4566	15 mm × 15 mm × 5 mm
Uni-Link-Einbettkassette	R. Langenbrinck	09-0503	Histology processing and embedding cassettes
Spezial-Tatowierfarbe Schwarz	H. Hauptnerund Richard Herberholz GmbH Co. KG	71492000	tattooing paste
Tatowierzange TZ1	EBECO E. Becker Co GmbH		tattooing device
Heparin	Carl Roth GmbH Co.	7692.1
Formaldehyde 37%	Carl Roth GmbH Co.	7386
Ketamine hydrochloride	Sigma-Aldrich Chemie GmbH	K2753-1G
Xylazine hydrochloride	Sigma-Aldrich Chemie GmbH	X1251-1G
sterile PBS	Biochrom	L182-50
digital camera	Nikon	Coolpix 5400
Syringe driven filter unit 0.45 μm	Millipore
Caliper	Mitutoyo 7309	1667338	Farnell (distributor)
disease scoring sheet			example enclosed