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 b....

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