1Department of Dermatology, University of Freiburg, 2Kepler High School Freiburg, 3Centre for Biological Signalling Studies (BIOSS), University of Freiburg
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Csorba, K., Sitaru, S., Sitaru, C. Granulocyte-dependent Autoantibody-induced Skin Blistering. J. Vis. Exp. (68), e4250, doi:10.3791/4250 (2012).
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.
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:
Concentration by ultrafiltration with Amicon tubes:
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.
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.
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:
When returning to the laboratory:
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).
4. Analysis of Skin and Plasma Samples
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 perilesional skin. In lesional skin subepidermal blisters and inflammatory infiltrate are seen by histology.
However, if the injection of pathogenic collagen VII-specific antibodies is discontinued, the disease activity gradually becomes lower, and within several weeks the lesions heal. Nevertheless, a certain degree of postinflammatory cicatricial alopecia may persist indefinitely.
Characterization of the recombinant autoantigen (and of pathogenic IgG)
Murine collagen specific IgG binds at the dermal -epidermal junction (Figure 2B). The specificity is assessed by immunoblot (Figure 2C, lanes 3, 4 and 7).
Scoring disease activity
The clinical evaluation of the disease is based on a scoring system we developed (Table 1): 0, no lesions; 1, less than 1% of the skin surface; 2, 1-5% of the skin surface; 3, 5-10% of the skin surface; 4, 10-20% of the skin surface is affected. IgG against murine collagen VII induces cutaneous lesions such as erythema, alopecia, blisters, erosions, crusts on the ears, eyes, snout, limbs and trunk of Balb/c mice (Figure 3).
Analysis of tissue-bound and circulating collagen-specific antibodies
Deposition of rabbit IgG, and mouse complement C3 are detected in frozen, perilesional tissue sections (Figure 4D and E respectively). The subepidermal blisters and the inflammatory infiltrate are seen in histological samples (Figure 4F). Circulating rabbit antibodies are analyzed by ELISA (Figure 5). Frozen tissue and/or organ extracts are analyzed by different assays for their protein and enzyme content (MPO assay).
Figure 1. Overall scheme of the in vivo blistering induced by the passive transfer of collagen VII-specific antibodies. Rabbits are immunized with murine collagen VII and rabbit IgG is purified from the immune sera. Subsequently, the specific autoantibodies are injected subcutaneously into mice following an injection/bleeding schedule. Mice are being checked for general health condition and disease signs daily.
Figure 2. Characterization of pathogenic collagen VII-specific IgG. Indirect IF analysis of salt-split normal mouse skin sections incubated with pre-immune rabbit serum and with murine collagen VII-specific immune rabbit serum results in no deposition (A) and deposition of autoantibodies at the dermal epidermal junction (B), respectively. The specific antibodies recognize the antigen(s) they were raised against when immunoblot with a set of overlapping recombinant murine collagen VII fragments is performed (C, lanes 3, 4 and 7).
Figure 3. Clinical evaluation of mice. IgG to murine collagen VII induces cutaneous lesions such as alopecia, blisters, erosions, crusts on the ears, eyes, snout, limbs and trunk of Balb/c mice (A-D). Mice injected with specific autoantibodies reach a score of 4, whereas the ones injected with NRIgG or Abs against an indifferent protein had a score of 0 (E). The clinical score was calculated as follows: 0, no lesions; 1, less than 1% of the skin surface; 2, 1-5% of the skin surface; 3, 5-10% of the skin surface; 4, 10-20% of the skin surface is affected. Weight loss of 5-10% of the total body weight during three consecutive days counts as an extra point in the final score.
Figure 4. Histo- and immunopathological findings in mice injected with collagen VII-specific IgG. Deposition of rabbit IgG (D), and mouse complement C3 (E) is detected by direct IF at the dermal epidermal junction in cryosections of perilesional skin, in vivo. In lesional skin subepidermal blisters and inflammatory infiltrate is seen by histology (F). No deposition of rabbit IgG (A), mouse complement C3 (B), nor is subepidermal blister formation seen in the controls (C).
Figure 5. Immunoassay of plasma from mice injected with collagen VII-specific IgG. Plasma levels of circulating rabbit antibodies were measured by ELISA.
Table 1. Skin blistering disease scoring sheet. Click here to view larger table.
Extra hints when scoring:
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 autoimmune dermatoses. It emerged also, as an exquisitely instructive model disease to study fundamental, biologically and clinically crucial aspects of antibody-mediated organ-specific autoimmune diseases that extend well beyond the limits of autoimmunity against collagen VII. Finally, this model may be used to study fundamental biological and pathophysiological processes, including basement membrane biology, granulocyte activation by immune complexes and complement activation.
Although several experimental settings, including ex vivo and animal models, are available for the autoimmune skin disease EBA 14, by far the most suitable to study the granulocyte-dependent inflammatory pathways is the passive transfer of autoantibodies into animals. In contrast to the ex vivo model, where the granulocytes are added to the skin sections previously incubated with the autoantibodies 15, here the infiltration of leukocytes is spontaneously reproduced by the mice. Furthermore, if we choose to work with specific antibodies raised against the antigen in other organisms, such as rabbit and goat, we avoid the problem of limited patient sera availability and we also have a better chance to reproduce complement activation and granulocyte recruitment 16.
For the successful reproduction of granulocyte-dependent blistering in the passive transfer model of EBA, it is important to be able to adequately score the signs of disease, starting with erythema and oedema through blisters, erosions with crusts and alopecia. The evaluation of mice is a process that needs to be previously learned. The scoring of the disease has to be performed by the same person, throughout the experiment and always seconded by a collaborator or aid person. The development and use of a standardized scoring system for each experimental disease is of the outmost importance. The enclosed scoring sheet should give the reader an insight how skin dermatoses are evaluated in mice.
When performed as shown in the video, the model of autoantibody-induced subepidermal blistering will greatly facilitate the further dissection of the granulocyte-dependent inflammatory pathways triggered by autoantibodies and resulting in tissue damage. Its value in medical research is supported by the promising perspective of developing and testing new antigen-specific T and B cell targeted anti-inflammatory therapies. Finally, it will help in answering fundamental biologically and clinically essential autoimmunity questions.
No conflicts of interest declared.
The authors acknowledge support by grants from the Deutsche Forschungsgemeinschaft SI-1281/4-1 and BIOSS from the Medical Faculty of the Freiburg University (to CS).
|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|
|digital camera||Nikon||Coolpix 5400|
|Syringe driven filter unit 0.45 μm||Millipore|
|Caliper||Mitutoyo 7309||1667338||Farnell (distributor)|
|disease scoring sheet||example enclosed|