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).

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:
<ol>
 <li>Thaw the rabbit serum ON at 4 &deg;C, mix it 1:1 with 1xPBS (running buffer) and centrifuge it at 1260 x g for 10 min at 20 &deg;C. Optionally, a filtering step with filter paper might be included after centrifugation in case the serum is fatty.</li>
 <li>Meanwhile, wash the protein G matrix filled column with 10 bed volumes (bed volume=matrix volume) of 1XPBS (running buffer).</li>
 <li>Apply the diluted and filtered serum to the column and incubate 1 hr on the rocking platform at room temperature (RT).</li>
 <li>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.</li>
 <li>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).</li>
 <li>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.</li>
 <li>Collect few drops of IgG eluate and using the elution buffer as etalon measure the OD at 280 nm. If the OD is &lt;0.1 stop collecting.</li>
 <li>Regenerate the protein G matrix and store it following the manufacturer's instructions.</li>
</ol>
Concentration by ultrafiltration with Amicon tubes:
<ol>
 <li>Wash the Amicon Ultra (15 ml /30KDa) ultrafiltration tubes with 1XPBS by centrifuging 15-20 min at 3,220 x g at 4 &deg;C. Discard the FT from the Amicon.</li>
 <li>Fill the Amicons with 15 ml of eluted IgG fraction and centrifuge for 25-30 min at 3,220 x g and 4 &deg;C; centrifugation time always depends on the protein content of the eluate.</li>
 <li>Discard FT and repeat ultrafiltration till you have no eluted fraction left.</li>
 <li>Wash the concentrated IgG extensively, twice, with 1XPBS 25-30 min by centrifugation.</li>
 <li>Collect the "sticky", yellowish antibody solution from the Amicon filter in a final volume of 1,500-2,000 &mu;l of 1XPBS.</li>
 <li>Measure concentration of IgG preparation using a spectrophotometer or NanoDrop: 
 Conc. (mg/ml) = A(280 nm) x dilution factor /1,4 </li>
 <li>Wash and store the Amicons following the manufacturer's instructions. </li>
</ol>
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.
<ol>
 <li>Prepare the data sheets for the disease scoring, ELISAs, immunofluorescence (IF) analysis and myeloperoxidase (MPO) assay 4.</li>
 <li>Label the tubes for blood and organs, cryomolds as well as histology processing and embedding cassettes. </li>
 <li>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 &mu;l. Make sure that you have enough IgG to perform the entire experiment. </li>
 <li>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. </li>
 <li>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. </li>
</ol>
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. 
<ol>
 <li>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). </li>
 <li>Register observed values and changes in the clinical evaluation sheet. </li>
 <li>Collect 20-30 &mu;l (2-3 drops) blood from the tail vein in a syringe containing the same volume of anticoagulant. </li>
 <li>Disinfect the fur and skin using 80% ethanol.</li>
 <li>Inject the antibody solution subcutaneously into the back using an insulin syringe. (For certain sites (e.g., the ears) only small volumes (10-50 &mu;l) are feasible to be injected.) </li>
</ol>
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 &mu;g/g body weight/injection autoantibodies. 

When finishing the experiment:
<ol>
 <li>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. </li>
 <li>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. </li>
</ol>
When returning to the laboratory:
<ol>
 <li>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.</li>
 <li>Embed the skin biopsy in Optimal Cut Temperature medium using cryomolds, properly label, and store them at -80 &deg;C.</li>
 <li>Place the tissue samples meant for histology in the labeled embedding molds and keep them in 3.7% formaldehyde till paraffin embedding.</li>
</ol>
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).
<ol>
 <li>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. </li>
 <li>Right and left eyes are controlled for signs of erythema, alopecia, erosions and/or crust. </li>
 <li>Forehead and snout area are brushed through next, continuing with the ventral part of the snout, lip and the mucous membrane of the mouth. </li>
 <li>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. </li>
 <li>Brush through the fur on the neck and back area with a curved fine point forceps. </li>
 <li>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.</li>
 <li>Check the tail. </li>
 <li>Take pictures of relevant body parts where the mice develop disease. Attention must be paid to the quality and quantity of the pictures. </li>
 <li>Calculate the clinical disease scores. </li>
</ol>
4. Analysis of Skin and Plasma Samples
<ol>
 <li>Prepare, store, and/or process all collected tissues according to the plan. </li>
 <li>Cut frozen tissue sections for IF detection of rabbit antibodies and mouse complement system components.</li>
 <li>Analyze the different protein and enzyme content (MPO assay) of frozen tissue and/or organ extracts.</li>
 <li>Section the paraffin embedded tissue and stain with H&amp;E to visualize the extent of tissue damage. </li>
 <li>Analyze the plasma by ELISA, immunoblot. </li>
</ol>

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	<li>Sitaru, C., Mihai, S., Otto, C., Chiriac, M. T., Hausser, I., Dotterweich, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+dermal-epidermal+separation+in+mice+by+passive+transfer+of+antibodies+specific+to+type+VII+collagen.">Induction of dermal-epidermal separation in mice by passive transfer of antibodies specific to type VII collagen.</a> J. Clin. Invest. 115, 870-878 (2005).</li><li>Kopecki, Z., Arkell, R. M., Strudwick, X. L., Hirose, M., Ludwig, R. J., Kern, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpression+of+the+Flii+gene+increases+dermal-epidermal+blistering+in+an+autoimmune+ColVII+mouse+model+of+epidermolysis+bullosa+acquisita.">Overexpression of the Flii gene increases dermal-epidermal blistering in an autoimmune ColVII mouse model of epidermolysis bullosa acquisita.</a> J. Pathol. 225, 401-413 (2011).</li><li>Kulkarni, S., Sitaru, C., Jakus, Z., Anderson, K. E., Damoulakis, G., Davidson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PI3K&#946;+plays+a+critical+role+in+neutrophil+activation+by+immune+complexes.">PI3K&#946; plays a critical role in neutrophil activation by immune complexes.</a> Sci. Signal. 4, 23-23 (2011).</li><li>Chiriac, M. T., Roesler, J., Sindrilaru, A., Scharffetter-Kochanek, K., Zillikens, D., Sitaru, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NADPH+oxidase+is+required+for+neutrophil-dependent+autoantibody-induced+tissue+damage.">NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage.</a> J. Pathol. 212, 56-65 (2007).</li><li>Mihai, S., Chiriac, M. T., Takahashi, K., Thurman, J. M., Holers, V. M., Zillikens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+alternative+pathway+of+complement+activation+is+critical+for+blister+induction+in+experimental+epidermolysis+bullosa+acquisita.">The alternative pathway of complement activation is critical for blister induction in experimental epidermolysis bullosa acquisita.</a> J. Immunol. 178, 6514-6521 (2007).</li><li>Sesarman, A., Sitaru, A. G., Olaru, F., Zillikens, D., Sitaru, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neonatal+Fc+receptor+deficiency+protects+from+tissue+injury+in+experimental+epidermolysis+bullosa+acquisita.">Neonatal Fc receptor deficiency protects from tissue injury in experimental epidermolysis bullosa acquisita.</a> J. Mol. Med. 86, 951-959 (2008).</li><li>Huang, X. R., Holdsworth, S. R., Tipping, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Th2+responses+induce+humorally+mediated+injury+in+experimental+anti-glomerular+basement+membrane+glomerulonephritis.">Th2 responses induce humorally mediated injury in experimental anti-glomerular basement membrane glomerulonephritis.</a> J. Am. Soc. Nephrol. 8, 1101-1108 (1997).</li><li>Matsumoto, I., Staub, A., Benoist, C., Mathis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arthritis+provoked+by+linked+T+and+B+cell+recognition+of+a+glycolytic+enzyme.">Arthritis provoked by linked T and B cell recognition of a glycolytic enzyme.</a> Science. 286, 1732-1735 (1999).</li><li>Oswald, E., Sesarman, A., Franzke, C., W&#246;lfle, U., Bruckner-Tuderman, L., Jakob, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+flavonoid+luteolin+inhibits+Fc&#947;-dependent+respiratory+burst+in+granulocytes,+but+not+skin+blistering+in+a+new+model+of+pemphigoid+in+adult+mice.">The flavonoid luteolin inhibits Fc&#947;-dependent respiratory burst in granulocytes, but not skin blistering in a new model of pemphigoid in adult mice.</a> PLoS One. 7, e31066 (2012).</li><li>Liu, Z., Diaz, L. A., Troy, J. L., Taylor, A. F., Emery, D. J., Fairley, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+passive+transfer+model+of+the+organ-specific+autoimmune+disease,+bullous+pemphigoid,+using+antibodies+generated+against+the+hemidesmosomal+antigen,+BP180.">A passive transfer model of the organ-specific autoimmune disease, bullous pemphigoid, using antibodies generated against the hemidesmosomal antigen, BP180.</a> J. Clin. Invest. 92, 2480-2488 (1993).</li><li>Anhalt, G. J., Labib, R. S., Voorhees, J. J., Beals, T. F., Diaz, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+of+pemphigus+in+neonatal+mice+by+passive+transfer+of+IgG+from+patients+with+the+disease.">Induction of pemphigus in neonatal mice by passive transfer of IgG from patients with the disease.</a> N. Engl. J. Med. 306, 1189-1196 (1982).</li><li>Toyka, K. V., Brachman, D. B., Pestronk, A., Kao, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myasthenia+gravis:+passive+transfer+from+man+to+mouse.">Myasthenia gravis: passive transfer from man to mouse.</a> Science. 190, 397-399 (1975).</li><li>Rose, N. R., Bona, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+criteria+for+autoimmune+diseases+(Witebsky's+postulates+revisited.">Defining criteria for autoimmune diseases (Witebsky's postulates revisited.</a> Immunol. Today. 14, 426-430 (1993).</li><li>Sitaru, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+models+of+epidermolysis+bullosa+acquisita.">Experimental models of epidermolysis bullosa acquisita.</a> Exp. Dermatol. 16, 520-531 (2007).</li><li>Sitaru, C., Kromminga, A., Hashimoto, T., Br&#246;cker, E. B., Zillikens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibodies+to+type+VII+collagen+mediate+Fcgamma-dependent+neutrophil+activation+and+induce+dermal-epidermal+separation+in+cryosections+of+human+skin.">Autoantibodies to type VII collagen mediate Fcgamma-dependent neutrophil activation and induce dermal-epidermal separation in cryosections of human skin.</a> Am. J. Pathol. 161, 301-311 (2002).</li><li>Sesarman, A., Oswald, E., Chiriac, M. T., Csorba, K., Vuta, V., Feldrihan, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+human+pemphigoid+autoantibodies+do+not+trigger+disease+by+the+passive+transfer+into+mice.">Why human pemphigoid autoantibodies do not trigger disease by the passive transfer into mice.</a> Immunol. Lett. , (2012).</li><li>Ishii, N., Recke, A., Mihai, S., Hirose, M., Hashimoto, T., Zillikens, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Autoantibody-induced+intestinal+inflammation+and+weight+loss+in+experimental+epidermolysis+bullosa+acquisita.">Autoantibody-induced intestinal inflammation and weight loss in experimental epidermolysis bullosa acquisita.</a> J. Pathol. 224, 234-244 (2011).</li></ol>

No conflicts of interest declared.

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...

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

granulocyte-dependent autoantibody-induced skin blistering

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.

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...

Watch this Scientific Journal Video about Granulocyte-dependent Autoantibody-induced Skin Blistering at JoVE.com

Granulocyte-dependent Autoantibody-induced Skin Blistering

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.

Autoimmune phenomena occur in healthy individuals, but when self-tolerance fails, the autoimmune response may result in specific pathology. According to ...

granulocyte-dependent-autoantibody-induced-skin-blistering

Research

JoVE Journal

Immunology and Infection

10.4K Views.  University of Freiburg. 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.

Video: Granulocyte-dependent Autoantibody-induced Skin Blistering

Cutaneous Leishmaniasis in the Dorsal Skin of Hamsters: a Useful Model for the Screening of Antileishmanial Drugs

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of lesion size is a hard procedure and animals show difficulty to eat, breathe and move because of the lesion. In order to optimize the hamster model for cutaneous leishmaniasis, young adult male and female golden hamsters (Mesocricetus auratus) were injected intradermally at the dorsal skin with 1 to 1.5 x l07 promastigotes of Leishmania species and progression of subsequent lesions were evaluated for up to 16 weeks post infection. The golden hamster was selected because it is considered the adequate bio-model to evaluate drugs against Leishmania as they are susceptible to infection by different species. Cutaneous infection of hamsters results in chronic but controlled lesions, and a clinical evolution with signs similar to those observed in humans. Therefore, the establishment of the extent of infection by measuring the size of the lesion according to the area of indurations and ulcers is feasible. This approach has proven its versatility and easy management during inoculation, follow up and characterization of typical lesions (ulcers), application of treatments through different ways and obtaining of clinical samples after different treatments. By using this method the quality of animal life regarding locomotion, search for food and water, play and social activities is also preserved.

Optimization of the experimental hamster model for cutaneous leishmaniasis by intradermal injection of Leishmania promastigotes at the dorsal skin. This approach is useful during inoculation, follow-up, characterization of lesions, application of treatments and obtaining of clinical samples. Locomotion, search for food and water, play and social activities are preserved.

Traditionally, hamsters are experimentally inoculated in the snout or the footpad. However in these sites an ulcer not always occurs, measurement of ...

Imaging of HIV-1 Envelope-induced Virological Synapse and Signaling on Synthetic Lipid Bilayers

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between CD4+ T cells involves the formation of a virological synapse (VS), which is an F-actin-dependent cell-cell junction formed upon the engagement of HIV-1 envelope gp120 on the infected cell with CD4 and the chemokine receptor (CKR) CCR5 or CXCR4 on the target cell 8. In addition to gp120 and its receptors, other membrane proteins, particularly the adhesion molecule LFA-1 and its ligands, the ICAM family, play a major role in VS formation and virus transmission as they are present on the surface of virus-infected donor cells and target cells, as well as on the envelope of HIV-1 virions1,4,5,6,7,13. VS formation is also accompanied by intracellular signaling events that are transduced as a result of gp120-engagement of its receptors. Indeed, we have recently showed that CD4+ T cell interaction with gp120 induces recruitment and phosphorylation of signaling molecules associated with the TCR signalosome including Lck, CD3&zeta;, ZAP70, LAT, SLP-76, Itk, and PLC&gamma;15.
In this article, we present a method to visualize supramolecular arrangement and membrane-proximal signaling events taking place during VS formation. We take advantage of the glass-supported planar bi-layer system as a reductionist model to represent the surface of HIV-infected cells bearing the viral envelope gp120 and the cellular adhesion molecule ICAM-1. The protocol describes general procedures for monitoring HIV-1 gp120-induced VS assembly and signal activation events that include i) bi-layer preparation and assembly in a flow cell, ii) injection of cells and immunofluorescence staining to detect intracellular signaling molecules on cells interacting with HIV-1 gp120 and ICAM-1 on bi-layers, iii) image acquisition by TIRF microscopy, and iv) data analysis. This system generates high-resolution images of VS interface beyond that achieved with the conventional cell-cell system as it allows detection of distinct clusters of individual molecular components of VS along with specific signaling molecules recruited to these sub-domains.

This article describes a method to visualize formation of an HIV-1 envelope-induced virological synapse on glass supported planar bilayers by total internal reflection fluorescence (TIRF) microscopy. The method can also be combined with immunofluorescence staining to detect activation and redistribution of signaling molecules that occur during HIV-1 envelope-induced virological synapse formation.

Human immunodeficiency virus type 1 (HIV-1) infection occurs most efficiently via cell to cell transmission2,10,11. This cell to cell transfer between ...

Transplantation of Tail Skin to Study Allogeneic CD4 T Cell Responses in Mice

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and host T cells, to easily monitor the graft, and to adapt the system in order to answer different immunological questions. Medawar and colleagues established allogeneic tail-skin transplantation in mice in 1955. Since then, the skin transplantation model has been continuously modified and adapted to answer specific questions. The use of tail-skin renders this model easy to score for graft rejection, requires neither extensive preparation nor deep anesthesia, is applicable to animals of all genetic background, discourages ischemic necrosis, and permits chemical and biological intervention. 
In general, both CD4+ and CD8+ allogeneic T cells are responsible for the rejection of allografts since they recognize mismatched major histocompatibility antigens from different mouse strains. Several models have been described for activating allogeneic T cells in skin-transplanted mice. The identification of major histocompatibility complex (MHC) class I and II molecules in different mouse strains including C57BL/6 mice was an important step toward understanding and studying T cell-mediated alloresponses. In the tail-skin transplantation model described here, a three-point mutation (I-Abm12) in the antigen-presenting groove of the MHC-class II (I-Ab) molecule is sufficient to induce strong allogeneic CD4+ T cell activation in C57BL/6 mice. Skin grafts from I-Abm12 mice on C57BL/6 mice are rejected within 12-15 days, while syngeneic grafts are accepted for up to 100 days. The absence of T cells (CD3-/- and Rag2-/- mice) allows skin graft acceptance up to 100 days, which can be overcome by transferring 2 x 104 wild type or transgenic T cells. Adoptively transferred T cells proliferate and produce IFN-&#947; in I-Abm12-transplanted Rag2-/- mice.

Tail-skin transplantation is a powerful model for studying T cell-dependent rejection and tolerance induction during allogeneic immune responses in mice. The advantages of this protocol are minor invasive surgery, and ease of monitoring with no need to sacrifice the recipient mouse.

The study of T cell responses and their consequences during allo-antigen recognition requires a model that enables one to distinguish between donor and ...

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the skin is relatively easy to access, it provides an ideal platform to study peripheral immune regulatory mechanisms. Immune resident cells in healthy skin conduct immunosurveillance, but also play an important role in the development of inflammatory skin disorders, such as psoriasis. Despite emerging insights, our understanding of the biology underlying various inflammatory skin diseases is still limited. There is a need for good quality (single) cell populations isolated from biopsied skin samples. So far, isolation procedures have been seriously hampered by a lack of obtaining a sufficient number of viable cells. Isolation and subsequent analysis have also been affected by the loss of immune cell lineage markers, due to the mechanical and chemical stress caused by the current dissociation procedures to obtain single cell suspension. Here, we describe a modified method to isolate T cells from both healthy and involved psoriatic human skin by combining mechanical skin dissociation using an automated tissue dissociator and collagenase treatment. This methodology preserves expression of most immune lineage markers such as CD4, CD8, Foxp3 and CD11c upon the preparation of single cell suspensions. Examples of successful CD4+ T cell isolation and subsequent phenotypic and functional analysis are shown.

Lymphocyte Isolation from Human Skin for Phenotypic Analysis and Ex Vivo Cell Culture

We describe a protocol to efficiently isolate skin resident T cells from human skin biopsies. This protocol yields sufficient numbers of viable human skin resident lymphocytes for flow cytometric analysis and ex vivo culture.

Human skin has an important barrier function and contains various immune cells that contribute to tissue homeostasis and protection from pathogens. As the ...

Preparation of Single-cell Suspensions for Cytofluorimetric Analysis from Different Mouse Skin Regions

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and epidermis home a variety of immune cells in both homeostatic and inflammatory conditions. Tools to obtain skin cell release for cytofluorimetric analyses are, therefore, very useful in order to study the complex network of immune cells residing in the skin and their response to microbial stimuli. Here, we describe an efficient methodology for the digestion of mouse skin to rapidly and efficiently obtain single-cell suspensions. This protocol allows maintenance of maximum cell viability without compromising surface antigen expression. We also describe how to take and digest skin samples from different anatomical locations, such as the ear, trunk, tail, and footpad. The obtained suspensions are then stained and analyzed by flow cytometry to discriminate between different leukocyte populations.

The skin is home to a complex immune cell network. We describe an efficient methodology for the digestion of mouse skin, from different parts of the animal's body, in order to obtain a single-cell suspension and analyze the different leukocyte populations resident in the skin by flow cytometry.

The skin is a barrier organ that interacts with the external environment. Being continuously exposed to potential microbial invasion, the dermis and ...

Isolation of Infiltrating Leukocytes from Mouse Skin Using Enzymatic Digest and Gradient Separation

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune cells in rodent models of skin inflammation. Here, we describe a protocol for the digestion of mouse skin in a nutrient-rich solution with collagenase D, followed by separation of hematopoietic cells using a discontinuous density gradient. Cells thus obtained can be used for in vitro studies, in vivo transfer, and other downstream cellular and molecular analyses including flow cytometry. This protocol is an effective and economical alternative to expensive mechanical dissociators, specialized separation columns, and harsher trypsin- and dispase-based digestion methods, which may compromise cellular viability or density of surface proteins relevant for phenotypic characterization or cellular function. As shown here in our representative data, this protocol produced highly viable cells, contained specific immune cell subsets, and had no effect on integrity of common surface marker proteins used in flow cytometric analysis.

This protocol describes enzymatic digestion of mouse skin in nutrient-rich medium followed by gradient separation to isolate leukocytes. Cells thus derived can be used for diverse downstream applications. This is an effective, economical, and improved alternative to tissue dissociation machines and harsher trypsin and dispase-based tissue digestion protocols.

Dissociating murine skin into a single cell suspension is essential for downstream cellular analysis such as the characterization of infiltrating immune ...

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly motile parasite not only reaches the liver to invade hepatocytes and transform into erythrocyte-infective form. It also migrates into the skin and to the proximal lymph node draining the injection site, where it can be recognized and degraded by resident and/or recruited myeloid cells. Intravital imaging reported the early recruitment of brightly fluorescent Lys-GFP positive leukocytes in the skin and the interactions between sporozoites and CD11c+ cells in the draining lymph node. We present here an efficient procedure to recover, identify and enumerate the myeloid cell subsets that are recruited to the mouse skin and draining lymph node following intradermal injection of immunizing doses of sporozoites in a murine model. Phenotypic characterization using multi-parametric flow cytometry provides a reliable assay to assess early dynamic cellular changes during inflammatory response to Plasmodium infection.

Myeloid Cell Isolation from Mouse Skin and Draining Lymph Node Following Intradermal Immunization with Live Attenuated Plasmodium Sporozoites

We describe here a protocol for isolating myeloid cells from mouse skin and draining lymph node following intradermal injection of Plasmodium sporozoites. Flow cytometry of collected cells provides a reliable assay to characterize the skin and draining lymph node inflammatory response to the parasite.

Malaria infection begins when the sporozoite stage of Plasmodium is inoculated into the skin of a mammalian host through a mosquito bite. The highly ...

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides.
IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 &plusmn; 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 &#181;g/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 &#181;g/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

We describe the isolation of cardiac extracellular matrix from C57Bl/6J control mice, tight-skin mice, and tight-skin mice treated with the IRF5 inhibitory peptide. We also describe the vasodilation studies on the isolated vessels from C57Bl/6J, tight-skin mice and tight-skin mice treated with the IRF5 inhibitory peptide.

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ...

Murine Full-thickness Skin Transplantation

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited application to humans, skin transplantation in mice has been widely employed for transplantation research. The procedure is easy to learn and perform, and it does not require delicate microsurgical techniques nor extensive training. Moreover, graft rejection in this model occurs in a very reproducible immunological reaction and is easily monitored by direct inspection and palpation. In addition, secondary skin transplantation with donor-matched or third-party skin grafts can be performed on more complex transplant models as an alternative and uncomplicated method to assess donor-specific tolerance. The complications are low and are in general limited to anesthesia overdose or respiratory distress after the procedure. Graft failure, on the other hand, occurs commonly as a result of poor preparation of the graft, incorrect positioning in the graft bed, or inappropriate placement of the bandage. In this article, we present a protocol for full-thickness skin transplantation in mice and describe the important steps necessary for a successful procedure.

Murine full-thickness skin transplantation is a well-established model to study rejection in an alloimmune setting. Here, we provide a tutorial of each step involved in performing a BALB/c--&#62;C57BL/6 full-thickness skin transplant.

Murine full-thickness skin transplantation is a well-established in vivo model to study alloimmune response and graft rejection. Despite its limited ...

Development of an Economical DNA Delivery System by "Acufection" and its Application to Skin Research

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a fascinating approach to investigate immune modulation of cutaneous inflammation in mouse models of human diseases. Here we present a cost-effective protocol that delivered naked DNA in mouse skin and leads to transgene expression. The method is coined &#34;acufection&#34;, denoting acupuncture-mediated DNA transfection. To perform acufection, mouse skin was first infused with DNA in phosphate-buffered saline (PBS) and then pricked lightly with a bundle of acupuncture needles to facilitate the absorption of DNA and transfection into cells. The plasmid DNA is presumably taken up by the keratinocyte and dendritic cells (DCs) in the skin and expressed into protein. Mechanical prick with the needles per se did not cause skin damage or induce keratinocyte activation. The expression of the transfected genes was detected in the skin at both transcriptional and translational levels following acufection for 2 days and maintained up to 7 days. The primary goal for the development of this acufection method was to investigate a previously undefined isoform of IL-15. Using this method, an alternatively spliced IL-15 isoform with partially deleted exon 7 (IL-15&#916;E7) was expressed in the skin and subsequently treated with a Toll-like receptor 7 (TLR7) agonist, imiquimod (IMQ), to induce inflammation. Acufection-delivered IL-15&#916;E7 in skin suppressed keratinocyte proliferation, epidermal thickness and neutrophil recruitment in IMQ-induced cutaneous inflammation. With increasing interest in identifying the regulatory mechanisms of cutaneous inflammation, the protocol described here provides a cost effective and versatile alternative to the gene gun system or microseeding for DNA delivery in vivo. It may potentially allow discovery of the function of a novel gene in the skin or for investigating new treatment for cutaneous diseases.

Development of an Economical DNA Delivery System by &quot;Acufection&quot; and its Application to Skin Research

This protocol is a cost-effective alternative for expressing naked plasmid DNA in mouse skin. The overall goal of the protocol is to deliver immune-related genes into skin tissue to delineate the functional role of a specific gene in cutaneous inflammation.

Dysregulation of immune response in skin is associated with numerous human skin disorders. Direct transfer of immune-related genes into skin tissue is a ...