Abstract
Disease models represent powerful tools used by biomedical researchers to address various questions related to the pathogenesis and the possible therapeutic targets in different diseases. To get a complete picture of the process one needs complementary animal and ex vivo disease models. While subsequent chapters dwell upon animal models, this chapter describes an ex vivo model for granulocyte-dependent dermal-epidermal separation induced by autoantibodies to the basal membrane. The strength of the described ex vivo model resides in the fact that it uses human material (the skin, autoantibodies, and cells), thus better mimicking the pathogenesis as it occurs in patients. The reproducibility rate of the protocol is about 85–90% when using a good combination of the three main ingredients. This rate can drop dramatically when changing one of the three. Nevertheless, being a one-day model, this procedure enables investigators to have a quick readout system when investigating multiple sera for their potential to activate granulocytes and induce dermal-epidermal separation.
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References
Gammon WR, Merritt CC, Lewis DM, Sams WM, Carlo JR, Wheeler CE (1982) An in vitro model of immune complex-mediated basement membrane zone separation caused by pemphigoid antibodies, leukocytes, and complement. J Invest Dermatol 78:285–290
Gammon WR, Merritt CC, Lewis DM, Sams WM Jr, Wheeler CE Jr, Carlo J (1981) Leukocyte chemotaxis to the dermal-epidermal junction of human skin mediated by pemphigoid antibody and complement: mechanism of cell attachment in the in vitro leukocyte attachment method. J Invest Dermatol 76:514–522
Sitaru C, Schmidt E, Petermann S, Munteanu LS, Brocker EB, Zillikens D (2002) Autoantibodies to bullous pemphigoid antigen 180 induce dermal-epidermal separation in cryosections of human skin. J Invest Dermatol 118:664–671
Sitaru C, Kromminga A, Hashimoto T, Brocker EB, Zillikens D (2002) Autoantibodies to type VII collagen mediate Fcgamma-dependent neutrophil activation and induce dermal-epidermal separation in cryosections of human skin. Am J Pathol 161:301–311
Sitaru C, Zillikens D (2005) Mechanisms of blister induction by autoantibodies. Exp Dermatol 14:861–875
Shimanovich I, Mihai S, Oostingh GJ, Ilenchuk TT, Brocker EB, Opdenakker G, Zillikens D, Sitaru C (2004) Granulocyte-derived elastase and gelatinase B are required for dermal-epidermal separation induced by autoantibodies from patients with epidermolysis bullosa acquisita and bullous pemphigoid. J Pathol 204:519–527
Mihai S, Chiriac MT, Herrero-Gonzalez JE, Goodall M, Jefferis R, Savage CO, Zillikens D, Sitaru C (2007) IgG4 autoantibodies induce dermal-epidermal separation. J Cell Mol Med 11:1117–1128
Chiriac MT, Roesler J, Sindrilaru A, Scharffetter-Kochanek K, Zillikens D, Sitaru C (2007) NADPH oxidase is required for neutrophil-dependent autoantibody-induced tissue damage. J Pathol 212:56–65
Recke A, Sitaru C, Vidarsson G, Evensen M, Chiriac MT, Ludwig RJ, Zillikens D (2009) Pathogenicity of IgG subclass autoantibodies to type VII collagen: induction of dermal-epidermal separation. J Autoimmun 34:435–444
Csorba K, Sesarman A, Oswald E, Feldrihan V, Fritsch A, Hashimoto T, Sitaru C (2010) Cross-reactivity of autoantibodies from patients with epidermolysis bullosa acquisita with murine collagen VII. Cell Mol Life Sci 67:1343–1351
Boyum A (1968) Isolation of leucocytes from human blood. Further observations. Methylcellulose, dextran, and ficoll as erythrocyte aggregating agents. Scand J Clin Lab Invest Suppl 97:31–50
Boyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Isolation of monuclear cells by one centrifugation, and of granulocytes by combining centrifugation and sedimentation at 1 g. Scand J Clin Lab Invest Suppl 97:77–89
Boyum A (1976) Isolation of lymphocytes, granulocytes and macrophages. Scand J Immunol Suppl 5:9–15
Boyum A, Lovhaug D, Tresland L, Nordlie EM (1991) Separation of leucocytes: improved cell purity by fine adjustments of gradient medium density and osmolality. Scand J Immunol 34:697–712
Yeo C, Saunders N, Locca D, Flett A, Preston M, Brookman P, Davy B, Mathur A, Agrawal S (2009) Ficoll-Paque versus Lymphoprep: a comparative study of two density gradient media for therapeutic bone marrow mononuclear cell preparations. Regen Med 4:689–696
Acknowledgements
The author received financial support from the Sectoral Operational Programme for Human Resources Development 2007–2013, cofinanced by the European Social Fund, under the project number POSDRU 89/1.5/S/61104 (“Social Sciences and Humanities in the context of globalised evolution—Development and Implementation of the Postdoctoral Research Program”).
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Chiriac, M.T. (2013). Induction of Granulocyte-Dependent Dermal-Epidermal Separation by Autoantibodies Ex Vivo. In: Has, C., Sitaru, C. (eds) Molecular Dermatology. Methods in Molecular Biology, vol 961. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-227-8_14
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DOI: https://doi.org/10.1007/978-1-62703-227-8_14
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