Publikationsserver der Universitätsbibliothek Marburg

Titel:Klonale experimentelle autoimmune Enzephalomyelitis: Charakterisierung myelinspezifischer Antikörper in einem TH2-induzierten adoptiven Transfermodell
Autor:Sieren, Michael
Weitere Beteiligte: Sommer, Norbert (Prod. Dr.)
Veröffentlicht:2012
URI:https://archiv.ub.uni-marburg.de/diss/z2012/0851
DOI: https://doi.org/10.17192/z2012.0851
URN: urn:nbn:de:hebis:04-z2012-08517
DDC:610 Medizin, Gesundheit
Titel(trans.):Clonal experimental autoimmune encephalomyelitis: characterization of myelinspecific antibodies in a TH2 adoptive transfer model
Publikationsdatum:2012-09-14
Lizenz:https://rightsstatements.org/vocab/InC-NC/1.0/

Dokument

Schlagwörter:
Transfermodell, Adoptives, EAE, Antikörper, TH2, PLP, Adoptive, TH2, Antibodies, Antikörper, Transfermodell, EAE, T-Lymphozyt, Allergische Encephalomyelitis

Zusammenfassung:
Die Multiple Sklerose ist in Europa und Nordamerika die häufigste Entmarkungserkrankung des zentralen Nervensystems. Sie ist neben Inflammation und Demyelinisierung auch durch neurodegenerative Prozesse gekennzeichnet. Hierbei spielen T-Zell-vermittelte Mechanismen eine wesentliche Rolle. Aufgrund der Heterogenität ihres klinischen Verlaufs sind weder individuelle Voraussagen bezüglich der Progression der neurologischen Defizite noch bezüglich des therapeutischen Ansprechens möglich. Diese klinische Heterogenität spiegelt sich in unterschiedlichen histopathologischen sowie liquorzytologischen Mustern wieder. Es finden sich Subtypen, bei denen humorale Effektormechanismen eine wesentliche Rolle zu spielen scheinen. Die experimentelle autoimmune Enzephalomyelitis (EAE) ist ein Tiermodell der Multiplen Sklerose und kann in suszeptiblen Tierstämmen durch aktive Immunisierung mit Myelinpeptiden oder durch den adoptiven Transfer myelinspezifischer CD4 positiver TH1-Zellen induziert werden. Die EAE galt lange als eine im Wesentlichen TH1-vermittelte Erkrankung, während TH2-Zellen eher protektive Funktionen zugeschrieben wurden. Dieses TH1-TH2-Paradigma stellt jedoch eine zu starke Vereinfachung der Immunantwort im Rahmen der EAE-Induktion dar. Zum einen spielen weitere T-Zell-Subpopulationen eine wesentliche Rolle bei der Pathogenese. So gelingt z.B. durch den adoptiven Transfer von TH17-Zellen eine sehr effiziente EAE-Induktion auch ohne eine vorherige in vitro Stimulation. Zum anderen kann in einem neuartigen adoptiven T-Zell-Transfermodell durch den Transfer klonaler Proteolipidprotein (PLP) 139-151 spezifischer CD4+ TH2-Zellen bei immunkompetenten SJL-Mäusen eine EAE mit chronifizierendem Verlauf induziert werden, deren entzündliche Läsionen in der chronischen Phase durch Infiltrate aus Makrophagen, B-Zellen, Plasmazellen sowie massiven Antikörperablagerungen gekennzeichnet sind. Im Serum der Mäuse ist eine hochtitrige Antikörperantwort gegen PLP139-51 nachweisbar. Ziel der vorliegenden Arbeit ist eine nähere Charakterisierung der Antikörperantwort in diesem Modell. Nach EAE-Induktion durch adoptiven Transfer von Zellen des PLP 139-151 spezifischen TH2-Zell-Klons 3-3 wurden in der chronischen Phase der Erkrankung Milzzellen entnommen und mit Zellen der Sp2-Maus-Myelom-Zelllinie fusioniert. Hierdurch wurden antikörperproduzierende Hybridomzellen generiert. Durch Klonierung mittels des Limiting-Dilution-Verfahrens und anschließender chromatograpischer Aufreinigung wurden monoklonale, myelinspezifische Antikörper gewonnen. Deren weitere Charakterisierung erfolgte mittels ELISA, Western-Blot, an Gewebsschnitten sowie in adoptiven Transferexperimenten. Die Besonderheit dieses Modells liegt darin, dass die monoklonalen Antikörper ohne vorherige aktive Immunisierung generiert wurden. Es handelt sich somit um einen neuen, physiologischeren Ansatz zur Charakterisierung der T-Zell-B-Zell-Interaktion bzw. der Rolle von Antikörpern in einem neuen TH2-induzierten EAE-Modell. Im ersten Teil der Arbeit wurden ausgewählte monoklonale PLP139-151 spezifische Antikörper der Isotypen IgG1 sowie IgG2b aufgereinigt. Zwar konnten diese PLP139-151 in einem ELISA binden. Weder in einem Western Blot mit Myelinproteinen noch in Kryo- oder Paraffinschnitten von ZNS-Gewebe zeigte sich hingegen eine spezifische Bindung an Myelin. Der adoptive Transfer in mit Pertussis Toxin vorbehandelte SJL-Mäuse konnte keine klinischen Symptome induzieren. Im zweiten Teil der Arbeit wurden die Untersuchungen daher auf Antikörper gegen weitere Myelinpeptide ausgedehnt. Hierbei konnte der Myelin Oligodendrozyten Glykoprotein (MOG) 35-55 spezifische monoklonale IgM Antikörper 4E6-2 identifiziert werden, der Myelin in Paraffinschnitten spezifisch färbte. Zudem zeigte sich im Western Blot eine Bindung an MOG. Der Antikörper wurde in einem Transferversuch an Mäusen getestet, bei denen zuvor eine EAE durch adoptiven Transfer eines TH1-Klons induziert worden war. Hierbei fand sich kein Hinweis auf eine krankheitsmodifizierende Wirkung des Antikörpers. Zur weiteren Klärung der Rolle antikörpervermittelter Mechanismen im hier beschriebenen Modell ist eine weitere Charakterisierung der Antikörper hinsichtlich ihrer Effektorfunktionen, wie z.B. ihrer Fähigkeit zur Komplementaktivierung, erforderlich.

Summary:
Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system. Clinical manifestation and progression of neurological deficits are highly heterogeneous and unpredictable. Clinical heterogeneity is reflected by four distinct histopathological patterns and defined patterns of cerebrospinal fluid pathology. T-cell mediated mechanisms seem to play a pivotal role in the course of disease. Furthermore humoral immunity seems to contribute essentially to inflammation in several subtypes of MS. Experimental autoimmune encephalomyelitis (EAE) is an animal model of MS. It can be either induced by active immunization with myelin components or by adoptive transfer of myelin specific CD4+ TH1-cells. A shift towards a TH2-response however has been considered to lead to an amelioration of the clinical course. Recent developments revealed that this TH1-TH2-paradigm is an oversimplification of the complex immune response during the course of EAE. Further T-cell subpopulations seem play a pivotal role in the pathogenesis of EAE. Adoptive Transfer of TH17-cells e.g. is a very efficient way of EAE-induction without prior in-vitro stimulation. In addition adoptive transfer of clonal proteolipidprotein (PLP) 139-151 specific TH2-cells to immunocompetent non-transgenic SJL-mice in a new model leads to induction of an EAE with a chronifying course of disease. Demyelinating lesions in the chronic phase of disease contain B-cells, plasma cells, macrophages and massive deposits of antibodies. Furthermore PLP139-151 specific antibodies as well as antibodies against other myelin components can be detected in the serum of the animals. The aim of this study is the further characterization of the antibody response in this new TH2-induced EAE model. After EAE-induction by adoptive transfer of the prior established PLP139-151 specific TH2 clone 3-3 spleen cells were obtained during the chronic phase of disease approximately 90 days after T-cell transfer. By fusion of these cells with cells of the Sp2 myeloma cell line antibody producing hybridoma cells were created. By cloning in limiting dilution technique followed by affinity chromatography of cell culture supernatants, monoclonal myelin specific antibodies were purified. For further characterization the so gained monoclonal antibodies were tested in myelin specific ELISAs and Western Blots, in immunohistochemistry on central nervous system (CNS) tissue samples and in adoptive transfer experiments. A distinctive feature of this model is that monoclonal antibodies were generated without prior active immunization with myelin antigens. This offers a completely new more physiologic approach to characterize T-cell-B-cell interactions and the role of antibodies in a new TH2-induced EAE-Modell. In the first part of the study selected monoclonal PLP139-151 specific antibodies of isotype IgG1 and IgG2b were purified and characterized as described before. PLP139-151 specifity could be shown in a peptide coated ELISA. Neither in Western Blots nor in immunohistochemistry with CNS tissue samples could reactivity against PLP or other myelin components be observed. Adoptive transfer of antibodies in SJL-mice pre-treated with Pertussis toxin could not induce clinical symptoms of EAE. In the second part the study therefore was extended to monoclonal antibodies specific for other myelin components. The monoclonal myelin oligodendroglia glycoprotein (MOG) 35-55 specific antibody 4E6-2 stained MOG in a Western Blot as well as myelin on paraffin embedded CNS tissue sections. The antibody was tested in adoptive co-transfer experiments in SJL-mice after prior EAE-induction by adoptive transfer of cells of the myelin specific TH1 clone cPLP 25. This experiment did not reveal a disease modifying effect of 4E6-2. For clarification of the role of antibody mediated mechanisms in our new TH2 mediated model of EAE in SJL-mice further characterization of these antibodies in regard to their effector functions like e.g. their ability to fix complement has to be carried out. Nevertheless the findings support the concept of pathogenetic heterogeneity of EAE and MS and might have an impact on the struggle for new therapeutic approaches.

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