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Die Wirkung des Zytokins BAFF auf die Expression von pro- und anti-apoptotischen bcl-2 Familienmitgliedern in B-Zellen des Huhnes
Die Wirkung des Zytokins BAFF auf die Expression von pro- und anti-apoptotischen bcl-2 Familienmitgliedern in B-Zellen des Huhnes
Effect of B-cell activating factor of the tumor necrosis factor family (BAFF) on the expression of pro- and anti-apoptotic bcl-2 family members in chicken B cells The recently discovered chicken cytokine BAFF (B-cell activating factor of the tumor necrosis factor family) was characterised as an important regulator of chicken B-cell homeostasis. Besides regulating B-cells in secondary lymphatic organs BAFF seems to have a significant impact on chicken B-cell development in the Bursa of Fabricius, too. Past studies already showed that chicken BAFF plays a vital role in the survival of B-cells both in vitro and in vivo. Yet molecular correlation for this effect has still to be established in the chicken. In mouse and man, the antiapoptotic effect of BAFF was linked to a regulation of certain bcl-2 family members. Thus this study focused on the identification of bcl-2 family members in the chicken and their regulation by BAFF at transcriptional level. By means of RT-PCR the expression of both anti-apoptotic (e.g. bcl-2, bcl-xL and Nr13) as well as pro-apoptotic (e.g. bak, bid, bim and bok) transcripts was shown in bursa, spleen and heart muscle at various developmental stages. To enable further studies quantitative RT-PCR assays were established for both anti-apoptotic (e.g. bcl-2, bcl-xL and Nr13) and pro-apoptotic (e.g. bak, bid, bim and bok) bcl-2 family members as well as for the B-cell specific marker chB6 and chBAFF. During bursal development, transcripts for pro-apoptotic bok and anti-apoptotic bcl-xL are increased while the level of bcl-2 mRNA is decreased. Considering the vast raise in B-cell number within the developing bursa, a means of correlating this with characterised changes in transcription levels had to be established. This was done based on expression levels of the B-cell marker chB6. Thus it could be shown that transcription of both anti-apoptotic genes like bcl-2 and Nr13 and pro-apoptotic genes such as bak and bim were decreased based on the amount of B-cell. In contrast, levels of bok transcript remained unchanged in B-cells during bursal development. Isolated lymphocytes taken from the spleen were used for inital studies on the impact of BAFF in vitro. In agreement with published data, the anti-apoptotic effect of BAFF could be demonstrated in this study, too. At transcriptional level, this was linked to a decrease in the transcription of pro-apoptotic bim. In contrast, incubation of spleen cells with chicken CD40-ligand resulted in the vast proliferation of B-cells from both juvenile and mature birds. However, age-related differences in the survival of lymphocytes were observed in this study, which correlated with a lower increase of anti-apoptotic bcl-xL in mature cells than in juvenile in response to CD40-ligand stimulation. To further analyse the effect of BAFF on bursal B-cell development in vivo a previously published retroviral vector system (RCAS) was utilized. Both the effect of overexpression of BAFF as well as its neutralization using a soluble decoy receptor (BCMA) were characterised at transcriptional level. Overexpressing BAFF led to insignificant changes during the development of the bursa. Since BAFF is expressed at high levels during all stages of bursal development, gene overexpression may not exert additional effects. Neutralization of BAFF on the other hand caused distinct changes among bcl-2 family members at the transcriptional level. It again proofed necessary to correlate these changes with B-cell numbers represented by the level of chB6 transcription. By this method genes highly expressed within the remaining B-cell population were characterised. Anti-apoptotic bcl-2 along with Nr13 was shown to be significantly increased in comparison to control cells.
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Dieckmann, Jenny
2008
Deutsch
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Dieckmann, Jenny (2008): Die Wirkung des Zytokins BAFF auf die Expression von pro- und anti-apoptotischen bcl-2 Familienmitgliedern in B-Zellen des Huhnes. Dissertation, LMU München: Tierärztliche Fakultät
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Abstract

Effect of B-cell activating factor of the tumor necrosis factor family (BAFF) on the expression of pro- and anti-apoptotic bcl-2 family members in chicken B cells The recently discovered chicken cytokine BAFF (B-cell activating factor of the tumor necrosis factor family) was characterised as an important regulator of chicken B-cell homeostasis. Besides regulating B-cells in secondary lymphatic organs BAFF seems to have a significant impact on chicken B-cell development in the Bursa of Fabricius, too. Past studies already showed that chicken BAFF plays a vital role in the survival of B-cells both in vitro and in vivo. Yet molecular correlation for this effect has still to be established in the chicken. In mouse and man, the antiapoptotic effect of BAFF was linked to a regulation of certain bcl-2 family members. Thus this study focused on the identification of bcl-2 family members in the chicken and their regulation by BAFF at transcriptional level. By means of RT-PCR the expression of both anti-apoptotic (e.g. bcl-2, bcl-xL and Nr13) as well as pro-apoptotic (e.g. bak, bid, bim and bok) transcripts was shown in bursa, spleen and heart muscle at various developmental stages. To enable further studies quantitative RT-PCR assays were established for both anti-apoptotic (e.g. bcl-2, bcl-xL and Nr13) and pro-apoptotic (e.g. bak, bid, bim and bok) bcl-2 family members as well as for the B-cell specific marker chB6 and chBAFF. During bursal development, transcripts for pro-apoptotic bok and anti-apoptotic bcl-xL are increased while the level of bcl-2 mRNA is decreased. Considering the vast raise in B-cell number within the developing bursa, a means of correlating this with characterised changes in transcription levels had to be established. This was done based on expression levels of the B-cell marker chB6. Thus it could be shown that transcription of both anti-apoptotic genes like bcl-2 and Nr13 and pro-apoptotic genes such as bak and bim were decreased based on the amount of B-cell. In contrast, levels of bok transcript remained unchanged in B-cells during bursal development. Isolated lymphocytes taken from the spleen were used for inital studies on the impact of BAFF in vitro. In agreement with published data, the anti-apoptotic effect of BAFF could be demonstrated in this study, too. At transcriptional level, this was linked to a decrease in the transcription of pro-apoptotic bim. In contrast, incubation of spleen cells with chicken CD40-ligand resulted in the vast proliferation of B-cells from both juvenile and mature birds. However, age-related differences in the survival of lymphocytes were observed in this study, which correlated with a lower increase of anti-apoptotic bcl-xL in mature cells than in juvenile in response to CD40-ligand stimulation. To further analyse the effect of BAFF on bursal B-cell development in vivo a previously published retroviral vector system (RCAS) was utilized. Both the effect of overexpression of BAFF as well as its neutralization using a soluble decoy receptor (BCMA) were characterised at transcriptional level. Overexpressing BAFF led to insignificant changes during the development of the bursa. Since BAFF is expressed at high levels during all stages of bursal development, gene overexpression may not exert additional effects. Neutralization of BAFF on the other hand caused distinct changes among bcl-2 family members at the transcriptional level. It again proofed necessary to correlate these changes with B-cell numbers represented by the level of chB6 transcription. By this method genes highly expressed within the remaining B-cell population were characterised. Anti-apoptotic bcl-2 along with Nr13 was shown to be significantly increased in comparison to control cells.