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.