Charakterisierung des Zytokins BAFF als wichtiger Regulator der B-Zellfunktion beim Haushuhn

This investigation is based on the identification of cDNA sequence
information for a putative chicken homologue of the mammalian
cytokine “B cell activating factor belonging to the TNF family”
(BAFF) in a chicken EST-database. The cDNAs encoded for the
complete open reading frame of chicken BAFF (chBAFF) including a
transmembrane domain and a potential furine cleavage side. ChBAFF
shows an uncommon high amino-acid sequence identity with 76%
identity to human BAFF in comparison with other chicken cytokines.
To investigate its biochemical and functional properties,
recombinant prokaryotic chBAFF was generated in E. coli and
eukaryotic chBAFF was obtained from transfected 293T cells. A
polyclonal rabbit antiserum raised against His-chBAFF reacted with
both, the prokaryotic and the eukaryotic cytokine and was used to
quantify 293T cell derived chBAFF in an ELISA system. Studies by
northern blot analysis revealed a strong expression signal in the
Bursa of Fabricius and a weak signal in the spleen, while all other
tissues including thymus and gut tissue were negative. In situ
hybridisation detected a wide distribution pattern for chBAFF-mRNA
in all areas of the bursa of Fabricius. However, the strongest
expression was seen in the medulla from bursal follikels. These
studies indicate that both the bursal stroma and bursal B-cells
must be considered as potential sources for chBAFF. To identify
target cells for this cytokine, chBAFF receptor(s) expression was
investigated in binding studies with Flag tagged chBAFF
(Flag-chBAFF) and binding was analysed by flow cytometry. Receptor
expression was clearly restricted to B-cells including mature
B-cells from peripheral lymphoid organs as well as immature bursal
B-cells. Subsequent studies on the ontogeny of chBAFF receptor
expression showed a positive correlation between the cytokine
receptor expression and the expression of the B-cell antigen
receptor in the developing embryonic bursa.
Receptor-ligand-interaction ELISA and co-immunoprecipitation
experiments demonstrated that chBAFF binds to all three mammalian
BAFF receptors identified thus far. Importantly, studies of chBAFF
binding to the chicken B-cell line DT40 showed that an engineered
soluble form of the human BAFF-receptor BCMA (huBCMA-Fc) inhibited
chBAFF binding. The addition of 293T cell derived chBAFF to splenic
lymphocyte cultures led to a significant increase in B-cell
viability. This dose-dependent effect was also observed in
lymphocyte cultures from ceacal tonsils and in cultures of purified
(>95%) splenic B-cell preparations. While the rapid apoptosis in
cultures of bursal lymphocytes could not be completely prevented,
chBAFF clearly increased the survival of these immature B-cells.
CFSE labelling experiments further showed that chBAFF did not
induce B-cell proliferation. Therefore, it is highly probable that
chBAFF, as its mammalian counterpart, is a potent inhibitor of
B-cell apoptosis. These in vitro studies were complemented by in
vivo experiments with purified prokaryotic chBAFF. Daily injection
of recombinant cytokine for 7 days induced a significant increase
in spleen weight and B-cell frequency in spleen and caecal tonsils.
Besides, the functional overexpression of chBAFF induced
significantly (4-fold) increased serum IgM levels. Therefore,
chBAFF does not only increase B cell numbers, it also influences
their differentiation to antibody secreting cells. However, no
effect was observed on the bursa of Fabricius prompting the reverse
experimental approach. With the availability of a soluble cytokine
specific receptor (huBCMA-Fc) in vivo knockdown studies could be
performed. Daily i.p. application of huBCMA-Fc to newly hatched
birds for 5 days led to decreased spleen weights and drastically
reduced the B-cell frequency in spleens and caecal tonsils. In
addition, bursal weights in treated birds were lower than in
untreated controls. This experiment in combination with the
observation of high expression levels of chBAFF-mRNA in the bursa
and functional BAFF-receptor(s) on bursal B-cells strongly
indicated a thus far unknown role for BAFF in early B-cell
development.