Hematopoiesis of immature myeloid dendritic cells in stroma-dependent spleen long-term cultures occurs independently of NF-KB/RelB function.
OBJECTIVE
The nuclear factor-kappaB (NF-KB)/RelB transcription factor plays an essential role in development of some dendritic cell (DC) subsets in mice. In this laboratory, immature myeloid DC are produced in vitro in a stroma-dependent murine spleen long-term culture (LTC) system. In LTC, DC differentiate from hematopoietic progenitors maintained within the stromal cell matrix. Expression and function of RelB in development of LTC-DC has been investigated, with a view to assessing the relationship between DC produced in this system and other known subsets of DC.


METHODS
RelB expression by LTC-DC was confirmed by detection of protein by Western blotting, RNA by reverse transcription polymerase chain reaction, and nuclear protein with DNA-binding function in electrophoretic mobility shift assays. The role of RelB in cell development was assessed by addition of antisense RelB oligonucleotides into LTC and colony assays established above STX3 stromal cells. RelB(-/-) mice were also examined for ability to produce LTC, and for presence of DC progenitors in spleen and bone marrow that can generate DC when overlaid on STX3 in cocultures.


RESULTS
Functional RelB was detected in both LTC-DC and in STX3 stromal cells. A critical role for RelB in DC differentiation from spleen progenitors was confirmed, because antisense RelB oligonucleotides specifically and completely inhibited production of large differentiated myeloid DC in LTC. Further investigation using RelB(-/-) mice revealed that RelB expression by stromal cells rather than hematopoietic cells was required for production of LTC-DC. This was evidenced by a combination of factors, including 1) inability to generate productive LTC from RelB(-/-) mice; 2) presence of DC precursors in RelB(-/-) bone marrow and spleen, which could produce DC in stromal cocultures; and 3) increased myeloid precursor frequency among RelB(-/-) spleen cells over RelB(+/+) control cell populations.


CONCLUSIONS
Specific development of fully differentiated, but immature myeloid CD11c(+)CD11b(+)MHC-CII(-)CD8alpha(-)CD40(-) DC in spleen LTC is dependent on expression of activated NF-kappaB/Rel-B. However, this appears to relate to stromal cell function rather than to the function of hematopoietic cells. Altogether these data confirm the importance of splenic stromal cells in myelopoiesis leading to development of immature DC as produced in LTC.
Tan
Jonathan K H
JK
School of Biochemistry and Molecular Biology, Faculty of Science, Australian National University, Canberra, Australia.
Ni
Keping
K
Le
Fei
F
O'Neill
Helen C
HC
D000818:Animals; D039481:CD11b Antigen; D019013:CD40 Antigens; D016827:CD8 Antigens; D018929:Cell Culture Techniques; D002454:Cell Differentiation / Q000502:physiology; D002478:Cells, Cultured; D018920:Coculture Techniques; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D000949:Histocompatibility Antigens Class II; D051379:Mice; D018345:Mice, Knockout; D022423:Myeloid Cells / Q000166:cytology / Q000378:metabolism*; D023461:Myeloid Progenitor Cells / Q000166:cytology / Q000378:metabolism*; D038042:Myelopoiesis / Q000502:physiology*; D013154:Spleen / Q000166:cytology / Q000378:metabolism*; D017154:Stromal Cells / Q000166:cytology / Q000378:metabolism; D051841:Transcription Factor RelB / Q000235:genetics / Q000378:metabolism*
D039481:CD11b Antigen; D019013:CD40 Antigens; D016827:CD8 Antigens; C409469:CD8 antigen, alpha chain; D000949:Histocompatibility Antigens Class II; C497594:Relb protein, mouse; D051841:Transcription Factor RelB