Rheumatoid arthritis (RA) is a chronic inflammatory disease of diarthrodial joints, characterized by infiltration of immune cells and local tissue hypoxia. Migrating monocytes and differentiated macrophages are forced to adapt their energy metabolism to hypoxia. Hypoxia-inducible factor (HIF)-1a is known to act as key regulator for the adaptation of migrating cells into hypoxic areas. The aim of this study was to investigate the mechanisms used by human monocytes and macrophages to adapt to hypoxia.
Method: Isolated human CD14+ monocytes were incubated for 5h under normoxic and hypoxic conditions with or without PMA stimulation, respectively. Cell survival rates were measured by 7-AAD staining and subsequent FACS analyses. Using a Nuclear Extract Kit (Actife Motiv), nuclear and cytosolic fractions were prepared in order to detect HIF-1a and NFkB by immunoblot. For the experiments with macrophages, primary human monocytes were differentiated into monocyte derived macrophages (hMDM) using M-CSF. The effects of normoxia and hypoxia on gene expression were compared between monocytes and hMDMs using quantitative PCR.
Result: Hypoxia was found to improve the survival of primary human monocytes. Furthermore, monocytes stabilize HIF-1a under hypoxic conditions in a time dependent manner. Interestingly HIF-1a was detected exclusively in the cytoplasm, but not in the nucleus of unstimulated cells. PMA stimulation, however, resulted in the translocation of HIF-1a into the nucleus with PKC-a/b1 being an essential regulator of this process. Also differentiation of monocytes into macrophages was found to be accompanied by the translocation of HIF-1a into the nucleus. In monocytes, hypoxia does induce the expression of glycolytic genes despite the absence of HIF-1a in the nucleus. NFkB1 turned out to be the transcription factor which mediates the regulatory effect under these conditions.
Conclusion: Monocytes migrate into inflamed and non-inflamed tissues where they differentiate into macrophages. During this differentiation process, HIF-1a translocates from the cytoplasm (monocytes) into the nucleus (macrophages). Therefore, while in macrophages HIF-1a seems to serve as key regulator for the adaptation to hypoxia, this function is fulfilled by NFkB1 in monocytes.
Disclosure: M. Fangradt, None; T. Gaber, None; M. Hahne, None; P. Hoff, None; M. Jakstadt, None; C. Strehl, None; G. R. Burmester, None; F. Buttgereit, None.