Submitted on December 3, 2007
Accepted on April 21, 2008
HYPOXIA ENHANCES LYSOSOMAL TNF
DEGRADATION IN MOUSE PERITONEAL MACROPHAGES
Nitza Lahat1, Michal Amit Rahat2*, Amalia Kinarty2, Lea Weiss-Cerem3, Sigalit Pinchevski2, and Haim Bitterman4
1 Immunology, Carmel Medical Center, Haifa, Israel; The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel
2 Immunology, Carmel Medical Center, Haifa, Israel
3 Ischemia-shock laboratory, Carmel Medical Center, Israel
4 Ischemia-shock laboratory, Carmel Medical Center, Israel; The Ruth and Bruce Rappaport Faculty of Medicine, Technion, Haifa, Israel
* To whom correspondence should be addressed. E-mail: rahat_miki{at}clalit.org.il.
Infection, simulated by LPS, is a potent stimulator of tumor necrosis factor 
(TNF) production, and hypoxia often synergizes with LPS to induce higher levels of the secreted cytokine. However, we show that in primary mouse peritoneal macrophages and in three mouse peritoneal macrophage cell lines (RAW 264.7, J774A.1 and PMJ-2R), hypoxia (O2 <0.3%) reduces the secretion of LPS-induced TNF (p<0.01). In RAW 264.7 cells this reduction was not regulated transcriptionally, as TNF mRNA levels remained unchanged. Rather, hypoxia and LPS reduced the intracellular levels of TNF by 2-fold (p<0.01) by enhancing its degradation in the lysosome and inhibiting its secretion via secretory lysosomes, as shown by confocal microscopy and verified by the use of the lysosome inhibitor Bafilomycin A1. In addition, although hypoxia did not change the accumulation of the soluble receptor TNFRII, it increased its binding to the secreted TNF by 2 fold (p<0.05). We suggest that these two post-translational regulatory checkpoints co-exist in hypoxia, and may partially explain the reduced secretion and diminished biological activity of TNF in peritoneal macrophages.
