Phagocytic removal of apoptotic cells occurs efficiently in vivo such that even in tissues with significant apoptosis, very few apoptotic cells are detectable 1 . This is thought to be due to the release of find-me signals by apoptotic cells that recruit motile phagocytes such as monocytes, macrophages, and dendritic cells, leading to the prompt clearance of the dying cells 2 . However, the identity and in vivo relevance of such find-me signals are not well understood. Here, through several lines of evidence, we identify extracellular nucleotides as a critical apoptotic cell find-me signal. We demonstrate the caspase-dependent release of ATP and UTP (in equimolar quantities) during the early stages of apoptosis by primary thymocytes and cell lines. Purified nucleotides at these concentrations were sufficient to induce monocyte recruitment comparable to apoptotic cell supernatants. Enzymatic removal of ATP and UTP (by apyrase or ectopic CD39 expression)Correspondence and requests for materials should be addressed to K.S.R. (ravi@virginia.edu). Supplementary Information is linked to the online version of the paper at www.nature.com/nature.Author Information Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing interests.Author Contributions M.R.E. designed, performed and analyzed most of the experiments in this study with input from K.S.R. F.B.C. performed ATP quantitation experiments. P.T.C. helped with in vivo thymic apoptosis experiments. E.R.L. carried out HPLC analysis of supernatants. S.F.W. generated the CD39 expression plasmid and stable Jurkat cell lines. D.P. conducted phagocytosis experiments. A.K. and N.L. carried out the MS analysis and provided critical support in establishing the air-pouch model system. R.I.W. and J.J.L. carried out immunohistochemical detection of apoptotic cells in the thymus. M.O. and P.S. assisted with the BMDM generation and macrophage chemotaxis experiments. T.K.H. provided critical intellectual input in the preparation of the manuscript. K.S.R. provided overall coordination with respect to conception, design and supervision of the study. K.S.R. and M.R.E. wrote the manuscript with comments from co-authors. NIH Public Access Author ManuscriptNature. Author manuscript; available in PMC 2010 April 8. Most developing thymocytes (95%) undergo apoptosis; yet in steady-state only 1-2% are detectable as apoptotic 4,5 . It is hypothesized that dying thymocytes secrete soluble factors that attract resident phagocytes to promote prompt clearance 2,6 . To determine if apoptotic thymocytes release such factors, cell-free supernatants after apoptosis induction (by antiFas/CD95 crosslinking) were assessed for their ability to attract THP-1 monocytesor primary human monocytes in a transwell migration assay ( Figure 1a and Supplemental Figure S2). Apoptotic supernatants caused a 3-fold increase in monocyte migration compared to supernatants of live thymocytes. Such release of chemotactic factors was also seen with Jurkat cells (...
Rapid and efficient removal of apoptotic cells by phagocytes plays a key role during development, tissue homeostasis, and in controlling immune responses1–5. An important feature of efficient clearance is the capacity of a single phagocyte to ingest multiple apoptotic cells successively, and to process the increased load of corpse-derived cellular material6–9. However, factors that influence sustained phagocytic capacity or how they in turn influence continued clearance by phagocytes are not known. Here we identify that the ability of a phagocyte to control its mitochondrial membrane potential is a critical factor in the capacity of a phagocyte to engulf apoptotic cells. Changing the phagocyte mitochondrial membrane potential (genetically or pharmacologically) significantly affected phagocytosis, with lower potential enhancing engulfment and higher membrane potential inhibiting uptake. We then identified that Ucp2, a mitochondrial membrane protein that acts to lower the mitochondrial membrane potential10–12, is upregulated in phagocytes engulfing apoptotic cells (but not synthetic targets, bacteria, or yeast). Loss of Ucp2 limited the capacity of phagocytes to continually ingest apoptotic cells, while overexpression of Ucp2 increased the capacity for engulfment and the ability to engulf multiple apoptotic cells. Mutational and pharmacological inhibition of Ucp2 uncoupling activity reversed the positive effect of Ucp2 on engulfment capacity, suggesting a direct role for Ucp2-mediated mitochondrial function in phagocytosis. Macrophages from Ucp2-deficient mice13, 14 were impaired in their capacity to engulf apoptotic cells in vitro, and Ucp2-deficient mice displayed profound in vivo defects in clearing dying cells in the thymus and the testes. Collectively, these data suggest that phagocytes alter the mitochondrial membrane potential during engulfment to regulate uptake of sequential apoptotic cells, and that Ucp2 is a key molecular determinant of this step in vivo. Since Ucp2 function has also been linked to metabolic diseases and atherosclerosis14–16, these data identifying a new role for Ucp2 in regulating apoptotic cell clearance may provide additional insights toward understanding the complex etiology and pathogenesis of these diseases.
Passage through the β-selection developmental checkpoint requires productive rearrangement of Tcrb gene segments and formation of a pre-T cell receptor (pre-TCR) on the surface of CD4–CD8– thymocytes. How other receptors influence β-selection is less well understood. Here, we define a new role for the chemokine receptor CXCR4 during T cell development. CXCR4 functionally associates with the pre-TCR and influences β-selection by regulating steady-state localization of immature thymocytes within thymic sub-regions, by facilitating optimal pre-TCR-induced survival signals, and by promoting thymocyte proliferation. We also characterize functionally relevant signaling molecules downstream of CXCR4 and the pre-TCR in thymocytes. These data designate CXCR4 as a co-stimulator of the pre-TCR during β-selection.
To investigate the unregulated Ras activation associated with cancer, we developed and validated a mathematical model of Ras signaling. The model-based predictions and associated experiments help explain why only one of two classes of activating Ras point mutations with in vitro transformation potential is commonly found in cancers. Model-based analysis of these mutants uncovered a systems-level process that contributes to total Ras activation in cells. This predicted behavior was supported by experimental observations. We also used the model to identify a strategy in which a drug could cause stronger inhibition on the cancerous Ras network than on the wild-type network. This system-level analysis of the oncogenic Ras network provides new insights and potential therapeutic strategies.
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