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 (...

Rationale: Macrophages change their phenotype and biological functions depending on the microenvironment. In atherosclerosis, oxidative tissue damage accompanies chronic inflammation; however, macrophage phenotypic changes in response to oxidatively modified molecules are not known.Objective: To examine macrophage phenotypic changes in response to oxidized phospholipids that are present in atherosclerotic lesions. Methods and Results:

Atherosclerosis is an inflammatory disease of large arteries. Flow cytometry of aortic cell suspensions showed that B and T lymphocytes and some macrophages and dendritic cells are already present in the adventitia of normal/noninflamed mouse aortas. Adoptively transferred lymphocytes constitutively homed to the aorta and resided within the adventitia up to 7 d after transfer. Lymphocyte trafficking into normal/noninflamed or atherosclerosis-prone aortas was partially L-selectin dependent. Antigen-activated dendritic cells induced increased T lymphocyte proliferation within the aorta 72 h after adoptive transfer. During progression of atherosclerosis in apolipoprotein-E–deficient mice, the total number of macrophages, T cells, and dendritic cells, but not B cells, increased significantly. This alteration in immune cell composition was accompanied by the formation of tertiary lymphoid tissue in the adventitia of atherosclerotic aortas. These results demonstrate that lymphocytes already reside within the normal/noninflamed aorta before the onset atherosclerosis as a consequence of constitutive trafficking. Atherosclerosis induces the recruitment of macrophages and dendritic cells that support antigen presentation.

Lipopolysaccharide (LPS), an outer-membrane component of Gram-negative bacteria, interacts with LPS-binding protein and CD14, which present LPS to toll-like receptor 4 (refs 1, 2), which activates inflammatory gene expression through nuclear factor kappa B (NF kappa B) and mitogen-activated protein-kinase signalling. Antibacterial defence involves activation of neutrophils that generate reactive oxygen species capable of killing bacteria; therefore host lipid peroxidation occurs, initiated by enzymes such as NADPH oxidase and myeloperoxidase. Oxidized phospholipids are pro-inflammatory agonists promoting chronic inflammation in atherosclerosis; however, recent data suggest that they can inhibit expression of inflammatory adhesion molecules. Here we show that oxidized phospholipids inhibit LPS-induced but not tumour-necrosis factor-alpha-induced or interleukin-1 beta-induced NF kappa B-mediated upregulation of inflammatory genes, by blocking the interaction of LPS with LPS-binding protein and CD14. Moreover, in LPS-injected mice, oxidized phospholipids inhibited inflammation and protected mice from lethal endotoxin shock. Thus, in severe Gram-negative bacterial infection, endogenously formed oxidized phospholipids may function as a negative feedback to blunt innate immune responses. Furthermore, identified chemical structures capable of inhibiting the effects of endotoxins such as LPS could be used for the development of new drugs for treatment of sepsis.

Lung epithelial cells can influence immune responses to airway allergens1,2. Airway epithelial cells also undergo apoptosis after encountering environmental allergens3; yet, relatively little is known about how these are cleared, and their effect on airway inflammation. Here we show that airway epithelial cells efficiently engulf apoptotic epithelial cells and secrete anti-inflammatory cytokines, dependent upon intracellular signalling by the small GTPase Rac1. Inducible deletion of Rac1 expression specifically in airway epithelial cells in a mouse model resulted in defective engulfment by epithelial cells and aberrant anti-inflammatory cytokine production. Intranasal priming and challenge of these mice with house dust mite extract or ovalbumin as allergens led to exacerbated inflammation, augmented Th2 cytokines and airway hyper-responsiveness, with decreased interleukin (IL)-10 in bronchial lavages. Rac1-deficient epithelial cells produced much higher IL-33 upon allergen or apoptotic cell encounter, with increased numbers of nuocyte-like cells1,4,5. Administration of exogenous IL-10 ‘rescued’ the airway inflammation phenotype in Rac1-deficient mice, with decreased IL-33. Collectively, these genetic and functional studies suggest a new role for Rac1-dependent engulfment by airway epithelial cells and in establishing the anti-inflammatory environment, and that defects in cell clearance in the airways could contribute to inflammatory responses towards common allergens.