Cells undergoing apoptosis are efficiently located and engulfed by phagocytes. The mechanisms by which macrophages, the professional scavenging phagocytes of apoptotic cells, are attracted to sites of apoptosis are poorly defined. Here we show that CX3CL1/fractalkine, a chemokine and intercellular adhesion molecule, is released rapidly from apoptotic lymphocytes, via caspase-and Bcl-2-regulated mechanisms, to attract macrophages. Effective chemotaxis of macrophages to apoptotic lymphocytes is dependent on macrophage fractalkine receptor, CX3CR1. CX3CR1 deficiency caused diminished recruitment of macrophages to germinal centers of lymphoid follicles, sites of high-rate B-cell apoptosis. These results provide the first demonstration of chemokine/chemokine-receptor activity in the navigation of macrophages toward apoptotic cells and identify a mechanism by which macrophage infiltration of tissues containing apoptotic lymphocytes is achieved. (Blood. 2008;112:5026-5036) IntroductionWhen apoptosis occurs at high rates in mammalian tissues, apoptotic cells are almost invariably encountered in situ in association with macrophages. 1 These professional scavengers are attracted to the dying cells and engage in their safe, nonphlogistic disposal by phagocytosis. Examples of this innate immune response to dying cells are readily apparent during normal organogenesis, in normal adult tissues, such as the germinal centers of lymphoid follicles, in inflammatory responses, and in pathologic conditions including tumors. The efficient clearance of apoptotic cells by phagocytes is a homeostatic mechanism that militates against histotoxic, proinflammatory, or immunogenic effects that may result from persistence of apoptotic cells. [1][2][3] In recent years, much progress has been made in improving our understanding of the molecular mechanisms underlying the interactions between apoptotic cells and macrophages and the immunologic implications of those interactions. [1][2][3][4][5][6] Before the tethering/ engulfment phases of macrophage-mediated apoptotic-cell clearance, phagocytes are required to navigate effectively to sites of apoptosis. Active release of chemoattractant ("find-me") signals from apoptotic cells at an early stage after engagement of the cell-death program would be predicted to underpin this process, but knowledge of the molecules involved is currently limited. Lysophosphatidylcholine (LPC) is released from apoptotic cells and functions in soluble form as a chemoattractant for mononuclear phagocytes. 7 Significantly, no chemokine family members have previously been implicated in this chemotactic process. Here we show that the chemokine and adhesion molecule CX3CL1, 8,9 also known as neurotactin or fractalkine (FKN), together with its cognate receptor CX3CR1, 10,11 plays an active role in the chemotaxis of macrophages to apoptotic cells. FKN is a type I transmembrane protein, the extracellular portion of which comprises the chemokine domain attached to a mucin stalk. Well known for its roles in inflammatory processe...

The Fc region of human IgG expresses interaction sites for many effector ligands. In this review the topographical distributions of ten of these sites are discussed in relation to functional requirement. It is apparent that interaction sites localised to the inter-CH2-CH3 domain region of the Fc allow for functional divalency, whereas sites localised to the hinge proximal region of the CH2 domain are functionally monovalent, with expression of the latter sites being particularly dependent on glycosylation. All x-ray crystal structures for Fc and Fc-ligand complexes report that the protein structure of the hinge proximal region of the CH2 domain is "disordered", suggesting "internal mobility". We propose a model in which such "internal mobility" results in the generation of a dynamic equilibrium between multiple conformers, certain of which express interaction sites specific to individual ligands. The emerging understanding of the influence of oligosaccharide/protein interactions on protein conformation and biological function of IgG antibodies suggests a potential to generate novel glycoforms of antibody molecules having unique profiles of effector functions.

Here we consider the impact of the physiological cell-death programme on normal tissue homeostasis and on disease pathogenesis, with particular reference to evolution and progression of neoplasia. We seek to describe the direct contributions played by apoptosis in creating the microenvironments of normal and malignant tissues and to discuss the molecular mechanisms underlying the elements of the '3Rs' that define the meaning of apoptosis: recognition, response, and removal. Apoptotic cells elicit responses in other cell types-both phagocytic and non-phagocytic-through short-and long-range signalling modes that range from direct contact to intercellular communication via membrane-bound microparticles. Such cellular responses include migration, proliferation, and differentiation, as well as production of immunomodulatory and anti-inflammatory mediators together with, in the case of phagocytes, engulfment, and breakdown of apoptotic cells. In normal tissues, the removal of apoptotic cells is rapid and typically non-phlogistic. We discuss the importance of this clearance process in tissue homeostasis and the consequences of its failure in disease pathogenesis. Using the typical cell culture environment in vitro as an illustrative example in which apoptosis occurs commonly in the absence of the removal mechanisms, we also discuss the inhibitory effects of persistent apoptotic cells on their otherwise viable neighbours. Since apoptosis is a common and sustained event in high-grade malignancies, we hypothesize on its purposeful role in conditioning the tumour microenvironment. We propose that apoptosis subserves several pro-tumour functions-trophic, anti-inflammatory, and immunomodulatory-and we identify strategies targeting host responses to apoptotic cells as promising modes of future therapies that could be applied to multiple cancer types in which tumour-cell apoptosis is active.

Apoptosis is a noninflammatory, programmed form of cell death. One mechanism underlying the non-phlogistic nature of the apoptosis program is the swift phagocytosis of the dying cells. How apoptotic cells attract mononuclear phagocytes and not granulocytes, the professional phagocytes that accumulate at sites of inflammation, has not been determined. Here, we show that apoptotic human cell lines of diverse lineages synthesize and secrete lactoferrin, a pleiotropic glycoprotein with known antiinflammatory properties. We further demonstrated that lactoferrin selectively inhibited migration of granulocytes but not mononuclear phagocytes, both in vitro and in vivo. Finally, we were able to attribute this antiinflammatory function of lactoferrin to its effects on granulocyte signaling pathways that regulate cell adhesion and motility. Together, our results identify lactoferrin as an antiinflammatory component of the apoptosis milieu and define what we believe to be a novel antiinflammatory property of lactoferrin: the ability to function as a negative regulator of granulocyte migration.

SummaryBackgroundCells undergoing apoptosis are known to modulate their tissue microenvironments. By acting on phagocytes, notably macrophages, apoptotic cells inhibit immunological and inflammatory responses and promote trophic signaling pathways. Paradoxically, because of their potential to cause death of tumor cells and thereby militate against malignant disease progression, both apoptosis and tumor-associated macrophages (TAMs) are often associated with poor prognosis in cancer. We hypothesized that, in progression of malignant disease, constitutive loss of a fraction of the tumor cell population through apoptosis could yield tumor-promoting effects.ResultsHere, we demonstrate that apoptotic tumor cells promote coordinated tumor growth, angiogenesis, and accumulation of TAMs in aggressive B cell lymphomas. Through unbiased “in situ transcriptomics” analysis—gene expression profiling of laser-captured TAMs to establish their activation signature in situ—we show that these cells are activated to signal via multiple tumor-promoting reparatory, trophic, angiogenic, tissue remodeling, and anti-inflammatory pathways. Our results also suggest that apoptotic lymphoma cells help drive this signature. Furthermore, we demonstrate that, upon induction of apoptosis, lymphoma cells not only activate expression of the tumor-promoting matrix metalloproteinases MMP2 and MMP12 in macrophages but also express and process these MMPs directly. Finally, using a model of malignant melanoma, we show that the oncogenic potential of apoptotic tumor cells extends beyond lymphoma.ConclusionsIn addition to its profound tumor-suppressive role, apoptosis can potentiate cancer progression. These results have important implications for understanding the fundamental biology of cell death, its roles in malignant disease, and the broader consequences of apoptosis-inducing anti-cancer therapy.

Key points• Exosomes are vesicles that are released from the kidney into the urine. They contain RNA and protein from the cell of origin and can track changes in renal physiology non-invasively.• Current methods for the identification and quantification of urinary exosomes are time consuming and only semi-quantitative.• In this study, we applied nanoparticle tracking analysis to human urine and identified particles with a range of sizes, including a subpopulation of characteristic exosomal size that labelled positively with antibodies to exosome proteins. • Nanoparticle tracking analysis was able to track an increase in exosomal aquaporin 2 concentration following desmopressin treatment of a kidney cell line, a rodent model and a patient with central diabetes insipidus.• With appropriate sample storage, nanoparticle tracking analysis has potential as a tool for the rapid characterization and quantification of exosomes in human urine. This new method can be used to develop urinary extracellular vesicles further as a non-invasive tool for investigating human renal physiology.Abstract Exosomes are vesicles that are released from the kidney into urine. They contain protein and RNA from the glomerulus and all sections of the nephron and represent a reservoir for biomarker discovery. Current methods for the identification and quantification of urinary exosomes are time consuming and only semi-quantitative. Nanoparticle tracking analysis (NTA) counts and sizes particles by measuring their Brownian motion in solution.In this study, we applied NTA to human urine and identified particles with a range of sizes. Using antibodies against the exosomal proteins CD24 and aquaporin 2 (AQP2), conjugated to a fluorophore, we could identify a subpopulation of CD24-and AQP2-positive particles of characteristic exosomal size. Extensive pre-NTA processing of urine was not necessary. However, the intra-assay variability in the measurement of exosome concentration was significantly reduced when an ultracentrifugation step preceded NTA. Without any sample processing, NTA tracked exosomal AQP2 upregulation induced by desmopressin stimulation of kidney collecting duct cells. Nanoparticle tracking analysis was also able to track changes in exosomal AQP2 concentration that followed desmopressin treatment of mice and a patient with central diabetes insipidus. When urine was stored at room temperature, 4• C or frozen, nanoparticle concentration was reduced; freezing at −80• C with the addition of protease inhibitors produced the least reduction. In conclusion, with appropriate sample storage, NTA has potential as a tool for the characterization and quantification of extracellular vesicles in human urine.

Burkitt’s lymphoma (BL) is typified by frequent tumor cell apoptosis and significant macrophage infiltration. Since BL cells have an inherent tendency to undergo apoptosis at a high rate, we reasoned that macrophages in BL are functionally enhanced in at least two activities that have implications for tumor pathogenesis: 1) engulfment of apoptotic cells, an anti-inflammatory process known to suppress immune responses, and 2) production of BL cell survival factors that limit the extent of tumor cell apoptosis. In this study, we show that the microenvironment of BL is rich in the pleiotropic cytokine IL-10, which can be produced by both tumor cells and macrophages, and that IL-10-activated human macrophages have enhanced capacity to engulf apoptotic cells in vitro. This was found to be dependent on the macrophage tethering receptor of apoptotic cells, CD14. Furthermore, IL-10-activated macrophages were found to produce markedly higher levels of the B cell survival factor, B cell-activating factor of the TNF family/B lymphocyte stimulator (BAFF/BLyS) than macrophages matured in the absence of IL-10. Coculture of macrophages with BL cells further enhanced BAFF secretion. Significantly, we show that enhancement of BL cell survival by IL-10-activated macrophages is mediated by a BAFF-dependent component and that BAFF is produced at high levels by tumor-associated macrophages in situ. These results indicate that macrophages, regulated by IL-10, have the potential to promote BL pathogenesis, first, through suppression of antitumor immunity following enhanced engulfment of apoptotic tumor cells and, second, through increased production of tumor cell growth/survival factors.

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