Fractalkine-induced MFG-E8 leads to enhanced apoptotic cell clearance by macrophages

Clearance of apoptotic cells is crucial to maintain cellular function under normal and pathological conditions. We have recently shown that administration of immature dendritic cell-derived exosomes to septic animals promotes phagocytosis of apoptotic cells and improves survival by providing milk fat globule epidermal growth factor-factor VIII (MFG-E8). MFG-E8 acts as an opsonin for apoptotic cells to be engulfed by phagocytosis. In the present study we investigated whether the CX(3)C-chemokine fractalkine (CX(3)CL1) promotes apoptotic cell clearance through the induction of MFG-E8 in peritoneal macrophages. Cultured rat peritoneal macrophages (pMphi) and RAW264.7 macrophages were stimulated with LPS and CX(3)CL1. MFG-E8 expression was assessed by Western blot, cytokine secretion was assessed by ELISA, and phagocytosis of apoptotic thymocytes was determined by microscopy. For in vivo studies, cecal ligation and puncture (CLP) was used to induce sepsis in rats and mice. LPS significantly decreased MFG-E8 levels and phagocytosis of apoptotic cells, whereas CX(3)CL1 induced MFG-E8 expression in both nonstimulated and LPS-stimulated pMphi, without affecting TNF-alpha and IL-6 release. Anti-MFG-E8 blocking antibodies completely abrogated the prophagocytic effect of CX(3)CL1. Twenty hours after the induction of sepsis in rats via CLP, plasma CX(3)CL1 levels as well as MFG-E8 production in peritoneal macrophages decreased by 21% and 56%, respectively. Administration of CX(3)CL1 on the other hand induced MFG-E8 and prevented tissue injury. We conclude that CX(3)CL1 induces MFG-E8 in vitro and in vivo and enhances clearance of apoptotic cells in an MFG-E8-dependent manner. These findings suggest a possible novel treatment for patients in sepsis.     

The opsonin MFG-E8 is a ligand for the alphavbeta5 integrin and triggers DOCK180-dependent Rac1 activation for the phagocytosis of apoptotic cells

Opsonization of apoptotic cells facilitates recognition by phagocytes for the rapid clearance of potentially inflammatory cellular material. The secreted glycoprotein Milk Fat Globule Factor-E8 (MFG-E8) is a member of this family of bridging molecules and is believed to bind phosphatidylserine (PS) on the dying cell, linking it to integrin receptors on the phagocyte. Here we report the characterization of a functional signaling module involving MFG-E8, alphavbeta5 integrin, and DOCK180 for the activation of Rac1. We show that MFG-E8 and DOCK180 are both expressed in phagocytic-competent primary immature dendritic cells (DCs) and DC2.4 cells, and are potently down-regulated upon DC maturation, consistent with their role in phagocytosis and antigen capture. Coexpression of MFG-E8 with alphavbeta5 integrin potentiated integrin-mediated Rac1 activation, which was abrogated by mutagenesis in the RGD motif in MFG-E8. Moreover, expression of antisense DOCK180 abrogated MFG-E8-alphavbeta5-mediated Rac activation and impaired the phagocytosis of apoptotic cells. These data demonstrate a biochemical link between an opsonin of apoptotic cells, the alphavbeta5 integrin, and the Crk-DOCK180-Rac1 pathway, and importantly, show that MFG-E8 and DOCK180 are expressed according to the functional status of the phagocyte.     

Essential Role of MFG-E8 for Phagocytic Properties of Microglial Cells

Milk fat globule factor-E8 (MFG-E8) has been regarded as a key factor involved in the phagocytosis of apoptotic cells. We induced a lentivirus into the microglial cells for the augmentation or abrogation of MFG-E8 expression in mouse microglial cells, and investigated phagocytosis of phosphatidylserine tagged human red blood cells (hRBCs) in co-cultures. Increased MFG-E8 levels were associated with a significant increase in phagocytic activity compared to the controls. Conversely, phagocytosis dramitically decreased due to the abrogation of MFG-E8. In addition, the expression of the inflammatory cytokines, TNF-α and IL-1β, also increased or decreased in the microglial cells with the augmentation or abrogation of MFG-E8, respectively. Our findings indicate that the enhanced expression of MFG-E8 could increase phagocytosis of apoptotic cells; conversely, the rate of phagocytosis and the expression of inflammatory cytokines decreased when MFG-E8 expression was knocked down. Our results confirm that MFG-E8 plays an important role in phagocytosis, and possibly serves as an essential signal molecule for microglial cells.     

Milk fat globule-epidermal growth factor-factor 8 attenuates neutrophil infiltration in acute lung injury via modulation of CXCR2

Excessive neutrophil infiltration to the lungs is a hallmark of acute lung injury (ALI). Milk fat globule epidermal growth factor-factor 8 (MFG-E8) was originally identified for phagocytosis of apoptotic cells. Subsequent studies revealed its diverse cellular functions. However, whether MFG-E8 can regulate neutrophil function to alleviate inflammation is unknown. We therefore aimed to reveal MFG-E8 roles in regulating lung neutrophil infiltration during ALI. To induce ALI, C57BL/6J wild-type (WT) and Mfge8(-/-) mice were intratracheally injected with LPS (5 mg/kg). Lung tissue damage was assessed by histology, and the neutrophils were counted by a hemacytometer. Apoptotic cells in lungs were determined by TUNEL, whereas caspase-3 and myeloperoxidase activities were assessed spectrophotometrically. CXCR2 and G protein-coupled receptor kinase 2 expressions in neutrophils were measured by flow cytometry. Following LPS challenge, Mfge8(-/-) mice exhibited extensive lung damage due to exaggerated infiltration of neutrophils and production of TNF-α, MIP-2, and myeloperoxidase. An increased number of apoptotic cells was trapped into the lungs of Mfge8(-/-) mice compared with WT mice, which may be due to insufficient phagocytosis of apoptotic cells or increased occurrence of apoptosis through the activation of caspase-3. In vitro studies using MIP-2-mediated chemotaxis revealed higher migration of neutrophils of Mfge8(-/-) mice than those of WT mice via increased surface exposures to CXCR2. Administration of recombinant murine MFG-E8 reduces neutrophil migration through upregulation of GRK2 and downregulation of surface CXCR2 expression. Conversely, these effects could be blocked by anti-α(v) integrin Abs. These studies clearly indicate the importance of MFG-E8 in ameliorating neutrophil infiltration and suggest MFG-E8 as a novel therapeutic potential for ALI.     

Expression, purification and characterization of C2 domain of milk fat globule-EGF-factor 8-L

Milk fat globule-EGF-factor 8-L (MFG-E8L) is secreted by activated macrophages and functions as a linker protein or opsonin between the dying cells and phagocytes. MFG-E8L recognizes the apoptotic or dying cells by specifically binding to Phosphatidylserine (PS) exposed on the outer cell surface and enhances the engulfment of the apoptotic cells by phagocytes, thereby preventing the inflammation and autoimmune response against intracellular antigens that can be released from the dying cells. MFG-E8L contains two EGF-like domains, P/T (proline/threonine) rich domain followed by two discoidin-like domains (C1 and C2). Recent studies have shown that the C2 domain of MFG-E8L is specifically involved in interaction with PS exposed on the apoptotic cells. Towards understanding this specific molecular interaction between the MFG-E8L C2 domain and PS, we expressed, purified the C2 domain of MFG-E8L and performed the binding studies with phospholipids by (31)P NMR experiment. We demonstrated that our recombinant construct and expression system were effective and allowed us to obtain the C2 domain and also showed that the purified C2 domain was stable and properly folded, and our (31)P NMR studies indicated that the C2 domain had specific binding with PS.     

Review: milk fat globule-EGF factor 8 expression,function and plausible signal transduction in resolving inflammation

Although the cloning and molecular characterization of MFG-E8 was first reported in the early 90s, breakthrough on MFG-E8 research came into light when it was explored as an outstanding factor for phagocytosis of apoptotic cells by professional macrophages in 2002. Since then numerous studies have been performed on MFG-E8 not only to demonstrate the role of phagocytic clearance of apoptotic cells, but also to focus on a wide range of aspects, even emphasizing on a direct link to innate-immune systems. In terms of its role as therapeutic potentials, our group, as well as others, has shown MFG-E8 to be an essential factor in attenuating inflammation and improving prognosis in several animal models of life threatening diseases. Considering these versatile functions of MFG-E8, several in vitro and in vivo studies were embarked on to explore the mechanistic pathways exerted by MFG-E8 during inflammation. With the relevant cumulative findings, herein we reviewed the potential roles of MFG-E8 in pathophysiological conditions by highlighting its plausible signal-transduction mechanisms.     

Prolactin induces MFG-E8 production in macrophages via transcription factor C/EBPβ-dependent pathway

The lactogenic hormone prolactin (PRL) regulates milk protein gene expression in mammary glands. To maintain homeostatic balance in the body, milk fat globule epidermal growth factor 8 (MFG-E8) is vital for phagocytic clearance of apoptotic cells. We investigated the effects of PRL on MFG-E8 expression in macrophages by evaluating its promoter function. Macrophages were stimulated with PRL, and the expression of MFG-E8 was determined using real-time PCR and Western blotting. The role of MFG-E8 on phagocytosis of apoptotic cells in PRL-treated macrophages was assessed using microscopy, while the response of PRL to MFG-E8 expression was evaluated using luciferase assay. Following treatment with PRL, significant up-regulations of the PRL receptor and MFG-E8 were observed in macrophages, though PRL-treated macrophages more efficiently engulfed apoptotic cells. The results of MFG-E8 promoter analysis showed considerable up-regulation of promoter activity in macrophages following PRL treatment and results from mutation analysis of the MFG-E8 promoter suggested that the C/EBPβ binding site was responsible for PRL-induced activation of the MFG-E8 promoter. C/EBPβ activity was found to be up-regulated in PRL-treated cells as revealed by an electrophoretic mobility shift assay (EMSA). In conclusion, PRL is a potent inducer of MFG-E8 expression in macrophages, while its effect is mediated by the presence of a responsive element in the MFG-E8 promoter.     

Phagocytosis of apoptotic cells

Removal of apoptotic cells by phagocytes plays an important role in many biological processes, including embryological development and tissue remodelling. In addition, it has become apparent that one of the key mechanisms for the successful resolution of inflammation is the orchestrated clearance of apoptotic inflammatory cells by phagocytes (e.g., macrophages and dendritic cells) and other cells known to have phagocytic capacity (e.g., hepatocytes, endothelial cells, epithelial cells, etc.). Furthermore, phagocytosis of apoptotic cells is an active and highly regulated process that not only serves to remove potentially histotoxic cells from the inflammatory milieu, but also directs the phenotype of the phagocytic cell to be anti-inflammatory. Convincing evidence has been presented that reduced or dysregulated phagocytosis of apoptotic cells contributes to the development and propagation of inflammatory disorders. Conversely, enhanced phagocytosis of apoptotic cells may be exploited for therapeutic gain. Indeed, powerful anti-inflammatory drugs such as the glucocorticoids have been shown to augment clearance of apoptotic cells which may contribute to their therapeutic effectiveness. In this chapter, we describe methods for studying phagocytosis of apoptotic cells.     

Expression of developmental endothelial locus-1 in a subset of macrophages for engulfment of apoptotic cells

A major function of macrophages is to engulf apoptotic cells to prevent them from releasing noxious materials as they die. Milk fat globule-EGF-factor 8 (MFG-E8) is a glycoprotein secreted by activated macrophages that works as a bridge between apoptotic cells and phagocytes by specifically recognizing phosphatidylserine exposed on apoptotic cells. In this study, we found that developmental endothelial locus-1 (Del-1), originally identified as an embryonic endothelial cell protein that binds alphavbeta3 integrin, is structurally and functionally homologous to MFG-E8. That is, both consist of a signal sequence, two epidermal growth factor domains and two factor VIII-homologous domains (C1 and C2). Del-1 bound to the apoptotic cells by recognizing phosphatidylserine via the factor VIII-homologous domains with an affinity similar to that of MFG-E8. The phagocytic activity of NIH 3T3 cells against apoptotic cells was enhanced by Del-1 through an interaction between the epidermal growth factor domain in Del-1 and alphavbeta3 integrin expressed in the NIH 3T3 cells. Screening of primary macrophages and macrophage cell lines for the expression of MFG-E8 and Del-1 indicated that MFG-E8 and Del-1 are expressed in different sets of macrophages. These results suggest the existence of macrophage subsets that use MFG-E8 or Del-1 differently to engulf apoptotic cells.     

Regulation and functional relevance of milk fat globules and their components in the mammary gland

Mammary gland and epithelial cells are unique to mammals and are under the control of lactogenic hormones such as prolactin. Recent findings indicated that major components of milk fat globule membrane (MFGM) are under the control of lactogenic hormones, and that the major components butyrophilin and xanthine oxidoreductase are indispensable for milk fat secretion. Further, prolactin signaling is negatively controlled by two highly related protein tyrosine phosphatases, PTP1B and TC-PTP. Milk fat globule EGF factor 8 (MFG-E8) is one of the major components of MFGM and is upregulated during lactation. MFG-E8 is further upregulated in the involuting mammary gland. MFG-E8 on exosome-like membrane vesicles in the milk recovered from post-weaning but not lactating mammary glands exhibits higher binding activity to phosphatidylserine and apoptotic mammary epithelial cells, and serves as a link between apoptotic mammary epithelial cells and phagocytes. Recent reports using MFG-E8 deficient mice support the view that MFG-E8 is indispensable for eliminating apoptotic mammary epithelial cells during involution.     

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: Therapeutic potential of mitochondrially targeted antioxidants

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury; however, its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explore the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2h of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6h of reperfusion and peaking at 24h). Mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.     

Milk fat globule-EGF factor 8 mediates the enhancement of apoptotic cell clearance by glucocorticoids

The phagocytic clearance of apoptotic cells is essential to prevent chronic inflammation and autoimmunity. The phosphatidylserine-binding protein milk fat globule-EGF factor 8 (MFG-E8) is a major opsonin for apoptotic cells, and MFG-E8^−/− mice spontaneously develop a lupus-like disease. Similar to human systemic lupus erythematosus (SLE), the murine disease is associated with an impaired clearance of apoptotic cells. SLE is routinely treated with glucocorticoids (GCs), whose anti-inflammatory effects are consentaneously attributed to the transrepression of pro-inflammatory cytokines. Here, we show that the GC-mediated transactivation of MFG-E8 expression and the concomitantly enhanced elimination of apoptotic cells constitute a novel aspect in this context. Patients with chronic inflammation receiving high-dose prednisone therapy displayed substantially increased MFG-E8 mRNA levels in circulating monocytes. MFG-E8 induction was dependent on the GC receptor and several GC response elements within the MFG-E8 promoter. Most intriguingly, the inhibition of MFG-E8 induction by RNA interference or genetic knockout strongly reduced or completely abolished the phagocytosis-enhancing effect of GCs in vitro and in vivo. Thus, MFG-E8-dependent promotion of apoptotic cell clearance is a novel anti-inflammatory facet of GC treatment and renders MFG-E8 a prospective target for future therapeutic interventions in SLE.     

Phagocytosis: coupling of mitochondrial uncoupling and engulfment

Clearance of apoptotic cells by phagocytes avoids triggering an inflammatory response. A new study reveals that phagocytes dissipate their mitochondrial proton electrochemical gradient to allow for the ingestion of more apoptotic corpses. Mitochondria are therefore involved in all aspects of apoptosis, from its activation through to the phagocytosis of dead cells.     

Milk fat globule-EGF factor 8 is a critical protein for healing of dextran sodium sulfate-induced acute colitis in mice

Milk fat globule-EGF factor 8 (MFG-E8) has been shown to play an important role in maintaining the integrity of the intestinal mucosa and to accelerate healing of the mucosa in septic mice. Herein, we (a) analyzed the expression of MFG-E8 in the gut of wild-type (WT) C57BL/6 (MFG-E8(+/+)) mice with and without dextran sulfate sodium (DSS)-induced colitis, (b) characterized the pathological changes in intestinal mucosa of MFG-E8(+/+) and MFG-E8(-/-) mice with DSS-induced colitis and (c) examined the therapeutic role of MFG-E8 in inflammatory bowel disease by using DSS-induced colitis model. Our data documented that there was an increase in colonic and rectal MFG-E8 expression in MFG-E8(+/+) mice during the development of DSS colitis. MFG-E8 levels in both tissues decreased to below baseline during the recovery phase in mice with colitis. Changes in MFG-E8 gene expression correlated to the levels of inflammatory response and crypt-epithelial injury in both colonic and rectal mucosa in MFG-E8(+/+) mice. MFG-E8(-/-)mice developed more severe crypt-epithelial injury than MFG-E8(+/+) mice during exposure to DSS with delayed healing of intestinal epithelium during the recovery phase of DSS colitis. Administration of MFG-E8 during the recovery phase ameliorated colitis and promoted mucosal repair in both MFG-E8(-/-) and MFG-E8(+/+) mice, indicating that lack of MFG-E8 causes increased susceptibility to colitis and delayed mucosal healing. These data suggest that MGF-E8 is an essential protective factor for gut epithelial homeostasis, and exogenous administration of MFG-E8 may represent a novel therapeutic target in inflammatory bowel disease.     

KIM-1-/TIM-1-mediated phagocytosis links ATG5-/ULK1-dependent clearance of apoptotic cells to antigen presentation

Phagocytosis of apoptotic cells by both professional and semi-professional phagocytes is required for resolution of organ damage and maintenance of immune tolerance. KIM-1/TIM-1 is a phosphatidylserine receptor that is expressed on epithelial cells and can transform the cells into phagocytes. Here, we demonstrate that KIM-1 phosphorylation and association with p85 results in encapsulation of phagosomes by lipidated LC3 in multi-membrane organelles. KIM-1-mediated phagocytosis is not associated with increased ROS production, and NOX inhibition does not block LC3 lipidation. Autophagy gene expression is required for efficient clearance of apoptotic cells and phagosome maturation. KIM-1-mediated phagocytosis leads to pro-tolerogenic antigen presentation, which suppresses CD4 T-cell proliferation and increases the percentage of regulatory T cells in an autophagy gene-dependent manner. Taken together, these data reveal a novel mechanism of epithelial biology linking phagocytosis, autophagy and antigen presentation to regulation of the inflammatory response.     

An Apoptosis-Associated Mammary Protein Deficiency Leads to Enhanced Production of IgM Antibodies against Multiple Damage-Associated Molecules

Milk fat globule epidermal growth factor 8 (MFG-E8) is a protein that binds to apoptotic cells by recognizing phosphatidylserine and enhances the engulfment of apoptotic cells by macrophages. Many apoptotic cells are left unengulfed in the germinal centers of the spleen in the MFG-E8-deficient (MFG-E8^−/−) mice, and these mice develop an autoimmune disease resembling human systemic lupus erythematosus. We found that the MFG-E8 deficiency was accompanied by the increased production of immunoglobulins. Further Western blot and ELISA analyses validated the increase in the IgM levels in the MFG-E8^−/− mice. It was also revealed that the sera from the MFG-E8^−/− mice cross-reacted with oxidation-specific epitopes generated upon incubation of serum albumin with the peroxidized lipids. Among the modified proteins with several unsaturated aldehydes of chain lengths varying from three to nine carbons, the MFG-E8^−/− mice sera exclusively cross-reacted with the protein-bound 4-oxo-2-nonenal (ONE), a highly reactive aldehyde originating from the peroxidation of ω6 polyunsaturated fatty acids. In addition, the IgM monoclonal antibodies (mAbs) that selectively cross-reacted with the ONE-modified proteins were generated from the MFG-E8^−/− mice. A subset of the ONE-specific IgM mAbs significantly recognized the late apoptotic and necrotic cells and enhanced the phagocytosis by macrophages. These data demonstrate that the impairment of the phagocytic clearance of apoptotic cells through MFG-E8 can lead to the generation of natural antibodies, which may play a critical role in removing multiple damage-associated molecules, including oxidation-specific epitopes and late apoptotic/necrotic cells.     

Mrp8 and Mrp14 are endogenous activators of Toll-like receptor 4, promoting lethal, endotoxin-induced shock

To identify new components that regulate the inflammatory cascade during sepsis, we characterized the functions of myeloid-related protein-8 (Mrp8, S100A8) and myeloid-related protein-14 (Mrp14, S100A9), two abundant cytoplasmic proteins of phagocytes. We now demonstrate that mice lacking Mrp8-Mrp14 complexes are protected from endotoxin-induced lethal shock and Escherichia coli-induced abdominal sepsis. Both proteins are released during activation of phagocytes, and Mrp8-Mrp14 complexes amplify the endotoxin-triggered inflammatory responses of phagocytes. Mrp8 is the active component that induces intracellular translocation of myeloid differentiation primary response protein 88 and activation of interleukin-1 receptor-associated kinase-1 and nuclear factor-kappaB, resulting in elevated expression of tumor necrosis factor-alpha (TNF-alpha). Using phagocytes expressing a nonfunctional Toll-like receptor 4 (TLR4), HEK293 cells transfected with TLR4, CD14 and MD2, and by surface plasmon resonance studies in vitro, we demonstrate that Mrp8 specifically interacts with the TLR4-MD2 complex, thus representing an endogenous ligand of TLR4. Therefore Mrp8-Mrp14 complexes are new inflammatory components that amplify phagocyte activation during sepsis upstream of TNFalpha-dependent effects.     

Epithelial cells as phagocytes: apoptotic epithelial cells are engulfed by mammary alveolar epithelial cells and repress inflammatory mediator release

Clearance of apoptotic cells is critical to tissue homeostasis and resolution of inflammatory lesions. Macrophages are known to remove dying cells and release anti-inflammatory mediators in response; however, many cells traditionally thought of as poor phagocytes can mediate this function as well. In the lactating mammary gland following weaning, alveolar epithelial cell death is massive, yet the gland involutes rapidly, attaining its prepregnancy state in a matter of days. We found histologic evidence of apoptotic cell phagocytosis by viable mammary epithelial cells (MEC) in the involuting mouse mammary gland. Cultured MEC were able to engulf apoptotic cells in vitro, utilizing many of the same receptors used by macrophages, including the phosphatidylserine receptor (PSR), CD36, the vitronectin receptor alpha(v)beta3, and CD91. In addition, MEC, like macrophages, produced TGFbeta in response to stimulation of the PSR by apoptotic cells or the anti-PSR ab 217G8E9, and downregulated endotoxin-stimulated proinflammatory cytokine production. These data support the hypothesis that amateur phagocytes play a significant role in apoptotic cell clearance and its regulation of inflammation.     

Leukocyte migratory responses to apoptosis: The attraction and the distraction

An expanding body of evidence demonstrates that cells undergoing apoptosis send out a selection of molecular navigational signals including proteins, lipids and nucleotides that serve to recruit phagocytes to the dying targets, which are subsequently engulfed and removed. This homeostatic process is essentially non-phlogistic, contrasting markedly with the acute inflammatory responses elicited in phagocytes by damaging or infectious agents. The “professional” scavengers of apoptotic cells are mononuclear phagocytes—the macrophages—and sites of high-rate apoptosis are clearly characterized by macrophages associated with the apoptotic cells. By contrast, members of the other class of professional phagocytes—the granulocytes—are not recruited to sites of apoptosis as a direct consequence of the cell-death program. Indeed, recent work indicates that apoptotic cells release a mixture of migratory cues to leukocytes in order to selectively attract mononuclear phagocytes but not granulocytes through functional balancing of positive and negative signals. Here we discuss these molecular mechanisms that not only serve as migratory cues but also may activate responding phagocytes to engulf apoptotic cells effectively. Finally, we speculate upon new therapeutic opportunities these mechanisms offer for a range of pathological conditions, including inflammatory disorders and cancer.Key words: apoptosis, migration, chemotaxis, macrophage, monocyte, granulocyte, phagocytosis, lactoferrin, ATP, fractalkine