AIP56, a novel plasmid-encoded virulence factor of Photobacterium damselae subsp. piscicida with apoptogenic activity against sea bass macrophages and neutrophils

A strategy used by extracellular pathogens to evade phagocytosis is the utilization of exotoxins that kill host phagocytes. We have recently shown that one major pathogenicity strategy of Photobacterium damselae subsp. piscicida (Phdp), the agent of the widespread fish pasteurellosis, is the induction of extensive apoptosis of sea bass macrophages and neutrophils that results in lysis of these phagocytes by post-apoptotic secondary necrosis. Here we show that this unique process is mediated by a novel plasmid-encoded apoptosis inducing protein of 56 kDa (AIP56), an exotoxin abundantly secreted by all virulent, but not avirulent, Phdp strains tested. AIP56 is related to an unknown protein of the enterohemorrhagic Escherichia coli O157:H7 and NleC, a Citrobacter rodentium type III secreted effector of unknown function. Passive immunization of sea bass with a rabbit anti-AIP56 serum conferred protection against Phdp challenge, indicating that AIP56 represents a key virulence factor of that pathogen and is a candidate for the design of an anti-pasteurellosis vaccine.     

The bacterial exotoxin AIP56 induces fish macrophage and neutrophil apoptosis using mechanisms of the extrinsic and intrinsic pathways

It has been previously shown that the exotoxin of the important fish pathogen Photobacterium damselae ssp. piscicida is a key pathogenicity factor and is responsible for the extensive systemic apoptosis of macrophages and neutrophils seen in acute fish photobacteriosis. The focus of the present study was to further characterize the AIP56-induced apoptosis of sea bass professional phagocytes by assessing the involvement of caspases, mitochondria and oxidative stress. The resulting data indicate that the apoptotic response in peritoneal macrophages and neutrophils treated ex vivo with AIP56 involves activation of caspase-8, -9 and -3, and mitochondria as shown by loss of mitochondrial membrane potential, release of cytochrome c and over-production of ROS. These results together with previous data from this laboratory suggest that both the extrinsic and intrinsic apoptotic pathways are involved in the AIP56-induced phagocyte apoptosis.     

Lyt-2+ T cell-mediated protection against listeriosis. Protection correlates with phagocyte depletion but not with IFN-gamma production

The transfer of listeria-immune splenocytes into non-immune mice markedly increases host resistance to listeriosis. To study the mechanism for this enhancement, we compared the inflammatory response to infection in nonimmune and adoptively immunized mice. Despite much better containment of bacterial growth, adoptively immunized animals accumulated significantly fewer phagocytes (neutrophils and macrophages) in the spleen and liver than controls. Immune T cells not only inhibited phagocyte accumulation but also reduced the in vitro anti-listerial activity of splenocytes. Significant differences in phagocyte accumulation were observed even when the initial listeria dose was adjusted to produce comparable spleen listeria loads in immune and non-immune animals. However, bone marrow and peripheral blood phagocyte counts were similar in both groups. Depletion of Lyt-2+ cells (using mAb and C) from donor splenocytes prevented the transfer of protection and increased phagocyte accumulation without altering listeria-dependent IFN-gamma production by donor or recipient splenocytes in vitro. L3T4 depletion did not affect host resistance or phagocyte accumulation even though it reduced in vitro interferon production by donor cells. Hence the different effects of L3T4+ and Lyt-2+ cells in vivo cannot be explained simply by variations in IFN production. We suggest this paradoxical suppression of phagocyte accumulation during adoptive transfer may reflect lysis of bacteria-laden phagocytes by listeria-specific Lyt-2+ cells in vivo. Selective destruction of older, heavily infected cells might contribute to host resistance by eliminating a potential site for intracellular proliferation of bacteria.     

Salmonella induces macrophage death by caspase-1-dependent necrosis

We provide evidence that Salmonella typhimurium kills phagocytes by an unusual proinflammatory mechanism of necrosis that is distinguishable from apoptosis. Infection stimulated a distinctly diffuse pattern of DNA fragmentation in macrophages, which contrasted with the marked nuclear condensation displayed by control cells undergoing chemically induced apoptosis. In apoptotic cells, DNA fragmentation and nuclear condensation result from caspase-3-mediated proteolysis; caspases also subvert necrotic cell death by cleaving and inactivating poly ADP-ribose polymerase (PARP). Caspase-3 was not activated during Salmonella infection, and PARP remained in its active, uncleaved state. Another hallmark of apoptosis is sustained membrane integrity during cell death; yet, infected macrophages rapidly lost membrane integrity, as indicated by simultaneous exposure of phosphatidylserine with the uptake of vital dye and the release of the cytoplasmic enzyme lactate dehydrogenase. During experimentally induced necrosis, lethal ion fluxes through the plasma membrane can be prevented by exogenous glycine; similarly, glycine completely blocked Salmonella-induced cytotoxicity. Finally, inhibition of the interleukin (IL)-1-converting enzyme caspase-1 blocked the death of infected macrophages, but not control cells induced to undergo apoptosis or necrosis. Thus, Salmonella-infected macrophages are killed by an unusual caspase-1-dependent mechanism of necrosis.     

Novel role of ICAM3 and LFA-1 in the clearance of apoptotic neutrophils by human macrophages

Apoptotic cells express eat-me signals which are recognized by several receptors mainly on professional phagocytes of the mononuclear phagocyte system. This “engulfment synapse” can define a safe and effective clearance of apoptotic cells in order to maintain tissue homeostasis in the entire body. We show that the expression of four genes related to apoptotic cell clearance is strongly up-regulated in human macrophages 30 min after administration of apoptotic neutrophils. Out of these the significant role of the up-regulated intercellular adhesion molecule 3 (ICAM3) in phagocytosis of apoptotic neutrophils could be demonstrated in macrophages by gene silencing as well as treatment with blocking antibodies. Blocking ICAM3 on the surface of apoptotic neutrophils also resulted in their decreased uptake which confirmed its role as an eat-me signal expressed by apoptotic cells. In macrophages but not in neutrophils silencing and blocking integrin alphaL and beta2 components of lymphocyte function-associated antigen 1 (LFA-1), which can strongly bind ICAM3, resulted in a decreased phagocytosis of apoptotic cells indicating its possible role to recognize ICAM3 on the surface of apoptotic neutrophils. Finally, we report that engulfing portals formed in macrophages during phagocytosis are characterized by accumulation of ICAM3, integrin alphaL and beta2 which show co-localization on the surface of phagocytes. Furthermore, their simultaneous knock-down in macrophages resulted in a marked deficiency in phagocytosis and a slight decrease in the anti-inflammatory effect of apoptotic neutrophils. We propose that ICAM3 and LFA-1 act as recognition receptors in the phagocytosis portals of macrophages for engulfment of apoptotic neutrophils.     

A potential new pathway for Staphylococcus aureus dissemination: the silent survival of S. aureus phagocytosed by human monocyte-derived macrophages

Although considered to be an extracellular pathogen, Staphylococcus aureus is able to invade a variety of mammalian, non-professional phagocytes and can also survive engulfment by professional phagocytes such as neutrophils and monocytes. In both of these cell types S. aureus promptly escapes from the endosomes/phagosomes and proliferates within the cytoplasm, which quickly leads to host cell death. In this report we show that S. aureus interacted with human monocyte-derived macrophages in a very different way to those of other mammalian cells. Upon phagocytosis by macrophages, S. aureus persisted intracellularly in vacuoles for 3-4 days before escaping into the cytoplasm and causing host cell lysis. Until the point of host cell lysis the infected macrophages showed no signs of apoptosis or necrosis and were functional. They were able to eliminate intracellular staphylococci if prestimulated with interferon-gamma at concentrations equivalent to human therapeutic doses. S. aureus survival was dependent on the alternative sigma factor B as well as the global regulator agr, but not SarA. Furthermore, isogenic mutants deficient in alpha-toxin, the metalloprotease aureolysin, protein A, and sortase A were efficiently killed by macrophages upon phagocytosis, although with different kinetics. In particular alpha-toxin was a key effector molecule that was essential for S. aureus intracellular survival in macrophages. Together, our data indicate that the ability of S. aureus to survive phagocytosis by macrophages is determined by multiple virulence factors in a way that differs considerably from its interactions with other cell types. S. aureus persists inside macrophages for several days without affecting the viability of these mobile cells which may serve as vehicles for the dissemination of infection.     

E. coli virulence factor hemolysin induces neutrophil apoptosis and necrosis/lysis in vitro and necrosis/lysis and lung injury in a rat pneumonia model

Enteric gram-negative bacilli, such as Escherichia coli are the most common cause of nosocomial pneumonia. In this study a wild-type extraintestinal pathogenic strain of E. coli (ExPEC)(CP9) and isogenic derivatives deficient in hemolysin (Hly) and cytotoxic necrotizing factor (CNF) were assessed in vitro and in a rat model of gram-negative pneumonia to test the hypothesis that these virulence factors induce neutrophil apoptosis and/or necrosis/lysis. As ascertained by in vitro caspase-3/7 and LDH activities and neutrophil morphology, Hly mediated neutrophil apoptosis at lower E. coli titers (1 x 10(5-6) cfu) and necrosis/lysis at higher titers (> or =1 x 10(7) cfu). Data suggest that CNF promotes apoptosis but not necrosis or lysis. We also demonstrate that annexin V/7-amino-actinomycin D staining was an unreliable assessment of apoptosis using live E. coli. The use of caspase-3/7 and LDH activities and neutrophil morphology supported the notion that necrosis, not apoptosis, was the primary mechanism by which neutrophils were affected in our in vivo gram-negative pneumonia model using live E. coli. In addition, in vivo studies demonstrated that Hly mediates lung injury. Neutrophil necrosis was not observed when animals were challenged with purified lipopolysaccharide, demonstrating the importance of using live bacteria. These findings establish that Hly contributes to ExPEC virulence by mediating neutrophil toxicity, with necrosis/lysis being the dominant effect of Hly on neutrophils in vivo and by lung injury. Whether Hly-mediated lung injury is due to neutrophil necrosis, a direct effect of Hly, or both is unclear.     

The role of phagocytes in the protective mechanisms of fish

Phagocytes are cells principally dedicated to the recognition and elimination of invading organisms and damaged tissue. Those described in fish are the granulocytes (particularly neutrophils) and mononuclear phagocytes (tissue macrophages and circulating monocytes). Their movement to sites of microbial invasion is an early event in the inflammatory response and the role of host-derived factors as attractants, such as eicosanoids, is discussed. Opsonins mediate the recognition between phagocyte and particle, and receptors for serum complement component C3 and the Fc fragment of opsonic antibody have been described. Fundamental to the protection offered by the phagocytes is their bactericidal larvacidal activity, which is closely associated with the production of oxygen free radicals. Phagocytes as accessory cells are discussed, including their role in antigen presentation. A knowledge of the modulation of phagocyte function, with activation by various substances and suppression by others, is important if protective responses are to be achieved by up-regulating phagocyte activity.     

Apoptosis of sea bass (Dicentrarchus labrax L.) neutrophils and macrophages induced by experimental infection with Photobacterium damselae subsp. piscicida

The infection of sea bass (Dicentrarchus labrax L.) by intraperitoneal (i.p.) injection of the agent of fish pasteurellosis Photobacterium damselae subsp. piscicida resulted in the apoptosis of peritoneal neutrophils and macrophages. All the eight virulent and none of the two non-virulent strains tested exhibited apoptogenic activity. A secreted bacterial protein(s) is a likely candidate as the factor(s) responsible for this activity, since no apoptosis was induced by i.p. injected UV-killed virulent strains and the virulent culture supernatants exhibited a thermo-labile apoptogenic activity identical to that of live bacteria. The apoptotic process was characterized by the occurrence of DNA fragmentation detected by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL) staining and DNA electrophoresis, and of typical ultrastructural alterations namely cell shrinkage, chromatin condensation, nuclear fragmentation and production of blebs with shedding of apoptotic bodies. In the apoptotic process induced by lethal doses of virulent bacteria or culture supernatants both peritoneal macrophages and neutrophils were extensively affected, the majority of these cells being apoptotic and reaching values around 10(7)per peritoneal cavity for each cell type at 24h post-injection. Moreover, the number of non-apoptotic macrophages was always below the initial number in the resting peritoneal cavity. Since macrophages are key cells in the elimination of both bacteria and apoptotic moribund cells and apoptotic bodies, the induction by Ph. damselae subsp. piscicida of simultaneous macrophage and neutrophil apoptosis results, on the one hand, in the destruction of the two phagocytic cell types involved in the restriction of multiplication of the bacteria and, on the other hand, in the uncontrolled progression of the apoptotic process towards secondary necrosis and eventual lysis of high numbers of moribund neutrophils and of neutrophilic apoptotic bodies, with the consequent extensive release of their highly cytotoxic components. Abundant apoptotic cells were also seen in sections of head-kidney from fish dying from experimental pasteurellosis. In contrast, no apoptosis was seen in vitro after the treatment with virulent culture supernatants of sea bass head-kidney macrophage cultures or after the treatment ex vivo of peritoneal exudate leukocytes with virulent bacteria or culture supernatants. The apoptotic process described here appears as a novel and very powerful microbial pathogenic strategy.     

Modulation of phagocyte apoptosis by bacterial pathogens

Phagocytic leukocytes such as neutrophils and macrophages are essential for the innate immune response against invading bacteria. Binding and ingestion of bacteria by these host cells triggers potent anti-microbial activity, including production of reactive oxygen species. Although phagocytes are highly adept at destroying bacteria, modulation of leukocyte apoptosis or cell death by bacteria has emerged as a mechanism of pathogenesis. Whereas induction of macrophage apoptosis by pathogens may adversely affect the host immune response to infection, acceleration of neutrophil apoptosis following phagocytic interaction with bacteria appears essential for the resolution of infection. This idea is supported by the finding that some bacterial pathogens alter normal phagocytosis-induced neutrophil apoptosis to survive and cause disease. This review summarizes what is currently known about modulation of phagocyte apoptosis by bacteria and describes a paradigm whereby bacteria-induced neutrophil apoptosis plays a role in the resolution of infection.     

The pivotal role of scavenger receptor CD36 and phagocyte-derived oxidants in oxidized low density lipoprotein-induced adhesion to endothelial cells

Adhesion of phagocytes to endothelial cells constitutes a crucial step in atherogenesis. Oxidized low density lipoproteins (LDL) are supposed to facilitate the adhesion process. We investigated the molecular mechanisms by which mildly and extensively hypochlorite-oxidized LDL force adhesion of murine macrophages and human neutrophils to human umbilical venous endothelial cells. After 1h of co-incubation of macrophages, endothelial cells, and lipoproteins adhesion significantly increased to 160+/-13% (S.E.M., n=5) in the presence of mildly oxidized lipoprotein, and 210+/-11% (S.E.M., n=5) in the presence of extensively oxidized lipoprotein. Similar results were obtained with neutrophils. CD36 antibody (FA6-152) significantly reduced adhesion to 102+/-7% (S.E.M., n=5) using mildly oxidized low density lipoprotein and to 179+/-16% (S.E.M., n=5) using extensively oxidized low density lipoprotein. Native high density lipoprotein and to a lesser extent methionine-oxidized high density lipoprotein significantly counteracted the effects of low density lipoprotein. Prior incubation of endothelial cells with modified lipoproteins followed by their removal and subsequent incubation with macrophages or neutrophils resulted in only minor changes of adhesion. This suggests that the direct contact of low density lipoprotein with phagocytes followed by activation of a respiratory burst with release of reactive oxygen species facilitates the adhesion process. Accordingly, the addition of antioxidants (superoxide dismutase and catalase) to the co-incubation medium was followed by a significant decrease in phagocyte adhesion. It is concluded that oxidized low density lipoprotein-induced respiratory burst activation of phagocytes with subsequent release of oxidants constitutes a crucial step in promoting the adhesion of phagocytes to endothelial cells.     

Comparison of the physiological and biochemical indices of the phagocytosis of ram erythrocytes by macro- and microphages]

Morita and Perkins' method was applied to the study of the stage of ingestion and destruction of an antigen (sheep erythrocytes) in the macrophages of peritoneal exudate of rabbits and rats and in the microphages of rabbit pleural exudate. Ingestion and intracellular destruction of the antigen were accompanied by intensified respiration and glycolysis of phagocytes. Respiration of the three types of phagocytes at two stages of phagocytosis and also the digestive capacity of microphages proved to be sensitive to cyanide and colchicine. The latter failed to influence the ingestion of the antigen by the three types of phagocytes and its digestion by macrophages. The differences in the metabolism routes of macro- and microphages in intracellular destruction of the antigen was postulated. An intensification of the phagocytic activity after the immunization was characteristic of rabbit and rat macrophages.     

Calpain-mediated X-linked inhibitor of apoptosis degradation in neutrophil apoptosis and its impairment in chronic neutrophilic leukemia

The number of neutrophils in the blood and tissues is controlled by constitutive apoptotic programmed cell death and clearance by phagocytes such as macrophages. Here, we found that calpains cleave the X-linked inhibitor of apoptosis (XIAP) in vitro, producing fragments that are unable to inhibit caspase-3. These fragments were detected in normal neutrophils but were unstable and rapidly degraded. Calpain inhibition delayed tumor necrosis factor-alpha-induced apoptosis of normal neutrophils, consistent with a role for calpains in regulating the onset of apoptosis. Interestingly, neutrophils from three patients with chronic neutrophilic leukemia, a rare syndrome characterized by accumulation of mature neutrophils, exhibited decreased mu-calpain expression, diminished calpain activity, and impaired XIAP degradation. Neutrophils from these patients displayed a delay in spontaneous, Fas-stimulated, and tumor necrosis factor-alpha-induced apoptosis. These observations suggest that calpain-mediated XIAP degradation contributes to initiation of apoptosis in normal neutrophils and dysfunction of this regulatory pathway can lead to pathological neutrophil accumulation.     

MHC-unrestricted transfer of antilisterial immunity by freshly isolated immune CD8 spleen cells

Immune CD8 cells, which play an essential role in the adoptive transfer of antilisterial immunity, can specifically lyse Listeria-bearing macrophages in vitro in an MHC-unrestricted manner. In contrast, the adoptive transfer of immunity by unseparated immune lymphocytes has been reported to be MHC-restricted. To address the restriction properties of CD8 effectors in vivo, we assessed their efficacy in protecting syngeneic and allogeneic recipients. Protection was determined by comparing the number of viable splenic Listeria in naive mice and in recipients of 60 million CD8-enriched, L3T4-depleted, Listeria-immune spleen cells, 2 days after the infusion of 10,000 Listeria. Donor cells from B6 (H-2b) mice transferred about 4 logs of protection in syngeneic recipients and more than 2 logs in allogeneic B10.A (H-2a) or B10.BR (H-2k) mice. Immune B10.A CD8 cells transferred equivalent protection to B6 mice. Protection was almost completely abrogated by the lysis or lethal irradiation of CD8 cells before transfer in vivo. On the other hand, the depletion of macrophages or NK cells did not impair adoptive transfer. By comparison, nonimmune CD8 cells from normal mice or from mice stimulated with an irrelevant Ag in vivo did not transfer substantial immunity to allogeneic recipients. We have noted previously that protective CD8 cells inhibit phagocyte accumulation in the spleen of Listeria-infected syngeneic recipients. In the present studies, we observed similar changes in adoptively immunized allogeneic mice. Reduced phagocyte accumulation may reflect Listeria-dependent lysis of infected phagocytes by immune CD8 cells. In support of this, we showed that Listeria-immune donor cells rapidly acquired the capacity to mediate Listeria-dependent, MHC-unrestricted lysis of macrophages after incubation with small amounts of IL-2 in vitro. In sum, our data establish that Listeria-immune CD8 cells can function in vivo in MHC incompatible hosts, and indirectly support the hypothesis that the destruction of infected phagocytes may be important in T cell-mediated immunity against Listeria and perhaps other intracellular pathogens.     

Statins enhance formation of phagocyte extracellular traps

Statins are inhibitors of 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-limiting enzyme in cholesterol biosynthesis. Recent clinico-epidemiologic studies correlate patients receiving statin therapy with having reduced mortality associated with severe bacterial infection. Investigating the effect of statins on the innate immune capacity of phagocytic cells against the human pathogen Staphylococcus aureus, we uncovered a beneficial effect of statins on bacterial clearance by phagocytes, although, paradoxically, both phagocytosis and oxidative burst were inhibited. Probing instead for an extracellular mechanism of killing, we found that statins boosted the production of antibacterial DNA-based extracellular traps (ETs) by human and murine neutrophils and also monocytes/macrophages. The effect of statins to induce phagocyte ETs was linked to sterol pathway inhibition. We conclude that a drug therapy taken chronically by millions alters the functional behavior of phagocytic cells, which could have ramifications for susceptibility and response to bacterial infections in these patients.     

Mechanisms of failed apoptotic cell clearance by phagocyte subsets in cardiovascular disease

Recent evidence in humans indicate that defective phagocytic clearance of dying cells is linked to progression of advanced atherosclerotic lesions, the precursor to atherothrombosis, ischemic heart disease, and leading cause of death in the industrialized world. During atherogenesis, apoptotic cell turnover in the vascular wall is counterbalanced by neighboring phagocytes with high clearance efficiency, thereby limiting cellularity and maintaining lesion integrity. However, as lesions mature, phagocytic removal of apoptotic cells (efferocytosis) becomes defective, leading to secondary necrosis, expansion of plaque necrotic cores, and susceptibility to rupture. Recent genetic causation studies in experimental rodents have implicated key molecular regulators of efferocytosis in atherosclerotic progression. These include MER tyrosine kinase (MERTK), milk fat globule-EGF factor 8 (MFGE8), and complement C1q. At the cellular level, atheromata are infiltrated by a heterogenous population of professional phagocytes, comprised of monocytes, differentiated macrophages, and CD11c⁺ dendritic-like cells. Each cell type is characterized by disparate clearance efficiencies and varying activities of key phagocytic signaling molecules. It is in this context that we outline a working model whereby plaque necrosis and destabilization is jointly promoted by (1) direct inhibition of core phagocytic signaling pathways and (2) expansion of phagocyte subsets with poor clearance capacity. Towards identifying targets for promoting efficient apoptotic cell clearance and resolving inflammation in atherosclerosis and during ischemic heart disease and post myocardial infarction, this review will discuss potential in vivo suppressors of efferocytosis at each stage of clearance and how these putative interventional targets may differentially affect uptake at the level of vascular phagocyte subsets.     

Caspase-1-dependent pore formation during pyroptosis leads to osmotic lysis of infected host macrophages

Salmonella enterica serovar Typhimurium invades host macrophages and induces a unique caspase-1-dependent pathway of cell death termed pyroptosis, which is activated during bacterial infection in vivo. We demonstrate DNA cleavage during pyroptosis results from caspase-1-stimulated nuclease activity. Although poly(ADP-ribose) polymerase (PARP) activation by fragmented DNA depletes cellular ATP to cause lysis during oncosis, the rapid lysis characteristic of Salmonella-infected macrophages does not require PARP activity or DNA fragmentation. Membrane pores between 1.1 and 2.4 nm in diameter form during pyroptosis of host cells and cause swelling and osmotic lysis. Pore formation requires host cell actin cytoskeleton rearrangements and caspase-1 activity, as well as the bacterial type III secretion system (TTSS); however, insertion of functional TTSS translocons into the host membrane is not sufficient to directly evoke pore formation. Concurrent with pore formation, inflammatory cytokines are released from infected macrophages. This mechanism of caspase-1-mediated cell death provides additional experimental evidence supporting pyroptosis as a novel pathway of inflammatory programmed cell death.     

Alcohol suppresses lipopolysaccharide-induced tumor necrosis factor activity in serum and lung

Ethanol intoxication has been shown to suppress selected functions of the immune system thereby compromising host defense against bacterial infections. Tumor necrosis factor, a secretory protein produced by the macrophage in response to lipopolysaccharide, mediates the inflammatory cascade and stimulates phagocyte functions. Acute ethanol intoxication markedly suppressed both serum and lung tumor necrosis factor elicited in response to lipopolysaccharide. Furthermore, ethanol inhibited intratracheal lipopolysaccharide-induced neutrophil recruitment into the alveoli and prevented the fall in circulating neutrophils in response to intravenous lipopolysaccharide. Thus, the anti-inflammatory effects of ethanol may be secondary to suppression of macrophage-derived tumor necrosis factor.     

Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal

Efficient engulfment of the intact cell corpse is a critical end point of apoptosis, required to prevent secondary necrosis and inflammation. The presentation of "eat-me" signals on the dying cell is an important part of this process of recognition and engulfment by professional phagocytes. Here, we present evidence that apoptotic cells secrete chemotactic factor(s) that stimulate the attraction of monocytic cells and primary macrophages. The activation of caspase-3 in the apoptotic cell was found to be required for the release of this chemotactic factor(s). The putative chemoattractant was identified as the phospholipid, lysophosphatidylcholine. Further analysis showed that lysophosphatidylcholine was released from apoptotic cells due to the caspase-3 mediated activation of the calcium-independent phospholipase A(2). These data suggest that in addition to eat-me signals, apoptotic cells display attraction signals to ensure the efficient removal of apoptotic cells and prevent postapoptotic necrosis.