Engulfment Of Apoptotic Cells Research Articles

Live-cell imaging in Caenorhabditis elegans reveals the distinct roles of dynamin self-assembly and guanosine triphosphate hydrolysis in the removal of apoptotic cells.

Dynamins are large GTPases that oligomerize along membranes. Dynamin's membrane fission activity is believed to underlie many of its physiological functions in membrane trafficking. Previously, we reported that DYN-1 (Caenorhabditis elegans dynamin) drove the engulfment and degradation of apoptotic cells through promoting the recruitment and fusion of intracellular vesicles to phagocytic cups and phagosomes, an activity distinct from dynamin's well-known membrane fission activity. Here, we have detected the oligomerization of DYN-1 in living C. elegans embryos and identified DYN-1 mutations that abolish DYN-1's oligomerization or GTPase activities. Specifically, abolishing self-assembly destroys DYN-1's association with the surfaces of extending pseudopods and maturing phagosomes, whereas inactivating guanosine triphosphate (GTP) binding blocks the dissociation of DYN-1 from these membranes. Abolishing the self-assembly or GTPase activities of DYN-1 leads to common as well as differential phagosomal maturation defects. Whereas both types of mutations cause delays in the transient enrichment of the RAB-5 GTPase to phagosomal surfaces, only the self-assembly mutation but not GTP binding mutation causes failure in recruiting the RAB-7 GTPase to phagosomal surfaces. We propose that during cell corpse removal, dynamin's self-assembly and GTP hydrolysis activities establish a precise dynamic control of DYN-1's transient association to its target membranes and that this control mechanism underlies the dynamic recruitment of downstream effectors to target membranes.

Cell Surface Externalization of Annexin A1 as a Failsafe Mechanism Preventing Inflammatory Responses during Secondary Necrosis

The engulfment of apoptotic cells is of crucial importance for tissue homeostasis in multicellular organisms. A failure of this process results in secondary necrosis triggering proinflammatory cytokine production and autoimmune disease. In the present study, we investigated the role of annexin A1, an intracellular protein that has been implicated in the efficient removal of apoptotic cells. Consistent with its function as bridging protein in the phagocyte synapse, opsonization of apoptotic cells with purified annexin A1 strongly enhanced their phagocytic uptake. A detailed analysis, however, surprisingly revealed that annexin A1 was hardly exposed to the cell surface of primary apoptotic cells, but was strongly externalized only on secondary necrotic cells. Interestingly, while the exposure of annexin A1 failed to promote the uptake of these late secondary necrotic cells, it efficiently prevented induction of cytokine production in macrophages during engulfment of secondary necrotic cells. Our results therefore suggest that annexin A1 exposure during secondary necrosis provides an important failsafe mechanism counteracting inflammatory responses, even when the timely clearance of apoptotic cells has failed.

Gadolinium-containing phosphatidylserine liposomes for molecular imaging of atherosclerosis

Exteriorized phosphatidylserine (PS) residues in apoptotic cells trigger rapid phagocytosis by macrophage scavenger receptor pathways. Mimicking apoptosis with liposomes containing PS may represent an attractive approach for molecular imaging of atherosclerosis. We investigated the utility of paramagnetic gadolinium liposomes enriched with PS (Gd-PS) in imaging atherosclerotic plaque. Gd-PS-containing Gd-conjugated lipids, fluorescent rhodamine, and PS were prepared and characterized. Cellular uptake in RAW macrophages (fluorescent uptake of rhodamine) was studied on a fluorescence plate reader, while Gd-PS-induced alteration in T1 relaxivity was evaluated using a 1.5 T MRI scanner. RAW cells demonstrate PS-dependent uptake of across a range of concentrations (2, 6, 12, and 20%) in comparison to control liposomes with no PS (0%). In vivo performance of Gd-PS was evaluated in the ApoE(-/-) mouse model by collection of serial T1 weighted gradient echo MR images using an 11.7 T MRI system and revealed rapid and significant enhancement of the aortic wall that was seen for at least 4 h after injection. Gd-PS-enriched liposomes enhance atherosclerotic plaque and colocalize with macrophages in experimental atherosclerosis.

PPAR-δ senses and orchestrates clearance of apoptotic cells to promote tolerance

Macrophages rapidly engulf apoptotic cells to limit the release of noxious cellular contents and to restrict autoimmune responses against self antigens. Although factors participating in recognition and engulfment of apoptotic cells have been identified, the transcriptional basis for the sensing and silently disposing of apoptotic cells is unknown. Here we show that peroxisome proliferator activated receptor-δ (PPAR-δ) is induced when macrophages engulf apoptotic cells and functions as a transcriptional sensor of dying cells. Genetic deletion of PPAR-δ decreases expression of opsonins, such as C1qb, resulting in impairment of apoptotic cell clearance and reduction in anti-inflammatory cytokine production. This increases autoantibody production and predisposes global and macrophage-specific PPARd−/− mice to autoimmune kidney disease, a phenotype resembling the human disease systemic lupus erythematosus. Thus, PPAR-δ plays a pivotal role in orchestrating the timely disposal of apoptotic cells by macrophages, ensuring that tolerance to self is maintained.

Autophagy is not universally required for phosphatidyl-serine exposure and apoptotic cell engulfment during neural development

Apoptosis and autophagy are physiological processes implicated in the maintenance of cell and tissue homeostasis. We took advantage of the existence of multiple phases ofdevelopmental cell death in the embryonic chick retina and of the availability of shortterm organotypic retinal cultures to approach the possible relationship betweenapoptosis and autophagy during neural development. We examined retinas at embryonic day 5, an early stage at which cell death is related to eye morphogenesis and to retinalganglion cell generation, as well as at embryonic day 9, when cell death is associated with neurotrophic support of the retinal ganglion cells. Exposure to 3-methyl-adenine, aclassical inhibitor of autophagy, elicited a selective accumulation of apoptotic bodies in the dorsotemporal area of embryonic day 5 retinas where neurogenesis is taking place.This accumulation was correlated with a blockage of phosphatidyl-serine presentation and, consequently, with a lack of engulfment of the dying cells by their neighbors. Instriking contrast, none of these phenomena were observed in association with cell death in the optic nerve and optic fissure at embryonic day 5, or in embryonic day 9 retinas.Our data suggest that autophagy is essential for phosphatidyl-serine presentation by apoptotic cells during the phase of cell death associated to neurogenesis, but this is not auniversal requirement for all phases of cell death occurring during retinal development.

Apoptotic Cells Promote Their Own Clearance and Immune Tolerance through Activation of the Nuclear Receptor LXR

Effective clearance of apoptotic cells by macrophages is essential for immune homeostasis. The transcriptional pathways that allow macrophages to sense and respond to apoptotic cells are poorly defined. We found that liver X receptor (LXR) signaling was important for both apoptotic cell clearance and the maintenance of immune tolerance. Apoptotic cell engulfment activated LXR and thereby induced the expression of Mer, a receptor tyrosine kinase critical for phagocytosis. LXR-deficient macrophages exhibited a selective defect in phagocytosis of apoptotic cells and an aberrant proinflammatory response to them. As a consequence of these defects, mice lacking LXRs manifested a breakdown in self-tolerance and developed autoantibodies and autoimmune glomerulonephritis. Treatment with an LXR agonist ameliorated disease progression in a mouse model of lupus-like autoimmunity. Thus, activation of LXR by apoptotic cells engages a virtuous cycle that promotes their own clearance and couples engulfment to the suppression of inflammatory pathways.

Detection and characterization of soluble CD93 released during inflammation

CD93 is a cell surface glycoprotein that is required for efficient engulfment of apoptotic cells via an unknown mechanism. Recently, it was demonstrated that CD93 is proteolytically cleaved from the surface of activated human monocytes and neutrophils in response to inflammatory signals in vitro and that a soluble form of CD93 (sCD93) exists in human plasma. The objective of this study was to examine the relationship among production of sCD93, inflammation, and engulfment of apoptotic cells. Sterile peritonitis was induced in C57BL/6 mice, and lavage fluid was analyzed for presence of sCD93 by ELISA and Western blot. Cellular infiltrate was assessed by flow cytometry and analyzed for its capacity to shed CD93 in vitro. Peritoneal lavage fluid (PLF) was examined for its ability to regulate engulfment of apoptotic cells. There was an 8.9-fold increase in sCD93 following induction of peritonitis. Macrophages accounted for the majority of infiltrating leukocytes into the peritoneum when sCD93 levels were highest. Inflammatory peritoneal macrophages constitutively shed CD93 in vitro suggesting that this cell type contributes to elevated levels of sCD93 in vivo. Inflammatory PLF from wild-type mice containing elevated sCD93 significantly enhanced engulfment of apoptotic cells in vitro when compared to inflammatory fluid from CD93-deficient mice. These data demonstrate that inflammation triggers release of sCD93 in vivo, identify the inflammatory macrophage as a source of sCD93, and provide insight into the mechanism by which CD93 contributes to engulfment of apoptotic cells.

Involvement of a functional NADPH oxidase in neutrophils and macrophages during programmed cell clearance: implications for chronic granulomatous disease

Resolution of inflammation requires clearance of activated neutrophils by macrophages in a manner that prevents injury to adjacent tissues. Surface changes, including phosphatidylserine (PS) exposure, may target neutrophils for phagocytosis. In this study, we show that externalization of PS is defective in phorbol myristate acetate (PMA)-activated neutrophils obtained from chronic granulomatous disease (CGD) patients with mutations in components of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Moreover, activated neutrophils from CGD patients failed to undergo clearance upon cocultivation with macrophages from normal donors. In line with these results, treatment of donor neutrophils with diphenyleneiodonium (DPI), an inhibitor of NADPH oxidase, blocked PMA-induced PS oxidation and externalization and prevented their engulfment by macrophages. Furthermore, primary macrophages from CGD patients or human gp91(phox)-deficient PLB-985 cells differentiated into macrophage-like cells were defective for engulfment of apoptotic target cells. Pretreatment of normal macrophages with DPI also suppressed the subsequent ingestion of PS-positive target cells. Together, these data demonstrate that NADPH oxidase plays an important role in the process of macrophage disposal of target cells (programmed cell clearance). Thus we speculate that the lack of a functional NADPH oxidase results in impaired neutrophil clearance and the exaggerated inflammation that is characteristic for CGD.

Single-walled carbon nanotubes impair human macrophage engulfment of apoptotic cell corpses

Single-walled carbon nanotubes (SWCNT) are being produced in increasing quantities and the application of these materials in a large number of new technologies and consumer products necessitates studies of their potential impact on human health and the environment. To determine whether SWCNT affect viability or function of macrophages, important components of the innate immune system, we performed in vitro studies using primary human monocyte-derived macrophages (HMDM). Our findings show that SWCNT with a low content of metal impurities do not exert direct cytotoxic effects on HMDM. However, SWCNT suppressed chemotaxis of primary human monocytes in a standard chemotaxis assay. Moreover, macrophage engulfment of apoptotic target cells was significantly impaired following pre-incubation of HMDM with SWCNT at non-cytotoxic concentrations. These results are in line with previous studies showing that ultrafine carbon particles and carbon nanotubes may impair alveolar macrophage ingestion of microorganisms, and suggest that tissue homeostasis may be compromised by SWCNT due to suppressive effects on macrophages.

An essential role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response

Cells undergo programmed cell death/apoptosis throughout the lifespan of an organism. The subsequent immunologically silent removal of apoptotic cells plays a role in the maintenance of tolerance; defects in corpse clearance have been associated with autoimmune disease. A number of receptors and signaling molecules involved in this process have been identified, but intracellular signaling downstream of corpse recognition is only now being defined. Calcium plays a key role as a second messenger in many cell types, leading to the activation of downstream molecules and eventual transcription of effector genes; however, the role of calcium signaling during apoptotic cell removal is unclear. Here, using studies in cell lines and in the context of a whole organism, we show that apoptotic cell recognition induces both an acute and sustained calcium flux within phagocytes and that the genes required for calcium flux are essential for engulfment. Furthermore, we provide evidence that both the release of calcium from the endoplasmic reticulum and the entry of extracellular calcium through CRAC channels into the phagocytes are important during engulfment. Moreover, knockdown in Caenorhabditis elegans of stim-1 and jph-1, two genes linked to the entry of extracellular calcium into cells, led to increased persistence of apoptotic cells in the nematode. Loss of these genes seemed to affect early signaling events, leading to a decreased enrichment of actin adjacent to the apoptotic cell during corpse removal. We also show that calcium is crucial for the secretion of TGF-beta by the phagocytes during the engulfment of apoptotic cells. Taken together, these data point to an earlier unappreciated and evolutionarily conserved role for calcium flux at two distinguishable steps: the formation of the phagocytic cup and the internalization of the apoptotic cell, and the anti-inflammatory signaling induced in phagocytes by contact with apoptotic cells.

IgM Antibodies to Apoptosis-Associated Determinants Recruit C1q and Enhance Dendritic Cell Phagocytosis of Apoptotic Cells

Natural Abs, which arise without known immune exposure, have been described that specifically recognize cells dying from apoptosis, but their role in innate immunity remains poorly understood. Herein, we show that the immune response to neoantigenic determinants on apoptotic thymocytes is dominated by Abs to oxidation-associated Ags, phosphorylcholine (PC), a head group that becomes exposed during programmed cell death, and malondialdehyde (MDA), a reactive aldehyde degradation product of polyunsaturated lipids produced following exposure to reactive oxidation species. While natural Abs to apoptotic cells in naive adult mice were dominated by PC and MDA specificities, the amounts of these Abs were substantially boosted by treatment of mice with apoptotic cells. Moreover, the relative amounts of PC and MDA Abs was affected by V(H) gene inheritance. Ab interactions with apoptotic cells also mediated the recruitment of C1q, which enhanced apoptotic cell phagocytosis by immature dendritic cells. Significantly, IgM Abs to both PC and MDA were primary factors in determining the efficiency of serum-dependent apoptotic cell phagocytosis. Hence, we demonstrate a mechanism by which certain natural Abs that recognize neoantigens on apoptotic cells, in naive mice and those induced by immune exposure to apoptotic cells, can enhance the functional capabilities of immature dendritic cells for phagocytic engulfment of apoptotic cells.

T1716 Apoptotic Cell Engulfment By Intestinal Epithelial Cells and Its Relevance to Crohn's Disease

T1716 Apoptotic Cell Engulfment By Intestinal Epithelial Cells and Its Relevance to Crohn's Disease

The costimulatory role of TIM molecules

The T-cell immunoglobulin domain and mucin domain (TIM) family, including TIM-1, TIM-2, TIM-3, and TIM-4, is a relatively newly described group of molecules with a conserved structure and important immunological functions, including T-cell activation, induction of T-cell apoptosis and T-cell tolerance, and the clearance of apoptotic cells. TIM-1 costimulates T-cell activation and enhances cytokine production. In humans, TIM-1 also serves as a susceptibility gene for allergy and asthma. TIM-3, expressed on T cells and dendritic cells, regulates T-cell apoptosis and immune tolerance. By contrast, TIM-4, which is expressed primarily on antigen-presenting cells and which is a receptor for phosphatidylserine, regulates T-cell activation and tolerance, in part by mediating the uptake and engulfment of apoptotic cells. The TIM molecules thus have surprisingly broad activities affecting multiple aspects of immunology.

Abl Kinase Inhibits the Engulfment of Apopotic Cells in Caenorhabditis elegans

The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway includes the adaptor protein CED-2 CrkII and the small GTPase CED-10 Rac, and acts to rearrange the cytoskeleton of the engulfing cell. The other pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Although many components required for engulfment have been identified, little is known about inhibition of engulfment. The tyrosine kinase Abl regulates the actin cytoskeleton in mammals and Drosophila in multiple ways. For example, Abl inhibits cell migration via phosphorylation of CrkII. We tested whether ABL-1, the C. elegans ortholog of Abl, inhibits the CED-2 CrkII-dependent engulfment of apoptotic cells. Our genetic studies indicate that ABL-1 inhibits apoptotic cell engulfment, but not through CED-2 CrkII, and instead acts in parallel to the two known engulfment pathways. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The loss of ABL-1 function partially restores normal DTC migration in the CED-10 Rac pathway mutants. We found that ABI-1 the C. elegans homolog of mammalian Abi (Abl interactor) proteins, is required for engulfment of apoptotic cells and proper DTC migration. Like Abl, Abi proteins are cytoskeletal regulators. ABI-1 acts in parallel to the two known engulfment pathways, likely downstream of ABL-1. ABL-1 and ABI-1 interact physically in vitro. We propose that ABL-1 opposes the engulfment of apoptotic cells by inhibiting ABI-1 via a pathway that is distinct from the two known engulfment pathways.

A critical role for calcium flux in phagocytes for apoptotic cell engulfment and the anti-inflammatory response (134.52)

Abstract Calcium plays a key role as a second messenger in many cell types, but a role for calcium signaling during apoptotic cell removal is unclear. Using studies in cell lines and in the context of a whole organism, we demonstrate that apoptotic cell recognition induces both an acute and sustained calcium flux within phagocytes and that such calcium flux is essential for engulfment. Furthermore, we provide evidence that both the release of calcium from the endoplasmic reticulum and the entry of extracellular calcium via CRAC channels into the phagocytes are important during engulfment. Moreover, knockdown in C. elegans of stim-1 and jph-1, two genes linked to the entry of extracellular calcium into cells, led to increased persistence of apoptotic cells in the nematode. Loss of these genes appeared to affect early signaling events, leading to decreased enrichment of actin adjacent to the apoptotic cell during corpse removal. We also show that calcium is crucial for the anti-inflammatory TGF-β response seen in phagocytes during engulfment of apoptotic cells. Taken together, these data point to a previously unappreciated and evolutionarily conserved role for calcium flux at two distinguishable steps: formation of the phagocytic cup and internalization of the apoptotic cell, and the anti-inflammatory signaling induced in phagocytes by contact with apoptotic cells.

The Role of Dynamin in the Clearance of Apoptotic Cells

In animals, cells undergoing apoptosis are rapidly engulfed and degraded by phagocytes. The removal of apoptotic cells provides a safe means for eliminating unwanted and dangerous cells from the body. Furthermore, it prevents tissue injury, and inflammatory and auto‐immune responses induced by the lingering dead cells. In a genetic screen for mutations that affect the removal of apoptotic cells in the nematode C. elegans. We isolated fourteen loss‐of‐function alleles of dyn‐1, which encodes the C. elegans ortholog of large GTPase dynamin. Our cell biological characterizations indicate that DYN‐1 is essential for both the engulfment and degradation of apoptotic cells. DYN‐1 promotes the incorporation of endosomes to the extending pseudopods during engulfment, and the recruitment and incorporation of both endosomes and lysosomes to phagosomes containing apoptotic cells. Phosphatidylinositol 3‐phosphate (PI(3)P) and the small GTPase RAB‐7 mediate the specific function of DYN‐1 in phagosome maturation. Our in vitro and in vivo analyses demonstrate that the GTP hydrolysis and self‐assembly activities of DYN‐1 control distinct aspects of DYN‐1's function in phagosome maturation. Furthermore, the phagocytic receptor CED‐1 recruits DYN‐1 to the site of engulfment. These findings indicate that DYN‐1 is a key component of a pathway that regulates vesicle transport during the removal of apoptotic cells.

Abl Is Able to Inhibit Cell Engulfment

Programmed cell death is essential to development—it's how the tiny mitten of a growing hand becomes a glove of individuated fingers, for instance. To dispose of all those cellular corpses, nearby cells (or roving specialists such as the immune system's macrophages) engulf their dead neighbors and digest the remains. Engulfment, therefore, is a valuable process, and it is known to be promoted by two parallel signaling pathways. But preventing engulfment is also important (one can almost hear a little cell's faint cry, “I'm not dead yet!”); however, little is known about how the process is inhibited. In a new study, Michael Hurwitz and colleagues describe a pathway that does just that. To wrap itself around another cell, the engulfing cell must recruit new a membrane and completely reorganize its cytoskeleton, and these processes are regulated by the two pro-engulfment pathways, known as the CED-1 pathway (thought to be responsible primarily for membrane recruitment) and the CED-10 Rac pathway (thought to be responsible primarily for cytoskeletal rearrangement). Cytoskeletal remodeling is also controlled by a host of other regulators outside these pathways, though, including a multi-talented protein called Abl kinase. It interacts with dozens of partners (creating effects that are mostly not understood in vivo), and its dysregulation is linked to at least two forms of cancer. To explore the effects of Abl kinase in engulfment, the authors turned to the roundworm Caenorhabditis elegans, which produces an Abl kinase homolog called ABL-1. They found that disabling ABL-1 through mutation in animals defective for engulfment led to even more cells being engulfed, which is exactly what would be expected from the loss of an engulfment inhibitor. To quantify defects in the engulfment process, the authors simply counted corpses in the worm larvae, and they recognized that, rather than having a direct effect on engulfment itself, ABL-1 mutation might reduce corpse number by other means, such as suppressing cell death. But through direct observation of the developing larvae, they ruled out this and other alternatives, leading them to conclude that ABL-1 directly inhibited engulfment. The protein appears to exert its effect on the engulfing cell, because it inhibited engulfment when it was expressed everywhere except the engulfed cell. For many years, two molecular pathways were known to regulate the engulfment of apoptotic cells. Now, a third pathway has been identified in C. elegans that is inhibited by the Abl tyrosine kinase oncoprotein. Mutation of abl-1 was able to partially overcome the effect of complete inhibition of the membrane recruitment pathway but not the cytoskeletal rearrangement pathway. However, the cytoskeletal rearrangement pathway is also required for certain cell migrations, and mutation of abl-1 did partially overcome the effect of complete inhibition of the that pathway on cell migrations. These data suggested that ABL-1 acts independently of both pathways, but the effect of its loss is simply too weak to overcome the engulfment defect of complete inhibition of the cytoskeletal regulation pathway. Thus, ABL-1 appeared to inhibit a third engulfment regulatory pathway. The authors then explored the function of a known interactor with ABL-1, called ABI-1. In contrast to mutant ABL-1, mutations in ABI-1 reduced engulfment, just as mutations in the two previously known pro-engulfment pathways did. And it did so whether or not ABL-1 was mutated, suggesting that its effect is downstream of ABL-1. The picture that emerges, then, is that ABL-1 acts directly on ABI-1 to inhibit it, and thus inhibits engulfment. Along with deepening the understanding of programmed cell death control, these results may have some direct therapeutic relevance. Engulfment of infected cells by macrophages is a key means for the body to maintain health, and engulfment of cancerous cells might also prevent cancer development. Abl kinase inhibitors might promote macrophage activity in these conditions. Impaired engulfment is a hallmark of systemic lupus erythematosus, suggesting that inhibiting Abl kinase might be of use here as well. Testing these ideas shouldn't take long—not only do such inhibitors exist, but at least two are already approved and marketed for other conditions.

D025 Phagocytosis is pivotal in the beneficial effect of bone marrow mononuclear cellsbased therapy for myocardial infarction

Cell-based therapy is a promising option for treatment of cardiovascular diseases. Based on experimental studies demonstrating that bone marrow-derived mononuclear cells (BMMNCs) improve the functional recovery after ischemia, clinical trials were initiated to address this new therapeutic concept. BMMNCs improve neovascularization of ischemic tissue by a broad repertoire of potential therapeutic actions. Whereas initial studies documented that the cells incorporate and differentiate to cardiovascular cells, other studies suggested that short-time paracrine mechanisms mediate the beneficial effects. Here, we hypothesized that BMMNCs have a phagocytic ability, and switch to a proangiogenic phenotype after engulfment of apoptotic cells. Activation of such angiogenic program may be pivotal in the beneficial effect of BMMNCs-based therapy. In vitro, wildtype (WT) BMMNCs ingestion of apoptotic cells upregulated the release of proangiogenic factors VEGF and HGF by 15- and 5-fold, respectively. In contrast, BMMNCs collected from mice deficient in MFG-E8, a protein that is required for attachment and engulfment of apoptotic cells by phagocytes, displayed lower phagocytic ability, leading to decrease in VEGF and HGF release. The capacity of BMMNCs to differentiate into cells with endothelial phenotype was similar in control and MFG-E8-deficient cells. In an in vivo model of mice myocardial infaction (MI), transplantation of WT BMMNCs increased fractionnal shortening (120 % of untreated control, p < 0.05), 14 days after MI. Size of the infarct scar was reduced by 30 % (p < 0.05 vs untreated control), and capillary density in the infarct border zone was raised by 10 % (p < 0.05 vs untreated control) in the WT BMMNCs group. In contrast, transplantation of MFG-E8 deficient BMMNCs displayed no significant effect on cardiac function, infarct size or angiogenesis in the ischemic myocardium. Four days after MI, VEGF protein levels were raised by 1.4 fold in the myocardium of WT BMMNCs treated animals compared to untreated controls (p < 0.05), while treatment with MFG-E8 deficient BMMNCs failed to raise VEGF levels. Taken together, these results suggest that phagocytosis of apoptotic cells reprograms BMMNCs into a proangiogenic phenotype. Hence, the therapeutic effect of transplanted BMMNCs depends, at least in part, on their phagocytic ability.

The Phosphatidylserine Receptor TIM-4 Does Not Mediate Direct Signaling

Engulfment of apoptotic cells is an active process coordinated by receptors on phagocytes and ligands on apoptotic cells [1]. Phosphatidylserine (PtdSer) is a key ligand on apoptotic cells, and recently three PtdSer recognition receptors have been identified, namely, TIM-4, BAI1, and Stabilin-2 [1-6]. Whereas BAI1 is dependent on the ELMO1/Dock180/Rac signaling module, and Stablilin-2 appears to use the intracellular adaptor GULP [2, 3, 7], little is known about how TIM-4 transduces signals downstream of PtdSer recognition [8]. To test the role of known engulfment signaling pathways in TIM-4-mediated engulfment, we used a combination of dominant-negative mutants, knockdown of specific signaling proteins, and knockout cell lines. TIM-4 appears to be largely independent of the two known engulfment signaling pathways [7, 9-17], yet the TIM-4-mediated uptake is inhibited by cytoskeleton disrupting drugs. Remarkably, a version of TIM-4 lacking its cytoplasmic tail promoted corpse uptake via PtdSer recognition. Moreover, replacement of the transmembrane region of TIM-4 with a glycophosphatidylinositol anchor still promoted engulfment comparable to wild-type TIM-4. Thus, the transmembrane region and cytoplasmic tail of TIM-4 are dispensable for apoptotic cell engulfment, and we propose that TIM-4 is a PtdSer tethering receptor without any direct signaling of its own.

Ferritin Expression in Rat Hepatocytes and Kupffer Cells after Lead Nitrate Treatment

Lead nitrate induces hepatocyte proliferation and subsequent apoptosis in rat livers. Iron is a constituent of heme and is also required for cell proliferation. In this study, the expression of ferritin light-chain (FTL), the major iron storage protein, was investigated in rat livers after a single intravenous injection of lead nitrate. Western blotting and immunohistochemistry revealed that FTL was increased in hepatocytes around the central veins and strongly expressed in nonparenchymal cells. Some FTL-positive nonparenchymal cells were identified as Kupffer cells that were positive for CD68. FTL-positive Kupffer cells occupied about 60% of CD68-positive cells in the periportal and perivenous areas. The relationships between FTL expression and apoptosis induction or the engulfment of apoptotic cells were examined. TUNEL-positive cells were increased in the treatment group, and enhanced expression of milk fat globule EGF-like 8 was demonstrated in some Kupffer cells and hepatocytes, indicating enhanced apoptosis induction and phagocytosis of apoptotic cells. FTL-positive Kupffer cells were not detected without lead nitrate treatment or in rat livers treated with clofibrate, which induces hepatocyte proliferation but not apoptosis. These results suggest that FTL expression in Kupffer cells after lead treatment is dependent on phagocytosis of apoptotic cells.