Engulfment Of Apoptotic Cells Research Articles

Involvement of COX-1 and up-regulated prostaglandin E synthases in phosphatidylserine liposome-induced prostaglandin E 2 production by microglia

After engulfment of apoptotic cells through phosphatidylserine (PS)-mediated recognition, microglia secrete prostaglandin E 2 (PGE 2), a potent anti-inflammatory molecule in the central nervous system. Despite the clinical significance, the mechanism underlying PGE 2 production by phagocytosis of apoptotic cells is poorly understood. In the present study, we used PS liposomes to elucidate the phagocytic pathway for PGE 2 production in microglia, because PS liposomes mimic the effects of apoptotic cells on microglia/macrophages. The level of PGE 2 in the culture medium of primary cultured rat microglia was significantly increased by PS liposomes treatment but not by phosphatidylcholine liposomes treatment. The specific ligand for class B scavenger receptor (SR-B), high density lipoprotein, significantly suppressed PS liposome-induced PGE 2 production. PS liposomes were immediately phagocytosed by microglia and sorted to endosomes/lysosomes. Cyclooxygenase (COX)-2 and membrane-bound prostaglandin E synthase-1 (mPGES-1) were induced by treatment with lipopolysaccharide (LPS) but not with PS liposomes. On the other hand, mPGES-2 and cytosolic PGES (cPGES) that are functionally coupled with COX-1 were upregulated after treatment with PS liposomes or LPS. Furthermore, PS liposome-induced PGE 2 production was significantly suppressed by indomethacin, a preferential COX-1 inhibitor, but not by NS-398, a selective COX-2 inhibitor. PS liposomes induced activation of p44/p42 extracellular signal-regulated kinase (ERK) but not p38 mitogen-activated protein kinase in SR-BI independent manner. These observations strongly suggest that the up-regulation of terminal PGESs that are preferentially coupled with COX-1, especially mPGES-2, plays the pivotal role in PS liposome-induced PGE 2 production by microglia. Although SR-BI plays an essential role in PS liposome-induced PGE 2 production, other PS-recognizing receptors, possibly PS-specific receptor, could also promote PGE 2 production by transducing intracellular signals including p44/p42 ERK after PS liposomes treatment.

Molecular Determinants of Kinase Pathway Activation by Apo2 Ligand/Tumor Necrosis Factor-related Apoptosis-inducing Ligand

Apo2 ligand/tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) mainly activates programmed cell death through caspases. By contrast, TNF primarily induces gene transcription through the inhibitor of kappaB kinase (IKK), c-Jun N-terminal kinase (JNK), and p38 mitogen-activated protein kinase pathways. Apo2L/TRAIL also can stimulate these kinases, albeit less strongly; however, the underlying mechanisms of this stimulation and its relation to apoptosis are not well understood. Here we show that Apo2L/TRAIL activates kinase pathways by promoting the association of a secondary signaling complex, subsequent to assembly of a primary, death-inducing signaling complex (DISC). The secondary complex retained the DISC components FADD and caspase-8, but recruited several factors involved in kinase activation by TNF, namely, RIP1, TRAF2, and NEMO/IKKgamma. Secondary complex formation required Fas-associated death domain (FADD), as well as caspase-8 activity. Apo2L/TRAIL stimulation of JNK and p38 further depended on RIP1 and TRAF2, whereas IKK activation required NEMO. Apo2L/TRAIL induced secretion of interleukin-8 and monocyte chemoattractant protein-1, augmenting macrophage migration. Thus, Apo2L/TRAIL and TNF organize common molecular determinants in distinct signaling complexes to stimulate similar kinase pathways. One function of kinase stimulation by Apo2L/TRAIL may be to promote phagocytic engulfment of apoptotic cells.

Involvement of PKC Activation in Mer Tyrosine Kinase-Mediated Phagocytosis of Apoptotic Cells.

Efficient phagocytosis of apoptotic cells is crucial for many cellular processes. One of earliest signals to the phagocyte is the expression of phosphatidylserine (PS) on the outer surface of the apoptotic cell that provides a potent ‘eat-me' signal. Mer-family tyrosine kinases and αvβ5(3) integrin are major receptors recognizing PS through the opsonizing proteins MFG-E8, or Gas6, respectively. The previous studies have suggested Mer and integrin αvβ5 signaling pathways converge on tyrosine phosphorylation of FAK and p130Cas, as well as downstream CrkII/Dock180/Rac1 activation, evolutionally-conserved for engulfment. In this study, we aimed at investigating how Mer initiates early signaling events to mediate phaogcytosis and activation of integrin αvβ5, and found that Gas6-Mer-induced tyrosine phosphorylation of FAK and p130Cas was inhibited by calphostin C, and engulfment of apoptotic cells by 293T cells overexpressing wild-type Mer was also suppressed by membrane-permeable PKC inhibitory peptide, suggesting that PKC activation is an early signaling event in Mer-mediated phagocytosis. Consistently, we found that Mer activation stimulates tyrosine phosphorylation of PLCγ2, an upstream event of PKC activation. More interestingly, using constitutively active form for Mer (CDMer) and CDMer mutants, we further found that Tyr 867 of Mer is predominantly responsible for PLCγ2 phosphorylation. In addition, Mer activation stimulated complex formation between integrin αvβ5 and Rack1, a receptor for activated PKC. Taken together, PKC is required for Mer-mediated activation signals for phagocytosis, and might be involved in the crosstalk between Mer and integirn αvβ5 via interaction with Rack1.

The Role of Lactadherin in the Phagocytosis of Phosphatidylserine-Expressing Sickle Red Blood Cell by Macrophages.

Sickle cell anemia, the most common serious hemoglobinopathy, is associated with a markedly reduced life span of red blood cells due to their preferential clearance by macrophages. During polymerization of sickle hemoglobin, phosphatidylserine, an anionic phospholipid normally present exclusively on the inner leaflet of the membrane bilayer is exteriorized to outer leaflet. This exposure of phosphatidylserine is thought to be a tag for macrophage recognition. Lactadherin, also known as milk fat globule-EGF factor 8, is a phosphatidylserine-binding glycoprotein secreted by macrophages that promotes the engulfment of apoptotic cells. Here, we investigated the role of lactadherin in the phagocytosis of sickle red blood cells.The binding of fluorescein-lactadherin to normal and sickle red blood cells was studied by flow cytometry. We quantified the effect of lactadherin on phagocytosis of red blood cells by monocyte-derived macrophage.In normal individuals, less than 0.5% of red blood cells showed any binding to lactadherin when analyzed by flow cytometry. However, in sickle cell patients, circulating red blood cells showed 2 to 10- fold increase in lactadherin binding (P<0.0002). Lactadherin stimulated the phagocytosis of resting sickle red blood cells by macrophages but had no significant effect on the phagocytosis of normal red blood cells. Deoxygenation of sickle red blood cells further increased the lactadherin binding and phagocytosis. Antibodies to integrin αVβ3 also inhibited macrophage binding and phagocytosis.These results show lactadherin may play a major role in sickle red cell clearance by anchoring the phosphatidylserine-expressing sickle red blood cells to integrins on tissue macrophages.

Impaired involution of mammary glands in the absence of milk fat globule EGF factor 8

During the involution of mammary glands, epithelial cells undergo apoptosis and are cleared for the next cycle of lactation. The clearance of apoptotic epithelial cells is mediated by neighboring epithelial cells and by macrophages that migrate into the mammary glands. Here, we report that milk fat globule EGF factor 8 (MFG-E8), a secreted glycoprotein that binds to apoptotic cells by recognizing phosphatidylserine, was expressed by epithelial cells and macrophages in mammary glands and was involved in engulfment of apoptotic cells. A deficiency of MFG-E8 caused the accumulation of a large number of milk fat globules (MFGs) in the mammary ducts during involution, indicating that the excess MFGs were cleared by an MFG-E8-dependent mechanism. The MFG-E8(-/-) mice developed mammary duct ectasia with periductal mastitis, and the redevelopment of the mammary gland for their second litter was impaired. These results demonstrate that MFG-E8-mediated phagocytosis of apoptotic epithelial cells and MFGs is important for efficient involution of mammary glands.

Calreticulin Serves as an "Eat Me" Signal

Apoptotic cells are removed by phagocytosis upon recognition of the dying cells. The exact signals for phagocytosis of apoptotic cells remain to be determined. Gardai et al. suggest that on apoptotic cells, cell surface calreticulin (Crt), although it is also present on healthy cells, becomes recognized by the engulfing cells, thereby contributing to the phagocytosis signal. Phagocytosis of apoptotic cells was decreased if the cells were first exposed to an antibody against Crt. Furthermore, Crt-deficient apoptotic cells were not phagocytosed efficiently in vitro or in vivo. In vitro phagocytosis of the Crt-deficient apoptotic cells was restored if the Crt was added exogenously. Both professional (macrophages) and nonprofessional (fibroblasts) phagocytic cells were dependent on cell surface Crt for recognition of apoptotic cells. Crt is known to bind LRP [low-density lipoprotein (LDL) receptor-related protein], and exposure of macrophages to an antibody against the extracellular, but not the intracellular, domain of LRP inhibited phagocytosis of apoptotic neutrophils. Crt also stimulated macropinocytosis and membrane ruffling through activation of Rac in macrophages, and these responses were absent in LRP –/– mouse embryo fibroblasts (MEFs), which also did not phagocytose apoptotic cells and did not bind exogenously added Crt. Receptor-associated protein (RAP) is a known antagonist of LRP, and treatment of macrophages with exogenous RAP inhibited engulfment of apoptotic cells. Furthermore, RAP and Crt reciprocally displaced each other from the surface of macrophages, which suggests competitive binding for the LRP. Because Crt is present on the surface of nonapoptotic cells, a mechanism must exist to allow the Crt "eat me" signal to be recognized at the proper time. One difference is that Crt was more abundant on the surface and redistributed into patches that were also abundant in phosphatidylserine and the ganglioside GM1 on the surface of apoptotic cells. Also, CD47 (also known as integrin-associated protein), which interacts with SIRPα (signal regulatory protein α) to inhibit phagocytosis, was either decreased at the surface of apoptotic cells or redistributed to patches away from the patches containing Crt. Thus, the "eat me" and "don't eat me" signals appear to segregate in apoptotic cells, which may allow the "eat me" signals to prevail. S. J. Gardai, K. A. McPhillips, S. C. Frasch, W. J. Janssen, A. Starefeldt, J. E. Murphy-Ullrich, D. L. Bratton, P.-A. Oldenborg, M. Michalak, P. M. Henson, Cell-surface calreticulin initiates clearance of viable or apoptotic cells through trans -activation of LRP on the phagocyte. Cell 123 , 321-334 (2005). [PubMed]

Efficient migration of dendritic cells toward lymph node chemokines and induction of TH1 responses require maturation stimulus and apoptotic cell interaction

AbstractDendritic cells (DCs) have the unique ability to initiate primary immune responses, and they can be conditioned for vaccinal purposes to present antigens after the engulfment of apoptotic cells. To recruit the rare antigen-specific naive T cells, DCs require a maturation step and subsequent transport toward lymph node (LN). To date, prostaglandin E2 (PGE2) is the best-characterized compound inducing this LN-directed migration in vitro, but PGE2 may skew the immune responses in a TH2 direction. We demonstrate here that on incubation with apoptotic tumor cells and tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS), human monocyte-derived DCs become fully mature and acquire high migratory capacities toward LN-directing chemokines. The migration of TNF-α-treated DCs occurs only after cotreatment with apoptotic cells but not with necrotic cells. DC migration requires CD36 expression and incubation with apoptotic cells in the presence of heat-labile serum components. Moreover, on treatment with apoptotic cells and LPS, the migrating DCs are able to recruit naive T cells to generate TH1 immune responses. Our results show that the cotreatment of DCs with apoptotic tumor cells and inflammatory signals is promising for the design of an antitumoral DC-based vaccine. (Blood. 2005;106:1734-1741)

Transepithelial Migration, Necrosis and Apoptosis as Silent and Pro- Inflammatory Fates of Airway Granulocytes

In inflammatory airway diseases, granulocytes such as eosinophils and neutrophils infiltrate the tissue where they are thought to exert pathogenic activities. To avoid a catastrophic accumulation of activated granulocytes, their recruitment must be balanced by efficient cell clearance mechanisms. In this regard, the focus has been on elimination through apoptosis and subsequent engulfment of apoptotic cells by phagocytes. However, novel data suggest that in the airways, powerful non-apoptotic mechanisms are also critically involved in cell clearance. One such mechanism is transepithelial migration into the airway lumen where the mucociliary escalator executes the final elimination. The physiological clearance of tissue granulocytes is normally silent, but can under certain situations shift to become a pro-inflammatory event. For example, apoptotic cells that escape phagocytosis, disintegrate in a pro-inflammatory process called secondary necrosis. In other situations, granulocytes are triggered to undergo an active and violent cytolytic death. This delicate balance in vivo, between silent and violent properties of granulocyte demise, complicates the design of pro-apoptotic pharmacological interventions. On the other hand, this insight may also open possibilities to new exciting treatment strategies. In this context, promoting transepithelial migration, prevention of secondary necrosis, and boosting the macrophage phagocytic system appear as exciting treatment avenues.

Autologous Apoptotic Cell Engulfment Stimulates Chemokine Secretion by Vascular Smooth Muscle Cells

Apoptosis of vascular smooth muscle cells (VSMCs) occurs in vivo under both physiological and pathological settings. The clearance of apoptotic cells may be accomplished in part by the surrounding normal VSMCs. However, the fate of internalized apoptotic cells, the rate of intracellular degradation, and the consequences of these processes to VSMC biology are unknown. Electron microscopy and confocal fluorescence imaging showed that rat VSMCs effectively bound and internalized autologous apoptotic VSMCs in vitro. Within 2 hours, the internalized apoptotic cells were delivered to lysosomes, and the majority of these internalized cells and their proteins were efficiently degraded by 24 hours. After degradation was completed, the phagocytic VSMCs remained viable with normal rates of proliferation. Clearance of apoptotic cells by VSMCs did not induce the release of vascular wall matrix proteases but was associated with a 1.6-fold increase in transforming growth factor-beta1 release. Interestingly, clearance of apoptotic cells stimulated VSMCs to secrete monocyte-chemoattractant protein-1 and cytokine-induced neutrophil chemoattractant. The coordinated release of transforming growth factor-beta1 and chemokines suggests that autologous apoptotic cell clearance stimulates VSMCs to release molecules that specifically recruit professional phagocytes while simultaneously dampening the inflammatory response and preventing vascular injury.

Taking insult from injury: Lipoxins and lipoxin receptor agonists and phagocytosis of apoptotic cells

Phagocytic clearance of apoptotic cells plays a pivotal role in the resolution of inflammation. Recent evidence has shown that such processes can be regulated by endogenous mediators, suggesting that specific mimetics may have therapeutic potential in chronic inflammation and autoimmune disorders. Here we review the mechanisms underlying recognition and engulfment of apoptotic cells and regulation of these processes by lipoxins and lipoxin receptor agonists.

Transglutaminase Type II Is a Key Element in the Regulation of the Anti-Inflammatory Response Elicited by Apoptotic Cell Engulfment

A key feature of the macrophage-dependent clearance of apoptotic cells is the down-regulation of proinflammatory cytokines. Deficiency in the phagocytosis of apoptotic cells is often associated with the development of inflammatory reactions, resulting in chronic inflammatory and autoimmune diseases. The molecular mechanisms that regulate the engulfment process and particularly the immunomodulatory factors involved are still largely unknown in mammals. We have previously reported that the ablation of transglutaminase type II (TG2) in mice results in the defective clearance of apoptotic cells associated with the development of splenomegaly, autoantibodies, and glomerulonephritis. In this study we have investigated the mechanisms at the basis of the development of inflammation/autoimmunity associated with the defective clearance of apoptotic cells characterizing TG2 knockout mice. To this aim we compared the macrophage response to apoptotic cell exposure in wild-type vs TG2-null mice. We demonstrated that the lack of TG2 results in an impaired capacity of macrophages to engulf, but not to bind, apoptotic cells, which is paralleled by an abnormal inflammatory response both in vivo and in vitro. We have identified a differential response in the release of several cytokines in TG2(-/-) vs wild-type mice. Particularly relevant is the finding that both TGF-beta and IL-12 regulations were significantly altered in the absence of TG2. These results help explain the autoimmune phenotype developed by these mice and suggest that TG2 is a key regulatory element of the anti-inflammatory features of apoptosis.

Enhanced Apoptotic Cell Clearance Capacity and B Cell Survival Factor Production by IL-10-Activated Macrophages: Implications for Burkitt’s Lymphoma

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.

C‐terminal SH3 domain of CrkII regulates the assembly and function of the DOCK180/ELMO Rac‐GEF

Genetic studies in Caenorhabditis elegans identified an evolutionarily conserved CED-2 (CrkII), CED-5 (DOCK180), CED-12 (ELMO), CED-10 (Rac1) module important for cell migration and phagocytosis of apoptotic cells. Previous studies have shown that DOCK180 and ELMO comprise an unconventional bipartite Dbl homology domain-independent Rac guanine nucleotide exchange factor (Rac-GEF); but it is still unclear how CrkII functions in Rac-GEF activity. In this study, we have characterized a unique function of CrkII in phagocytosis and Rac activation mediated by the C-terminal SH3 domain, a region of CrkII that has no clear cellular or biochemical function. We found that mutations that disrupt the C-terminal SH3 domain of CrkII (CrkII-SH3-C) abrogate engulfment of apoptotic cells and impair cell spreading on extracellular matrix. Surprisingly, despite the effects on engulfment, W276K CrkII strongly potentiated Rac-GTP loading when ectopically expressed in HEK 293T cells. Contrary to the effects of the true dominant negative SH2 domain mutants (R38K CrkII) and SH3-N domain mutants (W170K CrkII) that prevent macromolecular assembly of signaling proteins, W276K CrkII increases association between DOCK180 and CrkII as well as constitutive tethering of the Crk/DOCK180/ELMO protein complex that interacted with RhoG. Our results indicate that while N-terminal SH3 of CrkII promotes assembly between CrkII and DOCK180, the C-terminal SH3 of CrkII regulates the stability and turnover of the DOCK180/ELMO complex. Studies with W276K CrkII may offer a unique opportunity to study the structure and function of the DOCK180/ELMO Rac-GEF.

A Steric-Inhibition Model for Regulation of Nucleotide Exchange via the Dock180 Family of GEFs

CDM (CED-5, Dock180, Myoblast city) family members have been recently identified as novel, evolutionarily conserved guanine nucleotide exchange factors (GEFs) for Rho-family GTPases . They regulate multiple processes, including embryonic development, cell migration, apoptotic-cell engulfment, tumor invasion, and HIV-1 infection, in diverse model systems . However, the mechanism(s) of regulation of CDM proteins has not been well understood. Here, our studies on the prototype member Dock180 reveal a steric-inhibition model for regulating the Dock180 family of GEFs. At basal state, the N-terminal SH3 domain of Dock180 binds to the distant catalytic Docker domain and negatively regulates the function of Dock180. Further studies revealed that the SH3:Docker interaction sterically blocks Rac access to the Docker domain. Interestingly, ELMO binding to the SH3 domain of Dock180 disrupted the SH3:Docker interaction, facilitated Rac access to the Docker domain, and contributed to the GEF activity of the Dock180/ELMO complex. Additional genetic rescue studies in C. elegans suggested that the regulation of the Docker-domain-mediated GEF activity by the SH3 domain and its adjoining region is evolutionarily conserved. This steric-inhibition model may be a general mechanism for regulating multiple SH3-domain-containing Dock180 family members and may have implications for a variety of biological processes.

Clearance of apoptotic cells in Caenorhabditis elegans

Programmed cell death, or apoptosis, is a genetically controlled process of cell suicide that is a common fate during an animal's life. In metazoans, apoptotic cells are rapidly removed from the body through the process of phagocytosis. Genetic analyses probing the mechanisms controlling the engulfment of apoptotic cells were pioneered in the nematode Caenorhabditis elegans. So far, at least seven genes have been identified that are required for the recognition and engulfment of apoptotic cells and have been shown to function in two partially redundant signaling pathways. Molecular characterization of their gene products has lead to the finding that similar genes act to control the same processes in other organisms, including mammals. In this paper, we review these exciting findings in C. elegans and discuss their implications in understanding the clearance of apoptotic cells in mammals.

Quantifying macrophage defects in type 1 diabetes

Macrophages from animals prone to autoimmune (type 1) diabetes differ from those of diabetes-resistant animals in processing and clearing apoptotic cells. Using in vitro time-course assays of the number of engulfed apoptotic cells observed within macrophages, we quantified these differences in non-obese diabetic (NOD) versus Balb/c mice. Simple models lead to several elementary parameter estimation techniques. We used these to compute approximate rates of macrophage engulfment and digestion of apoptotic cells from basic features of the data (such as initial rise-times, phagocytic index and percent phagocytosis). Combining these estimates with full fitting of a sequence of model variants to the data, we find that macrophages from normal (Balb/c) mice engulf apoptotic cells up to four times faster than macrophages from the diabetes-prone (NOD) mice. Further, Balb/c macrophages appear to undergo an activation step before achieving their high engulfment rate. In NOD macrophages, we did not see evidence for this activation step. Rates of digestion of engulfed apoptotic cells by macrophages are similar in both types. Since macrophage clearance is an important mechanism of disposal of self-antigen, these macrophage defects could potentially be a factor in predisposition to type 1 diabetes.

Phosphatidylserine Receptor in Chronic Pancreatitis

This study analyzes the role of phosphatidylserine receptor (PSR) in chronic pancreatitis. In chronic pancreatitis, destruction of parenchyma comes along with infiltration of lymphocytes and macrophages. The phosphatidylserine receptor is expressed on the surface of macrophages and is crucial for the recognition and engulfment of apoptotic cells. In the present study, we investigated the role of this receptor and its relation to apoptosis in chronic pancreatitis. The expression and localization of PSR were analyzed by Northern blot analysis, RT-PCR, Western blot analysis and immunohistochemistry. Apoptosis was detected by the TUNEL method, and the RNA protection assay (RPA) was used to compare activation of apoptosis with PSR mRNA expression levels. In addition, the molecular data were related to clinicopathological parameters. PSR mRNA expression was low to absent in normal pancreatic tissue samples. In human chronic pancreatitis, increased expression of PSR mRNA was present in 12 of 29 samples (41%). Up-regulation of PSR could be confirmed by Western blot analysis. In chronic pancreatitis tissue, PSR immunoreactivity was present in all islets, in some ductal cells and in macrophages. The RNA protection assay revealed high mRNA levels of the antiapoptotic genes bcl-2 and bfl-1 (P < 0.05) in chronic pancreatitis tissues with high PSR mRNA expression. The TUNEL apoptosis in situ detection method showed positive signals in some redifferentiating acinar cells and focally in acinar cells adjacent to stromal fibroblasts in chronic pancreatitis tissue samples. The distribution pattern of PSR on pancreatic cells in chronic pancreatitis corresponded to a great extent with regions of high apoptotic activity. We show for the first time the presence of PSR in chronic pancreatitis on pancreatic cells other than macrophages in regions with high apoptotic activity. The coexpression and colocalization of this gene with other apoptosis mediators suggest its involvement in apoptotic processes. However, in chronic pancreatitis PSR is not only involved in phagocytosis of apoptotic cells by macrophages.

Thermotolerance of pulp cells and phagocytosis of apoptotic pulp cells by surviving pulp cells following heat stress

Apoptosis is known to be associated with wound healing and regeneration of dental pulp. We examined the effects of heat stress on clonal dental pulp cell line (RPC-C2A cells) to clarify the pulp wound healing process. RPC-C2A cells were exposed to heat stress at 43 degrees C for 45 min. After several time intervals, the inhibition of cell proliferation and apoptosis induction were analyzed by cell viability assay, DNA gel electrophoresis, nuclear staining, and terminal deoxynucleotidyl transferase mediated labeling assay. RPC-C2A cells showed the thermotolerance following heat stress. We found that apoptosis was induced in some RPC-C2A cells, whereas others remained alive, and observed the engulfment of apoptotic cells by scavenger-like RPC-C2A cells following heat stress. We also analyzed the phagocytotic activity of RPC-C2A cells and found that they had an ability to engulf apoptotic RPC-C2A cells, which was stimulated by heat stress. These results suggest that heat stress induces apoptosis of RPC-C2A cells, which are phagocytosed by the surviving RPC-C2A cells.

Draper-mediated and Phosphatidylserine-independent Phagocytosis of Apoptotic Cells by Drosophila Hemocytes/Macrophages

The mechanism of phagocytic elimination of dying cells in Drosophila is poorly understood. This study was undertaken to examine the recognition and engulfment of apoptotic cells by Drosophila hemocytes/macrophages in vitro and in vivo. In the in vitro analysis, l(2)mbn cells (a cell line established from larval hemocytes of a tumorous Drosophila mutant) were used as phagocytes. When l(2)mbn cells were treated with the molting hormone 20-hydroxyecdysone, the cells acquired the ability to phagocytose apoptotic S2 cells, another Drosophila cell line. S2 cells undergoing cycloheximide-induced apoptosis exposed phosphatidylserine on their surface, but their engulfment by l(2)mbn cells did not seem to be mediated by phosphatidylserine. The level of Croquemort, a candidate phagocytosis receptor of Drosophila hemocytes/macrophages, increased in l(2)mbn cells after treatment with 20-hydroxyecdysone, whereas that of Draper, another candidate phagocytosis receptor, remained unchanged. However, apoptotic cell phagocytosis was reduced when the expression of Draper, but not of Croquemort, was inhibited by RNA interference in hormone-treated l(2)mbn cells. We next examined whether Draper is responsible for the phagocytosis of apoptotic cells in vivo using an assay for engulfment based on assessing DNA degradation of apoptotic cells in dICAD mutant embryos (which only occurred after ingestion by the phagocytes). RNA interference-mediated decrease in the level of Draper in embryos of mutant flies was accompanied by a decrease in the number of cells containing fragmented DNA. Furthermore, histochemical analyses of dispersed embryonic cells revealed that the level of phagocytosis of apoptotic cells by hemocytes/macrophages was reduced when Draper expression was inhibited. These results indicate that Drosophila hemocytes/macrophages execute Draper-mediated phagocytosis to eliminate apoptotic cells.

Oxidation of phosphatidylserine: a mechanism for plasma membrane phospholipid scrambling during apoptosis?

Selective oxidation of phosphatidylserine (PS) during apoptosis precedes its externalization in plasma membrane and is essential for the engulfment of apoptotic cells. To experimentally test whether PS oxidation stimulates its externalization via its effects on aminophospholipid translocase (APT) or by enhanced PS scrambling, action of oxidized PS (PSox) was studied using leukemia HL-60 cells and lymphoma Raji cells. Both PS and PSox were equally well recognized by APT. PSox did not inhibit APT. Rate of transmembrane PS diffusion was fourfold higher in cells with integrated PSox than with PS. Thus, PSox acts as a “non-enzymatic scramblase” likely contributing to PS externalization.