Activation of PI3-kinase/PKB contributes to delay in neutrophil apoptosis after thermal injury

Neutrophil apoptosis is delayed under trauma and/or sepsis injury conditions. The molecular mechanism for the delay in apoptosis has not been well defined. We investigated whether activation of phosphatidyl inositol 3-kinase (PI3-kinase)/PKB signaling pathway contributes to the delay in neutrophil apoptosis with thermal injury. Rats were subjected to burns (30% total body surface area, 98°C for 10 s), and euthanized 24 h later. Blood neutrophils were isolated with the use of Ficoll gradient centrifugation and cultured for the indicated time periods. Apoptosis was determined using annexin V and PI labeling and flow cytometry. NF-B activation was examined using gel mobility shift assay and confocal microscopy. Expression levels of inhibitory apoptosis proteins (IAPs), including cellular IAP1 (cIAP1), cIAP2, X-linked IAP (XIAP), and survivin, and Bcl-2 family members such as Bcl-xl and Bad, were determined by Western blot analysis and/or RT-PCR, real-time PCR. The results showed that in culture, the decrease in apoptosis of neutrophils from thermally injured rats was prevented in the presence of PI3-kinase inhibitors wortmannin and LY-294002. There was upregulation of PKB and Bad phosphorylation and NF-B activation in N-formyl-L-methionyl-L-leucyl-L-phenylalanine-stimulated neutrophils from thermally injured rats compared with the sham injured group. Increased Bad phosphorylation and NF-B activation were also attenuated by wortmannin. Bcl-xl expression in neutrophils was upregulated with thermal injury and inhibited in the presence of wortmannin. However, the expression of IAP family members was neither affected by thermal injury nor inhibited by wortmannin. These data suggest that the delay in neutrophil apoptosis with thermal injury is partly caused by activation of PI3-kinase/PKB signaling and NF-B, which appeared to be related to the increased Bcl-xl expression and phosphorylation of Bad, but not IAP expression. polymorphonuclear neutrophils; nuclear factor-B; Bcl-xl; Bad; inhibitory apoptosis protein; burn injury     

Methylglyoxal induces mitochondria-dependent apoptosis in sarcoma

In the preceding paper (A. Ghosh et al. (2011) Biochemistry (Moscow), 76, 1051–1060), using several comparable tissue materials, it has been convincingly demonstrated that methylglyoxal, a normal metabolite, inhibits mitochondrial complex I of specifically malignant cells. This suggests a distinct alteration of complex I, a highly important enzyme for energy (ATP) production, in malignancy. The present paper shows that as a consequence of this inhibition mitochondrial membrane potential is drastically reduced in sarcoma tissue but not in normal skeletal muscle. This was estimated spectrofluorimetrically using the dye rhodamine 123. As a consequence, cytochrome c was released from the sarcoma mitochondria as evidenced by Western blot analysis. Moreover, on treatment with methylglyoxal membrane potential collapse of sarcoma 180 cells was also indicated by fluorescence-activated cell sorter analysis. Atomic force microscopic study demonstrated gross structural alteration specifically of tumor mitochondria on methylglyoxal treatment. All these studies suggest that methylglyoxal might initiate an apoptotic event in malignant cells.     

PAF receptor antagonist modulates neutrophil responses with thermal injury in vivo

The role of platelet-activating factor (PAF) inCa²⁺ signaling and Ca²⁺-related enhancement ofreactive oxygen intermediate (ROI) generation in neutrophils ofburn-injured rats was ascertained by evaluating the effect of treatmentof the rats with a PAF receptor antagonist. The treatment of rats withthe antagonist also allowed us to evaluate the role of PAF in thepriming of neutrophil ROI response with burn in vivo. A full skinthickness burn injury was produced in anesthetized rats by exposing30% of total body surface area to 98°C water for 10 s. Sham andburn rats were killed 1 day later, and their blood was collected toobtain neutrophils. Fluorescence-activated cell sorter analysis wasused to quantify ROI production by the neutrophils. Cytosolic-freeCa²⁺ concentration ([Ca²⁺]_i)imaging technique was employed to measure neutrophil[Ca²⁺]_i in individual cells andmicrofluorometry for the assessment of[Ca²⁺]_i responses in suspensions ofneutrophils. There was an overt enhancement of ROI generation by burnrat neutrophils. ROI release was accompanied by a marked elevation of[Ca²⁺]_i signaling. The treatment of rats withPAF receptor antagonist before burn prevented the upregulation of both[Ca²⁺]_i and ROI generation in neutrophils.These studies indicate that enhanced ROI production in neutrophils inthe early stages after burn injury results from a PAF-mediated primingof the [Ca²⁺]_i signaling pathways in vivo.

     

PTEN: A crucial mediator of mitochondria-dependent apoptosis

The highly frequent mutation of phosphatase and tensin homologue deleted on chromosome 10 (PTEN) in various cancers has attracted much attention to study its role in tumorigenesis. As an important tumor suppressor, the pro-apoptotic function of PTEN has been linked to its capacity antagonizing the PI3K/Akt signaling pathway. However, less data are available concerning its role in neurodegeneration in which apoptotic processes are also involved. In the present study, we attempted to study the role and the underlying mechanism of PTEN in neuronal apoptosis. Using primary rat hippocampal cultures, staurosporine (STS, 100 nM) induced a time-dependent apoptosis, accompanied by a marked production of reactive oxygen species (ROS), release of cytochrome c and activation of caspase 9 and 3. However, the expression of PTEN, and the levels of phospho-PTEN and phospho-Akt were not changed at all time points tested (0.5–24 h) after STS stimulation, suggesting that the protein level as well as the phosphorylation status of PTEN were not related to the procession of apoptosis. Interestingly, immunostaining revealed a punctate intracellular distribution of PTEN from 2 to 8 h after adding STS. Double labeling and Western blotting of mitochondrial fraction demonstrated a mitochondrial location and accumulation of PTEN, respectively, after challenging with STS. Furthermore, we provide evidence for the first time that PTEN was associated with Bax in the absence and the presence of STS. Of note, the STS-induced marked increase in the cellular ROS level, release of cytochrome c and activation of caspase 3 were inhibited in cultured hippocampal cells when PTEN was knocked down by a specific antisense. Moreover, knockdown of PTEN significantly protected hippocampal cells from apoptotic damage. These findings demonstrated that PTEN is a crucial mediator of mitochondria-dependent apoptosis, and thus could become a molecular target for interfering with neurodegenerative diseases.     

Cadmium induces mitochondria-dependent apoptosis of normal human hepatocytes

The heavy metal cadmium, an environmental pollutant, has been widely demonstrated to be toxic, in particular for liver. In murines, cadmium induces apoptosis of hepatocytes and hepatomas. In human cells, apoptosis induced by cadmium has been exclusively demonstrated in tumoral cell lines. Nothing was known in normal liver, in vitro or in vivo. In the present study, we examined the effects of cadmium in nonmalignant human hepatocytes. For that purpose, we investigated whether cadmium was able to induce apoptosis of normal human hepatocytes (NHH) in primary culture and of a SV40-immortalized human hepatocyte (IHH) cell line. Treatment of IHH and NHH with cadmium induced the presence of a sub-G₁ population at 10 and 100 μmol/L, respectively. DAPI staining of both cell types treated with cadmium 100 μmol/L revealed the induction of nuclear apoptotic bodies, supporting the hypothesis of apoptosis. In IHH and NHH, cadmium 100 μmol/L induced PARP cleavage into a 85 kDa fragment. In order to investigate the involvement of mitochondria in cadmium-induced apoptosis, we measured the mitochondrial membrane potential (Δ_Ψm). We observed that in IHH and NHH, cadmium 100 μmol/L induced a decrease of Δ_Ψm. As expected, cadmium under the same conditions enhanced caspase-9 and caspase-3 activities. In addition, cadmium from 1 to 100 μmol/L induced the expression of p53 and phosphorylation of its Ser15 in IHH and NHH. In conclusion, we showed in this study that human hepatocytes were sensitive to cadmium and apoptosis induced at concentrations suggested in the literature to inhibit p53 DNA-binding and DNA repair.     

Reactive oxygen and nitrogen species induce cell apoptosis via a mitochondria-dependent pathway in hyperoxia lung injury

Hyperoxia-induced lung injury limits the application of mechanical ventilation on rescuing the lives of premature infants and seriously ill and respiratory failure patients, and its mechanisms are not completely understood. In this article, we focused on the relationship between hyperoxia-induced lung injury and reactive oxygen species (ROS), reactive nitrogen species (RNS), mitochondria damage, as well as apoptosis in the pulmonary epithelial II cell line RLE-6TN. After exposure to hyperoxia, the cell viability was significantly decreased, accompanied by the increase in ROS, nitric oxide (NO), inflammatory cytokines, and cell death. Furthermore, hyperoxia triggered the loss of mitochondrial membrane potential (▵Ψm), thereby promoting cytochrome c to release from mitochondria to cytoplasm. Further studies conclusively showed that the Bax/Bcl-2 ratio was enlarged to activate the mitochondria-dependent apoptotic pathway after hyperoxia treatment. Intriguingly, the effects of hyperoxia on the level of ROS, NO and inflammation, mitochondrial damage, as well as cell death were reversed by free radical scavengers N-acetylcysteine and hemoglobin. In addition, a hyperoxia model of neonatal Sprague-Dawley (SD) rats presented the obvious characteristics of lung injury, such as a decrease in alveolar numbers, alveolar mass edema, and disorganized pulmonary structure. The effects of hyperoxia on ROS, RNS, inflammatory cytokines, and apoptosis-related proteins in lung injury tissues of neonatal SD rats were similar to that in RLE-6TN cells. In conclusion, mitochondria are a primary target of hyperoxia-induced free radical, whereas ROS and RNS are the key mediators of hyperoxia-induced cell apoptosis via the mitochondria-dependent pathway in RLE-6TN cells.     

Modulation of mercury-induced mitochondria-dependent apoptosis by glycine in hepatocytes

Mercury (Hg) is one of the universal environmental pollutants and is responsible for various organ pathophysiology including oxidative stress-induced hepatic disorders. In the present study, we aimed to explore the protective role of glycine in Hg-induced cytotoxicity and cell death in murine hepatocytes. Exposure of mercury (20 μM), in the form HgCl₂ for 1 h, significantly enhanced the ALT and ALP leakage, increased reactive oxygen species production, reduced cell viability and distorted the antioxidant status of hepatocytes. Flow cytometric analyses shows that Hg-induced apoptotic death in hepatocytes. Mechanism of this pathophysiology involves reduced mitochondrial membrane potential, variations in Bcl-2/Bad proteins, activation of caspases and cleavage of PARP protein. In addition, Hg distinctly increased NF-κB phosphorylation in association with IKKα phosphorylation and IκBα degradation. Concurrent treatment with glycine (45 mM), however, reduced Hg-induced oxidative stress, attenuated the changes in NF-κB phosphorylation and protects hepatocytes from Hg-induced apoptotic death. Hg also distinctly increased the phosphorylation of p38, JNK and ERK mitogen-activated protein kinase (MAPKs). Glycine treatment suppressed these apoptotic events, signifying its protective role in Hg-induced hepatocyte apoptosis as referred by reduction of p38, JNK and ERK MAPK signaling pathways. Results suggest that glycine can modulate Hg-induced oxidative stress and apoptosis in hepatocytes probably because of its antioxidant activity and functioning via mitochondria-dependent pathways and could be a beneficial agent in oxidative stress-mediated liver diseases.     

A simultaneous release of SOD1 with cytochrome c regulates mitochondria-dependent apoptosis

To elucidate the significance of mitochondrial localization of Cu/Zn-SOD (SOD1), we studied the relationship between the release of mitochondrial SOD1 and apoptosis. Kinetic analysis using HL-60 cells showed that both mitochondria-dependent and mitochondria-independent pro-apoptotic drugs, such as staurosporine and actinomycin D, increased the generation of reactive oxygen species (ROS) and decreased mitochondrial membrane potential (Δψ). ROS generation by these drugs was inhibited by Mn (III) tetrakis (5,10,15,20-benzoic acid) porphyrin (MnTBAP), a cell membrane-permeable SOD mimetic. However, MnTBAP inhibited the apoptosis induced by staurosporine but not by actinomycin D. MnTBAP failed to inhibit Δψ decrease and release of SOD1 and cytochrome c induced by actinomycin D. Moreover, 4,4′-diisothiocyanatostilbene-2,2′-disulfonic acid (DIDS), an inhibitor of voltage-dependent anion channel (VDAC), inhibited the release of the two proteins and apoptosis induced by staurosporine but not actinomycin D. These results suggest that ROS plays an important role in mitochondria-dependent but not mitochondria-independent apoptosis and that the release of SOD1 increases the susceptibility of mitochondria to oxidative stress, thereby enhancing a vicious cycle leading to apoptosis.     

A mitochondria-dependent pathway mediates the apoptosis of GSE-induced yeast

Grapefruit seed extract (GSE), which has powerful anti-fungal activity, can induce apoptosis in S. cerevisiae. The yeast cells underwent apoptosis as determined by testing for apoptotic markers of DNA cleavage and typical chromatin condensation by Terminal Deoxynucleotidyl Transferase-mediated dUTP Nick End Labeling (TUNEL) and 4,6'-diaminidino-2-phenylindole (DAPI) staining and electron microscopy. The changes of ΔΨmt (mitochondrial transmembrane potential) and ROS (reactive oxygen species) indicated that the mitochondria took part in the apoptotic process. Changes in this process detected by metabonomics and proteomics revealed that the yeast cells tenaciously resisted adversity. Proteins related to redox, cellular structure, membrane, energy and DNA repair were significantly increased. In this study, the relative changes in the levels of proteins and metabolites showed the tenacious resistance of yeast cells. However, GSE induced apoptosis in the yeast cells by destruction of the mitochondrial 60 S ribosomal protein, L14-A, and prevented the conversion of pantothenic acid to coenzyme A (CoA). The relationship between the proteins and metabolites was analyzed by orthogonal projections to latent structures (OPLS). We found that the changes of the metabolites and the protein changes had relevant consistency.     

Macrophage-induced neutrophil apoptosis

Macrophages (Mphi) contribute to the resolution of early inflammation by recognizing and ingesting apoptotic polymorphonuclear neutrophils (PMN). In addition, experiments reported here demonstrated that Mphi can actively induce PMN apoptosis. Coculture of cells from 2- or 5-day-old wounds in rats, or of Mphi purified from such preparations, with PMN-rich wound cell populations obtained 1 day after wounding increased PMN apoptosis by >3-fold. Neither resident- nor Proprionibacterium acnes-elicited peritoneal Mphi-induced PMN apoptosis. Apoptosis was not mediated by a soluble factor and required E:T contact. Fixed wound-Mphi and membrane isolates from viable Mphi were as effective as intact cells in inducing PMN apoptosis. Mphi-induced apoptosis was inhibited by peptide Arg-Gly-Asp-Ser, anti-beta3 (CD61) Ab, CD36 peptide, or anti-TNF-alpha Ab. Soluble TNF-alpha did not induce PMN apoptosis. In additional studies, K562 cells (negative for beta3, TNF-alpha, and Fas ligand) transfected to express either alphavbeta3 integrin, an uncleavable membrane form of TNF-alpha, or both were used in cocultures with wound PMN. Only the double transfectants were able to induce PMN apoptosis, an effect inhibited by anti-beta3 (CD61) or anti-TNF-alpha Abs. These results demonstrate that wound Mphi induce PMN apoptosis through a constitutive effector mechanism requiring both intercellular binding through integrin-ligand interactions and membrane-bound TNF-alpha.     

Disruption of Smad5 gene induces mitochondria-dependent apoptosis in cardiomyocytes

Our previous studies have shown that SMAD5, an important intracellular mediator of transforming growth factor beta (TGF-beta) family, is required for normal development of the cardiovascular system in vivo. In the current study, we reported that the lack of the Smad5 gene resulted in apoptosis of cardiac myocytes in vivo. To further investigate the mechanism of the Smad5 gene in cardiomyocyte apoptosis, the embryonic stem (ES) cell differentiation system was employed. We found that the myotubes that differentiated from the homozygous Smad5ex6/ex6 mutant ES cells underwent collapse and degeneration during the late stages of in vitro differentiation, mimicking the in vivo observation. By electron microscopy, abnormal swollen mitochondria were observed in cardiomyocytes both from Smad5-deficient embryos and from ES-differentiated cells. There was also a significant reduction in mitochondrial membrane potential (Deltapsi m) and a leakage of cytochrome c from mitochondria into the cytosol of myocytes differentiated from Smad5 mutant ES cells. The expression of p53 and p21 was found to be elevated in the differentiated Smad5 mutant myocytes, and this was accompanied by an up-regulation in caspase 3 expression. These results suggest that the Smad5-mediated TGF-beta signals may protect cardiomyocytes from apoptosis by maintaining the integrity of the mitochondria, probably through suppression of p53 mediated pathways.     

Lyn- and ERK-mediated vs. Ca2+ -mediated neutrophil O responses with thermal injury

We evaluated the dependency of neutrophil O production on PTK-Lyn and MAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn was assessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessed by Western blot analysis. O production was measured by isoluminol-enhanced luminometry. Imaging technique was employed to measure neutrophil [Ca2+](i) in individual cells. Thermal injury caused marked upregulation of Lyn and ERK1/2 accompanying enhanced neutrophil O production. Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478) before burn injury caused complete inhibition of the respective kinase activation. Both AG556 and AG1478 produced an ~66% inhibition in O production. Treatment with diltiazem (DZ) produced an ~37% inhibition of O production without affecting Lyn or ERK1/2 activation with burn injury. Ca2+ mobilization was upregulated with burn injury but not affected by treatment of burn rats with AG556. Unlike the partial inhibition of burn-induced O production by AG556, AG1478, or DZ, platelet-activating factor antagonist (PAFa) treatment of burn rats produced near complete inhibition of O production. PAFa treatment also blocked activation of Lyn. The findings suggest that the near complete inhibition of O production by PAFa was a result of blockade of PTK as well as Ca2+ signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result of potentiation of Ca2+ -linked and -independent signaling triggered by inflammatory agents such as PAF.     

Methylene blue-mediated photodynamic therapy induces mitochondria-dependent apoptosis in HeLa cell

Methylene blue (MB), a widely studied reagent, is investigated in this work for its usage in photodynamic therapy (PDT). PDT has been proved to be highly effective in the treatment of different types of cancers. Previous studies showed MB has both high affinity for mitochondria and high photodynamic efficiency. To elucidate the effects of MB in PDT, we analyzed PDT-induced apoptosis in HeLa cells by introducing different doses of MB into the culture media. Our data showed that MB-mediated PDT triggered intense apoptotic cell death through a series of steps, beginning with photochemical generation of reactive oxygen species. The release of cytochrome c and activation of caspase-3 indicated that MB-PDT-mediated apoptosis in HeLa cells was executed by the mitochondria-dependent apoptotic pathway. Importantly, proteomic studies confirmed that expression levels of several mitochondrial proteins were altered in MB-PDT-induced apoptosis, including TRAP1, mitochondrial elongation factor Tu and peroxiredoxin 3 isoform b. Western blot data showed that phosphorylation of ERK1/2 and PKA were reduced in MB-PDT treated cells, indicating several signal molecules participating in this apoptotic cascade. Moreover, MB-PDT induced an increase in the strength of interaction between Bcl-xL and dephosphorylated Bad. This led to loss of the pro-survival function of Bcl-xL and resulted in mitochondria-mediated apoptosis. This study provides solid evidence of a strong induction by MB-PDT of a mitochondria-dependent apoptosis cascade in HeLa cells.     

Hyperosmotic stress induces metacaspase- and mitochondria-dependent apoptosis in Saccharomyces cerevisiae

During the last years, several reports described an apoptosis-like programmed cell death process in yeast in response to different environmental aggressions. Here, evidence is presented that hyperosmotic stress caused by high glucose or sorbitol concentrations in culture medium induces in Saccharomyces cerevisiae a cell death process accompanied by morphological and biochemical indicators of apoptotic programmed cell death, namely chromatin condensation along the nuclear envelope, mitochondrial swelling and reduction of cristae number, production of reactive oxygen species and DNA strand breaks, with maintenance of plasma membrane integrity. Disruption of AIF1 had no effect on cell survival, but lack of Yca1p drastically reduced metacaspase activation and decreased cell death indicating that this death process was associated to activation of this protease. Supporting the involvement of mitochondria and cytochrome c in caspase activation, the mutant strains cyc1Deltacyc7Delta and cyc3Delta, both lacking mature cytochrome c, displayed a decrease in caspase activation associated to increased cell survival when exposed to hyperosmotic stress. These findings indicate that hyperosmotic stress triggers S. cerevisiae into an apoptosis-like programmed cell death that is mediated by a caspase-dependent mitochondrial pathway partially dependent on cytochrome c.     

Molecular control of neutrophil apoptosis

Human neutrophils constitutively undergo apoptosis and this process is critical for the resolution of inflammation. Whilst neutrophil apoptosis can be modulated by a wide variety of agents including GM-CSF, LPS and TNF-alpha, the molecular mechanisms underlying neutrophil death and survival remain largely undefined. Recent studies have shown the involvement of members of the Bcl-2 protein family (especially Mcl-1 and A1) and caspases in the regulation and execution of neutrophil apoptosis. Cell surface receptors and protein kinases, particularly mitogen-activated protein kinases, also play critical roles in transducing the signals that result in neutrophil apoptosis or extended survival. This review summarises current knowledge on the molecular mechanisms and components of neutrophil apoptosis.     

The thermal treatment effects on bovine blood neutrophil granulocytes apoptosis and necrosis in vitro

The aim of the study was to evaluate the effect of selected temperatures on viability (apoptosis and necrosis) of bovine blood neutrophil granulocytes (neutrophils) in vitro. The following temperatures were tested: -80, -20, 4, 23, 37 degrees C. Heparinised bovine blood was incubated for 1, 4 and 24 h under respective temperature. Apoptosis and necrosis of neutrophils were detected by light microscopy, transmission electron microscopy (TEM) and flow cytometry (FCM). From selected temperatures, 4 degrees C impaired the neutrophil viability least. The proportion of apoptotic and necrotic neutrophils amounted to (mean +/- SD) 5.25 +/- 3.53% and 0.83 +/- 0.38%; 7.09 +/- 2.07% and 1.64 +/- 0.50%; 35.39 +/- 12.53% and 5.46 +/- 1.46%; after 1, 4 and 24 h incubation, respectively. The temperature (4 degrees C) is the best alternative for short-term storage.     

Lyn- and ERK-mediated vs. Ca2+-mediated neutrophil O2- responses with thermal injury

We evaluated thedependency of neutrophil O production on PTK-Lyn andMAPK-ERK1/2 in rats after thermal injury. Activation of PTK-Lyn wasassessed by immunoprecipitation. Phosphorylation of ERK1/2 was assessedby Western blot analysis. O production was measuredby isoluminol-enhanced luminometry. Imaging technique was employed tomeasure neutrophil [Ca²⁺]_i in individualcells. Thermal injury caused marked upregulation of Lyn and ERK1/2accompanying enhanced neutrophil O production.Treatment of rats with PTK blocker (AG556) or MAPK blocker (AG1478)before burn injury caused complete inhibition of the respective kinaseactivation. Both AG556 and AG1478 produced an ~66% inhibition inO production. Treatment with diltiazem (DZ) producedan ~37% inhibition of O production withoutaffecting Lyn or ERK1/2 activation with burn injury. Ca²⁺mobilization was upregulated with burn injury but not affected bytreatment of burn rats with AG556. Unlike the partial inhibition ofburn-induced O production by AG556, AG1478, or DZ,platelet-activating factor antagonist (PAFa) treatment of burn ratsproduced near complete inhibition of O production.PAFa treatment also blocked activation of Lyn. The findings suggestthat the near complete inhibition of O production byPAFa was a result of blockade of PTK as well as Ca²⁺signaling. Overall, our studies show that enhanced neutrophil O production after thermal injury is a result ofpotentiation of Ca²⁺-linked and -independent signalingtriggered by inflammatory agents such as PAF.

     

The novel neutrophil differentiation marker phosphatidylglucoside mediates neutrophil apoptosis

A new type of glycolipid, phosphatidylglucoside (PtdGlc), was identified as a component of raft-like membrane domains of the human leukemia cell line HL-60. In this study, we show that PtdGlc forms functional domains that are different from those produced by lactosylceramide (LacCer)-enriched lipid rafts. These rafts initiate neutrophil apoptosis. Neutrophils are the only type of human peripheral blood leukocyte or monocyte-derived dendritic cell to express large amounts of PtdGlc on their cell surfaces. PtdGlc was not colocalized with LacCer. Anti-PtdGlc IgM DIM21 did not induce neutrophil chemotaxis or superoxide generation, whereas anti-LacCer IgM T5A7 induced these activities. DIM21, but not T5A7, significantly induced neutrophil apoptosis. DIM21-induced apoptosis was inhibited by specific inhibitors of cysteine-containing aspartate-specific proteases (caspases)-8, -9, and -3 but not by the Src family kinase inhibitor PP1, PIP(3) kinase inhibitor LY294002, NADPH oxidase inhibitor diphenyleneiodonium, superoxide dismutase, or catalase. PtdGlc was colocalized with Fas on the neutrophil plasma membrane. DIM21 and the agonist anti-Fas Ab DX2 induced the formation of large Fas-colocalized clusters of PtdGlc on the plasma membrane. Furthermore, the antagonistic anti-Fas Ab ZB4 significantly inhibited DIM21-induced neutrophil apoptosis. These results suggest that PtdGlc is specifically expressed on neutrophils and mediates apoptosis of these cells, and that the Fas-associated death signal may be involved in PtdGlc-mediated apoptosis.