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(Journal of Leukocyte Biology. 2000;68:187-193.)
© 2000 by Society for Leukocyte Biology

Induction of neutrophil death resembling neither apoptosis nor necrosis by ONO-AE-248, a selective agonist for PGE2 receptor subtype 3

Jiajia Liu, Tohru Akahoshi, Shixu Jiang*, Rie Namai, Hidero Kitasato{dagger}, Hirahito Endo, Toru Kameya* and Hirobumi Kondo

Departments of Internal Medicine,
* Pathology, and
{dagger} Microbiology, Kitasato University School of Medicine, Kitasato, Sagamihara, Kanagawa, Japan

Correspondence: Tohru Akahoshi, M.D., Ph.D., Department of Internal Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa, 228-8555 Japan. E-mail: akahoshi{at}med.kitasato-u.ac.jp


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ABSTRACT
 
An increase of intracellular cAMP mediated by prostaglandin E2 (PGE2) has been shown to delay spontaneous apoptosis of neutrophils. It has been demonstrated that a selective agonist for PGE2 receptor subtype 3 (the EP3 receptor) is capable of decreasing cAMP and stimulating phosphoinositide turnover in various types of cells. We investigated the effect of a selective EP3 receptor agonist, ONO-AE-248, on neutrophil viability. ONO-AE-248 rapidly caused a unique form of neutrophil death. The agonist primarily induced morphological changes of the nucleus, including fusion of the lobules, decreased compactness of the chromatin, and blebbing and rupture of the nuclear membrane. This was followed by an increase of plasma membrane permeability and cell lysis. During these processes, neither apoptotic changes such as nuclear condensation, DNA fragmentation, and expression of phospholipid phosphatidylserine on the plasma membrane nor necrotic changes such as chromatin clumping and organelle destruction were apparent in the treated neutrophils. The fatal effect of the agonist might be specific for neutrophils because it failed to promote the rapid death of other types of cells. Although activation of neutrophils by ONO-AE-248 was not evident, experiments using metabolic inhibitors demonstrated that the agonist caused neutrophil death via the activation of protein kinase C in the presence of intracellular ATP. These findings indicated that EP3 receptor-mediated signals might promote a novel form of neutrophil death, which differs from typical apoptosis or necrosis.

Key Words: cell death • inflammation • protein kinase C • cAMP


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INTRODUCTION
 
Prostaglandin E2 (PGE2) has a broad range of physiological and pharmacological actions [1 ]. Specific receptors for PGE2 on the cytoplasmic membrane have been identified to belong to the seven-transmembrane-domain superfamily of receptors that associate with heteromeric G proteins [2 , 3 ]. PGE2 receptors are pharmacologically classified into four subtypes: EP1, EP2, EP3, and EP4. The EP1 subtype regulates calcium influx, the EP2 and EP4 subtypes are involved in stimulation of adenylyl cyclase, and the EP3 subtype modulates the inhibition of adenylyl cyclase [4 5 6 7 ]. The human EP3 subtype is composed of several different isoforms that have different sequences of the carboxy-terminal cytoplasmic tail derived from alternative RNA splicing [8 , 9 ]. A recent study using a selective agonist, such as M&B 28767 and sulprostone, demonstrated that EP3 receptor-mediated signaling promoted the depletion of intracellular cAMP and an increase of intracellular calcium [8 , 10 ]. It has also been reported that the EP3 receptor subtype mediates diverse actions, including water reabsorption in the kidneys, gastric acid secretion, neurotransmitter release, the generation of fever, and the reduction of myocardial infarct size [11 12 13 14 15 ].

Neutrophils are terminally differentiated cells that play an important role in host defenses against microbial infection. In addition, there is considerable evidence that activated neutrophils contribute to tissue damage through the production of various inflammatory mediators [16 , 17 ]. Neutrophils have the shortest lifespan among all types of circulating leukocytes. Senescent neutrophils die rapidly, exhibiting nuclear condensation, cellular shrinkage, cytoplasmic vacuolation, and DNA fragmentation, changes that are indicative of programmed cell death or apoptosis [18 ]. Neutrophils can also be killed by a wide range of stimuli such as toxins and viruses in a process known as necrosis, which is characterized by increased membrane permeability with subsequent cellular swelling and lysis [19 ]. Although a number of agents have been shown to regulate apoptosis or necrosis in neutrophils, the mechanism deciding the final outcome still remains to be clarified.

Rossi et al. recently reported that PGE2 inhibited neutrophil apoptosis by increasing the intracellular level of cAMP [20 ]. Because the EP3 receptor dominantly mediates the reduction of cAMP, we hypothesized that EP3 receptor signals might accelerate neutrophil apoptosis. Therefore, we investigated the effects of a selective EP3 receptor agonist, ONO-AE-248 [15 , 21 ], on in vitro neutrophil survival in the present study.


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MATERIALS AND METHODS
 
Reagents
Propidium iodide (PI), staurosporine, H-7, and oligomycin were purchased from Sigma (St. Louis, MO). The selective EP3 agonist, ONO-AE-248, was kindly provided by Ono Pharmaceutical (Osaka, Japan) [21 ]. A CellTiter 96® AQueous One Solution Cell Proliferation Assay kit containing MTS tetrazolium compound [22 ] was purchased from Promega (Madison, WI). Fluorescein isothiocyanate (FITC)-labeled annexin V and a DIG-High Prime DNA Labeling and Detection Kit were obtained from Roche Diagnostics (Mannheim, Germany).

Isolation of cells
Heparinized peripheral blood was obtained from healthy volunteers. Neutrophils were isolated using 3% dextran sedimentation followed by density gradient centrifugation with Ficoll-Paque (Pharmacia, Uppsala, Sweden). Erythrocytes were eliminated by hypotonic lysis with Gey’s solution. After washing with phosphate-buffered saline (PBS), neutrophils were suspended in RPMI 1640 medium (GIBCO, Detroit, MI) supplemented with 10% heat-inactivated fetal calf serum (FCS; Bioserum, Victoria, Australia), 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 2 mM L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. The neutrophils were shown to be 96% pure by microscopy.

Cell culture
Neutrophils (2 x 106 cells/mL: 1 mL per well) were cultured in 24-well flat-bottomed tissue culture dishes (NUNC, Roskilde, Denmark). Cells were incubated in the presence and absence of ONO-AE-248 for the indicated periods at 37°C in a humidified 5% CO2, 95% air incubator. To deplete cellular ATP stores, the neutrophils were incubated with oligomycin in glucose-free RPMI 1640 medium supplemented with 10% dialyzed FCS [23 ].

Expression of the EP3 receptor gene by neutrophils
Total cellular RNA was extracted from fresh cells by the acid guanidium-phenol-chloroform method, and EP3 gene expression was assessed by the reverse transcriptase-polymerase chain reaction (RT-PCR) followed by Southern blot hybridization. In brief, 1 µg of RNA was reverse-transcribed into single-stranded cDNA by incubation for 1 h at 42°C with 20 µL of a reverse transcription reaction mixture containing random hexadeoxynucleotide primer and Rous associated virus 2 reverse transcriptase (Takara, Kyoto, Japan). The PCR was done in a thermal cycler with a 25-µL reaction volume containing 5 µL of cDNA solution, 2.5 units of Taq DNA polymerase, and 0.5 µM each of the sense and antisense primers. Amplification was performed for 30 cycles (95°C for 1 min, 63°C for 30 s, and 72°C for 30 s) using the primers 5’-ACC CGCCTCAACCACTCCTACACA-3’ and 5’-ATGGCGCTGGCGATGAACAAC-3’. The expected PCR product obtained with these EP3 primers was 410 base pairs (bp) in size. The PCR products were electrophoresed on 2% agarose gel, transferred to a nylon membrane, and hybridized with a digoxigenin (DIG) end-labeled oligonucleotide probe (5’-ATGTGCTCCCAACGCTGCTT-3’). Three-primed end-labeling of the probe and its detection after hybridization were performed using a DIG-High Prime DNA Labeling and Detection Kit (Roche Diagnostics, Mannheim, Germany) [24 ]. The membrane was subsequently exposed to Fuji RX-U film.

Determination of neutrophil viability
The viability of neutrophils was determined by the MTS assay [22 ]. In brief, neutrophils (2 x 105 cells) were incubated in the presence or absence of ONO-AE-248 in 96-well plates in a final volume of 100 µL. At the indicated times, 20 µL of MTS reagent was added to each well. After incubation at 37°C for a further 2 h, the absorbance at 490 nm was determined. The quantity of formazan product as measured by the amount of 490-nm absorbance is directly proportional to the number of living cells in culture. In the ATP depletion experiment, cell viability was evaluated by the trypan blue dye exclusion assay.

Electron microscopy of neutrophils
To evaluate the morphological changes of neutrophils by electron microscopy, cells were fixed with 1% glutaraldehyde and then were fixed with 1% osmium tetroxide. Cells were subsequently stained en bloc with 2% uranyl acetate, dehydrated with a graded series of ethanol and propylene monoxide, and embedded in resin. Serial sections of the neutrophil specimens were cut on a diamond knife, mounted on formvar film-coated single-slot grids, and then stained with aqueous solutions of uranyl acetate and lead citrate.

Detection of apoptosis and necrosis
Apoptosis of neutrophils was detected by flow cytometric analysis of the nucleus as described by Nicoletti et al. [25 ]. In brief, neutrophils were suspended in 1 mL of a hypotonic fluorochrome solution (100 µg/mL of propidium iodide in 0.1% sodium citrate and 0.1% Triton X-100), and were stored in the dark overnight at 4°C. Then the fluorescence of each cell nucleus was measured using a FACScan flow cytometer (Becton Dickinson, Mountain View, CA). In some experiments, the cells were incubated in PBS containing FITC-labeled annexin V and propidium iodide (10 µg/mL) before analysis by flow cytometry.

Detection of DNA fragmentation
Neutrophils (7 x 106 cells) were incubated with 400 µL of cold hypotonic lysis buffer (10 µM Tris-HCl, pH 7.5, 1 mM EDTA, and 0.2% Triton X-100) for 20 min, and the lysate was centrifuged at 10,000 g for 10 min. Low-molecular-weight DNA in the supernatant was treated with proteinase K, extracted with a 1:1 phenol/chloroform mixture, and precipitated with 2-propanol. After digestion with RNase, the samples were electrophoresed on 1% agarose gel and stained with ethidium bromide.

Statistical analysis
Results are expressed as the mean and standard deviation (SD). Statistical analysis was performed using the paired Student’s t test. P values of less than 0.01 were considered significant.


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RESULTS
 
EP3 receptor gene expression by neutrophils
Expression of EP3 receptor mRNA was evaluated by RT-PCR using specific primers followed by Southern blot hybridization. As shown in Figure 1 , a PCR product of the predicted size for the EP3 receptor was detected in neutrophils as well as in peripheral blood mononuclear cells (PBMC), monocytic leukemia cell line (THP-1), and B cell lymphoma cell line (BALL-1). However, T cell lymphoma cells (Jurkat) did not express the EP3 receptor. The expression of the EP3 receptor in neutrophils was confirmed by neutrophils derived from four different healthy donors (two females and two males). This finding provided evidence of EP3 receptor expression by neutrophils.



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  		Figure 1. Expression of the EP3 receptor gene. Expression of the EP3 receptor by neutrophils, PBMC, and the three cell lines was analyzed using RT-PCR followed by Southern blot hybridization. Gene expression of the EP3 receptor was detected in neutrophils (lane 1), PBMC (lane 2), THP-1 cells (lane 4), and BALL-1 cells (lane 5), but not in Jurkat cells (lane 3).

Reduction of neutrophil viability by ONO-AE-248
The effect of a selective EP3 receptor agonist, ONO-AE-248, on the viability of cultured neutrophils was evaluated by the MTS assay. As shown in Figure 2A , this agonist decreased the viability of neutrophils in a concentration-dependent manner, and it was significant over the concentration of 5 µM (P < 0.01). Figure 2B shows time-dependent effect of ONO-AE-248 on neutrophil viability. This agonist decreased neutrophil viability in a time-dependent manner, and it was significant after 24 h of incubation (P < 0.01).



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  		Figure 2. Effect of ONO-AE-248 on neutrophil viability. (A) Neutrophils were incubated with various concentrations of ONO-AE-248 for 24 h. (B) Neutrophils were incubated with ONO-AE-248 at 5 x 10-5 M for the indicated periods. Cell viability was determined by the MTS assay as described in Materials and Methods. Results demonstrate absorbance at 490 nm and represent the mean ± SD of triplicate determinations. The results are representative of five experiments using neutrophils isolated from five different donors. *P < 0.01 compared with control.

Morphological changes of neutrophils
We analyzed the morphological changes of neutrophils by electron microscopy after treatment with ONO-AE-248 for 12 h. As shown in Figure 3A , most of the control cells retained a normal morphology. In contrast, ONO-AE-248 promoted dramatic changes of neutrophils (Fig. 3B) . Morphological changes primarily occurred in the nucleus. Nuclear lobules fused with each other and the nucleus became a large round structure similar to that seen in mononuclear cells. The compactness of the chromatin was also decreased, whereas separation of the nuclear membranes and blebbing of the nuclear envelope occurred and the nuclei finally underwent rupture (Fig. 3C and 3D) . However, the intracellular organelles of these cells maintained a normal appearance. Although most of the neutrophils treated with ONO-AE-248 predominantly exhibited these changes, a few cells showed either apoptotic changes (nuclear condensation and cellular shrinkage) or necrotic changes (cell membrane lysis; Fig. 3B ).



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  		Figure 3. Electron microscopy of neutrophils. Neutrophils were incubated with or without ONO-AE-248 at 5 x 10-5 M for 12 h. Cells were fixed with 1% glutaraldehyde and ultrathin sections were analyzed by electron microscopy. (A) Most neutrophils incubated with medium alone maintained a normal appearance; original magnification x3,200. Bar indicates 2 µm. (B) Most neutrophils incubated with the agonist exhibited a mononuclear cell-like nucleus and major morphological changes of the nuclear membrane. There were also some apoptotic cells (arrow) showing nuclear condensation, and necrotic cells (arrowhead) showing destruction of cell membrane and nuclear rupture; original magnification x3,000. Bar indicates 2 µm. (C) Neutrophils treated with the agonist exhibited blebbing of the nuclear envelope and rupture of the nucleus; original magnification x6,000. Bars indicate 1 µm. (D) Higher-magnification view of the cell in panel C; original magnification x15,000. Bars indicate 1 µm.

Effect of ONO-AE-248 on neutrophil apoptosis
Neutrophils were incubated in the presence or absence of ONO-AE-248 at a final concentration of 5 x 10-5 M for 24 h. Subsequently, cells were treated with a hypotonic fluorochrome solution containing PI, and apoptotic nuclei were detected by flow cytometry. Neutrophils cultured with medium alone exhibited a hypodiploid DNA peak, which is a characteristic feature of apoptosis (Fig. 4A ). In contrast, ONO-AE-248-treated neutrophils did not exhibit the hypodiploid DNA peak (Fig. 4B) . This finding suggested the absence of DNA fragmentation during the process of neutrophil death caused by ONO-AE-248.



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  		Figure 4. Effect of ONO-AE-248 on neutrophil apoptosis. Neutrophils (2 x 106 cells) were incubated in triplicate with (A) medium alone or (B) ONO-AE-248 at 5 x 10-5 M for 24 h. Cells were subsequently harvested, and apoptosis was investigated by flow cytometric analysis. A DNA histogram and the percentage of cells showing apoptosis are illustrated. Similar results were obtained in four separate experiments performed with neutrophils isolated from four different donors.

Agarose gel electrophoresis of low-molecular-weight DNA
To confirm that neutrophil death was not associated with DNA fragmentation, we performed agarose gel electrophoresis of low-molecular-weight DNA. Neutrophils (7 x 106 cells) were incubated with or without ONO-AE-248 (5 x 10-5 M) for 24 h. Cells were harvested, and low-molecular-weight DNA was extracted. As seen in Figure 5 , incubation with medium alone led to an increase of low-molecular-weight DNA, which showed a dense ladder pattern on electrophoresis. However, treatment with ONO-AE-248 led to a decrease of low-molecular-weight DNA and no DNA fragmentation.



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  		Figure 5. Agarose gel electrophoresis of low-molecular-weight DNA. Neutrophils were incubated with medium alone (lane 2) or ONO-AE-248 (lane 3) at a final concentration of 5 x 10-5 M for 24 h. DNA marker, {lambda}HindIII and Eco RI digest, is shown in lane 1. Low-molecular-weight DNA was isolated from the neutrophils and was detected by agarose gel electrophoresis. The results are representative of three separate experiments using neutrophils isolated from three different donors.

Double staining of neutrophils with annexin-V and PI
Expression of phospholipid phosphatidylserine (PS) on the cell surface is thought to be one of the early features of apoptosis [26 ]. Annexin-V has a high affinity for PS and is used in conjunction with PI to distinguish apoptotic cells from necrotic cells or cells that have already died of apoptosis. This is based on the fact that PI selectively penetrates the cytoplasmic membrane of necrotic cells, but not apoptotic cells [27 ]. Neutrophils cultured with 5 x 10-5 M of ONO-AE-248 for 12 or 24 h were resuspended in PBS, and were stained using FITC-labeled annexin-V and PI. As demonstrated in Figure 6 , treatment of neutrophils with the agonist did not enhance apoptotic changes revealed by annexin-V staining (Fig. 6 , top left panel) when compared with the untreated control cells. However, the changes of necrosis revealed by PI staining (Fig. 6 , top right panel and bottom right panel) were increased in the agonist-treated cells, especially at 24 h. This was representative of three separate experiments. These results indicate that ONO-AE-248 could not cause the apoptotic changes in cell-surface expression of phosphatidylserine, but it increased necrotic changes after incubation for 24 h.



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  		Figure 6. Double staining of ONO-AE-248-treated neutrophils with annexin-V and propidium iodide (PI). Neutrophils were incubated with (B and D) or without (A and C) ONO-AE-248 (5 x 10-5 M) for 12 (A and B) or 24 h (C and D). Then the cells were stained with FITC-labeled annexin-V for apoptotic changes and PI for necrotic changes, and the fluorescence intensity was determined by flow cytometry. Both a fluorescence dot plot and the percentage of positive cells are shown. The results are representative of three separate experiments using neutrophils isolated from three different donors.

Effect of metabolic inhibitors on neutrophil death caused by ONO-AE-248
Phorbol myristate acetate (PMA), a potent stimulator of protein kinase C (PKC), has been demonstrated to rapidly kill neutrophils [28 ], with the cells showing similar features to neutrophils killed by ONO-AE-248. Therefore, we examined the effects of PKC inhibitors on neutrophils incubated with the agonist. As shown in Figure 7A , neutrophil death was significantly reduced by two PKC inhibitors (staurosporine and H-7), indicating the possible involvement of PKC activation in the process of neutrophil death caused by ONO-AE-248.



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  		Figure 7. Inhibition of ONO-AE-248-induced neutrophil death by PKC inhibitors and ATP depletion. (A) Neutrophils were pretreated for 30 min with or without PKC inhibitor, staurosporine (STS), or H-7, both at a concentration of 1 x 10-6 M and were subsequently incubated with ONO-AE-248 (5 x 10-5 M) for 24 h. Cell viability was determined by the MTS assay. Data are shown as the mean ± SD of triplicate determinations. Either STS or H-7 significantly abolished the effect of ONO-AE-248. The results are representative of three separate experiments using neutrophils isolated from three different donors. *P < 0.01 compared with culture in the presence of STS or H-7. (B) Neutrophils were incubated with oligomycin (10 µg/mL) in glucose-free medium for 24 h in the presence or absence of ONO-AE-248 (5 x 10-5 M). Cell viability was determined by the trypan blue dye exclusion assay. Data show the mean ± SD (of triplicate determinations) of the percentage of living cells. Oligomycin significantly abrogated the effect of ONO-AE-248. The results are representative of three separate experiments. *P < 0.01 compared with culture in the presence of oligomycin.

It is well known that ATP is an essential energy source for cells. We used oligomycin to block mitochondrial F1F0 ATPase in neutrophils during incubation in glucose-free medium [23 ]. The viability of treated neutrophils was determined by the trypan blue dye exclusion assay. As shown in Figure 7B , ATP depletion completely blocked the effect of ONO-AE-248 on neutrophils. A number of cytotoxic agents have been shown to induce necrosis even in the absence of intracellular ATP [29 , 30 ]. Therefore, this result indicated that ONO-AE-248 was not cytotoxic for neutrophils.

Effect of ONO-AE-248 on various types of cells
We investigated the effect of ONO-AE-248 on other types of cells such as PBMC, a monocytic leukemia cell line (THP-1), and a T cell lymphoma cell line (Jurkat). The agonist failed to cause rapid killing of PBMC or the two tumor cell lines, even at a concentration of 5 x 10-5 M (Fig. 8 ), after 48 h of incubation.



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  		Figure 8. Effect of ONO-AE-248 on the viability of various types of cells. Neutrophils (filled circles), PBMC (filled squares), THP-1 cells (filled triangles), and Jurkat cells (filled diamonds) were incubated with ONO-AE-248 at a concentration of 5 x 10-5 M for the indicated time, and cell viability was determined by the MTS assay. Data are percentages relative to the negative control and represent the mean ± SD of triplicate determinations. The results are representative of three separate experiments. *P < 0.01 compared with the negative control.


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DISCUSSION
 
This study demonstrated that a selective EP3 receptor agonist, ONO-AE-248, caused neutrophil death without the typical features of apoptosis or necrosis. This conclusion was based on the nuclear DNA histograms obtained with PI staining, agarose gel electrophoresis of low-molecular-weight DNA, double staining of cells with annexin V and PI, and electron microscopy.

In multicellular organisms, two basic forms of cell death, necrosis and apoptosis, have been well documented [18 , 19 ]. Necrosis is death caused by sudden changes of the environment, leading to rupture of the plasma membrane, destruction of organelles, and extravasation of the cellular contents [19 ]. Apoptosis (or programmed cell death) refers to a more physiological form of death that features chromatin condensation and DNA fragmentation [18 , 31 ]. In the case of ONO-AE-248-induced death, neutrophils exhibited nuclear changes as early as 12 h after the start of incubation without showing typical apoptotic features, such as PS expression on the cytoplasmic membrane, chromatin condensation, and DNA fragmentation. An increase of membrane permeability and lysis of the cytoplasmic membrane occurred subsequently, becoming apparent at 24 h. However, characteristic features of necrosis such as chromatin clumping and organelle destruction could not be detected. Therefore, this EP3 receptor agonist apparently caused neutrophil death with primary changes in the nucleus followed by membrane destruction.

Incubation with PKC inhibitors (staurosporine or H-7) prevented this EP3 receptor agonist-induced neutrophil death. Recent studies have noted the possible involvement of PKC activation during EP3 receptor-mediated signaling [15 , 32 ]. The present study demonstrated that ONO-AE-248 promoted neutrophil death through the activation of PKC. In addition, ONO-AE-248 was unable to kill neutrophils in the absence of ATP, indicating that the process of neutrophil death mediated by this agonist was dependent on ATP. It has been reported that various cytotoxic agents promoting necrosis can exert an effect in the absence of intracellular ATP [29 , 30 ]. Thus, the need for ATP provides evidence that neutrophil death caused by ONO-AE-248 is distinct from necrosis.

Morphological changes of the agonist-treated neutrophils primarily occurred in the nucleus, beginning with the fusion of nuclear lobules to form a round structure, followed by blebbing of the nuclear envelope and eventual nuclear rupture. During these nuclear changes, the compactness of chromatin also showed a decrease. It has been demonstrated that the cellular changes of apoptosis first occur in the chromosomes and the nuclear membrane [33 , 34 ]. Therefore, neutrophil death caused by this EP3 receptor agonist has some similarity to the early stage of apoptosis. A hallmark of apoptosis is the cleavage of chromatin into nucleosomal fragments [18 ]. However, DNA fragmentation as a results of endonuclease activation was not observed in ONO-AE-248-treated cells. Moreover, the agonist inhibited DNA fragmentation occurring spontaneously in cultured neutrophils. Several studies have indicated that some dying cells, which lack the hallmarks of necrosis, may fail to generate chromosomal DNA ladders during the death process [35 , 36 ]. These reports have suggested the presence of a novel form of cell death, which is neither apoptosis nor necrosis. Based on these previous reports and the present findings, we presume that ONO-AE-248 induces neutrophil death by a different pathway from that of typical apoptosis or necrosis.

Takei et al. recently reported that rapid killing of human neutrophils by a potent PKC activator was accompanied by different cellular changes from those seen in typical apoptosis or necrosis [28 ]. They found that neutrophils died rapidly after exposure to PMA, with the main morphological changes beginning in the nucleus and no evidence of DNA fragmentation. These nuclear changes were followed by an increase of membrane permeability. The neutrophil death caused by PMA seems to be quite similar to that caused by ONO-AE-248. Because PMA is well known to activate neutrophils and because neutrophil activation by phagocytosis of opsonized zymosan also promotes a similar type of death [28 ], this may be a form of activation-induced death. Although there are some morphological and signaling similarities between PMA- and ONO-AE-248-induced neutrophil death, we failed to detect neutrophil activation by this agonist in the present study. ONO-AE-248 did not modulate the expression of adhesion molecules (CD62L and CD11b/ CD18) on neutrophils. Moreover, the agonist suppressed fMLP-induced O2- production (data not shown). Talpain and colleagues have reported that another EP1/EP3 receptor agonist, sulprostone, inhibited O2- production by neutrophils [37 ]. Thus, it is suggested that ONO-AE-248 might induce neutrophil death by a similar but distinct mechanism from that for PMA-induced neutrophil death.

PGE2 receptors are divided into four subtypes, and the EP3 receptor is the best-characterized among them [3 ]. It is composed of six isoforms with different carboxy-terminal tails that are generated by alternative mRNA splicing. The carboxy-terminal tail influences both the G protein specificity and the signaling pathway. It has been reported that EP3 isoforms are involved in inhibition or stimulation of adenylyl cyclase, and stimulation of phosphoinositide turnover [8 , 38 ]. The present study provided direct evidence of EP3 receptor expression on neutrophils by RT-PCR, suggesting that EP3 receptor-mediated signaling might play a role in the regulation of neutrophil function. ONO-AE-248 binds to EP3{alpha} receptor and has been shown to decrease intracellular cAMP levels [15 , 21 ]. Recent studies have revealed that PGE2 and agents increasing cAMP can inhibit the spontaneous apoptosis of neutrophils. This PGE2-induced delay of neutrophil apoptosis is presumably mediated by signals derived from the EP2 receptor or the EP4 receptor that increase intracellular cAMP [4 , 6 ]. In contrast, the EP3 receptor agonist decreased cAMP and activated the PKC pathway, but did not accelerate typical apoptosis and instead induced a unique form of neutrophil death. This indicates the diverse effects of PGE2 receptor-mediated signals on neutrophil.

This study demonstrated the expression of the EP3 receptor in neutrophils, PBMC, and THP-1, but not in Jurkat cells. However, ONO-AE-248 failed to promote killing of PBMC and THP-1 cells after the treatment for 48 h. This indicates that neutrophils may be a most susceptible cell type to the killing effect of ONO-AE-248. This is consistent with the observation that the apoptotic program in neutrophils is exquisitely sensitive to the influence of environmental factors [20 , 28 ]. Biological effects of ONO-AE-248 on PBMC and THP-1 cells were not elucidated well in this study. Our next study will be aimed at investigating it.

In conclusion, we showed that a selective EP3 receptor agonist, ONO-AE-248, could promote a unique form of neutrophil death that was different from either typical apoptosis or necrosis. Impairment of neutrophil survival by EP3 receptor-mediated signaling may be a regulatory mechanism that operates at sites of inflammation and tissue injury. In order to determine the pathophysiological role of the EP3 receptor, the influence of EP3 receptor agonists on in vivo models of inflammation should be investigated. The results may provide a basis for the therapeutic use of agonists or antagonists to PGE2 receptor in various human diseases.


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ACKNOWLEDGEMENTS
 
This work was supported by grants from the Ministry of Education and Culture of Japan (10670423) and from Kitasato University Graduate School of Medical Science (MSRP-01A, 1999). Jiajia Liu and Tohru Akahoshi contributed equally to this work.

Received October 21, 1999; revised February 22, 2000; accepted March 24, 2000.


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C.-Y. Liu, A. Takemasa, W. C. Liles, R. B. Goodman, M. Jonas, H. Rosen, E. Chi, R. K. Winn, J. M. Harlan, and P. I. Chuang
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