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(Journal of Leukocyte Biology. 2001;70:52-58.)
© 2001 by Society for Leukocyte Biology

Retardation of early-onset PMA-induced apoptosis in mouse neutrophils deficient in myeloperoxidase

Tetsuto Tsurubuchi*, Yasuaki Aratani*, Nobuyo Maeda{dagger} and Hideki Koyama*

* Kihara Institute for Biological Research, Yokohama City University, Totsuka, Yokohama 244-0813, Japan, and
{dagger} Department of Pathology and Laboratory Medicine, University of North Carolina, Chapel Hill

Correspondence: Yasuaki Aratani, Kihara Institute for Biological Research, Yokohama City University, Maioka-cho 641-12, Totsuka, Yokohama 244-0813, Japan. E-mail: yaratani{at}yokohama-cu.ac.jp


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ABSTRACT
 
Neutrophil apoptosis is a mechanism involved in the resolution of inflammation. To explore the role of hypochlorous acid (HOCl) produced by neutrophils while they are undergoing apoptosis, we compared the rates of apoptosis in neutrophils isolated from normal mice and from myeloperoxidase (MPO)-deficient mice, which are unable to generate HOCl. Apoptosis in MPO-deficient neutrophils stimulated by phorbol myristate acetate (PMA) was significantly slower than in normal neutrophils during 3 h of incubation. Exposure of normal neutrophils to H2O2 together with PMA resulted in a dramatic acceleration of apoptosis, and almost all of the cells revealed apoptotic morphology at 1 h. This acceleration was inhibited by cytochrome c, a superoxide scavenger. Conversely, in MPO-deficient neutrophils activated with PMA and H2O2, little acceleration was observed before 1 h, although it gradually increased thereafter. This retardation was almost completely reversed when MPO or HOCl was exogenously added. These results suggest that coexistence of HOCl and superoxide accelerates the early onset of neutrophil apoptosis.

Key Words: reactive oxygen species • hypochlorous acid • hydrogen peroxide • superoxide • inflammation


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INTRODUCTION
 
Neutrophils play a central role in host defense against infectious microorganisms [1 ]. They have also been implicated in the pathogenesis of tissue injury seen in inflammatory diseases of the lung, kidney, joints, and other organs [2 ]. Consequently, removal of activated and potentially dangerous neutrophils from an inflamed area is an important injury-limiting mechanism. Haslett et al. [3 ] first reported that effete neutrophils are removed from sites of inflammation by apoptosis. Aging neutrophils exhibit apoptotic characteristics such as nuclear condensation, DNA fragmentation, and exposure of new cell surface structures that can be recognized by macrophages [4 ]. Any significant delay of neutrophil apoptosis leads to excessive neutrophil accumulation and damage of healthy tissues [5 ].

Several lines of evidence indicate that reactive oxygen species (ROS) are involved in apoptosis of neutrophils. Neutrophil apoptosis is inhibited under hypoxic conditions, which dramatically decreases the generation of ROS [6 ]. The major source of ROS in neutrophils is NADPH oxidase, a multicomponent enzyme that catalyzes the transfer of electrons from NADPH to molecular oxygen to produce superoxide (O2-). Neutrophils isolated from patients with chronic granulomatous disease [7 ], which is characterized by a genetic deficiency in any one of the components of the NADPH oxidase [8 ], show a decreased rate of spontaneous cell death [9 ]. Neutrophil apoptosis is promoted by exogenous hydrogen peroxide (H2O2) but is delayed by antioxidants, including catalase [6 ]. These studies demonstrate an important role of O2- and H2O2 as major mediators of neutrophil apoptosis. However, whether there are several other ROS that might mediate neutrophil apoptosis remains to be determined.

Myeloperoxidase (MPO) (EC 1.11.1.7) is an enzyme found mainly in neutrophils and to a lesser degree in monocytes [10 ]. In chemoattractant-activated neutrophils, MPO transforms H2O2 generated during the oxidative burst into highly cytotoxic hypochlorous acid (HOCl) in the presence of chloride ions (Cl-) [11 ]. This MPO-H2O2-Cl- system appears to function in the killing of microbes by neutrophils [12 13 14 15 16 17 18 19 20 ]. It may also be involved in their cytotoxicity against tumor cells [21 , 22 ] and in tissue damage at sites of inflammation where neutrophils can release both MPO and H2O2 [23 24 25 26 27 28 ].

In this study, we attempted to define the involvement of HOCl produced by MPO from neutrophils in apoptosis by comparing neutrophils isolated from normal mice with those from MPO-deficient mice, which lack the ability to produce HOCl.


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MATERIALS AND METHODS
 
Mice
All mice used were 12–14-week-old female C57BL/6 mice purchased from the Japan SLC (Hamamatsu). MPO-null mutant mice previously bred by us [12 ] were backcrossed eight times with the C57BL/6 mice to ensure similar genetic backgrounds. All animals were housed under specific-pathogen-free conditions.

Isolation of neutrophils
Mice were injected intraperitoneally with 2 mL of 1.5% fluid thioglycollate medium (Difco Laboratories, Detroit, MI). After 4 h, peritoneal exudate cells containing ~80% neutrophils and ~20% macrophages were harvested by peritoneal lavage with 15 mL of phosphate-buffered saline. Total cell numbers were determined with a hemacytometer. Neutrophils were purified by adherence to a plastic dish for suspension culture. Then 1.5 x 106 peritoneal-exudate cells were plated into a 35-mm plastic dish and incubated at 37°C in 5% CO2 in air for 10 min in Hanks’ balanced salt solution (HBSS). The solution was removed, and the adhered cells were washed twice with HBSS. Although not all of the neutrophils adhered to the dish, most macrophages were eliminated by this procedure: Judging from cytochemical staining with the 3,3',5,5'-tetramethylbenzidine liquid substrate system (Sigma, St. Louis, MO), >95% of the adherent cells in wild-type mice were peroxidase positive and >75% of adherent cells exhibited the ring-shaped chromosomes that are characteristic of mouse neutrophils, regardless of their genotype.

Activation of neutrophils
Phorbol 12-myristate 13-acetate (PMA) (Wako Chemicals, Osaka, Japan) and/or H2O2 was added to the adherent neutrophil samples in HBSS at the final concentrations of 30 ng/mL and 0.1 mM, respectively, and the cells were incubated at 37°C in 5% CO2 in air. In some experiments, purified human MPO (Elastin Products, Owensville, MO) or cytochrome c (cyt C) (Wako Chemicals) was simultaneously added to the cells at final concentrations of 2.5 µg/mL or 20 mM, respectively.

Morphological assessment of apoptosis and cell viability
Cell samples in plastic dishes were stimulated as described above. At different time points, the HBSS was removed, and the cells were stained with Giemsa solution. Thereafter, at least 200 cells per sample were evaluated under a microscope, and the cells with condensed nuclei were defined as apoptotic. Apoptosis was also measured with a fluorescein isothiocyanate-conjugated annexin V apoptosis detection kit (Takara Co., Kyoto, Japan) according to the manufacturer’s instructions. In parallel, the trypan blue dye exclusion procedure was used to distinguish normal or apoptotic cells from necrotic cells.


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RESULTS
 
Light-microscopic observation of morphological changes in PMA- and H2O2-treated neutrophils
Approximately 75% of the mouse neutrophils that adhered to the dish possessed native ring-shaped nuclei, whereas the remaining cells exhibited condensed nuclei, a feature usually detected in typical apoptotic cells [29 ], probably because they had been activated within the peritoneal cavity by thioglycollate. No further increase in the number of cells with such condensed nuclei was detected after 1 h of in vitro incubation in HBSS alone (Fig. 1A and B ). Similar to a previous study of human neutrophils [30 ], activation of wild-type neutrophils with PMA (30 ng/mL) and H2O2 (0.1 mM) resulted in a rapid onset of nuclear condensation, and almost all of the cells had condensed nuclei at 1 h (Fig. 1C and 1E) . We were surprised, however, to find that these activators could not promote any morphological changes in the MPO-deficient cells within 1 h (Fig. 1D and 1F) .



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  		Figure 1. Light-microscopic observations of PMA- and H2O2-treated neutrophils. Wild-type (A, C, E) and MPO-deficient (B, D, F) neutrophils (1.5 x 106 each) were plated into 35-mm plastic dishes and incubated for 10 min as described in Materials and Methods. Adherent cells were exposed to PMA and H2O2 together (C–F) or HBSS alone (A, B) for 1 h, stained with Giemsa solution, and photographed at magnifications of 400x (A to D) or 1,000x (E, F).

Retardation of nuclear condensation in MPO-deficient neutrophils
Figure 2 shows time-dependent changes in the number of cells with condensed nuclei. In both the wild-type and the MPO-deficient mutant neutrophils, the nuclear condensation did not occur at all up to 3 h in HBSS (Fig. 2A) and scarcely proceeded in HBSS containing H2O2 alone (Fig. 2C) . Although PMA increased the number of cells with condensed nuclei in neutrophils of both genotypes, the increase in the number of mutant cells was markedly retarded compared with the increase in number of wild-type cells (Fig. 2B) . PMA plus H2O2 exhibited a dramatic condensation-inducing effect on the wild-type cells; >90% of them displayed condensed nuclei 1 h after activation (Fig. 2D) . This contrasts with the observation that either PMA (Fig. 2B) or H2O2 alone (Fig. 2C) could scarcely stimulate nuclear condensation at 1 h. Conversely, in the mutant cells, PMA plus H2O2 showed no significant effect on the early period of nuclear condensation, although these activators promoted apoptosis thereafter (Fig. 2D) .



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  		Figure 2. Time course of nuclear condensation activated by PMA and/or H2O2. At time zero, wild-type (circles) and MPO-deficient (squares) neutrophils were exposed to HBSS alone (A), PMA (B), H2O2 (C), or PMA plus H2O2 (D) and incubated for different periods of time. The number of cells displaying nuclear condensation (see Fig. 1C and 1D ) was determined under a microscope. A minimum of 200 cells were counted at each time point, and the relative number of cells with condensed nuclei to total cell number is expressed as the mean ± SD of data from five different experiments.

Cell surface exposure of PS in activated neutrophils
Loss of plasma membrane asymmetry resulting in cell surface exposure of phosphatidylserine (PS) is a biochemical hallmark of early apoptosis [29 ]. Microscopic observation using fluorescein isothiocyanate-labeled annexin V, which specifically binds to PS on the cell surface, showed that fewer MPO-deficient mutant neutrophils incubated with PMA and H2O2 had PS exposure at 1 h than the wild-type cells had at that time point (Fig. 3B and F ), although a similar number of cells was stained after 3 h (Fig. 3D and 3H) . This finding is consistent with the delayed nuclear condensation in the mutant cells, as shown in Fig. 1D . Trypan blue exclusion analysis revealed that >90% of both the wild-type and the mutant neutrophils were still viable 1 h after the treatment (data not shown). These results demonstrate that neutrophils activated by PMA plus H2O2 for 1 h proceeded to apoptosis, and that MPO deficiency retards the early onset of apoptosis. Therefore, in our experiments, we regarded nuclear condensation as an indicator of apoptosis. However, the number of trypan blue-positive cells gradually increased after 1 h, and about half of the cells became trypan blue permeable at 3 h (data not shown), indicating that at the later time points, many of the apoptotic cells proceeded into secondary necrosis.



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  		Figure 3. Time-dependent cell surface exposure of PS in PMA- and H2O2-treated neutrophils. The same number of wild-type (A–D) and MPO-deficient (E–H) neutrophils as in Fig. 1 was exposed to PMA and H2O2 for 0 h (A, E), 1 h (B, F), 2 h (C, G), and 3 h (D, H), and PS exposure was determined by microscopic observation of annexin V binding as described in Materials and Methods. Magnification, 400x.

Effect of exogenously added MPO and HOCl on the apoptosis of neutrophils
To confirm the involvement of MPO in neutrophil apoptosis, we examined the effect of exogenously added MPO enzyme on apoptosis. As shown in Figure 4 , the addition of human MPO to MPO-deficient mutant neutrophils activated with PMA and H2O2 could increase the number of apoptotic cells by 1 h to the same approximate number as the wild-type cells. However, heat-inactivated MPO showed no such activity, confirming that the catalytic activity of MPO is necessary for the rapid onset of apoptosis.



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  		Figure 4. Exogenously added MPO accelerates apoptosis of MPO-deficient neutrophils. Wild-type and MPO-deficient mutant neutrophils were incubated with PMA and H2O2 for 1 h in the absence (white bars) and presence of native (black bars) and heat-denatured (striped bars) human MPO enzyme, and the number of cells displaying apoptotic morphology was counted as described in Materials and Methods. Data from three different experiments are expressed as means ± SD.

MPO catalyzes the reaction producing HOCl from H2O2 and Cl-. Therefore, we next examined the effect of exogenously added HOCl on neutrophils activated by PMA alone. We found that HOCl dose-dependently increased the number of apoptotic cells in both normal and MPO-deficient neutrophils, indicating the ability of HOCl to replace MPO plus H2O2 and induce the rapid apoptosis (Fig. 5 ). These results strongly suggest that HOCl generated by MPO from neutrophils contributes to the apoptotic cell death of PMA-activated neutrophils.



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  		Figure 5. Exogenously added HOCl accelerates apoptosis of neutrophils. Wild-type (circles) and MPO-deficient (squares) neutrophils were incubated with various concentrations of HOCl for 1 h in the presence of PMA, and the number of cells with apoptotic morphology was counted as described in Materials and Methods. Data from three different experiments are expressed as means ± SD.

Effect of the antioxidant cyt C on the apoptosis of neutrophils
PMA has been widely used as a potent activator of the respiratory-burst enzyme NADPH oxidase [31 , 32 ]. During the respiratory burst, neutrophils generate O2- via the NADPH oxidase system. Indeed, we previously reported that PMA is able to activate neutrophils isolated from normal and MPO-deficient mice to produce O2- at almost the same rate [12 ]. To clarify the role of O2- in PMA-induced neutrophil apoptosis, we examined the effect of cyt C an O2- scavenger, on apoptosis. As shown in Figure 6 , cyt C strongly inhibited the apoptosis of wild-type neutrophils activated with PMA plus H2O2 at 1 h. The residual number of apoptotic cells was almost as same as the number in MPO-deficient mutant cells. Conversely, little inhibitory effect on the apoptosis of mutant cells was found. These results strongly suggest that coexistence of HOCl and O2- is required to accelerate the early onset of apoptosis.



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  		Figure 6. cyt C inhibits PMA- and H2O2-induced apoptosis of normal neutrophils. Wild-type and MPO-deficient mutant neutrophils were incubated with PMA and H2O2 for 1 h in the presence (black bars) and absence (white bars) of cyt C, and the number of cells with apoptotic morphology was counted as described in Materials and Methods. Data from three different experiments are expressed as means ± SD.


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DISCUSSION
 
In this study, we observed that the PMA-activated neutrophils isolated from MPO-deficient mice retard their own apoptosis compared with neutrophils from normal mice and that this retardation is most evident 1 h after activation with H2O2 and PMA (Fig. 1 2 3) . The delay in apoptosis is reversed by addition of purified MPO enzyme (Fig. 4) or HOCl (Fig. 5) , strongly suggesting that neutrophil apoptosis is partly mediated by HOCl generated by MPO from neutrophils.

Many researchers have focused on the role of ROS in regulating the life span of inflammatory cells. Hannah et al. [6 ] reported that apoptosis of neutrophils is inhibited under hypoxic conditions, which extremely decrease the generation of ROS. Lundqvist-Gustafsson and Bengtsson [30 ] showed that PMA causes a rapid onset of apoptosis in human neutrophils. Narayanan et al. [33 ] suggested that the onset of apoptosis in PMA-activated neutrophils is partly due to oxidative stress induced by down-regulation of the antioxidants O2- dismutase and glutathione, leading to intracellular accumulation of O2-. Oishi and Machida [34 ] reported that exogenously added O2- dismutase retards spontaneous apoptosis in human neutrophils. Unlike normal neutrophils, neutrophils from patients with chronic granulomatous disease exhibit significantly retarded apoptosis in vitro [9 ]. From these results, the hypothesis that O2- generated by neutrophils is responsible for the neutrophils’ own apoptosis has been well accepted. We observed in this study that PMA induces apoptosis of both wild-type and MPO-deficient mutant neutrophils (Fig. 2B) . However, we also observed that apoptosis proceeds significantly more slowly in MPO-deficient neutrophils than in wild-type neutrophils (Fig. 2B) , in spite of the observation that both wild-type and MPO-deficient neutrophils treated with PMA are able to release O2- at almost equivalent rates [12 ]. This difference probably results from the defect in HOCl production by the mutant cells.

H2O2 has been considered another mediator of apoptosis [9 , 30 , 33 ]. This is supported by our findings that addition of H2O2, together with PMA, rapidly and dramatically induced the apoptosis of wild-type neutrophils and that almost all of the cells exhibited the apoptotic phenotype within 1 h after activation (Fig. 1 2D and 3) . Because H2O2 is poorly reactive as an oxidant [35 ], cytotoxicity associated with H2O2 is believed to be generally attributed to its role as a source of the more reactive oxygen free radicals such as hydroxyl radical [36 , 37 ]. In fact, this radical is generated from H2O2 in the presence of intracellular iron by the Fenton reaction and is cytotoxic by virtue of its ability to initiate lipid peroxidation, to damage membranes, and to inactivate enzymes. Indeed, inhibition of hydroxyl radical production has been reported to inhibit PMA-induced neutrophil autocytotoxicity [38 , 39 ]. However, in the MPO-deficient neutrophils activated with PMA and H2O2, we observed that no apoptotic death occurred before 1 h (Fig. 1 2D and 3) and that addition of purified MPO enzyme (Fig. 4) or HOCl (Fig. 5) compensated for the defect in apoptosis. Given those findings and the observation that apoptosis was not accelerated by H2O2 alone (Fig. 2C) , we concluded that HOCl largely contributes at least to the early onset of apoptosis of PMA-activated neutrophils. Because PMA induces dramatic neutrophil degranulation, exogenously added H2O2 might serve as the substrate of secreted MPO to produce HOCl. This view is supported by a more recent study of specific inhibitors of MPO that showed that H2O2-induced apoptosis in HL-60 human leukemia cells is mediated by MPO and is linked to a non-Fenton oxidative event [40 ]. It should be noted that HOCl treatment at 100 µM for 1 h was more effective for wild-type cells than for MPO-deficient cells (Fig. 5) . One possible explanation for the difference is that, in addition to the exogenous HOCl, HOCl generated from wild-type cells by the MPO-H2O2-Cl- system contributes to apoptosis.

We have previously reported that both wild-type and MPO-deficient neutrophils treated with PMA are able to release O2- at almost equivalent rates [12 ]. However, PMA alone scarcely accelerated the apoptosis during the first hour (Fig. 2B) , suggesting that O2- alone is not sufficient to induce the early onset of apoptosis. On the other hand, the PMA- and H2O2-induced apoptosis observed in the wild-type cells disappeared when cyt C was added (Fig. 6) . cyt C oxidizes O2- toO2; this results not only in reduced amounts of O2- but also in H2O2 formation from O2-. Because 0.1 mM H2O2 was exogenously added, it is more plausible that it was the reduced levels of O2- rather than H2O2 resulting from cyt C treatment that caused inhibition of the early period of apoptosis. Moreover, addition of HOCl to wild-type and MPO-deficient cells without activation by both PMA and H2O2 did not induce apoptosis (data not shown), indicating that HOCl alone has no dramatic effect on apoptosis. Therefore, it seems likely that both O2- and HOCl are indispensable in the acceleration of at least the early onset of apoptosis of PMA-activated neutrophils.

Although in the mutant cells apoptosis occurred scarcely 1 h after exposure to PMA or PMA plus H2O2, apoptosis occurred thereafter, and similar percentages of apoptotic cells were observed in both the normal and mutant cells after 3 h (Fig. 2B and 2D) . These results suggest that ROS other than HOCl or nonoxidative factors contribute to the later stage of apoptosis.

The PMA-activated apoptosis observed in this study and others [30 , 41 ] was much faster than the apoptosis observed in Fas ligand-triggered apoptosis [9 , 41 ]. Moreover, in agreement with the previous reports [38 , 41 ], we were unable to detect any DNA fragmentation in PMA-treated cells (data not shown). Therefore, the PMA-activated neutrophil cell death is difficult to classify as conventional apoptosis. That is, how the cell dies—whether by apoptosis, necrosis, or an atypical death process as described by Takei et al. [38 ]—may depend on the difference in stimulus. For example, Fadeel et al. have shown that caspase activation occurs in neutrophils undergoing Fas ligand-mediated apoptosis, but no such activation is found in neutrophils stimulated with PMA [41 ]. This difference may be due to the heterogeneity in biochemical and morphological changes, such as the absence of DNA fragmentation in apoptotic neutrophils [42 ]. However, PS exposure was markedly accelerated in treated neutrophils compared with untreated neutrophils (Fig. 3) . PS exposure is known as one of the earliest markers of apoptosis, and it precedes the morphologic appearances of apoptosis, changes in membrane permeability to agents such as trypan blue and propidium iodide, and the characteristic DNA fragmentation. Physiologically, PS exposure is one of the mechanisms by which apoptotic cells are recognized by macrophages and targeted for ingestion [4 ]. Cell death induced by PMA may therefore be considered as a type of apoptosis-like activation-induced death that occurs more rapidly than cell death by other processes. In the present experiments, neutrophils were incubated in the absence of autologous serum to avoid any influence of the undefined nature of serum components on the neutrophil apoptosis. The presence of serum in culture medium reduces the rate of neutrophil apoptosis, and in the absence of serum, neutrophils undergo secondary necrosis with large numbers of trypan blue-positive cells [43 ]. Also, we used the neutrophils adherent to plastic dishes. It generally is acknowledged that neutrophil apoptosis is modulated through adhesion to different substrates [44 ]. Such experimental conditions may also contribute to the higher rate of apoptosis than the rates observed by others [9 , 41 ] and to the occurrence of secondary necrosis.

Resolution of inflammation is poorly understood, and apoptosis is proposed as a candidate mechanism for elimination of excess neutrophils as inflammation resolves [5 ]. Because MPO-deficient neutrophils undergo delayed apoptosis in vitro, it is possible that these neutrophils remain alive longer at sites of inflammation. As a result, they would continue to release various ROS, inflammatory cytokines and cytotoxic enzymes for a longer time, eventually resulting in tissue damage. Further work with physiological stimuli other than PMA, such as Fas ligand [9 , 41 ] and tumor necrosis factor-{alpha}, [45 ] is required to confirm the role of MPO in neutrophil apoptosis and to determine whether such defective functions of neutrophils are involved in the pathology of various inflammatory conditions.


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ACKNOWLEDGEMENTS
 
This work was supported in part by grants-in-aid from the Ministry of Education, Science, Sports and Culture and from the Japan Health Sciences Foundation.

We thank Ayako Onuma for animal care.

Received July 31, 2000; revised December 10, 2000; accepted March 19, 2001.


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