
* Kihara Institute for Biological Research, Yokohama City University, Totsuka, Yokohama 244-0813, Japan, and
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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Key Words: reactive oxygen species hypochlorous acid hydrogen peroxide superoxide inflammation
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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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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 manufacturers
instructions. In parallel, the trypan blue dye exclusion procedure was
used to distinguish normal or apoptotic cells from necrotic cells.
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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 (CF) 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).
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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.
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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 (AD) and MPO-deficient (EH) 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.
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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.
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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.
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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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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 dieswhether 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-
, [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.
We thank Ayako Onuma for animal care.
Received July 31, 2000; revised December 10, 2000; accepted March 19, 2001.
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