Blood, Vol. 93 No. 11 (June 1), 1999:
pp. 4006-4010
Red Blood Cells Inhibit Apoptosis of Human Neutrophils
By
Kazutetsu Aoshiba,
Yuri Nakajima,
Shuji Yasui,
Jun Tamaoki, and
Atsushi Nagai
From First Department of Medicine, Tokyo Women's Medical University,
8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.
 |
ABSTRACT |
Oxidative stress has been implicated in the triggering of apoptosis
in neutrophils. Because red blood cells (RBCs) are well known to
scavenge oxidants including H2O2, we tested the
hypothesis that RBCs inhibit apoptosis of neutrophils by reducing
intracellular oxidative stress. Apoptosis of neutrophils was evaluated
by light microscopy and DNA gel electrophoresis. We found that
coculture with RBCs protected against neutrophil apoptosis. Neither
physical contact between RBCs and neutrophils nor the cellular
integrity of RBCs was required to protect against neutrophil apoptosis. Neutrophil apoptosis was promoted by exogenous
H2O2 but suppressed by catalase, indicating a
role for H2O2 as a mediator of apoptosis. The
protective effect of RBCs against apoptosis was due to catalase and
glutathione metabolism because blocking of these antioxidant systems in
RBCs attenuated the protective effect of RBCs. These results suggest
that neutrophils are protected against apoptosis in the circulation.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
MATURE NEUTROPHILS are short-lived cells.
They have a circulating half-life of less than 10 hours1,2
and once they have migrated into tissue they die via apoptosis within
several days without the requirement for any apparent inductive
stimuli.3,4 This rapid and spontaneous apoptosis in
neutrophils is believed to provide a neutrophil clearance mechanism
that would act to limit tissue injury and lead to resolution of
inflammation.4
Recent studies have suggested that circulating neutrophils can undergo
apoptosis in the vasculature.5,6 However, if neutrophils undergo apoptosis rapidly in the vasculature, they would die before responding to inflammatory stimuli and infiltrating tissue. This suggests the need for a mechanism in which neutrophil apoptosis is
inhibited in the circulation. Recently, platelets have been shown to
inhibit neutrophil apoptosis,7 supporting this view. However, little attention has been paid to interactions between neutrophils and red blood cells (RBCs), which are about 20-fold more
abundant than platelets in the circulation.
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.8 Apoptosis is also inhibited in
neutrophils obtained from patients with chronic granulomatous disease
who carry a mutation that limits ROS generation.9
Neutrophil apoptosis is promoted by exogenous
H2O2 but delayed by antioxidants including
catalase,8 indicating a role for
H2O2 as a mediator of neutrophil apoptosis.
In the circulation, RBCs are 1,000-fold more abundant than neutrophils.
RBCs have large amounts of antioxidants such as catalase, glutathione,
and superoxide dismutase,10-15 and RBC membranes are permeable to H2O2 and
O2
,10 suggesting that RBCs
can scavenge ROS. RBCs have been shown to protect against
H2O2-mediated damage to cells or
tissues.11-14 RBCs scavenge H2O2
generated by neutrophils16 and therefore block formation of
hydroxy radical (·OH) and hypochlorite
(HOCl).15 Because H2O2 can freely
cross the cell membrane and therefore readily diffuses out of cells if
generated intracellularly,17 the reduction of extracellular H2O2 by antioxidants is expected to decrease
intracellular H2O2.18
In this report, we show that RBCs inhibit apoptosis of neutrophils by
reducing intracellular oxidant stress. Our data suggest that
neutrophils may be protected against apoptosis in the circulation.
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MATERIALS AND METHODS |
Reagents.
All reagents for cell culture were obtained from GIBCO Life
Technologies, Inc (Gaithersburg, MD). 3-amino-1,2,4-triazole, 1-chloro-2,4-dinitrobenzene, mercaptosuccinate, and
4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS)
were purchased from Sigma-Aldrich Japan K.K. (Tokyo, Japan).
Blood samples.
Blood samples were obtained from healthy volunteers by venipuncture of
the forearm vein and were immediately anticoagulated with 10 mmol/L EDTA.
Preparation and treatment of RBCs.
RBCs were isolated by centrifugation of the blood samples at
200g for 10 minutes, gentle removal of platelet-rich plasma and leukocytes, washing three times with phosphate-buffered saline (PBS),
and resuspension in PBS at 50% hematocrit. For some experiments, RBC
antioxidant systems were blocked as previously described. RBC catalase
was irreversibly inhibited by incubating a cell suspension with 50 mmol/L aminotriazole (catalase inhibitor) and 0.4 mmol/L H2O2 for 1 hour at 37°C.19 RBC
glutathione was blocked by treating a cell suspension with either 2 mmol/L chlorodinitrobenzene (glutathione depleting
agent),20 or 0.2 mmol/L mercaptosuccinate (glutathione peroxidase inhibitor)21 for 1 hour at 37°C. If
necessary, RBCs were treated simultaneously with aminotriazole,
chlorodinitrobenzene, and mercaptosuccinate to inhibit both catalase
and glutathione metabolism, which account for the majority of the
H2O2-scavenging activity of RBCs. RBC transport
of O2 was blocked by incubating a cell
suspension with 0.1 mmol/L DIDS for 1 hour at 37°C.10
Before use, RBCs treated as above were washed three times in PBS to
remove excess reagents. In some experiments, RBCs were lysed by brief
sonication with an ultrasonic sonicator.
Preparation of neutrophils.
Human neutrophils were isolated by dextran sedimentation and
centrifugation on a Histopaque gradient (without endotoxin; Sigma) as
previously described.22 Contaminating erythrocytes were
removed by hypotonic water lysis. The isolated neutrophils were washed twice with PBS and resuspended in RPMI1640 containing 5%
heat-inactivated fetal calf serum (FCS), 100 U/mL penicillin, and 100 µg/mL streptomycin. To avoid accidental neutrophil activation and
clumping, neutrophil preparations were carefully washed by
centrifugation at 200g, the supernatant was aspirated, cells
were gently resuspended with a pipet, and sudden changes in temperature
were avoided. The purity of neutrophil populations was greater than
95% on May-Grünwald-Giemsa stain, and neutrophil viability was
greater than 98% as determined by trypan blue dye exclusion. Less than
5% of neutrophil preparations showed a polarized shape, a sensitive
marker for neutrophil activation.23
Coculture of neutrophils with RBCs.
Neutrophils (5 × 104) were mixed with autologous RBCs
at ratios of 1:10, 1:100, and 1:1000, and cultured in 96-well
round-bottom plates (Becton Dickinson, Lincoln Park, NJ) at 37°C in
a humidified incubator containing 5% CO2. In some
experiments, neutrophils and RBCs were separately cultured in a 24-well
plate using the cell culture insert (pore size, 0.4 µm; Becton
Dickinson Labwares, Franklin Lakes, NJ). Neutrophils (2.5 × 105) were placed in the lower chamber separated by a
membrane from the upper chamber in which RBCs were placed (2.5 × 106).
Apoptosis assay.
Aliquots of neutrophils were cytospun on glass slides and air-dried.
Slides were stained with May-Grünwald-Giemsa. Apoptosis was
assessed based on nuclear pyknosis or chromatin condensation together
with cytoplasmic vacuolation on oil-immersion microscopy.3 Three hundred cells were scored in each experiment to determine the
percentage of apoptotic cells.
DNA fragmentation assay.
DNA fragmentation was evaluated using Apoptosis Ladder Detection Kit
(Wako, Tokyo, Japan) according to the manufacturer's instructions.
Briefly, neutrophils (1 × 104) were resuspended in
lysis buffer containing RNase and proteases. Then, DNA was extracted in
extraction buffer, precipitated by adding isopropanol, washed in 70%
ethanol, air-dried, and resuspended in Tris-EDTA (TE) buffer. DNA was
electrophoresed on a 1% agarose gel, stained with SYBR Green I
(Molecular Probes, Inc, Eugene, OR), and visualized under ultraviolet light.
Statistics.
Results are presented as mean ± standard error of mean (SEM).
Comparisons were made by Student's t-test or analysis of
variance (ANOVA) with Scheffe's correction as appropriate. P < .05 was accepted as significant.
| |
RESULTS |
RBCs inhibit spontaneous apoptosis of neutrophils.
When cultured in RPMI 1640 containing 5% FCS, neutrophils
constitutively underwent apoptosis, with 62.8% of cells apoptotic at
24 hours and 81.3% apoptotic by 48 hours as assessed by light microscopy (Fig 1A). The constitutive rate
of apoptosis in neutrophils shown here is within the range reported
previously.3,9 When neutrophils were cultured with RBCs at
a ratio of 1:10, neutrophil apoptosis was significantly inhibited at
each time point evaluated (Fig 1A). Inhibition of apoptosis by RBCs was
also dose-dependent (Fig 1B). When neutrophils were cultured with RBCs
at a ratio of 1:1,000, a physiological ratio in the blood, apoptosis
was decreased by about 80%. Figure 2 shows
typical micrographs of neutrophils cultured for 24 hours with or
without RBCs at a ratio of 1:100. Neutrophil-RBC contact was not
required to inhibit apoptosis because apoptosis was inhibited even when
neutrophils and RBCs were cultured separately using culture inserts.
Percentage of apoptotic neutrophils after 24-hour culture with or
without RBCs at a ratio of 1:10 was 85.8% ± 1.0% for no RBCs
versus 55.8% ± 1.7% for contact culture and 68.1% ± 2.6%
for separate culture (P < .01). The integrity of RBCs was not
required because apoptosis was inhibited even when neutrophils were
cultured with RBC lysates (data not shown). The protection of
neutrophil apoptosis by RBCs was substantiated by the results of DNA
gel electrophoresis, which showed that DNA ladder formation was
inhibited when neutrophils were cultured with RBCs
(Fig 3).
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| Fig 1.
Effect of RBCs on neutrophil apoptosis. Neutrophils were
cultured for varying times (A) or 16 hours (B) in RPMI 1640 containing
5% FCS in the presence or absence of 1:10 to 1:1000 RBCs. Apoptotic
cells were identified by morphology on light microscopy and % apoptosis was obtained from 300 counts per experiment. Data represents
mean ± SEM of 6 experiments. ** P < .01 versus cells in the
absence of RBCs.
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| Fig 2.
Light microscopy of neutrophils cultured for 24 hours
without (A) or with (B) RBCs at a ratio of 1:100 (×400). Arrows
indicate apoptotic cells showing nuclear pyknosis or chromatin
condensation together with cytoplasmic vacuolation. Note apoptosis is
inhibited in the presence of RBCs.
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| Fig 3.
Effect of RBCs on nucleosomal DNA fragmentation in
neutrophils. Neutrophils were cultured for 24 hours in the presence or
absence of 1:1000 RBCs. Cells were harvested and DNA fragmentation was
analyzed by agarose gel electrophoresis. Lane 1, molecular weight
marker; lane 2, cells cultured without RBCs; lane 3, cells cultured
with RBCs; lane 4, cells isolated freshly.
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Role of H2O2 as a mediator of neutrophil
apoptosis.
To determine the role of H2O2 in neutrophil
apoptosis, we cultured neutrophils in the presence or absence of either
catalase or H2O2. As shown in
Fig 4, application of catalase inhibited neutrophil apoptosis whereas H2O2 promoted
apoptosis. These results are consistent with a previous
report9 suggesting that H2O2 is a
mediator of neutrophil apoptosis.
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| Fig 4.
Effects of catalase and H2O2 on
neutrophil apoptosis. Neutrophils were cultured for 24 hours in the
presence or absence of catalase and H2O2.
Apoptosis was evaluated as described in Fig 1. To avoid inactivation of
H2O2 by serum, serum-free medium was used to
assess the effect of H2O2 on neutrophil
apoptosis. This probably reflects increased apoptosis in
H2O2-untreated control cells (70.8% ± 2.0%)
compared with apoptosis in catalase-untreated control cells (62.8% ± 1.7%). Data represents mean ± SEM of 4 experiments. * P < .05, ** P < .01 versus control cells in the absence of
catalase or H2O2.
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Catalase and glutathione are responsible for protection of neutrophil
apoptosis by RBCs.
To determine the contribution of each of RBC antioxidant to protection
against neutrophil apoptosis, RBC antioxidant systems were
pharmacologically blocked before analysis of the protective effect on
apoptosis (Fig 5). Inhibition of RBC
catalase by pretreatment with aminotriazole partially suppressed the
protection against neutrophil apoptosis by RBCs. Likewise, inhibition
of glutathione metabolism with chlorodinitrobenzene or
mercaptosuccinate partially suppressed the protection against
apoptosis. Simultaneous inhibition of catalase and glutathione
metabolism, which account for the majority of the
H2O2-scavenging activity of RBCs, completely
abolished the protection. In contrast, pretreatment of RBCs with the
anion channel blocker DIDS, which blocks transport of
O2 across RBC membranes, had little
effect on the protection against apoptosis by RBCs. These results
suggest that the inactivation of H2O2 by
catalase and glutathione metabolism is the mechanism by which RBCs
protect neutrophils against apoptosis.
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| Fig 5.
Effects of antioxidant inhibitors on the ability of RBCs
to inhibit neutrophil apoptosis. RBCs were pretreated with the catalase
inhibitor aminotriazole (AMT), the glutathione metabolism inhibitors
chlorodonitrobenzene (CDNB) and mercaptosuccinate (MS), a combination
of AMT, CDNB, and MS, or the O2 transport
inhibitor DIDS. Excess reagents were removed by washing RBCs three
times in PBS. Then, RBCs were mixed with neutrophils at a
neutrophil/RBC ratio of 1:10. After 48 hours of culture, neutrophil
apoptosis was evaluated as described in Fig 1. Data represents mean ± SEM of five experiments. **P < .01 versus untreated RBCs.
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DISCUSSION |
There is increasing evidence to indicate that ROS play an important
role in spontaneous- and stimulus-mediated apoptosis of neutrophils.8,9,24-27 Our data showing that neutrophil
apoptosis is promoted by exogenous H2O2 but
blocked by catalase confirms a previous study9 indicating
H2O2 as a mediator of neutrophil apoptosis.
Although cellular sources of H2O2 are numerous
and difficult to determine, the major source of endogenous
H2O2 is a leak from the mitochondrial electron
transport chain. All aerobic cells constantly produce small amounts of
O2 via mitochondrial respiratory
enzymes. O2 is then dismutated to form
H2O2. Many types of cells also generate O2/H2O2 via
various enzyme systems including arachidonic acid metabolism, microsomal cytochrome systems, and DNA synthesis enzymes. Furthermore, phagocytes such as neutrophils are endowed with nicotinamide adenine dinucleotide phosphate (NADPH) oxidase that releases large amounts of
O2 and H2O2 on
stimulation.28 Thus, the exact source of
H2O2-mediating apoptosis of neutrophils remains
to be determined. Nevertheless, our data together with
others9 indicate that intracellular H2O2, at least in part, functions as a mediator
of neutrophil apoptosis.
The major finding in our study is that RBCs inhibit apoptosis of
neutrophils. A number of studies have suggested that RBCs can scavenge
H2O2 within their microenvironment. For
example, RBCs can protect against H2O2-mediated
damage to cells and tissues.12,13,15,16 Because
H2O2 can freely cross the cell
membrane17 and there are 1000-fold more RBCs than
neutrophils in the blood, the elimination of extracellular
H2O2 by RBCs is expected to decrease
H2O2 within neutrophils.
RBCs are endowed with high activities of catalase and glutathione
peroxidase, which together probably account for the majority of
H2O2-scavenging activity of
RBCs.11,13,15,29 Specific inhibition of these RBC
antioxidants in the present study showed that both catalase and
glutathione metabolism are responsible and sufficient for protection of
apoptosis by RBCs. In contrast, inhibition of
O2 transport across RBC membranes had no
protective effect against apoptosis. These data imply that inactivation
of H2O2 by catalase and glutathione within RBCs
mediates the protective effect against neutrophil apoptosis.
The fact that RBCs can inhibit neutrophil apoptosis in vitro suggests
that neutrophil apoptosis is inhibited in the circulation in vivo.
Because blood plasma contains few antioxidant enzymes,30,31 RBCs may constitute a major antioxidant system that suppresses neutrophil apoptosis in the blood. However, it should also be noted
that although we found that RBCs inhibited neutrophil apoptosis in
vitro, this effect was rather small at 6 hours of culture and became
apparent after this point of time. Because neutrophils have a
circulating half-life of less than 10 hours,1,2
extrapolation of our in vitro data to in vivo situations requires caution.
Apoptosis is believed to provide a mechanism of neutrophil clearance
that limits tissue injury and leads to resolution of inflammation.4 Recently, apoptosis of neutrophils has also been shown to occur in the circulation.5,6 However, it
remains unclear whether apoptosis occurs as rapidly in the circulation as in tissue. If so, a considerable amount of circulating neutrophils would die before reaching their target tissue on inflammatory stimulation. Thus, it seems reasonable to speculate that there is a
mechanism by which apoptosis of neutrophils is suppressed in the
circulation. A recent study has shown that platelets also inhibit
apoptosis of neutrophils although the mechanism is not well
defined.7 Taken together, our data suggest that neutrophils are protected against apoptosis in the circulation.
| |
FOOTNOTES |
Submitted July 24, 1998; accepted January 25, 1999.
Supported by a Grant-in-Aid for Scientific Research (#30147392) from
the Ministry of Education, Science, and Culture, Japan.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address correspondence to Atsushi Nagai, MD, First Department of
Medicine, Tokyo Women's Medical University, 8-1 Kawada-cho,
Shinjuku-ku, Tokyo 162-8666, Japan.
| |
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ABSTRACT
INTRODUCTION
