Divisions of
* Pathology II and
Medical Microbiology, Linköping University, Faculty of Health Sciences, Linköping, Sweden
Correspondence: Helen Lundqvist-Gustafsson, Division of Pathology II, Faculty of Health Sciences, S-581 85 Linköping, Sweden. E-mail: helen.lundqvist{at}pat.liu.se
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Key Words: bacterial toxins • DNA-fragmentation • morphology • inflammation
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The results of studies in vitro suggest that changes in the local
environment relay signals that either retard or accelerate neutrophil
apoptosis. Proinflammatory mediators, such as interferon-
,
granulocyte-macrophage colony-stimulating factor, lipopolysaccharide,
complement factor 5a [4
], leukotriene B4
[5
], and interleukin (IL)-1b [6
], have
been found to transduce survival signals to neutrophils, whereas IL-8
[7
] and tumor necrosis factor a [8
]
induce neutrophil apoptosis. Although the signal transduction pathways
involved in those effects remain largely unknown, recent findings have
indicated that tyrosine phosphorylation events play a role in the
signaling pathways that lead to neutrophil apoptosis
[9
]. The p38-mitogen-activated protein kinase (p38-MAPK)
is a serine/threonine kinase that is stimulated by phosphorylation of
tyrosine and threonine residues [10
] during cell
activation by proinflammatory cytokines, osmotic stress, and UV
irradiation [11
]. Because irradiation and other stress
stimuli are known to induce apoptosis in a variety of cell types,
p38-MAPK has been suggested to participate in the process leading to
apoptosis in response to these stimuli [12
]. In
neutrophils, constitutive phosphorylation and activation of p38-MAPK
has been suggested to participate in spontaneous apoptosis
[13
]. Another study claimed that spontaneous as well as
Fas-induced apoptosis occurs independently of p38-MAPK activation,
whereas stress-induced apoptosis (UV irradiation, hyperosmolarity, or
sphingosine) is inhibited by the p38-MAPK inhibitor SK&F 86002
[14
]. Together the functional significance of p38-MAPK
in neutrophils and its role in apoptosis are still obscure. Perhaps
divergent signals generated downstream of p38-MAPK activation can
control either cell death or survival [reviewed in ref. 15
].
Intracellular production of ROS induces neutrophil apoptosis [16 17 18 ], whereas the presence of antioxidants prolongs cell survival [19 ]. Phagocytosis of Escherichia coli induces neutrophil apoptosis through an oxygen-dependent mechanism [20 ], suggesting activation-induced regulation of the life span of neutrophils.
The aim of the work reported here was to study the effects of viable and heat-killed S. aureus on neutrophil survival by assaying phagocytosis, NADPH oxidase activity, and activation of p38-MAPK.
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Isolation of human neutrophils
Human neutrophils were isolated from freshly drawn heparinized
blood obtained from the blood bank at Linköping University
Hospital. Erythrocytes were removed by dextran sedimentation followed
by hypotonic lysis. The lysate was centrifuged on a Ficoll-Paque
gradient [21
], and the extracted granulocyte fraction
was washed twice and resuspended in Krebs-Ringer glucose buffer (KRG,
containing 10 mM glucose, 1.5 mM Mg2+, and 1 mM
Ca2+, pH 7.3). The suspended cells were placed on ice and
used within 2 h of preparation. Microscopic examination revealed
that at least 97% of the cells obtained in this way were neutrophils.
Cultivation and opsonization of S. aureus
S. aureus bacteria (strain WOOD 46; catalase
positive, protein-A free) were stored at -70°C. Cultures were grown
for 18 h in liquid growth medium and then transferred to fresh
medium and cultured for another 2 h. Thereafter, the bacteria were
washed and opsonized in 20% normal human serum (37°C, 20 min),
washed once, and subsequently resuspended at 108/mL in KRG
and kept on ice. Portions of the suspension were boiled for 15 min
(100% of the bacteria killed) before opsonization, and other portions
were not opsonized.
In some experiments, broth from the above-mentioned 2-h cultivation period was centrifuged and sterile filtered and then either added directly to neutrophils or boiled for 15 min and then added to neutrophils.
FITC-labeling of bacteria
Bacteria (109/mL) were labeled with FITC (0.25
mg/mL) in carbonate buffer [3.35% Na2CO3,
1.54% NaHCO3 (v/w), pH 10.0) for 30 min at 37°C. After
being washed four times in PBS, cells were resuspended in KRG and
stored on ice.
Phagocytosis
Neutrophils (106 cells) were mixed with FITC-labeled
opsonized bacteria (107) in KRG and incubated for 60 min.
At predetermined intervals, one drop of the reaction mixture was mixed
with two drops of crystal violet (0.5 mg/mL in KRG) and immediately
analyzed by fluorescence microscopy. Bacteria that were not ingested
were stained with the dye, and their fluorescence was quenched, whereas
ingested S. aureus remained fluorescent [22
].
Assessment of apoptosis
Neutrophils (106) in KRG were prewarmed at 37°C
for 5 min, and S. aureus bacteria
(106–108) in suspension were subsequently
added to a total volume of 1 mL. The samples were analyzed after 60 min
to assess apoptosis.
Morphology
Neutrophils stained with Türck’s reagent (0.02% gentian
violet in 6% acetic acid) were evaluated by light microscopy, and the
portion of cells with condensed nuclei was determined. In each sample,
200–400 cells were counted.
Exposure of phosphatidylserine
Neutrophils were stained with a combination of annexin V-FLUOS
and PI according to the instructions of the manufacturer. Cells
(104 per sample) were analyzed in a flow cytometer (Becton
Dickinson, Heidelberg, Germany) using Lysys II software. Cells
with an increased FITC fluorescence, corresponding to an increased
exposure of phosphatidylserine, were considered apoptotic
[23
], whereas cells that exhibited both increased FITC
and PI fluorescence were considered necrotic. Cell debris was excluded
by raising the forward scatter threshold.
Fragmented DNA
Fragmented DNA was analyzed by flow cytometry performed on
PI-stained, permeabilized neutrophils [24
]. Briefly
described, pelleted cells were resuspended in 1.5 mL of a hypotonic
solution containing 50 mg/mL of PI, 0.1% sodium citrate, and 0.1%
Triton X-100 and were then incubated overnight. The fluorescence of DNA
was detected in a flow cytometer (Becton Dickinson; 105
events per sample) using Lysys II software. The broad, low-fluorescence
intensity peaks from apoptotic neutrophil DNA were easily distinguished
from the narrow, high-intensity peak of intact DNA. The percentage of
low-fluorescence (fragmented) DNA was analyzed with CellQuest software.
Production of reactive oxygen species
Hydrogen peroxide production
Cellular H2O2 production was assayed as
an increase in oxidized pHPA, using a modified version of the method
published by Hyslop and Sklar [25
], as earlier described
[26
]. Neutrophils (106) were suspended in
KRG (0.9 mL) supplemented with 0.5 mg of pHPA, 4 U of HRP, and 1 mM
NaN3 to block the intracellular
H2O2-consuming enzymes myeloperoxidase and
catalase [27
]. Thereafter, the cells were prewarmed at
37°C for 5 min and challenged with S. aureus (100 µL
containing 107 bacteria). The cells were subsequently
removed by centrifugation (10 s at 13,000 g), and the
oxidized pHPA was detected in a Shimadzu spectrofluorimeter (RF-540;
ex, 317 nm; em, 400 nm). Fluorescence
values were converted to nanomoles of H2O2 by
comparison with a standard curve obtained using known amounts of
H2O2.
Chemiluminescence
Chemiluminescence (CL) was measured as previously described
[28
]. Neutrophils (106) were mixed in
polypropylene tubes with KRG supplemented with either 50 µM luminol
and 4 U of HRP (total CL) or 50 µM luminol, 2000 U of catalase, and
200 U of SOD (intracellular CL), prewarmed at 37°C for 5 min, and
then challenged with S. aureus (107). Light
emission was recorded continuously at 37°C in a Biolumat LB 9509
(Berthold, Wildbad, Germany).
Preparation of cell lysates and immunoblotting
Neutrophils (107) were challenged for 5 min with
viable or heat-killed serum-opsonized S. aureus
(108), after which 1 mL of ice-cold PBS (pH 7.3)
supplemented with 1 mM Na3VO4 was
added. Cells were pelleted and resuspended in 0.3 mL of lysis buffer
(1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate
(SDS) pH 7.4), supplemented with 1 mM Na3VO4,
10 µg/mL of aprotinin, 10 µg/mL of leupeptin, 1.4 µg/mL of
pepstatin, and 1 mM PMSF). After 15 min at 4°C, the lysate was
collected by centrifuging for 10 min at 4°C and 15,800 g,
heated in sample buffer at 95°C for 5 min [29
], and
then centrifuged for 2 min at 15,800 g. Samples of the
prepared lysates were subjected to SDS-10% polyacrylamide gel
electrophoresis (SDS-PAGE), and proteins were transferred onto a
nitrocellulose membrane using transfer buffer. The membranes were
subsequently blocked for 60 min in TBS-BSA-Tween buffer (200 mM Tris
and 137 mM NaCl [pH 7.6], supplemented with 0.1% Tween-20 and 5%
BSA) and then incubated for 60 min with anti-phospho-p38-MAPK antibody
(diluted 1:1,000 in TBS-BSA-Tween) which recognizes only p38-MAPK when
it is activated by dual phosphorylation at Thr-180 and Tyr-182. The
membranes were then washed and incubated for 60 min with an
HRP-conjugated goat anti-rabbit antibody (diluted 1:1,700 in
TBS-BSA-Tween) and thereafter washed and developed using the enhanced
CL detection system. To confirm the identity of the protein and to
ensure that each lane was loaded with the same amount of protein, the
blot was stripped and reprobed with an anti-p38-MAPK antibody (diluted
1:1,000) that recognized both phosphorylated and nonphosphorylated
p38-MAPK.
Statistics
The Mann-Whitney U test was used to evaluate
differences between groups of cells. P values were
considered significant when <0.05, and they are distinguished by a
system of asterisks: *, P < 0.05; **,
P < 0.01; ***, P < 0.001.
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 View larger version (17K): [in a new window] |
Figure 1. Correlation between number of S. aureus and neutrophil
apoptosis. Neutrophils were challenged with different numbers of viable
( ) or heat-killed ( ) S. aureus for 60 min. Thereafter,
the neutrophils were permeabilized and then PI stained overnight, and
fragmented DNA was subsequently analyzed by flow cytometry
(104 events/sample). Apoptosis is presented as percent
fragmented DNA of total. Data are means ± SD
(n=3).
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 View larger version (54K): [in a new window] |
Figure 2. Apoptotic morphology and exposure of phosphatidylserine in neutrophils
exposed to viable S. aureus. Neutrophils were challenged
with viable or heat-killed S. aureus at a ratio of 1:10 for
60 min. (A) The neutrophils were subsequently stained with gentian
violet and analyzed by light microscopy. Cells with condensed nuclei
characteristic of apoptosis are shown in the micrograph (arrowheads),
and the data in the diagram represent means ± SD
(n=5; *, P<0.05). (B) Alternatively, neutrophils
were washed in PBS and stained with annexin V-FLUOS in combination with
PI and then analyzed by flow cytometry. Cells exhibiting enhanced
fluorescence in both the FL1 and the FL3 channel (i.e., necrotic cells)
were gated away, and data on the rest of the neutrophils were plotted
as histograms. The results are representative of three separate
experiments.
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 View larger version (27K): [in a new window] |
Figure 3. Chemiluminescence in neutrophils interacting with S. aureus.
Neutrophils (106) were mixed with S. aureus at a
1:10 ratio in KRG buffer supplemented with 50 µM luminol and 4 U of
HRP [total CL (a and c)] or with 50 µM luminol, 2000 U of catalase,
and 200 U of SOD [intracellular CL (b and d)]. Results from one
representative experiment are shown.
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We noted that unopsonized bacteria, which were not as readily taken up by neutrophils as opsonized bacteria (47% phagocytosing cells with 12±7 bacteria/phagocyte for nonopsonized bacteria compared with 94% phagocytosing cells with 12±4 bacteria/phagocyte for opsonized bacteria), were also capable of inducing apoptosis, although they did so less effectively than the opsonized bacteria. Pretreatment of neutrophils with 5 µM cytochalasin b for 10 min inhibited phagocytosis (48% phagocytosing cells with 3±2 bacteria/phagocyte) but did not abrogate the capacity of viable bacteria to induce apoptosis (Fig. 4 ). Cytochalasin b alone did not provoke apoptosis (data not shown). These findings show that phagocytosis of the bacteria is not a prerequisite of apoptosis.
 View larger version (39K): [in a new window] |
Figure 4. S. aureus-induced neutrophil apoptosis is not dependent on
phagocytosis. Neutrophils were exposed to S. aureus for 60
min under different conditions. Flow cytometry was performed on
PI-stained nuclei, and the amount of fragmented DNA was determined.
Apoptosis is presented as percent fragmented DNA of total. The results
are shown as means ± SD. Significant differences from
control cells (open bar) are marked with asterisks (*,
P<0.05; **, P<0.01; ***, P<0.001).
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 View larger version (34K): [in a new window] |
Figure 5. S. aureus-induced neutrophil apoptosis is activated by a
soluble factor. Neutrophils were exposed for 60 min to fresh or boiled
(15 min, 100°C) sterile filtered supernatant from S.
aureus cultivated in nutrient broth for 2 h. Flow cytometry
was performed on PI-stained nuclei, and the amount of fragmented DNA
was determined. Apoptosis is presented as percent fragmented DNA of
total. The results are shown as means ± SD
(n=3; **, P<0.01).
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 View larger version (16K): [in a new window] |
Figure 6. Kinetics of S. aureus-induced apoptosis. Neutrophils were
exposed to viable or heat-killed S. aureus or sterile
filtered supernatant for different periods of time. Flow cytometry was
performed on PI-stained nuclei, and the amount of fragmented DNA was
determined. Apoptosis is presented as percent fragmented DNA of total.
The results are shown as means ± SD
(n=3).
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50% at the concentration
10 µM (Fig. 7b)
. Further experiments with this concentration showed
that SB203580 significantly reduced S. aureus-induced
apoptosis (P>0.05) as well as supernatant-induced apoptosis
(P>0.05) (Fig. 7c)
. SB203580 alone or the carrier dimethyl
sulfoxide had no effect on apoptosis (data not shown).
 View larger version (35K): [in a new window] |
Figure 7. Viable S. aureus and S. aureus-derived
supernatant induce phosphorylation of p38-MAPK. (A) Phosphorylation of
p38-MAPK in neutrophils stimulated with S. aureus was
analyzed by Western blotting with an anti-phospho-p38-MAPK antibody.
(B) Concentration inhibition curve of the p38-MAPK inhibitor SB203580.
Neutrophils were pretreated with SB203580 (1–10 µM) or with buffer
alone for 15 min on ice and then for 10 min at 37°C, and they were
subsequently incubated with viable S. aureus for 60 min.
Flow cytometry was performed on PI-stained nuclei, and the amount of
fragmented DNA was determined. The results are representative of two
separate experiments. (C) Neutrophils were or were not pretreated with
10 µM SB203580 and subsequently incubated with viable S.
aureus or with S. aureus-derived supernatant for 60
min. Apoptosis was analyzed as described in B. The results are shown as
means ± SD (n=3; *, P<0.05).
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12 h. After 18 h,
there was no longer any difference between samples due to substantial
spontaneous apoptosis in the control population. It is not yet known how S. aureus causes neutrophil apoptosis. The strong positive correlation between the number of viable bacteria and the extent of apoptotic neutrophils could indicate that apoptosis is initiated by phagocytosis-induced cellular activation (e.g., degranulation and NADPH oxidase activation). In a previous study of human neutrophils [16 ], we noted that activation of the plasma membrane-bound oxidase by N-formyl-methionyl-leucyl-phenylalanine did not induce apoptosis, whereas apoptosis was induced by intracellular formation of H2O2 during stimulation of NADPH oxidase with phorbol myristate acetate or ionomycin. It is interesting that only the viable S. aureus caused extracellular release of ROS. However, we found no difference between the levels of NADPH oxidase activity that occurred in response to viable and heat-killed bacteria in quantitative terms. Thus, we concluded that ROS derived from NADPH oxidase are not major signals in S. aureus-induced apoptosis. This is further supported by the fact that apoptosis was not blocked in neutrophils pretreated with the NADPH oxidase inhibitor DPI in concentrations that completely abrogated the respiratory burst.
Several of our observations indicated that S. aureus can
induce neutrophil apoptosis, even if the bacteria are not ingested.
Apoptosis is not blocked by pretreatment with a known inhibitor of
phagocytosis (cytochalasin b) or by exposure to unopsonized bacteria,
which are not readily taken up; thus, it is unlikely that phagocytosis
per se initiates proapoptotic signaling. It is interesting that the
supernatant from the 2-h cultivation of S. aureus also had a
strong apoptosis-inducing effect. Notably, that effect was lost when
the supernatant was boiled for 15 min, similar to what was seen on heat
inactivation of the bacteria. Thus, S. aureus-induced
apoptosis in neutrophils seemed to be largely caused by a soluble,
bacteria-derived factor. Other investigators have studied the role of
S. aureus toxins in regulating apoptosis in various cells
involved in the immune response. Staphylococcal enterotoxin B has been
observed to induce apoptosis in T lymphocytes in patients suffering
from atopic dermatitis and might thereby dictate the pathogenicity of
the disease [35
]. Furthermore, S.
aureus-derived 
-toxin induces apoptosis in T lymphocytes caused
by formation of small transmembrane pores [36
].
A number of recent studies have suggested that tyrosine phosphorylation is involved in signaling pathways that often lead to neutrophil apoptosis. It has been reported that apoptosis induced by stress (i.e., sphingosine, UV irradiation, or hyperosmolarity) initiates activation of p38-MAPK [14 15 ], but data on the role of p38-MAPK in spontaneous apoptosis are conflicting [13 14 ]. We found an interesting difference in signaling exhibited by neutrophils; phosphorylation of p38-MAPK occurred in neutrophils exposed to viable bacteria or bacteria-derived supernatant, but in those treated with heat-killed bacteria either no phosphorylation or a very modest one took place. Moreover, inhibition of p38-MAPK by SB203580 significantly reduced the apoptosis-inducing capacity of these stimuli. At least one S. aureus-derived toxin, namely the staphylococcal superantigen toxic shock syndrome toxin-1, has been implicated in activation of protein tyrosine kinases in target cells [37 ]. Our data suggest a similar scenario for S. aureus-induced apoptosis, although the activating factor is not yet known.
Together, our results show that interaction of human neutrophils with viable S. aureus rapidly induced apoptosis, a process that involves p38-MAPK. The causative factor(s) appeared to be released from the bacteria. Additional studies are needed to identify this factor or factors and to further investigate p38-MAPK regarding its role in apoptosis signal transduction in neutrophils and its involvement in the proapoptotic machinery. Based on our findings, we speculate that the ability of viable S. aureus to induce apoptosis may represent an active strategy to avoid being killed by cells of the immune system.
Received July 5, 2000; revised April 23, 2001; accepted April 24, 2001.
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