Download Pathogen-Induced Apoptotic Neutrophils Express Heat

Pathogen-Induced Apoptotic Neutrophils
Express Heat Shock Proteins and Elicit
Activation of Human Macrophages
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of June 18, 2017.
Limin Zheng, Min He, Min Long, Robert Blomgran and Olle
Stendahl
J Immunol 2004; 173:6319-6326; ;
doi: 10.4049/jimmunol.173.10.6319
http://www.jimmunol.org/content/173/10/6319
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References
The Journal of Immunology
Pathogen-Induced Apoptotic Neutrophils Express Heat Shock
Proteins and Elicit Activation of Human Macrophages1
Limin Zheng,2*† Min He,*† Min Long,† Robert Blomgran,† and Olle Stendahl†
N
eutrophils are the first cells to be recruited to the site of
bacterial infections, and they provide the first line of
host defense against invading microorganisms. Upon encountering bacteria, neutrophils initially react by using their microbicidal mechanisms to kill the microbes and/or prevent them
from spreading until the macrophages (M␾)3 can accumulate.
Thereafter, the neutrophils are programmed to die by apoptosis and
are rapidly removed by the M␾ (1–3). There is emerging evidence
that the clearance of apoptotic neutrophils not only prevents the
release of potentially toxic intracellular intracellular substances,
but also actively regulates the infection-induced inflammatory responses (4, 5). Uptake of apoptotic neutrophils has been shown to
inhibit the production of proinflammatory mediators in M␾ by
secretion of anti-inflammatory cytokines such as TGF-␤ (4 –10).
This active anti-inflammatory response in M␾ represents a mechanism for the safe clearance of apoptotic cells under physiological
conditions and serves as a key step in the resolution phase of
inflammation (4, 5). In contrast, such anti-inflammatory events can
inhibit Ag presentation and promote the growth of intracellular
*Key Laboratory of Gene Engineering of the Education Ministry, Department of
Biochemistry, College of Life Sciences, Sun Yatsen (Zhongshan) University, Guangzhou, China; and †Division of Medical Microbiology, Faculty of Health Sciences,
Linkoping University, Linkoping, Sweden
Received for publication October 22, 2003. Accepted for publication September
10, 2004.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by the Natural Science Foundation of China and Guangdong Province (Projects 30371600 and 031620), the Swedish Medical Research
Council (Projects 13026, 14689, and 5968), and the King Gustaf V Memorial Foundation, the “973” project (2004CB518801).
2
Address correspondence and reprint requests to Dr. Limin Zheng, Department of
Biochemistry, College of Life Sciences, Zhongshan University, Guangzhou 510 275,
People’s Republic of China. E-mail address: [email protected]
Abbreviations used in this paper: M␾, macrophage; HSP, heat shock protein; MPO,
myeloperoxidase; PI, propidium iodide.
3
Copyright © 2004 by The American Association of Immunologists, Inc.
parasites within M␾ (5, 11, 12), which implies that induction of
apoptosis in neutrophils represents a pathogenic strategy for microbes to eliminate these key immune cells and use them as “Trojan horses” to enter M␾. However, in the cited studies, neutrophil
apoptosis was induced by age or irradiation; thus, the results may
not apply to the very early phase of infection, when many neutrophils undergo pathogen-induced apoptosis.
Increasing numbers of pathogens have been found to modulate
host cell apoptosis and thereby influence the progression of diseases (13–20). Recent studies have shown that induction of phagocyte apoptosis may not necessarily represent a pathogenic strategy
to eliminate these key immune cells but rather a host defense
mechanism against invading microorganisms. For example, apoptosis of M␾ limits the intracellular growth of mycobacteria, and
uptake of these infected apoptotic cells by neighboring M␾ reduces the viability of intracellular bacteria and facilitates Ag
presentation (20 –22). We have previously observed that proinflammatory responses in M␾ are triggered by uptake of mycobacteria-induced apoptotic neutrophils, but not by ingestion of
uninfected apoptotic neutrophils (16). Other investigators have
shown that interactions between M␾ and apoptotic inflammatory
neutrophils prevent the growth of Leishmania major both in vitro
and in vivo, whereas phagocytosis of uninfected apoptotic cells by
M␾ promotes the intracellular growth of Trypanosoma cruzi (11,
19). These findings imply that interaction with apoptotic neutrophils primes M␾ so that they are not simply anti-inflammatory
actors, as previously suggested, but they also respond to danger
signals in a more complex way that may play a crucial role in host
defense. It is not yet known how M␾ distinguish between and react
differently to uninfected or inflammatory apoptotic neutrophils.
The role of apoptosis in modulating the pathogenesis of infectious diseases varies with the causative organisms, and the majority of pathogens that induce apoptosis are extracellular bacteria
(13, 14). Therefore, we used both Gram-negative and Gram-positive extracellular bacteria as model systems to determine whether
0022-1767/04/$02.00
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Ingestion of aged or irradiated apoptotic neutrophils actively suppresses stimulation of macrophages (M␾). Many bacterial
pathogens can also provoke apoptosis in neutrophils, but little is known about how such apoptotic cells influence M␾ activation.
We found that neutrophils undergoing apoptosis induced by UV irradiation, Escherichia coli, or Staphylococcus aureus could
either stimulate or inhibit M␾ activation. In contrast to M␾ that had ingested irradiated apoptotic neutrophils, M␾ that had
phagocytosed bacteria-induced apoptotic neutrophils exhibited markedly increased production of the proinflammatory cytokine
TNF-␣, but not the anti-inflammatory cytokine TGF-␤. Moreover, ingestion of bacteria, but not UV-induced apoptotic neutrophils, caused increased expression of Fc␥RI on M␾, and this effect was not provoked directly by bacteria associated with the
apoptotic neutrophils. Instead, we found that a link between pathogen-induced apoptotic neutrophils and up-regulation of the heat
shock proteins HSP60 and HSP70, and we also observed that recombinant HSP60 and HSP70 potentiated LPS-stimulated production of TNF-␣ in M␾. The opposing macrophage responses to neutrophils undergoing apoptosis induced in different ways may
represent a novel mechanism that regulates the extent of the immune response to invading microbes in two steps: first by aiding
the functions of M␾ at an early stage of infection, and subsequently by deactivating those cells through removal of uninfected
apoptotic neutrophils. HSP induction in neutrophils may provide the danger signals required to generate a more effective macrophage response. The Journal of Immunology, 2004, 173: 6319 – 6326.
6320
UPTAKE OF BACTERIA-INDUCED APOPTOTIC NEUTROPHILS ACTIVATES M␾
these pathogen-induced apoptotic neutrophils influence M␾ activation, and if so, how they exert that effect. We found that uptake
of neutrophils undergoing pathogen-induced apoptosis caused M␾
to increase both production of the proinflammatory cytokine
TNF-␣ and surface expression of Fc␥RI. This activation of M␾
was not provoked by the bacteria associated with apoptotic neutrophils. Instead, the danger signals needed to trigger the activation
of macrophages seemed to come from heat shock proteins (HSP)
expressed on the inflammatory apoptotic neutrophils.
Materials and Methods
Reagents
Preparation of bacteria
Escherichia coli strain ATCC 25922 and Staphylococcus aureus strain
Wood46 (both from American Type Culture Collection, Manassas, VA)
were cultured for 18 h (15, 17) and then washed and resuspended in RPMI
1640 medium containing 5% FCS and 2 mM L-glutamine (RPMI medium).
In some experiments the bacteria were fixed with 7.3% formalin for 30 min
and washed before use.
Cell isolation
Human neutrophils were isolated from peripheral blood of healthy donors
as previously reported (16). Neutrophils of ⬃96% purity were resuspended
in RPMI medium. Human M␾ were prepared from PBMC as described
previously (8, 16). The cells in DMEM alone were plated at 4 ⫻ 106/well
in 24-well plates for 1.5 h, washed, then cultured in DMEM containing
10% human AB serum. The medium was changed every 3 days and was
replaced with DMEM without serum before use in the experiments (8, 16).
For all experiments, M␾ had been cultured for 6 –9 days before use.
Induction of neutrophil apoptosis
Neutrophils (2 ⫻ 106/ml) were incubated with E. coli or S. aureus (cell to
bacteria ratio, 1:20) for 20 min at 37°C; when using fixed bacteria, the
neutrophils were also exposed to UV irradiation for 8 min. Thereafter, the
samples were washed twice (200 ⫻ g, 5 min each time) with PBS and
incubated in RPMI medium containing 50 ␮g/ml gentamicin at 37°C for
3 h in a humidified CO2 incubator. For UV-induced apoptosis, neutrophils
were exposed to UV irradiation for 8 min, followed by culture for 3 h. In
some experiments neutrophils were exposed to mild heat (43°C) for 30
min, then incubated at 37°C for 3 h (23). This methodology routinely
yielded populations comprising 40 –70% cells positively stained with annexin V and ⬍4% positive for propidium iodide (PI). Where indicated,
neutrophils were pretreated with 10 ␮g/ml cycloheximide for 10 min before exposure to bacteria or heat stress.
Assessment of neutrophil apoptosis
Neutrophil apoptosis was quantified by flow cytometry using FITC-conjugated annexin V and by morphological examination (16, 17). Specific
binding of annexin V was achieved by incubating 106 cells in 60 ␮l of
binding buffer saturated with annexin V for 15 min at 4°C in the dark. To
discriminate between early apoptosis and necrosis, the cells were simultaneously stained with annexin V and PI before analysis. The binding of
annexin V-FITC (FL1) and PI (FL2) to the cells was measured by flow
cytometry (FACSCalibur, BD Biosciences) using CellQuest software (BD
Biosciences, Mountain View, CA) (16). At least 10,000 cells were counted
in each sample, and a gate based on forward and side scatters was set to
exclude cell debris.
Morphological assessment of apoptosis was performed on Giemsa- or
Turck-stained cytocentrifuged neutrophils as previously described (16, 17).
Neutrophils were lysed, and the genomic DNA was extracted according to
the protocol for apoptotic DNA laddering kit (R&D Systems). Samples (2
␮g of DNA/lane) were analyzed by gel electrophoresis (1.8% agarose) and
ethidium bromide staining. The gel was visually examined under 305 nm
of UV illumination (16).
Flow cytometric analysis of Fc␥RI expression
M␾ were left untreated or were incubated for 1 h with neutrophils undergoing apoptosis induced in different ways and were subsequently washed,
then cultured for an additional 18 h. Fc␥RI expression on M␾ was determined by flow cytometry using R-PE-conjugated anti-Fc␥RI and an isotype-matched control mAb (DakoCytomation, Glostrup, Denmark), essentially as previously described (24).
Cytokine production in M␾ after phagocytosis of apoptotic
neutrophils
Neutrophils undergoing apoptosis induced by exposure to UV irradiation,
heat shock, S. aureus, or E. coli were washed twice with PBS and resuspended in DMEM without serum. Before use, M␾ growing in each well
were washed and replaced with DMEM without serum. Apoptotic neutrophils (4 ⫻ 106/well of M␾) were added, and the plates were incubated at
37°C for 1 h. Thereafter, the wells were washed vigorously to remove
uningested neutrophils (8, 16, 25), fresh DMEM without serum was added,
and the supernatants were collected 18 h later (8, 16). As a control, apoptotic neutrophils were cultured for 18 h in DMEM without serum. In some
experiments, M␾ were incubated with recombinant human HSP60, HSP70,
or LPS (from E. coli 055:B5) for 18 h. Where indicated, M␾ were preincubated with 10 ␮g/ml polymyxin B sulfate for 30 min. The supernatants
were centrifuged to remove particular debris and were stored in aliquots at
⫺70°C. Cytokine concentrations in the culture supernatants were determined by ELISA, using Quantikine immunoassay kits according to the
instructions provided by the manufacturer.
We performed a phagocytic assay (8, 16, 25) to ensure that the M␾
ingested equivalent numbers of neutrophils undergoing apoptosis induced
in different ways. In short, the monolayers were fixed with 1% formalin
overnight and stained for myeloperoxidase (MPO) as a marker of ingested
neutrophils (8, 16); the M␾ themselves were routinely negative for MPO
staining. Phagocytosis of apoptotic neutrophils was quantified under an
inverted phase contrast microscope essentially as previously described (8).
M␾ that showed discrete, round, MPO-positive inclusions were scored as
having ingested one or more apoptotic neutrophils (8, 25).
Western blotting
The neutrophils were washed three times with PBS, and the pellets were
resuspended in lysis buffer (16) for 20 min on ice. After centrifugation at
10,000 ⫻ g for 10 min, the supernatants were dissolved in Laemmli sample
buffer (26) and heated at 95°C for 5 min. Equal amounts of cellular proteins
(15 ␮g/lane for HSP70; 35 ␮g/lane for HSP60 and HSP90) were separated
on 10% SDS-PAGE and electrotransferred to nitrocellulose membranes.
The membranes were blocked with 5% milk, and the presence HSPs on the
blots was detected with specific Ab and a commercial ECL kit (16). To
confirm that each lane received the same amount of proteins, the blots were
stripped and reprobed with anti-actin (C-2) Ab (Santa Cruz Biotechnology,
Santa Cruz, CA).
LPS detection
To analyze the LPS content in M␾ containing ingested apoptotic neutrophils, the monolayers were lysed by vigorous pipetting in H2O for 1 h and
then collected and centrifuged (2500 ⫻ g, 10 min). LPS in the samples was
determined using a Limulus amebocyte lysate endotoxin assay kit.
Statistical analysis
The data on cytokine concentrations are given as the mean ⫾ SEM, and the
data on apoptotic rates are the mean ⫾ SD. Statistical significance was
determined by Student’s t test. A value of p ⬍ 0.05 was considered statistically significant.
Results
Neutrophil apoptosis induced by viable E. coli and S. aureus
In initial experiments, flow cytometry using FITC-conjugated annexin V revealed that neutrophils exposed to E. coli or S. aureus
underwent rapid apoptosis. This effect was positively correlated
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An annexin V apoptosis detection kit and Quantikine kits for immunoassays of TNF-␣, TGF-␤, and IL-10 were purchased from R&D Systems
(Abingdon, U.K.). Anti-HSP60 (SPA-806), HSP70 (SPA-810, detects the
inducible form of HSP70), and HPS90 (SPA-830) Ab and recombinant
human HSP60 (ESP-540) and HSP70 (ESP-555, the inducible form of
HSP70) were obtained from StressGen (Victoria, Canada). ESP-540 and
ESP-555 were the low endotoxin preparations containing ⬍50 endotoxin
U/mg proteins. The Limulus amebocyte lysate endotoxin assay kit (Charles
River Endosafe, Charleston, SC) and cell isolation and tissue culture reagents were obtained from Invitrogen Life Technologies (Lidingo, Sweden), and electrophoresis and ECL reagents were purchased from Amersham Biosciences (Uppsala, Sweden). All other reagents were obtained
from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated in the text.
DNA fragmentation assay
The Journal of Immunology
with exposure time and bacteria to cell ratio, and excessive apoptosis was associated with loss of membrane integrity in an increased portion of neutrophils, which indicates necrosis or late
apoptosis (data not shown). Apoptotic and necrotic cells have different effects on activation of M␾ (5, 25, 27); therefore, we exposed the neutrophils to bacteria at a ratio of 1:20 for 20 min, then
washed and cultured them for 3 h. Under these conditions, ⬃50%
of the neutrophils were in the early stage of apoptosis with preserved membrane integrity (lower right quadrants in Fig. 1A), and
⬍4% were positive for both annexin V and PI (upper right quadrants in Fig. 1A). Morphological examination showed typical ap-
6321
optotic changes in neutrophils, including decreased cell volume and
chromatin condensation with loss of multilobular nuclear structure
(Fig. 1B). Similar results were obtained in complementary experiments performed to assay DNA fragmentation (Fig. 1C).
Only viable bacteria induced appreciable apoptosis, although
low rates of such cell death were observed in uninfected neutrophils (6 ⫾ 3%) and in neutrophils exposed to fixed bacteria under
identical conditions (7 ⫾ 4 and 6 ⫾ 2% for fixed E. coli and S.
aureus, respectively).
Ingestion of bacteria-induced apoptotic neutrophils stimulates
TNF-␣ production in M␾
Uptake of bacteria-induced apoptotic neutrophils increases the
expression of Fc␥RI on M␾
To further test the hypothesis that M␾ are activated by bacteriainduced apoptotic neutrophils, we examined another response exhibited by M␾, namely expression of Fc␥RI (CD64). Flow cytometry revealed that Fc␥RI was expressed constitutively on the
surface of human M␾, and the level of expression was not affected
by ingestion of uninfected apoptotic neutrophils (mean fluorescence intensity, 433 ⫾ 36 and 420 ⫾ 28, respectively). In contrast,
FIGURE 1. Neutrophil apoptosis induced by exposure to UV irradiation, S. aureus, or E. coli. Neutrophils were left untreated (1) or were
exposed to UV irradiation (2), S. aureus (3), or E. coli (4). Thereafter, the
neutrophils were cultured for 3 h, washed, and then analyzed as indicated.
A, Flow cytometric analysis of binding of annexin V-FITC (FL1) and PI
(FL2) in neutrophils. The relative distribution of cells manifesting early
(annexin⫹PI⫺) and late (annexin⫹PI⫹) apoptosis is illustrated (percentage); for clarity, the fluorescence profiles of 10% of the analyzed events are
shown. B, Morphological analysis of apoptosis. Cytospin preparations of
neutrophils were stained with Turck’s reagent (gentian violet) and examined under a light microscope; the micrograph shows neutrophils with condensed nuclei characteristic of apoptosis. C, Agarose gel electrophoresis of
internucleosomal DNA fragmentation in neutrophils. The molecular markers are indicated to the left (M). The results shown are representative of at
least five independent experiments. The proportions of apoptotic cells
(mean ⫾ SD) are indicated in Table I.
FIGURE 2. Photomicrograph of human monocyte-derived M␾ that
have ingested apoptotic neutrophils. The neutrophils are stained for MPO
and appear as condensed black bodies within the MPO-negative
macrophages.
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Uptake of uninfected apoptotic neutrophils is known to suppress
M␾ activation (4 –9). To determine whether neutrophils undergoing apoptosis provoked by bacteria have the same impact on M␾,
we compared the influence of bacteria- and UV-induced apoptotic
neutrophils on the inflammatory responses of M␾. Approximately
40% of the M␾ stained positively for neutrophils, and each positive M␾ had ingested ⬃1.2 neutrophils (Fig. 2 and Table I). In
agreement with previous reports (4 –9), M␾ that ingested UV-induced apoptotic neutrophils in our experiments displayed inhibited
production of the proinflammatory cytokine TNF-␣, whereas they
produced increased levels of anti-inflammatory TGF-␤1. In contrast, M␾ that phagocytosed bacteria-induced apoptotic neutrophils showed a markedly increased level of TNF-␣ and a slightly
decreased amount of TGF-␤1 (Fig. 3). Ingestion of apoptotic neutrophils elicited by E. coli appeared to augment IL-10 production
in M␾, but this increase was not statistically significant, because
the IL-10 level produced by unstimulated macrophages was low
(Fig. 3). Moreover, apoptotic neutrophils incubated in the absence
of M␾ did not generate any measurable levels of cytokines (data
not shown); thus, the cytokines we detected must have been produced by the M␾.
UPTAKE OF BACTERIA-INDUCED APOPTOTIC NEUTROPHILS ACTIVATES M␾
6322
Table I. Induction and rates of neutrophil apoptosis and phagocytosis
of apoptotic neutrophils by M␾a
Treatment of Neutrophils
UV
E. coli
Fixed E. coli/UV
S. aureus
Fixed S. aureus/UV
Neutrophil Apoptosis
(%)
M␾ with Ingested
Neutrophils (%)
54 ⫾ 8.9
51 ⫾ 13.9
42 ⫾ 7.9
44 ⫾ 8.2
42 ⫾ 8.1
41 ⫾ 3.0
42 ⫾ 0.7
40 ⫾ 2.5
40 ⫾ 2.8
39 ⫾ 4.5
a
Apoptosis was induced in neutrophils by UV irradiation, exposure to viable
bacteria, or both fixed bacteria exposure and UV irradiation as described in Materials
and Methods. Rates of apoptosis were measured by flow cytometry using annexin V.
Phagocytosis of apoptotic neutrophils by M␾ was determined by MPO staining. Each
value represents the mean ⫾ SD of results from at least five paired experiments.
Activation of M␾ by inflammatory apoptotic neutrophils is not
caused by bacteria associated with the neutrophils
Intact bacteria or bacterial components can elicit proinflammatory
responses in M␾. Accordingly, we performed three sets of experiments to determine whether activation of M␾ by inflammatory
neutrophils undergoing apoptosis is actually caused by the bacteria
that are associated with the neutrophils.
First, neutrophils were incubated with fixed E. coli or S. aureus
under the same conditions as those used with viable bacteria, and
this did not trigger apoptosis in the neutrophils (Table I). M␾ that
were allowed to interact with the neutrophils exposed to fixed bacteria produced only a moderate amount of TNF-␣ (Fig. 4). Only
dead neutrophils are ingested by M␾; thus, the divergent effects on
M␾ activation exerted by neutrophils exposed to fixed vs viable
bacteria may have been due to differences in the rates of apoptosis
exhibited by the neutrophils. To address that possibility, we also
irradiated neutrophils exposed to fixed bacteria to increase the apoptosis (Table I), but the M␾ that ingested those neutrophils still
displayed much lower production of TNF-␣ than M␾ incubated
with neutrophils undergoing apoptosis induced by viable bacteria
(Fig. 4).
Even though the neutrophils were washed extensively before
incubation with M␾, they still may have carried a small number of
bacteria that could have activated the M␾. Therefore, we performed a second set of experiments in which a CFU assay was
used to determine the number of bacteria associated with neutrophils (28). No viable bacteria were detected in the suspension of
apoptotic neutrophils before it was added to M␾, and ⬍5 ⫻ 104
CFU were found in 106 neutrophils immediately after exposure to
viable E. coli or S. aureus. Each well on the culture plate contained
⬃106 M␾ (8, 16), and ⬃40% of the M␾ contained neutrophils;
thus, we estimated that, at most, 2 ⫻ 104 bacteria had interacted
with the M␾ in each well. As shown in Fig. 4, TNF-␣ production
was only marginally increased in M␾ exposed to 5 ⫻ 105 viable
bacteria.
In the third set of experiments conducted to gain further evidence that neutrophil-associated bacteria did not interfere with activation of M␾, we measured LPS present in M␾ that had been
exposed to apoptotic neutrophils. M␾ were incubated with neutrophils undergoing apoptosis induced by viable E. coli or by fixed
E. coli and UV irradiation, which led to LPS levels of 0.19 ⫾ 0.11
and 0.11 ⫾ 0.03 ng/well, respectively (n ⫽ 5). However, adding
0.2 ng of LPS/well caused the M␾ to produce 300 ⫾ 31 pg of
FIGURE 3. Phagocytosis of bacteria-induced apoptotic neutrophils
stimulates production of TNF-␣ by human M␾. M␾ were incubated for 1 h
with neutrophils undergoing apoptosis induced by exposure to UV irradiation, S. aureus, or E. coli. Thereafter, the preparations were washed, the
supernatants were collected 18 h later and cytokine concentrations were
determined by ELISA. M␾ incubated with medium alone served as controls. The illustrated data represent the mean ⫾ SE of results from 12
separate experiments. ⴱ, p ⬍ 0.05; ⴱⴱ, p ⬍ 0.01 (compared with controls).
TNF-␣/ml, which is only ⬃10% of the level observed in M␾ exposed to viable E. coli-induced apoptotic neutrophils. These results
clearly indicate that very little of the activation of M␾ caused by
inflamed apoptotic neutrophils could have been induced by the
bacteria associated with the neutrophils. Thus, these apoptotic neutrophils must generate some other signals that can be sensed by
M␾ and consequently elicit active immune responses.
Increased expression of HSP in inflammatory apoptotic
neutrophils
It has been shown that certain members of the HSP family (e.g.,
HSP60, HSP70, and HSP90) can directly stimulate cells of the
innate immune system and thereby act as danger-signaling molecules (29 –35). Therefore, we performed Western blot analysis to
determine the amounts of HSP60, HSP70, and HSP90 expressed in
apoptotic neutrophils. The results revealed that freshly isolated
neutrophils contained all three of these proteins, and the level of
expression was not altered in neutrophils undergoing UV-induced
apoptosis (Fig. 5). In contrast, the expression of HSP60 and
HSP70, but not HSP90, was significantly increased in pathogeninduced apoptotic neutrophils (Fig. 5). Reprobing the blots with
anti-actin Ab confirmed that equal amounts of the proteins had
been loaded in each lane (data not shown).
Effect of heat-stressed apoptotic neutrophils and recombinant
HSP on activation of M␾
We used both heat-stressed apoptotic neutrophils and recombinant
HSP to determine whether the increased expression of HSP60 and
HSP70 in bacteria-induced apoptotic neutrophils played a role in
activation of M␾. Neutrophils that were exposed to mild heat exhibited increased apoptosis (55 ⫾ 6% cells stained positively with
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Fc␥RI expression was increased by ⬃60% in M␾ that had phagocytosed apoptotic neutrophils induced by E. coli or S. aureus
(mean fluorescence intensity, 716 ⫾ 52 and 696 ⫾ 30, respectively; n ⫽ 5).
The Journal of Immunology
annexin V and ⬍3% stained positively for PI; n ⫽ 5) and upregulated expression of HSP60 and HSP70 (Fig. 5), and M␾ that
ingested these neutrophils showed markedly increased production
of TNF-␣ (Fig. 6A). Moreover, such generation of TNF-␣ was
only marginally affected by the addition of polymyxin B, whereas
in parallel experiments the activity of LPS was completely blocked
by polymyxin B (Fig. 6B). These results indicate that heat-stressed
apoptotic neutrophils and LPS stimulate M␾ activation in
different ways.
Numerous investigations have shown that both HSP60 and
HSP70 potently provoke proinflammatory responses (29, 33–36),
but two recent studies of murine M␾ have suggested that the TNF␣-inducing activities of recombinant HSP preparations are due entirely to contaminating LPS (37, 38). Accordingly, to clarify the
role of HSP in activation of human M␾, we used highly purified
recombinant HSP60 and HSP70 preparations (ESP-540 and ESP555; StressGen Biotechnologies, Victoria, Canada), which contain
low levels of endotoxin and do not induce release of TNF-␣ from
murine M␾ (37, 38). In our experiments, both recombinant HSP60
and HSP70, at concentrations up to 5 ␮g/ml, caused only a slight
increase in TNF-␣ production by human M␾ (Fig. 7). Interestingly, recombinant HSP60 synergistically increased the LPS-stimulated release of TNF-␣ from M␾, and recombinant HSP70 had
the same effect, albeit not as pronounced (Fig. 7). HSP60 and
HSP70 had no additive effect in the presence or the absence of LPS
(Fig. 7).
We used the protein synthesis inhibitor cycloheximide to further
confirm that HSPs from apoptotic neutrophils are involved in activation of M␾. Pretreatment of neutrophils with cycloheximide
almost completely blocked the increased expression of HSP60 and
HSP70 induced by exposure to viable E. coli or heat stress (Fig.
8A). Furthermore, TNF-␣ production was significantly lower in
M␾ that had ingested the cycloheximide-treated apoptotic neutrophils (Fig. 8B).
FIGURE 5. Increased expression of HSP60 and HSP70 in neutrophils
undergoing bacteria-induced apoptosis. Neutrophils were left untreated or
were exposed to heat stress, UV irradiation, E. coli, or S. aureus. Thereafter, the neutrophils were cultured for 3 h and lysed. Equal amounts of
cellular proteins (15 ␮g/lane for HSP70; 35 ␮g/lane for HSP60 and
HSP90) were separated on 10% SDS-PAGE and blotted with Ab specific
against HSP60, HSP70, and HSP90. Each of the illustrated blots is representative of four separate experiments.
Discussion
Neutrophils are the first cells attracted to the site of bacterial invasion. These leukocytes are responsible for direct bactericidal activities, such as phagocytosis and generation of the respiratory
burst, and they can also regulate inflammatory responses by undergoing apoptosis (2). In our experiments, apoptotic neutrophils
induced by exposure to UV irradiation, E. coli, or S. aureus either
stimulated or inhibited the activation of M␾. We found that phagocytosis of bacteria-induced apoptotic neutrophils, but not uptake of
UV-induced apoptotic neutrophils, caused M␾ to increase both
production of the proinflammatory cytokine TNF-␣ and surface
expression of Fc␥RI. These findings indicate that bacteria-induced
neutrophil apoptosis represents a novel host defense mechanism
that aids the activities of local M␾ and links innate and adaptive
immune responses to achieve control of infection. Moreover, we
found that HSPs expressed by inflammatory apoptotic neutrophils
may constitute the signals required to generate a more effective
immune response.
Our results give important new insights into the significance of
neutrophil-macrophage interactions during inflammation. The dynamics of inflammation and changes in the inflammatory environment over time may have a substantial impact on the way that
apoptosis is induced in neutrophils, which, in turn, can lead to
either pro- or anti-inflammatory responses in M␾. In agreement
with previous reports (4 – 8, 25, 27), we noted that uptake of UVinduced apoptotic neutrophils actively suppressed stimulation of
M␾. However, as mentioned above, we also found that ingestion
of bacteria-induced apoptotic neutrophils caused M␾ to increase
production of TNF-␣ and expression of Fc␥RI. Our finding that
M␾ were activated by neutrophils undergoing apoptosis induced
by either extracellular (present study) or intracellular (16) pathogens clearly indicates that apoptosis that occurs in neutrophils after
exposure to bacteria represents a general host defense mechanism,
but not a strategy that invading pathogens use to kill immune cells.
Inasmuch as the short-lived neutrophils are the first cells to temporarily infiltrate the site of microbial invasion, infection of M␾
can occur indirectly through uptake of bacteria-laden apoptotic
neutrophils, instead of directly (i.e., by entry of bacteria themselves into the M␾), which might lead to different immune responses. For example, it has been shown that direct infection of
M␾ with mycobacteria blocks the responses of these phagocytes to
stimulation with IFN-␥ (24, 39), but we have previously shown
that M␾ produce much more TNF-␣ after ingesting mycobacteriainduced apoptotic neutrophils than after being directly infected
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FIGURE 4. Activation of M␾ by neutrophils undergoing bacteria-induced apoptosis is not caused by the bacteria associated with the neutrophils. The neutrophils were incubated with viable or fixed E. coli (A) or S.
aureus (B), or they were exposed to fixed bacteria and UV irradiation as
described in Materials and Methods. M␾ were subsequently coincubated
with the neutrophils for 1 h, after which the cultures were washed, and the
supernatants were collected 18 h later. M␾ incubated in medium alone or
only with bacteria (5 ⫻ 105/well) served as controls. TNF-␣ concentrations
in the culture supernatants were determined by ELISA. Values represent
the mean ⫾ SE from 10 separate experiments. ⴱⴱ, p ⬍ 0.01 (compared with
viable E. coli-induced apoptotic neutrophils).
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UPTAKE OF BACTERIA-INDUCED APOPTOTIC NEUTROPHILS ACTIVATES M␾
FIGURE 7. Recombinant HSP60 and HSP70 synergistically increase
LPS-stimulated release of TNF-␣ from M␾. Human M␾ were incubated in
medium alone or in medium containing 3 ␮g/ml HSP60, HSP70, or HSP60
and HSP70 in the presence (f) or the absence (䡺) of 0.2 ng/ml LPS for
18 h. Thereafter, the TNF-␣ concentration in the culture supernatants was
determined. Values represent the mean ⫾ SE from four separate experiments. ⴱⴱ, p ⬍ 0.01 (compared with LPS alone).
with M. tuberculosis (16). These findings suggest that bacteriainduced neutrophil apoptosis represents a novel link between innate and adaptive immune responses. This conclusion is supported
by recent studies in which it was observed that interactions between M␾ and inflammatory apoptotic neutrophils promoted the
killing of Leishmania major both in vitro and in vivo, whereas
uninfected apoptotic lymphocytes amplified the intracellular
growth of L. major and T. cruzi within M␾ (11, 19). In addition,
it has been found that depletion of neutrophils is associated with a
decreased Th1 immune response and increased bacteria growth in
various tissues (40 – 42).
It is not yet known exactly how M␾ can distinguish between and
react differently to inflammatory apoptotic neutrophils and UVinduced apoptotic neutrophils. It has been speculated that the pattern recognition receptors on M␾ enable the phagocytic synapse in
these cells to perceive and interpret molecular patterns on apoptotic cells, which determine how the M␾ respond (4, 5, 43). However, the pattern recognition receptors involved in binding of apoptotic cells overlap with those that recognize pathogens or
necrotic cells (4, 5, 11, 43). This is illustrated by the findings that
activation of M␾ is inhibited by binding of the CD14 or phosphatidylserine receptor to apoptotic cells, whereas M␾ are actually
activated by binding of CD14 to bacteria or by linking phosphatidylserine receptors to necrotic cells (11, 27, 43– 45). Moreover,
we noted that in the absence of serum, stimulation with LPS or
intact bacteria alone or with neutrophils that had been exposed to
fixed bacteria caused M␾ to produce only moderate amounts of
TNF-␣ that corresponded to ⬃10 –20% of the levels seen in M␾
stimulated by incubation with neutrophils undergoing apoptosis
induced by viable bacteria (Fig. 4). These results indicate that very
little of the activation of M␾ by inflammatory apoptotic neutro-
FIGURE 8. Cycloheximide inhibits the expression of HSPs in neutrophils undergoing bacteria- or heat-stress-induced apoptosis and reduces the
ability of those cells to stimulate production of TNF in M␾. Neutrophils
were preincubated in medium alone or in medium containing 10 ␮g/ml
cycloheximide (CHX) for 10 min and were subsequently exposed to viable
E. coli or heat stress, then cultured for 3 h. A, Expression of HSPs in these
neutrophils was determined by Western blot analysis as described in Fig.
5, and each of the illustrated blots is representative of four separate experiments. B, M␾ were incubated for 1 h with apoptotic neutrophils that had
(f) or had not (䡺) been pretreated with cycloheximide, after which the
cultures were washed, and supernatants were collected 18 h later. Values
represent the mean ⫾ SE from five separate experiments. ⴱⴱ, p ⬍ 0.01
(compared with the M␾ exposed to cycloheximide-treated neutrophils).
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FIGURE 6. Uptake of heat-stressed apoptotic neutrophils stimulates
production of TNF-␣ in M␾. A, Neutrophils were left untreated or were
exposed to mild heat (43°C) for 30 min, then cultured for 3 h. Those
neutrophils were subsequently coincubated with M␾ for 1 h, after which
the cultures were washed, and supernatants were collected 18 h later. M␾
incubated in medium alone or with LPS (0.2 ng/ml) served as controls. B,
M␾ were preincubated with (f) or without (䡺) 10 ␮g/ml polymyxin B
(PB) for 10 min, after which LPS or neutrophils were added. Values represent the mean ⫾ SE from four separate experiments. ⴱⴱ, p ⬍ 0.01 (compared with control M␾ in A and with M␾ treated with polymyxin in B).
phils could have been induced by the bacteria that were associated
with the neutrophils. Thus, inflammatory apoptotic neutrophils
themselves must generate additional signals that can be sensed by
M␾ and consequently provoke active immune responses.
The deficiency of self/non-self paradigm has led to new hypotheses proposed by Janeway and Matzinger (46, 47), in that the immune system is more concerned with damage than with foreignness. According to this paradigm, the immune system is thought to
The Journal of Immunology
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