Decreased Alveolar Macrophage Apoptosis Is Associated with
Increased Pulmonary Inflammation in a Murine Model of
Helen M. Marriott, Paul G. Hellewell, Simon S. Cross, Paul G. Ince, Moira K. B. Whyte, and
David H. Dockrell2
Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature
of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial
infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have
explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO
synthase (iNOS)-deficient mice. iNOS?/?mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type
C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-?.
Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early
bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS?/?mice. Labeled apoptotic alveolar
decreased neutrophil recruitment in iNOS?/?mice and decreased TNF-? mRNA in lungs and protein in bronchial alveolar lavage, as
well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic.
This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo
demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during
pneumonia. The Journal of Immunology, 2006, 177: 6480–6488.
constitutive apoptosis (2). Modulation of macrophage lifespan is,
however, an important mechanism for regulation of macrophage
function. Although multiple pathogens induce macrophage apo-
ptosis as a mechanism of immune evasion, the existence of host
benefits from macrophage apoptosis has been more controversial
(3). Nevertheless, it has become apparent that macrophage apoptosis
can represent a host response that contributes to bacterial killing of
chronic intracellular pathogens (4) and organisms that do not persist
for prolonged periods intracellularly but are killed efficiently after
phagocytosis (5). However, the functional consequences of host-in-
duced macrophage apoptosis are incompletely characterized.
Infection with the Gram-positive diplococcus Streptococcus
pneumoniae represents the most frequent cause of community ac-
quired pneumonia (6). Although anti-pneumococcal Ab is critical
in determining the outcome of infection (7), the importance of
innate responses is increasingly recognized (8). Tissue macro-
phages, including alveolar macrophages (AM),3are critical to the
acrophages play a critical role during bacterial infec-
tion by coordinating the innate immune response (1)
and are long-lived tissue cells with a low incidence of
innate response, phagocytosing bacteria, and coordinating the
innate response to infection (1). AM depletion results in a reduc-
tion in the number of pneumococci that are required to trigger
neutrophil recruitment to the lung (9). During established pneu-
monia, AM also contribute to resolution of the inflammatory re-
sponse but are no longer critical for bacterial clearance because
neutrophils become the major cell phagocytosing bacteria (10).
Host-mediated macrophage apoptosis is a feature of pneumo-
coccal infection and its inhibition in vitro decreases pneumococcal
clearance (5). In murine models, decreased AM apoptosis is asso-
ciated with greater rates of invasive pneumococcal disease (9). We
have previously identified a key role for NO production in the
activation of the apoptotic cascade during pneumococcal disease
(11). Apoptosis is believed to contribute to resolution of inflam-
mation in the lung and pneumococcal pneumonia has been re-
garded as the paradigm for a host response that involves pulmo-
nary inflammation with subsequent complete resolution without
lung injury (12). The role of neutrophil apoptosis is well-estab-
lished but AM apoptosis may also facilitate resolution of the in-
flammatory response in the lung. Macrophages produce anti-
inflammatory cytokines after phagocytosing apoptotic bodies (13),
although the pattern of cytokines produced is modified in the pres-
ence of TLR ligation (14). It remains unclear whether macrophage
apoptosis influences the inflammatory response during pneumonia.
In view of the important roles of macrophages and of host-medi-
ated apoptosis in host defense against S. pneumoniae, pneumococ-
cal infection represents a relevant model with which to investigate
the effects of macrophage apoptosis on regulation of the inflam-
matory response. Because we have identified a critical role for NO
in regulating initiation of macrophage apoptosis during pneumo-
coccal infection, we chose to study the effect of decreasing mac-
rophage apoptosis in vivo using the well-characterized inducible
NO synthase (iNOS)-deficient mouse (15). On the basis of our
School of Medicine and Biomedical Sciences, University of Sheffield, Sheffield,
Received for publication March 22, 2006. Accepted for publication August 22, 2006.
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.
1This work was supported by Wellcome Trust Advanced and Senior Clinical Fel-
lowships (to D.H.D.), Nos. 065054 and 076945.
2Address correspondence and reprint requests to Dr. David H. Dockrell, Division of
Genomic Medicine, F-Floor, University of Sheffield, Beech Hill Road, Sheffield S10
2RX, U.K. E-mail address: firstname.lastname@example.org
3Abbreviations used in this paper: AM, alveolar macrophage; iNOS, inducible NO
synthase; BAL, bronchial alveolar lavage; DAPI, 4?,6?-diamidino-2-phenylindole;
qRT-PCR, quantitative RT-PCR; PMN, polymorphonuclear neutrophil.
The Journal of Immunology
Copyright © 2006 by The American Association of Immunologists, Inc.0022-1767/06/$02.00
prior in vitro findings, we predicted that these mice could have
decreased AM apoptosis and this model could then be examined to
determine the effects of decreased AM apoptosis, as opposed to
NO deficiency, on the regulation of the inflammatory response,
using a model of pneumococcal pneumonia.
We demonstrate that iNOS?/?mice have decreased rates of AM
apoptosis in vivo and that this is associated with a greater degree
of inflammation in the lung. Furthermore, we demonstrate that
instillation of apoptotic macrophages into mouse lungs decreases
the greater lung inflammation observed in iNOS?/?mice and de-
creases the likelihood of early invasive pneumococcal disease.
Materials and Methods
iNOS-deficient mice backcrossed onto a C57BL/6 background were ob-
tained from The Jackson Laboratory and maintained as a homozygous
colony. C57BL/6 mice (Harlan U.K.) were used as wild-type controls.
Female mice were used throughout. For aminoguanidine treatment,
C57BL/6 mice were treated with 2.5% aminoguanidine in their drinking
water, with 5% glucose added to increase palatability, starting 7 days be-
fore pneumococcal infection (16). Control mice were treated with an equiv-
alent dose of glucose. All animal experiments were conducted in accor-
dance with the Home Office Animals (Scientific Procedures) Act of 1986
and received local ethical committee approval.
In vivo pneumococcal infection model
Infection of mice with 107CFU of type 2 pneumococci (strain D39) or
mock infection with PBS was by direct tracheal instillation after anesthesia
with ketamine (100 mg/kg i.p.) and acepromazine (5 mg/kg i.p.) as previ-
ously described (9). In survival studies, mice showing physical signs of
severe illness were culled and their time of death recorded as the time at
which they were culled.
Collection of bronchial alveolar lavage, blood, and lungs
Mice were killed by an overdose of sodium pentobarbitone and exsangui-
nated by cardiac puncture. Bronchial alveolar lavage (BAL) was performed
as described (9). The cell differential was by review of cytospin prepara-
tions (9). Viable bacterial counts in lung and blood were obtained as de-
scribed previously (17).
Detection of apoptosis
Apoptosis detection was by Annexin V (BD Biosciences)/ToPro 3 (Mo-
lecular Probes) staining and flow cytometry or by nuclear morphology on
cytospin preparations as described (9). AM were identified by F4/80 flu-
orescence intensity (anti-mouse F4/80:FITC (CI:A3-1 clone); Serotec) and
forward vs side scatter characteristics and neutrophils by positive Ly6G
staining (anti-mouse Ly6G:FITC (1A8 clone); BD Biosciences) (9, 18).
Cytokine/chemokine mRNA detection
Whole lung was harvested and immediately stored in RNAlater (Sigma-
Aldrich). Total RNA was extracted from lung tissue by homogenization
into TRIzol (Invitrogen Life Technologies) following the manufacturer’s
protocol. RNase protection assays were performed using 5 ?g of RNA and
mCK-2b, mCK-5c, and custom probe sets according to the RiboQuant
protocol (BD Biosciences). Bands were quantitated using Image J 1.32
software (National Institutes of Health) and were normalized to GAPDH.
To perform TaqMan quantitative real-time PCR (qRT-PCR), RNA was
DNA digested (Ambion) following the manufacturer’s protocol for routine
DNase treatment. cDNAs were synthesized from RNA by reverse tran-
scription using random hexamer primers. Relative expression of TNF-?
normalized to ?-actin was by TaqMan gene expression assays (Applied
Biosystems) for TNF-? (Mm00443258_m1) and ?-actin (Mm00607939_s1)
following the manufacturer’s protocol.
Cytokines in BAL were measured using DuoSet ELISA development kits
(R&D Systems) for mouse TNF-?, KC (CXCL1), MIP-2 (CXCL2), and
IL-6 following the manufacturer’s protocols (19). Limits of detection were
Protein levels in BAL were measured using a BCA protein assay (Pierce),
following the manufacturer’s protocol (20).
Unlavaged lungs were fixed via the trachea with 10% buffered formalin at
20 cm H2O, paraffin-embedded sections prepared, sectioned, stained with
H&E and independently evaluated by two pathologists (P. G. Ince and S. S.
Cross) using a BH2 Olympus microscope.
Instillation of apoptotic bodies
Apoptotic AM were generated from AM obtained from BAL of donor
C57BL/6 mice. BAL was spun for 5 min at 1000 ? g and cells resuspended
in RPMI 1640 (Invitrogen Life Technologies) plus 10% heat-inactivated
FCS (Bioclear), 100 ?g/ml streptomycin, and 100 U/ml penicillin (Invitro-
gen Life Technologies) and incubated at 37°C for 4 h to allow macrophage
adherence. AM were washed and labeled with 2 ?M CellTracker Red
(Molecular Probes) for 30 min at 37°C. Apoptosis was induced by expo-
sure to 120 mJ/cm2irradiation (Stratalinker 1800; Stratagene). Twelve
hours post-UV treatment, AM were gently scraped and washed in PBS.
After this treatment, mean values for AM were 69% Annexin V?, 8%
ToPro3?, and 65% showed loss of ??m(9, 11). In additional experiments,
AM were made apoptotic by treatment with 10 ?M staurosporine for 16 h.
Cells were resuspended in PBS at 5 ? 106cells/ml. A total of 1 ? 104
apoptotic AM were delivered to the lungs by direct intratracheal instillation
immediately after pneumococcal infection. This number was chosen to
provide approximately enough apoptotic cells to restore the numbers of
apoptotic cells in the iNOS?/?mice to the level observed in the C57BL/6
mice. Twelve to 48 hours postinfection, mice were killed and BAL per-
formed. To confirm phagocytosis of apoptotic bodies, resident AM of some
C57BL/6 mice were labeled with PKH2 green fluorescent phagocytic cell
linker compound (Sigma-Aldrich) (18). PKH2 dye (stock 1 ? 10?3M) was
diluted 1/5 with diluent B and 100 ?l was administered i.v. Forty-two
hours, post-PKH2 treatment apoptotic AM were delivered to the lungs by
direct intratracheal instillation. Lungs were lavaged 30 min postinstillation
and cytospin preparations made from lung cells. Coverslips were mounted
with VectaShield containing 4?,6?-diamidino-2-phenylindole (DAPI; Vec-
tor Laboratories), and the cells imaged with a DeltaVision Microscope.
Ex vivo AM pneumococcal infection
C57BL/6 and iNOS-deficient mice were killed with an overdose of sodium
pentobarbitone and BAL was performed as described using RPMI 1640
(Invitrogen Life Technologies) plus 10% heat-inactivated FCS (Bioclear),
100 ?g/ml streptomycin, and 100 U/ml penicillin (Invitrogen Life Tech-
nologies) (9). BAL was spun for 5 min at 1000 ? g and cells resuspended
in RPMI 1640 plus 10% heat-inactivated FCS without antibiotics and in-
cubated at 37°C for at least 4 h to allow macrophage adherence. Infection
was with type 1 pneumococci opsonized with serum from mice immunized
with pneumovax vaccine at a multiplicity of infection of 10 (5). Killing was
assessed by analyzing colony counts in supernatants at 4 and 20 h postin-
fection (21). Apoptosis was assessed at 20 h postinfection by nuclear mor-
phology after DAPI staining as described (21).
Results are recorded as mean and SEM. Survival was calculated by
Kaplan-Meier followed by log-rank analysis. Parametric or nonparametric
testing was performed with the indicated tests using Prism 4.0 software
(GraphPad). Significance was defined as p ? 0.05.
AM apoptosis is reduced in iNOS?/?mice during pneumococcal
Because NO contributes to apoptosis of human macrophages dur-
ing pneumococcal infection in vitro (11), but important differences
exist between rodent and human macrophages with regard to the
level of NO produced (22), we first confirmed that NO also played
a role in apoptosis in murine AM when cultured ex vivo and chal-
lenged with pneumococci. As shown, murine AM from iNOS?/?
mice demonstrated lower levels of apoptosis (Fig. 1A) and de-
creased killing of bacteria (Fig. 1B) as compared with wild-type
cells, thus confirming a similar phenotype to that previously found
in human macrophages (11). We next examined levels of macro-
phage apoptosis in vivo in iNOS?/?mice (15). Pneumococcal-
infected C57BL/6 mice had significantly greater percentages of
apoptotic cells in BAL in comparison to mock-infected mice (Fig.
6481The Journal of Immunology
2A). Pneumococcal-infected iNOS?/?mice, however, had signif-
icantly fewer apoptotic cells in BAL than did pneumococcal-
infected C57BL/6 mice (Fig. 2A). Because the BAL fluid from the
pneumococcal-infected mice contained significant numbers of
neutrophils at each time point, we determined the level of apopto-
sis in the macrophage population by flow cytometry (9). There
were greater percentages of apoptotic macrophages in pneumococ-
cal-infected C57BL/6 mice than in mock-infected mice, but the
levels of apoptotic macrophages were significantly decreased in
iNOS?/?mice as opposed to C57BL/6 mice (Fig. 2B). When ap-
optosis was measured in neutrophils, we also found evidence of
decreased neutrophil apoptosis in iNOS?/?mice as opposed to
C57BL/6 mice after pneumococcal infection (Fig. 2C).
Effect of iNOS deficiency on neutrophil numbers in BAL
There was a significant increase in the number of neutrophils in
BAL from iNOS?/?mice as compared with C57BL/6 mice 24–48
h postinfection (Fig. 3A). Although there was evidence of in-
creased neutrophil viability in iNOS?/?mice (reduced neutrophil
apoptosis (Fig. 2C) and no increase in necrosis, data not shown),
increased recruitment was also likely to contribute to increased
neutrophil numbers. The iNOS?/?mice had no significant in-
crease in bacteria in the lung at 12–48 h (Table I), excluding the
possibility that a significantly greater bacterial load was the stim-
ulus for neutrophil recruitment. Similarly, peripheral blood neu-
trophil counts were similar between C57BL/6 mice and iNOS?/?
mice, suggesting an intrinsic difference in neutrophil numbers did
not explain the differences in numbers of BAL neutrophils after
infection, data not shown.
Neutrophil recruitment in pneumococcal infection demonstrates
important differences as compared with other stimuli such as LPS
with a prominent upstream role for TNF-? (23). We performed
RNase protection assays on lungs 12 h after infection to identify
cytokines and chemokines that were up-regulated in iNOS?/?
mice and were able to demonstrate significant up-regulation of
TNF-? message (Fig. 3, B and C). We also observed up-regulation
of mRNA for the chemokines KC and MCP3, (Fig. 3, B and C),
chemokines known to be associated with neutrophil recruitment in
pneumococcal infection (23) and in iNOS deficiency (24). Mea-
surement of cytokines in BAL revealed iNOS?/?mice had a sig-
nificant increase in TNF-? production relative to C57BL/6 mice
12 h postinfection and a nonsignificant trend toward greater pro-
duction at 24 h following pneumococcal infection (Fig. 3D). Lev-
els of TNF-? in PBS-treated mice of both strains were below the
level of detection. KC values in BAL fluid 12 h after infection
from iNOS?/?vs C57BL/6 mice were 803 ? 28.6 vs 578.8 ?
96.58 pg/ml (p ? 0.05, n ? 4).
Pharmacologic inhibition of iNOS modulates macrophage
apoptosis and neutrophil recruitment
To investigate whether these findings were related to decreased
production of NO and/or one of its reaction products or whether
they resulted from some nonspecific effect associated with iNOS
deficiency, for example, increased levels of a cofactor or metabolite
required for the functional activity of iNOS, we inhibited iNOS ac-
tivity pharmacologically. Inhibition with aminoguanidine, a specific
inhibitor of iNOS (25), replicated the findings in iNOS?/?mice with
decreased total or macrophage apoptosis and increased neutrophil
numbers or TNF-? levels in BAL all observed (Fig. 4).
iNOS?/?mice demonstrate enhanced levels of lung
inflammation and bacteremia
The increased numbers of neutrophils and proinflammatory cyto-
kines in the lung in iNOS?/?mice were associated with enhanced
early mortality in the first 42 h after infection, 22.0% iNOS?/?vs
4.9% C57BL/6 mice, p ? 0.05, Fisher’s exact test, but this was not
associated with a significant decrease in clearance of bacteria from
ptosis and bacterial killing cells after pneumococcal challenge ex vivo. A,
Percentage apoptotic cells in cultures of AM isolated from wild-type
(C57BL/6) and iNOS-deficient mice (iNOS?/?) 20 h after in vitro challenge
with opsonized type 2 pneumococci (Spn), n ? 6. B, Concentration of bacteria
challenge with opsonized type 2 pneumococci (Spn), n ? 3. Data are derived
from triplicate points and are representative of two independent experiments.
Mean ? SEM, ?, p ? 0.05, ???, p ? 0.001, t test; f, C57BL/6; ?, iNOS?/?.
iNOS deficiency is associated with a reduction in AM apo-
cells in bronchial lavage fluid after pneumococcal infection. A, The per-
centage of apoptotic events (apoptotic cells and bodies) in cytospins of
bronchial alveolar lavage (BAL) from wild-type controls (C57BL/6) and
iNOS-deficient mice (iNOS?/?) at the indicated time points after instilla-
tion of 107CFU of type 2 pneumococci (Spn) or PBS. Percentage of (B)
macrophage (AM) or (C) neutrophil (PMN) apoptosis (annexin V-PE?/
ToPro 3?, flow cytometry) in BAL from the same experiments as A; 12 h,
n ? 6–8, 24 h, n ? 3–9; 48 h, n ? 5–16. Mean ? SEM, ?, p ? 0.05, ??,
p ? 0.01, ???, p ? 0.001, t test;
, C57BL/6 PBS; ^, iNOS?/?PBS; f,
C57BL/6 Spn; ?, iNOS?/?Spn.
iNOS deficiency is associated with reduction in apoptotic
6482 MACROPHAGE APOPTOSIS REDUCES INFLAMMATION IN PNEUMONIA
the lung (Table I). However, by 10 days there was no difference in
overall mortality in the two groups of mice 54% iNOS?/?vs 48%
C57BL/6 mice (Fig. 5A). We have previously shown that de-
creased levels of macrophage apoptosis are associated with inva-
sive pneumococcal disease (5, 9) and in keeping with this finding
iNOS?/?mice had ?1 log higher bacterial colony counts in the
blood at 12–48 h after infection (Fig. 5B). IL-6 production is a
marker of sepsis-related mortality and poor outcomes in models of
pneumococcal disease (26) and increased IL-6 production in the
lungs of pneumococcal-infected mice was apparent 48 h after in-
fection (Fig. 5C). In both strains, the levels of IL-6 were below the
limit of detection after PBS treatment. iNOS?/?mice also dem-
onstrated increased levels of protein leak into the BAL, in keeping
with greater lung inflammation (Fig. 5D). In keeping with these
findings, lung histology showed a greater degree of neutrophilic
lung inflammation in the iNOS?/?mice, together with a greater
degree of disruption to the alveolar units, suggesting greater epi-
thelial cell injury (Fig. 6).
Macrophages in iNOS?/?mice contain fewer apoptotic bodies
Because there are fewer apoptotic cells in the lungs of iNOS?/?
mice, we addressed whether this translated into a difference in the
percentage of macrophages phagocytosing apoptotic bodies in
vivo. The phagocytic capacity of AM for apoptotic cells in vitro is
low in comparison to the capacity to ingest opsonized particles
(18) or the capacity of peritoneal macrophages to phagocytose ap-
optotic cells (27). Nevertheless, macrophages with internalized ap-
optotic cells are observed in BAL from mice with pneumococcal
infection (9). We estimated the percentage of extracellular apopto-
tic cells per cytospin, i.e., apoptotic cells that had not been phago-
cytosed by macrophages (Fig. 7A), and the percentage of intracel-
lular apoptotic cells (Fig. 7B). Although, over all the time points
studied, only a relatively low percentage of macrophages (C57BL/
6 4.4 ? 0.3%; iNOS?/?5.7 ? 0.5%) contained apoptotic cells
these apoptotic cells accounted for a significant percentage of the
total apoptotic events (C57BL/6 44 ? 2.4%; iNOS?/?55.7 ?
3.5%), arguing in favor of a relatively efficient clearance mecha-
nism for apoptotic cells in vivo during pneumococcal infection. As
illustrated, there were a lower percentage of intracellular apoptotic
bodies in iNOS?/?mice at all time points (Fig. 7B).
Instillation of apoptotic macrophages into the lung corrects
markers of greater lung inflammation in iNOS?/?mice
Because ingestion of apoptotic cells can down-regulate macro-
phage production of proinflammatory cytokines (13), we tested
whether exogenous apoptotic macrophages could decrease the pul-
monary inflammation observed during pneumococcal infection.
UV treatment induces high levels of macrophage apoptosis (5) and
efficiently induced AM apoptosis. A suspension of apoptotic AM
had no residual antimicrobial effect as there was no difference be-
tween bacterial colony counts in the lung 24 h after infection in the
presence or absence of these cells: median and interquartile range
for C57BL/6 mice in the presence of apoptotic cells 1.6 ? 106
CFU (0.5 103–1.3 ? 108) vs 1 ? 105CFU (8.3 ? 103–8.3 ? 107)
in the absence of apoptotic cells (n ? 11, p ? 0.81), Mann-Whit-
ney. Instilled Cell Tracker Red-labeled apoptotic AM were phago-
cytosed by resident AM labeled with the green fluorescent dye,
PKH2 (Fig. 8A). The greater numbers of recruited neutrophils in
the lungs of iNOS?/?mice could be the direct result of NO de-
ficiency increasing production of proinflammatory cytokines (28,
29). However, instillation of apoptotic AM decreased inflamma-
tion in the lung (Fig. 8B). There were decreased numbers of re-
cruited neutrophils in the lung 12–48 h after instillation of apo-
ptotic macrophages in iNOS?/?mice and to a lesser extent in
C57BL/6 mice (Fig. 8B). In the iNOS?/?mice, instillation of ap-
optotic AM reduced the numbers of neutrophils in the lung to a
level comparable to the number that were recruited into C57BL/6
Table I. Microbiological outcomes
12 h48 h
1.8 ? 105(1.5 ? 105? 3.1 ? 105), 100%, n ? 17
2.0 ? 105(1.2 ? 105?2.8 ? 105)a, 100%b, n ? 17
2.0 ? 104(5.0 ? 102? 3.0 ? 107), 81%, n ? 21
1.7 ? 106(8.3 ? 103? 2.5 ? 107), 87%, n ? 23
aMedian (interquartile range).
bPercent with detectable bacteria.
bronchial alveolar lavage from lungs of iNOS?/?mice
after pneumococcal infection. A, Numbers of neutro-
phils (#PMN) in bronchial alveolar lavage (BAL) from
wild-type controls (C57BL/6) and iNOS-deficient mice
(iNOS?/?) at the indicated time points after intratra-
cheal instillation of 107CFU of type 2 pneumococci. B,
Representative RNase protection assay of RNA from
whole lung from wild-type controls (C57BL/6) and
iNOS deficient mice (iNOS?/?) 12 h postinstillation of
107CFU of type 2 pneumococci. C, Levels of TNF-?,
?C, and monocyte chemotactic protein-3 (MCP3)
mRNA as quantified by densitometry and normalized to
GAPDH, n ? 4. D, TNF-? concentration in BAL in the
same experiments as A; 12 h, n ? 11–13; 24 h, n ?
8–14; 48 h, n ? 13–16. Mean ? SEM, ?, p ? 0.05, ??,
p ? 0.01, ???, p ? 0.001, t test; f, C57BL/6; ?,
Increased numbers of neutrophils in the
6483The Journal of Immunology
mice not instilled with apoptotic AM. Results were identical re-
gardless of whether AM were derived from iNOS?/?or C57BL/6
mice and in preliminary data if AM were made apoptotic by stauro-
sporine treatment as opposed to UV exposure (data not shown).
Therefore, decreased numbers of apoptotic macrophages contribute to
the increased lung inflammation during pneumococcal infection of
iNOS?/?mice and instillation of additional apoptotic macrophages
reverses the proinflammatory phenotype associated with NO defi-
ciency following pulmonary challenge with pneumococci.
Effect of apoptotic cells on TNF-? expression in iNOS?/?mice
In keeping with the pivotal role of TNF-? expression in recruit-
ment and activation of neutrophils during pneumococcal pneumo-
nia (23), we found that TNF-? mRNA expression in the lung was
reduced by instillation of apoptotic cells in iNOS?/?mice (Fig. 9,
A and B). Similar findings were observed by qRT-PCR (Fig. 9C).
This was confirmed by significant reduction in TNF-? expression
mococcal infection. A, Survival of wild-type iNOS-sufficient mice
(C57BL/6) and iNOS-deficient (iNOS?/?) mice after intratracheal instil-
lation of 107CFU of type 2 pneumococci, n ? 23–24/group. B, Concen-
tration of bacteria in blood of C57BL/6 and iNOS?/?mice 12 and 48 h after
intratracheal instillation of 107CFU of type 2 pneumococci. The percentage of
was 61 vs 81%. C and D, Concentration of (C) IL-6 and (D) protein in bron-
chial alveolar lavage (BAL) 48 h after intratracheal instillation of 107CFU of
type 2 pneumococci, n ? 8–16. Mean ? SEM, ?, p ? 0.05, t test, ??, p ?
0.01, ???, p ? 0.001, Mann-Whitney; f, C57BL/6; ?, iNOS?/?.
Increased lung inflammation in iNOS?/?mice after pneu-
iNOS?/?mice after pneumococcal infection. Representative appearances
of lung sections stained with H&E from C57BL/6 and iNOS-deficient mice
(iNOS?/?) 48 h after intratracheal instillation of 107CFU of type 2 pneu-
mococci as viewed by ?10 and ?40 objective. Images were obtained from
one of four mice in each group reviewed by two independent pathologists.
Histologic appearance of increased lung inflammation in
increases pulmonary inflammation after pneumococcal
infection. A, The percentage of apoptotic events (apo-
ptotic cells and bodies) in cytospins of bronchial alve-
olar lavage (BAL) from C57BL/6 mice 24 h after in-
stillation of 107CFU of type 2 pneumococci (Spn), in
the absence (H2O) or presence of aminoguanidine (AG)
treatment. B, Representative dot plots to show the per-
centage macrophage apoptosis (annexin V-PE?/ToPro
3?cells as determined by flow cytometry). C, Percent-
age of apoptotic macrophages. D, Number of recruited
neutrophils (#PMN); E, TNF-? concentration in BAL in
the same experiments as A; n ? 8. Mean ? SEM, ??, p ?
0.01, ??? p ? 0.001, t test; f, H2O; dark gray box, AG.
iNOS inhibition reduces apoptosis and
6484MACROPHAGE APOPTOSIS REDUCES INFLAMMATION IN PNEUMONIA
by ELISA, following instillation of apoptotic cells to iNOS?/?
mice (Fig. 9D). These alterations were persistent for up to 48 h
after infection (data not shown). Further screen of lung RNase
protection assays and BAL ELISAs showed other changes in early
cytokine/chemokine expression such as changes in IL-1 isoforms,
MCP3 (data not shown), KC (Fig. 10A) and MIP-2 (Fig. 10B), but
these differences were not as marked as for TNF-? expression.
IL-6 levels were markedly reduced following instillation of apo-
ptotic cells (Fig. 10C). Further support for the beneficial effect of
phagocytosis of apoptotic macrophages in iNOS?/?mice infected
with pneumococci comes from the observation that reduction in
the number of neutrophils and cytokines such as TNF-? and IL-6
expression was associated with a decreased likelihood of mice de-
veloping invasive pneumococcal disease after instillation of apo-
ptotic cells (Fig. 11).
We demonstrate decreased rates of macrophage apoptosis in the
lungs of iNOS?/?and aminoguanidine-treated mice during pneu-
mococcal infection. The decrease in macrophage apoptosis in
iNOS?/?mice is associated with development of earlier bactere-
mia and death, and also with increased markers of inflammation in
the lung. Instillation of apoptotic macrophages into the lungs of
iNOS?/?mice reversed the features of increased inflammation and
reduced the development of invasive bacterial disease.
The potential benefits of host-mediated macrophage apoptosis in
the innate response to infection are not fully understood. Apoptosis
is a mechanism which removes unwanted cells, thus limiting in-
flammation and tissue injury (30). It is plausible that macrophage
apoptosis during bacterial infection plays a role in down-regulating
the inflammatory response in addition to its role in enhancing mi-
crobial killing (5). The innate response to pneumococci involves
pulmonary inflammation (31), induction of host-mediated macro-
phage apoptosis (9) and a requirement for resolution of the inflam-
matory response for a successful outcome (12). Because iNOS?/?
mice have decreased levels of macrophage apoptosis during pneu-
mococcal infection, we anticipated this model would provide in-
sights into the relationship between regulation of inflammation and
induction of macrophage apoptosis.
The roles of NO in murine models of pneumococcal pneumonia
have been conflicting reflecting the variety of strains of mice stud-
ied, varying doses and strains of bacteria, and the use of pharma-
cologic agents as opposed to genetically modified mice (32–34).
Our findings with type 2 pneumococci have been reproduced with
type 1 pneumococci, demonstrating that the findings of less mac-
rophage apoptosis and greater neutrophil infiltration and TNF-?
expression in the lung were not specific to the strain we used (data
not shown). Although we have previously found that NO contrib-
utes to macrophage killing of pneumococci (11), we have also
demonstrated that during low-dose infection other elements of the
host response, including recruited neutrophils can compensate for
decreased bacterial killing by AM (9). Clearly NO is only one
factor involved in both microbicidal killing and AM apoptosis in-
duction and other factors also contribute. Furthermore, NO also
contributes to host defense by mechanisms independent of its ef-
fects on macrophage function (28, 35). On the basis of our findings
of increased inflammation in iNOS?/?mice, we cannot exclude a
role for NO produced in other cells for the phenotype we observed.
However, we show that removal of one factor involved in host
defense resets the AM response, and indeed the total innate host
response in the lung, leading to an altered threshold at which the
next element in the host response becomes critical to controlling
infection. As Kerr et al. (34) have clearly illustrated at intermediate
doses of pneumococci (albeit in mice of a different genetic back-
ground to those we studied), host defense is unable to compensate
for NO deficiency and larger numbers of bacteria are recovered
neutrophil numbers in bronchial alveolar lavage after pneumococcal infec-
tion. A, Resident AM of C57BL/6 mice were labeled in vivo with PKH2
green fluorescent phagocytic cell linker compound. AM from donor mice
were labeled in vitro with CellTracker Red and apoptosis induced by UV
irradiation. Apoptotic AM were delivered by intratracheal instillation.
Lungs were lavaged 30 min postinstillation and cytospin preparations made
from lung cells. AM nuclei were counterstained with DAPI. Arrows indi-
cate red apoptotic donor cells located in green resident AM. B, Numbers of
neutrophils (#PMN) in BAL from wild-type controls (C57BL/6) iNOS-defi-
cient mice (iNOS?/?) at the indicated time points after instillation of 107CFU
of type 2 pneumococci in the absence or presence of apoptotic AM (AC), n ?
3–7. Mean ? SEM, ? p ? 0.05, ?? p ? 0.01, t test; f, C57BL/6; dark gray
box, C57BL/6 AC; ?, iNOS?/?; light gray box, iNOS?/?AC.
Instillation of apoptotic macrophages into lungs reduces
coccal infection. A, The percentage of extracellular apoptotic cells (AC)
and (B) intracellular AC in cytospins of BAL from wild-type controls
(C57BL/6) and iNOS-deficient mice (iNOS?/?) at the indicated time points
after instillation of 107CFU of type 2 pneumococci (Spn) or PBS, n ? 4–16.
Mean ? SEM, ?, p ? 0.05,??, p ? 0.01, ???, p ? 0.001, t test;
PBS; o, iNOS?/?PBS; f, C57BL/6 Spn; ?, iNOS?/?Spn.
Macrophages phagocytose apoptotic cells during pneumo-
6485The Journal of Immunology
from the lungs of iNOS?/?mice. In the current study, we have
used a high dose of pneumococci and have been unable to docu-
ment any overall defect in bacterial killing in the lungs but have
demonstrated greater lung inflammation, consistent with prior ful-
minant infection models using NO inhibitors (33). Our inability to
demonstrate a clear microbiologic or survival difference in
iNOS?/?mice suggests significant redundancy and compensation
exists in this aspect of innate immunity in the lung and also that,
at the high doses we used, the compensatory measures are over-
whelmed so they are no longer the critical determinant of outcome
in the setting of iNOS deficiency.
Nevertheless, decreased macrophage apoptosis, in the setting of
decreased NO production, was associated with increased levels of
bacteremia, in keeping with our previous observation that inhibition
of macrophage apoptosis is associated in particular with increased
levels of invasive pneumococcal disease (5, 11). Importantly, instil-
lation of apoptotic AM in iNOS?/?mice reversed many of the fea-
tures of increased inflammation, even though these mice had the same
defects in NO production in macrophages and other cells as the
iNOS?/?mice that did not receive apoptotic cells. This suggests that
a relative deficiency in numbers of apoptotic cells and their clearance
is a significant contributory factor to increased lung inflammation and
is multifactorial and many other factors play a role.
NO has a wide range of actions including cell signaling function
(36). The proinflammatory effects of NO deficiency in the lung
have been demonstrated both by pharmacologic inhibition of
iNOS, and also by study of iNOS?/?mice (37). NO modifies
mice reduces bacteremia after pneumococcal infection. Percentage of wild-
type controls (C57BL/6) and iNOS-deficient mice (iNOS?/?) with detectable
bacteremia 12 h postinstillation of 107CFU of type 2 pneumococci in the
absence or presence of apoptotic AMs (AC), n ? 4–5. f, C57BL/6; dark gray
box, C57BL/6 AC; ?, iNOS?/?; light gray box, iNOS?/?AC.
Instillation of apoptotic macrophages into lungs of iNOS?/?
iNOS?/?mice reduces TNF-? levels in lung and bronchial alveolar lavage
after pneumococcal infection. A, Representative RNase protection assay of
RNA from whole lung from wild-type controls (C57BL/6) and iNOS-de-
ficient mice (iNOS?/?) 12 h postinstillation of 107CFU of type 2 pneu-
mococci in the absence or presence of apoptotic AMs (AC). B, Levels of
TNF-? mRNA as quantified by densitometry and normalized to GAPDH,
n ? 3–4. C, Levels of TNF-? mRNA as quantified by RT-PCR and nor-
malized to ?-actin, in the same mice as B. D, Concentration of TNF-? in BAL
12 h after instillation of 107CFU of type 2 pneumococci with and without AC,
n ? 3–4. Mean ? SEM, ?, p ? 0.05, ??, p ? 0.01, t test; f, C57BL/6; dark
gray box, C57BL/6 AC; ?, iNOS?/?, light gray box, iNOS?/?AC.
Instillation of apoptotic macrophages into lungs of
iNOS?/?mice reduces proinflammatory cytokine/chemokine levels in
bronchial alveolar lavage after pneumococcal infection. Concentration of
(A) MIP-2, (B) KC in BAL 12 h after, or (C) IL-6 24 h after instillation of
107CFU of type 2 pneumococci with and without AC; n ? 3–4. Mean ?
SEM, ?, p ? 0.05, t test; f, C57BL/6; dark gray box, C57BL/6 AC; ?,
iNOS?/?; light gray box, iNOS?/?AC.
Instillation of apoptotic macrophages into lungs of
6486MACROPHAGE APOPTOSIS REDUCES INFLAMMATION IN PNEUMONIA
levels of the transcription factor NF-?? (38) and decreases NF-??
binding to the regulatory region of proinflammatory cytokine
genes such as TNF-? and IL-6 (29). It remains possible the effects
we observed are mediated by a reaction product of NO not by NO
itself. However, pharmacologic inhibition of iNOS replicated the
phenotype of iNOS?/?mice, arguing in favor of mediation by NO
or a reactive nitrogen species rather than a nonspecific result of
deletion of the iNOS gene (35).
In addition to its signaling role, NO also plays a role in the
regulation of apoptosis (39). Ingestion of apoptotic bodies by mac-
rophages modifies cytokine production following stimulation with
microbial products (14). Although phagocytosis of apoptotic bod-
ies in the unstimulated macrophage results in production of anti-
inflammatory cytokines such as TGF-? (13), the effect of their
ingestion in the context of stimulation of TLRs in vitro is to
shorten the duration of proinflammatory cytokine production (14).
We instilled apoptotic AM to address whether decreased levels of
phagocytosis of apoptotic macrophages contributes to decreased
pulmonary inflammation during pneumococcal infection and con-
firmed this was the case. We conclude that one mechanism by
which NO deficiency contributes to lung inflammation is by de-
creasing macrophage apoptosis and one consequence of this is that
macrophages are less likely to phagocytose apoptotic cells and
therefore reset their cytokine expression profile. Interestingly, we
previously demonstrated that AM depletion also increased neutro-
phil recruitment during pneumococcal infection in vivo (9). Al-
though AM depletion would be anticipated to decrease total NO
production it would also decrease the number of AM undergoing
apoptosis during infection and the number of AM phagocytosing
apoptotic cells. Although absolute NO deficiency or high level AM
deficiency is unlikely during bacterial pneumonia, our findings
raise the possibility that subtle manipulation of NO generation or
levels of macrophage apoptosis could exert beneficial effects on
the degree of pulmonary inflammation during bacterial pneumonia.
Although the clearance of apoptotic cells alters cytokine expres-
sion profiles, these data have been generated in vitro using apo-
ptotic neutrophils or lymphocytes (40, 41). This study provides, to
our knowledge, the first data on the role of apoptotic macrophages
in the regulation of the inflammatory phenotype during a model of
pneumonia. Importantly, we have replaced the same cell type as
that which is undergoing apoptosis in the pathologic condition we
are studying rather than using transformed cell lines. Although our
study used UV-treated AM rather than pneumococcal-exposed
AM, we believe this was reasonable as it increased the yield of
apoptotic cells and it excluded the possibility of instilling addi-
tional bacteria with the apoptotic cells, a potential confounding
effect in prior studies (41). We show that these exogenous apo-
ptotic cells down-regulate pulmonary inflammation during pneu-
monia. This provides clear evidence that, in the context of pneu-
monia, macrophage apoptosis helps decrease the production of
inflammation which is of potential benefit to the host and raises the
possibility that enhancing levels of macrophage apoptosis or the
clearance of apoptotic cells could modulate levels of lung inflam-
mation in other pulmonary diseases.
The mechanism by which apoptotic macrophages exert this ef-
fect during pneumococcal pneumonia involves decreased TNF-?
production. TNF-? production is a critical upstream cytokine in
pneumococcal pneumonia which in combination with IL-1 iso-
forms mediates neutrophil recruitment in murine models of pneu-
mococcal pneumonia via the CXC chemokines KC and MIP-2
(23). TNF-? production peaks at 12 h after infection and contrib-
utes to microbiologic outcome and survival (32, 42), while en-
hanced generation of TNF-? compensates for absence of IL-1 sig-
naling during pneumococcal pneumonia (43). Nevertheless,
excessive TNF-? production, via its effects on neutrophil activa-
tion, contributes to lung injury in a variety of diseases (44–46) and
may worsen disease outcome during septic shock (47, 48). AM are
the major source of TNF-? during pneumococcal pneumonia (49),
thus it is not surprising that phagocytosis of apoptotic macro-
phages might alter the expression of this cytokine during pneumo-
coccal pneumonia. Prior studies have shown that macrophage
TNF-? expression is altered by phagocytosis of apoptotic cells (13,
50). Downstream consequences of phagocytosis of apoptotic mac-
rophages, such as chemokine expression profiles, might be less
obvious, even though there was clearly a sustained effect on neu-
trophil numbers to 48 h, because the experimental design involved
the instillation of apoptotic cells at the same time as bacteria. In-
stillation of iNOS?/?mice with apoptotic AM reduced TNF-?
(and other cytokines/chemokines) expression. In addition to its po-
tential to contribute to cytotoxicity, TNF-? expression can cause
up-regulation of receptors implicated in tissue invasion of pneumo-
cocci such as the platelet-activating factor receptor (51). Macrophage
apoptosis can therefore exert two important effects on microbiologic
outcome. The direct effect is to enhance macrophage killing of phago-
cytosed bacteria, as we have observed in vitro (11, 52) and confirmed
in low-dose infection models in which AM killing of bacteria is crit-
ical to preventing clinical disease (5, 11). A second indirect effect is
mediated by the phagocytosis of apoptotic macrophages, and is asso-
ciated with down-regulation of TNF-?. This decreases the develop-
ment of bacteremia by decreasing inflammation and/or the cytokine
mediated expression of receptors required for tissue invasion.
The clinical picture is likely to be complex with an optimal
number of apoptotic cells and cytokine profile required. This is
illustrated by the observation that restoration of apoptotic numbers
in iNOS?/?mice reduced inflammation and invasive disease, but
when numbers of apoptotic macrophages were optimal, as seen in
the C57BL/6 mice, instillation of additional apoptotic cells had
less impact on microbiologic and inflammatory outcomes. Al-
though our data supports a beneficial role for AM apoptosis in the
lung, apoptosis in other settings may be harmful. Inhibition of
lymphocyte apoptosis in sepsis improves disease outcome (53, 54).
In the current study, the increased levels of bacteremia in iNOS?/?
mice did not worsen the outcome, in keeping with the observation
that iNOS?/?mice are better able to tolerate bacteremia (34). Be-
cause NO contributes to lymphocyte apoptosis in a variety of set-
tings (55, 56), these findings may illustrate how modulation of
apoptosis in one anatomic location can be harmful and yet in an-
other distinct location can benefit host defense.
In conclusion, we provide evidence, in a model of pneumonia,
that phagocytosis of apoptotic AM is associated with reduced
TNF-? expression, neutrophil recruitment, and invasive pneumo-
coccal disease. These studies further define the role of host-medi-
ated macrophage apoptosis during bacterial infection and highlight
its impact on microbiologic and inflammatory outcomes.
We are grateful to Vanessa Singleton for help with the RNase protection
assay and Drs. Sarah Walmsley and Isobel Gowers for help with the
The authors have no financial conflict of interest.
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