Am J Physiol Lung Cell Mol Physiol 287: L706 –L714, 2004.
First published March 12, 2004; 10.1152/ajplung.00015.2004.
Myeloperoxidase deficiency enhances inflammation after allogeneic marrow
transplantation
Carlos Milla,1 Shuxia Yang,1 David N. Cornfield,1,2 Marie-Luise Brennan,3 Stanley L. Hazen,4
Angela Panoskaltsis-Mortari,1,2 Bruce R. Blazar,2 and Imad Y. Haddad1,2
1
Division of Pulmonary and Critical Care, Department of Pediatrics, and 2Division of Bone Marrow Transplantation
and Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455; 3Department of Cell Biology
and 4Department of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland, Ohio 44195
Submitted 20 January 2004; accepted in final form 8 March 2004
BONE MARROW TRANSPLANTATION (BMT) is a widely accepted
therapeutic modality for a number of malignant, hematologic,
immunologic, and genetic diseases. The success of BMT is
often compromised by the development of noninfectious diffuse lung injury termed idiopathic pneumonia syndrome (IPS)
(9). IPS occurs in 12–20% of all allogeneic BMT recipients,
with mortality rate in excess of 50% (19). Human and animal
studies have established that IPS injury is the result of severe
immune responses and is exacerbated by conditioning regimens (radiochemotherapy) (10, 11, 35). Recent evidence implicates, in this process, the generation of large amounts of
reactive oxygen/nitrogen species and the depletion of antioxidant potential during the course of irradiation, conditioning
drugs, and allogeneity (2, 24, 38). Bhalla and Folz (1) have
shown that treatment of recipient mice with N-acetylcysteine,
which repletes glutathione stores, attenuates chemotherapyinduced lipid peroxidation and suppresses lung dysfunction
after BMT. In rodents, nitric oxide production contributes to
the pathophysiology of graft-vs.-host disease (GVHD), which
affects the skin, liver, gastrointestinal tract, and the lung (16).
In humans, serum nitrite, the stable byproduct of nitric oxide,
and urinary F2-isoprostane, an indicator of in vivo lipid peroxidation, correlate with the severity of GVHD and IPS (3, 44).
In our established IPS model in irradiated mice, we have
shown that lung injury is mainly caused by donor T cells and
host macrophages/monocytes and is potentiated by conditioning drugs (35). Lung injury was associated with the generation
of large amounts of reactive oxygen and nitrogen species,
depletion of reduced glutathione, and detection of high levels
of nitrated proteins (24, 28). In addition, we have shown that
oxidant-induced lipid peroxidation in the lung correlated with
IPS severity in murine BMT recipients (50).
Oxidant/antioxidant imbalance, also referred to as oxidative
stress, promotes immune responses, including activation of T
cells (26, 39). The oxidizing environment enhances the activation and translocation of nuclear factor (NF)-␬B and increases
the production of proinflammatory cytokines such as TNF-␣
(29). However, oxidative stress is a potent inducer of programmed cell death or apoptosis (14) and may control inflammation by increasing oxidant-mediated elimination of activated T cells and macrophages (43). The effects of suppressed
oxidative stress on donor T cell-dependent inflammation and
lung injury after transplantation have not been fully elucidated.
MPO present in neutrophils and to a lesser extent in monocytes and macrophages catalyzes the reaction between hydrogen peroxide (H2O2) derived by phagocytic respiratory burst
and chloride to yield hypochlorous acid (HOCl), a potent
oxidant ⬃100 times more reactive than H2O2 (22). HOCl is a
component of the innate host defense against bacterial infections. Indeed, MPO-deficient (MPO⫺/⫺) mice exhibit increased mortality in a polymicrobial sepsis model (21). However, excessive generation of MPO-derived oxidants inactivates
proteins, oxidizes lipids, and damages DNA. MPO-derived oxidative stress is implicated in pathogenesis of lung injury in cystic
fibrosis (42), the acute respiratory distress syndrome (33), and
lung allograft rejection (31). MPO also facilitates nitration reactions (7, 20) that contribute to lung dysfunction after BMT (23).
Address for reprint requests and other correspondence: I. Y. Haddad, Univ.
of Minnesota, Dept. of Pediatrics, 420 Delaware St. SE, Minneapolis, MN
55455 (E-mail: [email protected]).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
nitrotyrosine; apoptosis; alveolar type II cells; idiopathic pneumonia
syndrome
L706
1040-0605/04 $5.00 Copyright © 2004 the American Physiological Society
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Milla, Carlos, Shuxia Yang, David N. Cornfield, Marie-Luise
Brennan, Stanley L. Hazen, Angela Panoskaltsis-Mortari, Bruce
R. Blazar, and Imad Y. Haddad. Myeloperoxidase deficiency enhances inflammation after allogeneic marrow transplantation. Am J
Physiol Lung Cell Mol Physiol 287: L706 –L714, 2004. First published March 12, 2004; 10.1152/ajplung.00015.2004.—Myeloperoxidase (MPO)-derived oxidants participate in the respiratory antimicrobial defense system but are also implicated in oxidant-mediated
acute lung injury. We hypothesized that MPO contributes to lung
injury commonly observed after bone marrow transplantation (BMT).
MPO-sufficient (MPO⫹/⫹) and -deficient (MPO⫺/⫺) mice were
given cyclophosphamide and lethally irradiated followed by infusion
of inflammation-inducing donor spleen T cells at time of BMT.
Despite suppressed generation of nitrative stress, MPO⫺/⫺ recipient
mice unexpectedly exhibited accelerated weight loss and increased
markers of lung dysfunction compared with MPO⫹/⫹ mice. The
increased lung injury during MPO deficiency was a result of donor T
cell-dependent inflammatory responses because bronchoalveolar lavage fluids (BALF) from MPO⫺/⫺ mice contained increased numbers of inflammatory cells and higher levels of the proinflammatory
cytokine TNF-␣ and the monocyte chemoattractant protein-1 compared with wild-type mice. Enhanced inflammation in MPO⫺/⫺ mice
was associated with suppressed apoptosis of BALF inflammatory
cells. The inflammatory process in MPO⫺/⫺ recipients was also
associated with enhanced necrosis of freshly isolated alveolar type II
cells, critical for preventing capillary leak. We conclude that suppressed MPO-derived oxidative/nitrative stress is associated with
enhanced lung inflammation and persistent alveolar epithelial injury.
INCREASED T CELL-DEPENDENT INFLAMMATION IN MPO⫺/⫺ MICE
The role of MPO in the pathogenesis of IPS has not been
investigated.
We hypothesized that the absence of MPO will attenuate
lung dysfunction after allogeneic BMT. Our results show,
however, that despite suppression of overall nitrative stress,
MPO⫺/⫺ mice exhibit enhanced inflammation in the periBMT period that was associated with persistent lung dysfunction. Data indicate that a potential reason for enhanced inflammation in the absence of MPO-derived oxidants is suppressed
apoptosis of lung-infiltrating inflammatory cells.
MATERIALS AND METHODS
AJP-Lung Cell Mol Physiol • VOL
Bronchoalveolar lavage. Mice were killed on day 7 post-BMT after
an intraperitoneal injection of pentobarbital sodium, and the thoracic
cavity was partially dissected. The trachea was cannulated with a
20-gauge angiocatheter and infused with 1 ml of ice-cold sterile PBS
and withdrawn. This was repeated several times, and the bronchoalveolar lavage fluid (BALF) was immediately centrifuged at 500 g for
10 min at 4°C to pellet cells. The initial 1.5 ml of BALF was used for
biochemical analysis and surfactant function, and the remaining fluid
was used to increase the yield of recovered cells. BALF and blood cell
count were determined using a Coulter Counter (model ZF; Coulter,
Miami, FL) after lysis of red blood cells by Zap-Oglobin II lytic
reagent (Coulter).
BALF biochemical analysis. Individual mouse cell-free BALF
levels of TNF-␣, monocyte chemoattractant protein (MCP)-1, IFN-␥,
and IL-10 were determined by sandwich ELISA using murine-specific
commercial kits (sensitivity 1.5–3 pg/ml; R&D Systems, Minneapolis, MN). Nitrite in BALF was measured according to the Greiss
method after the conversion of nitrate to nitrite with the NADHdependent enzyme nitrate reductase (Calbiochem, La Jolla, CA).
BALF total protein was determined by the bicinchoninic acid (Sigma,
St. Louis, MO) method with bovine serum albumin (BSA) as the
standard.
Peroxidase activity in macrophages/monocytes. BALF cell pellets
from each group of mice were combined, and cell differential was
determined in samples cytospun onto glass slides and stained with
Wright-Giemsa. Total BALF cells/well (2 ⫻ 105) were added to
flat-bottom 96-well microtiter plates, and macrophages/monocytes
were allowed to adhere for 1 h at 37°C in 5% CO2 air, followed by
removal of unbound cells. Peroxidase activity by adherent macrophages/monocytes was assessed by addition of 100 ␮l of tetramethyl
benzidine (TMB peroxidase substrate solution containing 0.01%
H2O2; Sigma) for 1 h at 23°C. Substrate color reaction was stopped by
the addition of stop solution to the microwell plate. Absorbance of the
yellow color representing oxidized TMB was measured at 450 nm. If
peroxidases were present, H2O2 would decompose at the expense of
an electron donor to generate, in the presence of chloride, the potent
oxidant HOCl, which can oxidize TMB (4).
Nitrative stress in BALF cells. For nitrotyrosine staining, cytospun
BALF cells were permeabilized and fixed with methanol at ⫺20°C for
7 min. Endogenous peroxidase activity was quenched by treatment
with 0.3% H2O2 in cold methanol for 30 min followed by three
washes with PBS. Nonspecific binding was blocked with 10% goat
serum for 30 min. The primary antibody, polyclonal rabbit antinitrotyrosine antibody (NTAb; Upstate Biotechnology, Lake Placid,
NY), at 0.01 mg/ml in 10% goat serum and 2% BSA in PBS, was
applied to the cells for 30 min. Control measurements included rabbit
IgG (Upstate Biotechnology) and NTAb in the presence of excess
nitrotyrosine (10 mM; NT block). To visualize specific NTAb binding, cells were incubated with secondary antibody, goat anti-rabbit
IgG conjugated with horseradish peroxidase (1:500 dilution), followed by the addition of 3,3⬘-diaminobenzidine (Vector Laboratories)
chromogenic substrate. The sections were counterstained with hematoxylin, dehydrated, overlaid with Permount (Sigma), and sealed with
coverslips. All slides were exposed to the primary/secondary antibodies and color development solutions for the same length of time.
Surfactant function in BALF using capillary surfactometer. The
ability of pulmonary surfactant contained in cell-free BALF to prevent
airway closure was evaluated with a glass capillary simulating a
terminal conducting airway as previously described (17). Surfactant in
the BALF was concentrated 10⫻ by centrifugation at 40,000 g for 1 h
at 4°C. A volume of supernatant (90%) of the centrifuged liquid was
removed from the test tube, and the remaining 10%, containing
pelleted surfactant, was vortexed before analysis using the capillary
surfactometer (Calmia Medical, Toronto, Ontario, Canada). Concentrated BALF (0.5 ␮l) was loaded into the narrow section of the
capillary (0.25-mm internal diameter), and its ability to maintain
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Mice. B10.BR (H2k) and C57BL/6J (termed B6; H2b) were purchased from Jackson Laboratories (Bar Harbor, ME). MPO⫺/⫺ mice
were generated by targeted disruption of the MPO gene as previously
described (6) and backcrossed ⬎10 generations to B6 background.
Mice were housed in microisolator cages in the specific pathogen-free
(SPF) facility of the University of Minnesota and cared for according
to the Research Animal Resources guidelines of our institution. In
SPF units, MPO⫺/⫺ mice breed and develop normally with no
evidence of bacterial infections. For BMT, donors were 6 – 8 wk of
age and recipients were used at 8 –12 wk of age. Sentinel mice were
found to be negative for 15 known murine viruses, including cytomegalovirus, K-virus, and pneumonia virus of mice.
Pre-BMT conditioning. B6 wild-type or MPO⫺/⫺ mice received
intraperitoneal injection of cyclophosphamide (Cytoxan; Bristol Myers Squibb, Seattle, WA) at 120 mg 䡠 kg⫺1 䡠 day⫺1 on days ⫺3 and ⫺2
pre-BMT. All mice were lethally total body irradiated (TBI) 1 day
before BMT by X-ray (7.5 Gy) at a dose rate of 0.41 Gy/min (47).
BMT. Our BMT protocol has been previously described (46).
Briefly, donor B10.BR bone marrow (BM) was T cell depleted (TCD)
with antithymocyte 1.2 monoclonal antibody (clone 30-H-12, rat
IgG2b, kindly provided by Dr. David Sachs, Massachusetts General
Hospital, Boston, MA) plus complement (Neiffenegger, Woodland,
CA). For each experiment, a total of 5–10 recipient mice per treatment
group were transplanted via caudal vein with 20 ⫻ 106 B10.BR TCD
BM cells with 15 ⫻ 106 spleen T cells as a source of GVHD/IPScausing T cells (BMS⫹Cy).
Pulmonary function analysis. Pulmonary mechanics in pentobarbital-anesthetized ventilated mice on day 7 after BMT were measured in
a cohort of MPO-sufficient (MPO⫹/⫹) and MPO⫺/⫺ mice following
the method described by Diamond and O’Donnell (13), with slight
modifications. In brief, after careful dissection of the neck, a short
metal cannula was inserted into the trachea and secured with 3.0 silk.
A polyethylene catheter was inserted orally into the lower third of the
esophagus to estimate pleural pressure. The animal was then placed
into a plethysmograph (model PLY3111; Buxco Electronics, Sharon,
CT) and connected to a mouse ventilator (Harvard Apparatus, MarchHugstetten, Germany) set at a respiratory rate of 150 breaths/min and
a tidal volume of 200 ␮l. Respiratory flow signal was measured
through a flow transducer (Sen Sym SCXL004, Buxco Electronics)
connected to the plethysmograph. Lung volume was obtained by
electric integration of the flow signal. Intraesophageal and airway
pressures were measured with a pressure transducer (Validyne DP45,
Buxco Electronics) directly connected to their respective ports. These
data were fed into a computer through a preamplifier (MaxII, Buxco
Electronics), and the data were analyzed with the Biosystem XA
software (Buxco Electronics). When the signal was stable, delivered
tidal volume was varied from 350 to 100 ␮l in 50-␮l decrements, and
for each delivered volume, the effective tidal volume, transpulmonary
pressure, and dynamic compliance were measured. Volume-pressure
plots were constructed for each treatment group. Body temperature
was maintained at 37°C throughout the experiment.
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L708
INCREASED T CELL-DEPENDENT INFLAMMATION IN MPO⫺/⫺ MICE
AJP-Lung Cell Mol Physiol • VOL
RESULTS
Lung dysfunction in MPO⫺/⫺ mice after allogeneic BMT.
To determine the role of MPO in acute lung injury after
allogeneic BMT, high-dose Cy/TBI-conditioned B6 wild-type
and MPO⫺/⫺ mice were given inflammation-inducing
B10.BR spleen T cells and killed on day 7 after BMT during
time of peak lung injury (24). The means ⫾ SE pre-BMT body
weights of MPO⫹/⫹ and MPO⫺/⫺ mice were 18.9 ⫾ 0.50 g
and 19.32 ⫾ 0.72 g, respectively (P ⬎ 0.05). After allogeneic
BMT, however, early weight loss was accelerated in MPO⫺/⫺
mice compared with MPO⫹/⫹ mice (Table 1).
Lung dysfunction was assessed by 1) measurement of BALF
total protein levels, 2) effectiveness of surfactant contained in
BALF to maintain capillary patency, and 3) lung mechanics
analysis in anesthetized-ventilated mice. BALF return volumes
collected on day 7 after BMT were similar in all groups (⬎90%
of instilled volume). BALF levels of total protein in untreated
control MPO⫹/⫹ and MPO⫺/⫺ were not different (⬍0.2
mg/ml). However, BALF protein levels collected on day 7 after
BMT were significantly higher in MPO⫺/⫺ compared with
MPO⫹/⫹ mice (Table 1).
Compared with surfactant in BALF of non-BMT MPO⫹/⫹
and MPO⫺/⫺ controls, the effectiveness of surfactant contained in day 7 after BMT pooled and concentrated BALF from
MPO⫹/⫹ BMS⫹Cy mice to maintain capillary patency was
significantly decreased. The magnitude of surfactant dysfunction was even more severe in BALF from MPO⫺/⫺ BMS⫹Cy
mice compared with MPO⫹/⫹ BMS⫹Cy mice, although the
difference was not statistically significant (Table 1). Lung
compliance at tidal volume of 200 ␮l in non-BMT control
MPO⫹/⫹ and MPO⫺/⫺ was similar (0.034 ⫾ 0.004 and
0.038 ⫾ 0.006 ml/cmH2O in MPO⫹/⫹ and MPO⫺/⫺ mice,
respectively; n ⫽ 3, P ⬎ 0.05). After allogeneic BMT, compliance was significantly decreased in both MPO⫹/⫹ and
MPO⫺/⫺ BMS⫹Cy mice (0.017 ⫾ 0.003 and 0.016 ⫾ 0.004
ml/cmH2O in MPO⫹/⫹ and MPO⫺/⫺ mice, respectively;
n ⫽ 3; P ⬍ 0.05 compared with non-BMT mice). VolumeTable 1. Day 7 after BMT weight loss, BALF total protein,
and BALF surfactant function
Group
Weight Loss
(% of Baseline Weight)
BALF Total Protein,
mg/ml
Surfactant Function
(% Open Time)
0
0
0.18⫾0.038
0.19⫾0.042
88.9⫾5.8
86.5⫾5.5
⫺21⫾1.7*
⫺26⫾1.9†
0.49⫾0.12*
0.81⫾0.07†
16.7⫾7.2*
9.6⫾4.3*
Control
MPO⫹/⫹
MPO⫺/⫺
BMS⫹Cy
MPO⫹/⫹
MPO⫺/⫺
Values of body weight and bronchoalveolar lavage fluid (BALF) total
protein are means ⫾ SE for n ⫽ 25–35 mice/group obtained from 5 independent bone marrow transplantation (BMT) experiments. Values of % open time
in capillary surfactometer are means ⫾ SE for pooled BALF samples from 2
mice/group per experiment from 2 independent experiments. B6 MPO⫹/⫹ and
MPO⫺/⫺ mice were given cyclophosphamide (Cy), lethally irradiated, and
infused with B10.BR bone marrow with inflammation-inducing donor spleen
T cells (BMS⫹Cy). Control mice were nonirradiated and nontransplanted.
Surfactant function was assessed in 10⫻ concentrated BALF samples by
capillary surfactometer. *P ⬍ 0.05 compared with non-BMT control
MPO⫹/⫹ mice. ⴱ†P ⬍ 0.05 comparing the effects of MPO deficiency in
BMS⫹Cy BMT mice.
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airflow was measured in response to increased pressure at one end of
the capillary. After initial extrusion of liquid, the percentage of the
following 120 s that the recorded pressure equaled zero indicated the
percentage of time that the capillary was open to free airflow. BALF
with well-functioning surfactant will return to the narrow section less
often and, therefore, the percentage of time the capillary remains open
will be more than BALF containing injured or suppressed surfactant.
Alveolar type II cell isolation. Alveolar type II (ATII) cells were
isolated from anesthetized control and experimental mice according to
methods described by Corti et al. (12). The abdominal cavity was
opened, and mice were exsanguinated by severing the inferior vena
cava and the left renal artery. The diaphragm was cut, and the chest
plate and the thymus were removed. The trachea was cannulated with
a 20-gauge catheter, and bronchoalveolar lavage was performed as
described above. The lungs were then perfused via the pulmonary
artery with 10 –20 ml of 0.9% normal saline using a 21-gauge needle
fitted on a syringe. Three milliliters of dispase (BD Biosciences,
Bedford, MA) were rapidly instilled through the cannula in the
trachea. Lungs were removed from the animal and incubated in a 1-ml
dispase for 45 min at 23°C. Lungs were then transferred to a 60-mm
culture dish containing 7 ml of HEPES-buffered DMEM and 100
U/ml DNase I (Sigma). The lung tissue was gently teased from the
airways and swirled for 5–10 min. The cell suspension was successively filtered through 100- and 40-␮m Falcon cell strainers and then
through 20-␮m nylon mesh. Cells were collected by centrifugation at
130 g for 8 min at 4°C and placed on biotinylated anti-CD45 and
biotinylated anti-CD16/CD32 precoated culture plates. After incubation for 2 h at 37°C, ATII cells were gently panned from the plate and
collected by centrifugation at 130 g for 8 min. ATII cells were
resuspended in PBS, washed 2⫻, and resuspended in PBS (1–2 ⫻ 106
cells/ml).
Prosurfactant protein C staining. Freshly purified lung cells and
BALF cells were stained with prosurfactant protein C (pro-SP-C)
polyclonal antibody (Chemicon, Temecula, CA) to confirm ATII cell
type. Pro-SP-C is specific to ATII cells and is present in abundance.
Cells (2 ⫻ 105 cells/ml) were cytospun onto glass slides and fixed in
methanol at ⫺20°C for 7 min. After blocking nonspecific sites with
normal donkey serum, cells were incubated with pro-SP-C primary
antibody (1:1,500) for 30 min at 23°C and rinsed 6⫻ with PBS.
Controls included replacement of primary antibody with rabbit IgG.
Cells were then incubated with fluorescein-conjugated donkey antirabbit secondary antibody (Jackson ImmunoResearch, West Grove,
PA) 1:100 in PBS for 1 h at 23°C in the dark. Cells were stained with
4⬘,6-diamidino-2-phenyllindone dihydrochloride hydrate (Sigma) to
show nuclei, rinsed briefly with PBS, and mounted with ProLong
Antifade kit (Molecular Probes).
Detection of apoptosis. Day 7 after BMT, BALF cells and freshly
purified ATII from each group of mice were combined and diluted to
a concentration of 1–2 ⫻ 106 cells/ml using annexin V binding buffer.
Cells were double stained with annexin V-FITC (BD PharMingen,
San Diego, CA) and propidium iodide (PI) following the manufacturer’s instructions. Annexin V recognizes phosphatidylserine on the
outer surface of cell membranes. This translocation of phosphatidylserine from the inner to outer surface of cell membranes occurs during
early/intermediate stages of apoptosis. Staining with PI was used to
simultaneously monitor cell necrosis. Analysis of cell fluorescence
intensity was determined by FACSCaliber flow cytometer (BD Biosciences, San Jose, CA) using CellQuest applications (BD Biosciences) with a total of 10,000 events counted. Values were reported
as the percentage of positive events or mean fluorescence (arbitrary
units).
Statistical analysis. Results are expressed as means ⫾ SE. Data
were analyzed by ANOVA or Student’s t-test. Statistical differences
among group means were determined by Tukey’s Studentized test. P
values ⱕ0.05 were considered statistically significant.
INCREASED T CELL-DEPENDENT INFLAMMATION IN MPO⫺/⫺ MICE
Fig. 1. Effective tidal volume-transpulmonary pressure plots in anesthetized
and ventilated mice measured on day 7 after allogeneic bone marrow transplantation (BMT). Control (nonirradiated and nontransplanted, E) and total
body irradiated myeloperoxidase (MPO)⫹/⫹ (Œ) or MPO⫺/⫺ (‚) mice given
cyclophosphamide (Cy) and inflammation-inducing donor spleen T cells
(BMS⫹Cy) were placed in a single-chamber plethysmograph and ventilated
with delivered tidal volume of 100 –350 ␮l. Effective tidal volume was
measured, and transpulmonary pressure was calculated using airway and
intraesophogeal pressures. Body temperature was maintained at 37°C throughout the experiment. Markers shown represent 3 different mice in each experimental group. *P ⬍ 0.05 comparing the slopes of MPO⫹/⫹ (line with longer
dashes) and MPO⫺/⫺ (line with shorter dashes) BMS⫹Cy mice with the
slope of MPO⫹/⫹ non-BMT control mice (solid line).
AJP-Lung Cell Mol Physiol • VOL
Fig. 2. Decreased nitrotyrosine immunostaining in bronchoalveolar lavage
fluid (BALF) cells from MPO⫺/⫺ mice after allogeneic transplantation.
Cy/total body irradiated (TBI) recipient MPO⫺/⫺ and wild-type (MPO⫹/⫹)
B6 mice were given B10.BR bone marrow plus donor spleen T cells
(BMS⫹Cy). Day 7 after BMT, BALF cells from the indicated group of mice
were centrifuged onto glass slides and incubated with nitrotyrosine antibody
(NTAb) or NTAb in the presence of 10 mM nitrotyrosine (NT block).
Nitrotyrosine was estimated based on the brown reaction product in the cell
cytoplasm. Shown is a representative figure from pooled cells obtained from
5– 8 mice per group per experiment. Two separate experiments were
performed.
MPO⫹/⫹ and MPO⫺/⫺ mice before and after BMT did not
differ (Fig. 3A). Consistent with the higher number of BALF
cells in MPO⫺/⫺ vs. MPO⫹/⫹ BMS⫹Cy mice, the levels of
the chemoattractant MCP-1 and the proinflammatory cytokine
TNF-␣ were higher in BALF from the former (Fig. 4). Also of
note, IFN-␥ levels were modestly, but not significantly, higher
in BALF of MPO⫺/⫺ vs. MPO⫹/⫹ mice. In contrast, BALF
levels of nitrite plus nitrate, the stable byproducts of nitric
oxide, were not significantly different in these groups of mice
(Fig. 4). BALF levels of IL-10, an anti-inflammatory cytokine,
were below detection limits by ELISA in all control and
experimental mice (data not shown). Together, these results are
consistent with exaggerated inflammation in MPO⫺/⫺ mice
after allogeneic BMT.
Suppressed apoptosis in BALF cells from MPO⫺/⫺ mice.
We hypothesized that suppressed oxidative/nitrative stressdependent apoptosis of lung-infiltrating inflammatory cells
may represent one mechanism for increased inflammation in
MPO⫺/⫺ mice after allogeneic BMT. Apoptosis/necrosis of
BALF cells obtained on day 7 after allogeneic BMT was
determined by flow cytometry after double staining the cells
with annexin V antibody and PI. Apoptosis in BALF cells from
unmanipulated control MPO⫺/⫺ and MPO⫹/⫹ mice was not
different (data not shown). Figure 5 shows increased apoptosis
and cellular necrosis in BALF cells from B6 Cy/TBI donor T
cell-recipient mice (BMS⫹Cy) compared with cells from B6
controls. Apoptosis and necrosis were less in BALF cells from
BMS⫹Cy MPO⫺/⫺ vs. MPO⫹/⫹ mice, consistent with a
major role of MPO-derived oxidants in the apoptosis of inflammatory cells after allogeneic BMT (mean fluorescence
intensity of 827 ⫾ 55 in MPO⫹/⫹ BMS⫹Cy mice vs. 368 ⫾
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pressure plots for tidal volumes ranging from 100 to 350 ␮l
demonstrated a rightward shift in MPO⫹/⫹ and MPO⫺/⫺
BMT mice compared with non-BMT control mice (Fig. 1),
consistent with development of lung injury after BMT. Together, these results indicate increased or persistent markers of
lung dysfunction in MPO-deficient mice after allogeneic BMT.
Suppressed nitrative stress in BALF cells from MPO⫺/⫺
mice. In contrast to cells from MPO⫹/⫹ mice, alveolar macrophages and lung-infiltrating monocytes collected from BALF
of MPO⫺/⫺ non-BMT and BMT recipients lacked peroxidase
activity as assessed by addition of TMB (TMB peroxidase
substrate solution containing 0.01% H2O2; data not shown).
Intracellular nitrative stress by macrophages/monocytes was
assessed by detection of antigenic sites related to nitrotyrosine.
Nitration of monocytes/macrophages obtained from MPO⫺/⫺
vs. MPO⫹/⫹ Cy/TBI mice given donor spleen T cell
(BMS⫹Cy) was decreased (Fig. 2). Nitration was specific
since staining was completely blocked in the presence of
excess antigen, 10 mM nitrotyrosine. Cells from non-BMT
control mice and Cy/TBI MPO⫹/⫹ and MPO⫺/⫺ mice given
bone marrow without donor T cells, a setting in which IPS
injury is mild rather than severe, exhibited baseline levels of
staining (data not shown).
MPO⫺/⫺ mice exhibit increased inflammation after allogeneic BMT. To begin to understand reasons of increased lung
dysfunction despite suppressed oxidative/nitrative stress in
MPO⫺/⫺ mice, the severity of donor T cell-dependent inflammation was assessed on day 7 after transplantation in
MPO⫺/⫺ and MPO⫹/⫹ mice. BALF collected from
MPO⫺/⫺ recipient mice contained significantly more inflammatory cells (Fig. 3A), although the cell differential was not
different as assessed by Wright-Giemsa stain of cytospun
samples (Fig. 3B and Table 2). This increased BALF cellularity
from MPO⫺/⫺ mice was not due to increased inflammatory
cells in the blood, since total white blood cell count in
L709
L710
INCREASED T CELL-DEPENDENT INFLAMMATION IN MPO⫺/⫺ MICE
Fig. 3. Enhanced cellularity in BALF, but not in circulation, of MPO⫺/⫺
mice after allogeneic BMT. Cell number in the first 1.5 ml of BALF collected
from control (C; unmanipulated) and Cy/TBI donor spleen T cell-recipient
MPO⫹/⫹ (filled bars) and MPO⫺/⫺ (open bars) mice (BMS⫹Cy). BALF
was collected on day 7 after BMT. Total cell count in BALF before centrifugation to pellet cells and in whole blood was determined using a Coulter
counter after lysis of red blood cells (A). B: cell differential assessed in
Giemsa-Wright-stained cytospun BALF samples from MPO⫹/⫹ (left)
BMS⫹Cy and MPO⫺/⫺ (right) BMS⫹Cy mice on day 7 after BMT were not
different. Values are means ⫾ SE for 15–20 mice per group. *P ⬍ 0.05
compared with control (nonirradiated and nontransplanted). ⫹P ⬍ 0.05 comparing the effects of MPO deficiency in each group. WBC, white blood cells.
47 in MPO⫺/⫺ BMS⫹Cy mice; P ⬍ 0.05 from 2 separate
experiments).
ATII cells are an important target of IPS, and ATII cell death
may represent a major mechanism of lung injury. Therefore,
the apoptosis/necrosis of ATII cells freshly isolated on day 7
DISCUSSION
Table 2. Day 7 after BMT BALF cell differential
in MPO⫹/⫹ and MPO⫺/⫺
Cell Differential
Group
Control
MPO⫹/⫹
MPO⫺/⫺
BMS⫹Cy
MPO⫹/⫹
MPO⫺/⫺
after allogeneic BMT was also evaluated. ATII cells were
identified by the presence of a precursor of SP-C using proSP-C antibody (Fig. 6). Pro-SP-C immunostaining was detected in ATII cells isolated from both control and experimental MPO⫹/⫹ and MPO⫺/⫺ mice and was absent in alveolar
macrophages and lung-infiltrating monocytes (data not shown).
Baseline apoptosis/necrosis of ATII cells from control
MPO⫹/⫹ and MPO⫺/⫺ mice was not different. After exposure to Cy/TBI and allogeneity, ATII cells from MPO⫹/⫹ and
MPO⫺/⫺ recipients exhibited increased apoptosis and necrosis (Fig. 7). Although the extent of apoptosis assessed by
incorporation of annexin V in ATII cells from MPO⫹/⫹ and
MPO⫺/⫺ BMS⫹Cy recipients was similar, ATII cells from
MPO⫺/⫺ mice exhibited increased incorporation of PI, consistent with enhanced cellular necrosis on day 7 after allogeneic BMT during MPO deficiency (Fig. 7).
%Macrophages
%Lymphocytes
%Neutrophils
⬎95
⬎95
⬍5
⬍5
0
0
65⫾6
67⫾5
34⫾4
32⫾5
1
1
Values are means ⫾ SE for n ⫽ 4 – 6 mice/group obtained from 2 experiments. Cell differential of day 7 after BMT BALF cells were assessed in
cytospun samples stained with Wright-Giemsa. B6 MPO⫹/⫹ and MPO⫺/⫺
mice were given Cy, lethally irradiated, and infused with B10.BR bone marrow
with inflammation-inducing donor spleen T cells (BMS⫹Cy).
AJP-Lung Cell Mol Physiol • VOL
Results of this study show that the absence of MPO in the
peri-BMT period causes persistent lung dysfunction associated
with enhanced donor T cell-dependent inflammation. Manifestations of exuberant IPS injury persisted in MPO-deficient
mice despite suppressed nitrative stress, supporting a dominant
role of accelerated immune responses in the early lung injury
after allogeneic BMT. The data indicate that MPO or MPOderived oxidants may represent an important homeostatic inflammatory control mechanism during lung injury after BMT.
MPO has also been shown to modulate the course of nonpulmonary inflammatory diseases. Brennan and coworkers (5)
reported that MPO-deficient mice are more susceptible to
experimental autoimmune encephalitis (EAE), a T cell-dependent neuronal disease. Interestingly, heterozygotes (MPO⫹/⫺)
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Fig. 4. Increased BALF levels of monocyte chemoattractant protein (MCP)-1
and TNF-␣ from MPO⫺/⫺ mice. Control (unmanipulated) and Cy/TBI recipient B6 MPO⫹/⫹ (filled bars) and MPO⫺/⫺ (open bars) mice were given
15 ⫻ 106 B10.BR donor spleen T cells (BMS⫹Cy) and BALF collected on day
7 after transplantation. TNF-␣, MCP-1, and IFN-␥ levels were measured by
sandwich ELISA. Nitrite was determined by the Greiss reaction after converting nitrate to nitrite by nitrate reductase. Shown are mean values ⫾ SE for
10 –15 mice per group pooled from 3 separate experiments. *P ⬍ 0.05 vs.
control. *⫹P ⬍ 0.05 comparing the effect of MPO deficiency within each BMT
group.
INCREASED T CELL-DEPENDENT INFLAMMATION IN MPO⫺/⫺ MICE
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Fig. 5. Suppressed apoptosis of BALF cells from MPO⫺/⫺ mice after
allogeneic BMT. BALF was collected from control (A; unmanipulated), on day
7 after BMT from Cy/TBI donor spleen T cell-recipient (BMS⫹Cy) MPO⫹/⫹
(B), and from MPO⫺/⫺ BMS⫹Cy mice (C). BALF cells from 2 mice per
group were pooled and stained with annexin V (x-axis) and propidium iodide
(PI) (y-axis). Two-color incorporation was assessed by flow cytometry. The
bottom left quadrants indicate the unstained cells, the bottom right quadrants
indicate the cells binding annexin V FITC (early apoptosis), the top left
quadrants indicate the cells that were PI stained (cells undergoing necrosis),
and the top right quadrants indicate the cells that were PI and annexin V
positive (late apoptosis). Percentages of apoptotic cells are indicated in each
quadrant. For each measurement, data from 10,000 events were collected with
a FACSCalibur flow cytometer. Fluorescence signals were collected in relative
fluorescence units. Events were analyzed with CellQuest software. Shown is a
representative experiment that was performed twice.
closely resembled wild type (MPO⫹/⫹) with respect to the
incidence of EAE, suggesting that a complete absence of
MPO-derived oxidants is necessary for the increased incidence
of EAE. Moreover, the proliferation rate of lymphocytes from
immunized MPO⫺/⫺ mice was increased by 50% compared
with wild-type mice. The cause of the accelerated EAE in
MPO⫺/⫺ mice may be the absence of MPO-dependent inhibition of lymphocyte proliferation. Similarly, we reasoned that
a potential cause of increased inflammation in MPO-deficient
mice after allogeneic BMT is suppression of the MPO-induced
cell death of lung-infiltrating inflammatory cells. Indeed, our
results show that MPO deficiency suppressed the apoptosis of
inflammatory cells contained in BALF, which may, at least in
part, explain the increased number of T cells and monocytes
and the high BALF levels of MCP-1 and TNF-␣. Consistent
with our data, Tsurubuchi et al. (41) reported that phorbol
myristate acetate-induced apoptosis of neutrophils from
MPO⫺/⫺ mice was significantly slower than in normal neutrophils.
On day 7 after allogeneic BMT, the generation of nitric
oxide in the lung as assessed by BALF levels of nitrite plus
nitrate was not modified by MPO deficiency. However, nitrated
AJP-Lung Cell Mol Physiol • VOL
Fig. 6. Freshly isolated alveolar type II (ATII) cell staining using prosurfactant
protein C (SP-C) antibody. Cells (2 ⫻ 105/ml) from control mice were
cytospun onto glass slides and stained with nonspecific IgG (A) or pro-SP-C
antibody (B) followed by fluorescein-conjugated secondary antibody; fluorescence was detected using a fluorescent microscope. Shown is a representative
experiment that was reproduced twice.
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proteins were decreased in BALF cells obtained from
MPO⫺/⫺ mice compared with wild-type recipients. These
results are in agreement with a major role of MPO in nitration
reactions in vivo and confirm that MPO significantly contributes to nitrotyrosine formation via the oxidation of nitrite to
nitrogen dioxide, as previously shown (15, 20). Nitration of
proteins can inhibit critical protein functions and may play a
central and causative role in acute lung injury (25, 49). Yet,
despite decreased detection of nitrated proteins in BALF cells
from MPO-deficient mice, lung dysfunction assessed on day 7
after BMT was at least as severe as wild-type mice.
Among possible explanations for persistent lung dysfunction
during MPO deficiency after allogeneic BMT is sustained
generation of inflammatory responses that can induce apoptotic/necrotic pathways in alveolar epithelial cells (30). Alloactivated T cells and TNF-␣, abundantly present in the lung of
MPO⫺/⫺ recipient mice, can trigger death signals in alveolar
epithelial cells (40, 45). We observed high levels of cell death
in freshly isolated ATII cells from the lungs of MPO⫺/⫺ on
day 7 after allogeneic BMT. Injury to these cells may disrupt
epithelial barrier integrity and cause enhanced permeability
edema that may result in decreased lung compliance and
increased levels of total proteins in BALF of MPO⫺/⫺ recipient mice. The presence of proteins in the alveolar space may
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INCREASED T CELL-DEPENDENT INFLAMMATION IN MPO⫺/⫺ MICE
also explain the abnormal surfactant function observed via the
capillary surfactometer in experimental transplanted mice compared with unmanipulated control mice (17).
Enhanced inflammation during noninfectious inflammatory
diseases is not limited to MPO deficiency but also observed in
mice lacking phagocytic nicotinamide adenine dinucleotide
phosphate oxidase (NADPH-oxidase or phox), a major source
of reactive oxygen species (37). For example, NADPH-oxidase-deficient (phox⫺/⫺) mice exhibit exaggerated inflammatory responses to sterile antigens (36). In our murine IPS
model, we observed exuberant pulmonary inflammation in
irradiated phox⫺/⫺ mice compared with wild-type recipient
mice (48). Exaggerated immune responses in phox⫺/⫺ mice
were associated with suppression of oxidative/nitrative stress
and impaired clearance of MCP-1 from the circulation (48).
Together, these results are consistent with the notion that
despite its well-documented damaging effects, oxidative stress
may function as an inflammatory control mechanism by inactivation of proinflammatory chemokines and by promoting the
apoptosis of inflammatory cells. Therefore, we speculate that
during inflammatory lung diseases, an optimal level of oxidative stress may exist where oxidant-induced damage is minimal
and oxidant-mediated inactivation of proinflammatory mediators and apoptosis are maximal. Inhibition of oxidative stress
below this threshold level may impair oxidant-dependent elimination of inflammatory cells and exacerbate inflammation.
Total inhibition of oxidant production may also be detrimental
because of the important physiological roles of reactive oxygen
species in regulating the redox state, which is critical for cell
growth/differentiation (34). The challenge remains to accurately estimate the extent of oxidative stress required to limit
inflammation without causing significant effector oxidant-induced injury.
Humans with inherited disorders caused by defects in respiratory burst oxidase, termed chronic granulomatous disease
(CGD), also develop severe noninfectious inflammatory granAJP-Lung Cell Mol Physiol • VOL
ulomas in lung, skin, and gastrointestinal tract (18) and exhibit
severe inflammatory complications after allogeneic BMT (27).
In addition, MPO levels are sixfold higher in humans than
rodents, and although humans deficient in MPO are not at
unusual risk of infection, they occasionally develop immunemediated noninfectious diseases, including pustular skin lesions and diabetes mellitus (32). Although the mechanisms of
these immune complications during NADPH-oxidase and
MPO deficiency are under investigation, our data support a role
for oxidant-mediated apoptosis of lung-infiltrating T cells and
monocytes in regulating the severity of inflammatory responses. Consistent with our results, Brown and coworkers (8)
have shown that neutrophils isolated from CGD patients are
more resistant to spontaneous apoptosis and produce less
anti-inflammatory mediators, including prostaglandin D2 and
IL-10. Levels of IL-10, however, were not upregulated in our
IPS model in BALF collected on day 7 after BMT.
In summary, we have shown that mice lacking MPO exhibit
enhanced donor T cell-dependent inflammation after allogeneic
transplantation. Experimental evidence indicates that one of
the mechanisms of increased inflammation is suppressed apoptosis leading to accumulation of activated inflammatory cells
that may generate high levels of proinflammatory mediators.
As a result, lung dysfunction and injury to alveolar epithelium
persist. Further experiments will be required to determine
whether partial inhibition of oxidant stress will maintain an
optimal level of oxidative/nitrative stress where oxidant-induced damage is minimal and oxidant-dependent elimination
of inflammatory cells is maximal. These results may also
clarify the reason for exuberant inflammation and immunemediated complications observed in CGD and MPO-deficient
patients. Moreover, these findings may improve our current
strategies for using antioxidants by avoiding extreme inhibition
of reactive species to preserve their ability to limit inflammatory responses.
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Fig. 7. Increased apoptosis/necrosis after allogeneic transplantation in freshly isolated ATII cells from MPO⫹/⫹ and
MPO⫺/⫺ mice. ATII cells were isolated on day 7 after BMT
from control MPO⫹/⫹ (A), control MPO⫺/⫺ (B), Cy/TBI
donor spleen T cell-recipient (BMS⫹Cy) MPO⫹/⫹ (C), and
MPO⫺/⫺ BMS⫹Cy mice (D). ATII cells from 2 mice per
group were pooled and stained with annexin V (x-axis) and PI
(y-axis). Two-color incorporation was assessed by flow cytometry. Percentage of necrotic cells (top left quadrants) were
higher in ATII from BMS⫹Cy MPO⫺/⫺ compared with
BMS⫹Cy MPO⫹/⫹ mice. For each measurement, data from
10,000 events were collected with a FACSCalibur flow cytometer. Events were analyzed with CellQuest software. Shown is
a representative experiment that was performed twice.
INCREASED T CELL-DEPENDENT INFLAMMATION IN MPO⫺/⫺ MICE
GRANTS
This work was supported by National Heart, Lung, and Blood Institute
Grants R01-HL-67334, HL-55209, and HL-62526.
19.
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