This information is current as
of June 18, 2017.
Exuberant Inflammation in Nicotinamide
Adenine Dinucleotide
Phosphate-Oxidase-Deficient Mice After
Allogeneic Marrow Transplantation
Shuxia Yang, Angela Panoskaltsis-Mortari, Mayank Shukla,
Bruce R. Blazar and Imad Y. Haddad
J Immunol 2002; 168:5840-5847; ;
doi: 10.4049/jimmunol.168.11.5840
http://www.jimmunol.org/content/168/11/5840
Subscription
Permissions
Email Alerts
This article cites 45 articles, 20 of which you can access for free at:
http://www.jimmunol.org/content/168/11/5840.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2002 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
References
The Journal of Immunology
Exuberant Inflammation in Nicotinamide Adenine Dinucleotide
Phosphate-Oxidase-Deficient Mice After Allogeneic Marrow
Transplantation1
Shuxia Yang,* Angela Panoskaltsis-Mortari,† Mayank Shukla,* Bruce R. Blazar,† and
Imad Y. Haddad2*†
I
diopathic pneumonia syndrome (IPS),3 a noninfectious lung
dysfunction after bone marrow transplantation (BMT), and
graft-vs-host disease (GVHD) account for the majority of
deaths after allogeneic BMT (1, 2). Reactive oxygen and nitrogen
species generated during the course of irradiation, conditioning
drugs, and allogeneity are implicated in the pathogenesis of tissue
injury associated with IPS and GVHD (3). Total body irradiation
(TBI) and commonly used conditioning drugs such as cyclophosphamide (Cy) have been shown to deplete antioxidant enzymes
and enhance the generation of superoxide (O2. ) and O2. -derived
toxic species (4 –7). The lung is particularly sensitive to Cy/TBI
because of its oxygen-rich environment (8). In addition, allo-acti*Department of Pediatrics, Divisions of Pulmonary and Critical Care and †Bone Marrow Transplantation and Cancer Center, University of Minnesota, Minneapolis, MN
55455
Received for publication November 2, 2001. Accepted for publication March
27, 2002.
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 grants from the American Lung Association (JM-CIA)
and the National Institutes of Health (R0 –1 HL67334 and HL55209).
2
Address correspondence and reprint requests to Dr. Imad Y. Haddad, Department of
Pediatrics, University of Minnesota, 420 Delaware Street Southeast, Minneapolis,
MN 55455. E-mail address: [email protected]
3
Abbreviations used in this paper: IPS, idiopathic pneumonia syndrome; BM, bone
marrow; BMS, BM plus spleen; BMT, BM transplant/transplantation; BALF, bronchoalveolar lavage fluid; CGD, chronic granulomatous disease; Cy, cyclophosphamide; GVHD, graft-vs-host disease; 䡠OH, hydroxyl radical; HOCl, hypochlorous acid;
iNOS, inducible NO synthase; LDH, lactic dehydrogenase; MCP-1, monocyte chemoattractant protein-1; MIP, macrophage-inflammatory protein; MPO, myeloperoxidase; NT, nitrotyrosine; ONOO⫺, peroxynitrite; phox, phagocytic oxidase; TCD, T
cell depleted; TBI, total body irradiation.
Copyright © 2002 by The American Association of Immunologists
vated donor T cells up-regulate the expression of inducible NO
synthase (iNOS) and the production of NO (9). When simultaneously generated in large amounts, NO rapidly reacts with O2. to
form peroxynitrite (ONOO⫺), a potent oxidant and tissue-damaging nitrating species (10).
In our established murine BMT model, we observed that infusion of allogeneic T cells on the day of BMT into TBI recipients
induces NO generation, and the addition of Cy to irradiation regimen facilitates T cell-dependent oxidative/nitrative stress and depletes glutathione (11, 12), possibly via the formation of ONOO⫺
(13). Using iNOS deletional mutant mice (iNOS⫺/⫺) we reported
that iNOS-derived NO amplifies T cell-dependent inflammation
and mortality after allogeneic BMT (14). However, during Cyfacilitated oxidative stress mice lacking iNOS persist to generate
NO-independent potent oxidants and in fact exhibited increased
mortality compared with wild-type mice (14). These data indicated
that cellular redox state is a main determinant of whether inhibition
of NO is beneficial or detrimental after allogeneic BMT.
Phagocyte NADPH-oxidase is a multicomponent enzyme that
transfers electrons from NADPH to oxygen to generate O2. (15).
The main function of NADPH-dependent oxidative stress is host
defense against invading microorganisms. O2. is a weak oxidant,
but the formation of hydroxyl radicals (䡠OH) by the O2. -driven
fenton reaction and the formation of ONOO⫺ by the reaction of
O2. with NO generate potent oxidants that can oxidize fluorescent
probes (16). In addition, neutrophils and, to a lesser extent, monocytes can release myeloperoxidase (MPO) into the extracellular
space, where it reacts with hydrogen peroxide (H2O2) and chloride
to generate hypochlorous acid (HOCl), a strong oxidizing agent
(17). Although oxidative stress is effective in control of infections,
0022-1767/02/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
We have shown that NO and superoxide (O2. ) contribute to donor T cell-dependent lung dysfunction after bone marrow transplantation (BMT) in mice. We hypothesized that inhibiting O2. production during inducible NO synthase induction would suppress
oxidative/nitrative stress and result in less severe lung injury. Irradiated mice lacking the phagocytic NADPH-oxidase ( phoxⴚ/ⴚ),
a contributor to O2. generation, were conditioned with cyclophosphamide and given donor bone marrow in the presence or absence
of inflammation-inducing allogeneic spleen T cells. On day 7 after allogeneic BMT, survival, weight loss, and indices of lung injury
between phoxⴚ/ⴚ and wild-type mice were not different. However, the majority of macrophages/monocytes from phoxⴚ/ⴚ mice
given donor T cells produced fewer oxidants and contained less nitrotyrosine than cells obtained from T cell-recipient wild-type
mice. Importantly, suppressed oxidative stress was associated with marked infiltration of the lungs with inflammatory cells and
was accompanied by increased bronchoalveolar lavage fluid levels of the chemoattractants monocyte chemoattractant protein-1
and macrophage-inflammatory protein-1␣ and impaired clearance of recombinant mouse macrophage-inflammatory protein-1␤
from the circulation. Furthermore, cultured macrophages/monocytes from NADPH-deficient mice produced 3-fold more TNF-␣
compared with equal number of cells from NADPH-sufficient mice. The high NO production was not modified during NADPHoxidase deficiency. We conclude that phoxⴚ/ⴚ mice exhibit enhanced pulmonary influx of inflammatory cells after BMT. Although
NO may contribute to increased production of TNF-␣ in phoxⴚ/ⴚ mice, the data suggest that NADPH-oxidase-derived oxidants
have a role in limiting inflammation and preventing lung cellular infiltration after allogeneic transplantation. The Journal of
Immunology, 2002, 168: 5840 –5847.
The Journal of Immunology
Materials and Methods
D. 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 without spleen T cells (BM ⫹ Cy) or
with 15 ⫻ 106 spleen T cells (BM plus spleen (BMS) ⫹ Cy) as a source
of GVHD/IPS-causing T cells. Day 7 post-BMT white blood cell count was
determined using a Coulter Counter (Model ZF; Coulter, Miami, FL) after
lysis of RBCs by Zap-Oglobin II lytic reagent (Coulter).
Bronchoalveolar lavage
Mice were sacrificed on day 7 after BMT after an i.p. injection of sodium
pentobarbital, and the thoracic cavity was partially dissected. The trachea
was cannulated with a 22-gauge angiocatheter, infused with 1 ml of icecold 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 the remaining fluid was used to increase
the yield of recovered cells.
BALF analysis
Cell-free BALF monocyte chemoattractant protein-1 (MCP-1), macrophage-inflammatory protein (MIP)-1␣, MIP-1␤, and TNF-␣ levels 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 reduced NADH-dependent enzyme nitrate reductase (Calbiochem, La Jolla, CA). BALF total protein was determined by
the bicinchoninic acid (Sigma-Aldrich, St. Louis, MO) method with BSA
as the standard.
Macrophage culture
The BALF cell pellets from mice in each treatment group were combined,
washed twice in cold PBS, and resuspended in RPMI 1640 medium (Celox
Laboratories, St. Paul, MN) containing 5% FCS, penicillin (100 U/ml), and
streptomycin (100 ␮g/ml). Total cell number was determined with a hemacytometer. A total of 2 ⫻ 105 cells/well were added to flat-bottom
96-well microtiter plates (Costar, Cambridge, MA), and macrophages were
allowed to adhere for 1 h at 37°C in 5% CO2 in air, followed by removal
of unbound cells. More than 95% of adherent cells were macrophages. The
cells were maintained in culture at 37°C for 48 h in 5% CO2 in air. At
termination of cell culture, supernatants were aspirated from individual
culture wells for measurement of TNF-␣ by ELISA, nitrite by the Greiss
method, and lactic dehydrogenase (LDH) by the colorimetric CytoTox 96
assay (Promega, Madison, WI). Cells were washed twice with PBS and
lysed with lysis solution (10⫻, Triton X-100; Promega), and cellular LDH
release was measured. Total (supernatant plus cellular) LDH values were
used to correct for possible differences in adherent cell number between
groups. TNF-␣ and nitrite readings were adjusted accordingly using the
BM group as an assigned reference value for 2 ⫻ 105 cells (the number of
cells originally plated per well).
Macrophage-derived intracellular oxidants
B10.BR (H-2K), C57BL/6J (H-2b), and phox⫺/⫺ mice generated by deletion of a 91-kDa subunit of the oxidase cytochrome b (gp91; backcrossed
⬎10 generations to C57BL/6 mice) were purchased from The Jackson
Laboratory (Bar Harbor, ME). Mice were housed in microisolator cages in
the specific pathogen-free facility of the University of Minnesota (Minneapolis, MN) and cared for according to the Research Animal Resources
guidelines of our institution. For BMT, donors were 6 – 8 wk of age and
recipients were used at 8 –10 wk of age. Sentinal mice were found to be
negative for 15 known murine viruses including CMV, K-virus, and pneumonia virus of mice.
Alveolar macrophages obtained from day 7 post-BMT BALF were cultured in flat-bottom 24-well plates (Costar) for 1 h followed by removal of
nonadherent cells. Adherent cells (mainly macrophages/monocytes) were
detached from culture plates using trypsin (0.05%; Life Technologies,
Carlsbad, CA) and suspended in PBS. Cells were loaded with 2⬘,7⬘-dichlorofluorescin diacetate (10 ␮M; Molecular Probes, Eugene, OR) for 15 min
at 37°C. During loading the acetate groups are removed by intracellular
esterases, trapping the probe inside the cells. Following an oxidative burst,
dichlorofluorescin is oxidized to the fluorescent probe, dichlorofluorescein.
Fluorescence was quantitated 30 min after loading by FACScan flow cytometer (BD Biosciences, San Jose, CA) using CellQuest applications (BD
Biosciences). For each sample 5000 events were analyzed by measuring
the increase in FL1 fluorescence (530 nm).
Pre-BMT conditioning
Histology and immunohistochemistry
Mice
C57BL/6 wild-type or phox⫺/⫺ mice received i.p. injections of Cy (Cytoxan; Bristol-Myers Squibb, Seattle, WA) 120 mg/kg per day on days ⫺3
and ⫺2 pre-BMT. On the day before BMT, all mice were lethally TBI (7.5
Gy) by x-ray at a dose rate of 0.41 Gy/min.
BM transplant
Our BMT and IPS generation protocols have been described previously
(29). Briefly, donor B10.BR bone marrow (BM) was T cell depleted (TCD)
with anti-Thy 1.2 mAb (clone 30-H-12, rat IgG2b; kindly provided by Dr.
In some animals lungs were extracted without lavage and were perfused
with 1 ml PBS via the right ventricle of the heart. A mixture of 0.5–1 ml
optimal cutting temperature medium (Miles Laboratories, Elkhart, IN) and
PBS (3:1) was infused via the trachea into the lung. The lung was snapfrozen in liquid nitrogen and stored at ⫺80°C. Frozen sections were cut 6
␮m thick, mounted onto glass slides, and fixed for 5 min in acetone. Representative sections were stained with H&E for histopathologic assessment.
After a blocking step in 10% normal horse serum (Sigma-Aldrich), sections
were incubated for 30 min at 23°C with the following biotinylated mAbs
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
not infrequently it also causes injury to host proteins, lipids, and
DNA, culminating in tissue damage.
In addition to their tissue-destructive effector function, NADPHoxidase-derived reactive species can regulate cellular signal transduction pathways (reviewed in Ref. 18). For example, in a model
of alcoholic liver injury, NADPH-oxidase activates the proinflammatory transcriptional factor NF-␬B (19), known to up-regulate
the expression of a number of genes involved in immune and
inflammatory responses. In addition, O2. and H2O2 have been reported to contribute to inflammation by increasing leukocyte adhesion to endothelium (20), by altering the intracellular redox state
(21), and by induction of intracellular calcium (22). In a model of
influenza-induced lung injury, transgenic mice that overexpress
extracellular superoxide dismutase, an antioxidant enzyme that
decreases the steady state of O2. , exhibited resistance to injury associated with suppressed generation of oxidative stress and decreased production of TNF-␣ and NO (23). An in vivo antiinflammatory role for oxidative stress has not been described.
Chronic granulomatous disease (CGD) is an inherited disorder
caused by defects in NADPH-oxidase-dependent O2. production
(24). Recently, mouse models of CGD have been created by deletion of the phagocytic oxidase (phox) membrane-bound component gp91phox (25) or of the cytoplasmic component p47phox (26).
phox⫺/⫺ mice are susceptible to severe bacterial and fungal infections. A second feature of CGD in humans and mice is the frequent
development of inflammatory granulomas in lung, skin, liver, and
the lining of gastrointestinal and genitourinary tracts (27). Although incomplete resolution of active infection has been suggested as a possible reason for granuloma formation, the rapid
response to systemic steroid therapy suggests a noninfectious etiology (28). Potential mechanisms for the in vivo occurrence of
inflammatory granulomas have not been clearly defined.
The purpose of this study was to investigate the role of NADPHoxidase during noninfectious T cell-dependent inflammation after
transplantation. We hypothesized that Cy/TBI phox⫺/⫺ mice given
allogeneic T cells would exhibit decreased oxidative/nitrative
stress but persistent NO-dependent inflammation. Our results indicate phox⫺/⫺ mice have enhanced lung cellular infiltration associated with severe activation of macrophages/monocytes. Because NO production after allogeneic transplantation in phox⫺/⫺
and wild-type mice was comparable, the data suggest a role for
O2៛ -derived oxidative stress in modulation of the early post-BMT
inflammatory events.
5841
5842
EXAGGERATED INFLAMMATION IN phox⫺/⫺ MICE AFTER BMT
Multiplex quantitative RT-PCR
Total RNA was extracted from whole lungs obtained on day 7 after BMT
using the guanidium thiocyanate-phenol-chloroform method (Tri-Reagent;
Sigma-Aldrich). Reverse transcriptase was performed using a cDNA synthesis kit (First-Strand cDNA Synthesis kit; Amersham Pharmacia Biotech,
Uppsala, Sweden). MCP-1 cDNA were amplified using mouse MCP-1
gene-specific primers with 18S rRNA as an internal control (Gene Specific
Relative RT-PCR; Ambion, Austin, TX). The PCR products were electrophoresed through 1% agarose gel and amplified cDNA bands were visualized by ethidium bromide staining. MCP-1 PCR product was included as
positive control. Densitometry was used in relative semiquantitative assessment of RT-PCR product (NIH Image; Scion, Frederick, MD).
umes collected on day 7 after transplantation were similar in all
groups (⬎90% of instilled volume). BALF from Cy/TBI donor T
cell-recipient phox⫺/⫺ mice contained a significantly higher total
number of inflammatory cells compared with wild-type mice (Fig.
1A). This increase of cellularity in BALF from phox⫺/⫺ mice was
not due to an increased number of inflammatory cells in the blood
(Fig. 1B). Furthermore, H&E-stained lung sections from phox⫺/⫺
Cy/TBI-conditioned mice given donor T cells revealed severe interstitial infiltration with inflammatory cells (Fig. 2). As determined by immunohistochemistry, the increased number of lunginfiltrating cells in phox⫺/⫺ mice were positive for Mac-1, CD4,
and CD8 surface markers, consistent with enhanced monocytic and
lymphocytic influx during NADPH-oxidase deficiency (Fig. 3).
Cy/TBI NADPH-oxidase-deficient and -sufficient mice given BM
without T cells did not exhibit significant cellular infiltration in
the lung.
phox⫺/⫺ mice have increased expression of the CC chemokines
MCP-1, MIP-1␣, and MIP-1␤
Previous data showed that up-regulation of MCP-1 on day 7 after
BMT BALF and lung parenchyma of T cell-recipient mice preceded lung infiltration with host monocytes, whereas increased expression of MIP-1␣ and MIP-1␤ was accompanied by infiltration
with donor T cells (30). We reasoned that measurement of MCP-1,
MIP-1␣, and MIP-1␤ in BALF of T cell-recipient phox⫺/⫺ mice
may clarify, at least in part, the exuberant influx of monocytes and
T cells into the lungs. Day 7 after allogeneic BMT MCP-1, MIP1␣, and MIP-1␤ levels were significantly higher in the BALF of
Chemokine clearance
To assess the clearance of chemokines from the circulation after allogeneic
transplantation in the presence or absence of NADPH-oxidase-derived oxidative stress, Cy/TBI donor T cell-recipient wild-type and phox⫺/⫺ mice
were injected with recombinant mouse MIP-1␤ on day 6 after BMT. Recombinant MIP-1␤ (10 ng) or an equal volume of PBS was given i.p. and
a cohort of phox⫹/⫹ and phox⫺/⫺ mice were sacrificed at 1 and 4 h after
MIP-1␤/PBS administration (n ⫽ 3 per time point). Serum MIP-1␤ was
determined by sandwich ELISA (R&D Systems).
Lung weights
Mice were sacrificed on day 7 after BMT and the thoracic cavity was
partially dissected. To maximize use of mice, the right lung (bi-lobed) was
used for weight determinations while the left lobe was processed for tissue
staining. For each mouse, the wet weight was determined immediately after
removal from the thorax. Lungs were dried overnight to a constant weight
at 80°C followed by determination of dry weights and wet:dry weight ratio
was calculated. No correction for extravascular blood content was attempted in the calculations.
Statistical analysis
Results are expressed as means ⫾ SEM. Data were analyzed by ANOVA
or Student’s t test. Statistical differences among group means were determined by Tukey’s Studentized test. Values of p ⱕ 0.05 were considered
statistically significant.
Results
phox⫺/⫺ mice have exaggerated cellular lung infiltration after
allogeneic BMT
To examine the role of host phagocyte NADPH-oxidase during the
early inflammatory response and oxidative stress after allogeneic
BMT, conditioned B6 wild-type and phox⫺/⫺ mice were given
B10.BR donor spleen T cells at time of BMT. BALF return vol-
FIGURE 1. Increased cellularity in BALF of phox⫺/⫺ mice after allogeneic BMT. C57BL/6 NADPH-oxidase-sufficient and -deficient mice
were given 120 mg/kg Cy per day on days ⫺3 and ⫺2, lethally irradiated
on day ⫺1, and infused with B10.BR TCD BM either without donor spleen
T cells (BM ⫹ Cy) or with spleen T cells (BMS ⫹ Cy). A, Bronchoalveolar
lavage was performed on day 7 after BMT. Return BALF volume was
similar in all groups (⬎90% of instilled volume). Cell number in the first
1.5 ml of BALF before centrifugation to pellet cells was determined using
a hemacytometer. B, On day 7 after BMT white blood cell count was
determined using a Coulter Counter. Values are means ⫾ SE for 8 –10 mice
per group. ⴱ, p ⬍ 0.05 vs BM ⫹ Cy wild-type mice. ⫹, p ⬍ 0.05 comparing the effects of NADPH-oxidase deficiency in each group.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
(BD PharMingen, San Diego, CA): anti-CD4 (clone GK1.5), anti-CD8
(clone 2.43), anti-Gr-1 (clone RB6-8C5), and anti-Mac-1 (clone M1/70).
Immunoperoxidase staining was performed using avidin-biotin blocking
reagents, ABC-peroxidase conjugate, and diaminobenzidine chromogenic
substrate (Vector Laboratories, Burlingame, CA). In control measurements
the primary Ab was omitted. The sections were counterstained with hematoxylin, dehydrated, overlaid with Permount (Sigma-Aldrich), and
sealed with coverslips. The number of positive CD4/CD8 (T cells), positive
Mac-1 (macrophages/monocytes), and positive Gr-1 (neutrophils) cells in
the lung were quantitated as the percentage of nucleated cells at a magnification of ⫻50 (⫻20 objective lens). Four fields per lung were evaluated.
For nitrotyrosine (NT) staining, BALF cells were centrifuged onto glass
slides, 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
Ab, polyclonal anti-NT Ab (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 polyclonal IgG
(Upstate Biotechnology) and NT Ab in the presence of excess NT (10 mM;
NT block). To visualize specific NT Ab binding, sections were incubated
with secondary Ab, goat anti-rabbit IgG conjugated with HRP (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-Aldrich), and
sealed with coverslips. Cells were considered NT positive based on the
presence or absence of the brown reaction product in the cell cytoplasm.
The Journal of Immunology
5843
phox⫺/⫺ compared with wild-type mice (Fig. 4). The increased
level of chemokines was dependent on infusion of T cells, because
both phox⫺/⫺ and wild-type mice given BM without T cells had
low BALF levels of MCP-1, MIP-1␣, and MIP-1␤. The enhanced
MCP-1 protein during NADPH-oxidase deficiency was not due to
up-regulation of MCP-1 expression, because lung MCP-1 mRNA,
as assessed by multiplex gene-specific relative RT-PCR, in
phox⫺/⫺ and wild-type recipients was similar (Fig. 5). Taken together, these data are consistent with decreased clearance instead
FIGURE 4. Increased expression of the CC chemokines MCP-1, MIP1␣, and MIP-1␤ in BALF from phox⫺/⫺ mice after allogeneic transplantation. C57BL/6 phox⫺/⫺ and wild-type mice were preconditioned with
Cy/TBI and given B10.BR TCD BM without spleen cells (BM ⫹ Cy) or
with 15 ⫻ 106 spleen cells (BMS ⫹ Cy) on the day of BMT. BALF was
collected on day 7 after BMT. Chemokine expression was determined by
sandwich ELISA. Mean values ⫾ SE are indicated for 10 –15 mice per
group collected from two separate experiments. ⴱ, p ⬍ 0.05 vs BM ⫹ Cy.
⫹, p ⬍ 0.05 comparing the effect of NADPH-oxidase deficiency on chemokine levels.
of increased production as the cause of the elevated chemokine
BALF levels from Cy/TBI T cell-recipient phox⫺/⫺ mice.
phox⫺/⫺ mice exhibit impaired clearance of rMIP-1␤ from the
circulation after allogeneic BMT
FIGURE 3. NADPH-oxidase deficiency enhances the influx of monocytes and T cells in the lung after allogeneic transplantation. Expression of
Mac-1, CD4, CD8, and Gr-1 was determined by immunoperoxidase staining with biotinylated mAbs. Cy/TBI C57BL/6 NADPH-oxidase-deficient
and -sufficient mice were given B10.BR TCD BM with spleen donor cells
(BMS ⫹ Cy). Lung tissues were harvested on day 7 after BMT. Data are
expressed as the percentage of nucleated cells expressing the surface
marker in the lung as determined by counting four fields per lung section
under light microscope. Shown are mean values ⫾ SE from two to three
mice per group per experiment from two representative experiments.
ⴱ, p ⬍ 0.05 vs controls (nonirradiated and nontransplanted). ⫹, p ⬍ 0.05
comparing the effects of NADPH-oxidase deficiency in each group.
To confirm that oxidative stress facilitates chemokine clearance,
recombinant mouse MIP-1␤ or PBS were injected i.p. in wild-type
and phox⫺/⫺ mice on day 6 after allogeneic BMT, and serum
MIP-1␤ was measured 1 and 4 h later. Consistent with BALF
MIP-1␤, serum MIP-1␤ levels in PBS-injected mice were higher
in Cy/TBI donor T cell-recipient phox⫺/⫺ compared with wildtype mice. Injection of rMIP-1␤ increased serum MIP-1␤ levels
measured after 1 h in both wild-type and phox⫺/⫺ BMS⫹Cy mice.
However, 4 h after injection of the recombinant chemokine,
MIP-1␤ levels in wild-type mice had returned to baseline. In contrast, MIP-1␤ levels in MIP-1␤-treated phox⫺/⫺ mice remained
elevated (Fig. 6). These data establish the critical role of oxidative
stress in the in vivo clearance of chemokines.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 2. Mice deficient in phagocytic NADPH-oxidase (phox⫺/⫺)
exhibit increased cellular lung infiltration after allogeneic transplantation.
H&E and Mac-1 immunostaining of frozen lung sections taken on day 7
after BMT from Cy/TBI-conditioned phox⫺/⫺ C57BL/6 mice given TCD
BM from B10.BR mice (BM ⫹ Cy), or phox⫹/⫹ and phox⫺/⫺ C57BL/6
mice given B10.BR spleen T cells in addition to BM (BMS ⫹ Cy). A large
number of lung-infiltrating cells were Mac-1-positive, consistent with
monocytic influx. Resolution power: ⫻50 (right panel) and ⫻100 (middle
and left panels), equivalent to ⫻20 and ⫻40 objective lens, respectively.
5844
EXAGGERATED INFLAMMATION IN phox⫺/⫺ MICE AFTER BMT
FIGURE 5. NADPH-oxidase deficiency does not modify donor T celldependent MCP-1 mRNA expression in the lung after transplantation.
C57BL/6 phox⫺/⫺ and wild-type mice were preconditioned with Cy/TBI
and given B10.BR TCD BM plus 15 ⫻ 106 spleen cells (BMS ⫹ Cy).
MCP-1 cDNA were amplified using mouse MCP-1 gene-specific primers
with 18S rRNA as an internal control. The PCR products were electrophoresed through 1% agarose gel and amplified cDNA bands were visualized by ethidium bromide staining. MCP-1 PCR product was included as
positive control. C represents unmanipulated control C57BL/6 mice. Data
represent results of one experiment, which was reproduced two times.
Macrophages from phox⫺/⫺ mice exhibit enhanced production
of TNF-␣ but not NO
Macrophage/monocyte-derived oxidative/nitrative stress in
phox⫺/⫺ mice
The contribution of NADPH-oxidase to the generation of oxidative
and nitrative stress by monocytes/macrophages extracted from day
7 after BMT BALF was examined. The generation of strong oxidants by alveolar macrophages/monocytes was assessed using di-
FIGURE 6. Impaired clearance of recombinant mouse MIP-1␤ from the
circulation of phox⫺/⫺ mice after allogeneic transplantation. Cy/TBI
C57BL/6 phox⫺/⫺ and wild-type mice were given donor B10.BR TCD BM
plus donor T cells (BMS ⫹ Cy). On day 6 after BMT mice were injected
with MIP-1␤ (10 ng i.p.) or the same volume (0.2 ml) of PBS at time 0
(indicated by an arrow). A cohort of mice from each group were sacrificed
at 1 h (n ⫽ 3) and at 4 h (n ⫽ 3) after injection of MIP-1␤ or PBS. Values
are means ⫾ SE. ⴱ, p ⬍ 0.05 comparing the effect of NADPH-oxidase
deficiency in PBS- and MIP-1␤-injected BMS ⫹ Cy mice.
FIGURE 7. Increased production of TNF-␣, but not NO, by cultured
alveolar macrophages/monocytes from mice lacking phagocytic NADPHoxidase (phox⫺/⫺) mice after allogeneic transplantation. C57BL/6 phox⫺/⫺
and wild-type (WT) mice were preconditioned with Cy/TBI and given
B10.BR BM without (BM ⫹ Cy) or with inflammation-causing donor
spleen T cells (BMS ⫹ Cy). On day 7 after BMT, BALF cells (2 ⫻ 105
cells/well) were suspended in RPMI and macrophages were allowed to
adhere to flat-bottom 96-well plates, followed by removal of nonadherent
cells. A, Spontaneous TNF-␣ production in supernatant of cells cultured for
48 h. B, Spontaneous NO production in supernatant of macrophages/monocytes cultured for 48 h. Nitrite level was assessed using the Greiss reaction.
Values are mean ⫾ SE obtained from at least four wells of pooled macrophages obtained from four to six mice per group per experiment (total of
three experiments). ⴱ, p ⬍ 0.05 vs BM. ⫹, p ⬍ 0.05 comparing the effect
of NADPH-oxidase deficiency in each group.
chlorofluorescin as an intracellular fluorescent probe. Neither NO
nor O2. is able to oxidize dichlorofluorescin. In contrast, ONOO⫺
and other strong oxidants such as 䡠OH and HOCl oxidize dichlorofluorescin to form the highly fluorescent product dichlorofluorescein (31). Generation of oxidants was dependent on infusion of
donor T cells because macrophages from irradiated mice given
BM without T cells exhibited baseline fluorescence (data not
shown). Compared with cells from Cy/TBI wild-type mice given
donor T cells, the majority of monocytes/macrophages from Cy/
TBI phox⫺/⫺ T cell-recipient mice exhibited lower levels of fluorescence, quantified by flow cytometry (Fig. 8).
Intracellular nitrative stress was assessed by detection of antigenic sites related to NT. Nitration of monocytes/macrophages
from BMS ⫹ Cy phox⫺/⫺ mice was less than cells from BMS ⫹
Cy NADPH-oxidase-sufficient mice. Nitration was specific because staining was completely blocked in the presence of excess
Ag, 10 mM NT (Fig. 9). These data indicate that the majority of
alveolar monocytes/macrophages generate fewer oxidants and nitrating species during NADPH-oxidase deficiency after allogeneic
transplantation.
Survival, weight loss, and lung injury
The effects of NADPH-oxidase deficiency on day 7 after BMT
survival, weight loss, and indices of lung injury were determined.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
To determine whether NADPH-oxidase deficiency also altered the
production of inflammatory mediators by macrophages/monocytes, equal number of cells obtained from day 7 after allogeneic
BMT BALF were cultured for 48 h and supernatant was assessed
for TNF-␣ and nitrite, the stable byproduct of NO metabolism.
Macrophages/monocytes from Cy/TBI phox⫺/⫺ mice given donor
T cells (BMS ⫹ Cy) produced ⬃3-fold more TNF-␣ than cells
from BMS ⫹ Cy wild-type mice (Fig. 7A). In contrast, nitrite
levels in the supernatant of the same macrophages were not different (Fig. 7B). Similarly, day 7 after BMT BALF from T cellrecipient Cy/TBI mice lacking phagocytic NADPH-oxidase contained significantly higher levels of TNF-␣, but not nitrite plus
nitrate, than BALF from T cell-recipient wild-type controls (data
not shown).
The Journal of Immunology
5845
the presence of allogeneic T cells, because Cy/TBI wild-type and
phox⫺/⫺ recipients not given T cells exhibited 100% survival and
regained baseline weight by day 7 after BMT. The high levels of
day 7 after BMT BALF total protein, an index of lung permeability
edema, was not modified by NADPH-oxidase deficiency. Furthermore, day 7 after BMT lung wet weights, dry weights, and wet:dry
weights ratio between wild-type and phox⫺/⫺ mice were not significantly different (Table I).
Discussion
Forty-seven of 48 phox⫺/⫺ Cy/TBI T cell-recipient mice and 48 of
51 wild-type mice survived at least until day 7 after BMT ( p ⬎
0.05). Post-BMT weight loss in phox⫺/⫺ and wild-type mice was
also similar. On day 7 after BMT, weight loss was 25 ⫾ 1% and
24 ⫾ 0.5% of baseline in wild-type and phox⫺/⫺ mice, respectively. The early deaths and day-7 weight loss were dependent on
FIGURE 9. Decreased NT immunostaining in BALF cells from
phox⫺/⫺ mice after allogeneic transplantation. Cells were obtained from
day 7 after BMT BALF of C57BL/6 Cy/TBI-conditioned wild-type (WT)
mice given B10.BR BM (BM ⫹ Cy), and Cy/TBI-conditioned wild-type
and phox⫺/⫺ mice given BM plus 15 ⫻ 106 donor spleen T cells (BMS ⫹
Cy). BALF cells were centrifuged onto glass slides and incubated with
nonspecific rabbit IgG, NT Ab, or NT Ab in the presence of 10 mM NT
(NT block). Shown is a representative figure; data were reproduced two
times.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
FIGURE 8. Decreased production of oxidants by the majority of macrophages/monocytes from mice lacking phagocytic NADPH-oxidase
(phox⫺/⫺) after allogeneic transplantation. C57BL/6 phox⫺/⫺ and wildtype (WT) mice were preconditioned with Cy/TBI and given B10.BR BM
plus donor spleen T cells (BMS ⫹ Cy). Alveolar macrophages/monocytes
obtained from day 7 after BMT BALF were loaded with dichlorofluorescin-diacetate (10 ␮M) for 15 min at 37°C. Generation of intracellular oxidants was determined 30 min after loading by measuring intracellular
fluorescence of dichlorofluorescein, the oxidized product of dichlorofluorescin, using flow cytometry. Forward- and side-scattered light (FSC and
SSC) of cells from phox⫺/⫺ (left inset) and wild-type (right inset) after
allogeneic transplantation was similar. Shown is a representative experiment of duplicate samples from wild-type and phox⫺/⫺ BMS ⫹ Cy mice;
results were reproduced two times.
The main findings of these studies are that mice lacking NADPHoxidase exhibit increased inflammation despite suppression of oxidative/nitrative stress after cytoxan, irradiation, and allogeneic
BMT. Inflammation was characterized by markedly enhanced cellular infiltration with T cells and monocytes and increased activation of alveolar macrophages/monocytes. Enhanced donor T celldependent inflammation was observed in the absence of positive
surveillance cultures, suggesting that NADPH-oxidase has a role
in limiting the early inflammatory responses after BMT.
CGD mice are known to sequester abnormally large numbers of
inflammatory cells following Ag exposure. For example, tracheal
instillation of sterile Aspergillus fumigatus resulted in extensive
neutrophil infiltrate, but the reasons for the increase influx of cells
in the lungs are incompletely understood (32). Our previous studies show donor T cell-dependent induction of the CC chemokines
MCP-1, MIP-1␣, and MIP-1␤ after transplantation (30). In the
current study, chemokine levels were higher in BALF from Cy/
TBI donor T cell-recipient phox⫺/⫺ compared with wild-type
mice. The increased MCP-1 protein during NADPH-oxidase deficiency was observed in the absence of increased expression of
MCP-1 mRNA, consistent with inefficient MCP-1 clearance and
subsequent accumulation in the lungs. Oxidant-induced inactivation of chemokines was confirmed using recombinant mouse
MIP-1␤ injected in phox⫺/⫺ and phox⫹/⫹ mice on day 6 after
allogeneic BMT. Persistent elevation of MIP-1␤ levels in the serum of MIP-1␤-treated phox⫺/⫺ mice compared with similarly
treated wild-type mice strongly supports a critical role for oxidative stress in clearance of chemokines, and indicate oxidants may
have an important anti-inflammatory function in vivo.
Clark (33) reported that the ability of formyl-methionyl peptide
to attract inflammatory cells is abolished in the presence of PMAstimulated neutrophils obtained from healthy volunteers, but not
patients with CGD. Notably, incubation of PMA-stimulated normal neutrophils with the MPO inhibitor, azide, and catalase, but
not superoxide dismutase, prevented chemotactic factor inactivation, suggesting a more important role for MPO-catalyzed H2O2HOCl than O2. and ONOO⫺ in the inactivation process. These
results may clarify the reason for the Cy-facilitated elevation of
lung and BALF MCP-1, MIP-1␣, and MIP-1␤ levels despite
ONOO⫺ generation in Cy/TBI mice infused with allogeneic T
cells (30). Alternatively, ONOO⫺ has been shown to up-regulate
chemokine gene expression (34), offsetting the potential of
ONOO⫺ to inactivate MCP-1 as reported in an in vitro system
using chemically synthesized ONOO⫺ (35). Taken together, we
hypothesize that the lack of NADPH-oxidase-derived oxidative
stress during donor T cell-dependent inflammation limits the clearance of oxidant-sensitive chemoattractant proteins, leading to exaggerated migration of inflammatory cells into the lungs.
Not only were lung-infiltrating cells increased in number, but
monocytes/macrophages obtained from day 7 after BMT BALF of
T cell-recipient phox⫺/⫺ mice also secreted more TNF-␣ on a per
cell basis than similarly treated controls. Because we previously
reported that NO amplifies T cell-dependent TNF-␣ production
after allogeneic BMT (14), we initially expected to find increased
EXAGGERATED INFLAMMATION IN phox⫺/⫺ MICE AFTER BMT
5846
Table I. Lavage fluid total protein and lung weights after allogeneic BMTa
Lung Weights
BMS ⫹ Cy
BALF (total protein
in mg/ml)
Wet (mg)
Dry (mg)
Wet/dry ratio
NADPH-oxidase⫹/⫹
NADPH-oxidase⫺/⫺
0.36 ⫾ 0.03
0.35 ⫾ 0.02
140 ⫾ 30
125 ⫾ 10
26 ⫾ 6
28 ⫾ 6
6.75 ⫾ 1.4
6.9 ⫾ 2.2
a
C57BL/6 NADPH-oxidase-sufficient and -deficient mice were given 120 mg/kg Cy per day on days ⫺3 and ⫺2, lethally
irradiated on day ⫺1, and infused with B10.BR TCD BM plus donor spleen T cells (BMS ⫹ Cy). BALF collection and lung
weights were performed on day 7 after BMT. Values are means ⫾ SE for n ⫽ 6 –10 mice per group.
Kubo et al. (41) postulated that persistence of cobra venom factorinduced permeability edema in the lungs of CGD mice is caused
by ONOO⫺, formed during the simultaneous production of O2. by
xanthine oxidase and NO by iNOS. Although our results show
decreased oxidative burst and nitrative stress by the majority of
monocytes/macrophages of mice lacking NADPH-oxidase, we
cannot rule out in vivo formation of oxidative stress via endothelial
cell-derived xanthine oxidase and nonphagocytic NADPH-oxidase
(42). However, CGD mice are unable to clear infections, presumably because of inadequate generation of in vivo oxidative stress
(43). Taken together, we favor the hypothesis that the exuberant
inflammatory response in NADPH-oxidase-deficient mice is the
main culprit responsible for abolishing the potentially tissue-protective effects of decreased macrophage/monocyte-dependent
oxidants.
We used mice lacking membrane-bound component of
NADPH-oxidase (gp91). Although differential susceptibility of
gp91 and p47 NADPH-oxidase-deficient mice to the lethal effects
of hyperoxia has been suggested (44), it is important to note that
p47 knockout mice also manifest exuberant inflammation following i.p. injection with the sterile irritant thioglycolate (26). Notably, Koay et al. (45) recently reported increased neutrophil influx
and elevated MIP-2 levels in lung tissue from LPS-treated
p47phox⫺/⫺ mice compared with wild-type mice. Enhanced inflammation occurred despite inhibition of LPS-induced NF-␬B activation in NADPH-oxidase-deficient mice.
In summary, we have shown that mice lacking NADPH-oxidase
exhibit exuberant migration and activation of inflammatory cells
into the lungs after allogeneic transplantation. Based on our previous data (14), we initially hypothesized that inflammation in
phox⫺/⫺ during inhibition of phagocyte NADPH-oxidase-derived
oxidants is NO dependent. However, NO production in phox⫺/⫺
and phox⫹/⫹ was similar, consistent with NO-independent exacerbation of early inflammatory responses in mice lacking NADPHoxidase after allogeneic BMT. Data indicate that oxidative stress
facilitates the clearance of chemokines that contribute to the initiation and sustenance of donor T cell-dependent inflammation after BMT (Fig. 10). These results may also explain why CGD patients develop inflammatory lesions in the absence of infection.
Further studies will be necessary to determine whether scavenging
extracellular O2. without eliminating NADPH-oxidase will result in
modulation of the severe early inflammatory response and attenuation of IPS injury following allogeneic transplantation.
Acknowledgments
FIGURE 10. A model of the causes of enhanced inflammation in
NADPH-oxidase mutant mice. Cy/TBI phox⫺/⫺ mice given allogeneic T
cells exhibit suppressed production of NADPH-oxidase-derived oxidative/
nitrative stress but persistent production of NO, which may amplify donor
T cell-dependent inflammation. Inhibition of ONOO⫺, 䡠OH, and HOCl
impairs the clearance of chemokines that attract monocytes and T cells and
activate inflammatory cells. Exaggerated production of inflammatory mediators may lead to the formation of noninfectious inflammatory lesions.
We gratefully acknowledge the expert technical assistance of John Hermanson, Liz Taras, and Melinda Berthold. We also acknowledge the assistance of the Flow Cytometry Core facility of the University of Minnesota Cancer Center.
References
1. Clark, J. G., J. A. Hansen, M. I. Hertz, R. Parkman, L. Jensen, and H. H. Peavy.
1993. Idiopathic pneumonia syndrome after bone marrow transplantation. Am.
Rev. Respir. Dis. 147:1601.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
NO production by macrophages from phox⫺/⫺ mice. O2. is known
to limit the steady state of NO (36), and scavengers of O2. can
enhance NO production (37). However, NO generation in BALF
and by macrophages from Cy/TBI T cell-recipient phox⫺/⫺ mice
and genetically matched controls were not significantly different.
We concluded that although NO may have contributed to the early
donor T cell-dependent inflammatory responses, another NO-independent factor (or factors) is responsible for exaggerated inflammation and enhanced production of macrophage-derived proinflammatory cytokines in phox⫺/⫺ mice. Of note is that chemokines
have been shown to contribute to inflammatory cell activation. For
example, MIP-1␣ can stimulate TNF-␣ production during acute
lung injury in rats (38).
In contrast to our study, van der Veen et al. (39), using a model
of experimental allergic encephalomyelitis, reported that mice
lacking NADPH-oxidase exhibit NO-dependent suppression of T
cell proliferation associated with improvement in clinical score
and brain histopathology. NO is known to inhibit T cell-immune
responses in vivo (40). A potential explanation for the lack of
antiproliferative T cell effects of NO in our model is the complete
MHC mismatch, which supercedes the inhibitory effects of NO.
Weight loss, BALF indices of lung injury, wet lung weights, and
wet:dry lung weights ratio between phox⫺/⫺ and wild-type mice
after allogeneic BMT were not different. A potential reason for
persistence of lung dysfunction during NADPH-oxidase deficiency
is lung infiltration with high numbers of activated inflammatory
cells capable of secreting oxidant-independent tissue-damaging
mediators, such as perforin, TNF-␣, and a variety of metalloproteinases and proteases. Alternatively, NADPH-oxidase-independent generation of oxidative stress in phox⫺/⫺ mice may result in
persistent injury after allogeneic transplantation. For example,
The Journal of Immunology
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
linked chronic granulomatous disease, an inherited defect in phagocyte superoxide production. Nat. Genet. 9:202.
Jackson, S. H., J. I. Gallin, and S. M. Holland. 1995. The p47phox mouse knockout model of chronic granulomatous disease. J. Exp. Med. 182:751.
Foster, C. B., T. Lehrnbecher, F. Mol, S. M. Steinberg, D. J. Venzon, T. J. Walsh,
D. Noack, J. Rae, J. A. Winkelstein, J. T. Curnutte, and S. J. Chanock. 1998. Host
defense molecule polymorphisms influence the risk for immune-mediated complications in chronic granulomatous disease. J. Clin. Invest. 102:2146.
Danziger, R. N., A. T. Goren, J. Becker, J. M. Greene, and S. D. Douglas. 1993.
Outpatient management with oral corticosteroid therapy for obstructive conditions in chronic granulomatous disease. J. Pediatr. 122:303.
Panoskaltsis-Mortari, A., P. A. Taylor, T. M. Yaeger, O. D. Wangensteen,
P. B. Bitterman, D. H. Ingbar, D. A. Vallera, and B. R. Blazar. 1997. The critical
early proinflammatory events associated with idiopathic pneumonia syndrome in
irradiated murine allogeneic recipients are due to donor T cell infusion and potentiated by cyclophosphamide. J. Clin. Invest. 100:1015.
Panoskaltsis-Mortari, A., R. M. Strieter, J. R. Hermanson, K. V. Fegeding,
W. J. Murphy, C. L. Farrell, D. L. Lacey, and B. R. Blazar. 2000. Induction of
monocyte- and T-cell-attracting chemokines in the lung during the generation of
idiopathic pneumonia syndrome following allogeneic murine bone marrow transplantation. Blood 96:834.
Kooy, N. W., J. A. Royall, and H. Ischiropoulos. 1997. Oxidation of 2⬘,7⬘-dichlorofluorescin by peroxynitrite. Free Radical Res. 27:245.
Morgenstern, D. E., M. A. Gifford, L. L. Li, C. M. Doerschuk, and M. C. Dinauer.
1997. Absence of respiratory burst in X-linked chronic granulomatous disease
mice leads to abnormalities in both host defense and inflammatory response to
Aspergillus fumigatus. J. Exp. Med. 185:207.
Clark, R. A. 1982. Chemotactic factors trigger their own oxidative inactivation by
human neutrophils. J. Immunol. 129:2725.
Filep, J. G., M. Beauchamp, C. Baron, and Y. Paquette. 1998. Peroxynitrite
mediates IL-8 gene expression and production in lipopolysaccharide-stimulated
human whole blood. J. Immunol. 161:5656.
Sato, E., K. L. Simpson, M. B. Grisham, S. Koyama, and R. A. Robbins. 1999.
Effects of reactive oxygen and nitrogen metabolites on MCP-1-induced monocyte
chemotactic activity in vitro. Am. J. Physiol. 277:L543.
Gutierrez, H. H., B. Nieves, P. Chumley, A. Rivera, and B. A. Freeman. 1996.
Nitric oxide regulation of superoxide-dependent lung injury: oxidant-protective
actions of endogenously produced and exogenously administered nitric oxide.
Free Radical Biol. Med. 21:43.
Haddad, I. Y., J. P. Crow, P. Hu, Y. Ye, J. Beckman, and S. Matalon. 1994.
Concurrent generation of nitric oxide and superoxide damages surfactant protein
A. Am. J. Physiol. 267:L242.
Shanley, T. P., H. Schmal, H. P. Friedl, M. L. Jones, and P. A. Ward. 1995. Role
of macrophage inflammatory protein-1␣ (MIP-1␣) in acute lung injury in rats.
J. Immunol. 154:4793.
van der Veen, R. C., T. A. Dietlin, F. M. Hofman, L. Pen, B. H. Segal, and
S. M. Holland. 2000. Superoxide prevents nitric oxide-mediated suppression of
helper T lymphocytes: decreased autoimmune encephalomyelitis in nicotinamide
adenine dinucleotide phosphate oxidase knockout mice. J. Immunol. 164:5177.
Fu-Dong, S., M. Flodstrom, S. H. Kim, S. Pakala, M. Cleary, H.-G. Ljunggren,
and N. Sarvetnick. 2001. Control of the autoimmune response by type 2 nitric
oxide synthase. J. Immunol. 167:3000.
Kubo, H., D. Morgenstern, W. M. Quinian, P. A. Ward, M. C. Dinauer, and
C. M. Doerschuk. 1996. Preservation of complement-induced lung injury in mice
with deficiency of NADPH oxidase. J. Clin. Invest. 97:2680.
Mohazzab, K. M., P. M. Kaminski, and M. S. Wolin. 1994. NADH oxidoreductase is a major source of superoxide anion in bovine coronary artery endothelium.
Am. J. Physiol. 266:H2568.
Segal, B. H., N. Sakamoto, M. Patel, K. Maemura, A. S. Klein, S. M. Holland,
and G. B. Bulkley. 2000. Xanthine oxidase contributes to host defense against
Burkholderia cepacia in the p47phox⫺/⫺ mouse model of chronic granulomatous
disease. Infect. Immun. 68:2374.
Sundar, K. M., K. A. Sanders, K. A. Albertine, Z. M. Wang, I. S. Farrukh, and
J. R. Hoidal. 2001. Role of NADPH-oxidase in hyperoxic lung injury.
Am. J. Respir. Crit. Care Med. 163:A839.
Koay, M. A., J. W. Christman, B. H. Segal, A. Venkatakrishnan, T. R. Blackwell,
S. M. Holland, and T. S. Blackwell. 2001. Impaired pulmonary NF-␬B activation
in response to lipopolysaccharide in NADPH oxidase-deficient mice. Infect. Immun. 69:5991.
Downloaded from http://www.jimmunol.org/ by guest on June 18, 2017
2. Ho, V. T., E. Weller, S. J. Lee, E. P. Alyea, J. H. Antin, and R. J. Soiffer. 2001.
Prognostic factors for early severe pulmonary complications after hematopoietic
stem cell transplantation. Biol. Blood Marrow Transplant. 7:223.
3. Vora, A., J. Monaghan, P. Nuttall, and D. Crowther. 1997. Cytokine-mediated
nitric oxide release: a common cytotoxic pathway in host-versus-graft and graftversus-host reactions. Bone Marrow Transplant. 20:385.
4. Riley, P. A. 1994. Free radicals in biology: oxidative stress and the effects of
ionizing radiation. Int. J. Radiat. Biol. 65:27.
5. Weijl, N. I., F. J. Cleton, and S. Osanto. 1997. Free radicals and antioxidants in
chemotherapy-induced toxicity. Cancer Treat. Rev. 23:209.
6. Venkatesan, N., and G. Chandrakasan. 1994. Cyclophosphamide induced early
biochemical changes in lung lavage fluid and alterations in lavage cell function.
Lung 172:147.
7. Cooper, J. A. D., Jr., W. W. Merrill, and H. Y. Reynolds. 1986. Cyclophosphamide modulation of bronchoalveolar cellular populations and macrophage oxidative metabolism. Am. Rev. Respir. Dis. 134:108.
8. Hornsey, S., B. M. Cullen, R. Myers, H. C. Walker, and M. J. Hedges. 1986.
Radiation injury in mouse lung: dependence on oxygen levels in the inspired gas.
Int. J. Radiat. Biol. Relat. Stud. Phys. Chem. Med. 50:471.
9. Weiss, G., H. Schwaighofer, M. Herold, D. Nachbaur, H. Wachter,
D. Neiderweiser, and E. R. Werner. 1995. Nitric oxide formation as predictive
parameter for acute graft-versus-host disease after human allogeneic bone marrow transplantation. Transplantation 60:1239.
10. Beckman, J. S., and W. H. Koppenol. 1996. Nitric oxide, superoxide, and peroxynitrite: the good, the bad, and ugly. Am. J. Physiol. 271:C1424.
11. Haddad, I. Y., A. Panoskaltsis-Mortari, D. H. Ingbar, S. Yang, C. E. Milla, and
B. R. Blazar. 1999. High levels of peroxynitrite are generated in the lungs of
irradiated mice given cyclophosphamide and allogeneic T cells: a potential mechanism of injury after marrow transplantation. Am. J. Respir. Cell Mol. Biol. 20:
1125.
12. Ziegler, T. R., A. Panoskaltsis-Mortari, L. H. Gu, C. R. Jonas, C. L. Farrel,
D. L. Lacey, D. P. Jones, and B. R. Blazar. 2001. Regulation of glutathione redox
status in lung and liver by conditioning regimens and keratinocyte growth factor
in murine allogeneic bone marrow transplantation. Transplantation 72:1354.
13. Haddad, I. Y., A. Panoskaltsis-Mortari, D. H. Ingbar, E. R. Resnik, S. Yang,
C. L. Farrel, D. L. Lacey, D. N. Cornfield, and B. R. Blazar. 1999. Interactions
of keratinocyte growth factor with a nitrating species after marrow transplantation
in mice. Am. J. Physiol. 277:L391–L400.
14. Yang, S., V. A. Porter, D. N. Cornfield, C. Milla, A. Panoskaltsis-Mortari,
B. R. Blazar, and I. Y. Haddad. 2001. Effects of oxidant stress on inflammation
and survival of iNOS knockout mice after marrow transplantation. Am. J. Physiol.
281:L922.
15. Clark, R. A. 1999. Activation of the neutrophil respiratory burst oxidase. J. Infect.
Dis. 179:S309.
16. Possel, H., H. Noack, W. Augustin, G. Keilhoff, and G. Wolf. 1997. 2,7-Dihydrodichlorofluorescein diacetate as a fluorescent marker for peroxynitrite formation. FEBS Lett. 416:175.
17. Eiserich, J. P., M. Hristova, C. E. Cross, A. D. Jones, B. A. Freeman,
B. Halliwell, and A. van der Vliet. 1998. Formation of nitric oxide-derived inflammatory oxidants by myeloperoxidase in neutrophils. Nature 391:393.
18. Thannickal, V. J., and B. L. Fanburg. 2000. Reactive oxygen species in cell
signaling. Am. J. Physiol. 279:L1005.
19. Kono, H., I. Rusyn, M. Yin, E. Gabele, S. Yamashina, A. Dikalova,
M. B. Kadiiska, H. D. Connor, R. P. Mason, B. H. Segal, et al. 2000. NADPH
oxidase-derived free radicals are key oxidants in alcohol-induced liver disease.
J. Clin. Invest. 106:867.
20. Niu, X. F., C. W. Smith, and P. Kubes. 1994. Intracellular oxidative stress induced by nitric oxide synthesis inhibition increases endothelial cell adhesion to
neutrophils. Circ. Res. 74:1133.
21. Sen, C. K., and L. Packer. 1996. Antioxidant and redox regulation of gene transcription. FASEB J. 10:709.
22. Sen, C. K., S. Roy, and L. Packer. 1996. Involvement of intracellular Ca2⫹ in
oxidant-induced NF-␬B activation. FEBS Lett. 385:58.
23. Suliman, H. B., L. K. Ryan, L. Bishop, and R. J. Folz. 2001. Prevention of
influenza-induced lung injury in mice overexpressing extracellular superoxide
dismutase. Am. J. Physiol. 280:L69.
24. Segal, B. H., T. L. Leto, J. I. Gallin, H. L. Malech, and S. M. Holland. 2000.
Genetic, biochemical, and clinical features of chronic granulomatous disease.
Medicine 79:170.
25. Pollock, J. D., D. A. Williams, M. A. Gifford, L. L. Li, X. Du, J. Fisherman,
S. H. Orkin, C. M. Doerschuk, and M. C. Dinauer. 1995. Mouse model of X-
5847