1 Division of Pulmonary and Critical Care, Department of Pediatrics, and 2 Division of Bone Marrow Transplantation, Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455
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ABSTRACT |
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In a model of idiopathic
pneumonia syndrome after bone marrow transplantation (BMT), injection
of allogeneic T cells induces nitric oxide (·NO), and the addition of
cyclophosphamide (Cy) generates superoxide (O/
) given donor bone marrow and spleen T
cells (BMS) exhibited improved survival compared with matched BMS
controls. Bronchoalveolar lavage fluids obtained on day 7 post-BMT from iNOS(
/
) BMS mice contained less tumor necrosis
factor-
and interferon-
, indicating that ·NO stimulated the
production of proinflammatory cytokines. However, despite suppressed
inflammation and decreased nitrotyrosine staining, iNOS(
/
) mice
given both donor T cells and Cy (BMS + Cy) died earlier than
iNOS-sufficient BMS + Cy mice. Alveolar macrophages from iNOS(
/
)
BMS + Cy mice did not produce ·NO but persisted to generate strong
oxidants as assessed by the oxidation of the intracellular fluorescent
probe 2',7'-dichlorofluorescin. We concluded that ·NO amplifies T
cell-dependent inflammation and addition of Cy exacerbates
·NO-dependent mortality. However, the lack of ·NO during Cy-induced
oxidant stress decreases survival of T cell-recipient mice, most likely
by generation of ·NO-independent toxic oxidants.
nitric oxide; peroxynitrite; lymphocytes; macrophages; tumor
necrosis factor-; idiopathic pneumonia syndrome
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INTRODUCTION |
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IDIOPATHIC PNEUMONIA
SYNDROME (IPS) refers to diffuse and often fatal noninfectious
lung dysfunction that occurs after bone marrow transplantation (BMT;
see Ref. 4). IPS accounts for at least 40% of nongraft
vs. host disease (GVHD) deaths after allogeneic BMT. Human studies and
recently established murine BMT models have confirmed that IPS is the
result of persistent immune destructive events that is potentiated with
conditioning regimens (5, 7, 9, 32). Once infiltrating
donor T cells, alloactivated by antigen-presenting cells, encounter
pulmonary antigens, immune-mediated damage begins. The major mediators
responsible for killing by cytolytic T cells are the lytic protein
perforin (cytolysin: pore-forming protein) and serine proteases such as granzyme B (14) and the Fas ligand apoptotic pathway
(28). A second pathway for stimulating lung injury is via
the release of proinflammatory cytokines by activated macrophages and
lung-infiltrating monocytes. Consistent with this hypothesis, elevated
levels of tumor necrosis factor (TNF)- and interleukin (IL)-1 and
IL-6 are present in the bronchoalveolar lavage fluid (BALF) or
parenchyma during IPS injury (6).
In our allogeneic BMT model, lung dysfunction in lethally irradiated
mice is dependent on the infusion of donor spleen T cells and is
associated with T cell-dependent early production of proinflammatory cytokines, including TNF- and interferon (IFN)-
, and the
generation of large amounts of nitric oxide (·NO) by inducible nitric
oxide synthase (iNOS) (17, 32). The high-output
iNOS-derived ·NO may serve several immunoregulatory functions that
can modify T cell immune responses. Macrophage-derived ·NO has been
shown to prevent T cell-dependent cytolysis by suppressing T cell
proliferation (24) and induction of apoptosis
(1). In addition, ·NO limits recruitment of neutrophils
into sites of inflammation (26) and suppresses the
expression of adhesion molecules (10). Furthermore, ·NO
has been shown to directly upregulate or downregulate the expression of
several cytokines and chemokines (2, 34, 40). Attempts at
determining the role of ·NO by administration of iNOS inhibitors
during GVHD after allogeneic BMT have yielded conflicting results
(11, 20). Although the use of nonspecific drugs that also
inhibit beneficial constitutive nitric oxide synthase (cNOS)-derived ·NO may explain some of the results, the main reasons for the contradictory effects of ·NO post-BMT remain unclear.
In our IPS model, the addition of the commonly used conditioning drug
cyclophosphamide (Cy) potentiated lung dysfunction and accelerated
mortality in donor T cell-recipient irradiated mice. Cy-facilitated
injury was dependent on T cells, since the injection of Cy alone
(without T cells) did not cause lung dysfunction and did not affect
survival of BMT mice (32). In addition, lung dysfunction
in Cy/TBI T cell-recipient mice was associated with the detection of
nitrated proteins (17). Because Cy is known to deplete
antioxidants and to enhance the generation of superoxide (O) formed by the simultaneous
production of ·NO by T cell-activated macrophages/epithelial cells
and Cy-induced O
formation
clarifies the dependence of Cy-facilitated toxicity on the presence of
allogeneic T cells.
The reaction of ·NO with O, is
a potent oxidant and nitrating species. ONOO
can oxidize
sulfhydryl groups, including glutathione, the most abundant antioxidant
present in the epithelial lining fluid (3). Additional
potent oxidants that may be formed by the iron-catalyzed Haber-Weiss reaction are the hydroxyl radical (·OH) and hypochlorous acid (HOCl) generated by the interaction of myeloperoxidase with hydrogen peroxide and chloride. Although there is little doubt that
ONOO
formation enhances ·NO toxicity, credible
experimental evidence indicates that the reaction of ·NO with
O
We used the ability to alter oxidant stress by the injection of Cy into
T cell-recipient mice to investigate the in vivo pathobiological role
of the reaction of ·NO with O contribute to lung inflammation and mortality after
allogeneic BMT. Our results indicate that iNOS-derived ·NO stimulate
TNF-
and IFN-
production and that Cy-induced oxidative/nitrative
stress promotes ·NO-mediated lung dysfunction. However, despite
suppressed inflammation, ·NO deficiency during Cy-induced oxidative
stress and depletion of antioxidants worsened the survival of mice
post-BMT, consistent with the generation of
O
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MATERIALS AND METHODS |
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Mice.
Female B10.BR (H2K), fully congenic iNOS(/
), and inbred
matched wild-type mice on the C57BL/6 (H2b) background were
purchased from Jackson Laboratory (Bar Harbor, ME). In addition,
C57BL/6 NADPH oxidase(
/
) mice generated by deletion of the 91-kDa
subunit of the oxidase cytochrome b (Jackson Laboratories)
were used for comparison with iNOS(
/
) mice by nitrotyrosine
staining and production of oxidants. Mice were housed in microisolator
cages in the specific pathogen-free facility of the University of
Minnesota and were 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.
Bone marrow transplant.
BMT was performed as previously described (16, 17, 32).
Briefly, C57BL/6 wild-type and knockout mice were lethally total body
irradiated (TBI; 7.5 Gy TBI by X-ray at a dose rate of 0.41 Gy/min) on
the day before BMT. A parallel set of mice also received 120 mg · kg1 · day
1 of Cy
(Cytoxan; BristolMyers Squibb, Seattle, WA) as a conditioning regimen
on days
3 and
2. Donor B10.BR bone
marrow (BM) was T cell depleted with a monoclonal anti-Thy 1.2 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/treatment group were transplanted via the caudal vein
with 20 × 106 B10.BR marrow supplemented with (BMS
and BMS + Cy) or without (BM and BM + Cy) 15 × 106 spleen T cells as a source of IPS-causing T cells. A
cohort of mice from each group was monitored for survival. As per our
approved animal research protocol, survival of BMS mice was monitored
for 30 days, and survival of BMS + Cy mice was monitored for 7 days after transplantation.
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 22-gauge angiocatheter, infused with 1 ml of ice-cold sterile PBS, and withdrawn. This was repeated two times, and return fluid was combined. The BALF was immediately centrifuged at 500 g for 10 min at 4°C to pellet cells.
BALF biochemical analysis.
Nitrite in BALF was measured according to the Greiss method after the
conversion of nitrate to nitrite with the NADH-dependent enzyme nitrate
reductase (Calbiochem, La Jolla, CA). IFN- and TNF-
levels in the
cell-free BALF were determined by ELISA using commercial kits (R&D
Systems, Minneapolis, MN). BALF non-protein-bound sulfhydryl (
SH)
content as an estimate of alveolar lining fluid glutathione level
concentration was quantified by the reaction of the SH group with
5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), as previously described
(38). BALF proteins were precipitated with 5% TCA, and
non-protein-bound
SH in the supernatant was determined after the
addition of DTNB. The absorbance of the yellow anion
2-nitro-5-thiobenzoate formed was measured at 412 nm.
Macrophage culture.
The BALF cell pellets from mice in each treatment group were combined,
washed two times in cold PBS, and resuspended in RPMI 1640 medium
(Celox Laboratories, St. Paul, MN) containing 5% FCS, 100 U/ml
penicillin, and 100 µg/ml streptomycin. Total cell number was
determined with a hemacytometer. Total cells (2 × 105/well) were added to mouse IgG-coated, 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 the termination of cell
culture, supernatants were aspirated from individual culture wells for
measurement of TNF- by ELISA (PharMingen, San Diego, CA), nitrite by
the Greiss method, and lactic dehydrogenase (LDH) by the colorimetric
CytoTox 96 assay (Promega, Madison, WI). Cells were washed two times
with PBS and lysed with lysis solution (10×, Triton X-100; Promega), and cellular LDH release was measured. Total (supernatant + 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 no. of cells originally
plated/well).
Macrophage-derived intracellular oxidants. Alveolar macrophages obtained from day 7 post-BMT BALF were cultured on glass coverslips for 1 h followed by removal of nonadherent cells. Adherent cells were loaded with 2',7'-dichlorofluorescin (DCFH) diacetate (0.1 µM; Molecular Probes, Eugene, OR) for 20 min. During loading, the acetate groups were removed by intracellular esterases, trapping the probe inside the cells. After an oxidative burst, DCFH was oxidized to dichlorofluorescein (DCF), which can be visualized on a single-cell basis using fluorescence microscopy. After rinsing with PBS to remove excess probe, generation of oxidants was monitored over time using an inverted fluorescence microscope (Nikon Eclipse TE200) connected to an extended ISIS intensified charge-coupled device camera (Robertsbridge, UK) using Axon Instruments (Foster City, CA) image capture and analysis software. DCF fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.
Histology and immunohistochemistry.
In some animals, lungs were extracted without lavage and were perfused
with 1.0 ml of saline via the right ventricle of the heart. A mixture
of 0.5-1.0 ml optimal cutting temperature medium (Miles
Laboratories, Elkhart, IN)-PBS (3:1) was infused via the trachea into
the lung. The lung was snap-frozen in liquid nitrogen and stored at
80°C. Thin (4-µm) frozen sections were mounted on glass slides
and fixed for 10 min in 3% paraformaldehyde at 4°C (nitrotyrosine
staining) and for 5 min in acetone (Mac-1 staining). Representative
sections were stained with hematoxylin and eosin (H&E) for
histopathological assessment. Nonantigenic sites were blocked with 10%
goat serum (nitrotyrosine; Sigma Chemical, St. Louis, MO) or 10% horse
serum (Sigma; Mac-1 staining) followed by incubation overnight at 4°C
with the following antibodies: 1) rabbit polyclonal
anti-nitrotyrosine antibody (NT Ab; 1:100 dilution; Upstate
Biotechnology, Lake Placid, NY) and 2) biotinylated monoclonal CD11b/Mac-1 (clone M1/70; PharMingen) using avidin-biotin blocking reagents, avidin-biotin complex-peroxidase conjugate, and
diaminobenzidine chromogenic substrate purchased from Vector Laboratories (Burlingame, CA). In control measurements, the primary antibody was omitted, or tissues were incubated with the NT Ab in the
presence of excess antigen (10 mM nitrotyrosine). To visualize specific
NT Ab binding, sections 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. The number of positive cells in the lung was
quantitated as the percentage of nucleated cells at a magnification of
200 (×20 objective lens). Four to eight fields per lung were evaluated.
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. A comparison of
survival curves between the different groups was made using the
log-rank test. P 0.05 were considered statistically significant.
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RESULTS |
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Cy-induced oxidative stress in allogeneic T cell-recipient mice.
Previous data indicate that injection of Cy (120 mg · kg1 · day
1) on
days
3 and
2 as a conditioning regimen
pre-BMT in TBI mice given allogeneic T cells increased day 7 post-BMT oxidative stress associated with the generation of a nitrating
species (17) and depleted lung glutathione antioxidant
defense (44). To determine whether Cy also depleted
epithelial lining fluid free thiol groups, BALF non-protein-bound
SH
levels were determined. On day 7 post-BMT, BALF from Cy/TBI
T cell-recipient mice (BMS + Cy) contained significantly lower levels
of free
SH compared with BMT mice given T cells alone (without Cy;
BMS) or BMT mice not given T cells (BM). Injection of Cy alone (without
T cells) did not significantly decrease BALF non-protein-bound
SH
(Fig. 1). The generation of strong
oxidants by alveolar macrophages/monocytes from Cy/TBI T cell-recipient mice was confirmed using DCFH as an intracellular fluorescent probe.
Neither ·NO nor O
and other strong
oxidants such as ·OH and HOCl oxidize DCFH to form the highly
fluorescent product DCF (23). In contrast to cells from
TBI mice not given T cells (BM), which exhibited background fluorescence, macrophages/monocytes from BALF of BMS + Cy mice showed
time-dependent intense fluorescence (Fig.
2).
|
|
Undetectable BALF nitrite plus nitrate in iNOS knockout mice after
allogeneic transplantation.
The return BALF volumes were similar in all groups of mice (>90% of
instilled volume). As previously reported (17), day 7 post-BMT BALF from TBI mice infused with allogeneic T cells with
and without Cy contained increased numbers of inflammatory cells, and
iNOS deficiency did not modify the cellular number or profile (data not
shown). In wild-type mice, injection of donor T cells increased
day 7 post-BMT BALF nitrite plus nitrate levels (Fig.
3). The lower BALF nitrite level in
BMS + Cy mice compared with BMS mice is most likely related to
formation of ·NO-derived species, as previously reported
(17). BALF nitrite plus nitrate levels of all
iNOS-deficient mice were undetectable (Fig. 3) and significantly less
than in wild-type mice, including the value obtained from control mice
(nonirradiated and nontransplanted mice) of 2.1 ± 0.3 µM.
|
Decreased BALF TNF- and IFN-
in iNOS knockout mice after
allogeneic transplantation.
In wild-type mice, injection of donor T cells also increased day
7 post-BMT BALF TNF-
and IFN-
, and the addition of Cy
further enhanced the production of these proinflammatory cytokines.
iNOS deficiency was associated with decreased levels of BALF TNF-
and IFN-
in allogeneic T cell-recipient mice with or without Cy
injection (Fig. 4). Furthermore, alveolar
macrophages/monocytes obtained from iNOS(
/
) Cy/TBI mice given donor
T cells and cultured for 48 h did not produce ·NO and generated
less TNF-
compared with macrophages/monocytes from wild-type mice
(Fig. 5). These data suggest that
iNOS-derived ·NO is a major amplifier of TNF-
and IFN-
production during allogeneic T cell-dependent inflammation in our IPS
model.
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Histology and macrophage/monocyte immunostaining.
To determine whether the inhibition of cytokine production in
BMS + Cy iNOS(/
) mice was accompanied by lower numbers of lung-infiltrating inflammatory cells, lung sections obtained on day 7 post-BMT were immunostained with CD11b/Mac-1 antibody.
Compared with irradiated mice not given allogeneic T cells (BM), lung
sections of Cy/TBI T cell-recipient mice showed evidence of lung injury associated with infiltration of Mac-1-positive cells. The lack of
iNOS-derived ·NO did not modify the number or type of inflammatory cells in the lung (Fig. 6). Mac-1
expression in the lung increased from 8 ± 2% of nucleated cells
in the BM group to 43 ± 6% of nucleated cells in the BMS + Cy
group and was not modified in the iNOS(
/
) BMS + Cy group (41 ± 8%). Values are means ± SE determined by counting the
percentage of cells expressing Mac-1 in four to eight fields per lung
section under light microscopy. Two to three mice per group from two
representative experiments were assessed. Cy alone in the absence of T
cells (BM + Cy) did not increase the number or activation state of
lung-infiltrating macrophages/monocytes (16).
|
Survival of iNOS knockout mice.
Mortality of mice after allogeneic transplantation is dependent on
infusion of donor T cells, and the addition of Cy accelerates T
cell-dependent mortality. To determine the contribution of ·NO to
post-BMT mortality in the presence and absence of Cy-induced oxidant
stress, survival of iNOS-deficient and iNOS-sufficient mice was
compared. Early survival of mice lacking iNOS-derived ·NO and given
donor spleen T cells (BMS) was enhanced compared with T cell-recipient
wild-type mice (P = 0.008; Fig.
7A). Similarly, we expected
improved survival of BMS + Cy iNOS(/
) mice compared with
BMS + Cy littermates. However, 1 wk post-BMT, Cy/TBI T cell-recipient iNOS(
/
) mice exhibited significantly higher mortality compared with
BMS + Cy iNOS-sufficient mice (Fig. 7B). Cy-facilitated
mortality in all mice was dependent on the presence of allogeneic T
cells, since iNOS(
/
) and iNOS-sufficient mice that were given Cy
without allogeneic T cells exhibited 100% survival in the same
post-BMT period (data not shown). Taken together, these data indicate
that ·NO contributes to and Cy-induced oxidant stress accelerates
mortality of mice after allogeneic transplantation. However, in the
absence of ·NO, BMS + Cy mice die early, possibly related to the
generation of oxidative stress (see below).
|
Persistent oxidant production but less nitrotyrosine staining from
BMS + Cy iNOS knockout mice.
To begin to understand potential reasons for accelerated mortality of
Cy/TBI T cell-recipient iNOS(/
) mice despite decreased production
of inflammatory mediators, the generation of oxidants by macrophages
and nitrotyrosine immunostaining of lung sections from iNOS(
/
) and
wild-type BMS + Cy mice was compared. For these sets of experiments,
BMT was also performed in mice lacking phagocyte respiratory burst
oxidase [NADPH oxidase(
/
)]. In wild-type Cy/TBI T cell-recipient
mice, macrophages obtained on day 7 post-BMT and loaded with
DCFH exhibited intense intracellular fluorescence associated with
specific lung nitrotyrosine staining (Fig.
8). Macrophages from NADPH oxidase(
/
)
BMS + Cy mice exhibited less fluorescence (~50% of BMS + Cy
wild-type mice) and decreased nitrotyrosine staining, suggesting an
important role for NADPH oxidase in the generation of Cy-induced
oxidative/nitrative stress during T cell-dependent generation of ·NO.
Compared with BMS + Cy wild-type mice, lung sections of iNOS(
/
)
BMS + Cy mice exhibited decreased nitrotyrosine staining. However,
DCFH-loaded macrophages from iNOS(
/
) mice continued to exhibit
intense fluorescence, suggesting persistent production of
·NO-independent potent oxidants (Fig. 8).
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DISCUSSION |
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These studies demonstrate the important roles of ·NO and
O and IFN-
in the lung. The
addition of Cy to the conditioning regimen during allogeneic T
cell-dependent ·NO induction increases oxidative/nitrative stress,
which allowed us to investigate the pathobiological significance of the
reaction of ·NO and O
The administration of donor spleen T cells on the day of BMT (day
0) induced the production of ·NO, TNF-, and IFN-
in the absence of significant oxidative/nitrative stress or depletion of BALF
free
SH groups. Our studies indicate that the lack of ·NO in
iNOS(
/
) donor T cell-recipient mice (BMS) was associated with
suppressed production of TNF-
and IFN-
and improved survival, suggesting that iNOS-derived ·NO exacerbated inflammation and accelerated mortality in this murine model. We have not investigated the mechanisms by which ·NO amplifies the inflammatory response. Hierholzer et al. (18), using iNOS(
/
) mice subjected
to hemorrhagic shock, have shown that ·NO may exacerbate inflammation
by the activation of transcriptional nuclear factor-
B and signal
transducer and activator of transcription-3 (18).
Macrophage-derived ·NO has been described to protect tissues from T
cell immune responses by suppressing helper T cell proliferation and
cytotoxicity (27). However, the low level of IFN-
contained in post-BMT BALF from iNOS(
/
) mice does not support the
hypothesis that the absence of ·NO exacerbated T cell proliferation.
A potential explanation for the lack of antiproliferative T cell
effects of ·NO in our model is the complete major histocompatibility
complex (MHC) mismatch, rendering alloactivated lymphocytes
unresponsive to the inhibitory effects of ·NO.
The formation of a nitrating species in Cy/TBI T cell-recipient
mice (BMS + Cy) during the simultaneous generation of ·NO and
O generation.
ONOO
can rapidly oxidize thiol groups (36).
Alternative ONOO
-independent nitration reactions include
the oxidation of nitrite by myeloperoxidase or related peroxidases
(12). Irrespective of the mechanism of formation, the
generation of nitrative stress in BMS + Cy was associated with
accelerated T cell-dependent inflammation and mortality. Although
acrolein, a Cy metabolite, has been shown to deplete glutathione
(33), our data indicate that injection of Cy without T
cells did not significantly alter BALF
SH levels or cause lung
dysfunction (17). Hill et al. (19) recently reported that Cy/TBI potentiates allogeneic T cell-mediated injury to
the gastrointestinal tract, resulting in increased translocation of
lipopolysaccharide (LPS) into the systemic circulation and amplification of the inflammatory response. However, the absence of any
detectable LPS in mice given Cy/TBI and syngeneic T cells in the study
of Hill et al. and the presence of nitrated proteins and depletion of
the free thiol groups in BMS + Cy mice observed in our model favor
the hypothesis that oxidative/nitrative stress is one of the main
reasons for exacerbated injury and decreased survival of irradiated
mice given Cy and allogeneic T cells.
As discussed above, Cy-induced oxidant stress enhanced ·NO
toxicity. However, ·NO deficiency during severe oxidative stress and
depletion of antioxidants resulted in rapid death of Cy/TBI donor T
cell-recipient mice. Despite the absence of ·NO production, DCFH-loaded macrophages from iNOS(/
) mice given donor T cells and
Cy (BMS + Cy) persisted to show intense fluorescence. These data are
consistent with the generation of O
/
) mice exhibit amplified lung injury
during oxidative stress generated by hyperoxic exposure (22). A model of our hypothesis of the pathobiological
effects of ·NO and O
,
that induce host iNOS-derived ·NO. In the lung, excess ·NO
amplifies the early post-BMT destructive inflammatory response. The
addition of Cy enhances cytokine-induced O
, a potent oxidant that potentiates ·NO-mediated
inflammation and mortality. However, the generation of
O
|
Mice lacking iNOS have been used to determine the effects of iNOS
deficiency on LPS-induced mortality. MacMicking et al.
(29) and Wei et al. (42) reported that
iNOS(/
) mice are more resistant to LPS-induced death. In contrast,
Nicholson and colleagues (31) observed that fatality of
iNOS(
/
) mice after LPS injection was similar to genetically matched
mice. These differences were attributed to variations in the background
strains of mice or different preparations of endotoxin administered.
Based on our results, we suggest that a potential explanation for the
conflicting survival data after LPS injection in iNOS(
/
) mice is
the difference in the severity of oxidative stress between the various
animal models used.
Day 7 post-BMT, BALF nitrite levels from iNOS(/
)
mice were below detection limits and significantly lower than
normal control mice. In addition, culture supernatant of alveolar
macrophages from BALF of iNOS(
/
) mice injected with iNOS-sufficient
BM and spleen T cells did not produce ·NO. These observations
suggested the following: 1) iNOS is the source of ·NO
measured in BALF of normal and BMT mice not given donor T cells and
2) activated alveolar macrophages obtained from BALF of
iNOS(
/
) donor T cell-recipient mice are of host origin, as
previously demonstrated using antibodies to MHC class II
(32). Despite the inability to produce iNOS-derived ·NO,
weak nitrotyrosine staining from iNOS(
/
) BMS + Cy mice was
observed. Potential sources for nitration reactions in iNOS(
/
) animals may include cNOS and formation of ·NO-independent nitrating species.
In summary, we have shown that T cell-dependent induction of ·NO
contributes to IPS injury and mortality by several mechanisms. ·NO
amplifies the early post-BMT inflammatory response and contributes to
the formation of toxic effector species, such as ONOO.
Oxidative stress and oxidant/antioxidant balance are major determinants of whether inhibition of iNOS-derived ·NO is beneficial or
detrimental to the host. A safer and more effective strategy may be to
limit the availability of O
formation or to use specific scavengers of
ONOO
.
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ACKNOWLEDGEMENTS |
---|
We acknowledge the expert technical assistance of John Hermanson.
![]() |
FOOTNOTES |
---|
This work was supported by grants from the American Lung Association (Johnie Murphy Career Investigator Award), the American Heart Association (Minnesota Affiliate), the Viking Children's Fund, and National Heart, Lung, and Blood Institute Grants R01-HL-67334 and HL-55209.
Address for reprint requests and other correspondence: I. Y. Haddad, Univ. of Minnesota, Dept. of Pediatrics, 420 Delaware St. S.E., Minneapolis, MN 55455 (E-mail: hadda003{at}tc.umn.edu).
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.
Received 29 March 2001; accepted in final form 17 May 2001.
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