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Abstract
Introduction
Materials and Methods
Results
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In a murine bone-marrow transplant (BMT) model designed to determine risk factors for lung dysfunction in irradiated mice, we reported that cyclophosphamide (Cy)-induced injury and lethality depended on the infusion of donor spleen T cells. In the study reported here, we hypothesized that alveolar macrophage (AM)-derived reactive oxygen/nitrogen species are associated with lung dysfunction caused by allogeneic T cells, which stimulate nitric oxide (·NO) production, and by Cy, which stimulates superoxide production. ·NO reacts with superoxide to form peroxynitrite, a tissue-damaging oxidant. On Day 7 after allogeneic BMT, bronchoalveolar lavage fluid (BALF) obtained from mice injected with T cells contained increased levels of nitrite, which was associated with increased lactate dehydrogenase and protein levels, both of which are indices of lung injury. The injury was most severe in mice receiving both T cells and Cy. Messenger RNA (mRNA) for inducible nitric oxide synthase was detected only in murine lungs injected with T cells ± Cy. AMs obtained on Day 7 after BMT from mice receiving T cells ± Cy spontaneously generated between 20 and 40 µM nitrite in culture, versus < 2 µM generated by macrophages obtained from mice undergoing BMT but not receiving T cells. The level of 3-nitrotyrosine, the stable byproduct of the reaction of peroxynitrite with tyrosine residues, was increased in the BALF proteins of mice injected with both T cells and Cy. We conclude that allogeneic T cells stimulate macrophage-derived ·NO, and that the addition of Cy favors peroxynitrite formation. Peroxynitrite generation clarifies the dependence of Cy-induced lung injury and lethality on the presence of allogeneic T cells.
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Introduction |
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Introduction
Materials and Methods
Results
Discussion
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A major complication limiting the success of bone-marrow transplantation (BMT) is the development of noninfectious diffuse lung injury, referred to as idiopathic pneumonia syndrome (IPS), with an overall mortality of more than 70% in affected patients. Although the etiology of IPS is probably multifactorial, and includes treatment-related toxicities caused by radiation or chemotherapy, recent evidence suggests that immunopathologic mechanisms are critical for development of lung dysfunction in this syndrome. First, the incidence of IPS is higher in patients undergoing allogeneic BMT than in those undergoing autologous BMT (1). Second, the severity of IPS is reduced in patients receiving immunosuppressive therapy for graft-versus-host disease (GVHD) (2). Although the risk of developing IPS may be directly related to the severity of GVHD after BMT (3, 4), the mechanisms by which allogeneic T cells and BMT conditioning regimens alter the course of development of IPS remain unclear.
To investigate the proinflammatory events induced by
allogeneity and by a common pre-BMT conditioning regimen, our group developed a model of IPS in which injection of alloresponsive splenic T cells into irradiated mice
results in lung dysfunction (5). As assessed histologically,
by wet/dry weights, and by specific lung compliance, lung
dysfunction in this model was most severe in mice that also
received cyclophosphamide (Cy; 120 mg/kg/d, on Days 
3
and 2) in addition to T cells. The administration of Cy
before BMT to mice given T cells accelerated mortality
(p < 0.0001) over that in mice given T cells alone. These
early Cy-induced deaths were dependent on the presence
of allogeneic T cells, since recipients of Cy plus syngeneic
T cells or Cy alone (without T cells) exhibited 100% survival in the same post-BMT period (5). The reason(s) for
the dependence of Cy-mediated injury and lethality on the
presence of allogeneic T cells remain(s) unknown.
Immunohistochemical studies of cryosections obtained
on Day 7 after BMT from mice injected with T cells showed
that donor T cells colocalized with host macrophages in
the lung. This was accompanied by an increase in the expression of major histocompatibility complex class II antigens and by the costimulatory B7-1 and B7-2 antigens, providing an ideal environment for the activation of donor T
cells (6). Activated T cells can produce interferon-gamma (IFN-
), which drives macrophage-derived nitric oxide
(·NO) production by inducible nitric oxide synthase (iNOS)
(7). The main functions of alveolar macrophage (AM)-
derived ·NO are killing of intracellular microbes (8), tumor
cell cytostasis (9), and regulation of T cell-mediated immune responses. Although at high concentrations ·NO can
be directly cytotoxic (10), its toxicity is greatly enhanced in
the presence of superoxide (O2· 
) (11). Because ·NO is a
free radical, it can undergo a radical-radical reaction with
O2· at near diffusion-limited rates to yield peroxynitrite
(ONOO), a potent oxidizing agent known to initiate lipid
peroxidation in biologic membranes, sulfhydryl oxidation
of proteins (12), and nitration of aromatic amino acid residues (13).
ONOO has been implicated as causal or contributing
to pathologic conditions marked by chronic inflammation
in humans, including rheumatoid arthritis (14), pulmonary
fibrosis (15), and obliterative bronchiolitis in lung transplant recipients (16). One way to demonstrate the in vivo
formation of ONOO is to detect the presence of stable byproducts of its reactions with biologic compounds. 3-Nitrotyrosine, the product of the addition of a nitro group
(NO2) to the ortho position relative to the hydroxyl group
of tyrosine, is one such stable compound. 3-Nitrotyrosine has been detected in the injured lungs of patients and animals (17). In vivo nitrating agents other than ONOO
have also been described, including nitrogen dioxide (20), as has also the myeloperoxidase-dependent oxidation of
nitrite by neutrophils (21).
Cy has been shown to stimulate O2· production in rat
lungs in vivo (22). Therefore, we hypothesized that the simultaneous generation of macrophage-derived ·NO by
donor T cells and O2· by Cy results in the formation of
toxic levels of ONOO in the lungs of mice undergoing
BMT. ONOO formation may clarify why Cy accelerates
lung dysfunction and mortality in BMT mice injected with
allogeneic but not syngeneic T cells. Our results show that
donor allogeneic spleen T cells induce ·NO production in
host macrophages. Increased nitrite levels in the bronchoalveolar lavage fluid (BALF) and in supernatant of cultured AMs were associated with lung dysfunction. Furthermore, increased 3-nitrotyrosine was found in the BALF
protein of BMT mice given both T cells and Cy, suggesting
increased generation of a nitrating species in this group of mice.
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Materials and Methods |
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Materials and Methods
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Mice
Female B10.BR (H2K) mice were purchased from the Jackson Laboratory (Bar Harbor, ME) and C57BL/6 (H2b) mice were purchased from the National Institutes of Health (Bethesda, MD). Mice were housed in microisolator cages in the specific pathogen free (spf) facility of the University of Minnesota, and were cared for according to the Research Animal Resources guidelines of our institution. For BMT, donors were 4 to 6 wk of age and recipients were used at 8 to 10 wk of age. Sentinel mice were found by our animal facility to be negative for 15 known murine viruses, including those that contribute to pneumonitis (e.g., cytomegalovirus [CMV], pneumonia virus of mice, K-virus) during repeated extensive evaluations over the study period. In addition, representative mice receiving cytotoxic therapy in combination with immune suppression (i.e., Cy/total body irradiation [TBI]) were examined and found to have no evidence of virus-induced pathology.
BMT
Our BMT protocol has been described previously (5, 23)
(Figure 1). Briefly, B10.BR mice were lethally irradiated
(7.5 Gy of TBI as X-irradiation at a dose rate of 0.41 cGy/
min) on the day before BMT, as previsously described (24).
Donor C57BL/6 bone marrow (BM) was T-cell-depleted
with a monoclonal anti-Thy 1.2 antibody (clone 30-H-12,
rat immunoglobulin [Ig]G2b, kindly provided by Dr. David
Sachs of the Massachusetts General Hospital, Boston, MA)
plus complement (Neiffenegger Co., Woodland, CA). For
each experiment, a total of 30 to 40 recipient mice per
treatment group underwent marrow transplantation via
the caudal vein with 20 × 106 C57BL/6 marrow cells supplemented with or without 15 × 106 natural killer (NK)-
cell-depleted (PK136, anti-NK 1.1 plus complement) spleen
cells (bone marrow/spleen cell [BMS] mice) as a source of
T cells causing GVHD. A parallel set of mice also received Cy (Cytoxan; Bristol Myers Squibb, Seattle, WA) at a
dose of 120 mg/kg/d as a conditioning regimen before
BMT on Days 3 and 2.
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Bronchoalveolar Lavage
Mice were killed after an intraperitoneal injection of sodium pentobarbital, and the thoracic cavity was partly dissected. The trachea was cannulated with a 19-gauge needle and infused with 1 ml of ice-cold sterile phosphate-buffered saline (PBS), which was then withdrawn. This was repeated three times, and the collected BALF was combined. The lavage fluid was immediately centrifuged at 500 × g for 10 min at 4°C to pellet cells. Lavage fluid total protein was determined with the bicinchoninic acid (BCA) method, with bovine serum albumin (BSA) used as a standard. Lactate dehydrogenase (LDH) levels were measured with the colorimetric CytoTox 96 assay (Promega, Madison, WI), and the LDH concentration (mU/liter) in BALF was calculated with bovine heart LDH as the standard. Nitrite in BALF was measured according to the Greiss method after the conversion of nitrate to nitrite with the reduced nicotinamide adenine dinucleotide (NADH)-dependent enzyme nitrate reductase (Calbiochem, La Jolla, CA).
Macrophage Cell Culture
The BALF cell pellets from each treatment group were
combined, washed twice, and resuspended in RPMI 1640 medium containing 5% fetal calf serum, penicillin (100 U/
ml), and streptomycin (100 µg/ml). Total cell number was
determined with a hemocytometer, and cell viability was
assessed by trypan blue exclusion. Cytospin samples were
prepared by centrifugation for 5 min on glass microscope slides. Slides were fixed with phosphate-buffered paraformaldehyde and treated with Wright-Giemsa stain. A total of
2 × 105 total cells/well were added to the bottoms of flat,
mouse IgG-coated, 96-well microtiter plates, and macrophages were allowed to adhere for 1 h at 37°C in 5%
CO2 in air, after which unbound cells were removed. Some
cells were cultured in the presence of lipopolysaccharide (LPS; 2 µg/ml) and IFN- (500 U/ml), or NG-monomethyl-
L-arginine (L-NMMA; 1 mM) added 30 min before exposure to LPS plus IFN-. 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 nitrite and LDH.
Cells were washed twice with PBS and lysed with lysis solution (Triton X-100; Promega, Madison, WI), and cellular
LDH content was measured. The percent cytotoxicity during culture of macrophages obtained from BMT mice was
calculated by dividing the concentration of LDH in cell-free supernatant by the total cellular plus supernatant
LDH in each well and multiplying by 100.
Northern Blots
The lungs of some animals were extracted without performing bronchoalveolar lavage (BAL), and were immediately frozen in liquid nitrogen. Total RNA was isolated
with Tri-Reagent (Sigma, St. Louis, MO). RNA samples
(10 µg) were electrophoresed on a formaldehyde-containing denaturing agarose gel, transferred to nitrocellulose filters, and crosslinked by exposure to ultraviolet (UV) radiation (120,000 µJ). The membrane was hybridized overnight
at 65°C with complementary DNA (cDNA) (Alexis, San
Diego, CA) for [32P]deoxycytosine triphosphate ([32P]dCTP)-
labeled murine macrophage iNOS. The hybridization filter was washed and autoradiography was performed by exposure to X-OMAT film (Kodak, Rochester, NY) at 70°C.
Messenger RNA (mRNA) levels were quantified by scanning densitometry with appropriate signals for housekeeping genes (
-actin).
In Situ Hybridization
The in situ hybridization procedure for mRNA of iNOS has been described in detail (25). Frozen sections (4 µm) were thaw-mounted onto baked glass slides and fixed in 3% formaldehyde (Fisher Scientific Co., Fairlawn, NJ) for 1 h. After acetylation (0.25% acetic anhydride) and treatment with 0.1 M triethanolamine-HCl (Boehringer Mannheim Biochemicals, Indianapolis, IN), sections were hybridized overnight at 50°C under a sealed coverslip with digoxigenin-labeled antisense RNA probes. The ribonucleotide probe sequences used were positions 2,016 to 2,322 for iNOS. Immunologic detection of digoxigenin-labeled RNA duplexes was accomplished with antidigoxigenin antibody (alkaline phosphatase conjugated; Boehringer Mannheim). Following color development, sections were mounted in Crystalmount (Biomeda Corp., Foster City, CA).
Western Blotting
To determine whether BALF contained nitrated proteins,
2 ml of cell-free fluid was incubated overnight at 4°C with
calcium chloride (10 mM) and centrifuged at high speed
(13,300 × g) at 4°C for 20 min. The protein pellet was resuspended in 100 µl of H2O. Protein content was determined with the BCA method, and equal amounts of protein (20 µg) were added to a 0.1 M Tris buffer containing 50 µM dithiothreitol, 0.01% bromophenol blue, 1% sodium dodecyl sulfate (SDS) and 10% glycerol and boiled
for 5 min. Proteins (20 µg/lane) were separated on 7.5%
SDS-polyacrylamide gel, transferred to nitrocellulose paper, and probed with a polyclonal antibody against nitrotyrosine (1:5,000 dilution; Upstate, Lake Placid, NY). In
control measurements, specific binding was blocked with 10 mM nitrotyrosine (Sigma). Samples of nitrated BSA
(BSA-NO2), nitrated human surfactant protein A (SP-A-
NO2), or nitrated BAL protein samples from control mice
(nontransplanted, nonirradiated; C-NO2), obtained by exposing BSA (1 mg/ml), SP-A (1 mg/ml), or BAL protein
(1 mg/ml) to ONOO (0.5 mM; Alexis) for 5 min at room
temperature, were used as positive controls. Bound antibody was detected with horseradish peroxidase-conjugated
goat antirabbit immunoglobulin antibodies, using a luminol-enhanced chemiluminescent detection kit (Pierce, Rockford, IL). SP-A was isolated from patients with alveolar
proteinosis as previously described (26).
Statistical Analysis
Results are expressed as means ± SEM. Statistical differences among group means were determined with one-way analysis of variance (ANOVA) and Bonferroni's modification of the t test.
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Introduction
Materials and Methods
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Effects of T Cells and Cy on Lung Injury and ·NO Production in BALF
The percent recoveries of BALF in terms of volume were similar in all groups (> 90%). Consistent with our previously reported histology and specific lung compliance data, analysis of BALF revealed evidence of lung injury in mice injected with donor spleen T cells. The injury was most severe in mice receiving both allogeneic spleen T cells and Cy conditioning (BMS + Cy group). LDH levels, a measure of cellular injury and lysis, were increased on Day 7 after BMT in the BALF of mice injected with T cells; LDH levels were even higher when mice were pretreated with Cy in addition to T cells (Figure 2, top panel ). BALF total protein, a measure of epithelial permeability, was significantly increased in mice treated with C57BL/6 T cells, and Cy further enhanced the injury (Figure 2, bottom panel ). Production of reactive nitrogen species was initially evaluated in BALF by measurement of levels of nitrite and nitrate, the stable byproducts of ·NO and ·NO-derived species. On Day 7 after BMT, nitrite and nitrate levels were increased in mice receiving T cells (Figure 3). The BALF of mice injected with both T cells and Cy contained less nitrite than that of mice injected with T cells alone, perhaps because of the generation of ·NO-derived species (Figure 3). The increased nitrite-plus-nitrate levels were predictive of lung injury, since they were always accompanied by increased LDH and total protein levels. Baseline levels of BALF nitrite, LDH, and total protein concentrations were found on Day 3 after BMT in all groups, and on Day 7 after BMT in mice given Cy/TBI alone (BM and BM + Cy). The lag period for ·NO production and evidence of significant lung injury in our mouse BMT model (Day 7, but not Day 3, after BMT) were probably due to the time required for in vivo activation of injected donor T cells (27).
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three mice per group per time point per experiment,
which was repeated three times. *P < 0.05 compared with control
(BM) group; +P < 0.05 for BMS + Cy versus BMS groups.
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Lung Tissue iNOS mRNA
Expression of mRNA for iNOS was examined with Northern blots and in situ hybridization. Northern blots of total RNA extracted from lung tissue on Day 7 after BMT were probed with cDNA for mouse iNOS. Specific iNOS cDNA binding was seen only in RNA isolated from lungs of mice injected with spleen T cells ± Cy (Figure 4). A much less intense band for iNOS mRNA was observed in lungs of mice not injected with T cells, even if Cy was administered. This was confirmed by in situ hybridization, which demonstrated intense iNOS mRNA expression in both AMs and epithelium in lung sections obtained from mice injected with T cells (not shown) and with T cells plus Cy (Figure 5), but not in sections obtained from mice not receiving T cells. These data suggest that iNOS is the likely source for the enhanced ·NO production in the lungs of mice injected with T cells ± Cy.
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Cellular Source and In Vitro Effects of Reactive Nitrogen Species
Figure 6 shows characteristic cytospin preparations with
Wright-Giemsa staining of cells obtained from BALF at
7 d after BMT. Lavaged cells obtained from mice injected
with T cells contained increased numbers of inflammatory
cells, and the cellular profile revealed a greater percentage
of lymphocytes than for mice not injected with T cells (Table 1). To determine whether AMs were the major source
of reactive nitrogen species, cells were isolated from
BALF and macrophages were selectively maintained in
culture on IgG-coated wells for 48 h. Measurement of total
LDH (supernatant plus cellular; mU/ml) at 48 h revealed a
similar number of cultured macrophages in each well (BM = 0.78 ± 0.05, BM + Cy = 0.80 ± 0.07, BMS = 0.76 ± 0.08, BMS +Cy = 0.75 ± 0.11). Therefore, differences betwen
the groups of mice in the lavage fluid cellular profile did
not affect the density of macrophages left in the wells.
AMs obtained on Day 7, but not Day 3 after BMT from
mice receiving allogeneic T cells or T cells plus Cy, spontaneously generated large amounts of nitrite measured in
the cell-free supernatant (Figure 7). In contrast, macrophages of mice receiving Cy, TBI, or Cy/TBI did not spontaneously generate reactive nitrogen species (Figure 7). To
quantify cell injury in vitro, we measured LDH levels in
the cell-free supernatant and intracellularly, and calculated the percent cytotoxicity as described in the MATERIALS AND METHODS section. Macrophages from mice receiving T cells ± Cy were injured because they spontaneously
released more LDH into the medium than did cells from
irradiated mice given Cy (Figure 8). Incubation of the
cultured macrophages with LPS (2 µg/ml) and IFN- (500 U/ml) significantly increased cytotoxicity only for macrophages obtained from mice injected with T cells and given
Cy conditioning (P < 0.05). The LPS/IFN--mediated toxicity was partly dependent on the generation of ·NO or
·NO-derived species, since inhibition of ·NO production by
L-NMMA restored LDH levels to their baseline values. Notably, L-NMMA suppressed nitrite levels in the supernatant to < 6 µM, but only restored cytotoxicity to pre-LPS/IFN-
levels. A possible explanation for this is that cells from mice
injected with T cells and Cy were irreversibly injured by in
vivo exposure to reactive nitrogen/oxygen species.
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BALF Proteins of BMS + Cy Mice Contained Increased Levels of 3-Nitrotyrosine
Western blots of BALF protein samples obtained from
mice injected with both allogeneic T cells and Cy (BMS + Cy) and probed with rabbit polyclonal anti-nitrotyrosine
antibody contained additional low- and high-molecular-weight nitrated proteins not observed in the other groups
of mice (Figure 9). This binding was specific, since it was
completely blocked in the presence of excess antigen (nitrotyrosine). These data are consistent with the production of a nitrating species mainly in the lungs of BMS + Cy
mice. Under our conditions, the most likely candidate capable of in vivo nitration of tyrosine residues was ONOO.
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In this study we focused on the production of reactive
oxygen/nitrogen species in murine lungs after BMT. The
major finding in the study was that reactive oxygen and
nitrogen species are generated during the course of lung
dysfunction associated with irradiation, exposure to Cy,
and treatment with allogeneic T cells. ·NO-derived species
play a critical role in the peritransplantation period that
may promote the generation of IPS. Conditions favoring the simultaneous production of ·NO and O2· are associated with the presence of nitrated proteins and severe lung dysfunction.
The lower nitrite levels in the BALF of mice receiving
T cells and Cy (BMS + Cy) than in those receiving T cells
alone (BMS) (Figure 3), in the absence of decreased iNOS
mRNA expression in the lungs (Figure 4), was our first
clue that ·NO-derived species were being generated in the
BMS + Cy-treated mice. The conditions favoring the relative amounts of ·NO versus ONOO in our BMT mice are
depicted in Figure 10. In this proposed model, allogeneic T
cells can damage the lung through two pathways: (1) direct
cytolytic activity by release of their stored intracellular contents (perforin, granzymes) to initiate the cascade of destructive events (28); and (2) stimulation of macrophage-derived ·NO, which in the presence of an O2· -generating
system leads to ONOO formation. The O2· -generation
potential of Cy was previously reported (22), and was confirmed in our laboratory with lucigenin-enhanced chemiluminescence (unpublished observation). The combined administration of allogeneic T cells and Cy therefore results
in the simultaneous generation of ·NO and O2· by macrophages, leading to ONOO formation in close proximity
to all components of the alveolar epithelium. Our model
clarifies the basic mechanisms for the dependence of Cy-induced lung dysfunction and lethality on the presence of allogeneic but not syngeneic T cells. In further support of
this model are the following experimental observations:
(1) The pre-BMT administration of Cy to irradiated mice
(BM + Cy) did not cause significant lung dysfunction, a
finding consistent with the concept that O2· is a weak oxidant (29). (2) The combined administration of Cy and syngeneic T cells was not injurious (5), probably because of the
inability of syngeneic T cells to activate host macrophages. This observation rules out a direct effect of Cy on the function of nonactivated T cells. (3) The detection of increased
levels of nitrated BALF proteins in irradiated BMT recipients given allogeneic T cells plus Cy. Because ·NO or reactive oxygen species alone are not capable of nitrating tyrosine residues of proteins (11), we conclude that the most
likely nitrating agent in our BMT mice was ONOO.
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Other nitrite-dependent reaction pathways may contribute to the in vivo nitration of proteins through the formation of nitryl chloride (NO2Cl) by the reaction of nitrite
with hypochlorous acid or myeloperoxidase (30). Although
such reactions may explain the background nitration of
our BALF protein samples, they are an unlikely source of
nitration in the experimental mice (BMS + Cy) for several
reasons. First, immunoperoxidase staining of BMS + Cy
recipient lungs from Day 7 after BMT revealed increased frequencies of T cells and monocytes, without infiltration
of neutrophils, the main source for myeloperoxidase. Second, pathways of nitration of BALF proteins were evident
mainly after the addition of a known O2· -generating agent
(Cy) to allogeneic T cells, an in vivo stimulator of epithelial cells- and macrophage-derived ·NO. Although we
cannot exclude the contribution of NO2Cl or other ·NO-derived species, we favor the explanation that the most
likely nitrating agent was ONOO.
Protein nitration may not be only a "footprint" for
ONOO (17), but also a mechanism by which reactive nitrogen species can damage the structure and function of
proteins. We have shown that SP-A is highly susceptible to
tyrosine nitration by ONOO (26), and that this specific
modification irreversibly impairs the function of this surfactant protein in vitro (31, 32). Although we did not quantitate 3-nitrotyrosine with high-pressure liquid chromatography (HPLC) (33), our immunoblotting technique had as
a main advantage the identification of additional nitrated proteins in the BALF of mice given both Cy and donor T
cells. The identity of these nitrated BALF proteins is not
certain. Of interest, however, is that one of the nitrated
proteins of mice treated with Cy and T cells had a molecular weight of ~ 30 kD, and may have represented SP-A.
Injury to SP-A in these mice may explain the severe surfactant dysfunction and decreased specific lung compliance noted in the BMS + Cy group (5).
Although ONOO is tissue damaging in vivo (18, 34),
high levels of ·NO, in the absence of enhanced O2· production, may serve diverse functions in pathogen-free mouse lungs. To protect delicate lung tissue from T-cell-mediated chronic immune responses, macrophages tightly regulate T-cell function and proliferation (35). Evidence indicates that ·NO serves this role by suppressing T-cell
proliferation and cytolysis (36). However, at high concentrations, ·NO binds to iron-sulfur centers of essential
cellular enzymes and renders them inactive (39, 40). In
vitro, we observed that inhibition of ·NO by L-NMMA during stimulation of cultured macrophages (from BMS + Cy-treated mice) with LPS plus IFN- protected cells
against further LPS/IFN--mediated injury. L-NMMA reduced nitrite levels in the cell supernatant to < 6 µM, but
only restored LDH levels to pre-LPS/IFN- values. Similarly, L-NMMA prevented spontaneous ·NO production
by cultured macrophages from BMS + Cy-treated mice,
but failed to prevent cytotoxicity (data not shown). One
possible explanation for this is that the AM from BMS + Cy-treated mice were already injured during in vivo exposure to ·NO-derived species and were destined to die even
if further ·NO production in vitro was modified.
The in vivo inhibition of ·NO production during systemic GVHD has yielded conflicting results. Hoffman and colleagues showed that ·NO synthesis during acute GVHD in unirradiated mice contributed to both lymphoid and erythroid host tissue destruction (41). The inhibition of ·NO synthesis by the administration of aminoguanidine decreased the lethality in these animals, with little or no improvement in hepatic, splenic, or intestinal correlates of GVHD (42). In contrast, Drobyski and associates observed enhanced weight loss and decreased survival following the administration of L-NMMA to irradiated mice after allogeneic marrow transplantation (43). Although these contradictory results may be related to the nonspecific nature of some of the inhibitors used, they also confirm the complexity of ·NO reactivity in vivo and emphasize the need to determine the major targets of ·NO before predicting its final biologic effects. We avoided in vivo inhibition of ·NO production because to prove our hypothesis, it was important to prevent the formation of a nitrating species without altering ·NO production.
Because most allogeneic BMT protocols in humans entail the use of Cy and TBI as a preconditioning regimen,
ONOO may be an important contributory molecule to
the pathophysiology of immune-mediated lung injury following BMT in humans. However, a notable point of controversy is the capacity of human AMs to produce ·NO. In
contrast to rodent macrophages, it is difficult to stimulate
human macrophages to produce nitrite in vitro (44). More
recent evidence is consistent with the production of reactive nitrogen species by human inflammatory cells, albeit
under different and tighter cytokine regulation than in rodent cells (45). A strong correlation has been observed between the induction of proinflammatory cytokines, levels
of serum nitrite and nitrate, and development of human
GVHD after allogeneic but not after autologous transplantation (46). Furthermore, serum ·NO concentrations were found to be predictive parameters for acute GVHD
after human allogeneic BMT (47).
In summary, the present study provides strong evidence
that detection of nitrated proteins in the BALF of marrow-transplanted mice during the simultaneous generation
of O2· and ·NO is associated with severe lung dysfunction. Further studies, using approaches designed to specifically inhibit ONOO, such as the use of transgenic mice
overexpressing extracellular superoxide dismutase, will be
required to convincingly prove the causality of nitrated
proteins in such lung dysfuntion. Meanwhile, macrophage-derived ·NO, as with systemic monocyte-derived ·NO (48),
remains an easily measurable marker for the extent of cell
activation, and a predictor of the development of an immune-mediated pulmonary attack.
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Footnotes |
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Address correspondence to: Imad Y. Haddad, M.D., University of Minnesota, Dept. of Pediatrics, 420 Delaware Street S.E., Minneapolis, MN 55455. E-mail: hadda003{at}tc.umn.edu
(Received in original form June 25, 1998 and in revised form October 9, 1998).
Abbreviations: alveolar macrophage, AM; bicinchoninic acid, BCA; bone marrow, BM; bovine serum albumin, BSA; bronchoalveolar lavage, BAL; cyclophosphamide, Cy; graft-versus-host disease, GVHD; inducible nitric oxide synthase, iNOS; interferon-, IFN-; idiopathic pneumonia syndrome, IPS; lactate dehydrogenase, LDH; lipopolysaccharide, LPS; NG-monomethyl-L-arginine, L-NMMA; surfactant protein-A, SP-A; total body irradiation, TBI.
Acknowledgments: This work was supported by grants from the Viking Children's Fund, The Minnesota Medical Foundation, and the American Heart Association, and by grants RO1 AI34495, HL56067, HL55209, PO1 AI35296, and Acute Lung Injury Grant P50 HL50152 from the National Institutes of Health. D.H.I. has a Career Investigator Award from the American Lung Association. The authors gratefully acknowledge the valuable comments of Drs. Sadis Matalon and Sha Zhu, the excellent secretarial assistance of Pam Vavra, and the technical assistance of Robert Bair.
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References |
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Materials and Methods
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Discussion
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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: 1601-1606 [Medline].
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