Effects of oxidant stress on inflammation and survival of iNOS knockout mice after marrow transplantation

Shuxia Yang1, Valerie A. Porter1, David N. Cornfield1,2, Carlos Milla1, Angela Panoskaltsis-Mortari2, Bruce R. Blazar2, and Imad Y. Haddad1,2

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>−</SUP></UP>·) and a tissue-damaging nitrating oxidant. We hypothesized that ·NO and O<UP><SUB>2</SUB><SUP>−</SUP></UP>· balance are major determinants of post-BMT survival and inflammation. Inducible nitric oxide synthase (iNOS) deletional mutant mice (-/-) 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-alpha and interferon-gamma , 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-alpha ; idiopathic pneumonia syndrome


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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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)-alpha 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-alpha and interferon (IFN)-gamma , 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<UP><SUB>2</SUB><SUP>−</SUP></UP>·) by respiratory burst oxidase (NADPH oxidase; see Refs. 8 and 41), the most likely nitrating species is peroxynitrite (ONOO-) formed by the simultaneous production of ·NO by T cell-activated macrophages/epithelial cells and Cy-induced O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. ONOO- formation clarifies the dependence of Cy-facilitated toxicity on the presence of allogeneic T cells.

The reaction of ·NO with O<UP><SUB>2</SUB><SUP>−</SUP></UP>· has become central to the understanding of oxidation reactions and generation of oxidative stress (13). ·NO or O<UP><SUB>2</SUB><SUP>−</SUP></UP>· alone are weak oxidants. However, their reaction product, ONOO-, 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<UP><SUB>2</SUB><SUP>−</SUP></UP>· may in fact lower the steady-state concentrations of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and therefore limit the formation of the extremely injurious ·OH and HOCl (43).

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<UP><SUB>2</SUB><SUP>−</SUP></UP>·. BMT experiments in the presence or absence of Cy conditioning were performed in irradiated mice lacking iNOS. We hypothesized that ·NO and ONOO- contribute to lung inflammation and mortality after allogeneic BMT. Our results indicate that iNOS-derived ·NO stimulate TNF-alpha and IFN-gamma 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<UP><SUB>2</SUB><SUP>−</SUP></UP>·-derived highly toxic oxidants.


    MATERIALS AND METHODS
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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 · kg-1 · 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-gamma and TNF-alpha 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-alpha 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-alpha 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.


    RESULTS
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ABSTRACT
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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 · kg-1 · 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<UP><SUB>2</SUB><SUP>−</SUP></UP>· is able to oxidize DCFH (35). In contrast, ONOO- 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).


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Fig. 1.   Cyclophosphamide (Cy) depletes non-protein-bound sulfhydryls (-SH) in bronchoalveolar lavage fluid (BALF) from allogeneic T cell-recipient mice after marrow transplantation. Day 7 post-bone marrow transplantation (BMT) BALF proteins were precipitated with 5% TCA, and free -SH content in the supernatant was determined by measurement of the absorbance at 412 nm after the addition of 5,5'-dithiobis(2-nitrobenzoic acid) (molar extinction coefficient of 13,600/cm). A solution of reduced glutathione was used as standard. Non-protein-bound -SH from BALF of control mice (nontransplanted, nonirradiated) was 11.5 ± 1.6 µM. BM, irradiated C57BL/6 mice given B10.BR bone marrow; BM + Cy, BM mice also given Cy conditioning (120 mg · kg-1 · day-1 on days -3 and -2); BMS, irradiated C57BL/6 mice given B10.BR bone marrow plus C57BL/6 spleen T cells; BMS + Cy, BMS mice also given Cy. Values are means ± SE obtained from the BALF of at least 5 mice/group. *P < 0.05 compared with control (BM).



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Fig. 2.   Cy enhances macrophage-derived production of oxidants in transplanted mice given allogeneic T cells. Day 7 post-BMT BALF macrophages 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) for 20 min. After cells were rinsed with PBS to remove excess probe, generation of intracellular oxidants capable of DCFH oxidation to dichlorofluorescein (DCF) was monitored (0 and 2 h) using an inverted fluorescence microscope (Nikon Eclipse TE200) combined with computerized image analysis. DCF fluorescence was measured at excitation wavelength of 480 nm and an emission wavelength of 520 nm. Shown is a representative experiment that was repeated 2 times with identical results. t, Time.

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.


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Fig. 3.   Nitrite levels in the cell-free BALF of irradiated C57BL/6 inducible nitric oxide synthase (iNOS) knockout and wild-type mice 7 days post-BMT. Mice receiving donor B10.BR spleen T cells (BMS or BMS + Cy) exhibited increased nitrite levels. Nitrite was barely detectable in all iNOS knockout mice. Nitrate was reduced with nitrate reductase before nitrite measurement by the Greiss reaction. Values are means ± SE for n = 12-20 mice/group. *P < 0.05 compared with control (BM). +P < 0.05 comparing the effect of iNOS deficiency in each group.

Decreased BALF TNF-alpha and IFN-gamma in iNOS knockout mice after allogeneic transplantation. In wild-type mice, injection of donor T cells also increased day 7 post-BMT BALF TNF-alpha and IFN-gamma , and the addition of Cy further enhanced the production of these proinflammatory cytokines. iNOS deficiency was associated with decreased levels of BALF TNF-alpha and IFN-gamma 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-alpha compared with macrophages/monocytes from wild-type mice (Fig. 5). These data suggest that iNOS-derived ·NO is a major amplifier of TNF-alpha and IFN-gamma production during allogeneic T cell-dependent inflammation in our IPS model.


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Fig. 4.   Suppressed production of proinflammatory cytokines in BALF of iNOS knockout mice after allogeneic transplantation. A: tumor necrosis factor (TNF)-alpha (pg/ml) measured in day 7 post-BMT BALF by ELISA. B: interferon (IFN)-gamma (pg/ml) measured day 7 post-BMT BALF by ELISA. iNOS knockout mice receiving donor spleen T cells with and without Cy (BMS or BMS + Cy) exhibited lower levels of TNF-alpha and IFN-gamma compared with wild-type matched recipients. Values are means ± SE for n = 7-14 mice/group. *P < 0.05 compared with control (BM). +P < 0.05 comparing the effect of iNOS deficiency in each group.



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Fig. 5.   Decreased production of nitric oxide (·NO) and TNF-alpha by cultured alveolar macrophages obtained from iNOS-deficient (-/-) mice after allogeneic transplantation. Spontaneous nitrite (y-axis on left) and TNF-alpha (y-axis on right) production in supernatant of alveolar macrophages obtained from irradiated BMT mice (BM), BMT mice given donor T cells (BMS), and BMT mice given T cells and Cy (BMS + Cy). Macrophages were cultured for 48 h as described in MATERIALS AND METHODS. Values are means ± SE obtained from at least 8 wells of pooled macrophages obtained from 4-6 mice/group for each experiment, which was repeated one time. nd, Not determined. *P < 0.05 compared with control (BM). +P < 0.05 comparing the effect of iNOS deficiency in each group.

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).


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Fig. 6.   iNOS deficiency does not modify the number of lung-infiltrating inflammatory cells after allogeneic transplantation. Hematoxylin and eosin (H&E) staining (top) and Mac-1 staining (bottom) of frozen lung sections taken on day 7 post-BMT from TBI C57BL/6 mice given BM from B10.BR mice (BM) or wild-type (WT) and iNOS(-/-) Cy/TBI C57BL/6 mice given B10.BR spleen T cells in addition to BM (BMS + Cy). Mac-1-positive macrophages/monocytes were manually counted as percentage of stained cells as described in MATERIALS AND METHODS. Shown is a representative figure (×100, resolution power equivalent to ×40 objective lens).

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).


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Fig. 7.   Post-BMT survival of irradiated knockout mice given allogeneic T cells (BMS) and T cells plus Cy (BMS + Cy). A: C57BL/6 iNOS knockout and matched wild-type mice (n = 10/group) were irradiated on day -1 and infused on day 0 with B10.BR marrow either with BMS or without spleen cells (BM) as indicated. Survival was monitored for 1 mo after transplantation. Mice lacking iNOS exhibited protection from allogeneic T cell-dependent mortality (*P < 0.05). B: irradiated C57BL/6 iNOS(-/-) (n = 16) and iNOS-sufficient (n = 22) mice received Cy (120 mg/kg) on days -3 and -2 and were infused on day 0 with B10.BR BM with B10.BR spleen cells (BMS + Cy). Additional wild-type mice (n = 10) were given Cy and BM without T cells (BM + Cy; n = 10). Mice lacking iNOS exhibited accelerated mortality compared with iNOS-sufficient mice (*P < 0.05).

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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Fig. 8.   Lung nitrotyrosine immunostaining (left) and macrophage-derived production of oxidants (right) from wild-type and knockout mice after allogeneic transplantation. Day 7 post-BMT, frozen sections from the indicated group of mice were incubated with nitrotyrosine antibody (NT Ab) or NT Ab in the presence of 10 mM nitrotyrosine (NT block). Macrophages obtained from day 7 post-BMT BALF cultured on glass coverslips were loaded with DCFH diacetate (0.1 µM) for 20 min. After cells were washed to remove excess probe (1 h), generation of intracellular oxidants was determined by measuring DCF fluorescence using an inverted microscope monitored as described in MATERIALS AND METHODS. Shown is a representative experiment that was repeated one time.


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ABSTRACT
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MATERIALS AND METHODS
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These studies demonstrate the important roles of ·NO and O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and their derived reactive species in the early post-BMT inflammatory and oxidant responses that lead to IPS-related injury and death. Results clearly indicate that iNOS-derived ·NO stimulates the production of TNF-alpha and IFN-gamma 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<UP><SUB>2</SUB><SUP>−</SUP></UP>· in vivo. The generation of oxidants was a major contributor to decreased survival after allogeneic transplantation. A concern is whether the absence of iNOS altered the efficacy of engraftment. In other studies, donor cell engraftment was seen in all recipients conditioned with either a lethal-dose total body irradiation alone or total body irradiation with Cy (39). In the present studies, wild-type and knockout recipients followed long term were healthy, indicating hematopoietic recovery that was likely of donor origin since the conditioning regimen used has been shown to fully ablate host marrow.

The administration of donor spleen T cells on the day of BMT (day 0) induced the production of ·NO, TNF-alpha , and IFN-gamma 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-alpha and IFN-gamma 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-kappa 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-gamma 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<UP><SUB>2</SUB><SUP>−</SUP></UP>· suggested ONOO- 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<UP><SUB>2</SUB><SUP>−</SUP></UP>·-derived, ·NO-independent oxidative stress in iNOS-deficient BMS + Cy mice. We hypothesize that, in the absence of ·NO, the formation of ·OH and HOCl during Cy-facilitated enhanced production of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· is favored. In vitro experiments using chemical donors of reactive oxygen and nitrogen species have shown that ·NO, depending on its relative fluxes, inhibits O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-dependent oxidation reactions (30, 37). Similarly, Kubes and associates (25) have shown that ·NO modulates oxidant-induced leukocyte adhesion to endothelial cells. Our results are in agreement with several reports suggesting that inhibition of ·NO production during oxidant stress exacerbates lung injury and accelerates mortality (15, 21). Furthermore, iNOS(-/-) 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<UP><SUB>2</SUB><SUP>−</SUP></UP>· balance after allogeneic transplantation is shown in Fig. 9. Alloactivated T cells secrete cytokines, the prototype being IFN-gamma , 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<UP><SUB>2</SUB><SUP>−</SUP></UP>· generation. ·NO reacts with O<UP><SUB>2</SUB><SUP>−</SUP></UP>· to generate ONOO-, a potent oxidant that potentiates ·NO-mediated inflammation and mortality. However, the generation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· in the absence of ·NO favors the formation of the extremely toxic O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-derived oxidants, including ·OH and HOCl.


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Fig. 9.   Proposed hypothesis for the pathobiological effects of ·NO and O<UP><SUB>2</SUB><SUP>−</SUP></UP>· interaction in our idiopathic pneumonia syndrome model. ·NO, induced by donor T cell-dependent proinflammatory cytokines, amplifies the early post-BMT inflammatory response. The addition of Cy enhances cytokine-induced O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generation. ·NO reacts with O<UP><SUB>2</SUB><SUP>−</SUP></UP>· to generate ONOO-, a potent oxidant that accelerates inflammation and mortality. The presence of donor T cells and Cy in the absence of iNOS-derived ·NO favors the formation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>·-derived toxic oxidants, including ·OH and HOCl.

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<UP><SUB>2</SUB><SUP>−</SUP></UP>· to prevent ONOO- formation or to use specific scavengers of ONOO-.


    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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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