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

Shuxia Yang, Valerie A. Porter, David N. Cornfield, Carlos Milla, Angela Panoskaltsis-Mortari, Bruce R. Blazar and Imad Y. Haddad
Am J Physiol Lung Cell Mol Physiol 281:L922-L930, 2001. You might find this additional info useful... This article cites 41 articles, 21 of which can be accessed free at: http://ajplung.physiology.org/content/281/4/L922.full.html#ref-list-1 This article has been cited by 8 other HighWire hosted articles, the first 5 are: An Official American Thoracic Society Research Statement: Noninfectious Lung Injury after Hematopoietic Stem Cell Transplantation: Idiopathic Pneumonia Syndrome Angela Panoskaltsis-Mortari, Matthias Griese, David K. Madtes, John A. Belperio, Imad Y. Haddad, Rodney J. Folz and Kenneth R. Cooke Am. J. Respir. Crit. Care Med., May 1, 2011; 183 (9): 1262-1279. [Abstract] [Full Text] [PDF]
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IFNγ differentially controls the development of idiopathic pneumonia syndrome and GVHD of the gastrointestinal tract Angela C. Burman, Tatjana Banovic, Rachel D. Kuns, Andrew D. Clouston, Amanda C. Stanley, Edward S. Morris, Vanessa Rowe, Helen Bofinger, Renae Skoczylas, Neil Raffelt, Olivier Fahy, Shaun R. McColl, Christian R. Engwerda, Kelli P. A. McDonald and Geoffrey R. Hill Blood, August 1, 2007; 110 (3): 1064-1072. [Abstract] [Full Text] [PDF] Absence of host tumor necrosis factor receptor 1 attenuates manifestations of idiopathic pneumonia syndrome Mayank Shukla, Shuxia Yang, Carlos Milla, Angela Panoskaltsis-Mortari, Bruce R. Blazar and Imad Y. Haddad Am J Physiol Lung Cell Mol Physiol, May 1, 2005; 288 (5): L942-L949. [Abstract] [Full Text] [PDF] Antioxidant defense responses to sleep loss and sleep recovery Carol A. Everson, Christa D. Laatsch and Neil Hogg Am J Physiol Regul Integr Comp Physiol, February 1, 2005; 288 (2): R374-R383. [Abstract] [Full Text] [PDF] Surfactant protein A is a required mediator of keratinocyte growth factor after experimental marrow transplantation Imad Y. Haddad, Carlos Milla, Shuxia Yang, Angela Panoskaltsis-Mortari, Samuel Hawgood, David L. Lacey and Bruce R. Blazar Am J Physiol Lung Cell Mol Physiol, September 1, 2003; 285 (3): L602-L610. [Abstract] [Full Text] [PDF] Updated information and services including high resolution figures, can be found at: http://ajplung.physiology.org/content/281/4/L922.full.html Additional material and information about AJP - Lung Cellular and Molecular Physiology can be found at: http://www.the-aps.org/publications/ajplung

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AJP - Lung Cellular and Molecular Physiology publishes original research covering the broad scope of molecular, cellular, and integrative aspects of normal and abnormal function of cells and components of the respiratory system. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2001 by the American Physiological Society. ISSN: 1040-0605, ESSN: 1522-1504. Visit our website at http://www.the-aps.org/.

of Minnesota. 2012 Address for reprint requests and other correspondence: I. NO limits recruitment of neutrophils into sites of inflammation (26) and suppresses the expression of adhesion molecules (10). Univ. 4). alloactivated by antigen-presenting IDIOPATHIC PNEUMONIA SYNDROME cells. 9. Effects of oxidant stress on inflammation and survival of iNOS knockout mice after marrow transplantation.1 DAVID N. Although the use of nonspecific drugs that also inhibit beneficial constitutive nitric oxide synthase (cNOS)-derived NO may explain some of the results. Effects of oxidant stress on inflammation and survival of iNOS knockout mice after marrow transplantation SHUXIA YANG. see Ref. most likely by generation of NO-independent toxic oxidants. 34. The high-output iNOS-derived NO may serve several immunoregulatory functions that can modify T cell immune responses.2 CARLOS MILLA.Am J Physiol Lung Cell Mol Physiol 281: L922–L930. Department of Pediatrics. NO has been shown to directly upregulate or downregulate the expression of several cytokines and chemokines (2. iNOS( / ) mice given both donor T cells and Cy (BMS Cy) died earlier than iNOS-sufficient BMS Cy mice. Dept.org on January 26.2 BRUCE R. S. Consistent with this hypothesis. David N. 7. and the generation of large amounts of nitric oxide ( NO) by inducible nitric oxide synthase (iNOS) (17.00 Copyright © 2001 the American Physiological Society . elevated levels of tumor necrosis factor (TNF). nitric oxide. lymphocytes. Minneapolis. However. IPS accounts for at least 40% of nongraft vs.1 ANGELA PANOSKALTSIS-MORTARI. immune-mediated damage begins. 40).C. BLAZAR. including TNF.7 -dichlorofluorescin. Haddad. Am J Physiol Lung Cell Mol Physiol 281: L922–L930.. the main reasons for the contradictory effects of NO post-BMT remain unclear. of Pediatrics. 2001.org Downloaded from ajplung. macrophages.S. Section 1734 solely to indicate this fact. 20). However. We hypothesized that NO and O2 balance are major determinants of post-BMT survival and inflammation.umn. injection of allogeneic T cells induces nitric oxide ( NO). Bronchoalveolar lavage fluids obtained on day 7 post-BMT from iNOS( / ) BMS mice contained less tumor necrosis factorand interferon. 32). 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.2 AND IMAD Y. and the addition of cyclophosphamide (Cy) generates superoxide (O2 ) and a tissue-damaging nitrating oxidant.and interferon (IFN). PORTER. A second pathway for stimulating lung injury is via the release of proinflammatory cytokines by activated macrophages and lung-infiltrating monocytes. In our allogeneic BMT model. idiopathic pneumonia syndrome (IPS) refers to diffuse and often fatal noninfectious lung dysfunction that occurs after bone marrow transplantation (BMT.. http://www. accepted in final form 17 May 2001 Yang. In addition. Carlos Milla. Blazar. lung dysfunction in lethally irradiated mice is dependent on the infusion of donor spleen T cells and is associated with T celldependent early production of proinflammatory cytokines. Once infiltrating donor T cells. MN 55455 (E-mail: hadda003@tc. Attempts at determining the role of NO by administration of iNOS inhibitors during GVHD after allogeneic BMT have yielded conflicting results (11. Cornfield. 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 . Shuxia. The article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U. tumor necrosis factor. the addition of the commonly used conditioning drug cyclophosphamide (Cy) potentiated lung dysfunction and accelerated mortality in donor T cell-recipient irradiated mice. and Imad Y.physiology. HADDAD1. Bruce R. 32).2 1 Division of Pulmonary and Critical Care.—In a model of idiopathic pneumonia syndrome after bone marrow transplantation (BMT). Cy-facilitated injury was The costs of publication of this article were defrayed in part by the payment of page charges. despite suppressed inflammation and decreased nitrotyrosine staining. L922 1040-0605/01 $5. the lack of NO during Cy-induced oxidant stress decreases survival of T cell-recipient mice.ajplung. In our IPS model. encounter pulmonary antigens. CORNFIELD. 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).E. Furthermore. 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. Macrophage-derived NO has been shown to prevent T cell-dependent cytolysis by suppressing T cell proliferation (24) and induction of apoptosis (1).1. Y.. Angela Panoskaltsis-Mortari. indicating that NO stimulated the production of proinflammatory cytokines. peroxynitrite. Minneapolis.and interleukin (IL)-1 and IL-6 are present in the bronchoalveolar lavage fluid (BALF) or parenchyma during IPS injury (6). Cancer Center. We concluded that NO amplifies T cell-dependent inflammation and addition of Cy exacerbates NO-dependent mortality. Porter. 2001. Valerie A. Haddad.edu). 420 Delaware St. University of Minnesota. and 2Division of Bone Marrow Transplantation. host disease (GVHD) deaths after allogeneic BMT.1 VALERIE A. Minnesota 55455 Received 29 March 2001..

and macrophages were allowed to adhere for 1 h at 37°C in 5% CO2 in air.and IFN. In addition. Minneapolis. donors were 6–8 wk of age. including glutathione.ajplung. For each experiment. Boston. Female B10. since the injection of Cy alone (without T cells) did not cause lung dysfunction and did not affect survival of BMT mice (32). Cambridge. Mice were killed on day 7 postBMT after an intraperitoneal injection of pentobarbital sodium. Seattle. MN). rat IgG2b. Massachusetts General Hospital.and nitrite readings were adjusted www. IFN. infused with 1 ml of ice-cold sterile PBS. A parallel set of mice also received 120 mg kg 1 day 1 of Cy (Cytoxan. credible experimental evidence indicates that the reaction of NO with O2 may in fact lower the steady-state concentrations of O2 and therefore limit the formation of the extremely injurious OH and HOCl (43). Promega). supernatants were aspirated from individual culture wells for measurement of TNF. We hypothesized that NO and ONOO contribute to lung inflammation and mortality after allogeneic BMT. 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 O2 . CA). kindly provided by Dr. As per our approved animal research protocol. MA) plus complement (Neiffenegger. and survival of BMS Cy mice was monitored for 7 days after transplantation. C57BL/6 wild-type and knockout mice were lethally total body irradiated (TBI. The reaction of NO with O2 has become central to the understanding of oxidation reactions and generation of oxidative stress (13).production and that Cy-induced oxidative/nitrative stress promotes NO-mediated lung dysfunction. Triton X-100. Total (supernatant cellular) LDH values were used to correct for possible differences in adherent cell number between groups. Paul.41 Gy/min) on the day before BMT. Our results indicate that iNOS-derived NO stimulate TNF. 100 U/ml penicillin. fully congenic iNOS( / ). The cells were maintained in culture at 37°C for 48 h in 5% CO2 in air. The BALF cell pellets from mice in each treatment group were combined. Madison. Bone marrow transplant. However. A cohort of mice from each group was monitored for survival. ONOO . CA). St. Total cell number was determined with a hemacytometer.5 -dithiobis(2-nitrobenzoic acid) (DTNB). their reaction product. and recipients were used at 8–10 wk of age. a total of 5–10 recipient mice/treatment group were transplanted via the caudal vein with 20 106 B10. Briefly. and return fluid was combined. and lactic dehydrogenase (LDH) by the colorimetric CytoTox 96 assay (Promega. More than 95% of adherent cells were macrophages. The absorbance of the yellow anion 2-nitro-5-thiobenzoate formed was measured at 412 nm. the most abundant antioxidant present in the epithelial lining fluid (3). MATERIALS AND METHODS Mice. The BALF was immediately centrifuged at 500 g for 10 min at 4°C to pellet cells. Bronchoalveolar lavage.BR (H2K). In addition.org Downloaded from ajplung. At the termination of cell culture. survival of BMS mice was monitored for 30 days. 2012 281 • OCTOBER 2001 • . Cells were washed two times with PBS and lysed with lysis solution (10 . BALF proteins were precipitated with 5% TCA. flat-bottom 96well microtiter plates (Costar. despite suppressed inflammation. For AJP-Lung Cell Mol Physiol • VOL BMT. and 100 g/ml streptomycin. WI). BALF biochemical analysis. David Sachs. is a potent oxidant and nitrating species.physiology. WA) as a conditioning regimen on days 3 and 2. followed by removal of unbound cells. 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. ONOO formation clarifies the dependence of Cy-facilitated toxicity on the presence of allogeneic T cells. Macrophage culture. 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 O2 . The trachea was cannulated with a 22-gauge angiocatheter.2 antibody (clone 30-H-12.org on January 26. BMT was performed as previously described (16. Although there is little doubt that ONOO formation enhances NO toxicity.by ELISA (PharMingen. San Diego. NO deficiency during Cy-induced oxidative stress and depletion of antioxidants worsened the survival of mice post-BMT. and nonprotein-bound SH in the supernatant was determined after the addition of DTNB. and the thoracic cavity was partially dissected. MA). lung dysfunction in Cy/TBI T cell-recipient mice was associated with the detection of nitrated proteins (17). NO or O2 alone are weak oxidants. and withdrawn. and inbred matched wild-type mice on the C57BL/6 (H2b) background were purchased from Jackson Laboratory (Bar Harbor. as previously described (38). Donor B10. CA). ME). However. see Refs. washed two times in cold PBS.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.NITRIC OXIDE/SUPEROXIDE BALANCE AFTER ALLOGENEIC TRANSPLANTATION L923 dependent on T cells. BMT experiments in the presence or absence of Cy conditioning were performed in irradiated mice lacking iNOS. MN) containing 5% FCS. BristolMyers Squibb. nitrite by the Greiss method. and cellular LDH release was measured. Total cells (2 105/well) were added to mouse IgG-coated.5 Gy TBI by X-ray at a dose rate of 0. 7. 17. 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. 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. This was repeated two times. La Jolla.and TNFlevels in the cell-free BALF were determined by ELISA using commercial kits (R&D Systems. consistent with the generation of O2 -derived highly toxic oxidants. Because Cy is known to deplete antioxidants and to enhance the generation of superoxide (O2 ) by respiratory burst oxidase (NADPH oxidase.BR bone marrow (BM) was T cell depleted with a monoclonal anti-Thy 1. 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. Woodland. TNF. ONOO can oxidize sulfhydryl groups. 8 and 41). 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. and resuspended in RPMI 1640 medium (Celox Laboratories. 32).

BALF non-protein-bound SH levels were determined. Lake Placid. 1. Undetectable BALF nitrite plus nitrate in iNOS knockout mice after allogeneic transplantation. Representative sections were stained with hematoxylin and eosin (H&E) for histopathological assessment. As previously reported (17). IN)-PBS (3:1) was infused via the trachea into the lung. PharMingen) using avidin-biotin blocking reagents. 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. the acetate groups were removed by intracellular esterases. and sealed with coverslips. macrophages/monocytes from BALF of BMS Cy mice showed time-dependent intense fluorescence (Fig.BR bone marrow plus C57BL/6 spleen T cells.600/cm). St. In some animals. A solution of reduced glutathione was used as standard. which can be visualized on a single-cell basis using fluorescence microscopy. Mac-1 staining) followed by incubation overnight at 4°C with the following antibodies: 1) rabbit polyclonal anti-nitrotyrosine antibody (NT Ab. Molecular Probes. Values are means SE obtained from the BALF of at least 5 mice/group. The sections were counterstained with hematoxylin. In wild-type mice. Adherent cells were loaded with 2 . DCFH was oxidized to dichlorofluorescein (DCF). BM. BMS) or BMT mice not given T cells (BM). To visualize specific NT Ab binding.5–1. of cells originally plated/well). The return BALF volumes were similar in all groups of mice ( 90% of instilled volume). trapping the probe inside the cells.5 1. NY) and 2) biotinylated monoclonal CD11b/Mac-1 (clone M1/ 70.5 -dithiobis(2-nitrobenzoic acid) (molar extinction coefficient of 13. BMS Cy. 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. overlaid with Permount (Sigma). day 7 post-BMT BALF from TBI mice infused with allogeneic T cells with and without Cy contained increased numbers of inflammatory cells. BMS mice also given Cy.m) 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). lungs were extracted without lavage and were perfused with 1. Alveolar macrophages obtained from day 7 post-BMT BALF were cultured on glass coverslips for 1 h followed by removal of nonadherent cells. In control measurements. DCF fluorescence was measured at an excitation wavelength of 480 nm and an emission wavelength of 520 nm.05 compared with control (BM). Nonantigenic sites were blocked with 10% goat serum (nitrotyrosine. Upstate Biotechnology. The lung was snap-frozen in liquid nitrogen and stored at 80°C. dehydrated. irradiated C57BL/6 mice given B10. A comparison of survival curves between the different groups was made using the log-rank test. MO) or 10% horse serum (Sigma. OR) for 20 min. After rinsing with PBS to remove excess probe.0 ml optimal cutting temperature medium (Miles Laboratories. and iNOS deficiency did not modify the cellular number or profile (data not shown). 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. 1). Results are expressed as means SE.L924 NITRIC OXIDE/SUPEROXIDE BALANCE AFTER ALLOGENEIC TRANSPLANTATION accordingly using the BM group as an assigned reference value for 2 105 cells (the no. goat anti-rabbit IgG conjugated with horseradish peroxidase (1:500 dilution).0 ml of saline via the right ventricle of the heart. Neither NO nor O2 is able to oxidize DCFH (35). P 0. followed by the addition of 3. or tissues were incubated with the NT Ab in the presence of excess antigen (10 mM nitrotyrosine). Cyclophosphamide (Cy) depletes non-protein-bound sulfhydryls ( SH) in bronchoalveolar lavage fluid (BALF) from allogeneic T cell-recipient mice after marrow transplantation. irradiated C57BL/6 mice given B10. injection of donor T cells increased day 7 post-BMT BALF nitrite plus nitrate levels (Fig. During loading. A mixture of 0. ONOO and other strong oxidants such as OH and HOCl oxidize DCFH to form the highly fluorescent product DCF (23). *P 0.05 were considered statistically significant. Injection of Cy alone (without T cells) did not significantly decrease BALF non-protein-bound SH (Fig. Day 7 post-bone marrow transplantation (BMT) BALF proteins were precipitated with 5% TCA.ajplung. 2012 Cy-induced oxidative stress in allogeneic T cell-recipient mice. and free SH content in the supernatant was determined by measurement of the absorbance at 412 nm after the addition of 5. 1:100 dilution. which exhibited background fluorescence. On day 7 post-BMT.org on January 26. Macrophage-derived intracellular oxidants.org 281 • OCTOBER 2001 • . Histology and immunohistochemistry. nonirradiated) was 11. 2). avidin-biotin complex-peroxidase conjugate. the primary antibody was omitted. sections were incubated with secondary antibody.BR bone marrow.6 M.physiology.3 -diaminobenzidine (Vector Laboratories) chromogenic substrate. Statistical analysis. Statistical differences among group means were determined by Tukey’s Studentized test.1 M. Louis. BM mice also given Cy conditioning (120 mg kg 1 day 1 on days 3 and 2).7 -dichlorofluorescin (DCFH) diacetate (0. To determine whether Cy also depleted epithelial lining fluid free thiol groups. The lower Downloaded from ajplung. 3). and diaminobenzidine chromogenic substrate purchased from Vector Laboratories (Burlingame. UK) using Axon Instruments (Foster City. BM Cy. Sigma Chemical. Non-protein-bound SH from BALF of control mice (nontransplanted. www. 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 AJP-Lung Cell Mol Physiol • VOL Fig. Elkhart. The number of positive cells in the lung was quantitated as the percentage of nucleated cells at a magnification of 200 ( 20 objective lens). In contrast to cells from TBI mice not given T cells (BM). BMS. Eugene. In contrast. RESULTS 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). CA). Four to eight fields per lung were evaluated. CA) image capture and analysis software. After an oxidative burst. Data were analyzed by ANOVA or Student’s t-test. Thin (4.

4).in allogeneic T cell-recipient mice with or without Cy injection (Fig. 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.05 comparing the effect of iNOS deficiency in each group. Nitrite was barely detectable in all iNOS knockout mice.(pg/ml) measured in day 7 post-BMT BALF by ELISA. injection of donor T cells also increased day 7 post-BMT BALF TNF. To determine whether the inhibition of cytokine production in BMS Cy iNOS( / ) mice was accompanied by lower numbers of lung-infiltrating inflam- Downloaded from ajplung.and IFN. Time.compared with macrophages/monocytes from wild-type mice (Fig. In wild-type mice. Cy enhances macrophage-derived production of oxidants in transplanted mice given allogeneic T cells. iNOS deficiency was associated with decreased levels of BALF TNF. Shown is a representative experiment that was repeated 2 times with identical results. DCF fluorescence was measured at excitation wavelength of 480 nm and an emission wavelength of 520 nm.BR spleen T cells (BMS or BMS Cy) exhibited increased nitrite levels.production during allogeneic T cell-dependent inflammation in our IPS model.1 M) for 20 min.05 comparing the effect of iNOS deficiency in each group.physiology. www.org on January 26.7 dichlorofluorescin (DCFH) diacetate (0. Mice receiving donor B10. A: tumor necrosis factor (TNF). Decreased BALF TNF.ajplung. Nitrate was reduced with nitrate reductase before nitrite measurement by the Greiss reaction. 2012 BALF nitrite level in BMS Cy mice compared with BMS mice is most likely related to formation of NOderived species. AJP-Lung Cell Mol Physiol • VOL Fig.05 compared with control (BM).org 281 • OCTOBER 2001 • .in iNOS knockout mice after allogeneic transplantation. Suppressed production of proinflammatory cytokines in BALF of iNOS knockout mice after allogeneic transplantation..1 0. t.NITRIC OXIDE/SUPEROXIDE BALANCE AFTER ALLOGENEIC TRANSPLANTATION L925 Fig. 3.(pg/ml) measured day 7 post-BMT BALF by ELISA. These data suggest that iNOS-derived NO is a major amplifier of TNF. B: interferon (IFN).05 compared with control (BM). Adherent cells were loaded with 2 . 3) and significantly less than in wild-type mice. Values are means SE for n 12–20 mice/group. and the addition of Cy further enhanced the production of these proinflammatory cytokines. P 0.and IFN. 2. as previously reported (17). After cells were rinsed with PBS to remove excess probe. 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. 4. *P 0. Day 7 post-BMT BALF macrophages were cultured on glass coverslips for 1 h followed by removal of nonadherent cells.and IFN. Histology and macrophage/monocyte immunostaining. Values are means SE for n 7–14 mice/group. BALF nitrite plus nitrate levels of all iNOS-deficient mice were undetectable (Fig. 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. Fig.and IFN. iNOS knockout mice receiving donor spleen T cells with and without Cy (BMS or BMS Cy) exhibited lower levels of TNF.compared with wild-type matched recipients. P 0. including the value obtained from control mice (nonirradiated and nontransplanted mice) of 2.3 M. *P 0. 5). Furthermore.and IFN.

Spontaneous nitrite (y-axis on left) and TNF. these data indicate that NO contributes to and Cy-induced oxidant stress accelerates mortality of mice after allogeneic transplantation. BMT mice given donor T cells (BMS). which was repeated one time. Downloaded from ajplung. nd. Mortality of mice after allogeneic transplantation is dependent on infusion of donor T cells. *P 0. 6).05 compared with control (BM).BR spleen T cells in addition to BM (BMS Cy).(y-axis on right) production in supernatant of alveolar macrophages obtained from irradiated BMT mice (BM). matory cells.BR mice (BM) or wild-type (WT) and iNOS( / ) Cy/TBI C57BL/6 mice given B10. 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%).org on January 26. and BMT mice given T cells and Cy (BMS Cy). 7B). Shown is a representative figure ( 100. 6. The lack of iNOS-derived NO did not modify the number or type of inflammatory cells in the lung (Fig. Cy alone in the absence of T cells (BM Cy) did not increase the number or activation state of lung-infiltrating macrophages/monocytes (16). 5. and the addition of Cy accelerates T cell-dependent mortality. 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. 2012 Fig. 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. 7A). Taken together. Cy/TBI T cellrecipient iNOS( / ) mice exhibited significantly higher mortality compared with BMS Cy iNOS-sufficient mice (Fig. Decreased production of nitric oxide ( NO) and TNF.ajplung.org .physiology. AJP-Lung Cell Mol Physiol • VOL 281 • OCTOBER 2001 • www. P 0. Two to three mice per group from two representative experiments were assessed. 1 wk post-BMT.008. lung sections obtained on day 7 post-BMT were immunostained with CD11b/Mac-1 antibody. 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. Mac-1-positive macrophages/monocytes were manually counted as percentage of stained cells as described in MATERIALS AND METHODS. iNOS deficiency does not modify the number of lung-infiltrating inflammatory cells after allogeneic transplantation. Cy-facilitated mortality in all mice was dependent on the presence of allogeneic T cells. Not determined. To determine the contribution of NO to post-BMT mortality in the presence and absence of Cy-induced oxidant stress. we expected improved survival of BMS Cy iNOS( / ) mice compared with BMS Cy littermates.by cultured alveolar macrophages obtained from iNOS-deficient ( / ) mice after allogeneic transplantation. resolution power equivalent to 40 objective lens).05 comparing the effect of iNOS deficiency in each group. lung sections of Cy/TBI T cell-recipient mice showed evidence of lung injury associated with infiltration of Mac-1-positive cells. Compared with irradiated mice not given allogeneic T cells (BM). 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). Values are means SE obtained from at least 8 wells of pooled macrophages obtained from 4–6 mice/group for each experiment. Similarly. survival of iNOSdeficient and iNOS-sufficient mice was compared. However.L926 NITRIC OXIDE/SUPEROXIDE BALANCE AFTER ALLOGENEIC TRANSPLANTATION Fig. Macrophages were cultured for 48 h as described in MATERIALS AND METHODS. Survival of iNOS knockout mice. Fig.

In the present studies. Persistent oxidant production but less nitrotyrosine staining from BMS Cy iNOS knockout mice. suggesting an important role for NADPH oxidase in the generation of Cy-induced oxidative/nitrative stress during T cell-dependent generation of NO. renderwww. 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. the low level of IFN.and IFN. possibly related to the generation of oxidative stress (see below). 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).B and signal transducer and activator of transcription-3 (18). We have not investigated the mechanisms by which NO amplifies the inflammatory response.BR spleen cells (BMS Cy). However. In wild-type Cy/TBI T cell-recipient mice. 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. Post-BMT survival of irradiated knockout mice given allogeneic T cells (BMS) and T cells plus Cy (BMS Cy). The addition of Cy to the conditioning regimen during allogeneic T cell-dependent NO induction increases oxidative/nitrative stress. wild-type and knockout recipients followed long term were healthy. Additional wild-type mice (n 10) were given Cy and BM without T cells (BM Cy. suggesting persistent production of NO-independent potent oxidants (Fig. 7. n 10). DCFH-loaded macrophages from iNOS( / ) mice continued to exhibit intense fluorescence.in the absence of significant oxidative/nitrative stress or depletion of BALF free SH groups.and improved survival.NITRIC OXIDE/SUPEROXIDE BALANCE AFTER ALLOGENEIC TRANSPLANTATION L927 fluorescence ( 50% of BMS Cy wild-type mice) and decreased nitrotyrosine staining. BMT was also performed in mice lacking phagocyte respiratory burst oxidase [NADPH oxidase( / )]. The generation of oxidants was a major contributor to decreased survival after allogeneic transplantation. However. DISCUSSION Fig. The administration of donor spleen T cells on the day of BMT (day 0) induced the production of NO. the generation of oxidants by macrophages and nitrotyrosine immunostaining of lung sections from iNOS( / ) and wild-type BMS Cy mice was compared. and IFN. Compared with BMS Cy wild-type mice.org Downloaded from ajplung. A potential explanation for the lack of antiproliferative T cell effects of NO in our model is the complete major histocompatibility complex (MHC) mismatch. A concern is whether the absence of iNOS altered the efficacy of engraftment. 8). BMS Cy mice die early. To begin to understand potential reasons for accelerated mortality of Cy/TBI T cell-recipient iNOS( / ) mice despite decreased production of inflammatory mediators. macrophages obtained on day 7 post-BMT and loaded with DCFH exhibited intense intracellular fluorescence associated with specific lung nitrotyrosine staining (Fig.. lung sections of iNOS( / ) BMS Cy mice exhibited decreased nitrotyrosine staining.and IFN.in the lung.physiology. Macrophages from NADPH oxidase( / ) BMS Cy mice exhibited less AJP-Lung Cell Mol Physiol • VOL These studies demonstrate the important roles of NO and O2 and their derived reactive species in the early post-BMT inflammatory and oxidant responses that lead to IPS-related injury and death. (18).ajplung. Our studies indicate that the lack of NO in iNOS( / ) donor T cell-recipient mice (BMS) was associated with suppressed production of TNF. which allowed us to investigate the pathobiological significance of the reaction of NO and O2 in vivo. TNF.BR BM with B10. Mice lacking iNOS exhibited accelerated mortality compared with iNOS-sufficient mice (*P 0. Survival was monitored for 1 mo after transplantation. in the absence of NO. 2012 281 • OCTOBER 2001 • .05). 8). Results clearly indicate that iNOS-derived NO stimulates the production of TNF.BR marrow either with BMS or without spleen cells (BM) as indicated. using iNOS( / ) mice subjected to hemorrhagic shock.contained in post-BMT BALF from iNOS( / ) mice does not support the hypothesis that the absence of NO exacerbated T cell proliferation.05). Macrophage-derived NO has been described to protect tissues from T cell immune responses by suppressing helper T cell proliferation and cytotoxicity (27). In other studies. indicating hematopoietic recovery that was likely of donor origin since the conditioning regimen used has been shown to fully ablate host marrow. have shown that NO may exacerbate inflammation by the activation of transcriptional nuclear factor. Hierholzer et al. However. Mice lacking iNOS exhibited protection from allogeneic T cell-dependent mortality (*P 0. For these sets of experiments. suggesting that iNOS-derived NO exacerbated inflammation and accelerated mortality in this murine model.org on January 26.

8. and the AJP-Lung Cell Mol Physiol • VOL Fig. the generation of nitrative stress in BMS Cy was associated with accelerated T cell-dependent inflammation and mortality. Hill et al. However. resulting in increased translocation of lipopolysaccharide (LPS) into the systemic circulation and amplification of the inflammatory response. ing alloactivated lymphocytes unresponsive to the inhibitory effects of NO. After cells were washed to remove excess probe (1 h). a Cy metabolite. 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). www. amplifies the early post-BMT inflammatory response. The addition of Cy enhances cytokine-induced O2 generation.L928 NITRIC OXIDE/SUPEROXIDE BALANCE AFTER ALLOGENEIC TRANSPLANTATION Downloaded from ajplung. Alternative ONOO -independent nitration reactions include the oxidation of nitrite by myeloperoxidase or related peroxidases (12). Macrophages obtained from day 7 post-BMT BALF cultured on glass coverslips were loaded with DCFH diacetate (0. NO. The presence of donor T cells and Cy in the absence of iNOS-derived NO favors the formation of O2 -derived toxic oxidants. 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. Irrespective of the mechanism of formation. 9. Day 7 post-BMT.org on January 26. our data indicate that injection of Cy without T cells did not significantly alter BALF SH levels or cause lung dysfunction (17). a potent oxidant that accelerates inflammation and mortality. Lung nitrotyrosine immunostaining (left) and macrophage-derived production of oxidants (right) from wild-type and knockout mice after allogeneic transplantation. (19) recently reported that Cy/TBI potentiates allogeneic T cell-mediated injury to the gastrointestinal tract. including OH and HOCl.1 M) for 20 min. ONOO can rapidly oxidize thiol groups (36). has been shown to deplete glutathione (33).ajplung. The formation of a nitrating species in Cy/TBI T cellrecipient mice (BMS Cy) during the simultaneous generation of NO and O2 suggested ONOO generation. NO reacts with O2 to generate ONOO . Although acrolein. 2012 Fig.physiology. Proposed hypothesis for the pathobiological effects of NO and O2 interaction in our idiopathic pneumonia syndrome model. the absence of any detectable LPS in mice given Cy/TBI and syngeneic T cells in the study of Hill et al. induced by donor T cell-dependent proinflammatory cytokines.org 281 • OCTOBER 2001 • .

NO-independent oxidative stress in iNOS-deficient BMS Cy mice. We acknowledge the expert technical assistance of John Hermanson. A model of our hypothesis of the pathobiological effects of NO and O2 balance after allogeneic transplantation is shown in Fig. van den Brink MR. 4. 7. Idiopathic pneumonia syndrome after bone marrow transplantation. in the absence of NO. DCFHloaded macrophages from iNOS( / ) mice given donor T cells and Cy (BMS Cy) persisted to show intense fluorescence. Lung injury induced by alloreactive Th1 cells is characterized by host-derived mononuclear cell inflammation and activation of alveolar macrophages. L-Arginine attenuates lipopolysaccharide-induced lung chemokine production. 1999. 9. Pulido EJ. In summary. These differences were attributed to variations in the background strains of mice or different preparations of endotoxin administered.physiology. and Martin PJ. These observations AJP-Lung Cell Mol Physiol • VOL 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.. Chen W. and National Heart. Lung. 1986. Am J Physiol Lung Cell Mol Physiol 280: L400–L408. including OH and HOCl. NO reacts with O2 to generate ONOO . Parkman R. www. Allione A. and Crystal RG. 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. Crit Care Med 27: 1800–1806. Meng XZ. 2012 281 • OCTOBER 2001 • . Idiopathic pneumonia after bone marrow transplantation: cytokine activation and lipopolysaccharide amplification in the bronchoalveolar compartment. Am Rev Respir Dis 134: 108– 114. and Ferrara JL. REFERENCES 1. 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