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281:L922-L930, 2001. Am J Physiol Lung Cell Mol PhysiolPanoskaltsis-Mortari, Bruce R. Blazar and Imad Y. HaddadShuxia Yang, Valerie A. Porter, David N. Cornfield, Carlos Milla, Angelaof iNOS knockout mice after marrow transplantationEffects of oxidant stress on inflammation and survival

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

SHUXIA YANG,1 VALERIE A. PORTER,1 DAVID N. CORNFIELD,1,2 CARLOS MILLA,1

ANGELA PANOSKALTSIS-MORTARI,2 BRUCE R. BLAZAR,2 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

Received 29 March 2001; accepted in final form 17 May 2001

Yang, Shuxia, Valerie A. Porter, David N. Cornfield,Carlos Milla, Angela Panoskaltsis-Mortari, Bruce R.Blazar, and Imad Y. Haddad. Effects of oxidant stress oninflammation and survival of iNOS knockout mice after mar-row transplantation. Am J Physiol Lung Cell Mol Physiol281: L922–L930, 2001.—In a model of idiopathic pneumoniasyndrome after bone marrow transplantation (BMT), injec-tion of allogeneic T cells induces nitric oxide (NO), and the

addition of cyclophosphamide (Cy) generates superoxide(O2

) and a tissue-damaging nitrating oxidant. We hypothe-sized that NO and O2

balance are major determinants of

post-BMT survival and inflammation. Inducible nitric oxidesynthase (iNOS) deletional mutant mice ( / ) given donorbone marrow and spleen T cells (BMS) exhibited improvedsurvival compared with matched BMS controls. Bronchoal-

veolar lavage fluids obtained on day 7 post-BMT fromiNOS( / ) BMS mice contained less tumor necrosis factor-and interferon-, indicating that NO stimulated the produc-tion of proinflammatory cytokines. However, despite sup-pressed inflammation and decreased nitrotyrosine staining,iNOS( / ) mice given both donor T cells and Cy (BMS Cy)died earlier than iNOS-sufficient BMS Cy mice. Alveolarmacrophages from iNOS( / ) BMS Cy mice did not pro-

duce

NO but persisted to generate strong oxidants as as-sessed by the oxidation of the intracellular fluorescent probe2,7-dichlorofluorescin. We concluded that NO amplifies Tcell-dependent inflammation and addition of Cy exacerbatesNO-dependent mortality. However, the lack of NO duringCy-induced oxidant stress decreases survival of T cell-recip-ient mice, most likely by generation of NO-independent toxicoxidants.

nitric oxide; peroxynitrite; lymphocytes; macrophages; tumornecrosis factor-; idiopathic pneumonia syndrome

IDIOPATHIC PNEUMONIA SYNDROME (IPS) refers to diffuseand often fatal noninfectious lung dysfunction that

occurs after bone marrow transplantation (BMT; seeRef. 4). IPS accounts for at least 40% of nongraft vs.host disease (GVHD) deaths after allogeneic BMT.Human studies and recently established murine BMTmodels have confirmed that IPS is the result of persis-tent immune destructive events that is potentiatedwith conditioning regimens (5, 7, 9, 32). Once infiltrat-ing donor T cells, alloactivated by antigen-presenting

cells, encounter pulmonary antigens, immune-mediated damage begins. The major mediators responsiblefor killing by cytolytic T cells are the lytic proteinperforin (cytolysin: pore-forming protein) and serineproteases such as granzyme B (14) and the Fas ligandapoptotic pathway (28). A second pathway for stimulating lung injury is via the release of proinflammatorycytokines by activated macrophages and lung-infiltrating monocytes. Consistent with this hypothesis, ele vated levels of tumor necrosis factor (TNF)- and interleukin (IL)-1 and IL-6 are present in thebronchoalveolar lavage fluid (BALF) or parenchymaduring IPS injury (6).

In our allogeneic BMT model, lung dysfunction inlethally irradiated mice is dependent on the infusion ofdonor spleen T cells and is associated with T celldependent early production of proinflammatory cytokines, including TNF- and interferon (IFN)-, and thegeneration of large amounts of nitric oxide (NO) byinducible nitric oxide synthase (iNOS) (17, 32). Thehigh-output iNOS-derived NO may serve several im

munoregulatory functions that can modify T cell immune responses. Macrophage-derived NO has beenshown 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 theexpression of adhesion molecules (10). FurthermoreNO has been shown to directly upregulate or down-regulate the expression of several cytokines and chemokines (2, 34, 40). Attempts at determining the role oNO by administration of iNOS inhibitors duringGVHD after allogeneic BMT have yielded conflictingresults (11, 20). Although the use of nonspecific drugsthat also inhibit beneficial constitutive nitric oxide

synthase (cNOS)-derived

NO may explain some of theresults, the main reasons for the contradictory effectsof NO post-BMT remain unclear.

In our IPS model, the addition of the commonly usedconditioning drug cyclophosphamide (Cy) potentiatedlung dysfunction and accelerated mortality in donor Tcell-recipient irradiated mice. Cy-facilitated injury was

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: [email protected]).

The costs of publication of this article were defrayed in part by thepayment of page charges. The article must therefore be herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. Section 1734solely to indicate this fact.

Am J Physiol Lung Cell Mol Physiol281: L922–L930, 2001.

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dependent on T cells, since the injection of Cy alone(without T cells) did not cause lung dysfunction and didnot affect survival of BMT mice (32). In addition, lungdysfunction in Cy/TBI T cell-recipient mice was asso-ciated with the detection of nitrated proteins (17).Because Cy is known to deplete antioxidants and toenhance the generation of superoxide (O2

) by respira-

tory 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 cellsand Cy-induced O2

. ONOO formation clarifies the

dependence of Cy-facilitated toxicity on the presence of allogeneic T cells.

The reaction of NO with O2

has become central tothe understanding of oxidation reactions and genera-tion of oxidative stress (13). NO or O2

alone are weak

oxidants. However, their reaction product, ONOO, isa potent oxidant and nitrating species. ONOO canoxidize sulfhydryl groups, including glutathione, themost abundant antioxidant present in the epitheliallining fluid (3). Additional potent oxidants that maybe formed by the iron-catalyzed Haber-Weiss reactionare the hydroxyl radical (OH) and hypochlorous acid(HOCl) generated by the interaction of myeloperoxi-dase with hydrogen peroxide and chloride. Althoughthere is little doubt that ONOO formation enhancesNO toxicity, credible experimental evidence indicatesthat the reaction of NO with O2

may in fact lower

the steady-state concentrations of O2

and thereforelimit the formation of the extremely injurious OHand HOCl (43).

We used the ability to alter oxidant stress by theinjection of Cy into T cell-recipient mice to investigatethe in vivo pathobiological role of the reaction of NO

with O2

. BMT experiments in the presence or absenceof Cy conditioning were performed in irradiated micelacking iNOS. We hypothesized that NO and ONOO

contribute to lung inflammation and mortality afterallogeneic BMT. Our results indicate that iNOS-de-rived NO stimulate TNF- and IFN- production andthat Cy-induced oxidative/nitrative stress promotesNO-mediated lung dysfunction. However, despite sup-pressed inflammation, NO deficiency during Cy-in-duced oxidative stress and depletion of antioxidantsworsened the survival of mice post-BMT, consistentwith the generation of O2

-derived highly toxic oxi-

dants.

MATERIALS AND METHODS

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 (BarHarbor, ME). In addition, C57BL/6 NADPH oxidase( / )mice generated by deletion of the 91-kDa subunit of theoxidase cytochrome b (Jackson Laboratories) were used forcomparison with iNOS( / ) mice by nitrotyrosine stainingand production of oxidants. Mice were housed in microisola-tor cages in the specific pathogen-free facility of the Univer-sity of Minnesota and were cared for according to the Re-search Animal Resources guidelines of our institution. For

BMT, donors were 6–8 wk of age, and recipients were used at8–10 wk of age.

Bone marrow transplant. BMT was performed as previously described (16, 17, 32). Briefly, C57BL/6 wild-type andknockout mice were lethally total body irradiated (TBI; 7.5Gy TBI by X-ray at a dose rate of 0.41 Gy/min) on the daybefore BMT. A parallel set of mice also received 120mg kg1 day1 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 IgG2bkindly provided by Dr. David Sachs, Massachusetts GeneraHospital, Boston, MA) plus complement (Neiffenegger, Woodland, CA). For each experiment, a total of 5–10 recipienmice/treatment group were transplanted via the caudal veinwith 20 106 B10.BR marrow supplemented with (BMS andBMS Cy) or without (BM and BM Cy) 15 106 spleen Tcells as a source of IPS-causing T cells. A cohort of mice fromeach group was monitored for survival. As per our approvedanimal 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 postBMT after an intraperitoneal injection of pentobarbital sodium, and the thoracic cavity was partially dissected. Thetrachea was cannulated with a 22-gauge angiocatheter, infused with 1 ml of ice-cold sterile PBS, and withdrawn. Thiwas repeated two times, and return fluid was combined. TheBALF was immediately centrifuged at 500 g for 10 min at 4°Cto pellet cells.

BALF biochemical analysis. Nitrite in BALF was measured according to the Greiss method after the conversion onitrate to nitrite with the NADH-dependent enzyme nitratereductase (Calbiochem, La Jolla, CA). IFN- and TNF-levels in the cell-free BALF were determined by ELISA usingcommercial kits (R&D Systems, Minneapolis, MN). BALFnon-protein-bound sulfhydryl (SH) content as an estimateof 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 nonprotein-boundSH in the supernatant was determined afterthe addition of DTNB. The absorbance of the yellow anion2-nitro-5-thiobenzoate formed was measured at 412 nm.

Macrophage culture. The BALF cell pellets from mice ineach treatment group were combined, washed two times incold PBS, and resuspended in RPMI 1640 medium (CeloxLaboratories, St. Paul, MN) containing 5% FCS, 100 U/mpenicillin, and 100 g/ml streptomycin. Total cell numberwas determined with a hemacytometer. Total cells (2 105 /well) were added to mouse IgG-coated, flat-bottom 96well 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% ofadherent cells were macrophages. The cells were maintainedin culture at 37°C for 48 h in 5% CO2 in air. At the termination of cell culture, supernatants were aspirated from indi

vidual culture wells for measurement of TNF- by ELISA(PharMingen, San Diego, CA), nitrite by the Greiss methodand lactic dehydrogenase (LDH) by the colorimetric CytoTox96 assay (Promega, Madison, WI). Cells were washed twotimes with PBS and lysed with lysis solution (10, Triton

X-100; Promega), and cellular LDH release was measuredTotal (supernatant cellular) LDH values were used tocorrect for possible differences in adherent cell number between groups. TNF- and nitrite readings were adjusted

L923NITRIC OXIDE/SUPEROXIDE BALANCE AFTER ALLOGENEIC TRANSPLANTATION

AJP-Lung Cell Mol Physiol • VOL 281 • OCTOBER 2001 • www.ajplung.org



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 mac-rophages obtained from day 7 post-BMT BALF were culturedon glass coverslips for 1 h followed by removal of nonadher-ent cells. Adherent cells were loaded with 2,7-dichlorofluo-rescin (DCFH) diacetate (0.1 M; Molecular Probes, Eugene,OR) for 20 min. During loading, the acetate groups wereremoved by intracellular esterases, trapping the probe inside

the cells. After an oxidative burst, DCFH was oxidized todichlorofluorescein (DCF), which can be visualized on a sin-gle-cell basis using fluorescence microscopy. After rinsingwith PBS to remove excess probe, generation of oxidants wasmonitored over time using an inverted fluorescence micro-scope (Nikon Eclipse TE200) connected to an extended ISISintensified charge-coupled device camera (Robertsbridge,UK) using Axon Instruments (Foster City, CA) image captureand analysis software. DCF fluorescence was measured at anexcitation wavelength of 480 nm and an emission wavelengthof 520 nm.

Histology and immunohistochemistry. In some animals,lungs were extracted without lavage and were perfused with1.0 ml of saline via the right ventricle of the heart. A mixtureof 0.5–1.0 ml optimal cutting temperature medium (Miles

Laboratories, Elkhart, IN)-PBS (3:1) was infused via thetrachea into the lung. The lung was snap-frozen in liquidnitrogen and stored at 80°C. Thin (4-m) frozen sectionswere mounted on glass slides and fixed for 10 min in 3%paraformaldehyde at 4°C (nitrotyrosine staining) and for 5min in acetone (Mac-1 staining). Representative sectionswere stained with hematoxylin and eosin (H&E) for his-topathological assessment. Nonantigenic sites were blockedwith 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 followingantibodies: 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 diamino-benzidine chromogenic substrate purchased from Vector Lab-oratories (Burlingame, CA). In control measurements, theprimary antibody was omitted, or tissues were incubatedwith the NT Ab in the presence of excess antigen (10 mMnitrotyrosine). To visualize specific NT Ab binding, sectionswere incubated with secondary antibody, goat anti-rabbitIgG conjugated with horseradish peroxidase (1:500 dilution),followed by the addition of 3,3-diaminobenzidine (VectorLaboratories) chromogenic substrate. The sections werecounterstained with hematoxylin, dehydrated, overlaid withPermount (Sigma), and sealed with coverslips. The numberof positive cells in the lung was quantitated as the percentageof nucleated cells at a magnification of 200 (20 objectivelens). 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. Statisti-cal differences among group means were determined byTukey’s Studentized test. A comparison of survival curvesbetween the different groups was made using the log-ranktest. P 0.05 were considered statistically significant.

RESULTS

Cy-induced oxidative stress in allogeneic T cell-recip-ient mice. Previous data indicate that injection of Cy(120 mg kg1 day1) on days 3 and 2 as a condi-tioning 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 (17and depleted lung glutathione antioxidant defense(44). To determine whether Cy also depleted epithelialining fluid free thiol groups, BALF non-protein-boundSH levels were determined. On day 7 post-BMTBALF from Cy/TBI T cell-recipient mice (BMS Cycontained significantly lower levels of free SH com

pared with BMT mice given T cells alone (without CyBMS) or BMT mice not given T cells (BM). Injection ofCy alone (without T cells) did not significantly decreaseBALF non-protein-bound SH (Fig. 1). The generationof strong oxidants by alveolar macrophages/monocytesfrom Cy/TBI T cell-recipient mice was confirmed usingDCFH as an intracellular fluorescent probe. NeitherNO nor O2

is able to oxidize DCFH (35). In contrast

ONOO and other strong oxidants such as OH andHOCl oxidize DCFH to form the highly fluorescentproduct DCF (23). In contrast to cells from TBI micenot given T cells (BM), which exhibited backgroundfluorescence, macrophages/monocytes from BALF oBMS Cy mice showed time-dependent intense fluo

rescence (Fig. 2).Undetectable BALF nitrite plus nitrate in iNOS

knockout mice after allogeneic transplantation. Thereturn BALF volumes were similar in all groups omice (90% of instilled volume). As previously reported (17), day 7 post-BMT BALF from TBI miceinfused with allogeneic T cells with and without Cycontained increased numbers of inflammatory cellsand iNOS deficiency did not modify the cellular num-ber or profile (data not shown). In wild-type miceinjection of donor T cells increased day 7 post-BMTBALF nitrite plus nitrate levels (Fig. 3). The lower

Fig. 1. Cyclophosphamide (Cy) depletes non-protein-bound sulfhydryls (SH) in bronchoalveolar lavage fluid (BALF) from allogeneicT cell-recipient mice after marrow transplantation. Day 7 post-bonemarrow transplantation (BMT) BALF proteins were precipitatedwith 5% TCA, and free SH content in the supernatant was determined by measurement of the absorbance at 412 nm after thaddition of 5,5-dithiobis(2-nitrobenzoic acid) (molar extinction coefficient of 13,600/cm). A solution of reduced glutathione was used asstandard. Non-protein-bound SH from BALF of control mice (nontransplanted, nonirradiated) was 11.5 1.6 M. BM, irradiatedC57BL/6 mice given B10.BR bone marrow; BMCy, BM mice alsogiven Cy conditioning (120 mg kg1 day1 on days 3 and 2)BMS, irradiated C57BL/6 mice given B10.BR bone marrow pluC57BL/6 spleen T cells; BMSCy, BMS mice also given Cy. Valuesare means SE obtained from the BALF of at least 5 mice/group* P 0.05 compared with control (BM).

L924 NITRIC OXIDE/SUPEROXIDE BALANCE AFTER ALLOGENEIC TRANSPLANTATION




BALF nitrite level in BMS Cy mice compared withBMS mice is most likely related to formation of NO-derived species, as previously reported (17). BALF ni-trite plus nitrate levels of all iNOS-deficient mice wereundetectable (Fig. 3) and significantly less than inwild-type mice, including the value obtained from con-

trol mice (nonirradiated and nontransplanted mice) of 2.1 0.3 M.

Decreased BALF TNF- and IFN- in iNOS knockout mice after allogeneic transplantation. In wild-typemice, injection of donor T cells also increased day 7post-BMT BALF TNF- and IFN-, and the addition oCy further enhanced the production of these proinflammatory cytokines. iNOS deficiency was associated withdecreased levels of BALF TNF- and IFN- in allogeneic T cell-recipient mice with or without Cy injection

(Fig. 4). Furthermore, alveolar macrophages/monocytes obtained from iNOS( / ) Cy/TBI mice givendonor T cells and cultured for 48 h did not produce NOand generated less TNF- compared with macrophages/monocytes from wild-type mice (Fig. 5). Thesedata suggest that iNOS-derived NO is a major amplifier of TNF- and IFN- production during allogeneic Tcell-dependent inflammation in our IPS model.

Histology and macrophage/monocyte immunostaining. To determine whether the inhibition of cytokineproduction in BMS Cy iNOS( / ) mice was accompanied by lower numbers of lung-infiltrating inflam

Fig. 4. Suppressed production of proinflammatory cytokines inBALF of iNOS knockout mice after allogeneic transplantation. Atumor necrosis factor (TNF)- (pg/ml) measured in day 7 post-BMTBALF by ELISA. B: interferon (IFN)- (pg/ml) measured day 7post-BMT BALF by ELISA. iNOS knockout mice receiving donospleen T cells with and without Cy (BMS or BMSCy) exhibitedlower levels of TNF- and IFN- compared with wild-type matchedrecipients. Values are means SE for n 7–14 mice/group. * P 0.05 compared with control (BM). P 0.05 comparing the effect oiNOS deficiency in each group.

Fig. 2. Cy enhances macrophage-derived production of oxidants intransplanted mice given allogeneic T cells. Day 7 post-BMT BALFmacrophages were cultured on glass coverslips for 1 h followed byremoval of nonadherent cells. Adherent cells were loaded with 2,7-dichlorofluorescin (DCFH) diacetate (0.1 M) for 20 min. After cellswere rinsed with PBS to remove excess probe, generation of intra-

cellular oxidants capable of DCFH oxidation to dichlorofluorescein(DCF) was monitored (0 and 2 h) using an inverted fluorescencemicroscope (Nikon Eclipse TE200) combined with computerized im-age analysis. DCF fluorescence was measured at excitation wave-length of 480 nm and an emission wavelength of 520 nm. Shown is arepresentative experiment that was repeated 2 times with identicalresults. t, Time.

Fig. 3. Nitrite levels in the cell-free BALF of irradiated C57BL/6inducible nitric oxide synthase (iNOS) knockout and wild-type mice7 days post-BMT. Mice receiving donor B10.BR spleen T cells (BMSor BMSCy) exhibited increased nitrite levels. Nitrite was barelydetectable in all iNOS knockout mice. Nitrate was reduced withnitrate reductase before nitrite measurement by the Greiss reaction. Values are means SE for n 12–20 mice/group. * P 0.05compared with control (BM). P 0.05 comparing the effect of iNOSdeficiency in each group.





matory cells, lung sections obtained on day 7 post-BMTwere immunostained with CD11b/Mac-1 antibody.Compared with irradiated mice not given allogeneic Tcells (BM), lung sections of Cy/TBI T cell-recipient miceshowed evidence of lung injury associated with infil-tration of Mac-1-positive cells. The lack of iNOS-de-rived NO did not modify the number or type of inflam-matory cells in the lung (Fig. 6). Mac-1 expression in

the lung increased from 8 2% of nucleated cells in theBM group to 43 6% of nucleated cells in theBMS Cy group and was not modified in theiNOS( / ) BMS Cy group (41 8%). Values aremeans SE determined by counting the percentage ofcells expressing Mac-1 in four to eight fields per lungsection under light microscopy. Two to three mice pergroup from two representative experiments were as

sessed. Cy alone in the absence of T cells (BM Cydid not increase the number or activation state olung-infiltrating macrophages/monocytes (16).

Survival of iNOS knockout mice. Mortality of miceafter allogeneic transplantation is dependent on infusion of donor T cells, and the addition of Cy acceleratesT cell-dependent mortality. To determine the contribution of NO to post-BMT mortality in the presence andabsence of Cy-induced oxidant stress, survival of iNOSdeficient and iNOS-sufficient mice was comparedEarly survival of mice lacking iNOS-derived NO andgiven donor spleen T cells (BMS) was enhanced compared with T cell-recipient wild-type mice ( P 0.008Fig. 7 A). Similarly, we expected improved survival oBMS Cy iNOS( / ) mice compared with BMS Cylittermates. However, 1 wk post-BMT, Cy/TBI T cell-recipient iNOS( / ) mice exhibited significantlyhigher mortality compared with BMS Cy iNOS-sufficient mice (Fig. 7 B). Cy-facilitated mortality in almice was dependent on the presence of allogeneic Tcells, since iNOS( / ) and iNOS-sufficient mice thatwere given Cy without allogeneic T cells exhibited100% survival in the same post-BMT period (data notshown). Taken together, these data indicate that NOcontributes to and Cy-induced oxidant stress accelerates mortality of mice after allogeneic transplantation

Fig. 5. Decreased production of nitric oxide (NO) and TNF- bycultured alveolar macrophages obtained from iNOS-deficient ( / )mice after allogeneic transplantation. Spontaneous nitrite ( y-axis onleft) and TNF- ( y-axis on right) production in supernatant of alve-olar macrophages obtained from irradiated BMT mice (BM), BMTmice given donor T cells (BMS), and BMT mice given T cells and Cy(BMSCy). Macrophages were cultured for 48 h as described inMATERIALS AND METHODS. Values are means SE obtained from atleast 8 wells of pooled macrophages obtained from 4–6 mice/group

for each experiment, which was repeated one time. nd, Not deter-mined. * P 0.05 compared with control (BM). P 0.05 comparingthe effect of iNOS deficiency in each group.

Fig. 6. iNOS deficiency does not modify the number of lung-infiltrating inflammatory cells after allogeneictransplantation. Hematoxylin and eosin (H&E) staining (top) and Mac-1 staining (bottom) of frozen lung sectionstaken on day 7 post-BMT from TBI C57BL/6 mice given BM from B10.BR mice (BM) or wild-type (WT) andiNOS( / ) Cy/TBI C57BL/6 mice given B10.BR spleen T cells in addition to BM (BMS Cy). Mac-1-positivemacrophages/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).





However, in the absence of NO, BMS Cy mice dieearly, possibly related to the generation of oxidativestress (see below).

Persistent oxidant production but less nitrotyrosinestaining from BMS Cy iNOS knockout mice. To be-gin to understand potential reasons for acceleratedmortality of Cy/TBI T cell-recipient iNOS( / ) mice

despite decreased production of inflammatory media-tors, the generation of oxidants by macrophages andnitrotyrosine immunostaining of lung sections fromiNOS( / ) and wild-type BMS Cy mice was com-pared. For these sets of experiments, BMT was alsoperformed in mice lacking phagocyte respiratory burstoxidase [NADPH oxidase( / )]. In wild-type Cy/TBI Tcell-recipient mice, macrophages obtained on day 7 post-BMT and loaded with DCFH exhibited intenseintracellular fluorescence associated with specific lungnitrotyrosine staining (Fig. 8). Macrophages fromNADPH oxidase( / ) BMS Cy mice exhibited less

fluorescence (50% of BMS Cy wild-type mice) anddecreased nitrotyrosine staining, suggesting an important role for NADPH oxidase in the generation oCy-induced oxidative/nitrative stress during T cell-dependent generation of NO. Compared with BMS Cywild-type mice, lung sections of iNOS( / ) BMS Cymice 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).

DISCUSSION

These studies demonstrate the important roles oNO and O2

and their derived reactive species in the

early post-BMT inflammatory and oxidant responsesthat lead to IPS-related injury and death. Resultsclearly indicate that iNOS-derived NO stimulates theproduction of TNF- and IFN- in the lung. The addition of Cy to the conditioning regimen during allogeneic T cell-dependent NO induction increases oxida

tive/nitrative stress, which allowed us to investigatethe pathobiological significance of the reaction of NOand O2

in vivo. The generation of oxidants was a

major contributor to decreased survival after allogeneic transplantation. A concern is whether the absenceof iNOS altered the efficacy of engraftment. In otherstudies, donor cell engraftment was seen in all recipients conditioned with either a lethal-dose total bodyirradiation 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 sincethe conditioning regimen used has been shown to fullyablate host marrow.

The administration of donor spleen T cells on the dayof BMT (day 0) induced the production of NO, TNF-and IFN- in the absence of significant oxidative/nitrative stress or depletion of BALF free SH groups. Ourstudies indicate that the lack of NO in iNOS( / donor T cell-recipient mice (BMS) was associated withsuppressed production of TNF- and IFN- and improved survival, suggesting that iNOS-derived NOexacerbated inflammation and accelerated mortality inthis murine model. We have not investigated the mechanisms by which NO amplifies the inflammatory response. Hierholzer et al. (18), using iNOS( / ) micesubjected to hemorrhagic shock, have shown that NO

may exacerbate inflammation by the activation of transcriptional nuclear factor-B and signal transducerand activator of transcription-3 (18). Macrophage-derived NO has been described to protect tissues from Tcell immune responses by suppressing helper T celproliferation and cytotoxicity (27). However, the lowlevel of IFN- contained in post-BMT BALF fromiNOS( / ) mice does not support the hypothesis thatthe absence of NO exacerbated T cell proliferation. Apotential explanation for the lack of antiproliferative Tcell effects of NO in our model is the complete majorhistocompatibility complex (MHC) mismatch, render

Fig. 7. Post-BMT survival of irradiated knockout mice given alloge-neic T cells (BMS) and T cells plus Cy (BMSCy). A: C57BL/6 iNOSknockout and matched wild-type mice (n 10/group) were irradiatedon day 1 and infused on day 0 with B10.BR marrow either withBMS or without spleen cells (BM) as indicated. Survival was moni-tored for 1 mo after transplantation. Mice lacking iNOS exhibitedprotection 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 infusedon day 0 with B10.BR BM with B10.BR spleen cells (BMSCy). Additional wild-type mice (n 10) were given Cy and BM without Tcells (BMCy; n 10). Mice lacking iNOS exhibited acceleratedmortality compared with iNOS-sufficient mice (* P 0.05).





ing alloactivated lymphocytes unresponsive to the in-hibitory effects of NO.

The formation of a nitrating species in Cy/TBI T cell-recipient mice (BMS Cy) during the simultaneous gen-eration of NO and O2

suggested ONOO generation.

ONOO can rapidly oxidize thiol groups (36). AlternativeONOO-independent nitration reactions include the ox-idation of nitrite by myeloperoxidase or related peroxi-dases (12). Irrespective of the mechanism of formation,the generation of nitrative stress in BMS Cy was asso-

ciated with accelerated T cell-dependent inflammationand mortality. Although acrolein, a Cy metabolite, hasbeen shown to deplete glutathione (33), our data indicatethat injection of Cy without T cells did not significantlyalter BALF SH levels or cause lung dysfunction (17).Hill et al. (19) recently reported that Cy/TBI potentiatesallogeneic T cell-mediated injury to the gastrointestinaltract, resulting in increased translocation of lipopolysac-charide (LPS) into the systemic circulation and amplifi-cation of the inflammatory response. However, the ab-sence of any detectable LPS in mice given Cy/TBI andsyngeneic T cells in the study of Hill et al. and the

Fig. 9. Proposed hypothesis for the pathobiological effects of NO andO2

interaction in our idiopathic pneumonia syndrome model. NOinduced by donor T cell-dependent proinflammatory cytokines, amplifies the early post-BMT inflammatory response. The addition oCy enhances cytokine-induced O2

generation. NO reacts with O2

to

generate ONOO, a potent oxidant that accelerates inflammationand mortality. The presence of donor T cells and Cy in the absence ofiNOS-derived NO favors the formation of O2

-derived toxic oxidants

including OH and HOCl.

Fig. 8. Lung nitrotyrosine immunostaining (left) and macrophage-derived production of oxidants (right) fromwild-type and knockout mice after allogeneic transplantation. Day 7 post-BMT, frozen sections from the indicatedgroup 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 DCFHdiacetate (0.1 M) 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 inMATERIALS AND METHODS. Shown is a representative experiment that was repeated one time.





presence of nitrated proteins and depletion of the freethiol groups in BMS Cy mice observed in our modelfavor the hypothesis that oxidative/nitrative stress is oneof the main reasons for exacerbated injury and decreasedsurvival of irradiated mice given Cy and allogeneic Tcells.

As discussed above, Cy-induced oxidant stress en-hanced NO toxicity. However, NO deficiency during se-

vere oxidative stress and depletion of antioxidants re-sulted in rapid death of Cy/TBI donor T cell-recipientmice. Despite the absence of NO production, DCFH-loaded macrophages from iNOS( / ) mice given donor Tcells and Cy (BMS Cy) persisted to show intense fluo-rescence. These data are consistent with the generationof O2

-derived, NO-independent oxidative stress in

iNOS-deficient BMS Cy mice. We hypothesize that, inthe absence of NO, the formation of OH and HOClduring Cy-facilitated enhanced production of O2

is fa-

vored. In vitro experiments using chemical donors of reactive oxygen and nitrogen species have shown thatNO, depending on its relative fluxes, inhibits O2

-depen-

dent oxidation reactions (30, 37). Similarly, Kubes and

associates (25) have shown that NO modulates oxidant-induced leukocyte adhesion to endothelial cells. Our re-sults are in agreement with several reports suggestingthat inhibition of NO production during oxidant stressexacerbates lung injury and accelerates mortality (15,21). Furthermore, iNOS( / ) mice exhibit amplifiedlung injury during oxidative stress generated by hyper-oxic exposure (22). A model of our hypothesis of thepathobiological effects of NO and O2

balance after allo-

geneic transplantation is shown in Fig. 9. Alloactivated Tcells secrete cytokines, the prototype being IFN-, thatinduce host iNOS-derived NO. In the lung, excess NOamplifies the early post-BMT destructive inflammatory

response. The addition of Cy enhances cytokine-inducedO2

generation. NO reacts with O2

to generate ONOO,a potent oxidant that potentiates NO-mediated inflam-mation and mortality. However, the generation of O2

in

the absence of NO favors the formation of the extremelytoxic O2

-derived oxidants, including OH and HOCl.

Mice lacking iNOS have been used to determine theeffects of iNOS deficiency on LPS-induced mortality.MacMicking et al. (29) and Wei et al. (42) reported thatiNOS( / ) mice are more resistant to LPS-induceddeath. In contrast, Nicholson and colleagues (31) ob-served that fatality of iNOS( / ) mice after LPS injection was similar to genetically matched mice. Thesedifferences were attributed to variations in the back-

ground strains of mice or different preparations of endotoxin administered. Based on our results, we sug-gest that a potential explanation for the conflictingsurvival data after LPS injection in iNOS( / ) mice isthe difference in the severity of oxidative stress be-tween the various animal models used.

Day 7 post-BMT, BALF nitrite levels from iNOS( / )mice were below detection limits and significantlylower than normal control mice. In addition, culturesupernatant of alveolar macrophages from BALF of iNOS( / ) mice injected with iNOS-sufficient BM andspleen T cells did not produce NO. These observations

suggested the following: 1) iNOS is the source of NOmeasured in BALF of normal and BMT mice not givendonor T cells and 2) activated alveolar macrophagesobtained from BALF of iNOS( / ) donor T cell-recipient mice are of host origin, as previously demonstrated using antibodies to MHC class II (32). Despitethe inability to produce iNOS-derived NO, weak nitrotyrosine staining from iNOS( / ) BMS Cy mice was

observed. Potential sources for nitration reactions iniNOS( / ) animals may include cNOS and formationof NO-independent nitrating species.

In summary, we have shown that T cell-dependentinduction of NO contributes to IPS injury and mortality by several mechanisms. NO amplifies the earlypost-BMT inflammatory response and contributes tothe formation of toxic effector species, such as ONOO

Oxidative stress and oxidant/antioxidant balance aremajor determinants of whether inhibition of iNOSderived NO is beneficial or detrimental to the host. Asafer and more effective strategy may be to limit theavailability of O2

to prevent ONOO formation or to

use specific scavengers of ONOO.

We acknowledge the expert technical assistance of John HermansonThis work was supported by grants from the American Lung

Association (Johnie Murphy Career Investigator Award), the American Heart Association (Minnesota Affiliate), the Viking Children’Fund, and National Heart, Lung, and Blood Institute Grants R01HL-67334 and HL-55209.

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