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SynthaseMonocyte-Derived Inducible Nitric Oxide Resolution of Experimental Lung Injury by

Crow and Landon S. KingVenkataramana K. Sidhaye, Paul M. Hassoun, Michael T.Waickman, Sekhar P. Reddy, David B. Pearse, Files, Claudia R. Avalos, Jackie V. Rodriguez, Adam T.Jason R. Mock, Yoshiki Eto, Brian T. Garibaldi, Daniel C. Franco R. D'Alessio, Kenji Tsushima, Neil R. Aggarwal,

http://www.jimmunol.org/content/189/5/2234doi: 10.4049/jimmunol.1102606July 2012;

2012; 189:2234-2245; Prepublished online 27J Immunol 

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6.DC1http://www.jimmunol.org/content/suppl/2012/07/27/jimmunol.110260

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The Journal of Immunology

Resolution of Experimental Lung Injury byMonocyte-Derived Inducible Nitric Oxide Synthase

Franco R. D’Alessio,1 Kenji Tsushima,1 Neil R. Aggarwal, Jason R. Mock,

Yoshiki Eto, Brian T. Garibaldi, Daniel C. Files, Claudia R. Avalos, Jackie V. Rodriguez,

Adam T. Waickman, Sekhar P. Reddy, David B. Pearse, Venkataramana K. Sidhaye,

Paul M. Hassoun, Michael T. Crow, and Landon S. King

Although early events in the pathogenesis of acute lung injury (ALI) have been defined, little is known about the mechanisms

mediating resolution. To search for determinants of resolution, we exposed wild type (WT) mice to intratracheal LPS and assessed

the response at intervals to day 10, when injury had resolved. Inducible NO synthase (iNOS) was significantly upregulated in the

lung at day 4 after LPS. When iNOS2/2 mice were exposed to intratracheal LPS, early lung injury was attenuated; however,

recovery was markedly impaired compared with WT mice. iNOS2/2 mice had increased mortality and sustained increases in

markers of lung injury. Adoptive transfer of WT (iNOS+/+) bone marrow-derived monocytes or direct adenoviral gene delivery of

iNOS into injured iNOS2/2 mice restored resolution of ALI. Irradiated bone marrow chimeras confirmed the protective effects of

myeloid-derived iNOS but not of epithelial iNOS. Alveolar macrophages exhibited sustained expression of cosignaling molecule

CD86 in iNOS2/2 mice compared with WT mice. Ab-mediated blockade of CD86 in iNOS2/2 mice improved survival and

enhanced resolution of lung inflammation. Our findings show that monocyte-derived iNOS plays a pivotal role in mediating

resolution of ALI by modulating lung immune responses, thus facilitating clearance of alveolar inflammation and promoting lung

repair. The Journal of Immunology, 2012, 189: 2234–2245.

Acute lung injury (ALI) remains an important clinicalproblem, affecting .190,000 patients annually in theUnited States, with an estimated 75,000 deaths/year (1).

Despite extensive investigation of the underlying mechanisms inALI pathogenesis, therapy remains mainly supportive, with onlylung-protective ventilation offering survival benefits (2).The pathogenesis of ALI is notable for the activation of in-

flammatory cells, including neutrophils and alveolar macrophages(AMs), with increased production of pro- and anti-inflammatorymediators. An acute exudative phase, lasting for up to 5–7 d, ismanifest pathologically as diffuse alveolar damage, with protein-rich edema fluid, neutrophilic infiltration of the interstitium andairspaces, and variable amounts of hyaline membranes (3). Fol-lowing the acute phase of ALI, some patients progress to a fibro-

proliferative phase (4–6), which has been correlated with an in-creased risk for death (7). Although it has long been recognizedthat ALI may evolve through these stages, investigation haslargely focused on defining and targeting early steps in the path-ogenesis. Little attention has been devoted to identification of themechanisms responsible for resolution of lung injury.NO has been implicated in the pathophysiology of ALI in

animals and humans (8–13). NO participates in the regulation ofevery organ system in normal and pathologic situations (14, 15).Although NO in the normal lung is produced by the constitutiveisoforms of NO synthase (neuronal NO synthase [nNOS] andendothelial NO synthase [eNOS]), its production during inflam-mation is mainly due to the inducible isoform (inducible NOsynthase [iNOS]) (16). In the lung, the major cells expressingiNOS are AMs, alveolar epithelial cells, and inflammatory infil-trating cells (17).ALI and sepsis are associated with increased iNOS-derived NO

(18–20). Several groups noted that deletion or inhibition of iNOSin mice is protective from LPS-induced mortality (21, 22), indi-cating a proinflammatory role for iNOS. Other investigators foundthat iNOS deletion had no effect (23). NO can contribute to mi-crovascular injury, pulmonary edema, and neutrophilic infiltrationin mouse models of sepsis (22, 23), with variable effects onmortality. However, NO can also have beneficial effects on theimmune system (24), acting as an antimicrobial (25–28) or anti-inflammatory/immunosuppressive agent (29–32), and it canmodulate signaling pathways, including NF-kB (33–36).In contrast to the effect on acute responses, little is known about

the impact of iNOS on the resolution of lung inflammation andinjury. We examined the effects of iNOS deletion on the resolutionof lung injury in a mouse model of LPS-induced ALI. We foundthat iNOS2/2 mice had a biphasic response, with reduced severityof lung injury on day 1 after intratracheal (i.t.) LPS, but with a sub-sequent marked delay in ALI resolution, indicating a previously

Division of Pulmonary and Critical Care Medicine, The Johns Hopkins UniversitySchool of Medicine, Baltimore, MD 21224

1F.R.D. and K.T. contributed equally to this work.

Received for publication September 9, 2011. Accepted for publication June 28, 2012.

This work was supported by National Heart, Lung, and Blood Institute GrantHL089346 and the Johns Hopkins Bayview Scholars Program (to L.S.K.), as well asby Grant K99HL103973 from the National Institutes of Health-National Heart, Lung,and Blood Institute (to F.R.D.).

Address correspondence and reprint requests to Dr. Franco R. D’Alessio, Division ofPulmonary and Critical Care Medicine, Johns Hopkins Asthma and Allergy Center,The Johns Hopkins University School of Medicine, Room 4A.62, 5501 Hopkins Bay-view Circle, Baltimore, MD 21224. E-mail address: fdaless2@jhmi.edu

The online version of this article contains supplemental material.

Abbreviations used in this article: 7-AAD, 7-aminoactinomycin D; ALI, acute lunginjury; AM, alveolar macrophage; BAL, bronchoalveolar lavage; BM, bone marrow;DCF, 29,79-dichlorofluoroscein; eNOS, endothelial NO synthase; h, human; iNOS,inducible NO synthase; i.t., intratracheal(ly); MFI, mean fluorescence intensity;MHC-II, MHC class II; nNOS, neuronal NO synthase; NOx, mono-nitrogen oxidesNO and NO2; PI, phagocytic index; ROS, reactive oxygen species; Treg, regulatoryT cell; WT, wild type.

Copyright� 2012 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/12/$16.00

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undescribed role for iNOS in lung repair. The defect in resolutionin iNOS2/2 mice was abrogated by adoptive transfer of naiveCD11b+iNOS+/+ monocytes or direct adenoviral delivery of iNOSto the lung. Furthermore, epithelial-derived iNOS did not con-tribute significantly to the resolution of ALI. We found thatmacrophage-derived iNOS regulates CD86 expression, whichactively participates in the regulation of lung inflammation.Monocyte-derived iNOS plays a pivotal role in the resolution oflung inflammation and lung repair.

Materials and MethodsAnimals

Male C57BL/6 wild type (WT) mice, 8–10 wk old and C57BL/6 back-ground iNOS2/2 male mice and congenic CD45.1 male mice were pur-chased from The Jackson Laboratory (Bar Harbor, ME). All mice werehoused at the Johns Hopkins University Asthma and Allergy Center, andexperiments were conducted under a protocol approved by the JohnsHopkins Animal Care and Use Committee.

Animal preparation

Using our published model (37), mice were anesthetized i.p. withketamine/acetylpromazine (150/13.5 mg/kg) before exposure of thetrachea. Escherichia coli LPS (O55:B5 Sigma-Aldrich L2880; 3.75 mg/gmouse weight) or sterile water (control) was then instilled i.t. (total volume35 ml) via a 20-gauge catheter. At 1, 4, 7, and 10 d after instillation, groupsof mice were anesthetized i.p. with ketamine/acetylpromazine and eutha-nized by exsanguination from the inferior vena cava. The lungs wereperfused with 1 ml PBS, and bronchoalveolar lavage (BAL) was performedon the right lung; the left lung was processed for histology. BAL sampleswere routinely cultured to assess for bacterial infection.

Macrophage cell lines

An AM immortalized cell line (MH-S; American Type Culture Collection)was cultured in six-well plates until 80% confluence before experimentalchallenge. Peritoneal macrophages were lavaged from C57BL/6 WT mice(The Jackson Laboratory) 6 d after injection of 3 ml sterile thioglycollate(Sigma-Aldrich). Cells were allowed to adhere overnight in RPMI 1640supplemented with 10% heat-inactivated low-LPS FBS and 1% penicillin-streptomycin/1% glutamine before use. Cells were harvested by gentlescraping and resuspended in a single-cell suspension for flow cytometricstaining.

Analysis of BAL fluid

BAL fluid was obtained by cannulating the trachea with a 20-gauge catheter.The right lung was lavaged with two aliquots of 0.7 ml PBS withoutcalcium. BAL fluid was centrifuged at 7003 g for 10 min at 4˚C. The cell-free supernatants were stored at 280˚C until further analysis. The cellpellet was diluted in PBS, and total cell number was counted with a he-mocytometer after staining with Turk’s Stain Solution (Merck, Tokyo,Japan). Cell differentials (300 cells/sample) were counted on a cytocen-trifuge preparation with Diff-Quik stain (Baxter Diagnostics, McGaw Park,IL). Total protein and albumin were measured in the cell-free supernatantusing bicinchoninic acid assay (Pierce, Rockford, IL) and a mouse serumalbumin ELISA kit (Alpha Diagnostic International, San Antonio, TX),respectively.

Measurement of cytokines

The levels of TNF-a, IL-10, MIP-2, and activated TGF (TGF-b1) weremeasured in BAL fluid by ELISA (R&D Systems, Minneapolis, MN).

Lung histology, immunofluorescence, and lung injury scoring

Lungs (n = 5/time point) were inflated to a pressure of 25 cm H2O using1% low-melting agarose (Invitrogen, Carlsbad, CA) for histologic evalu-ation by H&E staining (38). iNOS expression was assessed as previouslydescribed (13). Left lungs were fixed in 10% formalin neutral buffer for 8–12 h, embedded in paraffin, and sectioned. Briefly, for lung immunofluo-rescence, frozen sections were fixed with 4% paraformaldehyde, per-meabilized with Triton X-100 (Fisher), and subsequently blocked with10% donkey serum. Tissue sections were then incubated with FITC-conjugated iNOS mAb (BD Biosciences) for 1 h and then washed threetimes with cold PBS. Coverslips were mounted using Fluoromount-G(Southern Biotech) and analyzed using a fluorescent microscope.

For lung injury scoring, two blinded investigators analyzed the samplesand determined the levels of lung injury according to the semiquantitativescoring system outlined below. All lung fields (320 magnification) wereexamined for each sample. Assessment of histological lung injury wasperformed using the following scoring: 1, normal; 2, focal (,50% lungsection) interstitial congestion and inflammatory cell infiltration; 3, diffuse(.50% lung section) interstitial congestion and inflammatory cell infil-tration; 4, focal (,50% lung section) consolidation and inflammatory cellinfiltration; and 5, diffuse (.50% lung section) consolidation and in-flammatory cell infiltration. The mean score was used for comparisonbetween groups.

Immunoblotting

To assess the relative abundance of NO synthase isoforms (iNOS, eNOS,and nNOS) during the acute and resolution phases of lung injury, weperformed immunoblots of lung homogenates, as described (39). Thirty-twomicrograms of total protein/sample in 1.5% (w/v) SDS was resolved on 8%SDS-PAGE gels; transferred to nitrocellulose membranes (Bio-Rad, Her-cules, CA); incubated with Abs to iNOS, total eNOS (BD Pharmingen),phosphorylated eNOS (Cell Signaling, Danvers, MA), and nNOS (BDPharmingen); and detected using ECL Plus (Amersham, Piscataway, NJ).Equivalence of samples was confirmed using b-actin (Sigma-Aldrich) asa loading control. Relative band intensities were determined by densi-tometry using UN-SCAN-IT imaging software (Bio-Medicine, Orem, UT).

Determination of nitrate/nitrite by Griess reaction

NO formation was quantified colorimetrically in BAL fluid and lunghomogenates with the Griess reaction (World Precision Instruments, Sar-asota, FL) in duplicate wells following enzymatic conversion of nitrate tonitrite by nitrate reductase (World Precision Instruments). Fifty microlitersof BAL sample and 50 ml buffer were incubated with 100 ml Griess reagentfor 20 min at room temperature. Lung tissue was homogenized usinga Polytron homogenizer in 1 ml homogenization buffer containing 0.25 Msucrose, 1 mM EDTA, 1 mM sodium azide, and protease inhibitors(Complete Protease Inhibitors; Roche). One hundred microliters of lunghomogenate was incubated with 100 ml Griess reagent for 20 min at roomtemperature. Absorbance was measured at 570 nm using a microreader,and NOx concentration was assessed against a sodium nitrite standardcurve (0–100 mM analyzed in duplicate).

Measurement of reactive oxygen species generation

Reactive oxygen species (ROS) were monitored by measurement of H2O2

generation, based on the oxidation of 29,79-dichlorodihydrofluorescein(Sigma-Aldrich) to fluorescent 29,79-dichlorofluorescein (DCF). In brief,BAL cells were incubated with 100 mM 29,79-dichlorodihydrofluoresceinfor 20 min at 37˚C. After washing twice with FACS buffer and surfacestaining with CD11c, the intensity of DCF fluorescence was determinedusing a FACSCalibur flow cytometer (Becton Dickinson, San Jose, CA), withan excitation wavelength of 480 nm and an emission wavelength of 530 nm.

Phagocytic index

AMs were obtained from WT and iNOS2/2 mice by BAL. AMs (105/wellin DMEM without FCS) were allowed to adhere in eight-well tissueculture-treated plastic slides (Lab-Tek) for 30 min. The cells were washedgently, and latex microbeads labeled with FITC (1.7 mm in diameter;Polysciences) were added to the wells (8 3 106 beads/well). After 1 h ofincubation at 37˚C, the wells were washed gently with PBS, fixed withmethanol at 220˚C for 20 min, and washed extensively.

The cells were viewed by a blinded observer using a Nikon fluorescentmicroscope. The fraction of cells containing labeled beads and thephagocytic index (PI) were determined by counting$200 cells in each wellin random high-power fields. PI was calculated as follows: PI = fraction ofAM with beads 3 mean number of beads/positive AM. Three replicatewells were evaluated for each condition.

Bone marrow isolation, adoptive transfer, and chimeras

iNOS2/2 mice and CD45.1+ mice were used to harvest bone marrow (BM)cells, obtained from femurs and tibias by flushing with 50 ml PBS througha 26-gauge needle. After centrifuging, RBCs were lysed, and the remainingcells in RPMI 1640 medium were counted. CD11b+ cells were isolated fromthe BM cells using magnetic bead separation with CD11b MicroBeads(Miltenyi Biotec, Auburn, CA). Magnetically retained CD11b+ cells wereeluted, and the purity was checked using a FACSCalibur flow cytometer(Becton Dickinson, Franklin Lakes, NJ). The purity of CD11b+ BM cellfractions was always .99%. Purification of donor BM cells with magnetic

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CD11b beads yielded isolated CD11b+ cells: ∼40% monocytes and 60%neutrophils. Each single, purified cell suspension (25–30 3 106 cells in200 ml PBS) was adoptively transferred into iNOS2/2 mice (n = 4–5) at thedesignated time after i.t. LPS via tail vein injection, and samples wereharvested at days 2, 4, and 10 after i.t. LPS. Mice were separated into thefollowing groups: WTwithout adoptive transfer, PBS 200 ml into iNOS2/2

mice, WT BM into iNOS2/2 mice, and iNOS2/2 BM into iNOS2/2 mice.Recipient mice, B6.SJL-Ptprca Pep3b/BoyJ (congenic CD45.1 WT), and

iNOS2/2 mice (6–7 wk of age), were lethally irradiated with one exposure to9 Gy (Gammacell 40; Atomic Energy of Canada, Ottawa, ON). Immediatelyafter irradiation, 2 3 106 BM cells were transferred via retro-orbital injec-tion. Mice were housed in microisolators for 8 wk before ALI challenge andfed autoclaved food and water containing 5 mM sulfamethoxazole and 0.86mM trimethoprim. Reconstitution was assessed by flow cytometry.

Flow cytometry

Cells were first incubated with Fc Block-2.4G2 (BD Pharmingen) Ab to blockFcgRIII/II before staining with a specific Ab. The following Abs were pur-chased from BD Pharmingen (San Diego, CA) and BioLegend: anti–annexinV-PE, anti–7-aminoactinomycin D (7-AAD), anti–LY6G-FITC, anti–IA-IE-PE, anti–CD11b-allophycocyanin-e780, anti–CD45.1-PE, anti–CD45.2-PerCy5.5, anti–CD14-FITC, anti–CD86-allophycocyanin, anti–CD80-PE,and anti–F4/80-allophycocyanin; relevant isotype Abs were also pur-chased. Monocytes, AMs, and lymphocytes were gated with characteristicforward scatter/side scatter using a FACSAria instrument, with Cell Divaused for data acquisition (Becton Dickinson, San Jose, CA) and FlowJoused for analysis (Tree Star, San Carlos, CA).

Cell sorting

WT and iNOS2/2 mice were sacrificed 4 d after i.t. LPS. A 20 gaugecatheter was used to instill 1 ml dispase (Roche), followed by 0.5 ml 1%liquid agarose, which was solidified with ice. The lungs were removed andincubated in DMEM with 100 U DNase I (Roche) per milliliter, separatedinto single cells, and filtered through a 70-mm cell strainer (BectonDickinson). CD11b+FSChi (monocytes) and CD326+CD11b2 (lung epi-thelial cells) subsets were isolated by FACS using a Becton DickinsonFACSAria machine. After cytospin, cells were fixed with methanol, per-meabilized with Triton X, stained with mouse iNOS-FITC (BD Bio-sciences) for 1 h, and analyzed by fluorescent microscopy.

Adenoviral iNOS gene construct and delivery

To generate an adenovirus expressing red-shifted GFP-tagged iNOS (NOS2-rsGFP), we obtained the vector pcDNA3.1neo-iNOS-rsGFP from Dr. N.T.Eissa (Baylor College of Medicine, Houston, TX), subcloned the cDNA forthe tagged iNOS, and transferred iNOS-rsGFP into the shuttle vectorpAdCMV.DEST-V5 (Invitrogen). Replication-defective virus was preparedby transfecting pAdCMV-DEST-NOS2-rsGFP into HEK293A cells. Extractsfrom cultures showing cytopathic effects were collected and purified usingthe Adenopure Filter system (Puresyn). Control virus was constructed sim-ilarly. Viral titer is determined inHEK293A cells by counting GFP+ cells afterlimiting dilution. Pharyngeal aspiration of 50 ml/mouse (109 IU/ml) adeno-viral stock was performed at designated time points after injury.

In vivo Ab-mediated blockade

iNOS2/2 animals were given i.p. injections (250 mg/dose/mouse) of anti-mouse CD86 mAb (GL-1; Bio X Cell) or isotype (rat IgG; Sigma-Aldrich)on days 2, 4, and 6 after i.t. LPS challenge. CD86 blockade was confirmedin BAL fluid and lung cells by flow cytometry.

Statistical analysis

All values are reported as mean 6 SEM. Parametric or nonparametrictesting was performed as indicated. Results between groups were com-pared using the Mann–Whitney U test. Baseline and pre- and posttreatmentdata within a group were compared using repeated-measures one-wayANOVA (Fisher protected least-significant difference test). The survivalcurve was established with Kaplan–Meier survival analysis. A p value ,0.05 was used as the cut-off point for significance.

ResultsResolution of lung injury is impaired in iNOS2/2 mice

We examined the response of WTand iNOS2/2mice to i.t. LPS over10 d. Survival was significantly reduced in iNOS2/2 mice, whichwas evident after day 5 (Fig. 1A). Surviving WTand iNOS2/2 mice

appeared ill, with lethargy, piloerection, and huddling behavior afteri.t. LPS. These clinical signs resolved by day 5–6 in WT mice butpersisted in iNOS2/2 mice. In addition, although both WT andiNOS2/2 mice lost weight after i.t. LPS, WT mice regained the lostweight by day 10, whereas weight loss was sustained in iNOS2/2

mice (Fig. 1B). BAL protein (Fig. 1C) and albumin (Fig. 1D) in-creased in both groups to more than four to eight times the baselinelevels by day 1. In WT mice, BAL protein and albumin peaked atday 4 and returned to baseline by day 10. In iNOS2/2 mice, BALprotein and albumin continued to increase until day 7 and remainedelevated at day 10, suggesting impaired resolution of alveolarcapillary injury.On day 1 after i.t. LPS, BAL total cell count was lower in iNOS2/2

mice than in WT mice, whereas it was higher in iNOS2/2 mice ondays 7 and 10 (Fig. 1E). BAL macrophages increased in bothgroups, but they remained higher in iNOS2/2 mice on days 7 and10 (Fig. 1F). Lymphocytes increased similarly in both WT andiNOS2/2 mice at day 4 and remained elevated through day 10(Fig. 1F). BAL neutrophil numbers were significantly lower iniNOS2/2 mice than in WT mice on day 1 after i.t. LPS (Fig. 1F),but the neutrophil number was increased in iNOS2/2 mice on days7 and 10. In WT mice, neutrophils were nearly undetectable in thealveolar compartment by day 10 after i.t. LPS. The pattern ofhistological changes in the lung was consistent with patterns forBAL protein and cells (Fig. 1G). Specifically, interstitial thick-ening and cellular infiltration were worse in WT mice at day 1compared with iNOS2/2 mice. However, at day 7 the histologicalchanges were more pronounced in iNOS2/2 mice than in WTmice. By day 10, lung histology appeared near normal in the WTmice, yet histological changes persisted in iNOS2/2 mice. Blindedlung injury scoring confirmed a biphasic injury response iniNOS2/2 mice; despite an attenuated early injury, iNOS2/2 micedisplayed a significant impairment in the resolution of lung in-flammation (Fig. 1H). These observations suggest an importantrole for iNOS in the resolution of lung injury.

iNOS expression and source during ALI

We sought to examine the patterns of NO generation in lung andBAL fluid using the Griess reaction, which measures total nitrateand nitrite (NOx). Both BAL (Fig. 2A) and lung NOx (Fig. 2B)were significantly reduced in iNOS2/2 mice compared with WTmice at all time points. In WT mice, NOx production peaked atday 4 after i.t. LPS in both BAL and lung homogenate. Proteinimmunoblots revealed that iNOS had increased 5-fold in the lungof WT mice on day 4 after i.t. LPS, but it had returned to baselinelevels by day 7 (Fig. 2C, 2D). iNOS protein was not detectable iniNOS2/2 mice. The pattern of expression of total eNOS andphosphorylated eNOS was not different between WTand iNOS2/2

mice at any time point (Supplemental Fig. 1A). nNOS was higherin iNOS2/2 mice at day 4 after LPS and was similar to WT miceat all other time points. iNOS labeling was evident by immuno-fluorescence in alveolar cells only from WT mice on day 4 afteri.t. LPS. No iNOS labeling was detected in injured iNOS2/2 mice(Supplemental Fig. 1B). To better define cellular sources for iNOSduring lung inflammation, we harvested lungs from WT andiNOS2/2 mice 4 d after i.t. LPS. Lungs were minced and enzy-matically digested to obtain a single-cell suspension. Lung cellswere surface stained with markers for macrophages (CD11b) andepithelial cells (CD326) and subsequently sorted using a FACSA-ria cytometer. WT CD11b+FSChi macrophages showed intenseiNOS labeling by fluorescent microscopy, whereas iNOS2/2

macrophages did not show any labeling. WT CD326+CD11b2

epithelial cells also labeled positive for iNOS; in contrast, iNOS2/2

epithelial cells did not show any labeling (Fig. 2E). These data

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indicate that iNOS is upregulated in inflammatory macrophages andlung epithelial cells during LPS-induced lung inflammation.

Alveolar inflammatory milieu is altered in iNOS2/2 mice

To begin examining the mechanisms that might contribute to thedifference in LPS response in iNOS2/2 mice, we measured selectBAL cytokines in the two groups. The proinflammatory cytokineTNF-a was elevated in both WT and iNOS2/2 groups on day 1after LPS (Fig. 3A), but it was greater in iNOS2/2 mice on days 4,7, and 10. The neutrophil chemokine MIP-2 was not differentbetween WT and iNOS2/2 mice on day 1 after i.t. LPS; however,it was greater in iNOS2/2 mice on day 4 (Fig. 3B). Active TGF-b1increased similarly in both groups on day 4 after i.t. LPS, but it

only remained elevated on days 7 and 10 in iNOS2/2 mice (Fig.3C). The anti-inflammatory cytokine IL-10 increased in bothgroups on day 1 after i.t. LPS and was not different between thetwo groups at any time (Fig. 3D).

Alveolar neutrophil clearance is impaired in iNOS2/2 miceafter ALI

The persistent elevation of neutrophils in iNOS2/2 mice on days 7and 10 after i.t. LPS was striking, in contrast to WT animals inwhich alveolar neutrophils were almost completely cleared.iNOS2/2 mice displayed higher levels of neutrophil chemokineMIP-2 (Fig. 3B) by day 4 and had persistent disruption of theiralveolar capillary membrane (elevated BAL albumin; Fig. 1D),

FIGURE 1. Resolution of lung injury is impaired in iNOS2/2 mice. WT and iNOS2/2 mice (n = 8/group/time point) were challenged with i.t. LPS.

Survival (A) and body weight relative to baseline (B) were plotted at intervals after injury. BAL protein (C), BAL albumin (D), BAL total cell counts (E),

and BAL differential cell counts (F) were determined in WT and iNOS2/2 mice after i.t. water (control) or i.t. LPS. (G) Histological sections from WT and

iNOS2/2 mice were stained with H&E (original magnification 320; insets, 3100). (H) Histopathological mean lung injury scores from low-power (320)

sections (n = 4–6 animals/group/time point). Data are expressed as mean 6 SEM. *p , 0.05, log-rank test (A) and unpaired Student t test (B–F). †p , 0.05

versus iNOS2/2 within the same time point.

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suggesting roles for barrier disruption and chemokine gradients inneutrophil recruitment. We also evaluated neutrophil apoptosisand macrophage efferocytosis as potential contributors to the in-crease in alveolar neutrophils. To determine whether changes inapoptosis may contribute to the differences in neutrophil number,we harvested BAL cells at intervals after i.t. LPS and assessedannexin V and 7-AAD labeling of neutrophils by flow cytometry.On day 1 after LPS, apoptosis (annexin V+, 7-AAD2) (Fig. 4) was

not different between the groups. By day 4, apoptosis was sig-nificantly greater in neutrophils of WT mice compared withiNOS2/2 mice, and it remained elevated at day 7. By day 10,neutrophil apoptosis was similar between the two groups.We also examined AM phagocytosis using BAL macrophages

harvested at intervals after i.t. LPS. Phagocytosis by AMs fromiNOS2/2 mice was significantly lower than macrophages from WTmice on days 1 and 4 after i.t. LPS (Supplemental Fig. 1C). The PIwas not different in the two groups on days 7 and 10 after i.t. LPS.

The absence of iNOS leads to sustained AM activation afterLPS

Themarked delay in the resolution of lung inflammation in iNOS2/2

mice, coupled with the persistent elevation of BALTNF-a, led us toexamine phenotypic differences between AMs in WT and iNOS2/2

mice. Using multiparameter flow cytometry, we observed thatCD11b+CD11c+ AMs in WT and iNOS2/2 mice exhibited a sim-ilar expression of accessory and costimulatory molecules CD40and CD86 on day 1 after LPS. In contrast, by day 7 after i.t. LPS,iNOS2/2 macrophages had sustained expression of CD40, CD86,and MHC class II (MHC-II) (Fig. 5A). Cell surface expression ofcostimulatory molecule CD80 (data not shown) and TLR2 andTLR4 was similar between the two groups at all intervals after i.t.LPS (Fig. 5A). The mean fluorescence intensity (MFI) of mac-rophage cell surface molecules was compared between WT andiNOS2/2 mice (n = 4 animals/interval/group) (Fig. 5B) and rein-forces the differences noted in the graphs (Fig. 5A).We also sought to determine whether CD11b+CD11c+ mono-

cytes were functionally different between the groups. Intracellularproduction of ROS, as measured by DCF, was elevated in both

FIGURE 2. iNOS expression and source during

ALI. BAL fluid (A) and lung (B) nitrate/nitrite (NOx)

concentrations were measured in WT and iNOS2/2

mice after i.t. water (control) or LPS, at designated

intervals. (C) iNOS immunoblotting of lung homoge-

nates by SDS-PAGE at intervals after i.t. LPS in WT

and iNOS2/2 mice. b-actin was used as a loading

control. (D) iNOS densitometry (iNOS/actin) was

expressed as the percentage increase over the WT

control. Data are mean 6 SEM. (E) Lungs were har-

vested 4 d after i.t. LPS, enzymatically digested into

a single-cell suspension, and surface stained for

CD11b–PE–Texas red (red staining, upper panels) and

CD326. Cells were subsequently sorted (FACSAria)

and placed in a cytospin for staining with iNOS-FITC

Ab (arrows) using a fluorescent microscope. In the

lower panels, TO-PRO-3 (Invitrogen) nuclear staining

(arrowhead) was used. *p , 0.05 versus WT within

same time point.

FIGURE 3. Alveolar inflammatory milieu is altered in iNOS2/2 mice.

WT and iNOS2/2 mice received i.t. LPS or sterile water (day 1 control).

BAL proinflammatory TNF-a (A) and MIP-2 (B) and anti-inflammatory

activated TGF-b1 (C) and IL-10 (D) were measured by ELISA at the

designated intervals. Data are mean 6 SEM. *p , 0.05 versus WT mice

within same time point.

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WT and iNOS2/2 monocytes at day 1, but it was significantlyhigher in iNOS2/2 monocytes by day 4 (Fig. 5C). We also assessedthioglycollate-elicited peritoneal macrophages (13 106 cells/well)from WT and iNOS2/2 mice following stimulation with LPS (100ng/ml). Supernatant TNF-a was higher in the LPS-activated WTmacrophages compared with iNOS2/2 macrophages at 6 and 24 hafter LPS. In contrast, by 48 h, WT macrophages exhibited a sig-nificant reduction in TNF-a secretion, whereas iNOS2/2 macro-phages had persistently elevated levels of TNF-a, comparable to

their 24-h secretion profile (Fig. 5D). MIP-2 levels were similar inboth groups at every interval (Supplemental Fig. 1D).These findings indicate a role for iNOS in modulating AM

phenotype and function.

Delivery of iNOS restores resolution of ALI in iNOS2/2 mice

Our findings suggest that iNOS plays an important role in theresolution of lung injury and that AMs are a potential cellularsource of iNOS. To further assess a potential role for monocyte/

FIGURE 4. Alveolar neutrophil clearance is im-

paired in iNOS2/2 mice after ALI. (A) Neutrophil

apoptosis (annexin V+/7-AAD2) in the BAL fluid

from WT and iNOS2/2 mice after i.t. LPS was

assessed by flow cytometry. BAL neutrophils were

gated by characteristic granulocyte forward and side

scatter, and the population was further subgated for

Gr-1+ neutrophils. (B) BAL neutrophil apoptosis

expressed as average percentage in WT and iNOS2/2

mice was measured at intervals after i.t. LPS (n = 4/

group/time point). Data are mean 6 SEM. *p , 0.05

versus WT within the same time point.

FIGURE 5. The absence of iNOS leads to sustained AM activation after LPS. BAL fluid was obtained from WTand iNOS2/2 mice at the designated time

points after i.t. water (control) or i.t. LPS. (A) Multicolor flow cytometry graphs for BAL macrophages (CD11c+CD11b+) and their relative expression

profiles for CD40, MHC-II, CD86, TLR2, and TLR4. (B) MFI for cell surface BAL macrophages CD40, CD86, and MHC-II. (C) WT and iNOS2/2 BAL

macrophage (CD11c+) intracellular production of ROS, as measured by fluorescent DCF. (D) Thioglycollate-elicited macrophages were obtained at day 6

from WT and iNOS2/2 mice, plated, and stimulated with LPS (100 ng/ml). Cell supernatants were collected and used to measure TNF-a at the designated

intervals. Data are mean 6 SEM. *p , 0.05 versus WT within same time point, †p , 0.05 versus iNOS2/2 within the same time point.

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macrophages in this response, we performed adoptive transfer ofBM-derived CD11b+ monocytes into iNOS2/2 mice and examinedthe effect on the resolution of lung injury. iNOS2/2 mice receivingtail vein injections of PBS or iNOS2/2 BM-derived CD11b+ cellsexhibited delayed resolution of lung injury and decreased survival.In contrast, iNOS2/2 mice receiving WT iNOS+/+CD11b+ BM-derived cells delivered 24 h after i.t. LPS had improved survival(Fig. 6A). Flow cytometry confirmed that following adoptivetransfer, congenic donor BM monocytes (CD45.1+) trafficked tothe alveolar compartment and lung interstitium after LPS-inducedALI (Supplemental Fig. 2A). Donor CD45.1+F4/80+CD11b+

macrophages peaked in the alveolar and interstitial compartmentat day 4 after adoptive transfer and injury (data not shown).iNOS2/2 mice receiving BM-derived iNOS+/+CD11b+ cells had

higher NOx levels in the BAL (Fig. 6B) and lung homogenate onday 4 after adoptive transfer than did iNOS2/2 mice receivingPBS or iNOS2/2CD11b+ cells (Supplemental Fig. 2B), confirmingthat adoptive transfer of WT CD11b+ cells led to increased localNO production in the alveolar space. iNOS2/2 mice receiving

iNOS+/+CD11b+ cells exhibited complete resolution of histologi-cal injury by day 10 (Fig. 6C, 6D). BAL protein (Fig. 6E), totalcell count (Fig. 6F), and BAL neutrophils (Fig. 6G) were elevatedon day 10 in iNOS2/2 mice receiving PBS or iNOS2/2CD11b+

cells, but they were reduced nearly to WT levels following transferof iNOS+/+CD11b+ cells. Mice receiving PBS or iNOS2/2 BMcells had elevated BALTNF-a levels through day 10 after i.t. LPS,whereas transfer of BM iNOS+/+CD11b+ cells resulted in a re-duction of BAL TNF-a to WT levels (Fig. 6H).To test the potentially therapeutic effects of iNOS, we challenged

iNOS2/2 mice with i.t. LPS; after 48 h of established injury,animals were assigned to one of two treatment modalities: pha-ryngeal aspiration of a single dose of adenovirus encoding GFPdriven by a CMV promoter (Ad5CMV-GFP) or an adenovirusencoding the human iNOS tagged to GFP (Ad5CMV-hiNOS-GFP). We measured outcomes at day 7 after i.t. LPS. Directlung transgene delivery was confirmed by flow cytometry. Differ-ential alveolar cell transduction of adenovirus was detected by GFPfluorescence (Supplemental Fig. 2C; macrophages . neutrophils

FIGURE 6. Adoptive transfer of WT monocytes restores resolution of ALI in iNOS2/2 mice. iNOS2/2 mice were challenged with i.t. LPS; 24 h after

injury they were given PBS (sham) or BM-derived CD11b+ monocytes isolated from iNOS+/+ or iNOS2/2 mice by tail vein injection. (A) Survival curves

for designated groups (n = 8–10/group). (B) BAL NOx levels 4 d after i.t. LPS after designated transfers into iNOS2/2 mice. Mean 6 SEM. Lung H&E-

stained sections (320 for panoramic view,3100 for insets) (C) and histopathological lung injury scoring (D) were assessed 10 d after i.t. LPS (n = 6/group).

BAL fluid protein (E), BAL fluid total cell counts (F), BAL fluid neutrophils (as a percentage of total BAL cells) (G), and BAL fluid TNF-a levels (H) were

measured 10 d after i.t. LPS are shown after designated transfers into injured iNOS2/2 mice. *p, 0.05 versus PBS to iNOS2/2, *,#p, 0.05 versus WT BM

to iNOS2/2.

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. lymphocytes). In contrast, unstimulated animals had no trans-duction of AMs (data not shown), whereas lung parenchyma waseffectively transduced in the absence of lung inflammation. Epi-thelial transduction increased when inflammation was present (Sup-plemental Fig. 2D). We found that in vivo transgene delivery ofiNOS given as rescue therapy (48 h after LPS) produced significanttransduction of AMs and drove resolution of lung inflammationin iNOS2/2 mice. Furthermore, delivery of adeno-hiNOS-GFPdownregulated the expression of macrophage costimulatory mol-ecules CD86 and CD40 (Fig. 7C). In contrast, adeno-GFP had noeffect on these cellular and molecular targets, and it did not alter thesustained expression of macrophage CD86 and CD40. In summary,direct delivery of iNOS to the air space rescued injured iNOS2/2

mice and promoted resolution of ALI.

Myeloid, but not epithelial-derived iNOS modulates resolutionof lung injury

We showed that iNOS is upregulated in inflammatory alveolar andlung epithelial cells during inflammation in WTanimals. To evaluatethe relative contributions of myeloid and epithelial-derived iNOS, weperformed irradiated BM chimeras between congenic WT (CD45.1)and iNOS2/2 mice. Transfer of WT BM into irradiated WT micewas used as control; this group achieved complete resolution of i.t.LPS-induced lung injury by day 11. We noticed that, in irradiatedanimals, the injury response was shifted after i.t. LPS, as evidencedby their peak weight loss ∼6 d after LPS compared with 4 d innonirradiated animals (data not shown). Similarly, WT BM transferinto irradiated iNOS2/2 mice achieved almost complete resolutionof ALI, as evidenced by the reduction in BAL total cells and protein(Fig. 8A). These mice had myeloid iNOS but no epithelial-derivediNOS. In marked contrast, the transfer of iNOS2/2 BM into irra-diated WT mice resulted in sustained alveolar lung inflammationeven 11 d after i.t. LPS, with elevated BAL cell counts and protein,as well as persistent histological lung injury (Fig. 8). This group hadepithelial iNOS but no myeloid iNOS.These results are consistent with our adoptive-transfer experi-

ments using BM-derived monocytes and indicate that epithelial-derived iNOS does not play an important role in the resolutionof experimental lung injury after LPS.

iNOS regulates CD86 expression to regulate lung inflammation

We sought to determine whether sustained CD86 expression inmacrophages contributed to the persistent lung inflammation seenin injured iNOS2/2 mice. To avoid attenuating the initial inflam-

matory response to i.t. LPS and, thus, altering the primary injury,we administered i.p. injections of monoclonal anti-CD86 (250 mg/mouse/dose; Bio X Cell) or isotype Ab (rat IgG; Sigma-Aldrich)2 d after LPS exposure and again on days 4 and 6. Seven daysafter i.t. LPS, survival was improved by 33% in anti-CD86–treatediNOS2/2 mice (Fig. 9A, 80 versus 60% in isotype-treated group).BAL protein (Fig. 9B) and total cell counts (data not shown) weremarkedly attenuated in the anti-CD86 group. Lung histology (Fig.9C) showed enhanced resolution of lung inflammation in iNOS2/2

mice that received anti-CD86 compared with the isotype-treatedmice (Fig. 9C, 9D). We confirmed blockade of CD86 of mono-nuclear cells in the lung by flow cytometry (data not shown).To confirm that iNOS modulates macrophage CD86 expression,

we challenged an AM cell line (MH-S) with LPS in the presence orabsence of a specific iNOS inhibitor (1400W); we analyzed cellsurface expression for CD86 by flow cytometry at 24 and 48 h.Density plots showed that, although CD86 expression on the cellsurface of CD11b+ macrophages was attenuated 24 h after LPS inthe iNOS-inhibited (1400W) cells, CD86 expression was higher inthe same group when analyzed at 48 h (Fig. 9E, 9F). To evaluate theex vivo macrophage response, we isolated and plated naive peri-toneal macrophages from WT and iNOS2/2 mice and then stimu-lated them with LPS. A group of WT macrophages was treated with1400W. Cells were surface stained for CD86 expression at specificintervals. iNOS2/2 and WT macrophages treated with 1400W hadhigher expression of CD86 at 24 h compared with WT macrophages(Fig. 9G). iNOS2/2 macrophages had sustained CD86 expressionafter 48 h of LPS stimulation, whereas the WT and WT 1400W-treated cells returned back to baseline expression. Our findingssupport iNOS as a key regulator of macrophage CD86 expressionduring lung inflammation.

DiscussionNO has been implicated in the pathogenesis of lung inflammationand injury, principally as having a deleterious role in early injury.The role of iNOS as a proinflammatory mediator in the lung hasbeen described in numerous animal models, including OVA-induced airway inflammation, carrageenan-induced pleuritis (40),ventilator-induced lung injury (41), direct infectious or LPS-inducedALI (12, 42, 43), and sepsis or indirect LPS-induced ALI (10, 19, 21,22). Consistent with reports of the proinflammatory role of iNOS(44), we found, in our established model of i.t. LPS-induced lunginjury, that the absence of iNOS attenuated early injury. In markedcontrast, the absence of iNOS significantly delayed resolution of lunginflammation. Our findings reinforce the need to distinguish earlyfrom late responses when seeking a role for a specific mediator orpathway during an inflammatory response.In this model, lung injury progressed and peaked on day 4 after

LPS. At that timewe observed an induction of iNOS and NOx in thealveolar space, after which resolution of lung inflammation pro-ceeded. As described by other investigators (45), we believe thatearly proinflammatory events are important for induction of repairmechanisms. Repair was significantly delayed in the absence ofiNOS. Similarly, we recently described a pivotal role for alveolarregulatory T cells (Tregs) in the resolution of experimental lunginjury, which peaked on days 4–7 after ALI (37). Potentialinteractions between iNOS and Tregs in this response are thesubject of ongoing investigation.The role of iNOS in inflammation and infection can vary,

depending on the setting, the organ involved, the cell type, and/orthe stage of the inflammatory response, ultimately enhancing in-flammation or abrogating it (46). Kristof et al. (10) found thatiNOS2/2 mice displayed attenuated ALI in a murine model ofseptic shock. Similarly, Shanley et al. (44) found that exogenous

FIGURE 7. Viral delivery of iNOS, but not inhaled NO, restores reso-

lution of ALI in iNOS2/2 mice. iNOS2/2 mice were challenged with i.t.

LPS; 48 h after injury they received either a 50-ml pharyngeal aspiration of

AdCMV-GFP or AdCMV-hiNOS-GFP (109 IU/ml). Outcomes were

measured 7 d after i.t. LPS. Lung H&E-stained sections (original magni-

fication 320) (A) and BAL protein (B) for designated treatments. *p ,0.05 versus iNOS2/2 Adeno-GFP. (C) Flow cytometry graphs gated for

iNOS2/2 mice alveolar CD11b+CD11c+ macrophages and their relative

expression of surface markers after pharyngeal aspiration of the designated

adenoviral transgene delivery.

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NO increased inflammation and vascular leak and that the absenceof iNOS was associated with an attenuated acute inflammatoryresponse to i.t. endotoxin. However, a few reports indicate a morecomplex role for iNOS in injury responses (sequestration, adhe-sion, and activation of neutrophils in the lung endothelium) (47).For example, iNOS-deficient mice are resistant to LPS-inducedhypotension, but they suffer as much LPS-induced liver damageas do their WT counterparts (22).

We observed a delay in epithelial–endothelial barrier repair iniNOS2/2 mice compared with WT mice, as evidenced by thepersistently elevated alveolar protein and albumin. Both iNOSexpression and the production of TGF-b play critical roles invascular function during inflammatory responses; however, animbalance can lead to pathology (48). In WT mice, active alveolarTGF-b peaked at day 4, concurrent with maximal iNOS expres-sion, and then decreased to baseline levels on subsequent days. In

FIGURE 8. Myeloid, but not epithelial-derived, iNOS modulates the resolution of lung injury. Irradiated BM chimeras were generated between congenic

WT (CD45.1) and iNOS2/2 mice. WT BM to WT mice were used as irradiated controls. Eight weeks postirradiation, mice were challenged with i.t. LPS

and followed for 11 d. (A) BAL fluid protein and total cell counts were measured in designated groups. (B) Lung H&E-stained sections are shown for

designated groups 11 d after i.t. LPS (original magnification 320; insets, 3100). (C) Lung injury scoring was done for low-power (320) lung histo-

pathological sections. †p , 0.05, WT BM to WT.

FIGURE 9. iNOS regulates CD86 expression to

modulate lung inflammation. iNOS2/2 mice were

challenged with i.t. LPS; 48 h after injury they re-

ceived either isotype Ab (rat IgG) or anti-mouse CD86

(clone GL-1) on days 2, 4, and 6 after ALI. Survival

(A) and BAL protein (B) were evaluated in iNOS2/2

mice after i.t. LPS. (C) Histological sections were

stained with H&E (original magnification 320). (D)

Histopathological mean lung injury scores from low-

power (320) sections (n = 4–6 animals/group). The

AM cell line (MH-S) was challenged with media or

LPS (100 ng/ml) in the presence of DMSO or specific

iNOS inhibitor 1400W (25 mM) at designated inter-

vals. (E) Flow cytometry density plots show the rela-

tive expression of surface CD86 at designated intervals

after LPS. MFI for CD86 expression was measured in

MH-S cells (F) and resident peritoneal macrophages

(G) and compared among groups. Data are mean 6SEM. †,*p , 0.05, log-rank test (A) and unpaired

Student t test (B, D–F).

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contrast, iNOS2/2 mice had persistently elevated levels of TGF-b,perhaps reflecting a negative-feedback loop between iNOS andTGF-b (49). Although TGF-b can act as a potent anti-inflammatoryand reparative mediator, uncontrolled and sustained elevation ofTGF-b can lead to increases in alveolar epithelial permeability(50) and fibrotic complications (51). This is supported by thestudy by Hochberg et al. (52), who noted increased fibroticcomplications with markedly elevated kidney TGF-b levels afterunilateral ureteral obstruction in iNOS2/2 mice compared withWT mice. Furthermore, although similar levels of alveolar colla-gen were present in WT and iNOS2/2 mice by day 4, the lattergroup had markedly elevated collagen levels at day 7, whereas thelevels in WT mice had returned to baseline (B.T. Garibaldi, un-published observations), supporting a role for iNOS in the fibro-proliferative response after ALI.Apoptosis of inflammatory cells is fundamental to the resolution

of inflammation, and failure to clear inflammatory cells leads toexcessive tissue damage and sustained injury (45, 53). In contrastto the complete alveolar neutrophil clearance in WT mice, alve-olar neutrophilia persisted in iNOS2/2 mice. Several factors maycontribute to the sustained elevation of alveolar neutrophils in theabsence of iNOS. MIP-2, a potent neutrophil chemokine, washigher by day 4 in the BAL fluid from iNOS2/2 mice; togetherwith the persistently damaged alveolar–endothelial barrier seen inlater stages in iNOS2/2 mice, likely contributed to the elevatednumber of neutrophils. In addition, neutrophil apoptosis andefferocytosis were both impaired in iNOS2/2 mice; NO can in-duce apoptotic neutrophil death (54). We cannot exclude that otherfactors contribute to persistent alveolar neutrophilia in iNOS2/2

mice (e.g., production of neutrophil chemokine KC and/or LIXby a persistently injured alveolar epithelium or enhanced Th17responses, which can be involved in LPS-induced lung inflam-mation) (55).AMs play a critical role in the pathogenesis of ALI (56, 57). We

observed similar number, phenotype, and function of AMs in WTand iNOS2/2 mice during early lung injury. In marked contrast,during the resolution stage (.4 d after i.t. LPS), iNOS2/2 animalshad increased AM numbers, with significant differences in phe-notype and function compared with WT macrophages. Macro-phage cosignaling molecules (CD86, CD40) and MHC-II weresustained at higher levels in the absence of iNOS, indicatinga persistently activated macrophage phenotype. Cosignalingmolecules are cell surface glycoproteins, usually expressed inmacrophages and other APCs, which direct, modulate, and fine-tune T cell responses (58, 59). Emerging data suggest thatcosignaling molecules may regulate innate immune responses,best described in the context of Ag-specific models. For instance,genetic deletion of the cosignaling molecule CD40 in mice de-creased susceptibility to direct injury with LPS and polymicrobialsepsis (60, 61). Additionally, cosignaling molecules (CD40 andCD80/86) regulate inflammation in a model of polymicrobialsepsis, and humans with septic shock have higher monocyte ex-pression of CD40 and CD80 compared with patients with sepsis orhealthy controls (62). Consistent with our findings, Shi et al. (32)described that, in a model of autoimmune myasthenia gravis,iNOS2/2 macrophages had a higher expression pattern of CD40and MHC-II compared with WT, as well as sustained productionof proinflammatory TNF-a and ROS.During lung inflammation, iNOS was reported to originate from

inflammatory cells (neutrophils,macrophages) or epithelial cells (63).Although we cannot exclude a potential role for epithelial-derivediNOS during the resolution of lung inflammation, our adoptive-transfer experiments support a central role for macrophage-derivediNOS in this model. BM-derived CD11b+ monocytes trafficked to

the inflamed lung and alveolar compartments following adoptivetransfer. CD11b+iNOS+/+, but not CD11b+iNOS2/2, BM-derivedmonocytes restored the resolution of ALI, even when delivered24 h after established injury, suggesting a potential rescue therapy.Similarly, delivery of iNOS into the lung via adenovirus restoredresolution. Importantly, adenoviral iNOS was readily taken up bymacrophages in the inflamed alveolus, whereas transduction of theresident AMs in the uninjured lung was minimal. In contrast, thelung epithelium was readily transduced in normal and inflamedconditions. To dissect the role of cellular iNOS in driving the res-olution of experimental lung inflammation, we created irradiatedchimeras that showed that the myeloid-derived iNOS was sufficientto restore the normal lung repair. Lung epithelial iNOS had no effectin mediating the resolution of ALI. Although BM chimeras betweenWT and iNOS2/2 were described in murine models of sepsis-induced lung injury (64, 65), these studies identified that myeloid-derived iNOS contributed to pulmonary oxidant stress and micro-vascular leak, whereas parenchymal-derived iNOS had no apparenteffect. Our results appeared to contradict these reports, althoughthere are two main differences. First, our model of ALI is direct,with a significant epithelial injury component, followed by an influxof inflammatory cells; other investigators reported results from in-direct models of ALI in which there is no or modest alveolitis.Second, the models reported focused on early events after ALI(hours); we focused on later stages, during the resolution and repairof ALI. Although inhaled NO has been tested in patients with ALI,and transient improvements were shown in physiological parame-ters, such as oxygenation and pulmonary vascular resistance, fourmulticenter, randomized, placebo-controlled trials failed to showa therapeutic role for inhaled NO in patients with acute respiratoryfailure (66–68). We find that upregulation of iNOS and NOx istightly regulated following LPS-induced lung injury, so that location,cellular source, timing, and duration of increased NO may be criticalto its overall effect and, therefore, may not be adequately reproducedthrough continuous inhaled delivery of NO. Further definition of thedifferences between inhaled NO and the upregulation of cell-basediNOS/NO may provide important mechanistic insights that could beleveraged for the design of therapy.Although iNOS-derived NO has a diverse range of immunological

effects, we found the sustained expression of AM cosignalingmolecule CD86 in the absence of iNOS to be a prominent feature.Weconsidered several factors with regard to the timing of CD86 Ab-mediated blockade. First, we wanted to intervene when ALI wasfully established and, thus, prove that our intervention can be usedas rescue therapy, which is directly relevant to strategies for therapyin most patients with ALI. Second, we sought to avoid attenuatingthe early inflammatory response with pretreatment or early inter-vention, because the focus of this line of investigation is onmechanisms of resolution. Our laboratory is pursuing subcellularmechanisms of iNOS-mediated CD86 regulation. Sustained CD86expression could also be explained by enhanced APC survival andresponsiveness to TLR agonist seen in the absence of iNOS, asdescribed recently (69). Sustained macrophage CD86 expression,in conjunction with high MHC-II expression observed in iNOS2/2

mice, could potentially lead to enhanced costimulation of Th1responses from CD4+ cells, which were abundant by days 4–7after alveolar inflammation and contribute to persistent lung im-munopathology. In addition to the persistent CD86 expression iniNOS2/2 monocytes, we observed sustained upregulation of CD40expression. Its regulation by iNOS and its role in the modulationof lung inflammation need further evaluation. We were unable touse Ab-mediated blockade, because available murine Abs areagonistic for CD40. We cannot exclude a synergistic role forsustained macrophage CD40 expression in iNOS-mediated mod-

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ulation of lung inflammation, although blockade of CD86 wassufficient to restore the resolution of ALI.ALI continues to be a major clinical problem, with significant

annual morbidity and mortality, for which therapy is largelysupportive. In this article, we demonstrate a critical role formacrophage-derived iNOS in promoting the resolution of lunginjury. iNOS has differential effects depending on the stage ofinjury, with a predominant early proinflammatory effect and a re-parative, anti-inflammatory late-phase effect. The late effects aremediated, at least in part, by AM activation and modulation ofCD86 expression, but theymay occur via effects on other pathways,including Tregs, lipid mediators, and Th17 responses, amongothers. The surprising identification of iNOS as a determinant ofresolution of lung injury provides new insights into the resolutionof inflammation and may create opportunities for the developmentof new approaches to therapy.

AcknowledgmentsWe thank Dr. Mark Soloski and Joe Chrest for assistance in the Johns Hop-

kins Bayview Flow Cytometry Core, James Watkins and Andre Robinson

for expert assistance with tissue processing for histologic studies, and Tony

Eissa (Baylor College of Medicine) for kindly providing the hiNOS-GFP

vector.

DisclosuresThe authors have no financial conflicts of interest.

References1. Rubenfeld, G. D., E. Caldwell, E. Peabody, J. Weaver, D. P. Martin, M. Neff,

E. J. Stern, and L. D. Hudson. 2005. Incidence and outcomes of acute lunginjury. N. Engl. J. Med. 353: 1685–1693.

2. The Acute Respiratory Distress Syndrome Network. 2000. Ventilation withlower tidal volumes as compared with traditional tidal volumes for acute lunginjury and the acute respiratory distress syndrome. N. Engl. J. Med. 342: 1301–1308.

3. Ware, L. B., and M. A. Matthay. 2000. The acute respiratory distress syndrome.N. Engl. J. Med. 342: 1334–1349.

4. Bachofen, M., and E. R. Weibel. 1982. Structural alterations of lung parenchymain the adult respiratory distress syndrome. Clin. Chest Med. 3: 35–56.

5. Anderson, W. R., and K. Thielen. 1992. Correlative study of adult respiratorydistress syndrome by light, scanning, and transmission electron microscopy.Ultrastruct. Pathol. 16: 615–628.

6. Meduri, G. U., M. Eltorky, and H. T. Winer-Muram. 1995. The fibroproliferativephase of late adult respiratory distress syndrome. Semin. Respir. Infect. 10: 154–175.

7. Martin, C., L. Papazian, M. J. Payan, P. Saux, and F. Gouin. 1995. Pulmonaryfibrosis correlates with outcome in adult respiratory distress syndrome. A studyin mechanically ventilated patients. Chest 107: 196–200.

8. Baron, R. M., I. M. Carvajal, X. Liu, R. O. Okabe, L. E. Fredenburgh,A. A. Macias, Y. H. Chen, K. Ejima, M. D. Layne, and M. A. Perrella. 2004.Reduction of nitric oxide synthase 2 expression by distamycin A improvessurvival from endotoxemia. J. Immunol. 173: 4147–4153.

9. Haddad, I. Y., J. P. Crow, P. Hu, Y. Ye, J. Beckman, and S. Matalon. 1994.Concurrent generation of nitric oxide and superoxide damages surfactant proteinA. Am. J. Physiol. 267: L242–L249.

10. Kristof, A. S., P. Goldberg, V. Laubach, and S. N. Hussain. 1998. Role of in-ducible nitric oxide synthase in endotoxin-induced acute lung injury. Am. J.Respir. Crit. Care Med. 158: 1883–1889.

11. Kooy, N. W., J. A. Royall, Y. Z. Ye, D. R. Kelly, and J. S. Beckman. 1995.Evidence for in vivo peroxynitrite production in human acute lung injury. Am. J.Respir. Crit. Care Med. 151: 1250–1254.

12. Okamoto, T., K. Gohil, E. I. Finkelstein, P. Bove, T. Akaike, and A. van derVliet. 2004. Multiple contributing roles for NOS2 in LPS-induced acute airwayinflammation in mice. Am. J. Physiol. Lung Cell. Mol. Physiol. 286: L198–L209.

13. Farley, K. S., L. F. Wang, H. M. Razavi, C. Law, M. Rohan, D. G. McCormack,and S. Mehta. 2006. Effects of macrophage inducible nitric oxide synthase inmurine septic lung injury. Am. J. Physiol. Lung Cell. Mol. Physiol. 290: L1164–L1172.

14. Moncada, S., R. M. Palmer, and E. A. Higgs. 1991. Nitric oxide: physiology,pathophysiology, and pharmacology. Pharmacol. Rev. 43: 109–142.

15. Nathan, C., and Q. W. Xie. 1994. Nitric oxide synthases: roles, tolls, and con-trols. Cell 78: 915–918.

16. van der Vliet, A., and C. E. Cross. 2000. Oxidants, nitrosants, and the lung. Am.J. Med. 109: 398–421.

17. Kobzik, L., D. S. Bredt, C. J. Lowenstein, J. Drazen, B. Gaston, D. Sugarbaker,and J. S. Stamler. 1993. Nitric oxide synthase in human and rat lung: immu-

nocytochemical and histochemical localization. Am. J. Respir. Cell Mol. Biol. 9:371–377.

18. Hotchkiss, R. S., and I. E. Karl. 2003. The pathophysiology and treatment ofsepsis. N. Engl. J. Med. 348: 138–150.

19. Razavi, H. M., R. Werhun, J. A. Scott, S. Weicker, F. Wang, D. G. McCormack,and S. Mehta. 2002. Effects of inhaled nitric oxide in a mouse model of sepsis-induced acute lung injury. Crit. Care Med. 30: 868–873.

20. Wong, J. M., and T. R. Billiar. 1995. Regulation and function of inducible nitricoxide synthase during sepsis and acute inflammation. Adv. Pharmacol. 34: 155–170.

21. Wei, X. Q., I. G. Charles, A. Smith, J. Ure, G. J. Feng, F. P. Huang, D. Xu,W. Muller, S. Moncada, and F. Y. Liew. 1995. Altered immune responses in micelacking inducible nitric oxide synthase. Nature 375: 408–411.

22. MacMicking, J. D., C. Nathan, G. Hom, N. Chartrain, D. S. Fletcher,M. Trumbauer, K. Stevens, Q. W. Xie, K. Sokol, N. Hutchinson, et al. 1995.Altered responses to bacterial infection and endotoxic shock in mice lackinginducible nitric oxide synthase. Cell 81: 641–650.

23. Laubach, V. E., E. G. Shesely, O. Smithies, and P. A. Sherman. 1995. Micelacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death. Proc. Natl. Acad. Sci. USA 92: 10688–10692.

24. Bogdan, C. 2001. Nitric oxide and the immune response. Nat. Immunol. 2: 907–916.

25. Nathan, C., and M. U. Shiloh. 2000. Reactive oxygen and nitrogen intermediatesin the relationship between mammalian hosts and microbial pathogens. Proc.Natl. Acad. Sci. USA 97: 8841–8848.

26. Bogdan, C., M. Rollinghoff, and A. Diefenbach. 2000. The role of nitric oxide ininnate immunity. Immunol. Rev. 173: 17–26.

27. Bogdan, C., M. Rollinghoff, and A. Diefenbach. 2000. Reactive oxygen andreactive nitrogen intermediates in innate and specific immunity. Curr. Opin.Immunol. 12: 64–76.

28. Diefenbach, A., H. Schindler, N. Donhauser, E. Lorenz, T. Laskay,J. MacMicking, M. Rollinghoff, I. Gresser, and C. Bogdan. 1998. Type 1 in-terferon (IFNalpha/beta) and type 2 nitric oxide synthase regulate the innateimmune response to a protozoan parasite. Immunity 8: 77–87.

29. Spiecker, M., H. Darius, K. Kaboth, F. Hubner, and J. K. Liao. 1998. Differentialregulation of endothelial cell adhesion molecule expression by nitric oxidedonors and antioxidants. J. Leukoc. Biol. 63: 732–739.

30. Bobe, P., K. Benihoud, D. Grandjon, P. Opolon, L. L. Pritchard, and R. Huchet.1999. Nitric oxide mediation of active immunosuppression associated with graft-versus-host reaction. Blood 94: 1028–1037.

31. Kahn, D. A., D. C. Archer, D. P. Gold, and C. J. Kelly. 2001. Adjuvant immu-notherapy is dependent on inducible nitric oxide synthase. J. Exp. Med. 193:1261–1268.

32. Shi, F. D., M. Flodstrom, S. H. Kim, S. Pakala, M. Cleary, H. G. Ljunggren, andN. Sarvetnick. 2001. Control of the autoimmune response by type 2 nitric oxidesynthase. J. Immunol. 167: 3000–3006.

33. Marshall, H. E., K. Merchant, and J. S. Stamler. 2000. Nitrosation and oxidationin the regulation of gene expression. FASEB J. 14: 1889–1900.

34. Vodovotz, Y., L. Chesler, H. Chong, S. J. Kim, J. T. Simpson, W. DeGraff,G. W. Cox, A. B. Roberts, D. A. Wink, and M. H. Barcellos-Hoff. 1999. Reg-ulation of transforming growth factor beta1 by nitric oxide. Cancer Res. 59:2142–2149.

35. Obermeier, F., V. Gross, J. Scholmerich, and W. Falk. 1999. Interleukin-1 pro-duction by mouse macrophages is regulated in a feedback fashion by nitricoxide. J. Leukoc. Biol. 66: 829–836.

36. Connelly, L., M. Palacios-Callender, C. Ameixa, S. Moncada, and A. J. Hobbs.2001. Biphasic regulation of NF-kappa B activity underlies the pro- and anti-inflammatory actions of nitric oxide. J. Immunol. 166: 3873–3881.

37. D’Alessio, F. R., K. Tsushima, N. R. Aggarwal, E. E. West, M. H. Willett,M. F. Britos, M. R. Pipeling, R. G. Brower, R. M. Tuder, J. F. McDyer, andL. S. King. 2009. CD4+CD25+Foxp3+ Tregs resolve experimental lung injury inmice and are present in humans with acute lung injury. J. Clin. Invest. 119:2898–2913.

38. Halbower, A. C., R. J. Mason, S. H. Abman, and R. M. Tuder. 1994. Agaroseinfiltration improves morphology of cryostat sections of lung. Lab. Invest. 71:149–153.

39. King, L. S., S. Nielsen, and P. Agre. 1996. Aquaporin-1 water channel protein inlung: ontogeny, steroid-induced expression, and distribution in rat. J. Clin. In-vest. 97: 2183–2191.

40. Cuzzocrea, S., E. Mazzon, G. Calabro, L. Dugo, A. De Sarro, F. A. van De LOO,and A. P. Caputi. 2000. Inducible nitric oxide synthase-knockout mice exhibitresistance to pleurisy and lung injury caused by carrageenan. Am. J. Respir. Crit.Care Med. 162: 1859–1866.

41. Peng, X., R. E. Abdulnour, S. Sammani, S. F. Ma, E. J. Han, E. J. Hasan,R. Tuder, J. G. Garcia, and P. M. Hassoun. 2005. Inducible nitric oxide synthasecontributes to ventilator-induced lung injury. Am. J. Respir. Crit. Care Med. 172:470–479.

42. Akaike, T., and H. Maeda. 2000. Nitric oxide and virus infection. Immunology101: 300–308.

43. Karupiah, G., J. H. Chen, S. Mahalingam, C. F. Nathan, and J. D. MacMicking.1998. Rapid interferon gamma-dependent clearance of influenza A virus andprotection from consolidating pneumonitis in nitric oxide synthase 2-deficientmice. J. Exp. Med. 188: 1541–1546.

44. Shanley, T. P., B. Zhao, D. R. Macariola, A. Denenberg, A. L. Salzman, andP. A. Ward. 2002. Role of nitric oxide in acute lung inflammation: lessonslearned from the inducible nitric oxide synthase knockout mouse. Crit. CareMed. 30: 1960–1968.

2244 RESOLUTION OF LUNG INJURY IN iNOS2/2 MICE

by guest on Novem

ber 4, 2018http://w

ww

.jimm

unol.org/D

ownloaded from

45. Serhan, C. N., and J. Savill. 2005. Resolution of inflammation: the beginningprograms the end. Nat. Immunol. 6: 1191–1197.

46. Nathan, C. 1997. Inducible nitric oxide synthase: what difference does it make?J. Clin. Invest. 100: 2417–2423.

47. Hickey, M. J., K. A. Sharkey, E. G. Sihota, P. H. Reinhardt, J. D. Macmicking,C. Nathan, and P. Kubes. 1997. Inducible nitric oxide synthase-deficient micehave enhanced leukocyte-endothelium interactions in endotoxemia. FASEB J.11: 955–964.

48. Berg, D. T., A. Gupta, M. A. Richardson, L. A. O’Brien, D. Calnek, andB. W. Grinnell. 2007. Negative regulation of inducible nitric-oxide synthaseexpression mediated through transforming growth factor-beta-dependent mod-ulation of transcription factor TCF11. J. Biol. Chem. 282: 36837–36844.

49. Jun, C. D., B. M. Choi, S. U. Kim, S. Y. Lee, H. M. Kim, and H. T. Chung. 1995.Down-regulation of transforming growth factor-beta gene expression by anti-sense oligodeoxynucleotides increases recombinant interferon-gamma-inducednitric oxide synthesis in murine peritoneal macrophages. Immunology 85: 114–119.

50. Pittet, J. F., M. J. Griffiths, T. Geiser, N. Kaminski, S. L. Dalton, X. Huang,L. A. Brown, P. J. Gotwals, V. E. Koteliansky, M. A. Matthay, and D. Sheppard.2001. TGF-beta is a critical mediator of acute lung injury. J. Clin. Invest. 107:1537–1544.

51. Letterio, J. J., and A. B. Roberts. 1998. Regulation of immune responses byTGF-beta. Annu. Rev. Immunol. 16: 137–161.

52. Hochberg, D., C. W. Johnson, J. Chen, D. Cohen, J. Stern, E. D. Vaughan, Jr.,D. Poppas, and D. Felsen. 2000. Interstitial fibrosis of unilateral ureteral ob-struction is exacerbated in kidneys of mice lacking the gene for inducible nitricoxide synthase. Lab. Invest. 80: 1721–1728.

53. Savill, J., I. Dransfield, C. Gregory, and C. Haslett. 2002. A blast from the past:clearance of apoptotic cells regulates immune responses. Nat. Rev. Immunol. 2:965–975.

54. Fortenberry, J. D., M. L. Owens, M. R. Brown, D. Atkinson, and L. A. Brown.1998. Exogenous nitric oxide enhances neutrophil cell death and DNA frag-mentation. Am. J. Respir. Cell Mol. Biol. 18: 421–428.

55. Ferretti, S., O. Bonneau, G. R. Dubois, C. E. Jones, and A. Trifilieff. 2003. IL-17,produced by lymphocytes and neutrophils, is necessary for lipopolysaccharide-induced airway neutrophilia: IL-15 as a possible trigger. J. Immunol. 170: 2106–2112.

56. Hollingsworth, J. W., B. J. Chen, D. M. Brass, K. Berman, M. D. Gunn,D. N. Cook, and D. A. Schwartz. 2005. The critical role of hematopoietic cells inlipopolysaccharide-induced airway inflammation. Am. J. Respir. Crit. Care Med.171: 806–813.

57. Koay, M. A., X. Gao, M. K. Washington, K. S. Parman, R. T. Sadikot,T. S. Blackwell, and J. W. Christman. 2002. Macrophages are necessary for

maximal nuclear factor-kappa B activation in response to endotoxin. Am. J.Respir. Cell Mol. Biol. 26: 572–578.

58. Croft, M. 2003. Co-stimulatory members of the TNFR family: keys to effectiveT-cell immunity? Nat. Rev. Immunol. 3: 609–620.

59. Chen, L. 2004. Co-inhibitory molecules of the B7-CD28 family in the control ofT-cell immunity. Nat. Rev. Immunol. 4: 336–347.

60. Hashimoto, N., T. Kawabe, K. Imaizumi, T. Hara, M. Okamoto, K. Kojima,K. Shimokata, and Y. Hasegawa. 2004. CD40 plays a crucial role inlipopolysaccharide-induced acute lung injury. Am. J. Respir. Cell Mol. Biol. 30:808–815.

61. Gold, J. A., M. Parsey, Y. Hoshino, S. Hoshino, A. Nolan, H. Yee, D. B. Tse, andM. D. Weiden. 2003. CD40 contributes to lethality in acute sepsis: in vivo rolefor CD40 in innate immunity. Infect. Immun. 71: 3521–3528.

62. Nolan, A., M. Weiden, A. Kelly, Y. Hoshino, S. Hoshino, N. Mehta, andJ. A. Gold. 2008. CD40 and CD80/86 act synergistically to regulate inflamma-tion and mortality in polymicrobial sepsis. Am. J. Respir. Crit. Care Med. 177:301–308.

63. Hoyt, J. C., J. Ballering, H. Numanami, J. M. Hayden, and R. A. Robbins. 2006.Doxycycline modulates nitric oxide production in murine lung epithelial cells. J.Immunol. 176: 567–572.

64. Razavi, H. M., F. Wang, S. Weicker, M. Rohan, C. Law, D. G. McCormack, andS. Mehta. 2004. Pulmonary neutrophil infiltration in murine sepsis: role of in-ducible nitric oxide synthase. Am. J. Respir. Crit. Care Med. 170: 227–233.

65. Wang, F., M. Patel, H. M. Razavi, S. Weicker, M. G. Joseph, D. G. McCormack,and S. Mehta. 2002. Role of inducible nitric oxide synthase in pulmonary mi-crovascular protein leak in murine sepsis. Am. J. Respir. Crit. Care Med. 165:1634–1639.

66. Taylor, R. W., J. L. Zimmerman, R. P. Dellinger, R. C. Straube, G. J. Criner,K. Davis, Jr., K. M. Kelly, T. C. Smith, and R. J. Small; Inhaled Nitric Oxide inARDS Study Group. 2004. Low-dose inhaled nitric oxide in patients with acutelung injury: a randomized controlled trial. JAMA 291: 1603–1609.

67. Dellinger, R. P., J. L. Zimmerman, R. W. Taylor, R. C. Straube, D. L. Hauser,G. J. Criner, K. Davis, Jr., T. M. Hyers, and P. Papadakos; Inhaled Nitric Oxide inARDS Study Group. 1998. Effects of inhaled nitric oxide in patients with acuterespiratory distress syndrome: results of a randomized phase II trial. Crit. CareMed. 26: 15–23.

68. Lundin, S., H. Mang, M. Smithies, O. Stenqvist, and C. Frostell; The EuropeanStudy Group of Inhaled Nitric Oxide. 1999. Inhalation of nitric oxide in acutelung injury: results of a European multicentre study. Intensive Care Med. 25:911–919.

69. Giordano, D., C. Li, M. S. Suthar, K. E. Draves, D. Y. Ma, M. Gale, Jr., andE. A. Clark. 2011. Nitric oxide controls an inflammatory-like Ly6C(hi)PDCA1+DC subset that regulates Th1 immune responses. J. Leukoc. Biol. 89: 443–455.

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