Intercellular adhesion molecule-I (ICAM-I, CD54) deficiency segregates the unique pathophysiological requirements for generating idiopathic pneumonia syndrome (IPS) versus graft-versus-host disease following allogeneic murine bone marrow transplantation.

Following allogeneic bone marrow transplantation (alloBMT), idiopathic pneumonia syndrome (IPS) and graft-versus-host disease (GVHD) caused by donor cell alloreactivity remain major obstacles to a successful outcome. Intercellular adhesion molecule-1 (ICAM-1) is an adhesion molecule that is involved in regulating lymphohematopoietic cell migration and facilitating T-cell responses. To determine whether ICAM-1 expression in the host would affect IPS or GVHD tissue injury responses, ICAM-1(-/-) mice were compared with ICAM-1(+/+) controls. ICAM-1(-/-) recipients did not exhibit the manifestations of IPS injury such as an increase in lung weights nor decreased lung function. The influx of T cells, macrophages, and neutrophils was dramatically dampened as was the production of the inflammatory cytokines interferon-gamma and tumor necrosis factor alpha and the chemokines monocyte chemotactic protein 1, macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta, and lymphotactin, normally upregulated in the lung during IPS. In contrast, systemic levels of these mediators were unaffected and GVHD-induced lesions in the liver and colon did not differ in severity regardless of ICAM-1 expression. GVHD-mediated mortality was accelerated in ICAM-1(-/-) recipients at doses of allogeneic spleen cells that are otherwise not uniformally lethal. These data implicate ICAM-1 as playing a critical role in the generation of IPS; therefore, ICAM-1 may be a discerning element, segregating IPS from GVHD injury post-alloBMT.

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

In a model of idiopathic pneumonia syndrome after bone marrow transplantation (BMT), injection of allogeneic T cells induces nitric oxide (.NO), and the addition of cyclophosphamide (Cy) generates superoxide (O.) and a tissue-damaging nitrating oxidant. We hypothesized that.NO and O. balance are major determinants of post-BMT survival and inflammation. Inducible nitric oxide synthase (iNOS) deletional mutant mice (-/-) given donor bone marrow and spleen T cells (BMS) exhibited improved survival compared with matched BMS controls. Bronchoalveolar lavage fluids obtained on day 7 post-BMT from iNOS(-/-) BMS mice contained less tumor necrosis factor-alpha and interferon-gamma, indicating that.NO stimulated the production of proinflammatory cytokines. However, despite suppressed inflammation and decreased nitrotyrosine staining, iNOS(-/-) mice given both donor T cells and Cy (BMS + Cy) died earlier than iNOS-sufficient BMS + Cy mice. Alveolar macrophages from iNOS(-/-) BMS + Cy mice did not produce.NO but persisted to generate strong oxidants as assessed by the oxidation of the intracellular fluorescent probe 2',7'-dichlorofluorescin. We concluded that.NO amplifies T cell-dependent inflammation and addition of Cy exacerbates.NO-dependent mortality. However, the lack of.NO during Cy-induced oxidant stress decreases survival of T cell-recipient mice, most likely by generation of.NO-independent toxic oxidants.

Human surfactant protein a suppresses T cell-dependent inflammation and attenuates the manifestations of idiopathic pneumonia syndrome in mice.

We have previously shown an association between growth factor-induced upregulation of surfactant protein (SP)-A and suppression of alveolar inflammation in our murine model of donor T cell-dependent lung dysfunction after bone-marrow transplantation, referred to as idiopathic pneumonia syndrome (IPS). We hypothesized that SP-A protects the lung in vivo from IPS injury by downregulation of alveolar inflammation. Human SP-A (100 microg), purified by n-butanol extraction or preparative isoelectric focusing, was transtracheally instilled on Day 4 after BMT during a time of in vivo donor T-cell activation. At 48 h after treatment, immunohistochemical staining of lung sections showed that SP-A did not alter T cell- dependent cellular infiltration. However, macrophages from SP-A-instilled mice were less injured and spontaneously produced less tumor necrosis factor-alpha than did cells from buffer-instilled mice. Although exogenous SP-A did not significantly alter bronchoalveolar lavage fluid (BALF) high levels of total protein (TP), an inverse correlation between BALF SP-A and TP concentrations (r = -0.65; P = 0.02) was observed in SP-A-treated but not in buffer-instilled mice. The only difference between the effects of the two sources of SP-A was that butanol-extracted SP-A, but not isoelectric focusing-purified SP-A, suppressed the interferon-gamma/nitric oxide pathway. We conclude that SP-A downregulates T cell-dependent alveolar inflammation by multiple pathways leading to decreased IPS injury.

Cyclophosphamide prevents systemic keratinocyte growth factor-induced up-regulation of surfactant protein A after allogeneic transplant in mice.

We reported that systemic keratinocyte growth factor (KGF) given before bone marrow transplantation (BMT) prevents allogeneic T cell-dependent lung inflammation assessed on Day 7 post-BMT, but the antiinflammatory effects of KGF were impaired in mice injected with both T cells and conditioning regimen of cyclophosphamide (Cy). Intratracheal KGF is known to stimulate the expression of surfactant protein A (SP-A), an oxidant-sensitive T cell immunomodulator produced by alveolar type II cells. We hypothesized that systemic KGF up-regulates SP-A after allogeneic BMT, and the addition of Cy may interfere with the ability of KGF to enhance SP-A production. The subcutaneous administration of recombinant human KGF (5 mg/kg on Days -6, -5, and -4 pre-BMT) increased SP-A protein and mRNA in allogeneic T cell-recipient irradiated mice measured on Day 7 post-BMT. In contrast, the same KGF treatment in irradiated mice given T cells and Cy failed to up-regulate SP-A mRNA and protein expression. In mixed lymphocyte reaction experiments designed to simulate the in vivo model, the addition of human SP-A (5-50 microg) to alloactivated T cells suppressed the production of interleukin-2 in a dose-dependent fashion. We conclude that the systemic pre-BMT injection of KGF in recipients of allogeneic T cells up-regulates SP-A, which may contribute to the early antiinflammatory effects of KGF. The protective KGF-mediated SP-A production is abolished in mice given alloreactive T cells plus Cy.

Voltage-gated K(+)-channel activity in ovine pulmonary vasculature is developmentally regulated.

To examine mechanisms underlying developmental changes in pulmonary vascular tone, we tested the hypotheses that 1) maturation-related changes in the ability of the pulmonary vasculature to respond to hypoxia are intrinsic to the pulmonary artery (PA) smooth muscle cells (SMCs); 2) voltage-gated K(+) (K(v))-channel activity increases with maturation; and 3) O(2)-sensitive Kv2.1 channel expression and message increase with maturation. To confirm that maturational differences are intrinsic to PASMCs, we used fluorescence microscopy to study the effect of acute hypoxia on cytosolic Ca(2+) concentration ([Ca(2+)](i)) in SMCs isolated from adult and fetal PAs. Although PASMCs from both fetal and adult circulations were able to sense an acute decrease in O(2) tension, acute hypoxia induced a more rapid and greater change in [Ca(2+)](i) in magnitude in PASMCs from adult compared with fetal PAs. To determine developmental changes in K(v)-channel activity, the effects of the K(+)-channel antagonist 4-aminopyridine (4-AP) were studied on fetal and adult PASMC [Ca(2+)](i). 4-AP (1 mM) caused PASMC [Ca(2+)](i) to increase by 94 +/- 22% in the fetus and 303 +/- 46% in the adult. K(v)-channel expression and mRNA levels in distal pulmonary arteries from fetal, neonatal, and adult sheep were determined through the use of immunoblotting and semiquantitative RT-PCR. Both Kv2.1-channel protein and mRNA expression in distal pulmonary vasculature increased with maturation. We conclude that there are maturation-dependent changes in PASMC O(2) sensing that may render the adult PASMCs more responsive to acute hypoxia.

KGF pretreatment decreases B7 and granzyme B expression and hastens repair in lungs of mice after allogeneic BMT.

We investigated keratinocyte growth factor (KGF) as a pretreatment therapy for idiopathic pneumonia syndrome (IPS) generated as a result of lung damage and allogeneic T cell-dependent inflammatory events occurring in the early peri-bone marrow (BM) transplant (BMT) period. B10.BR (H2(k)) recipient mice were transplanted with C57BL/6 (H2(b)) BM with spleen cells after lethal irradiation with and without cyclophosphamide conditioning with and without subcutaneous KGF pretreatment. KGF-pretreated mice had fewer injured alveolar type II (ATII) cells at the time of BMT and exhibited ATII cell hyperplasia at day 3 post-BMT. The composition of infiltrating cells on day 7 post-BMT was not altered by KGF pretreatment, but the frequencies of cells expressing the T-cell costimulatory molecules B7.1 and B7.2 and mRNA for the cytolysin granzyme B (usually increased in IPS) were decreased by KGF. Sera from KGF-treated mice had increases in the Th2 cytokines interleukin (IL)-4, IL-6, and IL-13 4 days after cessation of KGF administration (i.e., at the time of BMT). These data suggest that KGF hinders IPS by two modes: 1) stimulation of alveolar epithelialization and 2) attenuation of immune-mediated injury as a consequence of failure to upregulate cytolytic molecules and B7 ligand expression and the induction of anti-inflammatory Th2 cytokines in situ.

Interactions of keratinocyte growth factor with a nitrating species after marrow transplantation in mice.

We reported that allogeneic T cells given to irradiated mice at the time of marrow transplantation stimulated tumor necrosis factor (TNF)-alpha, interferon (IFN)-gamma, and nitric oxide (. NO) production in the lung, and the addition of cyclophosphamide (known to stimulate superoxide production) favored the generation of a nitrating species. Although keratinocyte growth factor (KGF) prevents experimental lung injury by promoting epithelial repair, its effects on the production of inflammatory mediators has not been studied. KGF given before transplantation inhibited the T cell-induced increase in bronchoalveolar lavage fluid protein, TNF-alpha, IFN-gamma, and nitrite levels measured on day 7 after transplantation without modifying cellular infiltration or proinflammatory cytokines and inducible. NO synthase mRNA. KGF also suppressed. NO production by alveolar macrophages obtained from mice injected with T cells. In contrast, the same schedule of KGF failed to prevent permeability edema or suppress TNF-alpha, IFN-gamma, and. NO production in mice injected with both T cells and cyclophosphamide. Because only epithelial cells respond to KGF, these data are consistent with the production of an epithelial cell-derived mediator capable of downregulating macrophage function. However, the presence of a nitrating agent impairs KGF-derived responses.

Nitric oxide inhibits heterologous CFTR expression in polarized epithelial cells.

Nitric oxide (. NO) has been implicated in a wide range of autocrine and paracrine signaling mechanisms. Herein, we assessed the role of exogenous. NO in the modulation of heterologous gene expression in polarized kidney epithelial cells (LLC-PK(1)) that were stably transduced with a cDNA encoding human wild-type cystic fibrosis transmembrane conductance regulator (CFTR) under the control of a heavy metal-sensitive metallothionein promoter (LLC-PK(1)-WTCFTR). Exposure of these cells to 125 microM DETA NONOate at 37 degrees C for 24 h (a chemical. NO donor) diminished Zn(2+)-induced and uninduced CFTR protein levels by 43.3 +/- 5.1 and 34.4 +/- 17.1% from their corresponding control values, respectively. These changes did not occur if red blood cells, effective scavengers of. NO, were added to the medium. Exposure to. NO did not alter lactate dehydrogenase release in the medium or the extent of apoptosis. Coculturing LLC-PK(1)-WTCFTR cells with murine fibroblasts that were stably transduced with the human inducible. NO synthase cDNA gene also inhibited CFTR protein expression in a manner that was antagonized by 1 mM N(G)-monomethyl-L-arginine in the medium. Pretreatment of LLC-PK(1)-WTCFTR with ODQ, an inhibitor of guanylyl cyclase, did not affect the ability of. NO to inhibit heterologous CFTR expression; furthermore, 8-bromo-cGMP had no effect on heterologous CFTR expression. These data indicate that. NO impairs the heterologous expression of CFTR in epithelial cells at the protein level via cGMP-independent mechanisms.

High levels of peroxynitrite are generated in the lungs of irradiated mice given cyclophosphamide and allogeneic T cells. A potential mechanism of injury after marrow transplantation.

In a murine bone-marrow transplant (BMT) model designed to determine risk factors for lung dysfunction in irradiated mice, we reported that cyclophosphamide (Cy)-induced injury and lethality depended on the infusion of donor spleen T cells. In the study reported here, we hypothesized that alveolar macrophage (AM)-derived reactive oxygen/nitrogen species are associated with lung dysfunction caused by allogeneic T cells, which stimulate nitric oxide (.NO) production, and by Cy, which stimulates superoxide production.NO reacts with superoxide to form peroxynitrite, a tissue-damaging oxidant. On Day 7 after allogeneic BMT, bronchoalveolar lavage fluid (BALF) obtained from mice injected with T cells contained increased levels of nitrite, which was associated with increased lactate dehydrogenase and protein levels, both of which are indices of lung injury. The injury was most severe in mice receiving both T cells and Cy. Messenger RNA (mRNA) for inducible nitric oxide synthase was detected only in murine lungs injected with T cells +/- Cy. AMs obtained on Day 7 after BMT from mice receiving T cells +/- Cy spontaneously generated between 20 and 40 microM nitrite in culture, versus < 2 microM generated by macrophages obtained from mice undergoing BMT but not receiving T cells. The level of 3-nitrotyrosine, the stable byproduct of the reaction of peroxynitrite with tyrosine residues, was increased in the BALF proteins of mice injected with both T cells and Cy. We conclude that allogeneic T cells stimulate macrophage-derived.NO, and that the addition of Cy favors peroxynitrite formation. Peroxynitrite generation clarifies the dependence of Cy-induced lung injury and lethality on the presence of allogeneic T cells.

Modulation of adenovirus-mediated gene transfer by nitric oxide.

We assessed the role of .NO in recombinant adenovirus-mediated gene transfer both in vitro and in vivo. NIH3T3 fibroblasts, stably transfected with the human inducible nitric oxide synthase, but lacking tetrahydrobiopterin (NIH3T3/iNOS [inducibile nitric oxide synthase]), were infected with replication-deficient adenovirus (E1-deleted), containing either the luciferase or the Lac Z reporter genes (AdCMV-Luc and AdCMV-Lac Z; 1-10 plaque forming units [pfu]/cell). Incubation of infected cells with sepiapterin (50 microM), a precursor of tetrahydrobiopterin, progressively increased nitrate/nitrite levels in the medium and decreased both luciferase and beta-galactosidase protein expression to approximately 60% of their corresponding control values, 24 h later. NIH3T3/iNOS cells had normal ATP (adenosine 5'-triphosphate) levels and did not release LDH(lactic dehydrogenase) into the medium. Pretreatment of these cells with N(G)-monomethyl-L-arginine (L-NMMA; 1 mM), an inhibitor of iNOS, prevented the sepiapterin-mediated induction of .NO and restored gene transfer to baseline values. Incubation of NIH3T3/iNOS with 8-bromo-cGMP (400 microM) in the absence of sepiapterin, or exposure of AdCMV-Luc to large concentrations of .NO, did not alter the efficacy of gene transfer. .NO produced by NIH3T3/iNOS cells also suppressed beta-galactosidase expression in NIH3T3 cocultured cells stably transfected with beta-galactosidase gene, suggesting .NO inhibited gene expression at either the transriptional or posttranscriptional levels. To investigate the effects of inhaled .NO on gene transfer in vivo, CD1 mice received an intratracheal instillation of AdCMV-Luc (4 x 10(9) pfu in 80 microl of saline) and exposed to .NO (25 ppm in room air) for 72 h. At that time, no significant degree of lung inflammation was detected by histological examination. However, lung luciferase activity decreased by 53% as compared with air breathing controls (P < 0.05; n > or = 8). We concluded that overproduction of .NO decreases the efficiency of adenovirus-mediated gene transfer in lung cells in the absence of cytotoxicity or inflammation.

Inhibition of surfactant function by copper-zinc superoxide dismutase (CuZn-SOD).

The efficacy of antioxidant enzymes to limit oxidant lung injury by instillation with surfactant mixtures in preterm infants with hyaline membrane disease is under investigation. However, there is concern that instillation of proteins in the alveolar space may inactivate pulmonary surfactant. We studied the effects of bovine copper-zinc superoxide dismutase (CuZn-SOD) on the biophysical properties of two distinct surfactant preparations. Incubation of calf lung surfactant extract (CLSE, 1 mg phospholipid/ml) and Exosurf (0.1 mg phospholipid/ml) with CuZn-SOD (1-10 mg/ml) prevented the fall of surface tension at minimal bubble radius (Tmin) to low values with dynamic compression in a pulsating bubble surfactometer. CuZn-SOD also enhanced the sensitivity to inactivation by albumin, normal human serum, and after treatment with peroxynitrite. The inhibitory effects of CuZn-SOD on CLSE, but not Exosurf, were abolished at high lipid concentrations (3 mg/ml) and after the addition of human surfactant protein A (by weight). We conclude that CuZn-SOD may interfere with the surface activity of surfactant mixtures, leading to decreased effectiveness of surfactant replacement therapy.

Nitration of surfactant protein A (SP-A) tyrosine residues results in decreased mannose binding ability.

Nitric oxide (.NO) is a signal transducing free radical which can modify oxidant stress by limiting superoxide (O2.-)-mediated injury. However, the product of .NO reaction with O2.-, peroxynitrite (ONOO-), is a potent oxidizing and nitrating agent. Exposure of a mixture containing phosphatidylcholine liposomes and surfactant apoprotein A (SP-A; 10% by weight) to increasing concentrations of .NO, generated by spermine NONOate, and constant O2.- levels, produced by the action of xanthine oxidase on lumazine, suppressed O2.(-)-induced lipid peroxidation in the presence of Fe3(+)- EDTA. On the other hand, an increase in the .NO/O2.- value resulted in nitration of SP-A tyrosine residues, located in the carbohydrate recognition domain (CRD), and decreased the ability of SP-A to aggregate lipids and bind mannose, two functions that require an intact CRD. SP-A was also nitrated to a large extent following exposure to 3-morpholinosydnonimine (SIN-1) or tetranitromethane at pH 8. In each case, increased nitrotyrosine content correlated in a monotonic fashion with inhibition of lipid aggregation and mannose binding, correlated with the extent of functional inhibition. Superoxide dismutase (2400 U/ml) and urate (100 microM; nonspecific scavenger of both ONOO- and hydroxyl radical), but not mannitol (50 mM; hydroxyl radical scavenger), prevented the SIN-1-induced injury to SP-A. In contrast, spermine NON-Oate or xanthine oxidase plus lumazine alone neither inhibited SP-A function nor nitrated the protein. These results indicate that at high concentrations, .NO inhibit O2.-induced lipid peroxidation. However, ONOO., formed by the reaction of .NO and O2.-, nitrates SP-A leading to decreased ability to aggregate lipids and bind mannose.

Inhibition of alveolar type II cell ATP and surfactant synthesis by nitric oxide.

Alveolar type II (ATII) cells, are often exposed to increased concentration of endogenous and exogenous nitric oxide (.NO). Exposure of freshly isolated rat ATII cells for 2 h to 1-3 microM .NO, generated by S-nitroso-N-penicillamine (SNAP), spermine NONOate, or 3-morpholino-sydnonimine (SIN-1) in the presence of superoxide dismutase, resulted in approximately 60% decrease in the rate of surfactant synthesis, as measured by the rate of incorporation of [methyl-3H]choline into phosphatidylcholine, and 60-80% inhibition of cellular ATP levels, as determined by bioluminescence. Similar results were obtained after incubation of ATII cells with authentic peroxynitrite (0.5 mM) but not SIN-1, a putative generator of peroxynitrite. Addition into the medium of oxyhemoglobin (20 microM), which scavenged .NO, or enhancement of ATII glutathione levels by preincubation with glutathione ester (5 mM) totally prevented the NONOate (100 microM) inhibition of cellular ATP. In contrast to the in vitro findings, normal levels of ATP and lipid synthesis were measured in ATII cells isolated from the lungs of rats that breathed .NO gas (80 ppm) in 21% O2 for 2 h (n = 4). This lack of effect may be due either to the presence of various antioxidants (such as glutathione) in the epithelial lining fluid or to the relatively low concentrations of .NO reaching the alveolar epithelium. We conclude that .NO and peroxynitrite, at concentrations likely to be encountered in vivo during inflammation, decrease ATII cell energy stores and surfactant synthesis, which may lead to derangement of important physiological functions.

Inhaled nitric oxide injures the pulmonary surfactant system of lambs in vivo.

Nitric oxide (.NO) is a free radical, and as such may damage the pulmonary surfactant system. To determine the potential toxicity of .NO in vivo, we exposed 35 newborn lambs to 0, 20, 80 or 200 ppm .NO in either 21 or 60% O2 for 6 h. At the end of the exposure, lambs had normal values of arterial Po2, Pco2, and pH; total protein concentration in the bronchoalveolar lavage was also at normal levels. There were no differences in the surface properties of surfactant among the air or 60% O2 groups. Pulmonary surfactant samples, isolated from the bronchoalveolar lavage of lambs breathing air or 20 ppm .NO and reconstituted at a lipid concentration of 3 mg/ml, reached a low minimum surface tension (Tmin < 3 mN/m) in a pulsating bubble surfactometer. On the other hand, abnormal surface properties were observed in 36 and 60% of surfactant samples isolated from lungs of lambs that breathed 80 or 200 ppm .NO, respectively. These findings were confirmed using a captive bubble surfactometer. Surfactant protein A, isolated from the lungs of lambs that breathed 200 ppm .NO, exhibited decreased ability to aggregate lipids in vitro. These data are consistent with injury to the surfactant apoproteins during inhalation of either 80 or 200 ppm .NO for 6 h.

Nitration of surfactant protein A results in decreased ability to aggregate lipids.

We assessed the extent to which nitration of surfactant protein (SP) A, isolated from the bronchoalveolar lavage of patients with alveolar proteinosis, alters its ability to enhance lipid aggregation, bind lipids, and act synergistically with surfactant apoproteins B and C (SP-B, SP-C) in lowering the surface activity of surfactant lipids. SP-A was treated with various concentrations of tetranitromethane (TNM) at pH 6, 7.4, 8, or 10. Depending on the pH, TNM acts either as a nitrating (pH > or = 7.4) or an oxidizing agent (pH < or = 6). Exposure of SP-A to TNM (0.1-1 mM) at pH 7.4 or 8 for 30 min resulted in dose-and pH-dependent increases in nitrotyrosine, detected by Western blotting, enzyme-linked immunosorbent assay, and direct amino acid analysis. Treatment of SP-A with 0.5 mM TNM decreased its ability to aggregate lipids by 30% at pH 7.4, and 90% at pH 8, but had no effect on the disulfide-dependent oligomeric state of SP-A. In contrast, SP-A exposed to 1 mM TNM at pH 6 had background levels of nitrotyrosine and exhibited normal lipid aggregation properties. TNM, but not a hydroxyl radical-generating system, resulted in a pH-dependent loss of SP-A fluorescence, suggesting that tryptophan also may have been nitrated. Nitration of SP-A did not affect its ability to bind lipids. In addition, SP-A (1-3% by weight), treated with 0.25-0.5 mM TNM at pH 8, restored the surface-active properties of calf lung surfactant extract, previously damaged by exposure to peroxynitrite. We conclude that tyrosine nitration selectively inhibits the SP-A-mediated lipid aggregation without affecting its ability to bind lipids.

Quantitation of nitrotyrosine levels in lung sections of patients and animals with acute lung injury.

Activated alveolar macrophages and epithelial type II cells release both nitric oxide and superoxide which react at near diffusion-limited rate (6.7 x 10(9) M-1s-1) to form peroxynitrite, a potent oxidant capable of damaging the alveolar epithelium and pulmonary surfactant. Peroxynitrite, but not nitric oxide or superoxide, readily nitrates phenolic rings including tyrosine. We quantified the presence of nitrotyrosine in the lungs of patients with the adult respiratory distress syndrome (ARDS) and in the lungs of rats exposed to hyperoxia (100% O2 for 60 h) using quantitative immunofluorescence. Fresh frozen or paraffin-embedded lung sections were incubated with a polyclonal antibody to nitrotyrosine, followed by goat anti-rabbit IgG coupled to rhodamine. Sections from patients with ARDS (n = 5), or from rats exposed to hyperoxia (n = 4), exhibited a twofold increase of specific binding over controls. This binding was blocked by the addition of an excess amount of nitrotyrosine and was absent when the nitrotyrosine antibody was replaced with nonimmune IgG. In additional experiments we demonstrated nitrotyrosine formation in rat lung sections incubated in vitro with peroxynitrite, but not nitric oxide or reactive oxygen species. These data suggest that toxic levels of peroxynitrite may be formed in the lungs of patients with acute lung injury.

Concurrent generation of nitric oxide and superoxide damages surfactant protein A.

The conditions under which nitric oxide (.NO) may modulate or promote lung injury have not been identified. We hypothesized that .NO-induced injury results from peroxynitrite, formed by the reaction of .NO with superoxide. The simultaneous generation of .NO and superoxide by 3-morpholinosydnonimine (SIN-1, 0.1-2 mM) resulted in oxidation of dihydrorhodamine, a marker of peroxynitrite production, and a dose-dependent decrease in the ability of SP-A to enhance lipid aggregation. Western blot analysis of SIN-1 exposed SP-A samples, overlaid with a polyclonal antibody against nitrotyrosine, were consistent with nitration of SP-A tyrosine residues. Superoxide dismutase (100 U/ml), L-cysteine (5 mM), xanthine oxidase (10 mU/ml) and xanthine (500 microM), or urate (100 microM) prevented the SIN-1-induced dihydrorhodamine oxidation and injury to SP-A. .NO alone, generated by S-nitroso-N-acetylpenicillamine plus 100 microM L-cysteine, or superoxide and hydrogen peroxide, generated by pterin and xanthine oxidase in the absence of iron, did not damage SP-A or oxidize dihydrorhodamine. We concluded that peroxynitrite, but not .NO or superoxide and hydrogen peroxide, in concentrations likely to be encountered in vivo, caused nitrotyrosine formation and decreased the ability of SP-A to aggregate lipids.