Myeloperoxidase deficiency enhances inflammation ...

Am J Physiol Lung Cell Mol Physiol 287: L706 –L714, 2004. First published March 12, 2004; 10.1152/ajplung.00015.2004.

Myeloperoxidase deficiency enhances inflammation after allogeneic marrow transplantation Carlos Milla,1 Shuxia Yang,1 David N. Cornfield,1,2 Marie-Luise Brennan,3 Stanley L. Hazen,4 Angela Panoskaltsis-Mortari,1,2 Bruce R. Blazar,2 and Imad Y. Haddad1,2 1

Division of Pulmonary and Critical Care, Department of Pediatrics, and 2Division of Bone Marrow Transplantation and Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455; 3Department of Cell Biology and 4Department of Cardiovascular Medicine, Cleveland Clinic Foundation, Cleveland, Ohio 44195

Submitted 20 January 2004; accepted in final form 8 March 2004

BONE MARROW TRANSPLANTATION (BMT) is a widely accepted therapeutic modality for a number of malignant, hematologic, immunologic, and genetic diseases. The success of BMT is often compromised by the development of noninfectious diffuse lung injury termed idiopathic pneumonia syndrome (IPS) (9). IPS occurs in 12–20% of all allogeneic BMT recipients, with mortality rate in excess of 50% (19). Human and animal studies have established that IPS injury is the result of severe immune responses and is exacerbated by conditioning regimens (radiochemotherapy) (10, 11, 35). Recent evidence implicates, in this process, the generation of large amounts of reactive oxygen/nitrogen species and the depletion of antioxidant potential during the course of irradiation, conditioning

drugs, and allogeneity (2, 24, 38). Bhalla and Folz (1) have shown that treatment of recipient mice with N-acetylcysteine, which repletes glutathione stores, attenuates chemotherapyinduced lipid peroxidation and suppresses lung dysfunction after BMT. In rodents, nitric oxide production contributes to the pathophysiology of graft-vs.-host disease (GVHD), which affects the skin, liver, gastrointestinal tract, and the lung (16). In humans, serum nitrite, the stable byproduct of nitric oxide, and urinary F2-isoprostane, an indicator of in vivo lipid peroxidation, correlate with the severity of GVHD and IPS (3, 44). In our established IPS model in irradiated mice, we have shown that lung injury is mainly caused by donor T cells and host macrophages/monocytes and is potentiated by conditioning drugs (35). Lung injury was associated with the generation of large amounts of reactive oxygen and nitrogen species, depletion of reduced glutathione, and detection of high levels of nitrated proteins (24, 28). In addition, we have shown that oxidant-induced lipid peroxidation in the lung correlated with IPS severity in murine BMT recipients (50). Oxidant/antioxidant imbalance, also referred to as oxidative stress, promotes immune responses, including activation of T cells (26, 39). The oxidizing environment enhances the activation and translocation of nuclear factor (NF)-␬B and increases the production of proinflammatory cytokines such as TNF-␣ (29). However, oxidative stress is a potent inducer of programmed cell death or apoptosis (14) and may control inflammation by increasing oxidant-mediated elimination of activated T cells and macrophages (43). The effects of suppressed oxidative stress on donor T cell-dependent inflammation and lung injury after transplantation have not been fully elucidated. MPO present in neutrophils and to a lesser extent in monocytes and macrophages catalyzes the reaction between hydrogen peroxide (H2O2) derived by phagocytic respiratory burst and chloride to yield hypochlorous acid (HOCl), a potent oxidant ⬃100 times more reactive than H2O2 (22). HOCl is a component of the innate host defense against bacterial infections. Indeed, MPO-deficient (MPO⫺/⫺) mice exhibit increased mortality in a polymicrobial sepsis model (21). However, excessive generation of MPO-derived oxidants inactivates proteins, oxidizes lipids, and damages DNA. MPO-derived oxidative stress is implicated in pathogenesis of lung injury in cystic fibrosis (42), the acute respiratory distress syndrome (33), and lung allograft rejection (31). MPO also facilitates nitration reactions (7, 20) that contribute to lung dysfunction after BMT (23).

Address for reprint requests and other correspondence: I. Y. Haddad, Univ. of Minnesota, Dept. of Pediatrics, 420 Delaware St. SE, Minneapolis, MN 55455 (E-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

nitrotyrosine; apoptosis; alveolar type II cells; idiopathic pneumonia syndrome

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Milla, Carlos, Shuxia Yang, David N. Cornfield, Marie-Luise Brennan, Stanley L. Hazen, Angela Panoskaltsis-Mortari, Bruce R. Blazar, and Imad Y. Haddad. Myeloperoxidase deficiency enhances inflammation after allogeneic marrow transplantation. Am J Physiol Lung Cell Mol Physiol 287: L706 –L714, 2004. First published March 12, 2004; 10.1152/ajplung.00015.2004.—Myeloperoxidase (MPO)-derived oxidants participate in the respiratory antimicrobial defense system but are also implicated in oxidant-mediated acute lung injury. We hypothesized that MPO contributes to lung injury commonly observed after bone marrow transplantation (BMT). MPO-sufficient (MPO⫹/⫹) and -deficient (MPO⫺/⫺) mice were given cyclophosphamide and lethally irradiated followed by infusion of inflammation-inducing donor spleen T cells at time of BMT. Despite suppressed generation of nitrative stress, MPO⫺/⫺ recipient mice unexpectedly exhibited accelerated weight loss and increased markers of lung dysfunction compared with MPO⫹/⫹ mice. The increased lung injury during MPO deficiency was a result of donor T cell-dependent inflammatory responses because bronchoalveolar lavage fluids (BALF) from MPO⫺/⫺ mice contained increased numbers of inflammatory cells and higher levels of the proinflammatory cytokine TNF-␣ and the monocyte chemoattractant protein-1 compared with wild-type mice. Enhanced inflammation in MPO⫺/⫺ mice was associated with suppressed apoptosis of BALF inflammatory cells. The inflammatory process in MPO⫺/⫺ recipients was also associated with enhanced necrosis of freshly isolated alveolar type II cells, critical for preventing capillary leak. We conclude that suppressed MPO-derived oxidative/nitrative stress is associated with enhanced lung inflammation and persistent alveolar epithelial injury.

INCREASED T CELL-DEPENDENT INFLAMMATION IN MPO⫺/⫺ MICE

The role of MPO in the pathogenesis of IPS has not been investigated. We hypothesized that the absence of MPO will attenuate lung dysfunction after allogeneic BMT. Our results show, however, that despite suppression of overall nitrative stress, MPO⫺/⫺ mice exhibit enhanced inflammation in the periBMT period that was associated with persistent lung dysfunction. Data indicate that a potential reason for enhanced inflammation in the absence of MPO-derived oxidants is suppressed apoptosis of lung-infiltrating inflammatory cells. MATERIALS AND METHODS

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Bronchoalveolar lavage. Mice were killed on day 7 post-BMT after an intraperitoneal injection of pentobarbital sodium, and the thoracic cavity was partially dissected. The trachea was cannulated with a 20-gauge angiocatheter and infused with 1 ml of ice-cold sterile PBS and withdrawn. This was repeated several times, and the bronchoalveolar lavage fluid (BALF) was immediately centrifuged at 500 g for 10 min at 4°C to pellet cells. The initial 1.5 ml of BALF was used for biochemical analysis and surfactant function, and the remaining fluid was used to increase the yield of recovered cells. BALF and blood cell count were determined using a Coulter Counter (model ZF; Coulter, Miami, FL) after lysis of red blood cells by Zap-Oglobin II lytic reagent (Coulter). BALF biochemical analysis. Individual mouse cell-free BALF levels of TNF-␣, monocyte chemoattractant protein (MCP)-1, IFN-␥, and IL-10 were determined by sandwich ELISA using murine-specific commercial kits (sensitivity 1.5–3 pg/ml; R&D Systems, Minneapolis, MN). Nitrite in BALF was measured according to the Greiss method after the conversion of nitrate to nitrite with the NADHdependent enzyme nitrate reductase (Calbiochem, La Jolla, CA). BALF total protein was determined by the bicinchoninic acid (Sigma, St. Louis, MO) method with bovine serum albumin (BSA) as the standard. Peroxidase activity in macrophages/monocytes. BALF cell pellets from each group of mice were combined, and cell differential was determined in samples cytospun onto glass slides and stained with Wright-Giemsa. Total BALF cells/well (2 ⫻ 105) were added to flat-bottom 96-well microtiter plates, and macrophages/monocytes were allowed to adhere for 1 h at 37°C in 5% CO2 air, followed by removal of unbound cells. Peroxidase activity by adherent macrophages/monocytes was assessed by addition of 100 ␮l of tetramethyl benzidine (TMB peroxidase substrate solution containing 0.01% H2O2; Sigma) for 1 h at 23°C. Substrate color reaction was stopped by the addition of stop solution to the microwell plate. Absorbance of the yellow color representing oxidized TMB was measured at 450 nm. If peroxidases were present, H2O2 would decompose at the expense of an electron donor to generate, in the presence of chloride, the potent oxidant HOCl, which can oxidize TMB (4). Nitrative stress in BALF cells. For nitrotyrosine staining, cytospun BALF cells were permeabilized and fixed with methanol at ⫺20°C for 7 min. Endogenous peroxidase activity was quenched by treatment with 0.3% H2O2 in cold methanol for 30 min followed by three washes with PBS. Nonspecific binding was blocked with 10% goat serum for 30 min. The primary antibody, polyclonal rabbit antinitrotyrosine antibody (NTAb; Upstate Biotechnology, Lake Placid, NY), at 0.01 mg/ml in 10% goat serum and 2% BSA in PBS, was applied to the cells for 30 min. Control measurements included rabbit IgG (Upstate Biotechnology) and NTAb in the presence of excess nitrotyrosine (10 mM; NT block). To visualize specific NTAb binding, cells were incubated with secondary antibody, goat anti-rabbit IgG conjugated with horseradish peroxidase (1:500 dilution), followed by the addition of 3,3⬘-diaminobenzidine (Vector Laboratories) chromogenic substrate. The sections were counterstained with hematoxylin, dehydrated, overlaid with Permount (Sigma), and sealed with coverslips. All slides were exposed to the primary/secondary antibodies and color development solutions for the same length of time. Surfactant function in BALF using capillary surfactometer. The ability of pulmonary surfactant contained in cell-free BALF to prevent airway closure was evaluated with a glass capillary simulating a terminal conducting airway as previously described (17). Surfactant in the BALF was concentrated 10⫻ by centrifugation at 40,000 g for 1 h at 4°C. A volume of supernatant (90%) of the centrifuged liquid was removed from the test tube, and the remaining 10%, containing pelleted surfactant, was vortexed before analysis using the capillary surfactometer (Calmia Medical, Toronto, Ontario, Canada). Concentrated BALF (0.5 ␮l) was loaded into the narrow section of the capillary (0.25-mm internal diameter), and its ability to maintain

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Mice. B10.BR (H2k) and C57BL/6J (termed B6; H2b) were purchased from Jackson Laboratories (Bar Harbor, ME). MPO⫺/⫺ mice were generated by targeted disruption of the MPO gene as previously described (6) and backcrossed ⬎10 generations to B6 background. Mice were housed in microisolator cages in the specific pathogen-free (SPF) facility of the University of Minnesota and cared for according to the Research Animal Resources guidelines of our institution. In SPF units, MPO⫺/⫺ mice breed and develop normally with no evidence of bacterial infections. For BMT, donors were 6 – 8 wk of age and recipients were used at 8 –12 wk of age. Sentinel mice were found to be negative for 15 known murine viruses, including cytomegalovirus, K-virus, and pneumonia virus of mice. Pre-BMT conditioning. B6 wild-type or MPO⫺/⫺ mice received intraperitoneal injection of cyclophosphamide (Cytoxan; Bristol Myers Squibb, Seattle, WA) at 120 mg 䡠 kg⫺1 䡠 day⫺1 on days ⫺3 and ⫺2 pre-BMT. All mice were lethally total body irradiated (TBI) 1 day before BMT by X-ray (7.5 Gy) at a dose rate of 0.41 Gy/min (47). BMT. Our BMT protocol has been previously described (46). Briefly, donor B10.BR bone marrow (BM) was T cell depleted (TCD) with antithymocyte 1.2 monoclonal antibody (clone 30-H-12, rat IgG2b, kindly provided by Dr. David Sachs, Massachusetts General Hospital, Boston, MA) plus complement (Neiffenegger, Woodland, CA). For each experiment, a total of 5–10 recipient mice per treatment group were transplanted via caudal vein with 20 ⫻ 106 B10.BR TCD BM cells with 15 ⫻ 106 spleen T cells as a source of GVHD/IPScausing T cells (BMS⫹Cy). Pulmonary function analysis. Pulmonary mechanics in pentobarbital-anesthetized ventilated mice on day 7 after BMT were measured in a cohort of MPO-sufficient (MPO⫹/⫹) and MPO⫺/⫺ mice following the method described by Diamond and O’Donnell (13), with slight modifications. In brief, after careful dissection of the neck, a short metal cannula was inserted into the trachea and secured with 3.0 silk. A polyethylene catheter was inserted orally into the lower third of the esophagus to estimate pleural pressure. The animal was then placed into a plethysmograph (model PLY3111; Buxco Electronics, Sharon, CT) and connected to a mouse ventilator (Harvard Apparatus, MarchHugstetten, Germany) set at a respiratory rate of 150 breaths/min and a tidal volume of 200 ␮l. Respiratory flow signal was measured through a flow transducer (Sen Sym SCXL004, Buxco Electronics) connected to the plethysmograph. Lung volume was obtained by electric integration of the flow signal. Intraesophageal and airway pressures were measured with a pressure transducer (Validyne DP45, Buxco Electronics) directly connected to their respective ports. These data were fed into a computer through a preamplifier (MaxII, Buxco Electronics), and the data were analyzed with the Biosystem XA software (Buxco Electronics). When the signal was stable, delivered tidal volume was varied from 350 to 100 ␮l in 50-␮l decrements, and for each delivered volume, the effective tidal volume, transpulmonary pressure, and dynamic compliance were measured. Volume-pressure plots were constructed for each treatment group. Body temperature was maintained at 37°C throughout the experiment.

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RESULTS

Lung dysfunction in MPO⫺/⫺ mice after allogeneic BMT. To determine the role of MPO in acute lung injury after allogeneic BMT, high-dose Cy/TBI-conditioned B6 wild-type and MPO⫺/⫺ mice were given inflammation-inducing B10.BR spleen T cells and killed on day 7 after BMT during time of peak lung injury (24). The means ⫾ SE pre-BMT body weights of MPO⫹/⫹ and MPO⫺/⫺ mice were 18.9 ⫾ 0.50 g and 19.32 ⫾ 0.72 g, respectively (P ⬎ 0.05). After allogeneic BMT, however, early weight loss was accelerated in MPO⫺/⫺ mice compared with MPO⫹/⫹ mice (Table 1). Lung dysfunction was assessed by 1) measurement of BALF total protein levels, 2) effectiveness of surfactant contained in BALF to maintain capillary patency, and 3) lung mechanics analysis in anesthetized-ventilated mice. BALF return volumes collected on day 7 after BMT were similar in all groups (⬎90% of instilled volume). BALF levels of total protein in untreated control MPO⫹/⫹ and MPO⫺/⫺ were not different (⬍0.2 mg/ml). However, BALF protein levels collected on day 7 after BMT were significantly higher in MPO⫺/⫺ compared with MPO⫹/⫹ mice (Table 1). Compared with surfactant in BALF of non-BMT MPO⫹/⫹ and MPO⫺/⫺ controls, the effectiveness of surfactant contained in day 7 after BMT pooled and concentrated BALF from MPO⫹/⫹ BMS⫹Cy mice to maintain capillary patency was significantly decreased. The magnitude of surfactant dysfunction was even more severe in BALF from MPO⫺/⫺ BMS⫹Cy mice compared with MPO⫹/⫹ BMS⫹Cy mice, although the difference was not statistically significant (Table 1). Lung compliance at tidal volume of 200 ␮l in non-BMT control MPO⫹/⫹ and MPO⫺/⫺ was similar (0.034 ⫾ 0.004 and 0.038 ⫾ 0.006 ml/cmH2O in MPO⫹/⫹ and MPO⫺/⫺ mice, respectively; n ⫽ 3, P ⬎ 0.05). After allogeneic BMT, compliance was significantly decreased in both MPO⫹/⫹ and MPO⫺/⫺ BMS⫹Cy mice (0.017 ⫾ 0.003 and 0.016 ⫾ 0.004 ml/cmH2O in MPO⫹/⫹ and MPO⫺/⫺ mice, respectively; n ⫽ 3; P ⬍ 0.05 compared with non-BMT mice). VolumeTable 1. Day 7 after BMT weight loss, BALF total protein, and BALF surfactant function Group

Weight Loss (% of Baseline Weight)

BALF Total Protein, mg/ml

Surfactant Function (% Open Time)

0 0

0.18⫾0.038 0.19⫾0.042

88.9⫾5.8 86.5⫾5.5

⫺21⫾1.7* ⫺26⫾1.9†

0.49⫾0.12* 0.81⫾0.07†

16.7⫾7.2* 9.6⫾4.3*

Control MPO⫹/⫹ MPO⫺/⫺ BMS⫹Cy MPO⫹/⫹ MPO⫺/⫺

Values of body weight and bronchoalveolar lavage fluid (BALF) total protein are means ⫾ SE for n ⫽ 25–35 mice/group obtained from 5 independent bone marrow transplantation (BMT) experiments. Values of % open time in capillary surfactometer are means ⫾ SE for pooled BALF samples from 2 mice/group per experiment from 2 independent experiments. B6 MPO⫹/⫹ and MPO⫺/⫺ mice were given cyclophosphamide (Cy), lethally irradiated, and infused with B10.BR bone marrow with inflammation-inducing donor spleen T cells (BMS⫹Cy). Control mice were nonirradiated and nontransplanted. Surfactant function was assessed in 10⫻ concentrated BALF samples by capillary surfactometer. *P ⬍ 0.05 compared with non-BMT control MPO⫹/⫹ mice. ⴱ†P ⬍ 0.05 comparing the effects of MPO deficiency in BMS⫹Cy BMT mice.

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airflow was measured in response to increased pressure at one end of the capillary. After initial extrusion of liquid, the percentage of the following 120 s that the recorded pressure equaled zero indicated the percentage of time that the capillary was open to free airflow. BALF with well-functioning surfactant will return to the narrow section less often and, therefore, the percentage of time the capillary remains open will be more than BALF containing injured or suppressed surfactant. Alveolar type II cell isolation. Alveolar type II (ATII) cells were isolated from anesthetized control and experimental mice according to methods described by Corti et al. (12). The abdominal cavity was opened, and mice were exsanguinated by severing the inferior vena cava and the left renal artery. The diaphragm was cut, and the chest plate and the thymus were removed. The trachea was cannulated with a 20-gauge catheter, and bronchoalveolar lavage was performed as described above. The lungs were then perfused via the pulmonary artery with 10 –20 ml of 0.9% normal saline using a 21-gauge needle fitted on a syringe. Three milliliters of dispase (BD Biosciences, Bedford, MA) were rapidly instilled through the cannula in the trachea. Lungs were removed from the animal and incubated in a 1-ml dispase for 45 min at 23°C. Lungs were then transferred to a 60-mm culture dish containing 7 ml of HEPES-buffered DMEM and 100 U/ml DNase I (Sigma). The lung tissue was gently teased from the airways and swirled for 5–10 min. The cell suspension was successively filtered through 100- and 40-␮m Falcon cell strainers and then through 20-␮m nylon mesh. Cells were collected by centrifugation at 130 g for 8 min at 4°C and placed on biotinylated anti-CD45 and biotinylated anti-CD16/CD32 precoated culture plates. After incubation for 2 h at 37°C, ATII cells were gently panned from the plate and collected by centrifugation at 130 g for 8 min. ATII cells were resuspended in PBS, washed 2⫻, and resuspended in PBS (1–2 ⫻ 106 cells/ml). Prosurfactant protein C staining. Freshly purified lung cells and BALF cells were stained with prosurfactant protein C (pro-SP-C) polyclonal antibody (Chemicon, Temecula, CA) to confirm ATII cell type. Pro-SP-C is specific to ATII cells and is present in abundance. Cells (2 ⫻ 105 cells/ml) were cytospun onto glass slides and fixed in methanol at ⫺20°C for 7 min. After blocking nonspecific sites with normal donkey serum, cells were incubated with pro-SP-C primary antibody (1:1,500) for 30 min at 23°C and rinsed 6⫻ with PBS. Controls included replacement of primary antibody with rabbit IgG. Cells were then incubated with fluorescein-conjugated donkey antirabbit secondary antibody (Jackson ImmunoResearch, West Grove, PA) 1:100 in PBS for 1 h at 23°C in the dark. Cells were stained with 4⬘,6-diamidino-2-phenyllindone dihydrochloride hydrate (Sigma) to show nuclei, rinsed briefly with PBS, and mounted with ProLong Antifade kit (Molecular Probes). Detection of apoptosis. Day 7 after BMT, BALF cells and freshly purified ATII from each group of mice were combined and diluted to a concentration of 1–2 ⫻ 106 cells/ml using annexin V binding buffer. Cells were double stained with annexin V-FITC (BD PharMingen, San Diego, CA) and propidium iodide (PI) following the manufacturer’s instructions. Annexin V recognizes phosphatidylserine on the outer surface of cell membranes. This translocation of phosphatidylserine from the inner to outer surface of cell membranes occurs during early/intermediate stages of apoptosis. Staining with PI was used to simultaneously monitor cell necrosis. Analysis of cell fluorescence intensity was determined by FACSCaliber flow cytometer (BD Biosciences, San Jose, CA) using CellQuest applications (BD Biosciences) with a total of 10,000 events counted. Values were reported as the percentage of positive events or mean fluorescence (arbitrary units). Statistical analysis. Results are expressed as means ⫾ SE. Data were analyzed by ANOVA or Student’s t-test. Statistical differences among group means were determined by Tukey’s Studentized test. P values ⱕ0.05 were considered statistically significant.


Fig. 1. Effective tidal volume-transpulmonary pressure plots in anesthetized and ventilated mice measured on day 7 after allogeneic bone marrow transplantation (BMT). Control (nonirradiated and nontransplanted, E) and total body irradiated myeloperoxidase (MPO)⫹/⫹ (Œ) or MPO⫺/⫺ (‚) mice given cyclophosphamide (Cy) and inflammation-inducing donor spleen T cells (BMS⫹Cy) were placed in a single-chamber plethysmograph and ventilated with delivered tidal volume of 100 –350 ␮l. Effective tidal volume was measured, and transpulmonary pressure was calculated using airway and intraesophogeal pressures. Body temperature was maintained at 37°C throughout the experiment. Markers shown represent 3 different mice in each experimental group. *P ⬍ 0.05 comparing the slopes of MPO⫹/⫹ (line with longer dashes) and MPO⫺/⫺ (line with shorter dashes) BMS⫹Cy mice with the slope of MPO⫹/⫹ non-BMT control mice (solid line).


Fig. 2. Decreased nitrotyrosine immunostaining in bronchoalveolar lavage fluid (BALF) cells from MPO⫺/⫺ mice after allogeneic transplantation. Cy/total body irradiated (TBI) recipient MPO⫺/⫺ and wild-type (MPO⫹/⫹) B6 mice were given B10.BR bone marrow plus donor spleen T cells (BMS⫹Cy). Day 7 after BMT, BALF cells from the indicated group of mice were centrifuged onto glass slides and incubated with nitrotyrosine antibody (NTAb) or NTAb in the presence of 10 mM nitrotyrosine (NT block). Nitrotyrosine was estimated based on the brown reaction product in the cell cytoplasm. Shown is a representative figure from pooled cells obtained from 5– 8 mice per group per experiment. Two separate experiments were performed.

MPO⫹/⫹ and MPO⫺/⫺ mice before and after BMT did not differ (Fig. 3A). Consistent with the higher number of BALF cells in MPO⫺/⫺ vs. MPO⫹/⫹ BMS⫹Cy mice, the levels of the chemoattractant MCP-1 and the proinflammatory cytokine TNF-␣ were higher in BALF from the former (Fig. 4). Also of note, IFN-␥ levels were modestly, but not significantly, higher in BALF of MPO⫺/⫺ vs. MPO⫹/⫹ mice. In contrast, BALF levels of nitrite plus nitrate, the stable byproducts of nitric oxide, were not significantly different in these groups of mice (Fig. 4). BALF levels of IL-10, an anti-inflammatory cytokine, were below detection limits by ELISA in all control and experimental mice (data not shown). Together, these results are consistent with exaggerated inflammation in MPO⫺/⫺ mice after allogeneic BMT. Suppressed apoptosis in BALF cells from MPO⫺/⫺ mice. We hypothesized that suppressed oxidative/nitrative stressdependent apoptosis of lung-infiltrating inflammatory cells may represent one mechanism for increased inflammation in MPO⫺/⫺ mice after allogeneic BMT. Apoptosis/necrosis of BALF cells obtained on day 7 after allogeneic BMT was determined by flow cytometry after double staining the cells with annexin V antibody and PI. Apoptosis in BALF cells from unmanipulated control MPO⫺/⫺ and MPO⫹/⫹ mice was not different (data not shown). Figure 5 shows increased apoptosis and cellular necrosis in BALF cells from B6 Cy/TBI donor T cell-recipient mice (BMS⫹Cy) compared with cells from B6 controls. Apoptosis and necrosis were less in BALF cells from BMS⫹Cy MPO⫺/⫺ vs. MPO⫹/⫹ mice, consistent with a major role of MPO-derived oxidants in the apoptosis of inflammatory cells after allogeneic BMT (mean fluorescence intensity of 827 ⫾ 55 in MPO⫹/⫹ BMS⫹Cy mice vs. 368 ⫾ 287 • OCTOBER 2004 •

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pressure plots for tidal volumes ranging from 100 to 350 ␮l demonstrated a rightward shift in MPO⫹/⫹ and MPO⫺/⫺ BMT mice compared with non-BMT control mice (Fig. 1), consistent with development of lung injury after BMT. Together, these results indicate increased or persistent markers of lung dysfunction in MPO-deficient mice after allogeneic BMT. Suppressed nitrative stress in BALF cells from MPO⫺/⫺ mice. In contrast to cells from MPO⫹/⫹ mice, alveolar macrophages and lung-infiltrating monocytes collected from BALF of MPO⫺/⫺ non-BMT and BMT recipients lacked peroxidase activity as assessed by addition of TMB (TMB peroxidase substrate solution containing 0.01% H2O2; data not shown). Intracellular nitrative stress by macrophages/monocytes was assessed by detection of antigenic sites related to nitrotyrosine. Nitration of monocytes/macrophages obtained from MPO⫺/⫺ vs. MPO⫹/⫹ Cy/TBI mice given donor spleen T cell (BMS⫹Cy) was decreased (Fig. 2). Nitration was specific since staining was completely blocked in the presence of excess antigen, 10 mM nitrotyrosine. Cells from non-BMT control mice and Cy/TBI MPO⫹/⫹ and MPO⫺/⫺ mice given bone marrow without donor T cells, a setting in which IPS injury is mild rather than severe, exhibited baseline levels of staining (data not shown). MPO⫺/⫺ mice exhibit increased inflammation after allogeneic BMT. To begin to understand reasons of increased lung dysfunction despite suppressed oxidative/nitrative stress in MPO⫺/⫺ mice, the severity of donor T cell-dependent inflammation was assessed on day 7 after transplantation in MPO⫺/⫺ and MPO⫹/⫹ mice. BALF collected from MPO⫺/⫺ recipient mice contained significantly more inflammatory cells (Fig. 3A), although the cell differential was not different as assessed by Wright-Giemsa stain of cytospun samples (Fig. 3B and Table 2). This increased BALF cellularity from MPO⫺/⫺ mice was not due to increased inflammatory cells in the blood, since total white blood cell count in

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Fig. 3. Enhanced cellularity in BALF, but not in circulation, of MPO⫺/⫺ mice after allogeneic BMT. Cell number in the first 1.5 ml of BALF collected from control (C; unmanipulated) and Cy/TBI donor spleen T cell-recipient MPO⫹/⫹ (filled bars) and MPO⫺/⫺ (open bars) mice (BMS⫹Cy). BALF was collected on day 7 after BMT. Total cell count in BALF before centrifugation to pellet cells and in whole blood was determined using a Coulter counter after lysis of red blood cells (A). B: cell differential assessed in Giemsa-Wright-stained cytospun BALF samples from MPO⫹/⫹ (left) BMS⫹Cy and MPO⫺/⫺ (right) BMS⫹Cy mice on day 7 after BMT were not different. Values are means ⫾ SE for 15–20 mice per group. *P ⬍ 0.05 compared with control (nonirradiated and nontransplanted). ⫹P ⬍ 0.05 comparing the effects of MPO deficiency in each group. WBC, white blood cells.

47 in MPO⫺/⫺ BMS⫹Cy mice; P ⬍ 0.05 from 2 separate experiments). ATII cells are an important target of IPS, and ATII cell death may represent a major mechanism of lung injury. Therefore, the apoptosis/necrosis of ATII cells freshly isolated on day 7

DISCUSSION

Table 2. Day 7 after BMT BALF cell differential in MPO⫹/⫹ and MPO⫺/⫺ Cell Differential Group

Control MPO⫹/⫹ MPO⫺/⫺ BMS⫹Cy MPO⫹/⫹ MPO⫺/⫺

after allogeneic BMT was also evaluated. ATII cells were identified by the presence of a precursor of SP-C using proSP-C antibody (Fig. 6). Pro-SP-C immunostaining was detected in ATII cells isolated from both control and experimental MPO⫹/⫹ and MPO⫺/⫺ mice and was absent in alveolar macrophages and lung-infiltrating monocytes (data not shown). Baseline apoptosis/necrosis of ATII cells from control MPO⫹/⫹ and MPO⫺/⫺ mice was not different. After exposure to Cy/TBI and allogeneity, ATII cells from MPO⫹/⫹ and MPO⫺/⫺ recipients exhibited increased apoptosis and necrosis (Fig. 7). Although the extent of apoptosis assessed by incorporation of annexin V in ATII cells from MPO⫹/⫹ and MPO⫺/⫺ BMS⫹Cy recipients was similar, ATII cells from MPO⫺/⫺ mice exhibited increased incorporation of PI, consistent with enhanced cellular necrosis on day 7 after allogeneic BMT during MPO deficiency (Fig. 7).

%Macrophages

%Lymphocytes

%Neutrophils

⬎95 ⬎95

⬍5 ⬍5

0 0

65⫾6 67⫾5

34⫾4 32⫾5

1 1

Values are means ⫾ SE for n ⫽ 4 – 6 mice/group obtained from 2 experiments. Cell differential of day 7 after BMT BALF cells were assessed in cytospun samples stained with Wright-Giemsa. B6 MPO⫹/⫹ and MPO⫺/⫺ mice were given Cy, lethally irradiated, and infused with B10.BR bone marrow with inflammation-inducing donor spleen T cells (BMS⫹Cy).


Results of this study show that the absence of MPO in the peri-BMT period causes persistent lung dysfunction associated with enhanced donor T cell-dependent inflammation. Manifestations of exuberant IPS injury persisted in MPO-deficient mice despite suppressed nitrative stress, supporting a dominant role of accelerated immune responses in the early lung injury after allogeneic BMT. The data indicate that MPO or MPOderived oxidants may represent an important homeostatic inflammatory control mechanism during lung injury after BMT. MPO has also been shown to modulate the course of nonpulmonary inflammatory diseases. Brennan and coworkers (5) reported that MPO-deficient mice are more susceptible to experimental autoimmune encephalitis (EAE), a T cell-dependent neuronal disease. Interestingly, heterozygotes (MPO⫹/⫺) 287 • OCTOBER 2004 •

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Fig. 4. Increased BALF levels of monocyte chemoattractant protein (MCP)-1 and TNF-␣ from MPO⫺/⫺ mice. Control (unmanipulated) and Cy/TBI recipient B6 MPO⫹/⫹ (filled bars) and MPO⫺/⫺ (open bars) mice were given 15 ⫻ 106 B10.BR donor spleen T cells (BMS⫹Cy) and BALF collected on day 7 after transplantation. TNF-␣, MCP-1, and IFN-␥ levels were measured by sandwich ELISA. Nitrite was determined by the Greiss reaction after converting nitrate to nitrite by nitrate reductase. Shown are mean values ⫾ SE for 10 –15 mice per group pooled from 3 separate experiments. *P ⬍ 0.05 vs. control. *⫹P ⬍ 0.05 comparing the effect of MPO deficiency within each BMT group.


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Fig. 5. Suppressed apoptosis of BALF cells from MPO⫺/⫺ mice after allogeneic BMT. BALF was collected from control (A; unmanipulated), on day 7 after BMT from Cy/TBI donor spleen T cell-recipient (BMS⫹Cy) MPO⫹/⫹ (B), and from MPO⫺/⫺ BMS⫹Cy mice (C). BALF cells from 2 mice per group were pooled and stained with annexin V (x-axis) and propidium iodide (PI) (y-axis). Two-color incorporation was assessed by flow cytometry. The bottom left quadrants indicate the unstained cells, the bottom right quadrants indicate the cells binding annexin V FITC (early apoptosis), the top left quadrants indicate the cells that were PI stained (cells undergoing necrosis), and the top right quadrants indicate the cells that were PI and annexin V positive (late apoptosis). Percentages of apoptotic cells are indicated in each quadrant. For each measurement, data from 10,000 events were collected with a FACSCalibur flow cytometer. Fluorescence signals were collected in relative fluorescence units. Events were analyzed with CellQuest software. Shown is a representative experiment that was performed twice.

closely resembled wild type (MPO⫹/⫹) with respect to the incidence of EAE, suggesting that a complete absence of MPO-derived oxidants is necessary for the increased incidence of EAE. Moreover, the proliferation rate of lymphocytes from immunized MPO⫺/⫺ mice was increased by 50% compared with wild-type mice. The cause of the accelerated EAE in MPO⫺/⫺ mice may be the absence of MPO-dependent inhibition of lymphocyte proliferation. Similarly, we reasoned that a potential cause of increased inflammation in MPO-deficient mice after allogeneic BMT is suppression of the MPO-induced cell death of lung-infiltrating inflammatory cells. Indeed, our results show that MPO deficiency suppressed the apoptosis of inflammatory cells contained in BALF, which may, at least in part, explain the increased number of T cells and monocytes and the high BALF levels of MCP-1 and TNF-␣. Consistent with our data, Tsurubuchi et al. (41) reported that phorbol myristate acetate-induced apoptosis of neutrophils from MPO⫺/⫺ mice was significantly slower than in normal neutrophils. On day 7 after allogeneic BMT, the generation of nitric oxide in the lung as assessed by BALF levels of nitrite plus nitrate was not modified by MPO deficiency. However, nitrated AJP-Lung Cell Mol Physiol • VOL

Fig. 6. Freshly isolated alveolar type II (ATII) cell staining using prosurfactant protein C (SP-C) antibody. Cells (2 ⫻ 105/ml) from control mice were cytospun onto glass slides and stained with nonspecific IgG (A) or pro-SP-C antibody (B) followed by fluorescein-conjugated secondary antibody; fluorescence was detected using a fluorescent microscope. Shown is a representative experiment that was reproduced twice.

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proteins were decreased in BALF cells obtained from MPO⫺/⫺ mice compared with wild-type recipients. These results are in agreement with a major role of MPO in nitration reactions in vivo and confirm that MPO significantly contributes to nitrotyrosine formation via the oxidation of nitrite to nitrogen dioxide, as previously shown (15, 20). Nitration of proteins can inhibit critical protein functions and may play a central and causative role in acute lung injury (25, 49). Yet, despite decreased detection of nitrated proteins in BALF cells from MPO-deficient mice, lung dysfunction assessed on day 7 after BMT was at least as severe as wild-type mice. Among possible explanations for persistent lung dysfunction during MPO deficiency after allogeneic BMT is sustained generation of inflammatory responses that can induce apoptotic/necrotic pathways in alveolar epithelial cells (30). Alloactivated T cells and TNF-␣, abundantly present in the lung of MPO⫺/⫺ recipient mice, can trigger death signals in alveolar epithelial cells (40, 45). We observed high levels of cell death in freshly isolated ATII cells from the lungs of MPO⫺/⫺ on day 7 after allogeneic BMT. Injury to these cells may disrupt epithelial barrier integrity and cause enhanced permeability edema that may result in decreased lung compliance and increased levels of total proteins in BALF of MPO⫺/⫺ recipient mice. The presence of proteins in the alveolar space may

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also explain the abnormal surfactant function observed via the capillary surfactometer in experimental transplanted mice compared with unmanipulated control mice (17). Enhanced inflammation during noninfectious inflammatory diseases is not limited to MPO deficiency but also observed in mice lacking phagocytic nicotinamide adenine dinucleotide phosphate oxidase (NADPH-oxidase or phox), a major source of reactive oxygen species (37). For example, NADPH-oxidase-deficient (phox⫺/⫺) mice exhibit exaggerated inflammatory responses to sterile antigens (36). In our murine IPS model, we observed exuberant pulmonary inflammation in irradiated phox⫺/⫺ mice compared with wild-type recipient mice (48). Exaggerated immune responses in phox⫺/⫺ mice were associated with suppression of oxidative/nitrative stress and impaired clearance of MCP-1 from the circulation (48). Together, these results are consistent with the notion that despite its well-documented damaging effects, oxidative stress may function as an inflammatory control mechanism by inactivation of proinflammatory chemokines and by promoting the apoptosis of inflammatory cells. Therefore, we speculate that during inflammatory lung diseases, an optimal level of oxidative stress may exist where oxidant-induced damage is minimal and oxidant-mediated inactivation of proinflammatory mediators and apoptosis are maximal. Inhibition of oxidative stress below this threshold level may impair oxidant-dependent elimination of inflammatory cells and exacerbate inflammation. Total inhibition of oxidant production may also be detrimental because of the important physiological roles of reactive oxygen species in regulating the redox state, which is critical for cell growth/differentiation (34). The challenge remains to accurately estimate the extent of oxidative stress required to limit inflammation without causing significant effector oxidant-induced injury. Humans with inherited disorders caused by defects in respiratory burst oxidase, termed chronic granulomatous disease (CGD), also develop severe noninfectious inflammatory granAJP-Lung Cell Mol Physiol • VOL

ulomas in lung, skin, and gastrointestinal tract (18) and exhibit severe inflammatory complications after allogeneic BMT (27). In addition, MPO levels are sixfold higher in humans than rodents, and although humans deficient in MPO are not at unusual risk of infection, they occasionally develop immunemediated noninfectious diseases, including pustular skin lesions and diabetes mellitus (32). Although the mechanisms of these immune complications during NADPH-oxidase and MPO deficiency are under investigation, our data support a role for oxidant-mediated apoptosis of lung-infiltrating T cells and monocytes in regulating the severity of inflammatory responses. Consistent with our results, Brown and coworkers (8) have shown that neutrophils isolated from CGD patients are more resistant to spontaneous apoptosis and produce less anti-inflammatory mediators, including prostaglandin D2 and IL-10. Levels of IL-10, however, were not upregulated in our IPS model in BALF collected on day 7 after BMT. In summary, we have shown that mice lacking MPO exhibit enhanced donor T cell-dependent inflammation after allogeneic transplantation. Experimental evidence indicates that one of the mechanisms of increased inflammation is suppressed apoptosis leading to accumulation of activated inflammatory cells that may generate high levels of proinflammatory mediators. As a result, lung dysfunction and injury to alveolar epithelium persist. Further experiments will be required to determine whether partial inhibition of oxidant stress will maintain an optimal level of oxidative/nitrative stress where oxidant-induced damage is minimal and oxidant-dependent elimination of inflammatory cells is maximal. These results may also clarify the reason for exuberant inflammation and immunemediated complications observed in CGD and MPO-deficient patients. Moreover, these findings may improve our current strategies for using antioxidants by avoiding extreme inhibition of reactive species to preserve their ability to limit inflammatory responses. 287 • OCTOBER 2004 •

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Fig. 7. Increased apoptosis/necrosis after allogeneic transplantation in freshly isolated ATII cells from MPO⫹/⫹ and MPO⫺/⫺ mice. ATII cells were isolated on day 7 after BMT from control MPO⫹/⫹ (A), control MPO⫺/⫺ (B), Cy/TBI donor spleen T cell-recipient (BMS⫹Cy) MPO⫹/⫹ (C), and MPO⫺/⫺ BMS⫹Cy mice (D). ATII cells from 2 mice per group were pooled and stained with annexin V (x-axis) and PI (y-axis). Two-color incorporation was assessed by flow cytometry. Percentage of necrotic cells (top left quadrants) were higher in ATII from BMS⫹Cy MPO⫺/⫺ compared with BMS⫹Cy MPO⫹/⫹ mice. For each measurement, data from 10,000 events were collected with a FACSCalibur flow cytometer. Events were analyzed with CellQuest software. Shown is a representative experiment that was performed twice.

INCREASED T CELL-DEPENDENT INFLAMMATION IN MPO⫺/⫺ MICE GRANTS This work was supported by National Heart, Lung, and Blood Institute Grants R01-HL-67334, HL-55209, and HL-62526.

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