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American Journal of Respiratory Cell and Molecular Biology. Vol. 27, pp. 297-305, 2002
© 2002 American Thoracic Society
DOI: 10.1165/rcmb.2002-0035OC

Surfactant Protein A Decreases Lung Injury and Mortality after Murine Marrow Transplantation

Shuxia Yang, Carlos Milla, Angela Panoskaltsis-Mortari, Samuel Hawgood, Bruce R. Blazar and Imad Y. Haddad

Department of Pediatrics, Divisions of Pulmonary and Critical Care, Bone Marrow Transplantation, and University of Minnesota Cancer Center, University of Minnesota, Minneapolis, Minnesota; and Department of Pediatrics, Cardiovascular Research Institute, University of California San Francisco, San Francisco, California

Address correspondence to: Imad Y. Haddad, M.D., Department of Pediatrics, University of Minnesota, 420 Delaware Street S.E., Minneapolis, MN 55455. E-mail: hadda003{at}tc.umn.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Surfactant protein A (SP-A), a collectin associated with surfactant lipids, can have immune modulatory effects. We hypothesized that exogenous and basal endogenous SP-A can function to suppress donor T-cell–dependent inflammation that occurs during the generation of idiopathic pneumonia syndrome after bone marrow transplantation (BMT). Wild-type and SP-A–deficient mice were conditioned with cyclophosphamide and lethal irradiation and then given allogeneic donor bone marrow plus inflammation-inducing spleen T cells. On Day 7 after BMT, bronchoalveolar lavage fluids from SP-A–deficient mice contained increased numbers of inflammatory cells and higher levels of proinflammatory mediators tumor necrosis factor-{alpha}, interferon-{gamma}, and nitric oxide than wild-type mice. Exaggerated inflammation in SP-A–deficient mice was associated with decreased dynamic lung compliance and increased donor T-cell–dependent mortality (P = 0.0007, n = 10). Nitrative stress in alveolar macrophages from SP-A-/--conditioned BMT recipients was higher than for SP-A+/+ mice. Similarly, mice treated with transtracheal human SP-A (50 µg), instilled on Day 4 after BMT during a time of in vivo donor T cell activation, exhibited decreased inflammation and improved early survival compared with buffer-instilled mice. We concluded that basal endogenous SP-A and enhanced alveolar SP-A level modulate donor T-cell–dependent immune responses and prolong survival after allogeneic BMT.

Abbreviations: Bronchoalveolar lavage fluid, BALF • bone marrow, BM • bone marrow supplemented with spleen cells, BMS • bone marrow transplant(ation), BMT • cyclophosphamide, Cy • dynamic compliance, Cdyn • graft-versus-host disease, GVHD • interferon, IFN • idiopathic pneumonia syndrome, IPS • lactic dehydrogenase, LDH • nitric oxide, NO • phosphate-buffered saline, PBS • surfactant protein-A, SP-A • T cell-depleted, TCD • tumor necrosis factor, TNF • total body irradiation, TBI


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The presence of pulmonary surfactant at the blood–gas interface is essential for the survival of air-breathing mammals. By reducing surface tension, surfactant decreases the work of breathing, maintains alveoli open at end of expiration, and keeps alveoli dry. Surfactant is composed of a complex mixture of lipids and at least four surfactant proteins (SP), named in the order of discovery. SP-A was first identified as a component of surfactant in 1972 by King and Clements (1). SP-A coisolates with surfactant; therefore, studies have focused on its ability to regulate extracellular surfactant homeostasis. Indeed, in vitro studies have shown that SP-A acts synergistically with the hydrophobic proteins SP-B and SP-C to facilitate the adsorption of surfactant lipids (2). In addition, SP-A binds to high-affinity receptors on alveolar type II pneumocytes to enhance uptake and inhibit surfactant secretion (3, 4). Mature SP-A is a rosette-like octadecamer with a molecular mass of ~ 650 kD, and it is unclear how such a large molecule can promote surfactant adsorption to the air–liquid interface. Furthermore, studies utilizing gene-mutated mice have failed to confirm a major role of SP-A in surfactant metabolism in vivo (5).

SP-A is a collectin. Collectins share an NH2-terminal collagen-like region that forms a triple helix and a COOH-terminal calcium-dependent globular carbohydrate-recognition domain (C-type CRD). Other members of the collectin family include SP-D, along with mannose-binding protein, bovine conglutinin, and bovine collectin-43 (6). Collectins are involved in the first line of innate host defense against invading microbial agents (7, 8). The carbohydrate-recognition domain region binds to specific carbohydrate structures present on the surface of bacteria and viruses. This binding initiates microbial aggregation and facilitates phagocytosis, oxidative burst, and killing by inflammatory cells (9). Consistent with an important role in host defense, mice lacking SP-A are susceptible to bacterial and viral pneumonia (10, 11).

Serum collectins can enhance immune responses. Mannose-binding protein and conglutinin activate complement (7). C1q, a noncollectin protein of similar structure to SP-A, is a critical component of the first protein (C1) of the classical pathway of complement activation (12). While SP-A itself does not activate complement (12), it has been shown to stimulate T cell activation in vitro and the production of proinflammatory cytokines in the monocytic cell line, THP-1 (13, 14). However, thus far, all in vivo studies support a general anti-inflammatory role for SP-A during inflammatory pulmonary diseases. For example, virus-infected SP-A–deficient mice exhibit increased inflammation in vivo (15), but whether the anti-inflammatory effects of SP-A are related to viral clearance or direct suppression of inflammation remains unclear. Moreover, SP-A can downregulate macrophage and T cell immune responses in vitro (16, 17).

T cell immune responses are a major cause of mortality and morbidity after allogeneic bone marrow transplantation (BMT). Allo-activated T cells are implicated in the pathogenesis of graft-versus-host disease (GVHD) and greatly contribute to the development of transplant-related noninfectious lung dysfunction, referred to as idiopathic pneumonia syndrome (IPS) (18–20). In our allogeneic BMT model, early lung inflammation and dysfunction in lethally irradiated mice are dependent on the infusion of donor T cells (21). In a prior study, to determine whether exogenous SP-A attenuates the manifestations of IPS, human SP-A was transtracheally injected on Day 4 after transplantation, a time of peak activation of T cells. Two days after administration, SP-A–treated mice demonstrated improved permeability edema and less cytolysis compared with buffer-injected controls (22). The effects of SP-A treatment on survival of mice after allogeneic transplantation and the role of the basal level of endogenous SP-A were not determined.

In the present study, we hypothesized that SP-A improves the early survival after allogeneic BMT. Our results indicate that exogenous human SP-A and basal levels of endogenous SP-A prevent donor T-cell–dependent early production of inflammatory mediators and prolong post-BMT survival.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice
Inbred B10.BR (H2K) and C57BL/6 (B6; H2b) were purchased from the Jackson Laboratories (Bar Harbor, ME). SP-A-/- mice were generated from embryonic stem cells derived from the 129J mouse strain in which the mouse SP-A gene was disrupted by homologous recombination as previously described (23). SP-A–deficient mice were backcrossed > 10 generations to C57BL/6 mice. In experiments utilizing gene mutant animals, C57BL/6J mice (Jackson Laboratories) were used as wild-type controls. Mice were housed in micro-isolator cages in the specific pathogen-free facility of the University of Minnesota and cared for according to the Research Animal Resources guidelines of our institution. For BMT, donors were 6–8 wk of age, and recipients were used at 8–10 wk of age. Sentinel mice were found to be negative for 15 known murine viruses, including CMV, K-virus, and pneumonia virus of mice.

Pre-BMT Conditioning
Mice received intraperitoneal injection of cyclophosphamide (Cy) (Cytoxan; Bristol Myers Squibb, Seattle, WA) 120 mg/kg/d on Days -3 and -2 pre-BMT. On the day before BMT, all recipient mice (wild type and SP-A deficient) were lethally total body irradiated (TBI, 7.5 Gray) by X-ray at a dose rate of 0.41 Gray/min.

Bone Marrow Transplant
Our BMT and IPS generation protocols have been described previously (21, 24). Briefly, donor bone marrow (BM) was T cell depleted (TCD) with anti-Thy 1.2 monoclonal antibody (clone 30-H-12, rat IgG2b; kindly provided by Dr. David Sachs, Massachusetts General Hospital, Cambridge, MA) plus complement (Neiffenegger Co., Woodland, CA). For each experiment, a total of 5–10 recipient mice per treatment group were transplanted via caudal vein with 20 x 106 TCD BM cells without (BM+Cy) or with (BMS+Cy) 15 x 106 allogeneic spleen T cells as a source of GVHD/IPS-causing T cells. BMT was performed in both mouse strain combinations. As per our original protocol, B6 -> B10.BR for experiments using exogenous SP-A (n = 2 experiments). To perform experiments using gene mutant mice, we had to reverse the direction of the strain combination (B10.BR -> B6), because SP-A-/- mice are available to us only as B6 (n = 2 experiments). A cohort of mice from each group was monitored for early survival (Day 8 after BMT) and clinical evidence of GVHD (i.e., weight loss).

SP-A Instillation
Human SP-A was purified from the bronchoalveolar lavage fluid (BALF) of patients with alveolar proteinosis (provided by Dr. Jo Rae Wright, Duke University, Durham, NC) by sequential extraction with n-butanol as previously described (25). SP-A was resuspended in 5 mM tris (hydroxymethyl) aminomethane (Tris), pH 7.4. SP-A preparations contained a very low level of endotoxin (0.056 pg/µg). On Day 4 after BMT, during the time of donor T cell activation (26), mice were anesthetized with intraperitoneal sodium pentobarbital (5 mg) and injected transtracheally with either SP-A (50 µg) dispersed in 50 µl of 5 mM Tris or an equal volume of sterile buffer alone using a 27-gauge needle attached to a tuberculin syringe. TBI/Cy B10.BR mice given TCD donor B6 bone marrow (BM+Cy) without donor spleen T cells served as controls.

Bronchoalveolar Lavage
Mice were sacrificed on Day 7 after BMT or 2 d after SP-A/buffer injection. The thoracic cavity was partially dissected, and the trachea was cannulated with a 22-gauge angiocatheter and infused with 1 ml of ice-cold sterile phosphate buffered saline (PBS) and withdrawn. This was repeated once, and the return fluid was combined. Ten microliters of BALF was used to count the number of inflammatory cells with a hemacytometer, and the remaining fluid was immediately centrifuged at 500 x g for 10 min at 4°C to pellet cells. BALF cells were combined, resuspended in PBS, and centrifuged onto glass slides. Inflammatory cell types were identified by Wright-Giemsa staining and counted based on four sample sets per group from two different experiments.

BALF Analysis
Cell-free BALF tumor necrosis factor (TNF)-{alpha} and interferon (IFN)-{gamma} levels were determined by sandwich ELISA using murine-specific commercial kits (R&D Systems, Minneapolis, MN; sensitivity 1.5–3 pg/ml). Nitrite in BALF was measured according to the Greiss method after the conversion of nitrate to nitrite with the reduced nicotinamide adenine dinucleotide-dependent 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 as the standard. Lactic dehydrogenase (LDH) levels were measured by the colorimetric CytoTox 96 assay (Promega, Madison, WI), and LDH concentration (U/ml) in the BALF was calculated using bovine heart LDH as standard.

Western Blots for SP-A
Equal amounts of protein (10 µg) of BALF were solubilized in 0.1 M Tris buffer containing 50 µM dithiothreitol, 0.01% bromophenol blue, 1% sodium dodecyl sulfate, and 10% glycerol, and boiled for 5 min. The proteins were resolved by 12% sodium dodecyl sulfate polyacrylamide gels, transferred to nitrocellulose paper, and probed with the anti-sheep SP-A antibody (1:10,000 dilution) followed by alkaline phosphatase-conjugated goat anti-rabbit IgG (1:7,500 dilution) as the secondary antibody. Bound antibody was detected with nitro blue tetrazolium and 5-bromo-4-chloro-3indolyl-1-phosphate kit (Sigma).

Pulmonary Function Analysis
Pulmonary mechanics in pentobarbital-anesthetized ventilated mice were measured following the method described by Diamond (27), 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 (Buxco Electronics Inc., Sharon, CT) and connected to a mouse ventilator (Harvard Apparatus, March-Hugstetten, Germany) set at a respiratory rate of 150 breaths per minute and a tidal volume of 200 µl. 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 pressure 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 µl in 50 µl decrements, and for each delivered volume, the effective tidal volume, transpulmonary pressure, and dynamic compliance were measured. Body temperature was maintained at 37°C throughout the experiment. Volume–pressure plots were constructed for each treatment group.

Histology and Immunohistochemistry
In one to two mice per group per experiment, a mixture of 1 ml optimal cutting temperature medium (OCT; Miles Laboratories Inc., Elkhart, IN)/PBS (3:1) was infused in the trachea. The lungs were snap frozen in liquid nitrogen and stored at -80°C. Frozen sections were cut 4 µm thick, mounted onto glass slides, and fixed for 5 min in acetone. After a blocking step in 10% normal horse serum (Sigma Co.), sections were incubated for 30 min at 23°C with the anti–Mac-1 (clone M1/70; rat IgG2b,{kappa}) biotinylated monoclonal antibody (PharMingen, San Diego, CA). In control measurements the primary antibody was replaced with nonspecific IgG. Immunoperoxidase staining was performed using avidin–biotin blocking reagents, ABC-peroxidase conjugate, and DAB chromogenic substrate (Vector Laboratories, Inc., Burlingame, CA). The number of Mac-1–positive cells in the lung was quantitated as the percent of nucleated cells at a magnification of x50 (20x objective lens). Four fields per lung were evaluated.

Nitrative Stress in BALF Cells
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 anti-nitrotyrosine 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. Cells were considered nitrotyrosine-positive based on the presence or absence of the brown reaction product in the cell cytoplasm.

Statistical Analysis
Results are expressed as means ± standard error. Data were analyzed by analysis of variance or Student's t test. Statistical differences among group means were determined by Tukey's Studentized test. A comparison of survival curves between the different groups was made using the log rank test. For all analyses performed, a type I error probability of 0.05 was used as the cutoff for statistical significance.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
BMT Experiments
To investigate the role of the basal level of endogenous SP-A during the early donor T-cell–dependent mortality and tissue-destructive inflammatory response after allogeneic BMT, conditioned B6 wild-type and SP-A gene mutant mice were given B10.BR TCD BM plus donor spleen T cells (BMS+Cy). In additional experiments, the effects of enhancing alveolar SP-A levels by transtracheal injection of exogenous human SP-A on weight loss and early survival of Cy/TBI B10.BR mice given B6 TCD BM plus donor T cells (BMS+Cy) were determined. Although the direction of the strain combination is reversed, we have generated data that the type and severity of donor T-cell–dependent inflammatory response and IPS injury are similar in both strain directions (24, 28).

SP-A Is Absent in BALF of SP-A-/- Mice after Allogeneic BMT
BALF return volumes collected on Day 7 after transplantation were similar in all groups (> 90% of instilled volume). Using anti-sheep SP-A antibody, SP-A monomers were observed at ~ 30 kD only in the BALF of SP-A+/+ mice (Figure 1) . SP-A was undetectable in BALF of SP-A-/- control and SP-A-/-–transplanted mice. The band at ~ 43 kD observed on Western blots of SP-A–deficient and SP-A–sufficient mice, most likely representing SP-D, indicates that SP-D was not upregulated during SP-A deficiency after allogeneic transplantation.



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  		Figure 1. SP-A is undetectable in the bronchoalveolar lavage fluid (BALF) of mutant mice. Surfactant protein (SP)-A+/+ and SP-A-/- B6 mice were given cyclophosphamide (Cy) (120 mg/kg on Days -3 and -2) and total body irradiated (7.5 Gray on Day -1), followed by infusion of T-cell–depleted (TCD) B10.BR bone marrow plus idiopathic pneumonia syndrome (IPS)-causing donor bone marrow supplemented with spleen T cells (BMS+Cy). Shown is a representative Western blot of BALF proteins (10 µg per lane) from Day 7 after bone marrow transplantation (BMT) BALF as indicated, probed with polyclonal rabbit anti-sheep SP-A antibody followed by horseradish peroxidase-conjugated goat anti-rabbit IgG as the secondary antibody. Control, nonirradiated and nontransplanted. First lane represents purified human SP-A (1 µg). The band at ~ 43 kD may represent SP-D. Western blot was repeated two times with similar results.

 
Lung Injury Is Exacerbated in SP-A-/- Mice after Allogeneic BMT
We reported that exogenous SP-A prevents permeability edema and cytolysis after allogeneic BMT (22). To determine the effects of endogenous SP-A on markers of lung injury, BALF total protein and LDH levels were measured. Unmanipulated (nonirradiated and nontransplanted) and transplanted SP-A-/- mice given TCD bone marrow without donor T cells (BM+Cy) exhibited baseline concentrations of protein and LDH in BALF (Figure 2) . On Day 7 after allogeneic transplantation, BALF from conditioned SP-A-/- mice contained significantly higher total protein and LDH compared with SP-A+/+ mice, consistent with exacerbated donor T-cell–dependent lung injury during SP-A deficiency (Figure 2). To assess lung mechanics during SP-A deficiency, lung dynamic compliance (Cdyn) was measured in anesthetized and ventilated mice on Day 7 after allogeneic BMT. At a delivered tidal volume of 200 µl, Cdyn decreased from 0.04 ± 0.004 ml/cm H2O in unmanipulated control mice to 0.016 ± 0.003 ml/cm H2O in wild-type Cy/TBI donor T-cell–recipient mice (SP-A+/+ BMS+Cy). Compared with SP-A+/+ BMS+Cy mice, Cdyn was further decreased to 0.008 ± 0.0003 ml/cm H2O in mice lacking SP-A after allogeneic BMT (SP-A-/- BMS+Cy; P = 0.01, n = 3 mice per group). A plot of effective tidal volume versus transpulmonary pressure using delivered tidal volume of 100–350 µl demonstrated marked differences in lung mechanics between the groups (Figure 3) . Compared with controls, Cy/TBI mice infused with IPS-causing allogeneic T cells exhibited a downward and rightward shift of volume–pressure relationship, consistent with decreased compliance. The lung stiffness was most severe in SP-A-/- mice given donor T cells, as indicated by the very high transpulmonary pressures at low/moderate tidal volumes (Figure 3).



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  		Figure 2. Exacerbated markers of lung injury in BALF of SP-A-/- mice after allogeneic BMT. B6 SP-A+/+ and SP-A-/- were conditioned with Cy/total body irradiation (TBI) and given TCD bone marrow (BM) from B10.BR mice without (BM+Cy) or with inflammation-inducing donor spleen T cells (BMS+Cy). (A) Total protein levels measured in BALF on Day 7 after BMT. (B) Lactic dehydrogenase (LDH) levels measures in BALF on Day 7 after BMT. Control, nonirradiated and nontransplanted. Values are means ± SEM for n >= 10 mice per group from two experiments. *P < 0.05 compared with control. +P < 0.05 between wild type and SP-A-/- mice. Open bars, SP-A+/+; closed bars, SP-A-/-.

 


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  		Figure 3. Effective tidal volume–transpulmonary pressure plots in anesthetized and ventilated mice measured on Day 7 after allogeneic BMT. Control (nonirradiated and nontransplanted) Cy/TBI donor T-cell–recipient SP-A+/+ and SP-A-/- (BMS+Cy) mice were placed in a single-chamber plethysmograph and ventilated with delivered tidal volume of 100–350 µl. 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 three different mice in each experimental group. Open circles, control mice; open triangles, SP-A+/+ (BMS+Cy) mice; closed triangles, SP-A-/- (BMS+Cy) mice.

 
SP-A Enhances Survival after Allogeneic BMT
We investigated whether the SP-A–mediated attenuation of lung dysfunction is associated with improved survival after transplantation. Mortality in this BMT model was dependent on the presence of allogeneic T cells, because Cy/TBI wild type and SP-A-/- recipients not given T cells exhibited 100% survival. Consistent with a critical role for endogenous SP-A, mortality was higher in SP-A–deficient mice compared with SP-A–sufficient mice after allogeneic transplantation (P = 0.0007, n = 10 mice per group) (Figure 4A) . In addition, mice treated with exogenous human SP-A on Day 4 after BMT exhibited improved survival compared with buffer-treated controls (P = 0.0021, n = 16 mice per group) (Figure 4B). Of note is that changes in alveolar SP-A levels in SP-A–treated mice prolonged survival of mice without modifying the weight loss associated with GVHD in this BMT model (-24.5 ± 2.1% in buffer-treated mice versus -23.8 ± 1.5% in SP-A–treated mice; P > 0.05). Similarly, although on Day 7 after allogeneic transplantation, weight loss was slightly exaggerated in SP-A–deficient mice (-26.5 ± 2.1%) compared with SP-A–sufficient mice (-23.4 ± 1.9%), the difference was not significantly different (P = 0.13). These results suggest that SP-A–induced attenuation of lung injury may be the major factor responsible for the prolonged survival of mice after allogeneic transplantation.



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  		Figure 4. Effects of SP-A on survival after allogeneic transplantation. (A) Cy/TBI B6 SP-A-/- and SP-A+/+ mice were given TCD donor bone marrow (from B10.BR mice) without (BM+Cy) or with donor spleen T cells at time of BMT (BMS+Cy). Survival was determined on Day 8 after BMT. SP-A deficiency accelerated early mortality after allogeneic BMT (n = 10 mice per group; *P = 0.0007). Closed circles, SP-A-/- (BM+Cy) mice; open triangles, SP-A+/+ (BMS+Cy) mice; closed triangles, SP-A-/- (BMS+Cy) mice. (B) Cy/TBI B10.BR mice were given TCD donor bone marrow (from B6 mice) without (BM+Cy) (closed circles) or with donor spleen T cells at time of BMT (BMS+Cy), and transtracheally instilled with human SP-A (50 µg) (closed triangles) or equal volume of buffer (open triangles) on Day 4 after BMT (indicated by arrow). Survival was determined 4 d after injection of SP-A/buffer. SP-A treatment enhanced early survival after allogeneic transplantation. (n = 16 mice per group; *P = 0.0021.)

 
Anti-Inflammatory Properties of SP-A
To begin to understand the mechanisms of SP-A protection during the course of IPS, BALF levels of proinflammatory cytokines after allogeneic BMT were determined. Cy/TBI SP-A–deficient mice given BM in the absence of donor T cells (BM+Cy) contained undetectable levels of TNF-{alpha} and IFN-{gamma} in BALF collected on Day 7 after BMT. Consistent with an anti-inflammatory role for endogenous SP-A, on Day 7 after BMT, BALF from Cy/TBI SP-A-/- mice given inflammation-causing donor T cells (BMS+Cy) contained increased levels of TNF-{alpha} and IFN-{gamma} compared with SP-A+/+ BMS+Cy mice (Figure 5A) . Furthermore, SP-A treatment on Day 4 after BMT significantly decreased TNF-{alpha} and IFN-{gamma} levels in BALF collected 48 h after SP-A/buffer injection into the trachea of donor T-cell–recipient conditioned mice (BMS+Cy) (Figure 5B).



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  		Figure 5. SP-A modulates the production of tumor necrosis factor (TNF)-{alpha} and interferon (IFN)-{gamma} after allogeneic BMT. (A) Cy/TBI-recipient B6 mice lacking SP-A and given donor spleen T cells (BMS+Cy) have increased levels of TNF-{alpha} and IFN-{gamma} in the BALF collected on Day 7 after BMT. Open bars, SP-A+/+; closed bars, SP-A-/-. (B) Exogenous SP-A treatment suppresses TNF-{alpha} and IFN-{gamma} levels in BALF of Cy/TBI B10.BR mice given B6 BM with donor 15 x 106 spleen cells (BMS+Cy) on day of BMT. Human SP-A (50 µg) (hatched bars) or buffer (open bars) was instilled in the trachea via a midline incision and BALF was collected 48 h later. Control, nonirradiated and nontransplanted. Shown are mean values ± SE for 10–15 mice per group pooled from two separate experiments using knockout mice and two separate experiments using exogenous SP-A. *P < 0.05 versus control or BM+Cy. +P < 0.05 comparing the effect of SP-A deficiency or excess within each BMT group.

 
Lung Cellular Infiltration in SP-A-/- Mice
SP-A can regulate BALF levels of proinflammatory cytokines by modifying cellular infiltration and/or activation of inflammatory cells in the lung. On Day 7 after allogeneic BMT, BALF from SP-A-/- mice contained increased total cell counts (14.5 ± 1.7 x 104 cells/ml versus 8.5 ± 1.3 x 104 cells/ml in BMS+Cy wild-type mice; P < 0.05, n = 7 mice per group). As assessed by Wright-Giemsa stain, the cell-differential of cytospun samples from pooled BALF cell-pellets from SP-A-/- mice demonstrated a higher percentage of lymphocytes (44.6 ± 2.2% compared with 31 ± 2.2% in BMS+Cy wild type mice; P < 0.05, n = 4 sample sets per group from two experiments). In addition, Mac-1–positive cells in lung sections obtained on Day 7 after allogeneic BMT from SP-A–deficient mice (58 ± 3%) were significantly higher than SP-A–sufficient mice (44 ± 2.5%; P < 0.05, n = 4 sections per group from two experiments), consistent with exaggerated monocytic cell-infiltration (Figure 6) . In contrast, the number and type of BALF cells from mice given transtracheal exogenous SP-A or buffer on Day 4 after allogeneic BMT were similar (data not shown), most likely because SP-A was administered after release of critical chemoattractant signals (22). Our previous studies demonstrated that macrophages from human SP-A–treated BMS+Cy mice produced less TNF-{alpha} compared with equal number of cells from buffer-injected BMS+Cy mice (22). Taken together, the data indicate that SP-A can inhibit the production of inflammatory mediators by multiple pathways, including modulation of cellular recruitment and downregulation of donor T-cell–dependent macrophage-derived generation of proinflammatory cytokines.



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  		Figure 6. Increased expression of Mac-1–positive cells in lungs of SP-A–deficient mice after allogeneic BMT. Cy/TBI B6 SP-A-/- and SP-A+/+ mice were given TCD donor bone marrow (from B10.BR mice) with donor spleen T cells at time of BMT (BMS+Cy). Shown are representative immunoperoxidase-stained lung sections obtained from the indicated group of mice on Day 7 after BMT. Lung sections were stained with nonspecific rat IgG (labeled IgG) or anti–Mac-1 (clone M1/70) biotinylated monoclonal antibody (all remaining sections). Sections were developed with peroxidase-conjugated ABC and DAB chromogen (methyl green counterstain). Micrographs were obtained at two magnifications: x20 to demonstrate increased number of Mac-1–positive cells in the lung of SP-A–deficient mice; and x50 to demonstrate location of Mac-1–positive including alveolar macrophages (arrow). Control, nonirradiated and nontransplanted SP-A-/- mice. Scale bar = 25 µm.

 
Increased Generation of Nitric Oxide and Nitrative Stress in SP-A-/- Mice
We next evaluated the role of SP-A in donor T-cell–depen- dent nitric oxide (·NO) production and generation of nitrative stress. On Day 7 after allogeneic BMT, BALF from SP-A-/- mice contained increased levels of nitrate and nitrite, the stable byproducts of ·NO (Figure 7A) . Similarly, 48 h after injection, SP-A–treated mice exhibited decreased BALF levels of nitrate plus nitrite compared with BALF from buffer-treated mice (Figure 7B). Intracellular nitrative stress by macrophages/monocytes was assessed by detection of antigenic sites related to nitrotyrosine. Although not quantified, nitration of monocytes/macrophages from SP-A–deficient BMS+Cy mice appeared increased compared with cells of donor T-cell–recipient SP-A–sufficient Cy/TBI mice (Figure 8) . Nitration was specific because staining was completely blocked in the presence of excess antigen, 10 mM nitrotyrosine. Cells from Cy/TBI SP-A–deficient mice given bone marrow without T cells exhibited baseline levels of staining (data not shown). Because ·NO and ·NO-derived nitrative stress are associated with amplification of lung inflammation and injury, these results suggest that one of the protective mechanisms of SP-A is inhibition of ·NO and nitrative stress generation after allogeneic BMT.



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  		Figure 7. Endogenous and exogenous SP-A suppresses allogeneic T-cell–dependent ·NO generation after BMT. (A) Cy/TBI B6 mice lacking SP-A and given donor spleen T cells at time of BMT (BMS+Cy) have increased levels of nitrite in BALF collected on Day 7 after BMT. Open bars, SP-A+/+; closed bars, SP-A-/-. (B) Cy/TBI T-cell–recipient B10.BR mice (BMS+Cy) treated with human SP-A have decreased levels of nitrite in BALF collected 2 d following treatment. SP-A (50 µg) (hatched bars) or equal volume of buffer (open bars) was transtracheally instilled on Day 4 after BMT. Nitrate was reduced to nitrite prior to measurement by the Greiss reaction. Control, nonirradiated and nontransplanted; BM+Cy, Cy/TBI mice given TCD bone marrow without donor T cells. Shown are mean values ± SE for 10–15 mice per group pooled from two separate experiments using knockout mice and two separate experiments using exogenous SP-A. *P < 0.05 versus control or BM+Cy. +P < 0.05 comparing the effect of SP-A deficiency or excess within each BMT group.

 


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  		Figure 8. Increased nitrotyrosine immunostaining in BALF cells from SP-A–deficient mice after allogeneic transplantation. Cy/TBI-recipient SP-A–deficient and wild-type B6 mice were given B10.BR bone marrow plus donor spleen T cells (BMS+Cy). On Day 7 after BMT, BALF cells from indicated group of mice were centrifuged onto glass slides and incubated with nonspecific rabbit IgG, nitrotyrosine antibody (NTAb), or nitrotyrosine antibody in the presence of 10 mM nitrotyrosine (NT block). Shown is a representative figure that was reproduced twice from pooled cells obtained from 5–8 mice per group per experiment. Two separate experiments were performed. Slides were evaluated by a blinded observer.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The major findings of these studies are that SP-A protects the lung from allo-activated T cell immune responses, prevents the manifestations of IPS, and prolongs survival after allogeneic BMT. Results clearly establish SP-A as a modulator of the cellular adaptive immune system. The protective effects were not limited to basal endogenous SP-A, but also were observed following exogenous treatment to enhance alveolar SP-A levels. Similar effects of SP-A were observed in the B10.BR -> B6 strain combination using SP-A-/- and in the B6 -> B10.BR strain combination using human SP-A, confirming that the protection was not limited to a specific strain of mice or source of SP-A.

IPS injury after allogeneic BMT in irradiated mice is dependent on infusion of donor T cells and exacerbated by chemotherapeutic agents (21). Allogeneic T cells infiltrate the lung and are activated by antigen-presenting cells to release injurious lytic proteins (perforin) and serine proteases including granzyme B (29). In addition, T cells secrete IFN-{gamma}, which can induce the release of tissue-damaging macrophage-derived inflammatory mediators, potent oxidants, and nitrating species (30). Results show that SP-A did not modify chemoradiotherapy-induced injury that is T cell independent. Instead, data indicate that the main protective mechanism of SP-A is downregulation of the early T cell immune responses. Data supporting a direct suppressive effect of SP-A on allo-activated T cells include the increased total number and percentage of lymphocytes in the BALF of SP-A-/- mice and the inverse relationship between SP-A and IFN-{gamma} levels. Inhibition of T cell activation/proliferation is associated with subsequent downregulation of T-cell–dependent stimulation of macrophages and lung-infiltrating monocytes. However, an additional direct suppressive effect of SP-A on macrophages/monocytes cannot be ruled out, especially since SP-A has been shown to directly suppress the activation of alveolar macrophages (31). Recent evidence indicates significant cross-talk between the innate and adaptive immune systems (32) and that innate immunity (e.g., macrophages) may have a major impact on the nature of response of the adaptive immune system (33). Although donor T cells are required for IPS injury, the complexity of cellular interactions in vivo precludes a definite determination of the cell type affected by SP-A.

The basic mechanisms for the in vivo anti-inflammatory effects of SP-A after allogeneic BMT remain speculative. SP-A may inhibit T cell proliferation and activation by binding to specific cell-surface receptors. For example, the addition of a polyclonal antibody against an SP-A receptor (SP-R210) completely blocked the inhibition of T cell proliferation by SP-A (34). SP-A may also interfere with the process of antigen presentation by the blocking of a costimulatory signal crucial for T cell activation (35). Similarly, SP-A may dampen the inflammatory response by binding to CD14, a myeloid cell membrane receptor that elicits lipopolysaccharide-induced cellular responses (36). More recent data support a direct anti-inflammatory and antioxidant role for SP-A by inhibition of complement activation and lipid peroxidation (37, 38).

The presence of increased markers of lung injury and decreased dynamic lung compliance in the absence of SP-A after allogeneic BMT are likely the result of exaggerated inflammation with or without surfactant dysfunction. Our studies did not address the contribution of surfactant injury and the impact of SP-A deficiency on surfactant metabolism and homeostasis in this BMT model. However, Ikegami and colleagues reported that surfactant pool sizes and function in SP-A–deficient and SP-A–sufficient mice exposed to sublethal hyperoxia for 3 d were similar (5, 39), consistent with a minor direct effect of SP-A on surfactant metabolism and biophysical properties in vivo. Further studies will be required to evaluate surfactant homeostasis and function in the presence and absence of SP-A after allogeneic BMT.

To our knowledge, this is the first report that correlates SP-A–induced suppression of T-cell–dependent inflammation with survival. SP-A has been shown to suppress lipopolysaccharide- and microbe-induced inflammation in SP-A-/- mice in vivo, but survival was not evaluated (15, 40). In contrast to our results, Ikegami and coworkers have shown that death caused by hyperoxia- and N-nitroso-N-methylurethane–induced lung injury were not different in SP-A-/- and SP-A+/+ mice (39, 41). Because surfactant dysfunction is the primary mechanism of lung injury and mortality during exposure to high concentrations of oxygen (42) or N-nitroso-N-methylurethane (43), the contrasting effect of SP-A on mortality rates between our study and that of Ikegami may support a more significant role for SP-A in modulation of T cell immune responses rather than prevention of surfactant inactivation.

Notably, exogenous and endogenous SP-A enhanced survival without significant changes in weight loss following allogeneic BMT. In general, weight loss in our BMT model correlates with systemic inflammation and development of GVHD (21). Because SP-A is mainly limited to the lung, we hypothesize that inhibition of pulmonary inflammation and dysfunction are sufficient for improving survival, at least short-term. These observations underscore the importance of modifications of the local milieu of the lung in determining outcome during the course of inflammatory pulmonary diseases.

As discussed above, SP-A deficiency in infected mice is associated with enhanced inflammation and synthesis of proinflammatory cytokines. Another characteristic of virus- and bacteria-infected SP-A-/- mice is inadequate generation of oxidative stress by alveolar macrophages (44). Levine and colleagues reported that phytohemagutinin-activated alveolar macrophages from SP-A-/- mice obtained 3 d after transtracheal instillation of respiratory syncytial virus produced low levels of superoxide and hydrogen peroxide compared with cells from SP-A+/+ respiratory syncytial virus-infected mice (10). However, because only data from infected mice were shown, it is possible that the infectious agent may have contributed to the defect in oxidative burst, as has been shown following exposure to influenza virus (45). Our data demonstrate that nitrative stress, generated by the simultaneous generation of ·NO and superoxide (30), is increased in irradiated SP-A–deficient mice given allogeneic T cells and Cy (BMS+Cy). Because nitrative stress is associated with severe lung injury and exacerbated mortality after allogeneic BMT (24), we conclude that, in addition to anti-inflammatory properties, SP-A prevents IPS injury by suppression of ·NO and nitrative stress.

In summary, the present study demonstrates a suppressive role for endogenous and exogenous SP-A in regulation of the adaptive immune response and modulation of T cell immune responses in the alveolar space, leading to attenuation of lung injury and enhanced survival. These results should encourage development of recombinant human SP-A for use in the treatment of T-cell–dependent inflammatory lung diseases.


   Acknowledgments
 
This work was supported by grants from the American Lung Association (JM-CIA), and NIH R0-1 HL67334, HL55209, HL24075, and HL58047. The authors thank Dr. Jo Rae Wright for providing human SP-A. The authors gratefully acknowledge the expert technical assistance of John Bob Hermanson, and Melinda Berthold.

Received in original form March 22, 2002

Received in final form April 29, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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