AJP - Lung AJP: Renal Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Lung Cell Mol Physiol 292: L861-L871, 2007. First published December 8, 2006; doi:10.1152/ajplung.00145.2006
1040-0605/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
292/4/L861    most recent
00145.2006v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Jain, D.
Right arrow Articles by Beers, M. F.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Jain, D.
Right arrow Articles by Beers, M. F.

SP-D-deficient mice are resistant to hyperoxia

Deepika Jain,1 Elena Atochina-Vasserman,1 Helchem Kadire,1 Yaniv Tomer,1 Adam Inch,1 Pamela Scott,2 Rashmin C. Savani,3 Andrew J. Gow,2 and Michael F. Beers1

1Pulmonary and Critical Care Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania; 2Department of Pharmacology, Rutgers University, Piscataway, New Jersey; and 3Department of Pediatrics, University of Texas Southwestern at Dallas, Dallas, Texas

Submitted 14 April 2006 ; accepted in final form 30 November 2006


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Surfactant protein D (SP-D), a member of the collectin superfamily, modulates pulmonary inflammatory responses and innate immunity. Disruption of the SP-D gene in mice induces peribronchiolar inflammation, accumulation of large, foamy macrophages, increased bronchoalveolar lavage (BAL) phospholipid, and pulmonary emphysema. We hypothesized that absence of SP-D aggravates hyperoxia-induced injury. To test this, SP-D-deficient (SP-D–/–) and wild-type (SP-D+/+) mice were exposed to 80% or 21% oxygen. Paradoxically, during 14 days of hyperoxia, SP-D–/– mice had 100% survival vs. 30% in SP-D+/+. The survival advantage in SP-D–/– mice was accompanied by lower histopathological injury scores at days 5 and 14, although total BAL cells (8.2 ± 1.4 x 105 in SP-D–/– vs. 4.04 ± 0.25 x 105 in SP-D+/+ mice) and neutrophils (1.2 ± 0.4 x 105 vs. 0.03 ± 0.02 x 105 in SP-D–/– and SP-D+/+, respectively) were increased. In addition, BAL protein and lung-to-body weight ratios were similarly elevated in both groups after 3, 5, and 14 days of continuous exposure. Biochemically, in contrast to SP-D+/+, SP-D–/– mice had higher levels of surfactant phospholipid and SP-B at baseline and 5 days after hyperoxia accompanied by a preservation of surfactant biophysical activity. From a multiplex assay of nine cytokines, we found elevated levels of IL-13 in BAL fluid of normoxic SP-D–/– mice compared with SP-D+/+. We conclude that the resistance of SP-D-deficient mice to hyperoxia reflects homeostatic changes in the SP-D–/– phenotype involving both phospholipid and SP-B-mediated induced resistance of surfactant to inactivation as well as changes in the immunomodulatory BAL cytokine profile.

surfactant protein D; inflammation; alveolar macrophages; collectin

AS A BYPRODUCT OF THE USE of high oxygen (O2) concentrations often required in management of patients with respiratory failure, reactive oxygen species produced during hyperoxic exposure can adversely affect the lung. It has been proposed that the balance between oxygen radical production and antioxidant capacities can modulate oxygen-mediated injury. The lung responds to hyperoxia by enhancing the expression of cytoprotective proteins including antioxidants (65), DNA repair enzymes (55), and regulators of cell survival. In addition, elevated expression of certain surfactant components occurs in lungs of adult (54) and neonatal rats (64) during adaptation to both sublethal (85%) and lethal (95%) oxygen (1).

Surfactant protein D (SP-D), an airway epithelial secretory product, belongs to the superfamily of mammalian C-type (Ca2+-binding) lectins (collectins), which also includes SP-A and serum mannose-binding lectin. Like all collectins, monomeric SP-D is distinguished by the presence of an NH2-terminal cysteine-rich domain, a collagen domain, a coiled-coil neck domain, and a lectin domain with calcium-dependent regulatory elements. SP-D monomers associate through their collagenous domains to form a basic functional trimer, which then associates into higher order multimers. This oligomeric assembly provides high avidity, affinity, and specificity to SP-D ligand recognition. In the lung, SP-D ligands include allergens, particles, bacterial cell wall components, and viral envelope proteins (26). SP-D is also known to be chemotactic for neutrophils and mononuclear phagocytes (25) and to modulate alveolar type II (61) and macrophage function in vitro (39).

SP-D is produced primarily by alveolar type II cells and nonciliated bronchiolar cells in the lung (24) and is constitutively secreted into the alveoli where it influences surfactant homeostasis (40), effector cell functions, and host defense. It is upregulated in a variety of inflammatory and infectious conditions including Pneumocystis pneumonia (5), asthma (2), and bleomycin injury (17). Mice or rats exposed to hyperoxic challenge also have increases in SP-D (13), suggesting that it may have a role in protection from this insult.

Targeted disruption of the SP-D gene in vivo in two genetically different backgrounds has been shown to result in mice with increased alveolar and cellular pools of surfactant phospholipid, accelerated development of age-related emphysema, and large, foamy macrophages (14, 40). These SP-D null mice (SP-D–/–) also exhibit an increase in the baseline level of inflammation in the lung, increases in metalloproteinase activity, and biochemical evidence of enhanced oxidative-nitrative stress (4, 63). In addition, SP-D deficiency also confers susceptibility to specific bacterial (45) and viral infections (46).

We have previously reported modulation of bleomycin-induced lung injury by SP-D. In SP-D–/– mice, we showed increased susceptibility to bleomycin and evidence of oxidative-nitrative stress (17). Similar findings were observed when the same mice were infected with Pneumocystis carinii (6). Based on the anti-inflammatory properties of SP-D and its association with altered nitric oxide metabolism in both infectious and noninfectious (bleomycin) models of lung injury, we hypothesized that the inflammatory response to pure hyperoxic injury could also be exacerbated by the absence of SP-D. Paradoxically, we found that chronic SP-D deficiency confers resistance to hyperoxia-mediated lung injury. Furthermore, our data indicate that the SP-D-deficient phenotype develops compensatory changes beneficial to survival from hyperoxic challenge including increased surfactant pool sizes, altered macrophage responsiveness, and differential cytokine responses.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
SP-D-deficient mice. SP-D-deficient mice produced by targeted ablation of the mouse SP-D locus have been previously described (14). Mice bred to homozygosity were backcrossed 10 generations onto the C57BL/6 background (19). Age-matched wild-type C57BL/6 mice (SP-D+/+) purchased from Charles River Laboratories were used as controls. All mice were maintained under specific pathogen-free conditions in the barrier facilities at the University of Pennsylvania. Experiments were performed on 7- to 8-wk-old male mice.

Exposure to hyperoxia. SP-D-deficient and age-matched male mice were exposed to normoxia (21%, control group) or hyperoxia (80% ± 2%) for up to 14 days in sealed Plexiglas chambers (Braintree Scientific, Braintree, MA) with continuous O2 monitoring using an oxygen analyzer (Pacifitech, Temecula, CA). Animals were housed in environment-controlled cages on a 12-h dark-light cycle and allowed food and water ad libitum during the exposure. Mice were observed at 8-h intervals throughout the exposure. Cages were opened every 2 days for change of water, food, and bedding and as required for removal of dead mice. The study was reviewed and approved by the Institutional Animal Care and Use Committee of the University of Pennsylvania.

Preparation and analysis of bronchoalveolar lavage. Lungs were lavaged with five 1-ml aliquots of sterile saline. A cell pellet was obtained from bronchoalveolar lavage (BAL) samples by centrifugation at 400 g for 10 min. After resuspending the cell pellet in 1 ml of PBS (with Ca2+ and Mg2+), total cell counts were performed using a Z1 particle counter (Beckman-Coulter, Miami, FL) as described previously (5). Cytospins prepared from an aliquot of cell suspension were stained with Diff-Quik, and differential cell counts were performed manually.

A 200-µl aliquot of cell-free BAL from the first 1 ml collected was stored at –80°C for cytokine analysis by SearchLight Technology multiplex cytokine assay (Pierce Biotechnology, Woburn, MA). The remaining aliquots were pooled together.

Three-hundred microliters of the pooled BAL sample was stored for nitric oxide estimation, and the rest was fractionated into large-aggregate (LA) and small-aggregate (SA) fractions by centrifugation at 20,000 g for 60 min at 4°C as described previously (5). Total protein content of both fractions was determined by the Bradford method with bovine IgG as a standard (15).

Total lipids were extracted from LA and SA surfactant fractions with chloroform-methanol as described previously (11), and total phospholipids in the lipid extracts in each fraction were determined by Bartlett's colorimetric assay of inorganic phosphorus (8).

Macrophage cell culture and activation assay. Cell pellets obtained from BAL of SP-D+/+ and SP-D–/– mice exposed to either 21% or 80% oxygen for 5 days were pooled, washed, and resuspended in RPMI 1640 cell culture medium with 10% FBS. Cells were plated at a density of 0.5 x 106 cells/well in 48-well tissue culture plates (Falcon, Becton Dickinson) and incubated for 1 h in a humidified incubator at 37°C. The adherent cells were then washed twice with serum-free RPMI and allowed to equilibrate in serum-free medium for 30 min before stimulation with either LPS (1 µg/ml) or zymosan (100 µg/ml) for 18 h. Cell supernates were collected and stored at –80°C until further analysis. TNF-{alpha} in cell-free culture supernates was assayed by sandwich ELISA using a BD OptEIA ELISA kit (BD Pharmingen) as per the manufacturer's instructions.

Polyacrylamide gel electrophoresis and immunoblotting. SP-B in BAL was assayed by SDS-PAGE and Western blotting using 10–20% Novex Tricine gel system (Invitrogen), whereas NuPAGE Novex –10% Bis-Tris gels (Invitrogen) were used for SP-A and SP-D Western analysis. Immunoblots were performed with monospecific, polyclonal surfactant protein antisera to SP-A, SP-B, and SP-D as previously described (5, 9). Bands were visualized using horseradish peroxidase-conjugated secondary goat anti-rabbit IgG polyclonal antibodies (Jackson Immunoresearch Laboratories) and enhanced ECL chemiluminescence (Amersham). Band intensity was quantitated by densitometric scanning of the exposed film or by direct acquisition on Kodak 440 Imaging System (New Haven, CT).

Lung histology and inflammation scoring. After lavage, lungs were inflation fixed with 0.5 ml of 10% neutral buffered formalin for histological analysis. Paraffin sections prepared from the lungs were stained with hematoxylin and eosin for evaluation of airway inflammation. Hyperoxic lung injury was evaluated using the scoring system modified from Christofidou-Solomidou et al. (18). Hematoxylin-eosin-stained lung sections were examined by light microscopy and scored based on the presence or absence of peribronchiolar infiltration, thickening of the alveolar septae, vascular congestion, alveolar edema, and cellular infiltration into the alveoli. Because lung damage was typically patchy and microscopically heterogeneous at the histological level, the histopathological score (HPS) was normalized to the % of area exhibiting the requisite abnormalities. Lungs were assigned HPS from 0 to 3, in increments of 0.5, based on the amount of injury. Complete obliteration of lung structure was scored as 3. Scores assigned by two independent, blinded observers were averaged to get the final HPS.

Surface tension measurement by capillary surfactometry. The biophysical activity of recovered surfactant from exposed mice was measured in a capillary surfactometer as described in detail elsewhere (28). The LA fraction of the BAL was diluted to 1 mg/ml phospholipid concentration. To assess the capacity of surfactant to maintain patency, surfactant preparations (0.5-µl samples) were introduced into the narrow section of a glass capillary and compressed for 120 s. This results in cyclic extrusion of the surfactant from the narrow end of the capillary, allowing airflow and opening of the capillary. Surfactant dysfunction in the sample results in loss of capillary openness. Data were expressed as the percentage of the 120-s study period that the capillary is open to a free airflow.

Nitric oxide measurements. The analysis of nitric oxide metabolites was performed by chemical reduction and chemiluminescence using the Ionics/Sievers Nitric Oxide Analyzer 280 (Ionics Instruments, Boulder, CO) as previously described (4). This method is based on the reduction of total nitrogen oxides including nitrate, nitrite, and S-nitrosothiol using vanadium chloride in hydrochloric acid at 95°C.

Detection of protein carbonyls. Protein carbonyl groups were measured by a solid phase ELISA as previously described (47) using derivatization with 2,4-dinitrophenyl-hydrazine (2,4-DNPH). To derivatize the carbonyls, 100 µg of protein in 10 mM sodium phosphate, pH 7.4, was reacted with an equal volume of 40 µM 2,4-DNPH in 2.5 M HCl. After incubating the samples for 30 min at room temperature, unbound material was removed by passing through a Sephadex G-25 spin column. Samples were analyzed by solid phase ELISA using an anti-DNPH antibody (Molecular Probes) at 1:2,500 dilution. For negative controls, the samples were mixed with 2.5 M HCl in the absence of 2,4-DNPH.

Reduced thiol determination. The macrophage cell pellet was washed and harvested in 200 µl of buffer containing 20 mM Tris (pH 7.4), 150 mM NaCl, 10% glycerol, 1% Triton X-100, and 4 mM EGTA. An aliquot was used to quantify both protein and nonprotein thiols using a thiol quantification kit (Molecular Probes) according to directions provided by the manufacturer. An aliquot of the cell lysate was used for DC-protein assay (Bio-Rad). The total reduced thiol concentration was normalized to cellular protein. The method has been described in detail elsewhere (34).

Data analysis. All parametric data from experimental and control groups were expressed as means ± SE. Groups were compared utilizing unpaired two-tailed Student's t-test or ANOVA analyzed with a standard statistical software package (Prism v. 4.02, GraphPad Software). In all cases, a P value of <0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
SP-D–/– mice are resistant to hyperoxia-induced mortality. In contrast to increased susceptibility to acute lung injury induced by pathogens (6, 45) and bleomycin (17), we found that exposure to 80% oxygen resulted in mortality exclusively in SP-D+/+. As shown in Fig. 1, hyperoxia-exposed wild-type mice exhibited a 14-day mortality of 70% compared with 0% mortality in similarly exposed age- and sex-matched SP-D-deficient mice. Both groups had a 100% survival under normoxic conditions. To assess the effect of constitutive absence of SP-D in the development of injury, we elected to look at earlier time points. From the survival curve, SP-D+/+ mice appear to succumb after 3–5 days of exposure to O2, with a median survival of 10 days in this group. So, further studies were done at these time points, specific to this acute phase of hyperoxic injury.


Figure 1
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 1. Surfactant protein D (SP-D)-deficient mice are more tolerant to hyperoxia than wild-type mice. SP-D-deficient mice and C57BL/6 control mice, 7–8 wk of age, were exposed to 80% oxygen as described in METHODS. Survival for each group was recorded throughout a 14-day observation period and plotted as Kaplan-Meier survival analysis. Comparison of mortality at days 7 and 14 revealed enhanced tolerance in the SP-D–/– (SP-D-deficient) mice (P < 0.005 vs. SP–/– hyperoxia group at day 7 and day 14). Data are sum of 2 separate experiments; n = 10–20 for each group. SP-D–/– hyperoxia group showed a 100% survival after 14 days, comparable to the SP-D+/+ and SP-D–/– normoxia groups.

 
Both SP-D+/+ and SP-D–/– mice show similar levels of vascular leak after hyperoxia. Under normoxic conditions, SP-D–/– mice had ~1.5-fold higher levels of total alveolar protein in BAL (782.6 ± 70.7 µg, n = 19) compared with the SP-D+/+ (543.8 ± 96.2 µg, n = 18; P < 0.01). These values are similar to baseline values as previously reported (4). Despite enhanced survival, we found that early during hyperoxia challenge, both the SP-D–/– and SP-D+/+ animals demonstrated similar levels of cellular damage and vascular leak. Both groups exhibited a comparable increase in lung-to-body weight ratio at day 5 (Fig. 2A). SP-D–/– and SP-D+/+ animals also exhibited comparable absolute increases in total alveolar protein after 3 and 5 days of hyperoxia (Fig. 2B). However, since baseline protein levels in SP-D–/– mice were significantly higher, the relative protein increase in this group was only 2.2-fold vs. 4.6-fold in SP-D+/+ animals (n = 9–13, P < 0.05) at 5 days and among 14-day survivors (3.4-fold in SP-D–/– vs. 6.1-fold in SP-D+/+ animals, P < 0.05).


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 2. Morbidity during hyperoxic exposure is depicted. A: weight changes. Body weights and lung weights following hyperoxic exposure were recorded for both SP-D+/+ and SP-D–/– mice. Data are expressed as a ratio of lung-to-body weight at terminal end points. Values are means ± SE; n = 10–20, *P < 0.005 vs. corresponding control groups. B: bronchoalveolar lavage (BAL) total protein from the same time points (n = 6–30/group) were determined as described in METHODS. Data for each group are all expressed as means + SE (% of SP-D+/+ controls). *P < 0.05 vs. SP-D+/+ 0-d exposure group; #P < 0.05 vs. SP-D–/– 0-d group. WT, wild type; BALF, BAL fluid; d, day.

 
Pulmonary surfactant in SP-D–/– mice is resistant to inactivation by hyperoxia. Hyperoxic exposure in SP-D+/+ mice results in a loss in surfactant activity at 3 and 5 days (Fig. 3A). Constitutive absence of SP-D has been shown to affect surfactant homeostasis with marked increases in surfactant phospholipid content and SP-B protein content (4, 14, 43). In the present study, analysis of BAL from SP-D-deficient mice under normoxic conditions confirmed elevated BAL phospholipid (Fig. 3B) and SP-B content (Fig. 3C) in the SP-D–/– model. Total BAL phospholipid in SP-D–/– mice (348 ± 27.3 µg, n = 18) was threefold higher than the SP-D+/+ mice (115.7 ± 11.8 µg, n = 20) receiving 21% O2 for 5 days. Hyperoxic challenge introduced biochemical changes to both groups. For SP-D+/+, this translated into a depletion of BAL phospholipid at days 3 and 5 (Fig. 3B), with a 70% concomitant reduction in SP-B levels (Fig. 3C) in the LA fraction of BAL. Among the wild-type survivors at 14 days, we saw a twofold increase in total BAL phospholipid (Fig. 3B) and a partial recovery of SP-B. In contrast, surfactant biophysical activity was preserved in SP-D–/– mice at all time points (Fig. 3A). While the BAL phospholipid also decreased in SP-D–/– mice after hyperoxia, the absolute levels of phospholipid in these mice were always higher than the similarly exposed SP-D+/+ mice (Fig. 3B). BAL phospholipid in SP-D–/– mice was 253.1 ± 37.3 µg and 215.8 ± 19.1 µg after 3 and 5 days, respectively, compared with 35.1 ± 4 µg and 63.3 ± 5.4 µg in similarly exposed SP-D+/+ mice (n = 10–12, P < 0.001 at both time points). Similarly, absolute BAL SP-B levels never decreased to less than 50% of absolute wild-type values, as reported in phenotypically normal SP-B heterozygous mice (20).


Figure 3
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 3. Surfactant biophysics and biochemistry during hyperoxic challenge. After exposure to 80% O2, total phospholipid and SP-B were estimated in BAL as described in METHODS. A: SP-D-deficient mice preserve their surfactant function after exposure to hyperoxia. Surface activity of the large aggregate fraction of BALF was determined by measuring %capillary openness in a capillary surfactometer as described. Values are obtained by averaging triplicate measurements of each sample. Data are expressed as %capillary openness, 100 being fully open. Results are presented as means ± SE of n = 6–25 samples in each time point. *P < 0.05 vs. SP-D+/+ control group. B: SP-D–/– mice have increased phospholipid levels in BAL. Total phospholipid in BAL was estimated using a modification of the colorimetric Bartlett method as described. Data normalized to %SP-D+/+ level are expressed as means ± SE of n = 10–20 samples. *P < 0.05 vs. SP-D+/+ control group; #P < 0.05 vs. SP-D–/– control group. C: SP-D-deficient mice have increased SP-B levels in BAL. Western Blot analysis for SP-B production in SP-D–/– mice and SP-D+/+ mice (n = 6–18) using 4 µg of total protein. SP-B band density was determined, and SP-B content per total BAL recovered was calculated. Data normalized to %SP-D+/+ level are expressed as means ± SE. *P < 0.05 vs. SP-D+/+ control group; #P < 0.05 vs. SP-D–/– control group.

 
Hyperoxia also alters lung collectin content of these mice (Table 1). Exposure to 80% oxygen for 3 and 5 days results in an acute decrease in BAL SP-D of SP-D+/+ mice. By 14 days, SP-D expression recovers in the SP-D+/+ survivors, actually resulting in twofold higher levels than the normoxic SP-D+/+ mice. In addition to being deficient in SP-D, SP-D–/– mice have ~50% lower SP-A in BAL compared with SP-D+/+ at baseline. Hyperoxic exposure results in a further significant loss in SP-A levels at all time points studied, with a partial recovery among 14-day survivors in SP-D+/+ animals.


View this table:
[in this window]
[in a new window]

  		Table 1. Semiquantitative estimation of collectins in bronchoalveolar lavage

 
Exposure to 80% oxygen increases BAL cellularity. Exposure to hyperoxia results in the recruitment of inflammatory cells to the lungs of both SP-D+/+ and SP-D–/– mice (Fig. 4). Unexposed SP-D–/– mice demonstrate a significantly higher number of total cells (4.6 ± 0.2 x 105 cells), macrophages (4.5 ± 0.2 x 105 cells), and neutrophils (0.1 ± 0.03 x 105 cells) in the BAL fluid compared with SP-D+/+ (3 ± 0.25 x 105 total cells, 3 ± 0.2 x 105 macrophages, and no neutrophils, n = 17–20; P < 0.001). Hyperoxia produces a time-dependent infiltration of cells into the air spaces. As shown in Fig. 4A, SP-D+/+ mice exhibited a modest but significant cellular influx in the lung after 5 days of exposure (4 ± 0.3 x 105 total cells, n = 13). In contrast, SP-D-deficient mice had almost twofold higher total BAL cells at the 5-day time point (8.2 ± 1.4 x 105 total cells, n = 11). There was no significant increase in the number of macrophages in either group (Fig. 4B), although SP-D–/– mice had 50% more macrophages at baseline (4.5 ± 0.2 x 105, n = 18 in SP-D–/– vs. 3 ± 0.2 x 105 cells, n = 20 in SP-D+/+). A large proportion of the cellular infiltrate consisted of polymorphonuclear cells (PMNs) (Fig. 4C). Compared with SP-D+/+, hyperoxia-exposed SP-D–/– had 12-fold higher number of BAL PMNs neutrophils after 3 days (0.5 ± 0.1 x 105 vs. 0.04 ± 0.03 x 105 cells in SP-D+/+, n = 9) and 30-fold more PMNs after 5 days of exposure (1.2 ± 0.4 x 105 vs. 0.04 ± 0.02 x 105 cells in SP-D+/+, n = 11–13). Survivors after 14 days of exposure in both groups had a comparable number of total BAL cells in the alveoli (8.2 ± 0.6 x 105, n = 20 in SP-D–/– vs. 7.6 ± 1.2 x 105 cells, n = 6 in SP-D+/+, P > 0.05) as well as a similar number of BAL neutrophils (1.2 ± 0.3 x 105, n = 20 in SP-D–/– vs. 1.1 ± 0.5 x 105 cells, n = 6 in SP-D+/+).


Figure 4
View larger version (11K):
[in this window]
[in a new window]

  		Fig. 4. BAL cells in the lungs during hyperoxia. BAL total (A) and differential cell counts (B and C) were performed as described in METHODS after exposure of SP-D–/– and SP-D+/+ mice to 80% O2 for 0, 3, or 5 days as indicated. Data for each group are expressed as means + SE (n = 9–20). *P < 0.05 vs. SP-D+/+ normoxia group; #P < 0.05 vs. SP-D–/– normoxia group.

 
SP-D-deficient mice have less parenchymal inflammation than SP-D+/+ mice after hyperoxia. Histological lung sections confirmed our previous observation that at baseline, SP-D–/– mice possess patchy perivascular and peribronchial infiltrates of mainly mononuclear cells within the lung parenchyma (Fig. 5A). Lung sections of SP-D+/+ mice exhibited uniformly normal lung morphology. Lungs of mice exposed to 80% O2 for 3 days showed relatively no change in baseline lung structure in both groups. After 5 days of exposure to 80% oxygen, lungs from both SP-D+/+ and SP-D–/– mice had developed diffuse vascular congestion with tissue infiltration of PMNs and red blood cells, which increased further by day 14 (Fig. 5A). Histopathological injury estimated by semiquantitative scoring shows (Fig. 5B) that despite increases in BAL cells, lungs from SP-D–/– mice exhibited less inflammatory cell infiltration in the parenchyma and less morphological damage after 5 and 14 days of hyperoxia exposure than the similarly exposed SP-D+/+ animals.


Figure 5
View larger version (95K):
[in this window]
[in a new window]

  		Fig. 5. Hyperoxia-exposed SP-D-deficient mice have attenuated histopathological lung injury. A: representative morphological changes in formalin-fixed, paraffin-embedded, hematoxylin and eosin-stained left lung sections of SP-D+/+ and SP-D–/– mice under basal (day 0) conditions (left) and after 5 days (middle) or after 14 days (right) of exposure to 80% O2. B: intensity of inflammation in the lung tissue. Histological sections of lungs from SP-D+/+ and SP-D–/– mice obtained 5 or 14 days posthyperoxia were scored blindly by 2 readers for the intensity of inflammation using a published scale as described in METHODS. Bars represent means ± SE, n = 6–20. *P < 0.05 vs. SP-D+/+ day 0 group.

 
BAL cytokine in hyperoxia-exposed SP-D–/– mice is distinct from SP-D+/+ mice. Activation and recruitment of inflammatory cells is dependent on the expression and release of chemokines and cytokines. Using a cytokine multiplex assay (SearchLight Technology), we surveyed an array of nine different cytokines in the BAL of mice exposed to 80% oxygen for 3 and 5 days (Table 2). The cytokines assayed were broadly grouped as chemotactic [KC, monocyte chemoattractant protein (MCP)-1, and granulocyte/macrophage colony-stimulating factor (GM-CSF)], inflammatory [IFN-{gamma}, TNF-{alpha}, and macrophage inflammatory protein (MIP)-2], and those known to induce adaptation to hyperoxia/immunosuppression (IL-6, IL-10, and IL-13). In general, baseline cytokine levels in both groups were comparable and similarly upregulated after exposure to oxygen. BAL from SP-D–/– mice had 1.5-fold higher level of MCP-1 and 2-fold more GM-CSF at baseline. Hyperoxia-exposed SP-D–/– mice exhibited a significant upregulation of MCP-1 compared with the SP-D+/+ mice. In addition, SP-D–/– mice demonstrated a significant, but transient, early elevation in BAL levels of proinflammatory TNF-{alpha} as well as the immunosuppressive cytokine IL-13 compared with similarly exposed SP-D+/+ mice (Fig. 6). MIP-2 levels in SP-D–/– mice were increased at 5 days.


View this table:
[in this window]
[in a new window]

  		Table 2. Cytokine multiplex assay in BAL

 

Figure 6
View larger version (24K):
[in this window]
[in a new window]

  		Fig. 6. Hyperoxia-exposed SP-D-deficient mice have altered cytokine profiles. A cytokine multiplex assay was performed using SearchLight Technology. Values are fold changes compared with SP-D+/+ normoxia group. Data for each group are expressed as means ± SE (n = 4–9; *P < 0.05 vs. SP-D+/+ normoxia group; #P < 0.05 vs. SP-D–/– normoxia group). MIP-2, macrophage inflammatory protein-2; MCP-1, monocyte chemoattractant protein-1.

 
Metabolism of nitric oxide during hyperoxia is differentially regulated between SP-D+/+ and SP-D–/– mice. The activation of macrophages is associated with enhanced production of nitric oxide and cytokines (4, 66). Biochemical analysis of total BAL nitric oxide indicated that SP-D–/– mice produced 1.5-fold more nitric oxide than SP-D+/+ mice at baseline (Fig. 7). However, these groups responded differently to hyperoxic challenge. BAL nitric oxide levels in SP-D+/+ mice increased at early time points after exposure. In contrast, BAL nitric oxide production in SP-D–/– acutely dropped by 40% after hyperoxia, with absolute levels similar to the baseline levels in SP-D+/+ (Fig. 7).


Figure 7
View larger version (15K):
[in this window]
[in a new window]

  		Fig. 7. Nitric oxide (NO) in BAL in the lungs of SP-D+/+ and SP-D–/– mice. NO was detected in the BAL of SP-D+/+ and SP-D–/– mice by chemical reduction and chemiluminescence, as described in METHODS. Data are means ± SE (in nanomoles; n = 5–20; *P < 0.05 vs. SP-D+/+ control mice; #P < 0.05 vs. unexposed SP-D–/– mice).

 
SP-D–/– mice demonstrate increases in protein carbonyls and decreases in total lung disulfides. Formation of carbonyl groups in some amino acid side chains is the most widely studied oxidative stress-induced modification of proteins. Carbonyl groups, which are normally absent from cellular proteins, are thought to be formed after free radical attack of amino acid residues. To measure oxidant-induced protein damage, we assessed the disruption of cysteinyl residues, which are critical to the maintenance of protein structure and enzyme activity, via oxidation of free thiol groups as described (34). We found that the SP-D–/– mice demonstrate a higher baseline level of oxidative stress as shown by a twofold higher level of total lung protein carbonyls (Table 3). Furthermore, as an independent assessment, we also measured total free thiols. Ninety-five percent of these free thiols comes from GSH. We found that SP-D–/– mice had ~40% lower content of protein thiols and mixed disulfides, consistent with a notion of increased oxidative burden.


View this table:
[in this window]
[in a new window]

  		Table 3. Estimation of markers of oxidative stress in the lung

 
Macrophages from SP-D–/– mice are less responsive in vitro. Since macrophages are an important source of reactive oxygen species and reactive nitrogen species in vivo, attenuation of nitric oxide production suggests that constitutive absence of SP-D results in hyporesponsive macrophages. To test this, macrophage function was assessed in vitro. Cells isolated from BAL of SP-D+/+ and SP-D–/– mice were cultured and stimulated in vitro with either LPS or zymosan as described in METHODS. Cell activation was estimated as the amount of TNF-{alpha} secreted in the medium. We observed that at baseline, cells from SP-D–/– mice were hyporesponsive to both stimuli and secreted significantly less TNF-{alpha}. BAL macrophages isolated from lungs of both SP-D+/+ and SP-D–/– mice exposed to hyperoxia for 5 days also secreted significantly less TNF-{alpha} (Fig. 8).


Figure 8
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 8. Alveolar macrophages from the lungs of SP-D–/– mice are hyporesponsive in vitro. A: 21% O2; B: 80% O2. BAL macrophages from SP-D+/+ and SP-D–/– mice exposed to either normoxia or hyperoxia for 5 days were cultured and stimulated with either LPS or zymosan as described in METHODS. TNF-{alpha} was measured in cell culture supernatants by ELISA. Data are means ± SE (n = 3–6; *P < 0.05 vs. SP-D+/+ control mice; #P < 0.05 vs. unexposed SP-D–/– mice). ND, not detected.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Ablation of the SP-D gene results in progressive alveolar proteinosis and emphysema along with alterations in surfactant homeostasis, peribronchiolar mononuclear infiltrates, increased BAL cellularity, macrophage activation, oxidative stress, and matrix metalloproteinase production (4, 14, 43, 63), a phenotype that can be reversed by targeted expression of SP-D (30). Increased susceptibility of SP-D-deficient mice to bacterial, viral, and fungal infections (3, 6, 45, 46) as well as to noninfectious bleomycin-induced lung injury (17) has been previously reported. Protection of surfactant phospholipids and macrophages from oxidative damage in vitro by direct antioxidant functions of SP-D has also been observed (16). Therefore, it was logical to anticipate that hyperoxic treatment of SP-D–/– mice would result in a similar response. Surprisingly, we have made the novel observation that SP-D–/– mice demonstrate a remarkable tolerance to oxygen exposure.

The pathogenesis of hyperoxia-induced lung injury is complex and associated with many factors, such as increased expression of proinflammatory cytokines, high levels of nitric oxide, and low levels of surfactant (10, 42, 52). In hyperoxia-induced acute lung injury, excessive oxidant stress is characterized by increased pulmonary capillary permeability, inflammation, lung destruction via reactive oxygen species-mediated epithelial and endothelial cell death, and ultimately death (23). On the basis of the results presented above, this must reflect a unique acquired adaptive response of the phenotype.

The potential mechanisms that have been shown to result in protection against hyperoxia-induced injury include preserved surfactant function, attenuation of inflammation, and an adaptive increase in antioxidant components. Survival of SP-D–/– mice to hyperoxia was accompanied by preservation of surfactant function. Unlike SP-D+/+ animals, SP-D–/– mice were able to maintain higher BAL phospholipid levels, phospholipid-associated SP-B, and surfactant function early in hyperoxic exposure. Several previous studies have shown that exposure of rodents to either prolonged periods of hyperoxia or to oxidizing agents such as paraquat to induce a relatively severe oxidative insult resulted in significant lung dysfunction that was associated with oxidative changes in endogenous surfactant (29, 37, 50, 68) and depletion of surfactant phospholipid and surfactant proteins (35). Instillation of exogenous surfactant has been shown to attenuate the degree of alveolar injury caused by hyperoxia resulting in improved survival (51). Tokeida et al. (59, 60) have shown earlier that heterozygous SP-B-deficient mice are more susceptible to hyperoxia than SP-B+/+ mice and that this susceptibility can be restored by intratracheal administration of exogenous SP-B. Induction of tolerance in rabbits and rats by preexposure to hyperoxia has been shown to be accompanied by an increased level of BAL phospholipid (7, 67). The SP-D–/– phenotype appears to naturally recapitulate these events since SP-D–/– mice have two- to three-fold higher pool sizes of lung surfactant than wild-type C57BL/6 mice. Kramer et al. (44) have earlier shown a twofold variation in saturated phospholipid pool sizes between C57BL/6 and Swiss Black mice representing strain-specific steady-state levels not limited by rate of surfactant catabolism. We have observed that the median survival rate for Swiss Black mice exposed to 80% O2 is 6 days vs. 10 days for C57BL/6 mice vs. 100% survival for SP-D–/– mice (41), suggesting a direct correlation between phospholipid pool sizes and survival after hyperoxia.

Another characteristic feature of the surfactant system in SP-D–/– mice is a major decrease in hydrophilic SP-A (Table 1). Apart from the complete absence of SP-D, these mice have also been found to have 50% less SP-A than the wild-type mice. Although SP-A has been shown to have both anti- and proinflammatory properties (32), the exact relevance of SP-A deficiency in the lungs of SP-D–/– mice and its role in hyperoxia-induced lung injury needs to be investigated further. One possible investigative mechanism could be by means of SP-A–/– mice. Ikegami et al. (38) did not find any differences in survival or lung function of SP-A+/+ mice vs. mice deficient in SP-A after 3 days of exposure to 95% O2. However, these studies were done with small sample size, and possible adaptive mechanisms need to be evaluated further. Thus the lung surfactant of SP-D–/– mice is unique in that it contains higher phospholipid, twice as much SP-B but only one-half of the SP-A compared with the wild type.

We found that despite increased survival, SP-D–/– mice exhibit higher numbers of mono- and polymorphonuclear cells into the alveolar space after hyperoxia (Fig. 4). Paradoxically, even though we found increased number of alveolar cells (both macrophages and PMNs) in the SP-D-deficient mice, these animals also exhibited less parenchymal cells and less histopathological evidence of parenchymal injury. Despite altered inflammatory cell kinetics, analysis of the BAL from SP-D–/– mice suggests activation of effector cells at baseline. However, in vitro assays at baseline and during hyperoxia showed a significant downregulation of response to LPS or zymosan stimulation in SP-D–/–-derived macrophages (Fig. 8), suggesting an inherent inability of these cells to respond to stimuli. Acquired inactivation of pulmonary macrophages as a result of infection (human immunodeficiency virus), chronic inflammation (sepsis), and environmental factors such as smoke inhalation, malnutrition, and drugs is well established. Despite notable hyporesponsiveness in vitro, we observed approximately threefold higher levels of TNF-{alpha} in BAL fluid of SP-D–/– mice after 5 days of hyperoxia. It is possible that other cell types such as T cells and fibroblasts contribute to TNF-{alpha} detected in BAL. PMNs have also been shown to contribute to TNF production in a model of ventilation-induced lung injury (53).

Although the source of production is not addressed in this study, we observed that unlike SP-D+/+, SP-D–/– mice demonstrate a significant downregulation of nitric oxide production in the lung after hyperoxic exposure (Fig. 7), further supporting the notion that these macrophages acquire hyporesponsiveness. It is therefore possible that this baseline immunoactivation along with the attenuation of stimulus-induced responses contribute to a higher threshold level of tolerance to oxidant stress in SP-D-deficient mice, resulting in less parenchymal lung damage (Fig. 5B) and increased survival of these mice in hyperoxic conditions (Fig. 1).

Neonatal rats, mice, and rabbits exhibit relative tolerance to the pulmonary toxicity of high oxygen exposure compared with adult animals associated with enhanced differential biochemical protection (42) or with a rapid increased antioxidant response to hyperoxic challenge including, but not limited to, IL-6 (62), growth factors (57), and antioxidant enzymes. In this study, we did not find any differences in the IL-6 (Table 2) or in antioxidant enzyme extracellular superoxide dismutase (EC-SOD) levels (data not shown) in the SP-D-deficient mice. EC-SOD was of particular interest to us because this enzyme has been shown to be secreted in hyperoxia-exposed neonatal rats, which are more tolerant to oxidant stress (49). Furthermore, hyperoxia depletes endogenous EC-SOD in mice (56). Previous studies have shown that the classic antioxidant enzymes catalase, MnSOD and Cu,ZnSOD, do not change in hyperoxia-exposed rats (21) or mice (57), suggesting that these enzymes may play an ambiguous role in tolerance to hyperoxia.

We also estimated total thiols (protein and nonprotein, also known as disulfides or mercaptans) as an indirect measure of the redox status. Glutathione is a ubiquitously distributed molecule and a major antioxidant defense against reactive oxygen species/reactive nitrogen species in the cell. Several proteins in the signaling and metabolic pathways are susceptible to oxidative alterations and such cellular changes may prove to be physiologically relevant. The exposure of cysteine residues on the protein surface to reactive oxygen species or reactive nitrogen species can lead to S-thiolation within minutes after oxidative burst generation. The S-thiolated proteins represent an inactive state and can be retrieved by thioredoxins or glutaredoxins. Alternatively, these proteins may form mixed disulfides and then be reduced back to their active forms (33). We found an increase in total lung protein carbonyls and a decrease in mixed disulfides (Table 3), suggesting an increased oxidant burden in SP-D–/– mice. Together, the SP-D–/– mouse does not develop an adaptive increase in antioxidant strategy as a basis for tolerance to hyperoxia.

During a wide-ranging survey of BAL cytokines performed in this study, we found significant elevation in levels of IL-13 in BAL fluid of SP-D–/– mice after 3 days of exposure (Fig. 6). IL-13 is a T cell-derived, pleiotropic immunosuppressive cytokine, possibly produced by activated T lymphocytes. Activation of pulmonary T lymphocytes with a marked elevation in the proportion of memory CD4+ lymphocytes of CD44hi/CD62Llo phenotype has been previously reported in SP-D–/– mice (31). Murine IL-13 is known for downregulating cytotoxic and inflammatory functions of activated macrophages but has no effect on the antigen processing and presenting functions (27). A balance between T helper type 1 (Th1) and Th2 cytokines such as IL-13 can modulate innate immune responses in inflammation. A role for IL-13 in tolerance to high concentrations of O2 is supported by data from Corne et al. (22) who have shown increased survival of transgenic IL-13-overexpressing mice exposed to hyperoxia. Although this effect may be mediated, in part, by IL-13-induced expression of VEGF (22), IL-13 also decreases the production of nitric oxide by activated macrophages at both the protein and mRNA level (12). Nitric oxide, a product of inducible nitric oxide synthase (iNOS) activation, is known to be involved in a variety of physiological as well as pathophysiological processes such as signal transduction, innate immunity, and destruction of microbes and tumor cells. This effect of IL-13 on nitric oxide production is consistent with our observation that SP-D–/– mice downregulate nitric oxide production after hyperoxic exposure (Fig. 7). In contrast, SP-D+/+ mice respond to hyperoxic stress with significant upregulation of nitric oxide production. Bogdan et al. (12) have suggested that IL-13 does not block nitric oxide production completely and irreversibly but is responsible for maintenance of physiological or less toxic levels of nitric oxide through modulation (attenuation) of iNOS expression. At the same time, IL-13-stimulated macrophages demonstrate increased arginase activity, and the balance between arginase and NOS activities is thought to be important in inflammation repair and fibrosis. In addition to the existence of a highly protective, immunosuppressive microenvironment in SP-D-deficient mice, which makes them relatively tolerant to oxidant stress, counterregulation by a differential protective response is possible.

Hokuto et al. (36) looked at possible mechanisms involved in cellular signaling during hyperoxia and found that targeted disruption of Stat-3 can increase susceptibility to 95% O2. These authors show that Stat-3 influences expression of surfactant proteins as well as cytoprotective cytokines like the IL-6, IL-11, and transforming growth factor-{alpha} involved in maintenance of lung function during hyperoxia. Lian et al. (48) have also shown recently that overexpression of Stat-3 increases survival in 95% O2 partially by delaying acute capillary leakage, neutrophil infiltration, and hyperoxia-induced synthesis and secretion of matrix metalloproteinases. The characterization of molecules and/or pathways involved in the pathophysiology of hyperoxia was, however, beyond the scope of this present study and needs to be investigated further.

In summary, this report demonstrates an inherent tolerance of SP-D–/– mice to hyperoxic lung injury. The resistance to hyperoxia occurs despite a robust proinflammatory response in these animals. On the basis of the data presented in this study as well as studies in the literature, hyperoxia exposure results in a multifaceted immune response leading to inflammatory cell damage and death. The tolerance to hyperoxia is likewise multifactorial and involves both the immune and the endogenous surfactant systems. We propose that the tolerance of SP-D–/– mice to hyperoxia-induced acute lung injury may be due to 1) presence of a larger pool of endogenous surfactant able to counteract deleterious effects of hyperoxia and maintain lung patency, 2) an inherent inability of the immune effector cells to respond to oxidative stress due to the phenotype-induced lipidosis and proteinosis, and 3) existence of a general nonresponsive/immunosuppressive microenvironment generated as a result of targeted disruption of immunomodulatory actions of SP-D in vivo.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants HL-64520 and AT-000428 (to M. F. Beers), HL-074115 (to A. J. Gow and M. F. Beers), and HL-073896 (to R. C. Savani).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. F. Beers, Pulmonary and Critical Care Division, Univ. of Pennsylvania School of Medicine, Room H410F, Hill Pavilion, 380 South University Ave., Philadelphia, PA 19107 (e-mail: mfbeers{at}mail.med.upenn.edu)

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.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aderibigbe AO, Thomas RF, Mercer RR, Auten RL Jr. Brief exposure to 95% oxygen alters surfactant protein D and mRNA in adult rat alveolar and bronchiolar epithelium. Am J Respir Cell Mol Biol 20: 219–227, 1999.[Abstract/Free Full Text]
  2. Atochina E, Beers M, Tomer Y, Scanlon S, Russo S, Panettieri R, Haczku A. Attenuated allergic airway hyperresponsiveness in C57BL/6 mice is associated with enhanced surfactant protein (SP)-D production following allergic sensitization. Respir Res 4: 15, 2003.[CrossRef][Medline]
  3. Atochina EN, Beck JM, Preston AM, Haczku A, Tomer Y, Scanlon ST, Fusaro T, Casey J, Hawgood S, Gow AJ, Beers MF. Enhanced lung injury and delayed clearance of Pneumocystis carinii in surfactant protein A-deficient mice: attenuation of cytokine responses and reactive oxygen-nitrogen species. Infect Immun 72: 6002–6011, 2004.[Abstract/Free Full Text]
  4. Atochina EN, Beers MF, Hawgood S, Poulain F, Davis C, Fusaro T, Gow AJ. Surfactant protein-D, a mediator of innate lung immunity, alters the products of nitric oxide metabolism. Am J Respir Cell Mol Biol 30: 271–279, 2004.[Abstract/Free Full Text]
  5. Atochina EN, Beers MF, Scanlon ST, Preston AM, Beck JM. P. carinii induces selective alterations in component expression and biophysical activity of lung surfactant. Am J Physiol Lung Cell Mol Physiol 278: L599–L609, 2000.[Abstract/Free Full Text]
  6. Atochina EN, Gow AJ, Beck JM, Haczku A, Inch A, Kadire H, Tomer Y, Davis C, Preston AM, Poulain F, Hawgood S, Beers MF. Delayed clearance of Pneumocystis carinii infection, increased inflammation, and altered nitric oxide metabolism in lungs of Surfactant Protein D knockout mice. J Infect Dis 189: 1528–1539, 2004.[CrossRef][ISI][Medline]
  7. Baker RR, Holm BA, Panus PC, Matalon S. Development of O2 tolerance in rabbits with no increase in antioxidant enzymes. J Appl Physiol 66: 1679–1684, 1989.[Abstract/Free Full Text]
  8. Bartlett GR. Phosphorus assay in column chromatography. J Biol Chem 234: 466–468, 1959.[Free Full Text]
  9. Beers MF, Bates SR, Fisher AB. Differential extraction for the rapid purification of bovine surfactant protein B. Am J Physiol Lung Cell Mol Physiol 262: L773–L778, 1992.[Abstract/Free Full Text]
  10. Bhandari V, Johnson S, Smith-Kirwin S, Vigliotta G, Funanage V, Chander A. Hyperoxia and nitric oxide reduce surfactant components (DSPC and surfactant proteins) and increase apoptosis in adult and fetal rat type II pneumocytes. Lung 180: 301–317, 2002.[CrossRef][ISI][Medline]
  11. Bligh EG, Dyer WJ. A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37: 911–917, 1959.[Medline]
  12. Bogdan C, Thuring H, Dlaska M, Rollinghoff M, Weiss G. Mechanism of suppression of macrophage nitric oxide release by IL-13: influence of the macrophage population. J Immunol 159: 4506–4513, 1997. [Corrigenda. J Immunol 162: March 1999, p. 3106.][Abstract]
  13. Boggaram V. Regulation of surfactant protein gene expression by hyperoxia in the lung. Antiox Redox Signal 6: 185–190, 2004.[CrossRef][ISI][Medline]
  14. Botas C, Poulain F, Akiyama J, Brown C, Allen L, Goerke J, Clements J, Carlson E, Gillespie AM, Epstein C, Hawgood S. Altered surfactant homeostasis and alveolar type II cell morphology in mice lacking surfactant protein D. PNAS 95: 11869–11874, 1998.[Abstract/Free Full Text]
  15. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[CrossRef][ISI][Medline]
  16. Bridges JP, Davis HW, Damodarsamy M, Kuroki Y, Howles G, Hui DY, McCormack FW. Pulmonary surfactant proteins A and D are potent endogenous inhibitors of lipid peroxidation and oxidative cellular injury. J Biol Chem 275: 38848–38855, 2000.[Abstract/Free Full Text]
  17. Casey J, Kaplan J, Atochina-Vasserman EN, Gow AJ, Kadire H, Tomer Y, Fisher JH, Hawgood S, Savani RC, Beers MF. Alveolar surfactant protein D content modulates bleomycin induced lung injury. Am J Respir Crit Care Med 172: 869–877, 2005.[Abstract/Free Full Text]
  18. Christofidou-Solomidou M, Scherpereel A, Solomides C, Christie J, Stossel T, Goelz S, DiNubile M. Recombinant plasma gelsolin diminishes the acute inflammatory response to hyperoxia in mice. J Investig Med 50: 54–60, 2002.[ISI][Medline]
  19. Clark H, Palaniyar N, Strong P, Edmondson J, Hawgood S, Reid KBM. Surfactant protein D reduces alveolar macrophage apoptosis in vivo. J Immunol 169: 2892–2899, 2002.[Abstract/Free Full Text]
  20. Clark JC, Weaver TE, Iwamoto HS, Ikegami M, Jobe AH, Hull WM, Whitsett JA. Decreased lung compliance and air trapping in heterozygous SP-B-deficient mice. Am J Respir Cell Mol Biol 16: 46–52, 1997.[Abstract]
  21. Clerch LB, Massaro D. Tolerance of rats to hyperoxia. Lung antioxidant enzyme gene expression. J Clin Invest 91: 499–508, 1993.[ISI][Medline]
  22. Corne J, Chupp G, Lee CG, Homer RJ, Zhu Z, Chen Q, Ma B, Du Y, Roux F, McArdle J, Waxman AB, Elias JA. IL-13 stimulates vascular endothelial cell growth factor and protects against hyperoxic acute lung injury. J Clin Invest 106: 783–791, 2000.[ISI][Medline]
  23. Crapo JD. Morphologic changes in hyperoxic lung injury. Annu Rev Physiol 48: 721–731, 1986.[CrossRef][ISI][Medline]
  24. Crouch E, Parghi D, Kuan SF, Persson A. Surfactant protein D: subcellular localization in nonciliated bronchiolar epithelial cells. Am J Physiol Lung Cell Mol Physiol 263: L60–L66, 1992.[Abstract/Free Full Text]
  25. Crouch EC, Persson A, Griffin GL, Chang D, Senior RM. Interactions of pulmonary surfactant protein D (SP-D) with human blood leukocytes. Am J Respir Cell Mol Biol 12: 410–415, 1995.[Abstract]
  26. Crouch E, Wright JR. Surfactant proteins A and D and pulmonary host defense. Annu Rev Physiol 63: 521–554, 2001.[CrossRef][ISI][Medline]
  27. Doherty TM, Kastelein R, Menon S, Andrade S, Coffman RL. Modulation of murine macrophage function by IL-13. J Immunol 151: 7151–7160, 1993.[Abstract]
  28. Enhorning G. Pulmonary surfactant function studied with the pulsating bubble surfactometer (PBS) and the capillary surfactometer (CS). Comp Biochem Physiol A 129: 221–226, 2001.[CrossRef][Medline]
  29. Fisher HK, Clements JA, Tierney DF, Wright RR. Pulmonary effects of paraquat in the first day after injection. Am J Physiol 228: 1217–1223, 1975.[Abstract/Free Full Text]
  30. Fisher JH, Sheftelyevich V, Ho YS, Fligiel S, McCormack FX, Korfhagen TR, Whitsett JA, Ikegami M. Pulmonary-specific expression of SP-D corrects pulmonary lipid accumulation in SP-D gene-targeted mice. Am J Physiol Lung Cell Mol Physiol 278: L365–L373, 2000.[Abstract/Free Full Text]
  31. Fisher J, Larson J, Cool C, Dow S. Lymphocyte activation in the lungs of SP-D null mice. Am J Respir Cell Mol Biol 27: 24–33, 2002.[Abstract/Free Full Text]
  32. Gardai SJ, Xiao YQ, Dickinson M, Nick JA, Voelker DR, Greene KE, Henson PM. By binding SIRP[alpha] or calreticulin/CD91, lung collectins act as dual function surveillance molecules to suppress or enhance inflammation. Cell 115: 13–23, 2003.[CrossRef][ISI][Medline]
  33. Ghezzi P, Bonetto V, Fratelli M. Thiol-disulfide balance: from the concept of oxidative stress to that of redox regulation. Antiox Redox Signal 7: 964–972, 2005.[CrossRef][ISI][Medline]
  34. Gow AJ, Chen Q, Gole M, Themistocleous M, Lee VMY, Ischiropoulos H. Two distinct mechanisms of nitric oxide mediated neuronal cell death show thiol dependency. Am J Physiol Lung Cell Mol Physiol 278: L1099–L1107, 2000.
  35. Hokuto I, Ikegami M, Yoshida M, Takeda K, Akira S, Perl AK, Hull WM, Wert SE, Whitsett JA. Stat-3 is required for pulmonary homeostasis during hyperoxia. J Clin Invest 113: 28–37, 2004.[CrossRef][ISI][Medline]
  36. Hokuto I, Ikegami M, Yoshida M, Takeda K, Akira S, Perl AK, Hull WM, Wert SE, Whitsett JA. Stat-3 is required for pulmonary homeostasis during hyperoxia. J Clin Invest 113: 28–37, 2004.[CrossRef][ISI][Medline]
  37. Holm BA, Notter RH, Siegle J, Matalon S. Pulmonary physiological and surfactant changes during injury and recovery from hyperoxia. J Appl Physiol 59: 1402–1409, 1985.[Abstract/Free Full Text]
  38. Ikegami M, Jobe AH, Whitsett J, Korfhagen T. Tolerance of SP-A-deficient mice to hyperoxia or exercise. J Appl Physiol 89: 644–648, 2000.[Abstract/Free Full Text]
  39. Ikegami M, Na CL, Korfhagen TR, Whitsett JA. Surfactant protein D influences surfactant ultrastructure and uptake by alveolar type II cells. Am J Physiol Lung Cell Mol Physiol 288: L552–L561, 2005.[Abstract/Free Full Text]
  40. Ikegami M, Whitsett JA, Jobe A, Ross G, Fisher J, Korfhagen T. Surfactant metabolism in SP-D gene-targeted mice. Am J Physiol Lung Cell Mol Physiol 279: L468–L476, 2000.[Abstract/Free Full Text]
  41. Jain D, Atochina-Vasserman E, Kadire H, Tomer Y, Inch A, Beers MF. Surfactant protein D protects against hyperoxia-induced acute lung injury in a murine model. FASEB J 20: A1441, 2006.[Free Full Text]
  42. Johnston CJ, Wright TW, Reed CK, Finkelstein JN. Comparison of adult and newborn pulmonary cytokine mRNA expression after hyperoxia. Exp Lung Res 23: 537–552, 1997.[ISI][Medline]
  43. Korfhagen TR, Sheftelyevich V, Burhans MS, Bruno MD, Ross GF, Wert SE, Stahlman MT, Jobe AH, Ikegami M, Whitsett JA, Fisher JH. Surfactant protein-D regulates surfactant phospholipid homeostasis in vivo. J Biol Chem 273: 28438–28443, 1998.[Abstract/Free Full Text]
  44. Kramer BW, Ikegami M, Jobe AH. Surfactant phospholipid catabolic rate is pool size dependent in mice. Am J Physiol Lung Cell Mol Physiol 279: L842–L848, 2000.[Abstract/Free Full Text]
  45. LeVine AM, Whitsett JA, Gwozdz JA, Richardson TR, Fisher JH, Burhans MS, Korfhagen TR. Distinct effects of surfactant protein A or D deficiency during bacterial infection on the lung. J Immunol 165: 3934–3940, 2000.[Abstract/Free Full Text]
  46. LeVine AM, Whitsett JA, Hartshorn KL, Crouch EC, Korfhagen TR. Surfactant protein D enhances clearance of influenza A virus from the lung in vivo. J Immunol 167: 5868–5873, 2001.[Abstract/Free Full Text]
  47. Levine RL, Williams JA, Startman ER, Shacter E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 233: 346–357, 1994.[ISI][Medline]
  48. Lian X, Qin Y, Hossain SA, Yang L, White A, Xu H, Shipley JM, Li T, Senior RM, Du H, Yan C. Overexpression of Stat3C in pulmonary epithelium protects against hyperoxic lung injury. J Immunol 174: 7250–7256, 2005.[Abstract/Free Full Text]
  49. Mamo LB, Suliman HB, Giles BL, Auten RL, Piantadosi CA, Nozik-Grayck E. Discordant extracellular superoxide dismutase expression and activity in neonatal hyperoxic lung. Am J Respir Crit Care Med 170: 313–318, 2004.[Abstract/Free Full Text]
  50. Matalon S, Holm BA, Loewen GM, Baker RR, Notter RH. Sublethal hyperoxic injury to the alveolar epithelium and the pulmonary surfactant system. Exp Lung Res Suppl: 1021–1033, 1988.
  51. Matalon S, Holm BA, Notter RH. Mitigation of pulmonary hyperoxic injury by administration of exogenous surfactant. J Appl Physiol 62: 756–761, 1987.[Abstract/Free Full Text]
  52. Narula P, Xu J, Kazzazz JA, Robbins CG, Davis JM, Horowitz S. Synergistic cytotoxicity from nitric oxide and hyperoxia in cultured lung cells. Am J Physiol 274: 537–552, 1998.
  53. Noda E, Hoshina H, Watanabe H, Kawano T. Production of TNF-alpha by polymorphonuclear leukocytes during mechanical ventilation in the surfactant-depleted rabbit lung. Pediatr Pulmonol 36: 475–481, 2003.[CrossRef][ISI][Medline]
  54. Nogee LM, Wispe J, Clark JC, Weaver TE, Whitsett JA. Increased expression of pulmonary surfactant proteins in oxygen-exposed rats. Am J Respir Cell Mol Biol 4: 102–107, 1991.[ISI][Medline]
  55. O'Reilly MA. DNA damage and cell cycle checkpoints in hyperoxic lung injury: braking to facilitate repair. Am J Physiol Lung Cell Mol Physiol 281: L291–L305, 2001.[Abstract/Free Full Text]
  56. Oury TD, Schaefer LM, Fattman CL, Choi A, Weck KE, Watkins SC. Depletion of pulmonary EC-SOD after exposure to hyperoxia. Am J Physiol Lung Cell Mol Physiol 283: L777–L784, 2002.[Abstract/Free Full Text]
  57. Perkowski S, Sun J, Singhal S, Santiago J, Leikauf GD, Albelda SM. Gene expression profiling of the early pulmonary response to hyperoxia in mice. Am J Respir Cell Mol Biol 28: 682–696, 2003.[Abstract/Free Full Text]
  58. Tokieda K, Ikegami M, Wert SE, Baatz JE, Zou Y, Whitsett JA. Surfactant protein B corrects oxygen-induced pulmonary dysfunction in heterozygous surfactant protein B-deficient mice. Pediatr Res 46: 708–714, 1999.[ISI][Medline]
  59. Tokieda K, Iwamoto HS, Bachurski C, Wert SE, Hull WM, Ikeda K, Whitsett JA. Surfactant protein-B-deficient mice are susceptible to hyperoxic lung injury. Am J Respir Cell Mol Biol 21: 463–472, 1999.[Abstract/Free Full Text]
  60. Van Iwaarden JF, Shimizu H, Van Golde PH, Voelker DR, Van Golde LM. Rat surfactant protein D enhances the production of oxygen radicals by rat alveolar macrophages. Biochem J 286: 5–8, 1992.[ISI][Medline]
  61. Ward NS, Waxman AB, Homer RJ, Mantell LL, Einarsson O, Du Y, Elias JA. Interleukin-6-induced protection in hyperoxic acute lung injury. Am J Respir Cell Mol Biol 22: 535–542, 2000.[Abstract/Free Full Text]
  62. Wert SE, Yoshida M, LeVine AM, Ikegami M, Jones T, Ross GF, Fisher JH, Korfhagen TR, Whitsett JA. Increased metalloproteinase activity, oxidant production, and emphysema in surfactant protein D gene-inactivated mice. PNAS 97: 5972–5977, 2000.[Abstract/Free Full Text]
  63. White CW, Greene KE, Allen CB, Shannon JM. Elevated expression of surfactant proteins in newborn rats during adaptation to hyperoxia. Am J Respir Cell Mol Biol 25: 51–59, 2001.[Abstract/Free Full Text]
  64. Wispe JR, Roberts RJ. Molecular basis of pulmonary oxygen toxicity. Clin Perinatol 14: 651–666, 1987.[ISI][Medline]
  65. Yoshida M, Korfhagen TR, Whitsett JA. Surfactant protein D regulates NF-