Surfactant Protein D Protects against Acute Hyperoxic
Deepika Jain1, Elena N. Atochina-Vasserman1, Yaniv Tomer1, Helchem Kadire1, and Michael F. Beers1
1Surfactant Biology Laboratories, Pulmonary and Critical Care Division, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania
Rationale: Surfactant protein D (SP-D) is a member of the collectin
lipid homeostasis, and increased oxidative-nitrative stress.
Objectives: To test the hypothesis that SP-D would protect against
acute lung injury from hyperoxia in vivo.
Measurements and Main Results: Compared with littermate control
mice (wild-type [WT]), SP-D OE mice exposed to 80% O2demon-
strated substantially increased survival accompanied by significant
in total BAL cells and neutrophilia in response to hyperoxia, the SP-D
OE group had lower levels of BAL proinflammatory cytokines and
chemokines, including IL-6, tumor necrosis factor-a, and monocyte
chemotactic protein-1; increased mRNA levels of the transcription
factor NF-E2 related factor-2 (NRF-2) and phase 2 antioxidants
hemoxygenase-1 (HO-1), glutathione peroxidase-2 (GPx-2) and
NAD(P)H quinone oxidoreductase-1 (Nqo-1); and decreases in lung
tissue thiobarbituric acid–reactive substances. As proof of principle,
Dox-on mice exposed to 85% O2demonstrated increased mortality
upon withdrawal of doxycycline.
Conclusions: Local expression of SP-D protects against hyperoxic lung
injury through modulationofproinflammatorycytokines andantiox-
idant enzymatic scavenger systems.
Keywords: innate immunity; inflammation; collectin; antioxidants;
The distal pulmonary airspaces perform the coordinated func-
tion of gas exchange through a delicate balance of ventilation
and perfusion matching requiring regulated blood flow to
functional alveolar-capillary units. Alveolar integrity during
the respiratory cycle in vivo is maintained by a functionally
active monolayer of lung surfactant produced by alveolar type 2
cells and deposited at the air–liquid interface (1). The role of
surfactant lipid and its hydrophobic surfactant protein (SP)
components, SP-B and SP-C, in reducing surface tension and
stabilizing alveolar structures at low lung volumes is well known
(2). However, in addition to its gas exchange functions, the
epithelial surface of the alveoli and small conducting airways
must contend with a continuous bombardment with pathogens,
particles, and toxins during the course of normal breathing.
Nonetheless, the lung surface is maintained in an immunolog-
ically quiescent state through a complex interaction of effector
immune cells and soluble mediators. Related to this, it is be-
coming increasingly recognized that two other surfactant pro-
tein components, SP-A and SP-D, are a critical part of this local
immunologic modulation during lung injury (3–5).
SP-D, a 43-kD member of the collectin superfamily, is
a relatively minor component of lung surfactant but is of critical
importance to lung homeostasis. From a large volume of in vitro
and in vivo studies, SP-D has been shown to be an immunolog-
ically multifunctional, innate immune molecule. In addition to
recognizing and binding allergens, particles, bacterial cell wall
components, and viral envelope proteins in a pattern-specific
manner (4), SP-D can influence recruitment and activation of
effector immune cells in the lung. SP-D enhances actin polymer-
ization (6), promotes chemotaxis of macrophages and monocytes
(7), and modulates recruitment and function of neutrophils (8, 9)
and lymphocytes (10, 11). In vivo, the use of transgenic mouse
models suggests a protective role for SP-D against damage by
inflammatory stimuli. Local up-regulation of SP-D protein levels
in wild-type mice occurs after a variety of infectious and
inflammatory lung injuries, including bacterial and fungal pneu-
monia, bleomycin, and hyperoxia (12–14), while mice constitu-
tively deficient in SP-D develop, at baseline, progressive lung
inflammation and time-dependent airspace remodeling (15).
Hyperoxic lung injury is mediated by reactive oxygen species
of the lung (cellular and extracellular pathways) to effect their
removal. The injury is characterized by molecular and structural
modifications to proteins, lipids, and nucleic acids, accompanied
by morphologic and physiologic damage to the lung. Breakdown
of epithelial–endothelial barrier function, accumulation of
edema, recruitment and activation of inflammatory cells, elabo-
ration of cytokines, and development of shunt physiology com-
seen in the acute respiratory distress syndrome. The exact role of
pulmonary surfactant components in the pathogenesis or evolu-
tion of hyperoxic lung injury is incompletely defined. Perturba-
tions in homeostasis and biophysical activity of lung surfactant
lipids after ventilation-induced or hyperoxic injury are well
established (16, 17). Although these hydrophobic surfactant
AT A GLANCE COMMENTARY
Scientific Knowledge on the Subject
Surfactant protein D (SP-D), a pulmonary collectin with
important immunomodulatory properties, is up-regulated
in response to various inflammatory and infectious stimuli.
The role of SP-D in modulating inflammatory events in
acute lung injury is undefined.
What This Study Adds to the Field
These results provide in vivo evidence for an antiinflam-
matory effect of SP-D in response to noninfectious acute
lung injury and suggest a potential new therapeutic role for
SP-D against hyperoxic lung injury.
(Received in original form April 18, 2008; accepted in final form July 17, 2008)
Supported by National Institutes of Health grant HL 64520 (M.F.B.).
Correspondence and requests for reprints should be addressed to Michael F.
Beers, M.D., Pulmonary and Critical Care Division, University of Pennsylvania
School of Medicine, SVM Hill Pavilion Room H410F, 380 S. University Avenue,
Philadelphia, PA 19104-4539. E-mail: email@example.com
Am J Respir Crit Care Med
Originally Published in Press as DOI: 10.1164/rccm.200804-582OC on July 17, 2008
Internet address: www.atsjournals.org
Vol 178. pp 805–813, 2008
at the mRNA and/or protein levels after exposure to O2concen-
trations greater than or equal to 80% (5, 18).
Although distinct from hyperoxia, the intratracheal adminis-
tration of bleomycin to rodents also produces subacute lung injury
followed by resolution, fibrosis, and/or repair via a mechanism that
involves free radicals and oxidant stress. We have previously
shown that SP-D null mice demonstrate enhanced susceptibility to
intratracheal administration of bleomycin, whereas SP-D over-
expressing mice were protected (14). Based on these results and
on data showing increased oxidative stress in SP-D null mice, we
hypothesized a protective role for SP-D in hyperoxic lung injury.
Using a transgenic model that constitutively overexpresses rat
SP-D (SP-D OE) and a doxycycline-regulated, conditional SP-D
expressing mouse, we characterized the effects of SP-D on the
pulmonary response to hyperoxia. We demonstrate that SP-D is
protective against hyperoxia and that the enhanced survival in
SP-D OE mice is accompanied by suppression of proinflammatory
cytokines and induction of key antioxidant response genes.
Collectively, these results demonstrate important novel functions
for SP-D in the local modulation of lung inflammation in the distal
airways. A preliminary report of some of these results has been
previously published in the form of an abstract (19).
SP-D OE Mice
Mice overexpressing the rat isoform of SP-D (rSP-D) in a lung-specific
fashion using the human 23.7 kb SP-C promoter on the Swiss Black
and have been described previously (14, 20). Heterozygous transgenic
mice carrying a single concatamer of the rSP-D transgene were crossed
with wild-type SWB mice to generate SP-D OE mice and littermate
control (wild-type [WT]) mice. All mice were maintained under specific
pathogen-free conditions in the barrier facilities at the University of
Triple Transgenic SP-D Conditionally Expressing Mice
Breeding pairs of conditional SP-D expressing mice were kindly provided
by Dr. Jeffery Whitsett and have been described previously (21). Briefly,
ratCCSP-rtTA transgenic mice were mated with (tetO)7-rSP-D trans-
genic mice to generate double transgenic mice (ratCCSP-rtTA 1,
(tetO)7-rSP-D 1). Double transgenic mice were mated with SP-D2/2
mice to generate heterozygous (ratCCSP-rtTA 1, (tetO)7-rSP-D 1,
mSP-D1/2) mice. Heterozygous triple transgenic mice expressing rat
SP-D on a SP-D2/2background were then bred to homozygosity in our
facility and maintained on a doxycycline-supplemented diet (625 ppm).
Mice were exposed to hyperoxia in the Core Facility at the Institute for
Environmental Medicine as described in detail previously (18). SP-D
OE and littermate WT mice were exposed to room air (normoxia
control group) or to 80% or 85 6 2% O2(hyperoxia group) in sealed
Plexiglas chambers (Braintree Scientific, Inc., Braintree, MA). O2
levels were continuously monitored using an O2analyzer (Pacifitech,
Temecula, CA). Animals were housed in environment-controlled cages
and allowed food and water ad libitum during the exposure. Cages
were opened periodically for change of water, food, and bedding and as
required for removal of dead mice. Conditional SP-D mice were kept
on a doxycycline-supplemented diet (z625 ppm) or transferred to
normal chow 2 weeks before hyperoxia exposure.
All study protocols, animal care, and procedures had been reviewed
before the initiation of work by the Institutional Animal Care and Use
Committee of the University of Pennsylvania.
Preparation and Analysis of Bronchoalveolar Lavage
Lungs were lavaged with five 1.0-ml aliquots of sterile saline. Process-
ing and analysis of bronchoalveolar lavage (BAL) has been described
previously (18). Briefly, cell pellets obtained by centrifuging BAL sam-
ples at 400 3 g for 10 minutes were re-suspended in 1 ml of PBS, and
total cell counts were determined using a Z1 particle counter (Beck-
man-Coulter, Inc., Miami, FL). Cytospins prepared from an aliquot of
each cell suspension were stained with Diff-Quik, and manual differ-
ential cell counts were performed.
A 200-ml aliquot of each cell-free BAL was removed from the first
by Pierce Biotechnology (Woburn, MA). The remaining BAL was sepa-
rated into large-aggregate (LA) and small aggregate fractions by centrifu-
gation at 20,000 3 g for 60 minutes at 48C as described previously (22).
Total protein content of both fractions was determined by the
Bradford method with bovine IgG as a standard (23). Total lipids were
extracted from LA and small aggregate surfactant fractions with
chloroform-methanol as described previously (24). Total phospholipid
content in each fraction was determined by Bartlett’s colorimetric
estimation of inorganic phosphorus (25).
PAGE and Immunoblotting
BAL samples were separated using NuPAGE NOVEX-10% Bis-Tris
gels (Invitrogen, Inc., Carlsbad, CA). Immunoblots were performed
with monospecific, polyclonal surfactant protein antisera to SP-A or
SP-D. Total SP-D was determined using a rabbit polyclonal antibody
that recognizes mouse and rat SP-D (22). Because the SP-D OE mice
express a rSP-D transgene, rat SP-D levels were determined using
a commercially available monoclonal antibody against rat SP-D (Clone
VIF9; HyCult Biotechnology, Uden, The Netherlands). Bands were
visualized using horeseradish peroxidase–conjugated secondary goat
anti-rabbit IgG (Jackson Immunoresearch Laboratories, West Grove,
PA) and enhanced chemiluminescence (Amersham, Inc., Arlington
Heights, IL). Band intensity was quantitated by densitometric scanning
of exposed films or by direct acquisition on a Kodak 440 Imaging
System (Eastman Kodak Co., New Haven, CT).
Lung Histology and Estimation of Lipid Peroxides
After lavage, the left lobe of the lung was inflation fixed with 0.5 ml of
10% neutral buffered formalin for histologic analysis. Paraffin sections
prepared from the lungs were stained with hematoxylin and eosin for
evaluation of airway inflammation and were scored based on the
presence or absence of peribronchiolar infiltration, alveolar septal
thickening, vascular congestion, alveolar edema, and cellular infiltra-
tion into the alveoli as described previously (18).
Total lipid peroxides in lung samples were estimated by the method of
Fisher and colleagues (26). Briefly, lungs snap frozen in liquid N2were
homogenized in ice-cold saline containing 0.01% butylatedhydroxyto-
luene, deproteinized using 15% trichloroacetic acid, and boiled with
thiobarbituric acid at 908C for 15 minutes. Samples were immediately
cooled on ice and centrifuged at 4,000 rpm for 30 minutes, and the
absorbance of the resulting supernatant was read at 535 nm. Thiobarbi-
turic acid reactive substances (TBARS) were estimated using an extinc-
tion coefficient of 1.56 3 105/M/cm and expressed as pmol/mg protein.
Measurements of Surface Tension
The LA fraction of BAL was diluted to 1 mg/ml phospholipid concen-
tration for assessment of biophysical activity of recovered surfactant in
a capillary surfactometer as previously described (27). Samples (0.5 ml)
introduced into a glass capillary were compressed for 120 seconds,
resulting in cyclic extrusion of the surfactant from the capillary permitting
detectable airflow. Biophysical dysfunction in the sample results in loss of
capillary ‘‘openness.’’ Data were expressed as the percentage of the 120-
second study period that the capillary is open to a free airflow.
Quantitative Real-Time Reverse Transcription–Polymerase
Isolation of total lung RNA was performed using Trizol reagent
(Invitrogen, Inc., Carlsbad, CA). Reverse transcription of the DNase-
treated total RNA was performed using a RETROscript First Strand
Synthesis kit for real-time reverse transcription–polymerase chain
reaction (Applied Biosystems, Foster City, CA) and random decamer
primers. Quantitative real-time reverse transcription–polymerase chain
reaction analyses of transcription factor NRF-2 (Mm00477784_m1) and
806AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 1782008
antioxidant genes GPx-2 (Mm00850074_g1), HO-1 (Mm00516004_m1),
and Nqo-1 (Mm00500821_m1) were performed with TaqMan Gene
Expression Assays (Applied Biosystems; Foster City, CA) using an ABI
7500 FAST real-time polymerase chain reaction system. 18S RNA
(Hs99999901_s1) was used for normalization.
Parametric data from experimental and control groups were expressed as
mean 6 SEM, and groups were compared using an unpaired, two-tailed
Student’s t test or analysis of variance (ANOVA) using Instat version 3.0
(GraphPad Software, Inc., La Jolla, CA). Analysis on all parametric data
was done using one-way ANOVA followed by Tukey-Kramer multiple
comparisons test or unpaired t tests. Nonparametric data were expressed
as group mean values 6 SEM, and statistical comparison was done using
Kruskal-Wallis test followed by Dunn’s multiple comparison test or Mann
Whitney U test. In all cases, P < 0.05 was accepted as significant.
SP-D OE Mice Demonstrate Increased Survival in Hyperoxia
SP-D OE mice survived considerably longer in 80% O2than
similarly exposed SWB littermates. The median survival for the
WT group subjected to hyperoxia was 6 days, with 100%
mortality by Day 14 (Figure 1). In contrast, over two-thirds
(68.8%) of SP-D OE mice survived at least 14 days under
identical conditions. When assessed just before the onset of
mortality (Day 5), WT mice were found to have a significantly
greater loss of body weight compared with the similarly exposed
SP-D OE group (84 6 2% of initial body weight vs. 90 6 1% of
initial body weight for SP-D OE; n 5 11–12; P , 0.05).
To gain insight into the underlying mechanisms mediating this
survival advantage, we elected to assess the kinetics of hyperoxia-
SP-D OE Mice Have Less Hyperoxia-induced Lung Leak
Lung edema was significantly worse in WT mice exposed to
hyperoxia. There was a 25% increase in wet to dry lung weight
ratio in the WT group after 5 days of exposure (Figure 2A).
Commensurate with this observation, the alveolar protein
leak seen during hyperoxic exposure was markedly attenuated
in the SP-D OE group (Figure 2B). Under normoxic conditions,
no differences were observed between the strains (375.6 6 49.4
mg in SP-D OE vs. 374.2 6 47.2 mg in WT). During hyperoxia,
BAL protein levels rose in both groups when assessed at 3 days
(639.7 6 77.9 mg in SP-D OE vs. 987.7 6 108.4 mg in WT; P ,
0.05) or at 5 days of exposure (2,678.2 6 267.4 mg in SP-D OE
vs. 3,984.3 6 503.6 mg in WT; P , 0.05).
Surfactant Composition and Function Is Altered in SP-D OE
Mice and Littermate Control Mice
Surfactant activity can be altered in various forms of lung injury
due to protein leak or altered component expression. Measure-
ment of total BAL phospholipids revealed that these were
similarly depleted to about half that of the normoxia-exposed
mice after 3 days of exposure in the SP-D OE and WT groups
(Figure 3A). There was a partial recovery to approximately
60% of baseline values by Day 5. Concomitant with this loss of
surfactant, each hyperoxic group had similar degrees of in-
hibition of surfactant biophysical function as assessed by
capillary surfactometer (capillary open time 5 47.6 6 12.6%
for WT and 31.6 6 10.7% for OE; P . 0.05). Therefore, the
differences in survival observed between the groups cannot be
attributed solely to differences in surfactant function.
models. BAL from SP-D OE mice contained six- to eightfold
higher amounts of SP-D than littermate control mice at baseline
(Figure 3B). During hyperoxia, WT mice developed anacute SP-
D deficiency, with significant depletion of BAL SP-D to about
5. Total BAL SP-D in SP-D OE mice continued to increase
throughout hyperoxic exposure, reaching levels four- to fivefold
survival under hyperoxic conditions. SP-D overexpressing mice (OE)
and littermate control mice (7–8 wk of age) were exposed to 80%
oxygen. 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 OE mice (P , 0.0001 vs. corresponding control groups).
Data are representative of two separate experiments. n 5 25 for
hyperoxia groups; n 5 5 for normoxia control mice. WT 5 wild type.
Effect of surfactant protein D (SP-D) overexpression on
pression on alveolar–capillary integrity. (A) Alveolar capil-
lary leak was determined as the ratio of wet to dry lung
weights (n 5 6–11) in wild-type (WT) and SP-D over-
expressing (SP-D OE) mice. Data for each group are
expressed as mean 1 SEM, expressed as % of WT
normoxia. **P , 0.01 vs. WT normoxia. (B) Bronchoal-
veolar lavage (BAL) total protein from the same time
points (n 5 9–12 per group) were determined as de-
scribed in METHODS. Data are mean 1 SEM, expressed as
mg protein in BAL. *P , 0.05; ***P , 0.0001 vs. WT
normoxia group;#P , 0.05;###P , 0.0001 vs. SP-D OE
Effect of surfactant protein D (SP-D) overex-
Jain, Atochina-Vasserman, Tomer, et al.: SP-D Is Protective against Hyperoxia807
higher than the SP-D OE normoxia group after 5 days. Using
a monoclonal antibody specific for rat SP-D, Western blotting
revealed that after 5 days of O2exposure rSP-D immunoreactiv-
ity increased in SP-D OE mice by approximately threefold (vs.
the normoxia SP-D OE group), indicating that the increased
levels observed were in part due to up-regulation of transgene
no major differences in SP-A levels in the LA fraction of BAL at
baseline (99.9 6 19.6% of normoxia control in WT and 72.8 6
control in WT and 63.8 6 7.9% in SP-D OE).
Inflammatory Cell Influx Is Similar in WT and SP-D OE Mice
Total and differential cell counts were performed on BAL cell
pellets (Figure 4). WT and SP-D OE mice had similar total cell
counts at baseline (3.5 6 0.13 3 105vs. 4.1 6 0.42 3 105cells,
respectively). Both groups exhibited approximately a twofold
increase after 5 days of exposure to 80% O2, but there was no
significant difference between groups (Figure 4A). Similarly,
BAL neutrophils in WT and SP-D OE mice increased to similar
levels after hyperoxia (2.3 6 0.4 3 105vs. 1.7 6 0.5 3 105cells,
respectively) (Figure 4B). There were no differences in BAL
macrophages, lymphocytes, and eosinophils before or after
exposure or between groups (not shown).
Histopathologic examination and semiquantitative scoring of
hematoxylin-and-eosin-stained lungs of WT mice exposed to
80% O2showed marked perivascular cuffing, interstitial thick-
was significantly attenuated in the SP-D OE mice (Figure 5).
Cytokine Production Is Substantially Repressed in SP-D OE
Mice after Hyperoxia
Cytokine profiles in BAL were assessed using a mutiplex assay.
We observed a significant repression of cytokine production in
SP-D OE group. IL-10, IFN-g, granulocyte/macrophage colony–
stimulating factor, and IL-8 levels in BAL of SP-D OE mice
were significantly lower after 5 days of hyperoxia, whereas IL-6
and monocyte chemotactic protein (MCP)-1 levels were re-
duced after 3 and 5 days of exposure (Table 1).
SP-D OE Mice Respond to Hyperoxia by Up-regulating the
Expression of Phase II Antioxidant Genes
Resistance to hyperoxia has been associated with selective up-
regulation of antioxidant genes and detoxifying enzymes. We
first looked at the transcription factor NRF-2, which acts via the
ubiquitous antioxidant response element (ARE) binding site in
the promoter region to regulate expression of various stress
response genes, such as HO-1, GPx-2, and Nqo1. We observed
a 2.5-fold increase in NRF-2 mRNA levels after 5 days of
hyperoxia compared with no change in similarly exposed
littermate control mice (Figure 6). Concomitant with this
increase, we observed a parallel three- to fivefold increase in
mRNA levels of stress-responsive genes HO-1, GPx-1, and
Nqo1. Oxidant-mediated tissue injury in these mice was
assessed by measuring TBARS in postlavage lung homogenates.
We observed a twofold increase in the level of TBARS in WT
mice exposed to 80% O2for 5 days, which was completely
abrogated in the SP-D OE group (Figure 7).
flammatory cell recruitment. Bronchoalveolar lavage (BAL)
(A) total and (B) neutrophil cell counts were performed as
described in METHODS after exposure of SP-D overexpress-
ing (OE) and wild-type (WT) mice to 80% O2. Data for
each group are expressed as mean 6 SEM (n 5 9–13)
(*P , 0.05 and ***P , 0.0001 vs. WT normoxia group;
#P , 0.05 and###P , 0.0001 vs. SP-D OE normoxia
group). SP-D OE and littermates demonstrate similar in-
filtration of proinflammatory cells in the lungs in response
Effect of surfactant protein D (SP-D) on in-
Figure 3. Effect of surfactant protein D (SP-D) over-
expression on surfactant components during hyper-
oxic injury. SP-D overexpressing (SP-D OE) mice and
littermate control mice were exposed to 80% O2,
and mice were killed at different time points. Total
phospholipid and SP-D in bronchoalveolar lavage
(BAL) was determined as described in METHODS. (Left)
Total phospholipid in BAL was estimated using
a modification of the colorimetric Bartlett method
as described. Data, normalized to % wild-type (WT)
level, are expressed as mean 6 SEM. (n 5 9–12; *P ,
0.05; ***P , 0.0001 vs. WT control group. BAL
surfactant phospholipid levels in SP-D OE and WT
mice are similarly altered after hyperoxia. (Right)
Immunoblots against SP-D in small aggregate of
BAL were performed as described in METHODS (n 5 5–12) and quantified by densitometry on a KODAK imaging system. Data are expressed as
mean 6 SEM, normalized to % WT level (*P , 0.05 vs. WT control group;##P , 0.01 vs. SP-D OE control group). Unlike WT mice, SP-D OE mice do
not demonstrate an initial depletion of BAL SP-D.
808AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINE VOL 1782008
Loss of Lung-specific Expression of SP-D Confers
Susceptibility to Hyperoxia
To confirm our hypothesis that SP-D plays a protective role in
hyperoxic lung injury, we used mice expressing SP-D under the
control of a doxycycline-dependent promoter on a SP-D–null
background. These mice, on an FVB background, were bred to
homozygosity in our laboratory. Western blots on BAL from
these mice demonstrate that the animals maintained on a doxy-
cycline-supplemented diet (SP-D Dox-on group) produce about
10- to 15-fold higher basal levels of SP-D than B6 mice. When
doxycycline is removed from the diet (Dox-off group), SP-D
levels decline precipitously (Figure 8A). Using prolonged expo-
sures, trace amounts of SP-D could be detected at Day 14 on
Western blots but account for less than 3% of the levels seen in
TABLE 1. BRONCHOALVEOLAR LAVAGE PROFILE AFTER HYPEROXIA
Cytokine3 d 5 d0 d 3 d5 d
8.7 6 1.9†
1.8 6 0.3
3.8 6 0.8†
1.4 6 0.3
50.0 6 19.4†
3.1 6 0.4
2.1 6 0.47
2.16 6 0.28
2.16 6 0.27
5,284.3 6 1,941.7*
7.1 6 2.7*
0.2 6 0.1
7.96 6 2.9*
1.7 6 0.7
4.8 6 1.5†
5.8 6 2.2†
12.6 6 5.37†
153.3 6 60.1†
0.97 6 0.15
0.99 6 0.17
1.0 6 0.15
0.94 6 0.18
1.0 6 0.15
0.96 6 0.22
0.92 6 0.15
0.98 6 0.1
0.95 6 0.15
1.8 6 0.1‡
1.4 6 0.2
2.04 6 0.9
1.69 6 0.15
2.57 6 0.9†
2.0 6 0.28
1.4 6 0.17
1.08 6 0.3
1.08 6 0.2
423.6 6 211.1† ‡
0.6 6 0.2†
0.1 6 0.07
1.1 6 0.65†
1.0 6 0.5
1.3 6 0.57‡
1.7 6 0.8
3.08 6 1.08‡
43.7 6 27.0‡
Definition of abbreviations: GM-CSF 5 granulocyte/macrophage colony-stimulating factor; KC 5 keratinocyte chemoattractant; MCP-1 5 monocyte chemotactic
protein-1; MIP-2 5 macrophage inflammatory peptide-2; SP-D OE 5 surfactant protein D–overexpressing mice; WT 5 wild-type mice.
Cytokine levels in bronchoalveolar lavage were estimated by Searchlight Multiplex cytokine assay. Values are fold changes as compared with WT normoxia group. Data for
each group are expressed as mean 6 SEM (n 5 9–12). Multiple-group comparisons were done using one-way analysis of variance, followed by t tests.
* P , 0.05 vs. WT 0 d.
†P , 0.05 vs. similarly exposed WT.
‡P , 0.05 vs. SP-D OE 0 d.
actant protein D (SP-D) on
inflammation. (A) Represen-
eosin–stained lung sections
(OE) and littermate wild-type
(WT) mice exposed to 21%
O2 (Day 0) or to hyperoxia
for 3 and 5 days. (B) Histo-
logic scoring was performed
as published previously (18).
Data for each group are
expressed as mean 6 SEM
(n 5 5–12). Multiple group
comparisons were done by
analysis of variance, followed
by Dunn’s multiple group
comparisons. *P , 0.05 vs.
WT normoxia group;
0.05 vs. SP-D OE control
(normoxia) group. Similarly
exposed WT and SP-D OE
hyperoxia groups were com-
Jain, Atochina-Vasserman, Tomer, et al.: SP-D Is Protective against Hyperoxia809
SP-D Dox-on mice. However, acute loss of SP-D did not alter
baseline total phospholipid levels (Dox-on group, 220.4 6
Subsequent hyperoxia exposure experiments were done on
age-matched SP-D Dox-on and Dox-off mice. Under hyperoxic
conditions (85% O2), the SP-D Dox-on group survived consid-
erably longer than the Dox-off SP-D–deficient group (median
survival 5 11 days for Dox-on vs. 7 days for Dox-off; P ,
0.0001) (Figure 8B). Dox-off mice developed 100% mortality by
Day 13, whereas more than 20% of the Dox-on mice survived
the 14-day exposure protocol.
It is increasingly recognized that factors intrinsic to the lung
play a role in the differential susceptibility to O2 toxicity
observed in patients and in animal models exposed to hyperoxia
(28). In the present study, we show that local overexpression of
SP-D resulted in an enhanced median survival for transgenic
mice exposed to high concentrations of O2that was accompa-
nied by preservation of alveolar capillary barrier integrity,
a decrease in proinflammatory cytokines, and an up-regulation
of NRF2 and its dependent phase 2 antioxidant genes. Further-
more, the effect of SP-D occurred in a dose-dependent fashion
as mice conditionally expressing SP-D and mice rendered
acutely SP-D–deficient through withdrawal of doxycycline were
more susceptible to hyperoxia.
Lung surfactant lipid content and in vitro biophysical activity
were mildly inhibited in the SP-D OE and littermate control
groups and could not account for observed changes seen in
response to hyperoxia. This is in contrast to our experience with
the effects of hyperoxia on a murine model constitutively de-
ficient in SP-D. The SP-D knockout mouse exhibits a variety of
baseline phenotypic alterations, including increased surfactant
phospholipid pools, enlarged but nonfunctional pulmonary mac-
flammatory cytokines. Paradoxically, that model had enhanced
survival when subjected to hyperoxic exposure (18). After an
attributed to secondary enlargement of alveolar surfactant phos-
pholipid pools and SP-B, which develop in this phenotype. In
contrast, the triple transgenic SP-D conditional mice used in this
study, when maintained on doxycycline from birth, have no
discernible phenotype (21). Unlike chronically SP-D–deficient
mice, SP-D conditional mice removed from doxycycline for 2
weeks before hyperoxia (Dox-off) did not demonstrate signifi-
we and others have found that constitutive local SP-D over-
expression in the SP-D OE line also does not affect alveolar
phospholipid homeostasis (20). Taken together, the use of these
two models has allowed us to investigate the protective effects of
SP-D independent of any mechanistic contributions from surfac-
tant phospholipids or other phenotypic alterations associated
with constitutive knockdown of the SP-D gene.
that found in identically exposed littermate control mice (Figure
4), the lungs of SP-D OE mice subjected to hyperoxia had
paradoxically less lung damage histologically (Figure 5) and
biochemically (Figure 2) in association with a highly immunosup-
pressive microenvironment. Inflammatory and chemotactic cyto-
kine levels in the BAL of these mice were attenuated (Table 1).
Because the measured BAL cytokines represent products of
multiple cell types present in the distal airways, including macro-
phages (tumor necrosis factor-a), epithelial cells (IL-6, IL-8
Figure 6. Gene expression of NF-E2 related factor-2 (NRF-
2) and downstream antioxidant genes in wild-type (WT)
and surfactant protein D overexpressing (SP-D OE) mice.
Total lung mRNA was extracted from SP-D OE and
littermate WT mice exposed to 21% or 80% O2 for 5
days. Quantitative real-time reverse transcription–poly-
merase chain reaction analysis was performed for NRF-2,
glutathione peroxidase-2 (Gpx-2), hemoxygenase-1 (HO-
1), and NAD(P)H quinone oxidoreductase-1 (Nqo-1) and
normalized to 18S RNA as described. Data are expressed
as mean 6 SEM (n 5 5–10), normalized to WT level (*P ,
0.05; ***P , 0.0001 vs. SP-D OE normoxia group).
Hyperoxia-exposed SP-D OE mice exhibit significantly
higher levels of NRF-2, GPx-2, HO-1, and Nqo-1 mRNA
in the lung as compared with similarly exposed WT group.
idation in wild-type (WT) and
surfactant protein D overex-
pressing (SP-D OE) mice after
hyperoxia. Total lung lipid
peroxides were measured in
SP-D OE and WT mice before
or after exposure to 80% O2
for 5 days. Data are expressed
as mean 6 SEM (n 5 5–10),
normalized to WT level (*P ,
0.05 vs. WT
increases in lung thiobarbitu-
ric acid reactive substances
are abrogated in SP-D OE
Lung lipid perox-
810 AMERICAN JOURNAL OF RESPIRATORY AND CRITICAL CARE MEDICINEVOL 178 2008
1), and T cells (IL-13), the data suggest widespread inflammatory
suppression by SP-D in these mice. From a number of in vitro
of many types of these same effector cells in part through
alterations in nuclear factor-kB signaling (29), a common mecha-
nism for cytokine elaboration and oxidative burst (11, 30–37).
Thus, teleologically, itappearsplausible that the rise in SP-Dseen
in mice exposed to a variety of inflammatory stimuli (18, 38, 39)
represents an acute phase response to suppress further inflamma-
tory damage through a direct effect on the local pulmonary
cytokine-producing cell types, such as alveolar or airway epithelia.
In addition to alterations in the local cytokine milieu, we
have shown that the observed protection against hyperoxia in
SP-D OE mice is associated with up-regulation of NRF-2 and
increased expression of phase 2 antioxidant genes. Transcrip-
tion of various antioxidant enzymes, including HO-1 (40) and
GPx (41), is regulated by cis-regulatory elements known as
antioxidant response elements (AREs). The ARE consensus
sequence TGA(G/C)nnnGC is recognized by a number of
basic leucine zipper proteins and nuclear erythroid factor 2
(NF-E2)–related factors 1 and 2 (NRF-1 and NRF-2). Recent
mRNA expression for ARE-bearing antioxidant anddetoxifying
enzymesis protectiveagainstoxidativestress invitro(42–45)and
in vivo (46–48). A threefold increase in lung NRF-2 mRNA
expression occurred in mice after exposure to O2(48) at higher
doses (.95% O2) than in the current study where quantitative
real-time reverse transcription–polymerase chain reaction anal-
ysis demonstrated that 80% O2 exposure induces a 2.5-fold
increase in NRF-2 and a concordant three- to fivefold increase
in downstream antioxidant enzyme expression (GPx-2, HO-1,
and Nqo-1) that was not observed in the littermate control mice
(Figure 6). Thus, because SP-D OE mice at baseline lack an
enhanced antioxidant phenotype and because the SWB litter-
mates did not up-regulate a phase 2 antioxidant response at this
can synergistically participate in a coordinated up-regulation of
this protective response at inspired concentrations lower than
previously reported to enhance NRF-2 gene responses (48).
in the balance between intracellular oxidants and antioxidants
mice with doxycline-de-
expression of surfactant
protein D (SP-D) show
increased survival under
(A) SP-D expression in
triple transgenic (condi-
tional SP-D expressing)
chow diminishes rapidly
after removal of doxycy-
cline. Western blots of
maintained from birth
on 625 ppm dox feed
normal feed pellets for
0, 3, 7, or 14 days
clonal SP-D antiserum.
Data are expressed as
mean 6 SEM, normal-
ized to % B6 wild-type
(WT) level (n 5 1 for
WT; n 5 2–7 for condi-
mice). (B) Triple trans-
genic mice at 8 weeks of
age maintained on 625
ppm doxycycline feed
pellets (‘‘on dox’’) or
feed pellets 2 weeks ear-
lier (‘‘off dox’’) were ex-
posed to 85% O2 as
described in METHODS. Comparison of Kaplan-Meier survival curves for each group revealed enhanced tolerance in the SP-D–expressing mice
(P , 0.0001 vs. corresponding control groups). Data are representative of two separate experiments (n 5 24 for hyperoxia groups; n 5 10 for
normoxia control mice). All normoxia mice survived at the end of the experiment (data lines are superimposed). (C) Total phospholipid (PL) levels in
BAL from ‘‘Dox on’’ and ‘‘Dox off’’ (2-week) groups at baseline were determined as described in METHODS. Data are expressed as mean 6 SEM (n 5
8–9). There were no significant differences between baseline BAL PL levels in ‘‘Dox On’’ and ‘‘Dox Off’’ groups.
Jain, Atochina-Vasserman, Tomer, et al.: SP-D Is Protective against Hyperoxia811
as responsible for tissue damage by lipid peroxidation, enzyme
inactivation, DNA oxidation, or altered gene expression (49). In
addition to induction of enzymatic detoxifying proteins for ROS
scavenging described above, protection against oxidant-medi-
ated cell death in the lung is also afforded by up-regulation of
nonenzymatic detoxifying agents like vitamins E and C (50).
Functionally, using total TBARS as an experimental readout,
overexpression of SP-D was associated with marked attenuation
a potential direct free radical scavenger in vitro, limiting perox-
idation of surfactant lipid mixtures and protecting a macrophage
chain terminator that could then provide rapid quenching of
diene formation, thereby limiting lipid peroxidation (51).
Beyond damage by ROS, exposure to inflammatory stimuli
results in the production of reactive nitrogen species (RNS).
Classically, inflammatory lung injury has been amplified by NO
formed during the inflammatory cascade reacting with ROS to
form highly reactive molecules such as peroxynitrite (52). In this
model of hyperoxia, we have shown complete attenuation in SP-
D OE mice of the two- to threefold increase in oxidative stress
in WT mice (Figure 7). Although this study did not directly
assess the affect of hyperoxia on NO metabolism, it is likely that
protection from hyperoxic injury in SP-D overexpressing mice
could also be partly related to secondary abrogation of nitrative
stress. This is based on the consideration that because RNS are
generated by the reaction of NO with O2radicals, and even if
NO were to increase modestly during hyperoxia (e.g., by z35%
as we have shown previously ), the marked SP-D–induced
attenuation in ROS alone could account for a reduction in RNS.
In addition to up-regulation of antioxidant enzymes, SP-D
expression in SP-D OE mice exposed to hyperoxia increased to
a much greater extent than in littermate control mice (Figure
3B). Analysis of the cis-active human SP-C gene promoter that
is used to drive rSP-D expression in our SP-D overexpressing
mice revealed a potential binding site for NRF-2 at 2,924 bp
upstream of the start codon. This promoter region also contains
at least three other AREs at position 22763, 21972, and 2866,
suggesting a potential for the transgene to be subject to
regulation by ROS through ARE. In contrast, the regulatory
region of the endogenous SP-D promoter is mainly character-
ized by the presence of TTF-1 and AP-1 binding sites (53) and is
not expected to be directly sensitive to oxidant stress. Hence,
we speculate that SP-D OE mice exposed to 80% O2demon-
strate significantly greater increases in BAL SP-D as a result of
an oxidant-induced activation of transgene expression. The
resultant 30- to 40-fold increase in SP-D achieved in SP-D
OE versus similarly exposed littermates would also be possible
pharmacologically via intratracheal instillation with or without
surfactant. We and others have measured BAL SP-D concen-
trations in humans (54, 55) and found them to be approximately
1 mg/ml. Levels found in rodents are of similar magnitude (0.25–
0.5 mg/ml BAL, which translates to 0.75–2.0 mg/mouse of total
alveolar SP-D) (56). Pharmacologic instillation of SP-D has
been reported by Clark and colleagues (57), who repetitively
instilled recombinant forms of SP-D intranasally at doses of 10
to 50 mg in 50 ml. Such an instillation enriches alveolar SP-D
content by 20- to 40-fold over the basal WT levels.
In summary, we have shown in two different animal models
that SP-D is an important regulatory molecule in the protection
from hyperoxic lung injury. This protection occurs through
a variety of pathways resulting in generalized proinflammatory
cytokine immunosuppression and effects on phase II antioxi-
dant expression. Our results raise the possibility that exogenous
replacement pulmonary surfactants (heretofore deficient in
collectins) that are replete in SP-D could offer an advantage
through protection from lung damage when administered to
patients at risk for hyperoxic pulmonary injury.
Conflict of Interest Statement: None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.
Acknowledgment: The authors thank Dr. Frank McCormack at the University of
Cincinnati for provision of breeding pairs of SP-D overexpressing transgenic mice
and Dr. Jeffrey Whitsett at the University of Cincinnati for provision of breeding
pairs of SP-D conditional transgenic mice. The authors thank Dr. Aron Fisher and
Mr. Donald Fisher at the Institute for Environmental Medicine at PENN for access
to the hyperoxic exposure facility, Jennifer Newitt for genotyping and mainte-
nance of the transgenic colony, and Dr. Elena Abramova for assistance with
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Jain, Atochina-Vasserman, Tomer, et al.: SP-D Is Protective against Hyperoxia 813