Surfactant Protein D Deficiency Increases Lung Injury
Brooke A. King1and Paul S. Kingma1
1Perinatal Institute, Section of Neonatology, Perinatal and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio
syndrome (ARDS), are major causes of acute respiratory failure with
high rates of morbidity and mortality. Although surfactant protein
clinical studies suggest that this protein may be implicated in the
pathophysiology of ARDS, little is known regarding the function of
intraperitoneal injection of LPS and direct lung injury by intra-
tracheal injection of LPS in wild-type and Sftpd2/2mice to elucidate
the role of SP-D during ALI/ARDS. Results indicate that pulmonary
with wild-type mice. However, the magnitude of this difference was
10-fold greater after indirect lung injury compared with direct lung
injury. After indirect lung injury, there was a 2-fold increase in the
number of pulmonary monocyte/macrophages in the Sftpd2/2mice
when compared with wild-type mice, whereas pulmonary neutro-
phils were not increased. After indirect injury, the concentration of
granulocyte-macrophage colony stimulating factor (GM-CSF) was
contrast, after direct injury, the concentration of GM-CSF was 20-
fold less in Sftpd2/2mice than wild-type mice. Despite increased
mice after indirect lung injury was paradoxically increased. In
conclusion, these results suggest that SP-D inhibits pulmonary
inflammation and migration of peripheral monocyte/macrophages
Keywords: surfactant protein D; acute respiratory distress syndrome;
Acute lung injury (ALI), and its severe form, acute respiratory
distress syndrome (ARDS), are characterized by acute pulmo-
nary inflammation and pulmonary edema. The inflammatory
response in ARDS originates from the loss of vascular endothe-
of protein-rich pulmonary edema, surfactant dysfunction, pul-
monary inflammation, damage to the lung parenchyma and
pulmonary epithelium, and respiratory insufficiency (1). The
outcome of this self-perpetuating process is excessive lung injury
and, in many cases, multiple organ dysfunction syndrome, organ
failure, and death.
ALI/ARDS develops from a variety of clinical disorders that
can be differentiated into those associated with direct lung injury
(e.g., pneumonia, aspiration) and those causing indirect lung
injury (e.g., sepsis, shock). Although the resulting pulmonary
inflammation and damage to the lung parenchyma are similar,
there is a growing body of evidence that suggests that the two
modes of injury have unique underlying pathophysiological
Analysis of bronchoalveolar lavage fluid (BALF) samples
from adult patients with ARDS has demonstrated that, in
addition to inflammation, alterations in lung surfactant function
and composition are present. Whereas pulmonary levels of
surfactant proteins A, B, and C decrease during ARDS, levels
of SP-D increase (3). Moreover, patients with ARDS who have
relatively higher levels of pulmonary SP-D have increased
survival rates, suggesting a vital role for SP-D in ARDS.
SP-D is a member of the collectin family of innate defense
a variety of viral, bacterial, and fungal pathogens (4). SP-D
binding facilitates the uptake and clearance of pathogens from
the lung by alveolar macrophages and neutrophils (5). Although
binding infectious microbes is a critical feature of SP-D function,
animal models of SP-D deficiency indicate that SP-D also
regulates pulmonary immune cells and decreases inflammation
normal surfactant structure and for uptake and recycling of
surfactant by alveolar type II cells (8, 9). Taken collectively, the
changesof SP-D expression during ARDS and the critical role of
SP-D in regulating immune cells and maintaining normal surfac-
tant homeostasis suggest that SP-D plays a critical role in the
body’s response to ARDS.
Therefore, we hypothesize that SP-D attenuates pulmonary
immune cell activation during ALI/ARDS and as a result
reduces pulmonary inflammation and limits lung injury. To
determine if SP-D decreased inflammation during ALI/ARDS,
we induced lung injury in wild-type mice and mice lacking SP-D
(Sftpd2/2) by intraperitoneal or intratracheal injection of LPS.
Whereas SP-D deficiency resulted in increased macrophage
recruitment and a marked increase in pulmonary inflammation
in mice after indirect lung injury, survival was improved in SP-D–
MATERIALS AND METHODS
Studies were performed on 6- to 10-week-old Sftpd2/2and age-
matched Sftpd1/1litter mate wild-type control mice (10). Mice were
During indirect lung injury surfactant protein (SP)-D
inhibits pulmonary inflammation and migration of peripheral
monocyte/macrophages into the lung through granulocyte/
macrophage colony–stimulating factor–dependent path-
ways. However, despite the decreased pulmonary inflam-
mation, SP-D was not associated with decreased mortality
in mice. This study raises the importance of SP-D during
acute lung injury/acute respiratory distress syndrome in-
duced by indirect lung injury and suggests a potential
therapeutic intervention in neonatal and adult patients
with this syndrome.
(Received in original form December 2, 2009 and in final form July 1, 2010)
Supported by National Institutes of Health grant HL089505 (P.S.K.) and Ikaria
Advancing Newborn Medicine Grant for Fellows in Neonatology (B.A.K.).
Correspondence and requests for reprints should be addressed to Paul S. Kingma,
M.D., Ph.D., Section of Neonatology, Perinatal and Pulmonary Biology. Cincin-
nati Children’s Hospital Medical Center, 3333 Burnet Ave. ML7009, Cincinnati,
OH 45229-3039. E-mail: email@example.com
Am J Respir Cell Mol Biol
Originally Published in Press as DOI: 10.1165/rcmb.2009-0436OC on July 16, 2010
Internet address: www.atsjournals.org
Vol 44. pp 709–715, 2011
housed in conditions approved by the Institutional Animal Care and
Use Committee at Cincinnati Children’s Hospital Medical Center and
maintained in the vivarium under barrier containment facilities.
Evidence of common murine pathogens was not detected in sentinel
mice in the colony.
Treatment with Lipopolysaccharide
Animals were anesthetized with isoflurane. LPS, serotype Escherichia
coli O111:B4, dissolved in PBS in doses of 20 and 40 mg/kg was injected
into the intraperitoneal space. For direct lung injury, PBS or 20 mg/kg
LPS was administered by tracheal aspiration.
Assessment of Pulmonary Injury
Mice were killed with intraperitoneal injection of pentobarbital. BAL
was performed by intratracheal instillion and recovery of a 1 ml aliquot
of PBS. The fluid was stored at 2208C until analysis or 2808C for
Blood samples were obtained by cardiac puncture at the time of
death and stored at 2208C until cytokine levels were determined.
IL-6 was quantified in BALF and plasma with a murine enzyme-
linked immunosorbent assay using a commercially available kit (R&D
Systems, Minneapolis, MN). Granulocyte-macrophage colony stimu-
lating factor (GM-CSF), IL-10, IL-12p70, IL1b, IL-2, and TNF-a were
quantified with a multiplex bead-based assay (Bio-Rad, Hercules, CA).
Chromogenic limulus amebocyte lysate test was used to quantify
endotoxin in BALF (Cambrex BioScience, Walkersville, MD). A BCA
Protein Assay kit (Pierce, Rockford, IL) was used to determine protein
concentration in BALF. Antioxidant activity was quantified by measur-
ing copper-reducing equivalents using a commercially available assay
(Oxford Biomedical Research, Oxford, MI).
Cell counts were determined in BALF samples. BALF cells were
centrifuged and resuspended in 500 mL of PBS. Cell counts were
performed on cytospin preparations of the cell pellet suspension after
staining with hematoxylin and eosin.
For histology, lungs were inflation fixed at 25 cm H2O. Immuno-
histochemistry for MAC-3 was performed at dilutions of 1:200 by using
rabbit polyclonal antibody. Immune complexes were detected using an
Assessment of Mortality
For the general assessment of mortality, wild-type and Sftpd2/2mice
were treated with 40 mg/kg of LPS injected into the intraperitoneal
space and followed hourly for 24 hours.
Mice containing the doxycylcine-inducible Sftpd gene (CCSPrtTA1
(tetO)7-rSP-D1, mSP-D2/2mice) were used to investigate the role of
surfactant lipid levels on survival in Sftpd2/2mice. The high-surfactant
lipid group was kept on a doxycycline-free diet until the time of the
mortality study. The low-surfactant lipid group was kept on doxycy-
cline from the time of delivery until 4 weeks of age and then
transitioned to a doxycycline-free diet. At 7 weeks of age, each group
was treated with 40 mg/kg of LPS injected into the intraperitoneal
space and followed hourly for 24 hours.
All results are given as the mean 6 SEM. The groups were compared
using two-tailed Student’s t test. Differences of P , 0.05 were con-
Previous results from both clinical studies in patients with
ARDS and from mouse models of SP-D deficiency led us to
the hypothesis that SP-D decreases pulmonary inflammation
and limits lung injury during ALI. Because ALI/ARDS can be
caused by indirect and direct lung injury, this hypothesis was
tested by intraperitoneal injection of LPS to induce indirect
lung injury and intratracheal aspiration of LPS to induce direct
lung injury in wild-type and Sftpd2/2mice.
Sftpd2/2Mice Have Increased Inflammation after Indirect
To evaluate the role of SP-D on inflammation during ALI,
pulmonary IL-6 concentrations were assessed after indirect lung
injury with intraperitoneal LPS. BALF from Sftpd2/2had 4-
fold greater concentrations of IL-6 compared with wild-type
mice 6 hours after treatment (P , 0.05) (Figure 1A). These
results suggest that SP-D limits pulmonary inflammation during
indirect lung injury; however, SP-D is present in serum during
sepsis, raising the possibility that the increase in pulmonary
inflammation seen in Sftpd2/2mice may result from elevated
systemic inflammation. Therefore, markers of systemic inflam-
mation were also assessed. Similar to BALF findings, plasma
IL-6 concentrations were increased after intraperitoneal LPS
injection in Sftpd2/2mice when compared with wild-type mice;
after lung injury. Sftpd2/2and wild-type mice were treated with PBS or
LPS via intraperitoneal (40 mg/kg LPS) or intratracheal (20 mg/kg LPS)
injection (n 5 5 for each group). BALF or blood was harvested 2, 6, and
16 hours after injection, and IL-6 levels were quantified by ELISA. (A) At
6 hours, BALF IL-6 levels were 4-fold greater in Sftpd2/2mice than in
wild-type mice after intraperitoneal LPS treatment (P < 0.01). (B)
Plasma IL-6 levels were increased in Sftpd2/2mice compared with wild-
type mice after intraperitoneal LPS treatment; however, this 30%
increase (P 5 0.12) was not as dramatic as the differences seen in
BALF samples. (C) After intratracheal LPS treatment, BALF IL-6 levels
were similar between wild-type and Sftpd2/2mice, suggesting that SP-D
does not significantly protect the lung from inflammation 6 hours after
direct lung injury.
Bronchoalveolar lavage fluid (BALF) and plasma IL-6 levels
710AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 442011
however, the increase was an order of magnitude less than the
4-fold difference observed in pulmonary samples (Figure 1B).
Taken together, these results indicate that SP-D inhibits
pulmonary inflammation during indirect lung injury.
known role of SP-D in binding inhaled pathogens, one would
direct lung injury (i.e., intratracheal administration of LPS) than
indirect lung injury (i.e., intraperitoneal injection of LPS). Pre-
vious studies have examined the role of SP-D after direct lung
injury and, in marked contrast to our findings, observed only
a 50% increase in pulmonary IL-6 levels in Sftpd2/2mice
compared with wild-type mice (11). To confirm that the differ-
of lung injury and not to a variation in experimental conditions,
we induced direct lung injury in Sftpd2/2and wild-type mice by
intratracheal aspiration of LPS. To ensure that we were inducing
similar levels of lung injury, we used a dose of intratracheal LPS
that achieved similar levels of pulmonary inflammation as that
concentrations were increased above baseline in Sftpd2/2and
wild-type mice after intratracheal LPS; however, the relative
increase in BALF IL-6 concentrations in Sftpd2/2mice was only
50% higher than wild-type mice 6 and 16 hours after treatment
(Figure 1C). Our results with direct lung injury are similar to
results obtained by Ikegami and colleagues and support the
concept that SP-D has a larger role in protecting the lungs from
indirect lung injury compared with direct lung injury.
Distinct abnormalities in lung structure and alveolar cell
morphology in SP-D–deficient animals have been well described
(10, 12). Consequently, the increase in pulmonary inflammation
observed after an indirect injury may be the result of increased
capillary-alveolar leakage of systemic LPS inducing an exagger-
ated local inflammatory response in the lung. To assess if the
pulmonary inflammation was the result of inherently incompe-
tent tissue structure and subsequent increased capillary-alveolar
leakage of systemic LPS into the lung, BALF LPS was quantified
after indirect and direct lung injury. There were no differences in
and wild-type mice after either method of lung injury, indicating
that the elevated pulmonary inflammation in Sftpd2/2mice after
indirect lung injury is not due to increased LPS leakage into the
lung (Figure 2).
Sftpd2/2Mice Have Increased Morbidity but
Although there is quantitative evidence of increased pulmonary
inflammation in Sftpd2/2mice during indirect lung injury,
markers of inflammation may not correlate with clinical morbid-
ity. Subjectively, LPS-treated mice were lethargic and exhibited
hunched backs, ruffled fur, and ocular discharge; these charac-
teristics were more severe in Sftpd2/2mice than in wild-type
to objectively measure the clinical effect of SP-D during indirect
lung injury. In support of the subjective observations, Sftpd2/2
mice were significantly more acidotic and had higher alveolar
protein levels when compared with wild-type mice after LPS-
induced indirect lung injury (Figure 3).
previous studies examining hyperoxia-induced lung injury in
Sftpd2/2mice demonstrated increased pulmonary inflammation
with a paradoxical decrease in mortality in Sftpd2/2mice when
compared with wild-type control mice. We found similar results
after indirect lung injury. Twenty-four hours after injection of
LPS, survival was 63% in Sftpd2/2mice and only 33% in wild-
type mice, indicating that although pulmonary inflammation and
morbidity are increased in Sftpd2/2mice, overall mortality is
decreased (Figure 4A).
Several previous studies have established that alveolar
surfactant lipid pools are increased in Sftpd2/2mice. Therefore,
to determine if the paradoxical decrease in mortality in Sftpd2/2
mice after indirect lung injury was due to elevated surfactant
lipid pool sizes, mice containing the doxycycline-inducible Sftpd
gene (CCSPrtTA1(tetO)7-rSP-D1, Sftpd2/2) were used. Zhang
and colleagues demonstrated that when these mice were placed
on a doxycycline diet during the first 2 weeks of life, lipid pool
sizes decreased to wild-type levels and remained at wild-type
levels after removal of doxycycline for up to 4 weeks (13). We
took advantage of this finding to generate Sftpd2/2mice with
high- and low-surfactant lipid pool sizes. The high-surfactant-
lipid group was kept on a doxycycline-free diet (i.e., Sftpd2/2
phenotype with high-surfactant lipids), whereas the low-surfactant
lipid-group remained on doxycycline from the time of delivery
until 4 weeks of age, at which time they were transitioned to
a doxycycline-free diet (i.e., Sftpd2/2phenotype with low-
surfactant lipids). After intraperitoneal LPS injection, survival
was similar in these two groups, suggesting that surfactant lipid
pool size does not affect mortality after indirect lung injury in
Sftpd2/2mice (Figure 4B).
Because similar unanticipated mortality results have been
demonstrated in a hyperoxia model, another plausible rationale
for improved survival in these animal models may be attribut-
able to increased levels of antioxidant enzymes. Sftpd2/2mice
demonstrated a 1.5- to 2- fold increase in antioxidant activity in
BALF when compared with wild-type control mice after in-
direct lung injury (Figure 5). This result may provide some
insight into the survival mechanism involved in Sftpd2/2mice
subjected to indirect lung injury.
Macrophages Implicated in Inflammation
Previous studies demonstrated that Sftpd2/2mice accumulate
enlarged foamy alveolar macrophages, and several studies have
implicated SP-D in macrophage regulation. However, results by
Ikegami and colleagues suggested that an influx of pulmonary
neutrophils was responsible for the increased inflammation
observed in Sftpd2/2mice after direct lung injury (11). Upon
examination of BALF after indirect lung injury, we discovered
that, although pulmonary neutrophils were not increased, there
Figure 2. LPS levels in BALF after indirect and direct lung injury. The
amount of LPS in BALF samples (n 5 5 for each group) was quantified
by Limulus assay after mice were treated with intraperitoneal (IP) or
intratracheal (IT) LPS to assess if the increased pulmonary inflammation
observed in Sftpd2/2mice was due to increased capillary-alveolar
leakage of LPS. The levels of LPS were similar between the wild-type
and Sftpd2/2mice after either route of administration.
King and Kingma: Surfactant Protein D in Lung Injury 711
was a 2-fold increase in the number of monocyte/macrophages
in the Sftpd2/2mice compared with wild-type mice (Figure 6A).
In contrast, after direct lung injury, Sftpd2/2and wild-type mice
had a marked increase in pulmonary neutrophils but no increase
in the number of monocyte/macrophages (Figure 6B). These
results suggest that SP-D inhibits the influx of circulating
monocytes into the lung or that SP-D inhibits the migration
of macrophages from the pulmonary interstitium to the alveoli
(and subsequently BALF) during indirect lung injury. To
differentiate between these two mechanisms of monocyte/
macrophage movement, mouse lungs were immunostained with
MAC-3, a macrophage-specific antibody. During indirect lung
injury, the number of MAC-3–positive cells increased approx-
imately 2-fold in Sftpd2/2mice, whereas there was no increase
in MAC-3–positive cells in wild-type mice (Figure 6C). These
results indicate that there is an increase in the total number of
pulmonary monocyte/macrophages in the absence of SP-D and
suggest that SP-D inhibits migration of monocyte/macrophages
into the lung after indirect lung injury.
Macrophages respond to a variety of innate and adaptive
stimuli after injury or infection. To identify the mechanism for
the observed influx of macrophages seen in Sftpd2/2mice after
indirect lung injury, a multiplex cytokine assay (which allows for
the identification and quantification of several cytokines/chemo-
kines simultaneously) was performed on BALF samples after
indirect or direct lung injury. After indirect injury, the concen-
tration of GM-CSF was approximately 5-fold greater in Sftpd2/2
mice than in wild-type mice (Figure 7A). In contrast, after
direct injury, the concentration of GM-CSF was 20-fold less in
Sftpd2/2mice than in wild-type mice (Figure 7B). Although not
statistically significant because of sample variability, TNF-a was
also greater in Sftpd2/2mice than in wild-type mice after
indirect injury, yet there was no difference between the two
groups in direct injury. There were no differences in IL-10, IL-
12p70, IL-1b, or IL-2 between the two groups after either type
of lung injury. Because GM-CSF is known to increase myeloid
adhesion, proliferation, and differentiation, these results raise
the possibility that SP-D regulates the migration of monocytes
into the lung by inhibiting GM-CSF secretion during indirect
lung injury. In addition, the suppression of GM-CSF may lead
to a decrease in TNF-a and other inflammatory cytokines,
further altering macrophage activation and regulation.
ARDS develops in patients of all ages from a variety of insults.
Although SP-D plays a critical role in pulmonary innate
immunity and several clinical studies suggest that this protein
may be implicated in the pathophysiology of ARDS, little is
known regarding the function of SP-D in ALI/ARDS (14). In
the present study, we induced lung injury in wild-type and Sftpd2/2
Blood was collected for blood gas analysis 6 hours after intraperitoneal
injection of PBS or LPS, and BALF was collected for protein analysis 2, 6,
or 16 hours after injection. Sftpd2/2mice were more acidotic than wild-
type mice (P < 0.02, 20 mg/kg; P 5 0.01, 40 mg/kg; n 5 5 in all
groups) and trended to have more alveolar protein (P < 0.02 at 6 h).
pH and alveolar protein values after indirect lung injury.
Figure 4. Survival after indirect lung injury. (A) Sftpd2/2and wild-type
mice were treated with intraperitoneal injection of LPS. (B) CCSPrtTA1
(tetO)7-rSP-D1, Sftpd2/2mice were kept on a doxycycline-free diet
(Sftpd2/2high-surfactant-lipid group) or CCSPrtTA1(tetO)7-rSP-D1.
Sftpd2/2mice remained on doxycycline from the time of delivery until
4 weeks of age, at which time they were transitioned to a doxycycline-
free diet (i.e., Sftpd2/2low-surfactant-lipid group). Survival for each
group (n 5 10 for each group) was recorded throughout a 24-hour
observation period and plotted as a Kaplan-Meier survival analysis.
Comparison at 24 hours revealed enhanced survival in the Sftpd2/2
mice compared with wild-type mice but no difference in Sftpd2/2mice
with low or high surfactant lipid levels.
712AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGYVOL 442011
mice to elucidate the role of SP-D during endotoxemia-induced
Based on inflammatory cytokine levels, pulmonary inflamma-
tion was significantly higher in Sftpd2/2mice when compared
with wild-type mice with ALI after indirect lung injury. Consid-
ering the primary role and location of SP-D is in the lung, it is
counterintuitive that the difference in pulmonary inflammation
between Sftpd2/2and wild-type mice was greater after indirect
for this discrepancy is increased systemic LPS leakage into the
lung through damaged alveolar capillary membranes in Sftpd2/2
mice. However, the quantity of LPS in the lung after indirect or
direct lung injury was similar, which contradicts this hypothesis
and raises the possibility that SP-D plays a more complex role in
regulating the interchange between systemic and pulmonary
inflammation during ALI/ARDS. BALF cell counts in Sftpd2/2
and wild-type mice after lung injury suggest that the mechanistic
details of this role entail monocyte/macrophage recruitment and
activation. Whereas clinical studies have shown that there is
a predominant increase in neutrophils in BALF obtained from
patients with ARDS and animal studies demonstrate significant
elevation of neutrophils in the pulmonary microcirculation after
endotoxin treatment, our results demonstrate that after indirect
lung injury SP-D deficiency increases extravasation of peripheral
blood macrophages into the alveolar spaces without a change in
neutrophil influx (15, 16). Conversely, BALF cell counts suggest
that, after direct lung injury, SP-D deficiency does not alter
monocyte/macrophage migration (11). Analysis of inflammatory
cytokine profiles suggests that SP-D may control inflammatory
cell migration by inhibiting GM-CSF secretion and consequently
altering TNF-a and other inflammatory cytokine expression, but
the specifics of this signaling cascade require clarification by
further experiments. Together, these changes in cellular and
cytokine response demonstrate the importance of SP-D during
indirect lung injury. Finally, although several studies have
suggested that SP-D regulates pulmonary host defense cells and
that SP-D preserves the integrity of the alveolar-capillary in-
terface by maintaining balance in the pulmonary inflammatory
cascade, this is the first study to suggest that SP-D protects this
interface from the effects of the systemic inflammatory cascade
We found that, despite increased inflammatory cells and
ical increase in survival. The explanation for this is unclear. This
same phenomenon was seen in another study in mice exposed to
survival in Sftpd2/2mice exposed to hyperoxia may be due to
increased surfactant pool sizes, preserved surfactant function, or
an adaptive increase in antioxidant components (18). Our results
with CCSPrtTA1(tetO)7-rSP-D1, Sftpd2/2mice indicate that
increased surfactant pool sizes do not improve survival after lung
injury in Sftpd2/2mice. In contrast, the discovery of increased
antioxidant activity in BALF of Sftpd2/2mice after indirect lung
injury suggests one possible adaptive process that may improve
survival in these mice. Given that antioxidants increase only 2-
fold in Sftpd2/2mice, it is plausible that additional factors may
influence survival in Sftpd2/2mice subjected to lung injury (10,
Although the majority of clinical research on ARDS has
focused on adult patients, premature neonates represent a clini-
cally important population. Every year, 12.9 million babies are
born premature (, 37 wk gestation) worldwide, representing
a prevalence of preterm birth of 9.6% (20). Unfortunately, this
activity in BALF samples from Sftpd2/2and wild-type mice was
quantified after treatment with intraperitoneal LPS (n 5 5 for each
group). Sftpd2/2mice demonstrated an increase in antioxidant activity
in BALF when compared with wild-type control mice after indirect lung
injury (P < 0.02 at 2 h).
Antioxidant activity after indirect lung injury. Antioxidant
Figure 6. Cell count after acute lung injury. BALF samples from Sftpd2/2
and wild-type mice after treatment with PBS or LPS were stained, and cell
counts were quantified (n 5 5 for each group). (A) After indirect lung
injury, there was a 2-fold increase in the number of monocytes/macro-
phages (M) in the Sftpd2/2mice compared with wild-type mice, whereas
pulmonary neutrophils (N) were not increased. (B) In contrast, after direct
lung injury, Sftpd2/2and wild-type mice had a 5-fold increase in
pulmonary neutrophils but no increase in the number of monocyte/
macrophages. (C) Mouse lungs were immunostained with MAC-3,
a macrophage-specific antibody. MAC-3–positive cells increased in
Sftpd2/2mice after LPS treatment, whereas there was no increase in
MAC-3–positive cells in wild-type mice.
King and Kingma: Surfactant Protein D in Lung Injury 713
rate continues to increase. Premature infants are known to have
immature lung structure as well as quantitative and functional
deficiencies in surfactant. Infants born premature also have
higher rates of respiratory illness and septicemia than infants
bornatterm(21).Knowingthattheseare allriskfactorsfor ALI/
ARDS, the cumulative effect is that ALI/ARDS is a leading
cause of morbidity and mortality in premature infants. SP-D is
first detected in the lung, but at low levels, at 16 to 18 weeks
gestation, with levels rising moderately from 32 weeks to term
(22). Thus, it is important to reveal the SP-D–dependent mech-
anisms of ALI/ARDS after endotoxemia to improve the prog-
nosis in SP-D–deficient states, such as premature birth.
Due to its success in the treatment of premature infants with
respiratory distress syndrome, surfactant replacement therapy is
under investigation as a potential therapy for ALI/ARDS in
pediatric and adult patients (23, 24). Initial studies using surfac-
tant have not been encouraging; however, these studies used an
aerosolized form of surfactant or protein-free phospholipid
preparations of surfactant that did not contain SP-D (25–27).
With the findings of this study and further investigation, it is
reverse or impede the pulmonary inflammation and injury that
develops in patients of all ages with ALI/ARDS caused by
on whether the paradoxical decreased mortality rate observed in
Sftpd2/2mice is also observed in human SP-D–deficient states.
In summary, during indirect lung injury, SP-D inhibits
pulmonary inflammation and migration of peripheral mono-
cytes/macrophages into the lung through GM-CSF–dependent
pathways. However, despite the decreased pulmonary inflam-
mation, SP-D was not associated with decreased mortality in
mice. This study raises the importance of SP-D during ALI/
ARDS induced by indirect lung injury and suggests a potential
therapeutic intervention in neonatal and adult patients with this
Author Disclosure: None of the authors has a financial relationship with a
commercial entity that has an interest in the subject of this manuscript.
Acknowledgments: The authors thank Dr. Jeffrey Whitsett for supplying Sftpd2/2
mice and critical reading of the manuscript.
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