Am J Physiol Lung Cell Mol Physiol 287: L999 –L1006, 2004;
doi:10.1152/ajplung.00111.2004.
Lipopolysaccharide increases alveolar type II cell number in fetal mouse
lungs through Toll-like receptor 4 and NF-␬B
Lawrence S. Prince,1 Victor O. Okoh,1 Thomas O. Moninger,2 and Sadis Matalon3
Departments of 1Pediatrics and 3Anesthesia, University of Alabama at Birmingham,
Birmingham, Alabama 35249; and 2Central Microscopy Research Facility, Roy J. and
Lucille A. Carver College of Medicine, University of Iowa, Iowa City, Iowa 52242
Submitted 25 March 2004; accepted in final form 7 July 2004
Prince, Lawrence S., Victor O. Okoh, Thomas O. Moninger,
and Sadis Matalon. Lipopolysaccharide increases alveolar type II
cell number in fetal mouse lungs through Toll-like receptor 4 and
NF-␬B. Am J Physiol Lung Cell Mol Physiol 287: L999 –L1006,
2004; doi:10.1152/ajplung.00111.2004.—Chorioamnionitis is a major
cause of preterm delivery. Infants exposed to inflammation in utero
and then born preterm may have improved lung function in the
immediate postnatal period. We developed a mouse model of chorioamnionitis to study the inflammatory signaling mechanisms that might
influence fetal lung maturation. With this in vivo model, we found that
Escherichia coli lipopolysaccharide (LPS) increased the number of
alveolar type II cells in the fetal mouse lung. LPS also increased type
II cell number in cultured fetal lung explants, suggesting that LPS
could directly signal the fetal lung in the absence of maternal influences. Using immunostaining, we localized cells within the fetal
mouse lung expressing the LPS receptor molecule Toll-like receptor 4
(TLR4). Similar to the signaling pathways in inflammatory cells, LPS
activated NF-␬B in fetal lung explants. Activation of the TLR4/
NF-␬B pathway appeared to be required, as LPS did not increase the
number of type II cells in C.C3H-Tlr4Lps-d mice, a congenic strain
containing a loss of function mutation in tlr4. In addition, the sesquiterpene lactone parthenolide inhibited NF-␬B activation following LPS
exposure and blocked the LPS-induced increase in type II cells. On
the basis of these data from our mouse model of chorioamnionitis, it
appears that LPS specifically activated the TLR4/NF-␬B pathway,
leading to increased type II cell maturation. These data implicate an
important signaling mechanism in chorioamnionitis and suggest the
TLR4/NF-␬B pathway can influence lung development.
rabbits and fetal sheep (5, 7, 21, 22). By understanding how
inflammatory signals influence lung development, we may
learn more about the basic developmental mechanisms in the
fetal lung.
During fetal life, the lung continuously aspirates amniotic
fluid during normal breathing movements (8, 23). Microbial
particles and substances in the amniotic fluid therefore have
access to the fetal airway. Within the airway, infectious particles can activate Toll-Like receptors (TLRs) on the epithelial
cell surface. TLR4 is the receptor for lipopolysaccharide (LPS)
from gram-negative bacteria (3). TLR activation leads to nuclear NF-␬B translocation and production of the innate immune response. In a sheep model of chorioamnionitis, injection
of bacterial LPS into the amniotic fluid increases surfactant
production and lung growth (21). Separation of the airway
lumen from the amniotic fluid prevented changes in the lung
following amniotic LPS injection (22). Therefore, LPS required direct interaction with the fetal lung to alter development. We wanted to better understand the molecular mechanisms by which inflammatory signaling could influence lung
development. To take advantage of the powerful genomic and
molecular tools available, we developed a murine model of
chorioamnionitis. With this model, we tested the hypothesis
that LPS increases alveolar type II cell number through directly
activating the TLR4/NF-␬B pathway in fetal mouse lung.
METHODS
surfactant; respiratory distress syndrome; endotoxin; innate immunity
MANY PREMATURE INFANTS ARE exposed to inflammation before
delivery. Chorioamnionitis is the most common identifiable
cause of preterm labor and premature rupture of the amniotic
membranes (10). Infants born to mothers with chorioamnionitis have increased morbidity and mortality (12). Despite the
negative effects of chorioamnionitis on neonatal outcomes,
premature infants exposed to chorioamnionitis have improved
lung function in the immediate perinatal period (13, 30). These
infants are less likely to display clinical signs of surfactant
deficiency than premature infants not exposed to chorioamnionitis. One possibility that might explain this clinical observation is that inflammatory mediators present in chorioamnionitis
can accelerate fetal lung maturation. Experimental data in
animals support this hypothesis, as exposure to cytokines and
bacterial endotoxin increased surfactant synthesis in neonatal
Antibodies and animals. Goat polyclonal anti-TLR4 (M-16) and
rabbit antithyroid transcription factor-1 (TTF-1, H-190) were obtained
from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit antisurfactant protein A (SP-A) and antisurfactant protein C (SP-C) were
purchased from Research Diagnostics (Flanders, NJ). Rabbit antisurfactant protein B (SP-B) was obtained from Chemicon (Temecula,
CA). BALB/cJ and C.C3H-Tlr4Lps-d mice were obtained from Jackson Laboratories (Bar Harbor, ME). Mice were housed in pathogenfree facilities and mated overnight with embryonic day (E) 0 defined
as the day following vaginal plug. All animal procedures and protocols were reviewed and approved by the Animal Care Review Committees at both institutions.
Fetal mouse lung explant culture. E16 mice were euthanized by
pentobarbital sodium injection. The fetal lungs were then dissected
away from adjacent tissues. Lung tissue was minced into 0.5- to
1-mm3 cubes with fine-tipped scissors under a stereomicroscope. The
pieces of lung were placed onto 24-mm clear polyester membrane
supports (Transwell, 0.4-␮M pore size; Corning, Corning, NY). Culture medium (DMEM) was added only to the basal compartment. The
Address for reprint requests and other correspondence: L. S. Prince, Dept. of
Pediatrics, Div. of Neonatology, Univ. of Alabama at Birmingham, 525 New
Hillman Bldg., 619 19th St. So., Birmingham, AL 35249 (E-mail:
lprince@peds.uab.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.
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TLR4 SIGNALING INCREASES FETAL TYPE II CELLS
explants were cultured in a humidified atmosphere of 95% air-5%
CO2 at 37°C.
In vivo chorioamnionitis model. For the establishment of chorioamnionitis, pregnant mice were injected with phenol-extracted, ion
exchange-purified LPS from Escherichia coli (055:B5, Sigma L4524;
Sigma-Aldrich, St. Louis, MO). E15 mice were anesthetized by
pentobarbital sodium injection (50 mg/kg ip). The abdominal wall was
infiltrated with 0.1– 0.2 ml of bupivacaine. A 1-cm midline abdominal
incision allowed externalization of the uterine horns. A fine-tipped
pulled glass pipette was used for direct intra-amniotic injection of 5 ␮l
of sterile, endotoxin-free saline or LPS (20 ng/ml) into the amniotic
sac of each fetus. After internalization of the uterus, the abdominal
wall was sutured in two layers. Mice were returned to their cages,
given food and water ad libitum, and monitored for signs of pain or
distress. Euthanasia 24 –72 h following surgery allowed procurement
of amniotic fluid, uterine wall with membranes, placenta, and intact
lungs.
During the development of this protocol, we determined that 100
pg/fetus of LPS allowed delivery at term and survival of ⬎95% of the
fetal mice. Doses of 5 ng or higher were associated with increased
miscarriage and fetal death. Injection of ⬍10 pg/fetus LPS did not
consistently cause histological inflammation or elevate cytokines in
the amniotic fluid. The number of fetuses in each litter did not seem
to correlate with survival or inflammatory response.
Immunohistochemistry. Formalin-fixed tissue was paraffin-embedded, sectioned, and affixed to glass slides. In addition to standard
immunohistochemical techniques, we routinely performed antigen
recovery in sodium citrate (10 mM, pH 6) and quenching of endogenous peroxidase activity using 3% H2O2 in methanol (20). Immunostaining was detected using avidin-biotin-horseradish peroxidase
complexes (Vector Laboratories, Burlingame, CA) and diaminobenzamidine. Images were captured under a Zeiss Axiovert microscope
coupled with a Micropublisher charge-coupled device (CCD) camera
(Q Imaging, Burnaby, BC, Canada). For type II cell determination,
control, saline-injected, and LPS-injected litters were euthanized at
E18. Six separate litters were used for each condition. Three fetal
lungs from each litter were processed and stained with the indicated
antibodies against surfactant proteins or TTF-1. The investigators
were blinded to identity of the samples by random numbering of the
slides. Positive cells were quantified with Histometrix software (Kinetic Imaging, Durham, NC). The number of positive cells in each
fetal lung was measured in four random fields, each field from a
different section of the same lung. Statistical analysis between conditions was performed by unpaired t-test.
Immunofluorescence. Explants were fixed for 1 h in 4% paraformaldehyde at room temperature. We blocked nonspecific binding by
incubating samples with SuperBlock (Pierce, Rockford, IL) for 2 h at
room temperature. Explants were incubated with primary antibodies
for 16 –20 h at 4°C. After extensive washing, Alexa 594-conjugated
donkey anti-goat secondary antibody (Molecular Probes, Eugene, OR)
was added for 3 h at room temperature. Nuclei were labeled with
4⬘,6⬘-diamidino-2-phenylindole. The explants and attached membrane
were mounted between glass slides and coverslips in Vectashield
mounting medium (Vector Laboratories, Burlingame, CA). For visualization, multiple Z-images were acquired under an automated epifluorescent microscope (DM RXA2; Leica, Wetzlar, Germany) with a
CCD camera (Hamamatsu Orca ER, Bridgewater, NJ) driven by
SimplePCI (Compix, Cranberry Township, PA). Stray, out-of-focus
light was removed by a nearest-neighbors deconvolution algorithm
(Compix).
Electron microscopy. Fetal lung explants cultured for 72 h were
washed and fixed in 2.5% glutaraldehyde and processed with standard
electron microscopic procedures. Samples were postfixed in 1%
osmium tetroxide followed by 2.5% aqueous uranyl acetate and then
dehydrated in a graded series of ethanol washes. Thin sections (70
nm) of the Eponate 12-embedded specimen were placed on 135-mesh
hexagonal copper grids and poststained with uranyl acetate and
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Reynolds lead citrate. Sections were visualized under a Hitachi
H-7000 transmission electron microscope, and the resulting film
negatives were scanned and converted to TIFF images. Histometrix
image analysis software identified the number of type II cells by
counting osmiophilic inclusions lining the airway lumen. A sizing
filter algorithm excluded small vesicles and particles. Secreted lamellar bodies in the airway lumen were also excluded from analysis.
Statistical significance was measured with an unpaired t-test.
NF-␬B luciferase measurements. Recombinant E1-deleted adenovirus expressing the firefly luciferase gene downstream of a synthetic
NF-␬B response element was kindly provided by Dr. Paul McCray
and produced by the Gene Transfer Vector Core at the University of
Iowa and at the Viral Vector Core of the University of Alabama at
Birmingham Cystic Fibrosis Center. E16 fetal mouse lung explants
were infected with 109 viral particles/ml. For luciferase activity
measurements, explants were lysed and clarified by centrifugation.
Total protein concentrations were determined by bicinchoninic acid
method (Pierce). Luciferase activity was measured following addition
of luciferase substrate (SteadyGlo; Promega, Madison, WI) with a
single tube luminometer (Turner Designs, Sunnyvale, CA). Sample
activity (light units/mg protein) was then normalized to data from
control explants following 24 h of culture.
RESULTS
Intra-amniotic injection of LPS causes chorioamnionitis in
mice. We established a murine model of chorioamnionitis to
study how inflammatory signals alter fetal lung development.
E15 BALB/cJ mice received 100 pg/fetus of E. coli LPS via
intra-amniotic injection. This dose of LPS did not cause significant mortality or fetal loss. We tested whether LPS injection would specifically recruit inflammatory cells to the uterus
and elevate cytokines within the amniotic fluid. Twenty-four
hours following injection, the placenta, amniotic membranes,
and uterine wall were fixed, sectioned, and stained with hematoxylin and eosin. Microscopy revealed inflammatory cells
along the uterine wall of LPS-exposed mice (Fig. 1B). We
recognized the possibility of nonspecific inflammation arising
from the injection procedure. However, injection with sterile,
endotoxin-free saline did not recruit inflammatory cells (Fig.
1A). LPS disrupted the normal placental structure and increased fibrin deposition, consistent with inflammatory
changes (Fig. 1D) (2). No changes were seen in the placentas
of saline-injected controls (Fig. 1C).
Having observed inflammatory changes in the placenta and
uterus, we next tested whether LPS could increase cytokine
levels in the amniotic fluid. Elevated cytokines in the amniotic
fluid might expose the fetal mice and particularly the fetal
lungs to an inflammatory environment. We measured IL-1␤
and TNF-␣ concentrations in the amniotic fluid of salineinjected and LPS-injected mice 24 h following exposure and
from E16 mice that did not undergo a surgical procedure. LPS
increased concentrations of each cytokine in the amniotic fluid
compared with control mice (Fig. 1, E and F), whereas saline
injection did not. These findings collectively demonstrated that
LPS injection specifically induced chorioamnionitis in timed
pregnant mice.
LPS increases alveolar type II cell number. In premature
infants, exposure to chorioamnionitis appears to improve lung
function in the immediate perinatal period (13, 30). We hypothesized that chorioamnionitis increases the number of type
II cells in the developing lung. More type II cells might
increase surfactant production, improving lung compliance. To
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TLR4 SIGNALING INCREASES FETAL TYPE II CELLS
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fluid. As shown in Fig. 2, LPS increased the number of
SP-A-positive alveolar cells lining the distal airways. LPS also
increased the number of cells expressing TTF-1, a marker of
alveolar type II cells (Fig. 2). Similar data were obtained using
antibodies against SP-B and SP-C (data not shown). LPS
Fig. 1. LPS injection into the amniotic fluid of embryonic day (E) 15 mice
induced chorioamnionitis. Escherichia coli LPS (100 pg) or sterile, endotoxinfree saline was injected into the amniotic fluid surrounding each fetus at E15.
A and B: 24 h following injection, sectioning and hematoxylin-eosin (H&E)
staining of the uterus confirmed the presence of inflammation. A: no cellular
infiltrate was seen in saline-injected controls. B: a mixed population of
inflammatory cells lined the uterine wall of LPS-exposed animals. C and D:
placentas from injected mice were also stained by H&E and examined for signs
of inflammation. C: saline injection did not alter the normal structural appearance of the placenta. D: LPS-exposed placentas contained abnormal villus
structures and fibrin deposition consistent with inflammatory changes. E and F:
LPS injection also increased IL-1␤ and TNF-␣ concentrations in the amniotic
fluid. Twenty-four hours following injection of LPS or saline, the animals were
killed, and amniotic fluid was aspirated from the uterus. The concentrations of
cytokines were measured by ELISA. Injection of sterile saline did not increase
IL-1␤ or TNF-␣ concentrations compared with E16 controls. LPS increased
IL-1␤ (E) and TNF-␣ (F) levels ⬃5-fold (*P ⬍ 0.05, n ⫽ 5).
test this hypothesis, we measured type II cell number in fetal
mouse lungs by immunohistochemistry. Using an antibody
against SP-A, we stained fetal mouse lung sections at E18, 3
days following injection of LPS or saline into the amniotic
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Fig. 2. LPS injection increased the number of alveolar type II (TII) cells in
fetal mouse lungs. A: TII cells from E18 BALB/cJ fetal mouse lungs were
identified by immunostaining using a polyclonal antibody against surfactant
protein (SP)-A. B: SP-A-positive TII cells were identified in E18 lungs
exposed to endotoxin-free saline on E15, 72 h before death, fixation, and
processing. C: increased staining for SP-A in E18 lungs exposed to E. coli LPS
(100 pg/fetus) on E15. D: background staining using secondary antibody alone
(2°). E: additional sections were stained using an antibody against the TII cell
marker thyroid transcription factor (TTF)-1. The number of TII cells per mm2
was then counted from either the SP-A- or TTF-1-labeled samples (*P ⬍ 0.05,
n ⫽ 6).
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TLR4 SIGNALING INCREASES FETAL TYPE II CELLS
therefore increased the number of surfactant-expressing alveolar type II epithelial cells within the fetal mouse lung.
LPS might directly signal the epithelia of the fetal lung, or it
could elicit a systemic inflammatory response leading to increased type II cell number. We tested whether LPS could
induce similar responses in fetal mouse lung explants removed
from the systemic circulation. Fetal mouse lung explants cultured for 2– 4 days developed alveolar structures closely resembling E18 fetal mouse lungs. Transmission electron microscopy of the explants showed alveolar type II cell differentiation and surfactant secretion (Fig. 3, A and B). We cultured
E16 fetal lung explants for 3 days in serum-free media in the
presence and absence of LPS (250 ng/ml). Adding LPS to the
media increased the number of cells expressing SP-B (Fig.
3D). We obtained similar results using antibodies against
SP-A, SP-C, and TTF-1 (not shown). These data indicate that
LPS might increase type II cell number by directly interacting
with fetal lung tissue.
LPS activates the TLR4/NF-␬B pathway in fetal lung. Previous studies in other tissues suggest that LPS initiates an
innate immune response through TLR4 (1, 3). If LPS can
increase alveolar type II cells by directly signaling the fetal
lung, then cells within the fetal lung might express TLR4. To
identify these potentially LPS-responsive cells, we stained
sections of fetal mouse lung tissue using an antibody against
TLR4. We detected immunostaining for TLR4 both in the
conducting airways and in distal epithelia and mesenchyme
(Fig. 4, A and B). Columnar epithelial cells lining the airway
expressed TLR4 at the apical and basal surfaces (Fig. 4A). The
distal air spaces and adjacent mesenchyme labeled with
slightly less intensity (Fig. 4B). Nonimmune rabbit IgG gave
no detectable staining (not shown). We also detected TLR4
expression in E16 fetal lung explants using immunofluorescence. In explants cultured for 3 days, antibodies against TLR4
labeled epithelial and mesenchymal cells in the distal air spaces
(Fig. 4C). On the basis of these results, it is possible that cells
within the proximal and distal lung express TLR4 and respond
to LPS.
In cells that express TLR4 and activate inflammatory pathways, binding of LPS to TLR4 leads to activation and nuclear
translocation of NF-␬B (32). We wanted to determine whether
a similar pathway was involved in our explant model. To
measure NF-␬B activation, we infected E16 fetal mouse lung
explants with recombinant adenovirus expressing the luciferase
gene downstream of an NF-␬B response element. LPS increased luciferase gene expression in fetal lung explants, with
maximal induction at 48 h following LPS exposure (Fig. 5C).
Parthenolide, a sesquiterpene lactone inhibitor of NF-␬B (9),
inhibited the increase in luciferase activity. These data suggest
LPS can activate the TLR4/NF-␬B signaling pathway in fetal
mouse lungs.
TLR4/NF-␬B activation increases type II cell number. We
next wanted to determine whether LPS increases type II cell
number through signaling the TLR4/NF-␬B pathway. To first
test whether functional TLR4 was required in our in vivo
chorioamnionitis model, we injected LPS into the amniotic
fluid of E15 C.C3H-Tlr4Lps-d mice. This congenic strain bears
a loss of function mutation in the tlr4 allele on a BALB/cJ
genetic background (28). In contrast to the BALB/cJ animals,
injecting LPS into the amniotic fluid of C.C3H-Tlr4Lps-d failed
to increase the number of type II cells (Fig. 6). These results
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Fig. 3. LPS increased the number of alveolar TII cells in fetal mouse lung
explants. E16 BALB/cJ lungs were minced and seeded onto permeable
supports and cultured in an air-liquid interface. The explants develop alveolar
structures and differentiated epithelia and secrete surfactant (A and B). After 3
days of culture, alveolar TII cells were labeled with a polyclonal antibody
against SP-B (C and D). E: including LPS (250 ng/ml) in the culture media
increased the number of SP-B-positive cells (*P ⬍ 0.05, n ⫽ 16).
suggest that LPS signaled through TLR4 to increase the number of type II cells in fetal lung.
Consistent with TLR4 activation, we observed that LPS
activated NF-␬B in fetal lung explants. Using the NF-␬B
inhibitor parthenolide, we tested the hypothesis that NF-␬B
activation is required for the increase in type II cells seen
following LPS exposure. Fetal lung explants were cultured for
3 days in the presence of LPS with or without parthenolide. As
an additional approach to immunohistochemistry for identifi-
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TLR4 SIGNALING INCREASES FETAL TYPE II CELLS
Fig. 4. Toll-like receptor (TLR) 4 was expressed in the proximal and distal
fetal lung epithelia. E18 fetal mouse lungs were fixed, sectioned, and labeled
with a polyclonal antibody against TLR4 (A and B). Antibody labeling was
detected by diaminobenzamidine staining and bright field microscopy. The
proximal airway epithelia stained positive for TLR4 expression at both the
apical and basolateral surfaces (A). Background staining using only the
secondary antibody is shown in C. Immunofluorescence detected TLR4 expression (magenta) in E16 fetal mouse lung explants cultured for 3 days (D).
Nuclei were labeled with 4⬘,6⬘-diamidino-2-phenylindole (blue).
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through the TLR4/NF-␬B pathway, similar to its action in
other tissues with innate immune function. This pathway is
required for the changes in type II cell number, as LPS had no
effect in fetal lung explants treated with an NF-␬B inhibitor or
in mice lacking functional TLR4.
Our findings suggest that LPS directly signals cells within
the fetal lung. We observed increased type II cells in fetal lung
explants removed from both maternal and fetal circulations.
Consistent with our data, experiments in sheep found that
isolation of the fetal airway from the amniotic fluid prevented
the changes in lung maturation seen with injection of endotoxin
(22). Studies in sheep also suggested that maternal hormone
production does not mediate the changes in lung development
seen with endotoxin (15). Inflammation may also increase the
local and systemic production of steroids or growth factors that
could influence alveolar development and type II cell differentiation (31). These findings and our present data support the
hypothesis that the fetal lung is capable of responding to LPS
exposure in the absence of maternal influences.
Cells responsive to LPS express TLR4. We detected TLR4
expression in the epithelia and mesenchyme of fetal mouse
lungs. TLR4 appeared to be required for the changes in type II
cell number we observed, as LPS had no effect in C.C3HTlr4Lps-d mice lacking functional TLR4. These experiments
illustrate how our mouse model might be useful for investigating the molecular components of fetal inflammatory signaling.
By studying mice containing specific targeted gene disruptions,
cation of type II cells, we used electron microscopy to quantify
type II cell number. Fixation and osmium tetroxide staining of
serial sections identified type II cells when examined by
transmission electron microscopy (Fig. 7). As we observed by
immunohistochemistry, LPS increased the number of type II
cells in fetal lung explants (Fig. 7, B and D). Parthenolide
blocked the increase in type II cell number (Fig. 7, C and D),
suggesting that NF-␬B activation is required for the LPSinduced increase in type II cells. Parthenolide alone did not
change type II cell number (not shown). Measuring type II cell
number in toluidine blue-stained sections by light microscopy
and by immunostaining sections for SP-A gave similar results
(not shown). Our findings indicate LPS signals the TLR4/
NF-␬B pathway within the fetal mouse lung, leading to an
increase in type II cell number.
DISCUSSION
Compared with premature infants of equal size and gestation
born in the absence of inflammation, newborns exposed to
chorioamnionitis have a lower incidence of respiratory distress
syndrome (13, 30). Consistent with these clinical observations,
newborn lambs and rabbits exposed in utero to bacterial endotoxin have increased lung compliance and larger lung volumes
(5, 21, 22). We established a mouse model of chorioamnionitis
to study the molecular mechanisms by which LPS could
increase lung maturation. Our data show that LPS can increase
the number of alveolar type II cells both in vivo and in a lung
explant model. In the fetal mouse lung, LPS appears to signal
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Fig. 5. LPS activated NF-␬B in fetal lung explants. E16 fetal lung explants
were infected with a recombinant adenovirus expressing the luciferase gene
downstream of an NF-␬B response element. After infection, the explants were
cultured in the absence or presence of LPS (250 ng/ml). An additional set of
LPS-treated explants was also treated with the NF-␬B inhibitor parthenolide (1
␮M) or with parthenolide alone. Recombinant adenovirus constitutively expressing green fluorescent protein verified transfection (A). Phase microscopy
image of the same explant is shown in B. LPS increased luciferase activity
⬃8-fold over controls at 48 h (n ⫽ 5, C). Parthenolide blocked the induction
of luciferase by LPS at both 48 and 72 h (n ⫽ 5). *P ⬍ 0.05 between control
and LPS; #P ⬍ 0.05 between LPS and LPS ⫹ parthenolide.
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Fig. 6. LPS did not increase the number of alveolar TII cells in C.C3HTlr4Lps-d mice. Congenic C.C3H-Tlr4Lps-d mice at E15 received either sterile
saline (A) or E. coli LPS (B) via direct intra-amniotic injection. On E18 (72 h
following surgery) the animals were killed and the fetal lungs were fixed and
sectioned. A polyclonal antibody against SP-A stained alveolar TII cells.
C: SP-A-positive cells per mm2 were counted (n ⫽ 9).
we might identify the molecular mechanisms of how chorioamnionitis influences fetal lung development.
Chorioamnionitis can arise from a variety of pathogens. We
have used E. coli LPS in our model of chorioamnionitis as a
potent stimulator of the innate immune system, as gramnegative bacteria may comprise a significant percentage of
intrauterine infections (27). In addition, the signaling pathways
activated by LPS through TLR4 have been well studied.
Gram-positive and atypical pathogens can activate common
innate immune pathways through TLRs and may induce similar inflammatory responses in the amniotic fluid (1). Ureaplasma species can be isolated in chorioamnionitis cases (17),
but the chronic inflammatory response to this pathogen may
prove difficult to study during the short gestation of the mouse.
Cells within the placenta, amniotic membranes, and uterus can
all participate in the innate immune response, secreting cytokines and chemokines including IL-8, IL-10, monocyte chemoattractant protein-1, and regulated on activation, normal
T-expressed and presumably secreted (RANTES) (6, 25). In
addition, fetus-derived neutrophils can occupy the amniotic
fluid in chorioamnionitis (18). Although we have shown the
fetal lung can directly respond to endotoxin, the contribution of
cells in the uterus, placenta, and amniotic membranes could
alter how the fetal innate immune system responds to both
microbial products and inflammatory mediators.
Intra-amniotic injection of E. coli LPS increased inflammatory cytokines, type II cell numbers, and lung compliance in
fetal sheep (16, 21). Injection of IL-1, but not TNF-␣, also
increased surfactant expression, suggesting that signaling
through either TLR4 or the IL-1 receptor can stimulate alveolar
differentiation. IL-1␤ also increased surfactant gene expression
in neonatal rabbits and rabbit lung explants (5, 7). Similar to
the studies in sheep, TNF-␣ did not increase surfactant expres-
Fig. 7. NF-␬B inhibition prevented the increase in TII
cells following LPS exposure. E16 BALB/cJ explants
were cultured in the absence (A) or presence (B) of LPS
(250 ng/ml). An additional set of LPS-treated explants
were also treated with the NF-␬B inhibitor parthenolide
(1 ␮M, C). After 72 h of culture, the explants were
fixed, stained with OsO4, and processed. Transmission
electron microscopy identified TII cells lining the distal
airways. The number of TII cells per mm2 for each
condition was counted by examining multiple sections
from each of 12 explants from 3 different litters of
pregnant mice (*P ⬍ 0.05).
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TLR4 SIGNALING INCREASES FETAL TYPE II CELLS
sion in fetal rabbit lungs (26). IL-1 also had a larger effect on
fetal rabbit lungs effects compared with newborn animals (11).
These findings further suggest inflammatory signaling can
influence fetal lung development. The effects of LPS on
alveolar type II cell number could also represent a response to
injury in the fetal lung. Type II cells proliferate in response to
injury in adult lungs. Hyperoxic injury increased type II cell
numbers, possibly through the formation of reactive oxygen
species and NF-␬B activation (19, 29). We have not yet
determined whether the effects of LPS in our system result
from increased proliferation of type II cells or increased maturation of type II cell precursors. In cultured cells, NF-␬B
activation can increase SP-A expression through binding upstream elements in the SP-A promoter (14). Inflammatory
signals and injury mechanisms could therefore both influence
alveolar development.
Rounioja et al. (24) detected changes in cardiac hemodynamics and increased cytokine expression in the myocardium
of fetal DBA/2 mice exposed in utero to LPS. They did not
report lung inflammation or TLR4 expression in the lungs at
days 15–16 of gestation. Differences in gestation and mouse
strain may contribute to differences in TLR4 expression during
lung development. We have detected TLR4 expression and
similar increases in type II cell number following LPS exposure using both BALB/cJ and C57BL/6 mice (not shown). Our
data suggest that signaling through a TLR can influence development. While we have used an inflammatory stimulus
(LPS) in a model of chorioamnionitis, increased type II cell
number occurred in the absence of lung neutrophil influx or
gross damage to the epithelia. Activation of the NF-␬B pathway in fetal lung cells may represent an additional therapeutic
target for increasing the production of surfactant in premature
infants.
ACKNOWLEDGMENTS
We thank our colleagues for helpful comments and Kathryn Fallon for
assistance. L. S. Prince is a Fellow of the Parker Francis Families Foundation.
GRANTS
This work was supported by The Children’s Hospital Research Institute
(L. S. Prince) and Child Health Research Center Grant NICHD43397 (L. S.
Prince).
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