Am J Physiol Lung Cell Mol Physiol 301: L285–L295, 2011.
First published June 10, 2011; doi:10.1152/ajplung.00446.2010.
Pulmonary and systemic inflammatory responses to intra-amniotic IL-1␣
in fetal sheep
Suhas G. Kallapur,1,3 Boris W. Kramer,2 Ilias Nitsos,3 J. Jane Pillow,3 Jennifer J. P. Collins,2
Graeme R. Polglase,3 John P. Newnham,3 and Alan H. Jobe1,3
1
Division of Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, University of Cincinnati, Cincinnati, Ohio;
School of Oncology and Developmental Biology, Department of Pediatrics, Maastricht University Medical Center,
Maastricht, The Netherlands; and 3School of Women’s and Infants’ Health, University of Western Australia, Perth, Australia
2
Submitted 13 December 2010; accepted in final form 2 June 2011
prematurity; bronchopulmonary dysplasia; chorioamnionitis; fetal inflammatory response syndrome
recognizes two major types of
inflammatory products: pathogen- and damage-associated molecular patterns. While the pathogen-associated molecular patterns are mainly recognized by different Toll-like receptors
(TLRs), recent studies demonstrate that IL-1 signaling is critically required to mediate inflammation induced by a variety of
endogenous damage-associated molecular patterns such as uric
acid, ATP, reactive oxygen species, heat-shock proteins, and
others (5). The IL-1-related cytokines can be pro- or antiinflammatory and are closely linked to innate immune reTHE INNATE IMMUNE SYSTEM
Address for reprint requests and other correspondence: S. G. Kallapur,
Cincinnati Children’s Hospital Medical Center, Univ. of Cincinnati, Division
of Pulmonary Biology, 3333 Burnet Ave., Cincinnati, OH 45229-3039 (e-mail:
suhas.kallapur@cchmc.org).
http://www.ajplung.org
sponses (6). The cytosolic IL-1 processing and secretion
machinery, called the NALP3 inflammasome, normally tightly
controls the release of active IL-1 from cells (39). Familial
Mediterranean fever and neonatal-onset multi-inflammatory
disease are examples of rare autoinflammatory diseases caused
by an unregulated systemic IL-1 signaling (7). Recent reports
also implicate IL-1 in the pathogenesis of type 2 diabetes
(36). These diseases can be effectively treated with IL-1
receptor antagonists or a specific neutralizing IL-1 antibody
(7), which emphasizes the concept that even small amounts of
unchecked IL-1 signaling can cause a severe systemic inflammatory disorder.
Chorioamnionitis, defined as inflammation of the fetal membranes, complicates up to 70% of preterm deliveries before 30
wk of gestation (13). The epidemiological associations with
preterm infants exposed to chorioamnionitis are fetal systemic
inflammation and lung, brain, and gastrointestinal injury (14,
45). Lung inflammation may initiate a progressive injury response that results in bronchopulmonary dysplasia (BPD) (21).
IL-1 is postulated to play a central role in the progression of
preterm labor and fetal inflammatory responses (12, 13, 47).
Preterm infants with increased levels of IL-1 in tracheal
aspirates in the first few days of life are at increased risk of
subsequently developing BPD (20, 44, 52). In mice, prenatal
overexpression of the IL-1 transgene causes postnatal pulmonary pathology similar to BPD (3). Furthermore, IL-1 signaling and inflammasome activation are critical in mediating
bleomycin-induced pulmonary fibrosis and inflammation in
mice (11). In fetal sheep, intra-amniotic injection of LPS
robustly induces IL-1 expression and causes lung, chorioamnion, and systemic inflammation (24, 26). Therefore, pre- and
postnatal signaling by IL-1 appears to be important in the
pathogenesis of fetal inflammation and lung injury.
To understand if LPS-induced fetal inflammatory responses
were IL-1-dependent in fetal sheep, we blocked IL-1 signaling
using a recombinant human IL-1 receptor (IL-1R) antagonist
and demonstrated reduced pulmonary and systemic inflammatory responses (27). However, IL-1 blockade did not change
LPS-induced chorioamnionitis, which is known to contribute
to systemic inflammation in the fetus (2). Furthermore, LPSinduced lung monocyte chemotactic protein 1 and serum amyloid A (SAA) expression in the fetal sheep was IL-1-independent (48, 55). These results demonstrate that chorioamnionitis-induced fetal inflammation is mediated by different
cytokine networks in different fetal organ systems. The
IL-1-dependent inflammatory pathways are important to
understanding the pathogenesis of chorioamnionitis-associated fetal organ injury. Therefore, we sought to determine
1040-0605/11 Copyright © 2011 the American Physiological Society
L285
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Kallapur SG, Kramer BW, Nitsos I, Pillow JJ, Collins JJP,
Polglase GR, Newnham JP, Jobe AH. Pulmonary and systemic
inflammatory responses to intra-amniotic IL-1␣ in fetal sheep. Am J
Physiol Lung Cell Mol Physiol 301: L285–L295, 2011. First published June 10, 2011; doi:10.1152/ajplung.00446.2010.—Clinical and
epidemiological studies implicate IL-1 as an important mediator of
perinatal inflammation. We tested the hypothesis that intra-amniotic
IL-1␣ would induce pulmonary and systemic fetal inflammatory
responses. Sheep with singleton fetuses were given an intra-amniotic
injection of recombinant sheep IL-1␣ (100 g) and were delivered 1,
3, or 7 days later, at 124 ⫾ 1 days gestation (n ⫽ 5– 8/group). A
separate group of sheep were given two intra-amniotic IL-1␣ injections (100 g dose each): 7 days and again 1 day prior to delivery.
IL-1␣ induced a robust increase in monocytes, neutrophils, lymphocytes, and IL-8 protein in bronchoalveolar lavage fluid. H2O2 secretion was increased in inflammatory cells isolated from lungs of
IL-1␣-exposed lambs upon LPS challenge in vitro compared with
control monocytes. T lymphocytes were recruited to the lung. IL-1,
cyclooxygenase-1, and cyclooxygenase-2 mRNA expression increased in the lung 1 day after intra-amniotic IL-1␣ exposure. Lung
volumes increased 7 days after intra-amniotic IL-1␣ exposure, with
minimal anatomic changes in air space morphology. The weight of the
posterior mediastinal lymph node draining the lung and the gastrointestinal tract doubled, inducible nitric oxide synthase (NOSII)-positive
cells increased, and Foxp3-positive T-regulatory lymphocytes decreased in the lymph node after IL-1␣ exposure. In the blood,
neutrophil counts and plasma haptoglobin increased after IL-1␣ exposure. Compared with a single exposure, exposure to intra-amniotic
IL-1␣ 7 days and again 1 day before delivery had a variable effect
(increases in some inflammatory markers, but not pulmonary cytokines). IL-1␣ is a potent mediator of the fetal inflammatory response
syndrome.
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IL-1 IN FETAL INFLAMMATION
the downstream organ-specific effects of intra-amniotic
IL-1␣ in preterm fetal sheep, with an emphasis on the lung.
Additionally, we asked if repeated exposures to intra-amniotic IL-1␣ induced tolerance to the agonist, a characteristic
of repetitive exposures to LPS (22).
MATERIALS AND METHODS
Table 1. Physiological variables of preterm lambs at birth after intra-amniotic exposure to IL-1␣ or saline
Group
Control saline
1 day ⫹ 7 days
7 days
1 day
Composite
IL-1␣
1 day
3 days
7 days
1 day ⫹ 7 days
n
Body Wt, kg
Lung Wt-to-Body Wt Ratio
BALF Protein, mg/kg
3
1
1
5
3.4, 2.7, 2.9
3.2
2.2
2.9 ⫾ 0.5
0.028, 0.035, 0.03
0.036
0.035
0.033 ⫾ 0.002
3.8, 10.8, 5.5
5.9
8.0
6.8 ⫾ 2.7
7
8
8
8
2.7 ⫾ 0.4
2.8 ⫾ 0.3
2.7 ⫾ 0.3
2.7 ⫾ 0.2
0.032 ⫾ 0.005
0.036 ⫾ 0.008
0.034 ⫾ 0.004
0.033 ⫾ 0.003
7.3 ⫾ 1.6
18.9 ⫾ 14.5
10.1 ⫾ 3.5
13.4 ⫾ 5.5
Individual value are shown for control animals; other values are means ⫾ SD. BALF, bronchoalveolar lavage fluid. All the lambs were delivered at 124 ⫾
1 days gestational age.
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Animals. The animals were studied in Western Australia with
approval from the Animal Care and Use Committees of the Cincinnati
Children’s Hospital and the University of Western Australia. In
separate protocols, time-mated Merino ewes with singleton fetuses
were randomly assigned to different study groups of five to eight
animals (Table 1). Gastrointestinal tissues from some of the animals
in this study were used to study the effects of intra-amniotic IL-1␣ on
fetal gut inflammation (57).
Treatments. Pregnant sheep were given ultrasound-guided injections of recombinant sheep IL-1␣ (100 g; Protein Express, Cincinnati, OH) in 2 ml of saline into the amniotic fluid 1, 3, or 7 days prior
to delivery. A group of animals were injected with 100 g of IL-1␣
7 days and again 1 day prior to delivery. The dose of IL-1␣ was
selected on the basis of higher inflammatory responses for a 150- than
a 15-g dose in this model (54). In pilot experiments, we demonstrated an equivalent lung inflammatory response to the 100- and
150-g doses. The control group received a single equivalent 2-ml
intra-amniotic saline injection 1 or 7 days prior to delivery or repeated
injections 7 days and again 1 day prior to delivery, and the comparison
group was this composite control group. All fetal injections were
given with ultrasound guidance and with electrolyte analysis to
confirm an injection into amniotic, rather than allantoic, fluid (19).
Each ewe was heavily anesthetized with ketamine and medetomidine prior to delivery of the fetus. The fetuses were given lethal
intravascular doses of pentobarbital sodium. All animals were
delivered at 124 ⫾ 1 days gestational age. There were no fetal
deaths.
Tissue collection at delivery. At autopsy, blood was collected for
plasma, automated total white blood cell counts, and differential
counts with correction for nucleated red blood cells. Fetal lung fluid
was allowed to drain passively. For determination of compliance, lung
volume was measured at 40 cmH2O pressure from the deflation limb
of an air pressure-volume curve with the chest open (28). Thereafter,
the right and left lungs were separated. The right upper lobe of the
lung was used for morphology and morphometry following airway
inflation with 10% buffered formalin at 30 cmH2O pressure. For
collection of bronchoalveolar lavage fluid (BALF), the left lung was
inflated with normal saline to total lung capacity and then the saline
was withdrawn; this procedure was repeated three times (19). BALFs
were pooled and used for cell counts and protein measurements (28).
BALF cell counts are expressed as total cells recovered from the
lavage normalized to body weight. Portions of the right lower lobe of
the lung, liver, spleen, and posterior mediastinal lymph node were
snap-frozen for RNA extraction.
Lung, monocyte culture, and H2O2 generation. After exsanguination of the fetus, the lung was chopped thoroughly into fine pieces and
incubated in RPMI medium (31). The lung suspension was gently
passed through a 100-m mesh filter, and the suspension was washed
twice with PBS. Cells from the suspension were layered over discontinuous Percoll gradients (1.085 and 1.046 g/ml; Amersham Pharmacia Biotech, Piscataway, NJ) to separate the monocytic cells from the
other cells at the interface between the Percoll densities (31). Cells
were counted using Trypan blue to evaluate viability and then plated
in culture dishes using media supplemented with 10% heat-inactivated
fetal calf serum (Sigma Chemical). After incubation at 37°C for 2 h,
nonadherent cells were removed, and plates were washed twice with
PBS. Production of H2O2 by the cultured lung monocytes challenged
with LPS (100 ng/ml) was measured with an assay based on oxidation
of ferrous iron (Fe2⫹) to ferric iron (Fe3⫹) by H2O2 under acidic
conditions (Bioxytech H2O2-560 assay, OXIS, Portland, OR) (22).
Cytokine mRNA quantitation. Total RNA was isolated from the
fetal tissues by a modified Chomzynski method, as described elsewhere (28), and mRNA quantitation was performed using real-time
PCR. The mRNA was reverse-transcribed to yield a single-strand
cDNA (Fisher Scientific, Pittsburgh, PA), which was used as a
template with primers and TaqMan probes (Applied Biosystems,
Carlsbad, CA) specific to sheep sequences. The values for each
cytokine were normalized to the internal 18S rRNA. Data are expressed as fold increase over the control value. Quantitation of SAA3
was performed using RNase protection analysis (28, 55), because
alternatively spliced forms of the gene amplified by primer sequences
complicate real-time PCR analysis. Briefly, solution hybridization
was performed for 16 h using a molar excess of [␣-32P]UTP-labeled
riboprobes. Unhybridized single-strand RNA was digested with
RNase A/T1 (Pharmingen, San Diego, CA). RNase was then inactivated, and protected RNA was precipitated using RNase protection
assay inactivation buffer (RPA III, Ambion, Austin, TX). The ribosomal protein mRNA L32 was used as an internal control (23). The
protected fragments were resolved on 6% polyacrylamide-8 mol/l
urea gels, visualized by autoradiography, and quantified on a PhosphorImager using ImageQuant version 1.2 software (Molecular Dynamics, Sunnyvale, CA).
Lung morphometry. For morphometric and morphological analyses, the right upper lobe of the lung was inflation fixed at 30 cmH2O
pressure using 10% buffered formalin. Formalin was removed from
the fixed tissue within 24 h by washing in PBS (pH 7.4); then the
tissue was transferred to 70% ethanol and embedded in paraffin. The
lung sections were stained with a modified Hart’s procedure using
Weigert’s iron hematoxylin and metanil yellow. Lung air space area
was measured and expressed relative to the total lung area using the
color threshold function of Metamorph (version 6.1r0, Molecular
Devices/Universal Imaging, Sunnyvale, CA). Alveolar secondary sep-
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AJP-Lung Cell Mol Physiol • VOL
ences among the groups. We then performed a post hoc multiplecomparisons analysis to assess the differences between the control
group and the different treatment groups and between the 1- or 7-day
single-exposure group and the group exposed to IL-1␣ 7 days and
again 1 day before delivery. All multiple-group comparisons were
conducted with Dunn’s multiple-comparison correction factor applied
to control for an inflated type I error rate that occurs with multiple
testing. Statistical significance was accepted at P ⬍ 0.05.
RESULTS
Pulmonary responses to intra-amniotic IL-1␣. Exposure to
IL-1␣ did not change body weight or lung weight-to-body
weight ratio (Table 1). Intra-amniotic IL-1␣ did not increase
BALF protein (the 2-fold increase at 3 days was not statistically significant), suggesting minimal lung injury. No neutrophils and very few monocytes or lymphocytes were detected in
the BALF of control lambs (Fig. 1, A–C). IL-1␣ recruited
neutrophils, monocytes, and lymphocytes to the air spaces by
3 days. Interestingly, the neutrophil and lymphocyte counts
further increased twofold after repeated exposure to intraamniotic IL-1␣ 7 days and again 1 day before delivery, but
monocyte numbers did not change. The ability of lung monocytes to respond to LPS in vitro was evaluated by measurement
of oxidant responses. While the immature lung monocytes
from control lambs secreted 8 ⫾ 1.4 mol H2O2/106 monocytes, intra-amniotic IL-1␣ exposure for 7 days increased H2O2
secretion three- to fourfold, indicating maturation of the cells
(Fig. 1D). Consistent with cell recruitment to BALF, repeated
exposure to IL-1␣ further increased H2O2 generation twofold.
To better define the leukocyte populations, flow cytometry
was performed to immunophenotype cells not recovered in the
BALF by elastase digestion of lung tissue and purification of
leukocytes using CD45 magnetic beads. CD11b, an integrin
that mediates leukocyte adhesion and migration, is expressed
on monocytes and activated neutrophils (41). Compared with
controls, expression of CD11b (Fig. 1E), CD11c (Fig. 1F), or
MHCII (Fig. 1G) did not change significantly after IL-1␣
exposure.
Next, we performed immunostaining with an antibody
against the T-cell receptor CD3, which demonstrated recruitment of T lymphocytes 1 day after exposure, with no further
increases after repeated IL-1␣ exposures (Fig. 2). Most of the
CD3-positive T lymphocytes were in the pulmonary interstitium. Expression of MPO in neutrophils and monocytes also
increased after IL-1␣ exposure (data not shown).
Intra-amniotic injection of IL-1␣ induced a threefold increase in expression of IL-1 and a 20-fold increase in the
cyclooxygenase (COX) enzymes COX-1 and COX-2 in the
lung 1 day after exposure (Fig. 3, A–C). IL-6, IL-8, and IL-1R
antagonist mRNA expression in the lung increased variably 1
day after IL-1␣, but these values were not significantly increased over controls (P ⫽ 0.2, 0.07, and 0.3 vs. control,
respectively; Fig. 3, D–F). IL-1␣ exposure also did not induce
TNF␣ (Fig. 3G) or mRNA expression of the interferon-inducible genes IP-10 (CXCL10) and MIG (CXCL9) in the lung
(data not shown). In contrast to the single exposure, cytokine
mRNA expression after repeated exposure to intra-amniotic
IL-1␣ was not different from controls. IL-8 protein was not
detectable in the BALF of the control lambs, while IL-8 was
present at 10 –20 ng/ml in the BALF of all the groups
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tal crests were identified by the typical morphology and elastin
staining using Hart’s procedure (37). The secondary septal crests are
expressed relative to air space area. Blinded measurements were
performed in five random nonoverlapping fields (⫻20 objective) for
each animal and for at least four animals per group. The average
measurement from each lamb was used to compute a group average
and standard deviation.
Immunohistochemistry and scoring of inflammation. Inflammatory
cells in BALF were counted using a hemocytometer, and the differential counts were performed using DiffQuick staining (Baxter Health
Care, Deerfield, IL). Lung and lymph node tissues were embedded
in paraffin. After deparaffinization and rehydration of fixed tissue,
antigen retrieval was carried out using citric acid buffer (pH 6.0)
with microwave boiling. Endogenous peroxidase activity was
blocked with methyl alcohol/H2O2. Nonspecific interactions were
inhibited with 2% goat serum for primary and secondary antibody
incubations. Sections were incubated with anti-CD3 antibody (catalog
no. A0452, Dako; 1:100 dilution), anti-inducible nitric oxide synthase
(iNOS) antibody (BD Biosciences; 1:250 dilution), anti-Foxp3 (catalog no. 14-7979-82, e-Biosciences; 1:100 dilution), or anti-myeloperoxidase (MPO) antibody (catalog no. CMC028, Cell Marque, Rocklin, CA; 1:400 dilution). After incubation with the primary antibody at
4°C overnight, sections were incubated with the appropriate secondary antibody for 30 min at room temperature (1:200 dilution). Immunostaining was visualized using an avidin-biotin complex (ABC)
peroxidase Elite kit (Vectastain, Vector Laboratories) to detect the
antigen-antibody complexes. Antigen detection was enhanced with
nickel-diaminobenzidine; then the section was incubated with Triscobalt to give a black precipitate. Nuclei were counterstained with
Nuclear Fast Red for photomicroscopy. Blinded scoring of CD3- or
Foxp3-positive cells was performed for 10 comparable nonoverlapping high-power fields (⫻40 objective) of each animal, and the mean
number of cells from each animal was used for calculations (n ⫽ 4
animals/group).
Flow cytometry. Blood cells and lung cells were immunophenotyped by flow cytometry. Blood cells were reacted with the antibodies
in whole blood, unbound antibody was washed three times, and red
cell lysis was performed using a hypotonic buffer (Sigma-Aldrich, St.
Louis, MO). Lung cells were recovered following fine mincing of 1 g
of lung tissue in the presence of elastase (3 ml, 4.3 U/ml; Worthington) for 30 min at 37°C. For purification of the leukocyte population,
25 l of CD45-biotin (MCA2220B, Serotec) were added to the 500-l
cell suspension (1 ⫻ 108/ml) for 30 min at 4°C; then the preparation
was washed three times and incubated with 100 l of anti-biotin
magnetic beads (Miltenyi Biotech, Auburn, CA) for 20 min. The
preparation was washed three times, and the cells were suspended in
500 l of buffer and passed through a presoaked column in the
presence of a magnet (mini MACS kit, Miltenyi Biotech). The eluate
was discarded, and the positively selected cells were rinsed from the
column after removal of the magnet, washed twice, and resuspended
in buffer for flow cytometry. Typically, the CD45 selection resulted in
70 – 80% purity. The following antibodies were used for lung and
blood: CD14-FITC, major histocompatability complex (MHC) class
II (MHCII)-FITC, CD8-R-phycoerythrin (RPE), and CD11b
(MCA1568F, MCA2228F, MCA2216F, and MCA1425G, respectively, AbD-Serotec, Raleigh, NC) and CD4 and CD11c (GC1A and
BAQ153A, VMRD, Pullman, WA). The CD11b and CD4 antibodies
were conjugated with RPE and AF647, respectively, using zenon kits
(Z25255 and Z25108, Invitrogen, Carlsbad, CA), while the CD11c
antibody was labeled with a secondary anti-IgM-FITC. All the antibodies were used for a single-color flow, except CD4 and CD8, before
the samples were run on a FACSCanto machine (BD Biosciences).
Analysis was performed using FCS 3.0 software, and the gating for
unstained cells was performed using the appropriate isotype antibodies.
Data analysis. Values are means ⫾ SD. We initially performed the
Kruskal-Wallis test (nonparametric ANOVA) to assess overall differ-
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Fig. 1. Intra-amniotic IL-1␣-induced lung
inflammation. Pregnant sheep were given
intra-amniotic injections of recombinant
sheep IL-1␣ 1, 3, or 7 days and repeat
injections 7 days and again 1 day before
delivery (1d, 3d, 7d, and 1⫹7d) prior to
delivery at 124 ⫾ 1 days. Controls received
intra-amniotic saline (see Fig. 2 legend for
symbols). A–C: bronchoalveolar lavage fluid
(BALF) cells (neutrophils, monocytes, and
lymphocytes) from fetal lungs. D: H2O2 generation in monocytes from fetal lung.
Monocytes were purified over Percoll gradients and then challenged in culture with
100 ng/ml LPS, and H2O2 generation was
measured and normalized to number of
cells in culture. E–G: immunophenotyping
for CD11b, CD11c, and major histocompatability complex class II (MHCII) in
lung leukocytes purified using a CD45biotin magnetically coupled antibody after
enzymatic digestion. *P ⬍ 0.05 vs. composite control group. §P ⬍ 0.05 vs. IL-1␣
1d. #P ⬍ 0.05 vs. IL-1␣ 7d. ND, not done;
IA, intra-amniotic.
subjected to single and repeated exposures to intra-amniotic
IL-1␣ (Fig. 3H).
Lung morphometry was analyzed on the right upper lobe.
Compared with controls, the air space fraction decreased (Fig. 4A)
and the tissue fraction increased (data not shown) 1 day after
intra-amniotic IL-1␣. However, the air space fraction returned
to control values at 3 and 7 days after IL-1␣ and after repeated
exposures to IL-1␣ 7 days and again 1 day before delivery.
Secondary alveolar septal crest density is a sensitive measure
of lung development during the alveolar stage of lung development (4). Compared with controls, secondary alveolar septal
crest density relative to air space area did not change significantly after a single exposure to intra-amniotic IL-1␣ (Fig. 4B;
secondary septal crest density tended to be lower 3 days after
IL-1␣ exposure, P ⫽ 0.07 vs. controls). However, repeated
AJP-Lung Cell Mol Physiol • VOL
exposure to intra-amniotic IL-1␣ 7 days and again 1 day before
delivery decreased secondary septal crest density by 33%
compared with controls. Lung gas volume increased 7 days
after intra-amniotic IL-1␣ exposure. Lung volumes were also
increased after repeated exposure to IL-1␣ 7 days and again 1
day before delivery, similar to a single exposure 7 days before
delivery (Fig. 4C).
Systemic inflammatory responses after intra-amniotic IL-1␣.
mRNA expression of the acute-phase reactant SAA tended to
increase 35- to 50-fold in the liver 1 and 3 days (P ⫽ not
significant) after IL-1␣ exposure (Fig. 5A). However, in the
fetal lambs exposed to IL-1␣ 7 days and again 1 day before
delivery, SAA expression in the liver increased. Another acutephase reactant, plasma haptoglobin, increased from nearly
undetectable levels in controls to 750 ⫾ 135 ng/ml at 1 day,
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IL-1 IN FETAL INFLAMMATION
with a decrease to near control levels by 7 days (Fig. 5D). In
the lambs exposed to IL-1␣ 7 days and again 1 day before
delivery, plasma haptoglobin levels were 308 ⫾ 460 ng/ml.
Intra-amniotic IL-1␣ did not induce hepatic expression of
IL-1 or IL-6 mRNA or increase plasma IL-8 protein levels
(Fig. 5, B, C, and E). IL-1␣ also did not increase expression of
TNF␣, IFN␥, IL-4, IL-10, or IL-17 mRNA in the fetal spleen
(data not shown). Intra-amniotic IL-1␣ tended to cause an
initial neutropenia (3-fold decrease at 1 day, P ⫽ not significant; Table 2). Compared with controls, repeated exposure to
IL-1␣ caused a 10-fold increase in neutrophil counts. Compared with controls, the monocyte counts increased sixfold 3
days after IL-1␣ exposure, but expression of CD14, a LPS
coreceptor, lymphocyte counts, or the CD4-to-CD8 ratio did
not change significantly after IL-1␣ exposure.
Inflammatory responses in the posterior mediastinal lymph
node. The posterior mediastinal lymph node receives afferent
lymphatics from the lung and the gastrointestinal tract. In all
the IL-1␣-exposed groups (single as well as repeated exposures), lymph node weight doubled relative to the controls
(Fig. 6A). iNOS (NOSII)-positive cells, most likely monocytes
and neutrophils, were detected in the subcapsular and parafolAJP-Lung Cell Mol Physiol • VOL
licular regions of the lymph node in the IL-1␣-exposed animals, but not controls (Fig. 6, B–E). In contrast, NOSIIpositive cells were observed in the medullary regions of the
lymph node in all groups. These results are consistent with
migration of activated inflammatory cells from the lung or the
gastrointestinal tract to this draining node (51). MPO-positive
cells were increased in the subcapsular and paracortical regions
after IL-1␣ exposure (data not shown). T-regulatory cells
suppress inflammatory responses and are characterized by
expression of the transcription factor Foxp3 (9). Foxp3-positive cells decreased by 75% at 1 and 3 days after intra-amniotic
IL-1␣, with return to baseline by 7 days (Table 3). In lambs
exposed to IL-1␣ 7 days and again 1 day before delivery,
numbers of Foxp3-positive cells in the posterior mediastinal
lymph node were similar to controls.
DISCUSSION
Inflammatory responses vary greatly on the basis of the route
of exposure to the inflammatory agonists. For instance, IL-1
given intravenously (5–10 ng/kg) in humans elicits a pyrogenic
response (8), and an intravascular injection of 10 g of IL-1␣
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Fig. 2. Intra-amniotic IL-1␣ recruited T lymphocytes to the lung. Pregnant sheep were
given 1 or 2 intra-amniotic injections of
recombinant sheep IL-1␣ for intervals shown
prior to delivery at 124 ⫾ 1 days; controls
received intra-amniotic saline [1 ⫹ 7 days
(□), 7 days (Œ), 1 day (÷)] prior to delivery
at 124 ⫾ 1 days. Lung sections from the
fetuses were immunostained with an antiCD3 antibody. A: quantitation of CD3-positive cells per high-power field (HPF). *P ⬍
0.05 vs. composite control group. B–E: representative images from controls and fetuses
exposed to IL-1␣ for 1 day, 7 days, and 1 ⫹
7 days. Scale bar, 50 m.
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in the sheep fetus is lethal (27), while a 10-fold-higher dose
given by intra-amniotic injection in the present study did not
result in lethality. We used recombinant sheep IL-1␣ for these
experiments, but recombinant sheep IL-1 also results in
similar lung inflammatory responses (54). In contrast to the
intravenous injections, fetal inflammation resulting from agonists injected into the amniotic fluid is mediated via contact of
the agents with the lung, chorioamnion, gastrointestinal, and
skin epithelia (29, 33, 56). A biologically important conclusion
from the present study is that intra-amniotic injection of IL-1␣
can induce a unique inflammatory response in which the
pulmonary inflammation is robust, while systemic inflammatory responses are more modest. The inflammatory responses
in fetal lambs are similar to that in human infants exposed to
chorioamnionitis caused by microorganisms signaling via different TLRs (14, 42).
A striking result of our study is that, as a consequence of
lung inflammation, lung volumes increased by about threefold
7 days after intra-amniotic IL-1␣ exposure, without significant
changes in lung structure. Acute lung inflammation following
IL-1␣ exposure induced increases in cytokine expression and
recruitment of leukocytes, resulting in decreased air space
fraction (and increased tissue fraction) 1 day after IL-1␣
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exposure. However, the air space fraction normalized at later
times after IL-1␣ exposure, presumably secondary to resolution of tissue edema. We previously reported increased surfactant pools 7 days after intra-amniotic IL-1␣ in the fetal sheep
(49, 54). Taken together, the increased lung volumes after
IL-1␣ are likely due to surfactant, rather than anatomic, effects.
The fetal sheep at 124 days gestation is in an alveolar stage
of lung development (1). Therefore, any perturbation in lung
development should be reflected in arrested development of
alveolar secondary septal crests. Interestingly, the secondary septal crest density did not decrease following a single
exposure to IL-1␣ but did decrease by ⬃33% following
repeated exposures to IL-1␣. We previously reported a more
significant impairment of alveolar development after intraamniotic LPS exposure in fetal sheep, with a similar increase in lung volume (53).
Unlike the adult, the preterm sheep fetus is able to respond
to a limited number of proinflammatory stimuli injected into
the amniotic fluid. While a TLR4 agonist, LPS, induced a
robust inflammatory response, a TLR2 agonist caused an
inconsistent inflammatory response and a TLR3 agonist did not
cause fetal inflammation (16). Similarly, intra-amniotic injection of IL-8 or TNF␣ induced mild or no inflammation (18,
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Fig. 3. Intra-amniotic IL-1␣ increased pulmonary cytokine expression. Pregnant sheep were given 1 or 2 intra-amniotic injections of recombinant sheep IL-1␣
for intervals shown prior to delivery at 124 ⫾ 1 days; controls received intra-amniotic saline (see symbols in Fig. 2 legend). A–G: after delivery, total RNA was
extracted from fetal lung, and cytokines were quantified using real-time PCR assays with sheep-specific primers and TaqMan probes [IL-1, cyclooxygenase
enzymes (COX-1 and COX-2), IL-6, IL-8, IL-1 receptor antagonist (IL-1RA), and TNF␣]. Values for each cytokine were normalized to 18S rRNA. Mean mRNA
signals in control animals were given the value of 1, and levels at each time point are expressed relative to controls. H: BALF was used for IL-8 protein
measurement using ELISA. *P ⬍ 0.05 vs. composite control group. §P ⬍ 0.05 vs. IL-1 1d. ND, not done.
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IL-1 IN FETAL INFLAMMATION
25). However, there appear to be some species differences:
Sadowsky et al. (47) demonstrated that intra-amniotic infusions of IL-1 and TNF␣, but not IL-6 or IL-8, induced
preterm labor in nonhuman primates. The present study demonstrated that IL-1␣ is a unique proinflammatory cytokine, in
that it can induce robust fetal inflammation after intra-amniotic
injection.
The IL-1 cytokine family includes IL-1␣, IL-1, IL-1R
antagonist, IL-18, IL-33, and others (6). IL-1␣ and IL-1 are
synthesized as precursor molecules. While IL-1␣ is a cellassociated cytokine that is active as a precursor, IL-1 is active
only after the precursor is cleaved and the cytokine is secreted.
The recombinant sheep IL-1␣ used in this study was the mature
protein and was used because previous dose-finding experiments demonstrated that IL-1␣ was more potent than IL-1 in
this model (54). Since both cytokines activate the common
IL-1R (6), the present study demonstrates that IL-1 signaling in
the fetus can induce a systemic fetal inflammatory response.
Fig. 5. Intra-amniotic IL-1␣ induced modest systemic fetal inflammation. Pregnant sheep were given 1 or 2 intra-amniotic injections of recombinant sheep IL-1␣
for intervals shown prior to delivery at 124 ⫾ 1 days; controls received intra-amniotic saline (see symbols in Fig. 2 legend). A: serum amyloid A3 (SAA3) in
fetal liver was measured using an RNase protection assay. Values were normalized to L32, a ribosomal protein mRNA. B and C: IL-1 and IL-6 mRNA
expression was measured using real-time PCR assays and TaqMan probes. Values were normalized to 18S rRNA. Mean mRNA signal in control animals was
given the value of 1, and levels at each time point are expressed relative to controls. All assays used sheep-specific probes. D and E: plasma levels of haptoglobin
and IL-8. *P ⬍ 0.05 vs. composite control group.
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Fig. 4. Intra-amniotic IL-1␣ increased lung
volumes with modest effects on morphology. Pregnant sheep were given 1 or 2 intraamniotic injections of recombinant sheep
IL-1␣ for intervals shown prior to delivery at
124 ⫾ 1 days; controls received intra-amniotic saline (see symbols in Fig. 2 legend).
Right upper lobe of the lung was inflationfixed at 30 cmH2O pressure for morphology.
A: air space fraction expressed relative to
total lung area. B: alveolar secondary septal
crest density expressed relative to air space
area. C: lung air volumes measured at 40
cmH2O pressure. *P ⬍ 0.05 vs. composite
control group. §P ⬍ 0.05 vs. IL-1␣ 1d. #P ⬍
0.05 vs. IL-1␣ 7d.
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IL-1 IN FETAL INFLAMMATION
Table 2. Changes in blood leukocytes and immunophenotype after intra-amniotic exposure to IL-1␣
IL-1␣
9
Total WBC, 10 /l
Neutrophils, 109/l
Lymphocytes, 109/l
Monocytes, 109/l
%CD14⫹
%MHCII⫹
CD4-to-CD8 ratio
Control Saline
1 day
3 days
7 days
7 days ⫹ 1 day
6.4 ⫾ 6.3
1.4 ⫾ 1.4
4.2 ⫾ 4.1
0.2 ⫾ 0.07
1.6 ⫾ 0.2
9.4 ⫾ 1.1
1.5 ⫾ 0.2
3.9 ⫾ 0.7
0.4 ⫾ 0.4†
3.1 ⫾ 0.7
0.1 ⫾ 0.08
7.0 ⫾ 5.1†
9⫾5
1.1 ⫾ 0.5
7.1 ⫾ 3.3
3.1 ⫾ 1.7
2.4 ⫾ 1.1
1.3 ⫾ 0.8*
3.5 ⫾ 1.6
5⫾2
1.8 ⫾ 0.3
4.4 ⫾ 1.7†
2.3 ⫾ 1.0
1.6 ⫾ 0.8
0.1 ⫾ 0.09
1.4 ⫾ 1.0
ND
0.8 ⫾ 0.2†
18.6 ⫾ 16.4*
14.9 ⫾ 14.8*
1.7 ⫾ 0.8
0.1 ⫾ 0.08
0.9 ⫾ 1.5
22 ⫾ 16
1.7 ⫾ 0.6
Values are means ⫾ SD. Control group is a composite of intra-amniotic saline 1 day ⫹ 7 days (n ⫽ 3), 7 days (n ⫽ 1), and 1 day (n ⫽ 1). WBC, white blood
cells; MCHII, major histocompatability complex class II; ND, not done. *P ⬍ 0.05 vs. control. †P ⬍ 0.05 vs. IL-1␣ (1 day ⫹ 7 days).
were activated because they expressed NOSII and MPO. IL-1␣
exposure induced the cyclooxygenase enzymes Cox-1 and
Cox-2 and caused the lung monocytes to mature. The maturation of lung monocytes was demonstrated by a minimal oxidative burst in the control monocytes, while IL-1␣ exposure in
vivo for 3 or 7 days increased H2O2 secretion by the lung
monocytes in response to an in vitro challenge with LPS. The
IL-1␣-induced lung inflammation resulted in increased lung
Fig. 6. Intra-amniotic IL-1␣ caused inflammation in posterior mediastinal lymph node.
Pregnant sheep were given 1 or 2 intraamniotic injections of recombinant sheep
IL-1␣ for intervals shown prior to delivery at
124 ⫾ 1 days; controls received intra-amniotic saline (see symbols in Fig. 2 legend).
A: weight of posterior mediastinal lymph
node normalized to body weight. B–E: representative images of immunostaining with
an anti-inducible nitric oxide synthase
(NOSII) antibody using lymph nodes from
controls and animals exposed to IL-1␣. Main
frame of each panel shows subcapsular region; inset shows parafollicular and medullary areas. Scale bar, 50 m. *P ⬍ 0.05 vs.
composite control group. Note NOSII-positive cells only in IL-1␣-exposed animals in
subcapsular regions and medullary NOSIIpositive cells in all groups.
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There are similarities between the IL-1␣- and LPS-induced
fetal inflammatory responses. This is not surprising, because
LPS receptor signaling via TLR4 and IL-1 signaling via IL-1R
share similar intracellular signaling pathways (38). Similar to
the previously reported responses to intra-amniotic LPS (28,
34), we demonstrate here that intra-amniotic IL-1␣ induced an
equivalent recruitment of neutrophils, monocytes, and T lymphocytes to the lung in sheep fetuses. These inflammatory cells
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Table 3. Expression of Foxp3 in posterior mediastinal lymph
node cells after intra-amniotic exposure to IL-1␣
Group
Control saline
1 day ⫹ 7 days
7 days
1 day
Composite
IL-1␣
1 day
3 days
7 days
1 day ⫹ 7 days
Foxp3⫹ Cells, %/HPF
16.3, 20.7
15.3
18.6
17.8 ⫾ 2.3
3.6 ⫾ 3.6*
4.0 ⫾ 3.6*
12.6 ⫾ 3.0
19.4 ⫾ 6.4
gas volumes, similar to LPS effects (28, 34). However, unlike
LPS, intra-amniotic IL-1␣ induction of IL-1, IL-6, and IL-8
mRNA in the lung was minimal. The reasons for the decreased
induction of proinflammatory cytokine expression by IL-1␣ are
not known. Lack of protein data for many cytokines may
obscure interpretation of inflammatory effects of intra-amniotic
IL-1␣. LPS induces activation of the transcription factor
NF-B via MyD88-dependent and Toll/IL-1 receptor domaincontaining adaptor-inducing IFN (TRIF)-mediated MyD88independent pathways (38). The TRIF-mediated NF-B activation induces interferon, which increases the expression of
interferon-inducible chemokines such as IP-10 and MIG. We
previously demonstrated that intra-amniotic LPS induces IP-10
and MIG in the fetal sheep lung (23), while no induction of
these interferon-inducible chemokines after IL-1␣ exposure
was observed in this study. Taken together, these results
demonstrate that although signaling of LPS and IL-1 in the
fetus is similar, some aspects of their inflammatory repertoire
are unique.
Fetal inflammatory response syndrome (FIRS) in the human
is a unique systemic inflammatory response defined as chorioamnionitis associated with cord plasma IL-6 levels ⬎11 pg/ml
(14, 45). Unlike the “cytokine storm,” which causes multiorgan
dysfunction associated with the systemic inflammatory response syndrome in adults (40), FIRS is a more subtle inflammatory response. Despite the modest inflammatory responses,
FIRS was postulated to be the proximate cause of multiple
adverse neonatal outcomes (15). Since ⬍2% of the preterm
infants exposed to chorioamnionitis have early-onset bacteremia (50), systemic inflammation in the fetus must be initiated
by inflammatory responses in the fetal organs in contact with
infected amniotic fluid. Indeed, we and others have demonstrated that the fetal chorioamnion, lung, gut, and skin contribute to the systemic inflammation induced by chorioamnionitis
(30, 33). Chorioamnionitis in humans is caused by a variety of
organisms, mostly of low-grade virulence that colonize the
female lower genital tract (42).
In the present study, IL-1␣ induced a systemic inflammatory
response. Compared with the pulmonary responses, the systemic inflammation was modest. The neutrophil counts in the
blood changed, and the expression of the acute-phase reactant
haptoglobin in plasma increased. Interestingly, while the total
lymphocyte counts in the blood did not change, the CD4-toAJP-Lung Cell Mol Physiol • VOL
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Individual value are shown for control animals; other values are means ⫾
SD. Immunohistology for Foxp3 was done using paraffin-embedded sections
of posterior mediastinal lymph nodes. HPF, high-power field. *P ⬍ 0.05 vs.
composite control group.
CD8 ratio appeared to decrease in the cord blood 7 days after
IL-1␣ exposure. A decrease in the CD4-to-CD8 ratio in the
peripheral blood can be induced by a number of chronic
infections (46). One caveat of interpretation of data from
FACSCanto analysis is that the blood leukocyte counts initially
decreased and subsequently increased. Therefore, some of the
reported change in expression of leukocyte markers could be
confounded by this changing fetal blood leukocyte count. One
question regarding systemic inflammation relates to its pathogenesis. Using fetal surgical techniques, we recently selectively infused LPS into the fetal airways or the amniotic fluid,
with occlusion of the airway to prevent LPS from contacting
the lung (33). Lung infusion and chorioamnion exposure to
LPS can induce a systemic fetal inflammatory response. These
experiments illustrate the complex signaling between different
fetal organs, resulting in fetal inflammation from chorioamnionitis.
IL-1␣ also caused inflammatory changes in the lymph node
that drains lymph from the lung and the gastrointestinal tract.
After IL-1␣ exposure, the weight of the lymph node doubled,
and NOSII- and MPO-staining cells were detected in the
subcapsular space and the parafollicular and medullary regions. These results suggest migration of activated inflammatory cells from the draining organs and activation of the
resident macrophages. Similar to an intra-amniotic LPS-induced reduction of Foxp3-positive T-regulatory lymphocytes
in the fetal sheep thymus (35), we demonstrated a timedependent decrease in Foxp3-staining cells in the posterior
mediastinal lymph node. Foxp3 is a master regulator of the
anti-inflammatory T-regulatory cells (10, 17). However, a
detailed characterization of T-regulatory cells was not possible
because of lack of appropriate cross-reactive antibodies for
sheep. Although the findings are preliminary, the experiment
suggests that IL-1␣ signaling in chorioamnionitis can induce a
proinflammatory fetal milieu, since T-regulatory lymphocytes
are known to dampen inflammatory responses (9). Despite the
changes in the blood, liver, and regional lymph node, we did not
observe significant increases in the Th1, Th2, or Th17 cytokines
in the spleen after exposure to intra-amniotic IL-1␣. Because of
limitations of reagents for sheep dendritic cells, we could not
determine if there was migration and signaling via dendritic cells.
Whether the inflamed lymph node mediates systemic inflammation or whether the changes in the lymph node are merely a
response to end-organ inflammation is unknown.
The duration of exposure to chorioamnionitis in humans is
often prolonged (43); therefore, exposure to proinflammatory
agonists can also be for extended periods. We previously
demonstrated that repeated exposures to LPS induced tolerance
to endotoxin and other TLR agonists (22, 32). We therefore
asked if repeated exposure to IL-1␣ would cause innate immune tolerance. Repeated injections of IL-1␣ decreased some
measurements of inflammatory response but not others. Repeated exposure to intra-amniotic IL-1␣ increased inflammatory cell recruitment into the fetal lung, induced the monocyte
oxidant response, and increased blood neutrophils, suggesting no
tolerance. However, unlike the single IL-1␣ exposure, repeated
exposure to IL-1␣ did not increase lung COX expression or
decrease T-regulatory lymphocytes in the posterior mediastinal
lymph node. Therefore, unlike our previous results with LPS (22),
this study demonstrated complex organ- and pathway-specific
effects after repeated exposure to IL-1␣. The present study dem-
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IL-1 IN FETAL INFLAMMATION
onstrates that IL-1␣, a cytokine that can be induced by multiple
different pathogens, is a mediator of the FIRS.
ACKNOWLEDGMENTS
The authors thank Dr. Kathy Heel and Tracey Lee-Pullen (Center for
Microscopy Characterization and Analysis, University of Western Australia)
for consultation on FACS experiments; Dr. Susan Wert for consultation on
morphometry and Dr. Jareen Meinzen-Derr for statistics consultation (University of Cincinnati); and Amy Whitescarver, Manuel Alvarez, Jr., and Avedis
Kazanjian for expert technical assistance.
GRANTS
This study was funded by National Institute of Child Health and Human
Development Grant HD-57869 (to S. G. Kallapur).
DISCLOSURES
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