SPECIAL TOPIC
Pre- and Postnatal Lung Development, Maturation, and
Plasticity
LPS-induced lung injury in neonatal rats: changes
in gelatinase activities and consequences on lung growth
Marie-Laure
Franco,
Paul
Waszak,
Gaëlle
Banalec,
Micheline
Levame,
Chantal
Lafuma,
Alain
Harf, and
Christophe
Delacourt
Unité Institut National de la Santé et de la Recherche
Médicale U492, Faculté de Médecine, 94000 Créteil, France
 |
ABSTRACT |
Postnatal lung growth disorders may
involve imbalance between metalloproteinases and their inhibitors.
Inflammatory cell 92-kDa gelatinase overactivity has been reported in
adults with lung injury but has not been looked for in neonates. We
compared gelatinase activity in neonatal and adult rats and evaluated
postnatal lung growth after lipopolysaccharide (LPS)-induced lung
injury. Significant intra-alveolar inflammatory cell recruitment
occurred in adults and neonates; cell counts increased 16-fold in
adults and 2.7-fold in neonates. Total 92-kDa gelatinase activity was
increased in neonates and adults and was significantly correlated to
inflammatory cell counts. For a given cell count, 92-kDa gelatinase
increased more in neonates than in adults. Morphometric neonatal lung
analysis showed that LPS-injured lungs had decreases in absolute values of lung volume (P < 0.03), alveolar surface
(P < 0.004), and air space volume (P < 0.03). Doxycycline, a nonspecific metalloproteinase inhibitor,
partly inhibited LPS-induced 92-kDa gelatinase overactivity but did not
improve LPS-induced alveolar growth disorders. LPS-mediated lung injury
in neonatal rats induced both gelatinase B overactivity and alveolar
growth disorders, although no causal link between these two effects was demonstrated.
metalloproteinases; doxycycline; bronchopulmonary dysplasia
 |
INTRODUCTION |
LUNG DEVELOPMENT is a
continuous process that extends into postnatal life and requires active
extracellular matrix remodeling (1, 3, 6). Increased
elastin synthesis (6), accelerated collagen turnover
(3), and basement membrane disruption
(1) have been shown to be pivotal physiological events
during postnatal lung growth. Furthermore, in neonatal rats,
experimental alterations in lung matrix turnover induced alveolar
growth disorders (23). Metalloproteinases (MMPs) may be
involved in the regulation of matrix turnover, as they can
synergistically digest the main macromolecules found in connective
tissue matrices. Studies in animal models showed physiological
variations in lung activities of MMPs or their inhibitors during
postnatal lung growth (10, 31). Thus alterations in the
balance between MMPs and their inhibitors may induce alveolar growth
disorders. Such alterations may occur during the airway inflammatory
response induced by lung injury. Inflammatory cells recruited into the
lung can release many pro-inflammatory agents, including MMPs. The main
MMP released by inflammatory cells, such as neutrophils and alveolar
macrophages, is 92-kDa gelatinase (22, 43). Increased
92-kDa gelatinase activity has been found in the airways of adult
animals with experimental lung injury and of humans with inflammatory
airway disease (12, 16, 26, 41). In neonates, however,
lung injury-induced changes in airway gelatinase activities and the
potential effects of these changes on lung growth remain unexplored. We
previously demonstrated that activated alveolar macrophages from
newborn rats secreted more 92-kDa gelatinase than those from adults
(10, 13). This suggests that increased airway 92-kDa
gelatinase activity may be significant during lung injury in neonates,
despite earlier evidence of limited inflammatory cell recruitment into
airways (28). We compared changes in gelatinase activity
after lipopolysaccharide (LPS)-induced lung injury in neonatal and
adult rats, and we evaluated the potential consequences of these
changes on postnatal lung growth.
 |
METHODS |
Reagents.
All reagents were purchased from Sigma-Aldrich (St. Louis, MO), unless
otherwise specified.
Study design.
First, we compared the lung inflammatory response to LPS in neonatal
and adult rats to test the hypothesis that neonatal inflammatory cells
would release more 92-kDa gelatinase. Six-day-old and adult rats each
received an intratracheal instillation of LPS or of 0.9% NaCl as the
control. Changes in bronchoalveolar lavage (BAL) cell counts,
gelatinase B activity, and histology were evaluated 24 h and 15 days after the intratracheal instillation.
In a second step of the study, lung development was assessed by
morphometric methods in another group of newborn rats, 15 days after
intratracheal instillation of LPS or 0.9% NaCl. To look for a
potential link between LPS-induced gelatinase B activity and altered
lung development, we examined rats given subcutaneous doxycycline,
which is a nonspecific anti-MMP agent (20).
Animals.
Sprague-Dawley adult and neonatal rats were obtained from Charles River
(Saint-Aubin les Elbeuf, France). The rats were housed in air-filtered,
temperature-controlled units and were allowed free access to food and
water. Adult rats were males weighing ~250 g. Newborn rats were
tested on postnatal day 6 and were left with their mother
until they were killed on day 7 or day 21. Each dam with its litter was in an individual plastic cage.
Intratracheal LPS administration.
LPS (from Escherichia coli 0111:B4) or 0.9% NaCl was
instilled intratracheally as previously described (14,
16). Briefly, adult and 6-day-old rats were anesthetized by
inhalation of isoflurane (Forene, Abbott, Queensborough, UK), and the
trachea was exposed. Two doses of LPS were used, 0.2 g/kg and 2 g/kg
dry lung wt (LPS 1× and LPS 10×, respectively). Using data obtained
previously in our laboratory (10), we estimated that dry
lung weight was 50 mg in 6-day-old rats and 250 mg in adult male rats.
Intratracheal instillation volumes were 20 and 125 µl in neonates and
adults, respectively. A Hamilton syringe fitted with a 25-gauge needle was used in neonates, and a 1-ml tuberculin syringe fitted with a
22-gauge needle was used in adults. Air was pulsed after the liquid
volume. After the injection, the neck incision was sutured with silk.
Doxycycline treatment.
Doxycycline was started in neonates immediately before the
intratracheal instillation of LPS, in a dosage of 0.5 mg injected subcutaneously twice a day for 5 days. Controls were injected with
0.9% NaCl. The interval from intratracheal LPS instillation to
evaluation was 24 h for in situ zymography and gelatinase B activity and 15 days for lung development. Four subgroups were used for
the morphometric analysis: LPS instillation with doxycycline (LPS+/Dox+), LPS instillation without doxycycline (LPS+/Dox
), doxycycline without LPS (LPS
/Dox+), and controls (LPS
/Dox
).
BAL.
BAL was performed as previously described (10). Briefly,
rats were exsanguinated after anesthesia with 5 mg/100 g body wt of
intraperitoneal pentobarbital sodium. The thorax was opened widely to
expose the lungs and trachea. A short length of tubing was inserted
into the trachea and ligated. Eight to 10 separate aliquots of warm
saline (37°C) were used for BAL. Volumes ranged from 0.4 ml/aliquot
for 7-day-old rats to 2.5 ml/aliquot for adults. Total BAL volumes were
4, 12.5, and 20 ml for 7-day-old, 21-day-old, and adult rats, respectively.
BAL fluid collection and cell identification.
Total cells were counted using a hemacytometer. Differential cell
counts were determined by examining 200 cells. After centrifugation of
the BAL fluid at 300 g for 7 min, the cell-free supernatant was frozen at
80°C. The cell pellet was resuspended in isotonic saline in a concentration of 1 × 106 cells/ml.
Cytospin preparations were obtained using a Shandon 3 cytocentrifuge
(Shandon). Cell smears stained using standard May-Grünwald-Giemsa
were identified and counted under a light microscope.
Total protein determination.
Total protein concentration was measured using the Bradford protein
assay with an albumin standard curve, in each fluid sample from each
animal in each group.
Zymographic analysis of total gelatinase activity.
The supernatant was subjected to electrophoresis in 8% (wt/vol)
polyacrylamide gels containing 1 mg/ml gelatin with sodium dodecyl
sulfate (SDS-PAGE; Sigma Chemical, St. Louis, MO), under nonreducing
conditions. After electrophoresis, the gels were washed in Triton X-100
2.5% for 1 h, rinsed briefly, and incubated at 37°C for 48 h in buffer containing 100 mM Tris · HCl, pH 7.40, and 10 mM
CaCl2. The gels were then stained with Coomassie brilliant blue R250 and restained in a solution of 7.5% acetic acid and 5%
methanol. Zones of enzymatic activity were indicated by negative staining: areas of proteolysis were seen as clear bands against a blue background.
Enzyme activities in the gel slabs were quantitated using image
analysis (image analysis program NIH Image 1.52 Macintosh) to measure
both the surface area and the intensity of lysis bands. Results were
expressed as arbitrary units (AU) per 48 h per milliliter BAL
fluid. To check that the image analysis system provided linear results
over the range of activities measured in unknown samples, we used it to
measure activities for increasing volumes of a given aliquot. The AU
values were linearly related to sample volume (r2 = 0.99).
Immunoblotting was used to evaluate 92-kDa gelatinase activity.
Supernatants of BAL fluid from five neonates were pooled and partly
purified using gelatin-Sepharose chromatography as previously described
(13). Samples were separated by SDS-PAGE and transferred electrophoretically to nitrocellulose. After saturation of excess protein binding sites with 5% cow's milk in 0.05 M Tris · HCl and 0.15 M NaCl, pH 7.6 (Tris-buffered saline) for 1 h at room temperature, the nitrocellulose was incubated for 1 h at room temperature with a specific rabbit antibody to 92-kDa gelatinase (Triple Point Biologics, Forest Grove, OR), diluted 1:200 in the same
buffer as above. After thorough washing with Tris-buffered saline, the
samples were incubated for 1 h at room temperature with a
peroxidase-labeled swine anti-rabbit antibody (1:1,000 dilution) in
Tris-buffered saline containing 5% cow's milk. After washing, the
immunoblots were visualized using an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, UK).
Determination of free gelatinase activity.
Gelatinolytic activity was assayed using radiolabeled
[3H]gelatin. The specific activity of this substrate was
37 kBq/100 µg. Radiolabeled [3H]gelatin substrate (50 µg) was incubated with the sample at 37°C for 48 h in a
reaction mixture containing 50 mM Tris · HCl, 5 mM
CaCl2, and 0.02% NaN3, pH 7.40 (final volume
500 µl). At the end of the assay, the samples were cooled to 4°C,
and the undigested substrate and large molecular weight fragments were
precipitated using a solution of 12.5% trichloracetic acid and 2.5%
tannic acid. After centrifugation at 10,000 g for 10 min, a
50-µl aliquot of the supernatant was counted in a liquid
scintillation counter (Wallac 1409, Turku, Finland). Gelatinolytic
activity was expressed as micrograms gelatin hydrolyzed per 48 h
per milliliter of BAL fluid at 37°C. Controls were done for
spontaneous degradation of radiolabeled substrate in the presence of
physiological saline.
Preparation of histologic sections of rat lungs.
For each animal, the lungs were fixed by tracheal infusion of neutral
buffered paraformaldehyde at 30 cmH2O pressure. After routine processing and paraffin embedding, 4-µm midcoronal sections through both lungs were cut and stained with
hematoxylin-phloxin-saffron (HPS). The inflammatory response in the
lungs was scored using a semiquantitative scale based on inflammatory
cell type and location (alveoli, bronchi, and blood vessels). Two
independent investigators unaware of group assignment assessed the lung
sections, and for each section the mean of the two values for each
score was calculated.
Morphometric evaluation of lung structures.
Lungs from 21-day-old rats were used to measure fixed lung volumes
using fluid displacement (36). For each rat, 4-µm
mediofrontal paraffin sections, stained with HPS and including both
lungs, were made. Each section permitted us to evaluate all
lobes of neonatal lungs. For each animal, two to three sections were
performed for both right and left lungs. Thus at least two sections for right and left lungs were used to obtain individual data. Morphometric evaluation of the whole lung was ensured by this method. All
morphometric evaluations were made by the same investigator (P. Waszak), who was unaware of group assignment. A microscope (Leitz)
connected to a TV screen by a color video camera (Sony) was used.
Volume densities of pulmonary parenchymal structures (alveolar air
space, airways, blood vessels >20 µm in diameter, and interstitial
tissues) and alveolar surface density were measured using point
counting and mean linear intercept methods described by Weibel and
Cruz-Drive (42). Light microscope fields were quantitated
at an overall magnification of ×500, using a 42-point, 21-line
eyepiece reticle to examine 20 fields/animal (10 per lung) according to
a systematic sampling method from a random starting point. To correct
area values for shrinkage associated with fixation and paraffin
processing, we used a factor of 1.22, calculated during a previous
study. All morphometric data were expressed as relative, absolute, and specific values, as described by Burri and coworkers (7).
The relative values (volume density or surface density) were those obtained directly from morphometric measurements of the tissue sections. Absolute values (total volume or surface area per lungs) were
calculated by multiplying the relative values by the lung volume, and
specific values were calculated by dividing absolute values by body
weight and multiplying the result by 100.
In situ zymography.
In situ zymography was performed as described previously
(19). Briefly, slides were dipped into photographic
emulsion (NTB-2, Kodak, Paris, France) diluted 1:3 in distilled water.
Emulsion-covered slides were placed horizontally in humidified chambers
and incubated for 48 h at 37°C. The emulsion was allowed to dry,
and the slides were developed using D 19 developer (Kodak) according to
the manufacturer's instructions. The specimens were examined under a
microscope in transmitted light. The duration of photographic film
exposure varied with the level of gelatinolytic activity in the sample.
Statistical analysis.
Values were expressed as means ± SE. Between-group differences
were evaluated using the nonparametric Mann-Whitney test. Differences across three groups or more were examined using ANOVA analysis, followed by Fisher's protected least significant difference test, in
case of significance. P values <0.05 were considered
statistically significant.
 |
RESULTS |
Acute inflammatory response to LPS in the lungs: dose-response
study.
BAL and lung histology were performed 24 h after intratracheal
instillation of LPS 1×, LPS 10×, or 0.9% NaCl. The total cell count
in BAL fluids was significantly smaller in control neonates than in
control adults (1.6 ± 0.2 vs. 2.6 ± 0.2 × 105 cells/ml, respectively; P = 0.0006). As
expected, in the adults, both LPS doses induced highly significant
increases in intra-alveolar total inflammatory cells (ANOVA
P < 0.006), neutrophils (P < 0.02), and alveolar macrophages (P = 0.0003) (Fig.
1). In the neonates, LPS significantly
increased BAL cells, with a dose-response effect (P < 0.002), but the response was much weaker than in adults (Fig. 1). The
higher LPS dose induced a 16-fold increase in total BAL cells in adults
and only a 2.7-fold increase in neonates. Furthermore, the total BAL
cell increase in neonates was ascribable only to neutrophil
recruitment: there was no significant increase in alveolar macrophages.

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Fig. 1.
Bronchoalveolar lavage (BAL) cell counts 24 h after
intratracheal instillation of 0.9% NaCl (control), lipopolysaccharide
(LPS) 1×, or LPS 10× in adults (A) and in neonates
(B). Solid bars, mean total cells ± SE; hatched bars,
mean neutrophils; open bars, mean alveolar macrophages.
* P < 0.05 vs. control group.
|
|
BAL protein concentration increased significantly only in the adults
given the higher LPS dose (Table 1). No
changes in BAL protein concentrations were observed in the neonates.
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Table 1.
BAL proteins in neonatal and adult rats and changes induced by LPS
24 h and 15 days after intratracheal instillation
|
|
Histology showed prominent inflammatory infiltrates in adult lung
tissue, whereas only a very mild inflammatory reaction was seen
in the neonates (Fig. 2).

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Fig. 2.
Histologic sections of rat lungs 24 h after intratracheal
instillation of LPS 10× (B and D) or 0.9% NaCl
(A and C). Experiments were performed in neonates
(A and B) and in adults (C and
D). Inflammation was marked in the adults exposed to LPS
(D) but very mild in the neonates exposed to LPS
(B).
|
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Late inflammatory response to LPS in the lungs.
Total BAL cell counts were still significantly elevated 15 days after
intratracheal LPS instillation both in neonates (ANOVA P < 0.0001) and in adults (P < 0.04)
(Fig. 3). The higher LPS dose increased
BAL cells 2.4-fold in adults and 1.8-fold in neonates. Neutrophils were
no longer present 24 h after intratracheal LPS instillation, so
that the BAL cell increase at that time point was related only to
recruitment of alveolar macrophages.

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Fig. 3.
BAL cell counts 15 days after intratracheal instillation
of 0.9% NaCl (control), LPS 1×, or LPS 10× in adults (A)
and in neonates (B). Columns are mean total cells ± SE. * P < 0.05 vs. control group;
§ P < 0.05 vs. LPS 1× group.
|
|
No significant changes in BAL proteins were observed (Table 1).
LPS-induced changes in gelatinase activity.
Spontaneous 72-kDa gelatinase activity was measured by zymography in
BAL fluid from control neonates and adults, 24 h after intratracheal saline instillation. LPS significantly increased total
72-kDa gelatinase activity in adults (ANOVA P < 0.04)
but not in neonates (Table 2). This
increase was transient: it was no longer present 15 days after
intratracheal LPS instillation.
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Table 2.
Total 72-kDa gelatinase activity in neonatal and adult rats and changes
induced by LPS 24 h and 15 days after intratracheal instillation
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In contrast to 72-kDa gelatinase, total 92-kDa gelatinase showed marked
changes in activity 24 h after LPS instillation, in both neonates
and adults (Fig. 4). Immunoblotting with
anti-gelatinase B antibody confidently identified the 92-kDa band as
gelatinase B (Fig. 4). Gelatinase B activity was low or undetectable in
control animals and was significantly increased in a dose-dependent
manner after intratracheal LPS instillation (Fig.
5). The total 92-kDa gelatinase activity
in BAL fluid was significantly correlated to the number of inflammatory
cells both in the neonates (r = 0.59; P < 0.004) and in the adults (r = 0.49;
P < 0.02). However, with the higher LPS dose, BAL
inflammatory cell counts were 10 times higher in the adults than in the
neonates, whereas BAL 92-kDa gelatinase activity was only about four
times higher. Fifteen days after intratracheal LPS instillation, 92-kDa
gelatinase activity in BAL fluid had returned to undetectable levels in
the neonates and to very low mean levels in the adults (0.5 ± 0.3, 0.2 ± 0.2, and undetectable levels in the control group, LPS
1× group, and LPS 10× group, respectively).

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Fig. 4.
LPS-induced changes in BAL gelatinase activity measured
by gelatin zymography. Six-day-old rats (lanes a-c) and
adults (lanes d-f) received intratracheal instillations
of 0.9% NaCl (lanes a and d), LPS 1×
(lanes b and e), or LPS 10× (lanes c
and f), and BAL was performed 24 h later. Gelatinase B
(92-kDa band) and gelatinase A (72-kDa band) activities were present.
Immunoblotting firmly identified the 92-kDa band as gelatinase B
(lane g).
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Fig. 5.
Total 92-kDa gelatinase activity in BAL fluid of neonates
(solid bars) and adults (open bars) 24 h after intratracheal
instillation of 0.9% NaCl (control; n = 10 neonates
and 11 adults), LPS 1× (n = 7 neonates and 8 adults),
or LPS 10× (n = 6 neonates and 5 adults). ANOVA:
P < 0.0001 for both neonates and adults.
* P < 0.05 vs. control group; § P < 0.05 vs. LPS 1× group.
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Free gelatinolytic activity was very low in BAL fluid and was
unchanged by intratracheal LPS instillation (Table
3).
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Table 3.
Free gelatinolytic activity in neonatal and adult rats and changes
induced by LPS 24 h and 15 days after intratracheal
instillation
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Tissue gelatinolytic activity evaluated by in situ zymography
was increased after LPS instillation (Fig.
6).

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Fig. 6.
Lung tissue gelatinolytic activity evaluated by in situ
zymography in 7-day-old rats given intratracheal and subcutaneous
saline (A), intratracheal LPS and subcutaneous saline
(B), or intratracheal LPS and subcutaneous doxycycline
(C).
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LPS-induced changes in lung growth.
Because significant changes in BAL cell counts and 92-kDa gelatinase
activity in neonates occurred mainly with the highest LPS dose,
morphometric studies and inhibitory studies were performed only with
this dose (LPS 10×).
Subjective light microscopy evaluation of 21-day-old rats (15 days
after LPS instillation) suggested that LPS-instilled neonates had
enlarged peripheral air spaces and fewer alveoli than controls (Fig.
7).

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Fig. 7.
Lung morphology in 21-day-old rats given intratracheal
and subcutaneous saline (A), intratracheal LPS and
subcutaneous saline (B), or intratracheal LPS and
subcutaneous doxycycline (C).
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|
Light microscopy morphometric data, shown in Table
4, were consistent with the subjective
evaluation. Neonatal rats that received LPS without doxycycline
(LPS+/Dox
) had significant reductions in absolute lung volume
(P < 0.03), alveolar surface density
(P < 0.03, Fig. 8),
absolute alveolar surface (P < 0.004), and absolute air space volume (P < 0.03) compared with the control
group (LPS
/Dox
). The specific alveolar surface was also smaller in
the LPS-instilled neonates, but the difference with the control group
was not significant: 1,150 ± 141 vs. 1,388 ± 111 cm2/100 g, respectively.

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Fig. 8.
Alveolar surface density (Sva) in 21-day-old rats given
intratracheal LPS (LPS+) or 0.9% NaCl (LPS ) and subcutaneous
injections of doxycycline (Dox+) or 0.9% NaCl (Dox ).
* P < 0.05 vs. LPS /Dox group;
§ P < 0.05 vs. LPS /Dox+ group.
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Effects of doxycycline.
Gelatinase B activity measured by zymography 24 h after
intratracheal LPS instillation was significantly reduced in newborn rats treated with doxycycline (LPS+/Dox+) compared with those given
0.9% NaCl (LPS+/Dox
): 3.18 ± 0.23 vs. 6.41 ± 0.73 × 104 AU · ml
1 · 48 h
1, respectively (P < 0.02). Similarly,
in situ zymography showed that twice daily subcutaneous administration
of doxycycline induced partial inhibition of gelatinolytic
activity (Fig. 6).
Unexpectedly, light microscopy suggested that administration of
doxycycline worsened LPS-induced lung growth disorders (Fig. 7).
Similarly, morphometric evaluations indicated that doxycycline failed
to improve LPS-induced alveolar growth disorders (Table 4). In
LPS+/Dox+ newborn rats, absolute alveolar surface (P < 0.004) and absolute air space volume (P < 0.03) showed
similar reductions as in the LPS+/Dox
neonates, and the decrease in
alveolar surface density was even more significant (P < 0.002) compared with the LPS
/Dox
control group. Furthermore,
compared with the controls (LPS
/Dox
group), the group given
doxycycline without LPS (LPS
/Dox+ group) showed significant decreases
in absolute lung volume (P < 0.004), absolute alveolar
surface (P < 0.002), and absolute air space volume
(P < 0.003). Alveolar surface density and specific
alveolar surface (1,164 ± 100 cm2/100 g) were lower
in the LPS
/Dox+ group than in the LPS
/Dox
group (controls), but
the difference was not significant. LPS instillation with doxycycline
induced a significantly larger decrease in alveolar surface density
than doxycycline alone (P < 0.03, Fig. 8).
 |
DISCUSSION |
We evaluated the inflammatory response to LPS-induced lung injury
in neonatal rats, with special attention to gelatinase activities, and
we assessed potential consequences on postnatal lung growth. Our
results showed that LPS-induced inflammatory cell recruitment into
alveoli was impaired in neonates compared with adults but that the
neonates had an increase in BAL fluid 92-kDa gelatinase activity with a
higher ratio of 92-kDa gelatinase activity over inflammatory cells than
the adults. Furthermore, LPS-induced lung injury was associated with
postnatal lung growth disorders. However, we found no evidence of a
causal link between these disorders and the increase in 92-kDa
gelatinase activity.
Inflammatory response to LPS-induced lung injury.
Intratracheal instillation of LPS is a well-validated model of lung
injury in adult animals (14, 16). LPS has thus been shown
to activate resident alveolar macrophages and epithelial cells and to
induce endogenous pathways that result in an intra-alveolar influx of
inflammatory cells, mainly neutrophils. Our finding in LPS-injured
adult rats of major intra-alveolar recruitment of inflammatory cells is
consistent with these earlier studies. However, few data have been
published on lung inflammatory responses to LPS in the neonatal period.
Martin et al. (28) reported age-dependent impairment of
LPS-induced neutrophil recruitment. The highest LPS dose instilled into
the trachea in this study was lower (~10 µg in adults and 2 µg in
7-day-olds) than in our experiments. This probably explains why no
significant neutrophil recruitment was observed in neonates up to 28 days of age. The higher LPS doses used in our study [10 µg (LPS 1×)
and 100 µg (LPS 10×)] induced significant neutrophil recruitment
into the alveoli of 6-day-old rats, in a dose-dependent manner. This
effect was considerably less marked than in adults instilled with LPS
doses adjusted for lung weight. Many factors may account for this
difference. One is impaired function in neonates of the mechanisms
involved in LPS recognition in the airways. Indeed, a lower basal level
of expression of the high-affinity CD14 receptor and failure to
upregulate this expression after LPS administration have been reported
in neonatal neutrophils (34). Neonatal
monocytes/macrophages and neutrophils were, however, found able to
respond to LPS via the CD14-dependent pathway, but not via the
CD14-independent pathway, requiring a non-CD14 plasma protein either
missing or nonfunctional in neonates (8). Furthermore, the
LPS-binding protein involved in LPS recognition by CD14 is present and
functional during the neonatal period, as shown by the normal ability
of sera from neonates to enhance the effects of LPS on adult alveolar
macrophages (28). Another possible explanation is that
neonatal effector cells stimulated by LPS may fail to produce
proinflammatory mediators that either are directly chemotactic or
activate generation of chemoattractants by other cells. A
characteristic of the neonatal period is impairment of some of the
secretory functions of alveolar macrophages, such as oxidant production
(11). In one study, secretion by stimulated monocytes of
interleukin-8 and leukotriene B4, two major neutrophil chemoattractants, was decreased in neonates compared with adults (35). However, others failed to replicate this finding
(37, 39). Furthermore, the supernatant of macrophages
stimulated by LPS exhibited similar chemotactic effects on neutrophils
in neonates and in adult rats (28). Finally, a third
possibility is that neutrophils may have a defective response to
chemoattractants. Neutrophil motility in response to chemoattractants
has consistently been found depressed in neonates (17,
24). Diminished adherence functions (24),
deficiencies in membrane deformability (17), and reduced
expression of C5a receptors (32) may contribute to this
decrease in chemotaxis.
Gelatinase activities induced by LPS.
In our study, LPS-induced lung injury was associated with increased
92-kDa gelatinase activity in BAL fluids from both neonates and adults.
The level of 92-kDa gelatinase activity was significantly correlated to
the inflammatory cell count, suggesting that 92-kDa gelatinase
originated mainly from activated alveolar macrophages and neutrophils,
which are known to secrete large amounts of 92-kDa gelatinase
(22, 43). Normal structural cells, such as bronchial and
alveolar epithelial cells, may also contribute to 92-kDa gelatinase secretion, especially after LPS-induced cell stimulation (15, 44). The LPS-induced increase in 92-kDa gelatinase activity was
dose dependent in both neonates and adults. However, in proportion to
the number of macrophages and neutrophils in BAL fluids, the increase
in 92-kDa gelatinase activity induced by the higher LPS dose was
greater in neonates than in adults. Alveolar macrophages may play a
central role in this difference. We previously demonstrated that
increased 92-kDa gelatinase secretion by neonatal alveolar macrophages
compared with adult cells could be induced by stimulating the protein
kinase C pathway (13), and LPS-mediated 92-kDa gelatinase production has been shown to involve protein kinase C
(40).
Gelatinase B is involved in beneficial effects such as the cell
spreading and migration observed during respiratory epithelium repair
(25). However, most studies focused on the potential deleterious role of an injury-induced imbalance between 92-kDa gelatinase and its inhibitors, and increased 92-kDa gelatinase activity
in airways has been found in experimental models of lung injury
(14, 16) and in human lung diseases (12, 26,
41). During the neonatal period, increased 92-kDa gelatinase
activity may contribute to direct lung tissue alterations and may
interfere with physiological processes such as collagen turnover
(3) and basement membrane disruption (1),
thus leading to lung growth disorders.
LPS-induced postnatal lung growth disorders.
Postnatal lung growth is characterized by intense remodeling of
peripheral air spaces with an increase in the number of alveolar septa,
thinning of interalveolar walls, and microvascular maturation. Postnatal events that have been shown in experimental models to interfere with normal growth, thereby leading to alveolar development disorders, include hyperoxia (4), mechanical ventilation
(2), and administration of corticosteroids
(30). In human disease, alveolar growth is impaired in
bronchopulmonary dysplasia (BPD) (27), which occurs mainly
in premature neonates with hyaline membrane disease and may result from
various injuries to the immature lung. In particular, infections and
airway inflammation have been suggested as key factors in the
development of BPD (33). To our knowledge, our study is
the first to look for a direct link between LPS-mediated lung injury
and the occurrence of postnatal lung growth disorders. We showed that
intratracheal instillation of LPS induced significant changes in lung
structure, including decreases in lung volume, alveolar surface
density, total alveolar surface, and total volume of alveolar air. Our
LPS-injured neonatal rats had a lower mean body weight than our
controls, indicating that somatic growth may be affected by LPS;
however, the difference was not significant. Alveolar surface relative
to body weight remained lower in LPS-instilled neonates than in
controls. Such lesions are very similar to those observed in other
experimental models (29, 30) and are strongly in favor of
diminished septation in LPS-injured neonates. Our hypothesis was that
the LPS-mediated increase in gelatinase B activity might be linked to
impaired alveolar growth. Because tetracycline derivatives have been
shown to suppress MMP activity via both direct and indirect mechanisms (20), we used doxycycline to determine whether 92-kDa
gelatinase overproduction significantly promoted the development of
alveolar growth disorders. Doxycycline has been shown to significantly inhibit 92-kDa gelatinase activity in abdominal aortic aneurysm wall
(5), osteoarthritic cartilage (38), and
periodontitis gingiva (21). In our model, in situ
zymography showed that doxycycline blunted the LPS-induced increase in
tissue gelatinolytic activity. However, no beneficial effect on lung
growth was detectable. As it is recognized that doxycycline acts in a
nonspecific fashion with regard to different MMPs and that it inhibits
gelatinase B as well as other MMP family members, our results suggest
that LPS-induced lung growth disorders may not be mediated by an
increase in MMP activity. Besides gelatinase B and other MMPs, a wide
variety of proinflammatory mediators, such as neutrophil elastase,
oxidants, and cytokines, are released by LPS-activated inflammatory
cells and are also susceptible to interfere with harmonious lung
growth. On the contrary, our data suggest that doxycycline per se may have a deleterious effect on lung growth. In particular, lung volume,
absolute alveolar surface, and absolute air space volume were
significantly decreased in doxycycline-treated neonates. Although the
aim of the present study was not to elucidate the effects of
doxycycline on lung growth, our findings invite the hypothesis that
doxycycline may alter normal growth by inhibiting MMPs different from
the LPS-induced 92-kDa gelatinase and physiologically involved in
alveolar growth regulation. Furthermore, lung growth may be affected by
nonantibiotic effects of doxycycline other than MMP inhibition, such as
interactions with cell proliferation and apoptosis
(18) and/or inhibition of nitric oxide synthase activity
(9). Thus our results argue for a significant role of MMPs
in normal lung growth regulation rather than in the pathogenesis of
LPS-induced lung growth disorders.
In conclusion, we found that LPS-induced lung injury in neonatal rats
was associated with abnormal alveolar growth. These results strongly
suggest a direct contribution of gram-negative bacterial infections and
airway inflammation in the alveolar growth disorders observed in human
BPD. The exact role of the LPS-induced increased 92-kDa gelatinase
activity in the occurrence of lung growth impairment remains to be determined.
 |
FOOTNOTES |
Address for reprint requests and other correspondence: C. Delacourt, Unité INSERM U492, Faculté de
Médecine, 8 rue du Général Sarrail, 94000 Créteil, France (E-mail:
christophe.delacourt{at}creteil.inserm.fr).
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.
10.1152/ajplung.00140.2001
Received 24 April 2001; accepted in final form 9 July 2001.
 |
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