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Am J Physiol Lung Cell Mol Physiol 282: L491-L500, 2002; doi:10.1152/ajplung.00140.2001
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Vol. 282, Issue 3, L491-L500, March 2002

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

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

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.

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

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).

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).

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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  		Table 4.   Light microscopy morphometry



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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.

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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Lung Cell Mol Physiol 282(3):L491-L500
1040-0605/02 $5.00 Copyright © 2002 the American Physiological Society


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