Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio 45229
Submitted 1 July 2003 ; accepted in final form 11 November 2003
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ABSTRACT |
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bronchopulmonary dysplasia; chorioamnionitis; cytokines; lung injury; lung inflammation
We hypothesized that ventilation of the inflamed/injured premature lung would result in further amplification of the lung injury and deterioration of lung function. Therefore, we induced fetal lung inflammation/injury with intra-amniotic endotoxin 4 days before preterm delivery. We then evaluated lung function and multiple markers of inflammation after 4 h of mechanical ventilation.
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METHODS |
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Delivery and ventilation of preterm lambs. At 4 days after the intra-amniotic injection at 126 days of gestation, the premature lambs were delivered by cesarean section at 130 days of gestation through maternal midline abdominal incisions after collection of amniotic fluid (13). Lambs were randomly assigned to 4 h of ventilation or no ventilation. The unventilated lambs were given a lethal dose of pentobarbital sodium (50 mg/kg iv) at delivery and before initiation of breathing. For the groups ventilated for 4 h, each preterm lamb was given ketamine (10 mg/kg im) and acepromazine (0.1 mg/kg im), and after administration of local anesthetic (2% lidocaine sc), a tracheotomy was performed and a 4.5-mm tracheal tube was secured in place. Lung fluid was removed from the tracheal tube by aspiration, and the tracheal tube was occluded with a clamp. After the umbilical cord was cut, the lamb was weighed, surfactant (100 mg/kg; Survanta, Ross Products, Columbus, OH) was instilled into the lungs, and ventilation was initiated.
The initial ventilator settings were as follows: a fraction of inspired O2 (FIO2) of 1.0, a respiratory rate of 40 breaths/min, an inspiratory time of 0.7 s, a positive end-expiratory pressure (PEEP) of 4 cmH2O, and a peak inspiratory pressure (PIP) sufficient to yield a tidal volume (VT) of 8-9 ml/kg (12). This VT was chosen to minimize lung injury with the initiation of ventilation (31). Changes in VT were monitored continuously during ventilation (model CP-100, Bicore Monitoring Systems, Anaheim, CA). VT was measured with a Fleisch pneumotachometer at 30 min, 1 h, and every hour thereafter. PIP and FIO2 were regulated to maintain a target PCO2 of 55 mmHg and a target PO2 of 150-200 mmHg to minimize possible effects of the patent ductus arteriosis. A 5-Fr catheter was advanced into the aorta via an umbilical artery, and filtered fetal blood (10 ml/kg) collected from the placenta was transfused within 10 min of delivery. Dynamic compliance was calculated as VT (ml) normalized to body weight (kg) and divided by the ventilatory pressure (PIP - PEEP, cmH2O) (16). The ventilatory efficiency index (VEI) was calculated as follows: VEI = 3,800/respiratory rate x (PIP - PEEP) x PCO2, where 3,800 is a CO2 production constant (28). The arterial catheter was used for blood gas analysis and blood pressure monitoring. Fluid containing 10% dextrose was infused through a leg vein at 4 ml·kg-1·h-1. Rectal temperature was monitored and maintained at 38-39°C, the normal body temperature for sheep, with heating pads and radiant heat. Supplemental ketamine and acepromazine were given to prevent spontaneous breathing. After 4 h, each animal was deeply anesthetized with pentobarbital sodium (25 mg/kg iv), and FIO2 was changed to 1.0. The endotracheal tube was clamped for 3 min to permit O2 absorption, and the animal was exsanguinated.
Pressure-volume curves and lung processing. The thorax of each lamb was opened, the lungs were inflated with air to 40 cmH2O for 1 min, and a maximum lung volume was recorded (24). The pressure was sequentially lowered to 20, 15, 10, 5, and 0 cmH2O, and lung volumes (ml/kg body wt) were recorded after 30 s at each pressure. Volumes were corrected for the compliance of the system. Left and right lung weights were recorded. Pieces of the right lower lobe were immediately frozen in liquid nitrogen for RNA isolation. BAL of the left lung was done with 0.9% NaCl at 4°C, and the process was repeated five times (21). The BAL fluid (BALF) was pooled, and aliquots were saved for measurement of total protein (23), cytokines, cell number and differential count, and H2O2 production. With the use of left-to-right lung weight ratios, results using BALF were calculated for the total lung and normalized to kilograms body weight.
Amniotic fluid was incubated for 30 min at 37°C with 20 mg/ml N-acetylcysteine and 55 U/ml hyaluronidase (Sigma) to reduce the high viscosity (21). Amniotic fluid and BALF were centrifuged at 500 g for 10 min, and the cell pellets were resuspended in PBS. Cells were stained with trypan blue and counted. After cells were stained with Diff-Quick (American Scientific Products, San Diego, CA), differential cell counts were performed on cytospin preparations. The activation state of the BALF cells was assessed by measuring H2O2 production for 1 x 106 cells using an assay based on the oxidation of Fe2+ to Fe3+ by H2O2 under acidic conditions (Bioxytech H2O2 560 assay, OXIS International, Portland, OR) (21). The results are expressed as total alveolar cells normalized to kilogram body weight.
Cytokine mRNA. Total RNA was isolated from tissue from the right lower lobe and from cell pellets of BALF by guanidinium thiocyanate-phenol-chloroform extraction. RNase protection assays were performed with total RNA from lung tissue and cell pellets (18). Briefly, cRNA transcripts of ovine interleukins (IL-1, IL-6, and IL-8) and ovine ribosomal protein L32 as a reference RNA were synthesized with [32P]UTP (Life Sciences Products, Boston, MA) using SP6 or T7 polymerase (RNase protection assay 111, Ambion, Austin, TX). Aliquots (10 µg) of RNA were incubated with excess radiolabeled probes for cytokines and L32 at 55°C for 18 h. The remaining single-stranded RNA was digested with RNase A/TI (Ambion). Protected fragments were electrophoresed on a 6% polyacrylamide-urea sequencing gel and visualized by autoradiography. Densities of the protected bands were quantified on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA) using ImageQuant Software (Molecular Dynamics).
ELISAs for IL-1, IL-6, and IL-8. Lung tissue was homogenized in saline and centrifuged at 2,000 g for 15 min. A sample of BALF was concentrated fourfold with Centriplus (Millipore, Bedford, MA). Capture ELISAs were used to measure IL-1
, IL-6, and IL-8 in the supernatant of centrifuged lung homogenate and concentrated BALF (14). IgG fractions prepared from rabbit anti-sheep IL-1
, IL-6, or IL-8 serum were the primary antibodies, and guinea pig anti-sheep IL-1
, IL-6, or IL-8 serum was the secondary antibody. Antibodies for IL-6 and IL-8 were purchased from Chemicon International (Temecula, CA), and antibody for IL-1
was made in our laboratory. Standard curves were constructed from the absorbance of known amounts of recombinant sheep cytokines (custom-made by Protein Express, Cincinnati, OH). Standard curves were sensitive at 0.1-80 ng/ml for IL-1
, 0.15-50 ng/ml for IL-6, and 1.5-100 ng/ml for IL-8, with a correlation coefficient of 0.99 for all assays.
Immunohistochemistry for cytokines. The right upper lobe was inflation fixed at 30 cmH2O pressure with 10% formalin. Paraffinembedded 5-µm-thick tissue sections were treated for antigen retrieval using 0.1 M citrate, and endogenous peroxidase activity was inactivated by incubation with H2O2. Nonspecific binding sites were blocked with serum. Lung sections were incubated with a rabbit polyclonal antibody for ovine IL-6 (Chemicon), mouse polyclonal antibody for ovine IL-8 (Chemicon), or rabbit polyclonal antibody for ovine IL-1 (14). After the slides were washed with PBS to remove unbound antibody, they were incubated with the secondary biotinylated antibody against rabbit (for IL-6 and IL-1
) or mouse (for IL-8) IgG (Vector Laboratories, Burlingame, CA). After addition of avidin (Vector Laboratories), staining of positive cells was developed with diaminobenzidine and cobalt with a nuclear fast red counterstain.
Data analysis. Values are means ± SE. Analysis of variance followed by the Student-Newman-Keuls multiple comparison procedure was used for comparisons of the four groups. Two-tailed unpaired t-tests were used for two-group comparisons. Significance was accepted at P < 0.05.
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RESULTS |
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Inflammation in the lungs. In the unventilated control lambs, no neutrophils and very few monocytes were recovered by BAL (Fig. 3). Both cell types were increased in the endotoxin-exposed lambs that were not ventilated. Ventilation for 4 h recruited 1.0 ± 0.3 x 106 neutrophils/kg to the BALF of the control lambs. Ventilation did not further increase the neutrophils in the BALF of the endotoxin-exposed animals from the unventilated values of 56 ± 19 x 106 neutrophils/kg. There are no differences in monocyte numbers in BALF between the groups with and without ventilation. H2O2 production by the cells from the BALF was low for unventilated lambs. Ventilation increased H2O2 production in the control and endotoxin-exposed lungs, and qualitatively more H2O2 was produced by the cells from the ventilated and O2-exposed group. Total protein in BALF of the control unventilated lambs was 6.3 ± 1.4 mg/kg, and the increase with endotoxin exposure to 12.7 ± 3.1 mg/kg was not significant. The protein values for the ventilated lambs were 17.0 ± 1.7 mg/kg without endotoxin and 21.8 ± 3 mg/kg with endotoxin, and both values were higher than those for unventilated controls (P < 0.05). The antenatal endotoxin exposure caused lung inflammation, but the added stimulus of ventilation did not result in increased cell numbers or alveolar protein.
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Immunohistochemistry for cytokines. Expression of IL-1, IL-8 (Fig. 4), and IL-6 was analyzed by immunohistochemistry for three sections for each lamb. The immunostaining pattern and intensity for IL-1
and IL-8 were similar for the lambs within each group, and the representative photomicrographs for IL-8 are shown in Fig. 4. IL-1
and IL-8 staining was not seen in control lambs (Fig. 4, A and B). IL-1
and IL-8 were found in inflammatory cells in the air space for endotoxin-exposed lambs only (Fig. 4, C and D). Ventilation for 4 h (Fig. 4D) did not change the immunostaining for IL-1
or IL-8 relative to the unventilated endotoxin-exposed group (Fig. 4C). IL-6 was not detected in any of the lung tissues (data not shown).
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Cytokine mRNAs and proteins. The mRNA for IL-1 was increased fourfold in cells from BALF by the antenatal endotoxin but did not increase further after 4 h of ventilation (Fig. 5). IL-8 mRNA in the BALF cells was not increased significantly by ventilation or endotoxin, and IL-6 mRNA was not detected in the BALF cells. In contrast, ventilation and antenatal endotoxin increased the three cytokine mRNAs, but there was not a further increase with the combination of endotoxin and mechanical ventilation.
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The cytokine proteins IL-1, IL-8, and IL-6 were measured in the supernatant of centrifuged lung tissue homogenates and expressed as amount per kilogram body weight (Fig. 6, A-C). The amounts of IL-1
and IL-8 were significantly increased by antenatal endotoxin exposure. Cytokine proteins were also measured in concentrated BALF. IL-6 was not detectable in the BALF of any of the groups. IL-1
was detectable in four of the samples from endotoxin-exposed lambs. IL-8 in BALF was significantly higher in unventilated endotoxin-exposed and ventilated endotoxin-exposed groups than in controls (Fig. 6D). Mechanical ventilation did not increase the amounts of the cytokines in the lung homogenate and BALF of the control lambs. The combination of mechanical ventilation and antenatal endotoxin resulted in increased IL-8 in lung homogenate relative to the unventilated control or endotoxin-exposed groups.
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DISCUSSION |
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Intra-amniotic endotoxin causes chorioamnionitis and lung inflammation that can be detected within 5 h and persists for 15 days (17, 18, 21). The highest number of granulocytes are in BALF at 3 days, and the highest expression of the proinflammation cytokines IL-1 and IL-8 is on day 2 (18, 20). Apoptosis is highest on day 1, and proliferation is evident by day 3 (20); similar responses occur after 4-20 mg of intra-amniotic endotoxin (21). In this experiment, 10 mg of endotoxin were given 4 days before preterm delivery for evaluation of the postnatal response to mechanical ventilation of lungs that are just beyond the peak of injury as indicated by apoptosis and proinflammatory cytokine expression. The preterm lambs were treated with surfactant before ventilation to avoid severe ventilator-mediated injury and to reflect clinical practice (12). Protein levels in BALF were low as a result of surfactant treatment and gentle ventilation (24, 31). The ventilation period of 4 h was selected, because acute inflammation as indicated by granulocyte recruitment and cytokine expression occurs within 4 h and was decreased by 7 h in ventilated preterm lambs (27).
In a previous report, we found that chorioamnionitis induced in sheep 30 days before preterm delivery resulted in minimal residual inflammation in the lungs (13). However, in contrast, mechanical ventilation induced a large increase in monocytes and lymphocytes, but not in granulocytes, in the BALF. This pattern of response was consistent with a memory response that favored innate host defenses over an augmented acute inflammatory response. Ventilation of adult rats exposed to endotoxin resulted in large increases in proinflammatory cytokines, but those effects were elicited by using ventilation styles known to severely injure the lung (5). Dreyfuss et al. (8) recently reviewed the cytokine responses to mechanical ventilation in adult lungs and commented that acute inflammatory responses are not consistently observed. For this experiment, we treated the lambs with surfactant at delivery and limited ventilation pressures and VT to minimize lung injury and to better reflect current clinical practice (12). The lack of an amplified inflammatory response to the combination of endotoxin-induced chorioamnionitis and mechanical ventilation may also have resulted from the normal response of the fetal lung to suppress inflammation within 4 days of the fetal endotoxin exposure (17). The major difference between these experiments is the history of inflammatory exposure, where exposure 30 days before ventilation caused an "immune" response with increased monocytes and lymphocytes, whereas ventilation superimposed on a lung that was resolving inflammation had little effect.
In humans and animal models with inflamed lungs, high-molecular-weight substances such as endotoxin and cytokines can translocate from the lungs to the systemic circulation and cause a systemic inflammatory response (22). This translocation occurred in adult animals only when excessive ventilation was used (26). In contrast, endotoxin appeared in the circulation of surfactant-treated preterm lambs with gentle mechanical ventilation comparable to that used in this experiment, but hyperventilation of the full-term lung was required for intratracheal endotoxin to have systemic effects (19). Thus the preterm lung was uniquely sensitive to developing shock and falling WBC counts when endotoxin or recombinant IL-1 was instilled into the airways. These endotoxin-exposed animals had modestly increased cytokine levels and high numbers of neutrophils in the BALF at birth. However, systemic WBC did not decrease and blood pressure was similar to that of the control lambs. The half-life of endotoxin in amniotic fluid was
1.7 days, and the lung inflammation resulted from direct contact of the lung with the endotoxin (25). The physiological stability of the animals indicated that <10 µg of the endotoxin could have entered the systemic circulation from the lungs with mechanical ventilation (19).
The effects of the antenatal endotoxin exposure on lung function, lung inflammation, or systemic responses were evaluated for only 4 h, because by design we were exploring responses to initial lung adaptation after birth. Very little inflammation was induced in this model of gentle ventilation in lambs not exposed to endotoxin. The tendency for increased inflammation may be an indicator that the antenatal inflammation would result in persistent inflammation with continued mechanical ventilation. Such an effect is predicted by the association of chorioamnionitis and mechanical ventilation with BPD (30).
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ACKNOWLEDGMENTS |
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This study is supported by National Institutes of Health Grants HD-12714 (M. Ikegami and A. H. Jobe) and KO8-70711 (S. G. Kallapur).
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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