1 Thoracic Surgery Research Laboratory, University Health Network, Toronto General Hospital, Toronto M5G 2C4; and 2 Division of Respiratory Medicine, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, and University of Toronto, Toronto, Ontario, Canada M5G 1X5
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ABSTRACT |
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Ventilation-induced lung injury has been related to cytokine production. Immaturity and barotrauma are important contributors to the development of bronchopulmonary dysplasia in infants. In the present study, stretch of organotypic cultured fetal rat lung cells was used to simulate ventilation of preterm newborns. Cells were stimulated with lipopolysaccharide (LPS; 100 ng/ml) and/or mechanical stretch. After 4 h, stretch enhanced LPS-induced macrophage inflammatory protein (MIP)-2 production in a force- and frequency-dependent manner. The maximal effect of stretch was seen with 5% elongation at 40 cycles/min. In contrast, after 1 h of stimulation, stretch alone significantly increased MIP-2 production, which was not blocked by cycloheximide, an inhibitor of protein synthesis. At both the 1- and 4-h time points, only LPS increased MIP-2 mRNA levels. Stretch-induced MIP-2 release was associated with cell injury as measured by lactate dehydrogenase release and was not inhibited by gadolinium, a stretch-activated ion channel blocker. Taken together, these results suggest that the major effect of stretch on MIP-2 production from fetal rat lung cells is to stimulate its secretion.
ventilation; acute lung injury; physical force; cytokine; chemokine
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INTRODUCTION |
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RECENT STUDIES
HAVE DEMONSTRATED that ventilation-induced lung injury may be
mediated through proinflammatory cytokines and other inflammatory
mediators (5, 35) that may cause or exacerbate lung injury
by an inflammatory response (15). For example, in a
surfactant-depletion rabbit model, conventional ventilation resulted in
progressive hypoxemia, decreased pulmonary compliance, hyaline membrane
formation, and neutrophil accumulation in the lung (10).
The number of polymorphonuclear leukocytes and the levels of
inflammatory chemical mediators were significantly increased in
bronchoalveolar lavage (BAL) fluid (10), and mRNA of tumor necrosis factor (TNF)-, a proinflammatory cytokine, was increased in
the cells isolated from BAL fluid after conventional ventilation (31). Blockade of the function of proinflammatory
cytokines such as interleukin (IL)-1 (20) and TNF-
(9) attenuated the severity of ventilation-induced lung
injury in surfactant-depleted rabbits.
To study the cellular and molecular mechanisms of ventilation-induced cytokine production from the lung, several groups (34, 38) have used isolated lungs as models and demonstrated that ventilation could increase cytokine production. Others have employed cell culture models to study mechanical stretch-induced production of IL-8, a C-X-C chemokine important for neutrophil recruitment and activation (1-3), in a human lung carcinoma cell line, A549 cells (23, 36, 37). In these studies (23, 36, 37), cells were cultured as a confluent monolayer on flexible membranes that could be stretched with negative pressure or other mechanisms. Stretch alone or in combination with other stimuli increased IL-8 production (23, 36, 37).
The preterm lung is highly susceptible to injury during resuscitation or mechanical ventilation because of the small gas volumes per kilogram of body weight (11). Excessive ventilator settings, oxygen, and prematurity have been suggested to be the most important factors for bronchopulmonary dysplasia (BPD) in preterm infants (32, 33), especially in very low birth weight (VLBW) infants (11). It has been reported that ventilation of the VLBW infant is associated with increases in proinflammatory mediators in air space samples (8).
We hypothesized that mechanical stretch may induce cytokine production from preterm lung cells, which could play a role in ventilation-induced lung injury and contribute to the development of BPD. In contrast to previous studies (23, 36, 37), we applied mechanical stretch to primary cultured fetal rat lung cells in an organotypic culture to simulate the effects of ventilation on premature lungs. The three-dimensional culture system allowed fetal lung cells to form alveolar-like structures. In addition, lipopolysaccharide (LPS), the major component of endotoxin, was used to determine whether there was a synergistic effect on cytokine release in combination with mechanical stretch. Macrophage inflammatory protein (MIP)-2 is a rodent homologue of human IL-8 and an important mediator in inflammatory reactions (6, 18, 30). Xavier et al. and Zhang et al. have recently found that primary cultured adult rat pneumocytes (40) and lung explants (42) produced MIP-2 in response to LPS stimulation. Therefore, this C-X-C chemokine was chosen as a representative cytokine for study. Our data suggest that the major effect of mechanical stretch on MIP-2 production from fetal lung cells is to stimulate its secretion.
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METHODS |
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Materials. Cell culture media, fetal bovine serum, and trypsin were obtained from GIBCO (Burlington, ON). DNase and collagenase were from Worthington (Freehold, NJ). Gelfoam sponges were from Upjohn (Toronto, ON). All other chemicals were from Sigma (St. Louis, MO).
Mechanical strain of fetal lung cells in organotypic culture. Female timed-pregnant Wistar rats (200-250 g) were purchased from Charles River (St. Constant, PQ). Pregnant rats were killed by an excess of halothane on day 19 of gestation (term = 22 days). Fetal lungs were pooled from two litters for every cell isolation procedure. Organotypic cultures of fetal lung cells were established as previously described (14). Briefly, fetal rat lungs were dissected out, minced, and resuspended in Hanks' balanced salt solution. The minced lung tissue was trypsinized [0.125% (wt/vol) trypsin and 0.4 mg/ml of DNase], filtered, and centrifuged. The cells were inoculated on 2 × 2 × 0.25-cm Gelfoam sponges at a density of 3 × 106 cells/sponge and incubated overnight in MEM plus 10% (vol/vol) fetal bovine serum. The sponges were washed two times with serum-free MEM, and the medium was replaced by MEM and changed daily. Three days after cell isolation, the cells were challenged by mechanical stretch and/or LPS.
Bio-Stretch, a mechanical strain device (ICCT, Markham, ON) used in these studies, has been described in detail elsewhere (13). It consists of a programmable Bio-Stretch controller, a personal computer, and three sets of solenoids. Each set contains five solenoids and can be programmed individually. A culture dish with a Gelfoam sponge was placed in front of each solenoid. One end of the sponge was fixed to the bottom of the dish, and the other end was attached to a movable metal bar that was wrapped and sealed in sterile plastic tubing. A magnetic force generated through the solenoids acted on the metal bar to apply strain to the organotypic cultures. The sponges were subjected to various degrees of elongation from their original length at a defined frequency for various time periods. The recoil properties of the sponge allowed it to return to its original length after each stretch. The percent of elongation of sponges was determined as previously described (13). In some experiments, cycloheximide (8 µM) or gadolinium (10 µM) was added to the culture medium 30 min before stretch and/or LPS stimulation. With these concentrations, no measurable cytotoxic effect on fetal rat lung cells was found either with the inhibitor alone or with the LPS and/or stretch stimulation as assessed by lactate dehydrogenase (LDH) assay (data not shown).MIP-2 measurements.
Cell culture media were collected and stored at 80°C. MIP-2
analysis was performed in a blinded fashion with ELISA kits (Biosource, Camarillo, CA) according to the manufacturer's instructions. The absorbance of each well was read at 450 nm with an MR600 microplate reader (Dynatech laboratories, Chantilly, VA). The final concentration was calculated by converting the optical density (OD) readings against
a standard curve (40).
Cytoxicity assay. A cytotoxicity detection kit (Boehringer Mannheim, Mannheim, Germany) was used to measure LDH activity as previously described (42). In principle, NAD can be reduced to NADH/H+ by LDH-catalyzed conversion of lactate and pyruvate. The catalyst (diaphorase) then transfers H/H+ to the tetrazolium salt 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride, which is reduced to formazan. The color of the formazan dye formed is proportional to the amount of LDH released from cells as assessed by spectrometry at 500 nm.
RNA extraction and semiquantitative RT-PCR.
At different time intervals (1 or 4 h), culture medium was removed
and cells in the sponges were lysed in 4 M guanidinium thiocyanate. The
lysates were centrifuged on a 5.7 M cesium chloride cushion to pellet
RNA. Total RNA concentration was measured with a spectrophotometer
(Beckman DU 640B, Fullerton, CA). Five micrograms of total RNA were
used for the RT reaction with a Superscript II kit (GIBCO BRL). Ten
percent of cDNA from the RT reaction was used for PCR. Sequences of the
primers for -actin and MIP-2 and the optimized PCR conditions have
been previously described (40), except that 20 cycles of
PCR were performed for
-actin mRNA in the present study. The PCR
mixture was set up in a total volume of 30 µl containing 3 µl of
10× PCR buffer (200 mM Tris-Cl, pH 8.4, and 500 mM KCl), 1 µl of 50 mM MgCl2, 0.7 µl of 10 mM deoxynucleotide triphosphate
mix, 0.5 µl of each PCR primer (10 µM), and 0.3 µl of
Taq polymerase (GIBCO BRL). PCR was performed with a
programmable thermal cycler (PTC-100, MJ Research, Watertown, MA). Ten
microliters of PCR product were loaded on a 1.5% agarose gel with
ethidium bromide staining for visualization. After electrophoresis, the gels were photographed and quantified with a gel documentation system
(Gel Doc 1000, Bio-Rad Laboratories, Hercules, CA). To ensure
comparability, RT-PCR was performed simultaneously on all samples
collected from each experiment. PCR products were analyzed on the same
gel. The OD of the PCR product bands was quantified with integrated
image analysis software (Molecular Analyst version 1.5, Bio-Rad). With
optimized PCR conditions, all data were collected without saturation or
missing bands. The background of the OD reading for each band was
subtracted locally. Each experiment was conducted at least two times to
ensure reproducibility.
Statistical analysis. All experiments were carried out with materials collected from at least two to three separate cell cultures in triplicate or more. All data are expressed as means ± SE from separate measurements and were analyzed by one-way ANOVA followed by Student-Newman-Keuls test (29), with significance defined as P < 0.05.
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RESULTS |
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Mechanical stretch enhanced LPS-induced MIP-2 production from fetal rat lung cells. Xavier et al. (40) have recently shown that primary cultured adult rat lung epithelial cells responded to LPS stimulation by producing MIP-2. The optimal concentration of LPS was 10 µg/ml, and the peak MIP-2 production was at 4 h (40). To determine whether fetal rat lung cells could produce MIP-2 in response to LPS, cells isolated from day 19 fetal lungs (canalicular stage of lung development) were cultured in Gelfoam sponges and subjected to LPS stimulation (0.1-1,000 ng/ml) for 4 h. MIP-2 concentration in the culture medium increased from 660 to 1,800 pg/ml in an LPS dose-dependent manner (data not shown).
To study the effects of mechanical stretch and/or LPS on MIP-2 production, a dose of LPS (100 ng/ml) that induced a submaximal increase in MIP-2 concentration in the culture medium after a 4-h incubation was chosen. The cells were stimulated with and without LPS for 4 h and continuously subjected to either 2 or 5% stretch at 40 cycles/min. In the presence of LPS treatment, mechanical stretch significantly increased MIP-2 production in a force-dependent fashion (P < 0.05; Fig. 1).
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Mechanical stretch induced immediate release of MIP-2 and cell
injury.
Using the selected stretch regimen, we then studied the time course of
LPS- and/or stretch-induced MIP-2 production. The cells were stimulated
for three different time periods: 1, 2, and 4 h. Interestingly, at
1 or 2 h of incubation, LPS treatment did not increase MIP-2
levels, whereas mechanical stretch induced a significant elevation in
MIP-2 (P < 0.05; Fig.
4). The combined effect of LPS and
stretch on MIP-2 production was mainly due to stretch (Fig. 4). After
4 h of incubation, the results (Fig. 4) looked very similar to the
results described in Mechanical stretch enhanced LPS-induced
MIP-2 production from fetal rat lung cells (Figs.
1-3); that is, LPS-induced MIP-2 was enhanced by mechanical stretch. We used the 1- and 4-h incubation periods for further studies.
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The effect of stretch on MIP-2 release is to stimulate its
secretion.
To determine whether stretch-induced MIP-2 production requires
increased gene expression of MIP-2, cells were treated for 1 or 4 h as described in Mechanical stretch induced immediate release of
MIP-2 and cell injury. Total RNA was extracted, and steady-state mRNA levels of MIP-2 and -actin were analyzed with a
semiquantitative RT-PCR as previously described (40). The steady-state mRNA of
-actin was not altered by LPS and/or stretch (Fig. 6); thus the ratio of MIP-2 to
-actin mRNA was used to present the alteration in MIP-2 gene
expression. As shown in Fig. 6, at both time periods, it was LPS
treatment that increased mRNA levels of MIP-2 (P < 0.05), whereas mechanical stretch had no such effect in either the
absence or presence of LPS treatment (Fig. 6). This suggested that the
effect of stretch on MIP-2 production from fetal lung cells is at the
posttranscriptional level.
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Stretch-induced MIP-2 release was not blocked by the
stretch-activated ion channel inhibitor gadolinium.
Liu et al. and Xu et al. have shown that a stretch-activated ion
channel blocker, gadolinium, inhibited intermittent mechanical stretch-induced fetal rat lung cell proliferation (17) as
well as intermittent stretch-induced release of glycosaminoglycan from these cells (41). Recently, it has been shown that
gadolinium ameliorated high airway pressure-induced permeability
increases in isolated rat lungs (22). To determine
whether stretch-induced MIP-2 release is related to stretch-activated
ion channels, the cells were treated with gadolinium (10 µM) and
subjected to LPS and/or stretch. Figure 8
shows that gadolinium did not block the stretch-induced immediate
release of MIP-2 at 1 h (Fig. 8A). Interestingly, it
reduced the MIP-2 production induced by LPS alone or in combination with stretch at 4 h (P < 0.05; Fig.
8B).
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DISCUSSION |
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Stretch-induced cytokine from the lung and lung cells.
Using an ex vivo rat lung ventilation model, Tremblay et al.
(34) demonstrated that injurious ventilation regimens
increased BAL fluid concentrations of several cytokines including
TNF-, IL-1
, IL-6, IL-10, MIP-2, and interferon-
. With an
isolated perfused mouse lung model, it has been shown that
hyperventilation induced release of TNF-
and IL-6 into the perfusate
(38). These experiments demonstrated that
ventilation-induced cytokines are from the lung, and cytokines induced
from the lung can be secreted into the alveolar space and released into
the pulmonary circulation. Interestingly, although ventilation
increased IL-10 in BAL fluid (34), anti-inflammatory
cytokines such as IL-10 and soluble TNF-
receptors in the perfusate
were either not measurable or not altered by the ventilation
(38). Thus the imbalance between pro- and
anti-inflammatory cytokines released from the lung into the circulation
may contribute to the development of multiple-organ dysfunction
syndrome (4, 28).
A model to study the effect of ventilation on premature lungs. In this study, an organotypic culture model to study the effect of fetal breathing movements on fetal lung cell proliferation and differentiation (14, 27) was adapted to study the effect of "over"stretch on cytokine production. Fetal lung cells were stretched to mimic the mechanical ventilation on premature lungs. The computerized Bio-Stretch apparatus allows us to apply a unidirectional stretch to cells. There are three sets of solenoids, and each can be programmed separately. Thus different stretch forces, frequencies, or experimental periods could be compared in the same experiment (13). Culturing cells and applying stretch to cells in a three-dimensional culture better simulate cellular functions seen in vivo than those seen in two-dimensional cultures (12). Mixed fetal lung cells can reorganize into "alveolar-like structures" (16, 26); thus mesenchymal-epithelial interactions are preserved, and each alveolar-like structure can be stretched as a unit (16).
There are few inflammatory cells in the fetal lung; thus the MIP-2 measured in our experiments should be primarily from lung parenchymal cells. Because mixed cells were used, we could not identify the specific source of MIP-2. Primary cultured rat alveolar epithelial cells produce large amounts of MIP-2 (40). MIP-2 gene expression is increased in a rat fibroblast cell line (RFL-6) on TNF-Stimulation of stretch on MIP-2 secretion. There are several lines of evidence to suggest that the major effect of stretch on MIP-2 production from fetal rat lung cells is to stimulate its secretion. The mRNA levels of MIP-2 were elevated only by LPS and not by stretch. A rapid release of MIP-2 was observed as soon as 1 h after the initiation of stretch. This suggests that stretch induced release of premade MIP-2. Furthermore, when we used cycloheximide to inhibit protein synthesis, this stretch-induced immediate release of MIP-2 was still observed. In the studies of IL-8 production from human lung A549 cells (23, 36, 37), stretch was conducted for at least 8 h; thus the early effect of stretch on cytokine production was not described. In the ex vivo rat lung model, hyperventilation induced significant increases in cytokine production in the BAL fluid within 2 h (34). With the mouse isolated perfused lungs, hyperventilation-induced cytokines in the perfusate also increased significantly by 30 min (38).
An additive effect between a submaximal concentration of LPS and mechanical stretch on MIP-2 production was observed after 4 h. A similar result was obtained by Vlahakis et al. (37), who found that stretch increased the effect of TNF-Mechanical stretch-induced cell injury. With LDH release as a marker, we have demonstrated that the selected stretch regimen used in most of our experiments induced cell injury. The amplitude of stretch we used (5% of elongation) was the same as our laboratory has used previously for cell proliferation (14, 16, 17) and differentiation studies (19, 41). The major difference is that a continuous cyclic stretch was used in the present study. Liu et al. (16) and Tanswell et al. (33) have found that an intermittent stretch regimen (5% elongation, 60 cycles/min, 15 min/h) stimulated cell proliferation, whereas a continuous stretch with the same amplitude and frequency induced cell injury as determined by [14C]adenine release. The continuous cyclic stretching could be a nonphysiological stress to fetal lung cells because fetal breathing movements are irregular and intermittent. Jobe and Ikegami (11) suggested that in VLBW infants, BPD could represent the maldevelopment sequence from interference or interruption of normal development (11). Continuous mechanical ventilation might be considered as interference by fetal lung cells that are normally stretched by intermittent fetal breathing movements. On the other hand, the clinical relevance of 5% elongation is not clear because the surface area-volume relationship of premature lungs is unknown. The absolute stretch amplitude of fetal rat lung cells in vitro should not be extrapolated directly to that of mechanical ventilation of the human infant lung in vivo. However, these experimental data may provide clues for clinical studies.
It is not clear whether stretch-induced MIP-2 secretion is related to cell injury. Because stretch increased release of LDH at both the 1- and 4-h time points but stretch alone only induced MIP-2 release at 1 h, we suspect that its effect on MIP-2 secretion is not a direct consequence of cell injury. In summary, in this study, primary cultured fetal rat lung cells were used as a model to simulate the effect of mechanical ventilation on premature lungs at the cellular level. LPS-induced production of MIP-2 was enhanced by mechanical stretch in a force- and frequency-dependent manner. Although the amplitude of stretch was the same as that used for physiological studies, changing from an intermittent to a continuous stretch regimen is sufficient to induce cytokine production and cell injury. This could be one of the mechanisms by which mechanical ventilation induces cytokine production and cell injury in premature lungs. Furthermore, in the present study, we demonstrated that the major effect of stretch on cytokine production is to stimulate secretion, not gene expression. ![]() |
ACKNOWLEDGEMENTS |
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We are grateful to Xiao-hui Bai for technical assistance.
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FOOTNOTES |
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This work was supported by operating grants from the Medical Research Council of Canada (MT-13270), Canadian Cystic Fibrosis Foundation, the National Sanitarium Association of Canada, Ontario Thoracic Society, and James H. Cumming's Foundation.
E. Mourgeon was a recipient of Fellowships from the Société Française d'Anesthesie et de Réanimation (French Anesthesiology and Critical Care Society) and the Faculty of Medicine (University of Toronto, Toronto, Ontario, Canada). N. Isowa was a recipient of a Fellowship from the Department of Surgery and Dean's Office, Faculty of Medicine (University of Toronto). M. Liu is a Scholar of the Medical Research Council of Canada.
Address for reprint requests and other correspondence: M. Liu, Toronto General Hospital, Rm. CCRW 1-816, 200 Elizabeth St., Toronto, Ontario, Canada M5G 2C4 (E-mail: mingyao.liu{at}utoronto.ca).
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.
Received 4 January 2000; accepted in final form 3 May 2000.
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