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Published ahead of print on January 7, 2004, doi:10.1164/rccm.200310-1417OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 739-748, (2004)
© 2004 American Thoracic Society

Original Article

High Tidal Volume Ventilation Causes Different Inflammatory Responses in Newborn versus Adult Lung

Ian B. Copland, Francisco Martinez, Brian P. Kavanagh, Doreen Engelberts, Colin McKerlie, Jaques Belik and Martin Post

Program in Lung Biology and Department of Critical Care Medicine, The Hospital for Sick Children; Departments of Laboratory Medicine and Pathobiology and Pediatrics, University of Toronto, Toronto, Ontario, Canada; and Department of Pediatrics, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil

Correspondence and requests for reprints should be addressed to Martin Post, Ph.D., Lung Biology Program, The Hospital for Sick Children, 555 University Avenue, Toronto, ON, M5G 1X8 Canada. E-mail: martin.post{at}sickkids.ca


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We investigated the effect of high VT ventilation on adult and newborn rats by examining pulmonary injury and cytokine messenger RNA (mRNA). On the basis of compliance, edema formation, and histology, ventilation with 25 ml·kg-1 was more injurious to adult rats than newborns. Ventilation with 40 ml kg-1 minimally affected compliance in newborns but caused death in adults. Ventilation of adults for 30 minutes at 25 ml kg-1 upregulated the mRNA expression of interleukin (IL)-1ß, IL-6, tumor necrosis factor-{alpha} (TNF-{alpha}), macrophage inflammatory protein-2 (MIP-2), and IL-10, whereas in newborns such ventilation only increased mRNA expression of MIP-2 and IL-10. When VT was raised to 40 ml kg-1 in newborns, IL-1ß mRNA levels were additionally increased at 30 minutes, whereas ventilation for 3 hours additionally increased IL-6 and TNF-{alpha} mRNA. In newborns, the addition of 100% oxygen (O2) to 30 minutes of ventilation blunted the high VT induction of IL-1ß, IL-10, and MIP-2 mRNA expressions, whereas at 3 hours, 100% O2 concentration synergistically increased the mRNAs for TNF-{alpha} and IL-6. Overall, adult rats are more susceptible to high VT–induced lung injury compared with newborns. In newborns, the inflammatory response is dependent on VT, duration, and supplemental O2. Thus, recommendations for VT limitation based on adult data may be inappropriate for newborns.

Key Words: VT • oxygen • cytokines • lung maturity

Mechanical ventilation is the primary supportive treatment for infants and adults suffering from severe respiratory failure. Ample experimental evidence (1–13) as well as clinical studies (14) indicate that adverse mechanical ventilation worsens patient outcome. In the adult lung, overdistension alone induces a proinflammatory response by increasing the expression of the proinflammatory cytokines interleukin (IL)-1ß, IL-6, IL-8, and tumor necrosis factor-{alpha} (TNF-{alpha}), which likely contribute to the pathogenesis of ventilator-associated lung injury (VALI) (15). Consistent with this evolving knowledge, clinical recommendations, aimed at reducing VALI in adults with adult respiratory distress syndrome (ARDS), suggest limiting VT (16). It has been suggested that approaches aimed at protective ventilation in adults be applied to infants requiring mechanical ventilation (17, 18). These suggestions are based on the premise that the mechanisms of VALI are similar in the adult and newborn lung; however, there is minimal information to support this assumption. In particular, little is known about the kinetics of cytokine expression in ventilated newborn animals.

Overdistension alone enhances the messenger RNA (mRNA) expression of proinflammatory cytokines (IL-1ß, IL-6, IL-8, and TNF-{alpha}) in the lungs of preterm lambs (19), but the magnitude of response of individual cytokines differed from that in adults. The latter finding raises doubts regarding the validity of extrapolating ventilatory strategies designed to protect adults with ARDS to newborns. There are several reasons—including evolving airway structure and vascularization (20), relative deficiency of surfactant (21), excess of lung water (22), an immature immune system (23), and the presence of a highly compliant chest wall (21)—to believe that immature lungs would respond differently to high VT ventilation compared with adult lungs. To test this idea, we compared a newborn and adult rat model of high VT ventilation in vivo. We profiled the proinflammatory cytokines IL-1ß, IL-6, macrophage inflammatory protein-2 (MIP-2) (an IL-8 homolog), and TNF-{alpha} as well as the antiinflammatory cytokine IL-10 because of their appearance/absence in both adult and newborn models of lung injury (7, 19, 24–31). We hypothesized that high VT ventilation would be more injurious in newborn versus adult rats and that this increased susceptibility would be reflected in a shift in the cytokine balance toward proinflammatory mediators, which would be influenced by the VT, the length of ventilation, and the concentration of oxygen (O2) in the inspired gas.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal Preparation
Adult male (aged 3–4 months) and newborn (aged 5–8 days) Sprague-Dawley rats were anesthetized by intraperitoneal injection of ketamine (60 mg kg-1) and xylazine (5 mg kg-1). A tracheostomy was performed, and ECG (in the newborns) and intraarterial blood pressure (in the adults) was used for monitoring. Animal temperature was maintained at 37°C with a thermal blanket.

Mechanical Ventilation
Adult animals were allocated to either a control group (not ventilated) or to one of two high VT protocols using a rodent ventilator (Harvard, St. Laurent, PQ, Canada):

  • High VT-1 (VT 25 ml kg-1, positive end-expiratory pressure [PEEP] 0 cm H2O, frequency 30 minute-1, 21% O2).
  • High VT-2 (VT 40 ml kg-1, PEEP 0 cm H2O, frequency 30 minute-1, 21% O2).

Newborn animals were allocated to a control group (not ventilated) or one of four high VT protocols using a previously described custom-made mechanical ventilator (32):

  • High VT-1 (VT 25 ml kg-1, PEEP 0 cm H2O, frequency 60 minute-1, 21% O2).
  • High VT-2 (VT 40 ml kg-1, PEEP 0 cm H2O, frequency 60 minute-1, 21% O2).
  • High VT-3 (VT 25 ml kg-1, PEEP 0 cm H2O, frequency 60 minute-1, 100% O2).
  • High VT-4 (VT 40 ml kg-1, PEEP 0 cm H2O, frequency 60 minute-1, 100% O2).

Ventilation was continued for 30 minutes (n = 4 per group), 90 minutes (n = 6 per group), or 180 minutes (n = 4 per group). Nonventilated animals served as controls (n = 4 per group).

Total respiratory system dynamic compliance was measured by recording the delivered VT and the difference between peak inspiratory pressure (PIP) and end-expiratory airway pressure. Timed compliance measurements were compared with compliance measured after 5 minutes of ventilation, as adults and newborns show an initial rise in dynamic compliance.

Analysis of Lung Water
The upper right lobe was removed, weighed, and then dried at 80°C. Lung wet to dry weight ratio was used as an index of pulmonary edema formation.

Histologic Lung Injury
The lungs were pressure fixed and processed for paraffin embedding. Histologic scoring was performed by a blinded pathologist, as described previously (33).

RNA Preparation
One hundred micrograms of frozen left lung tissue was placed in lysis buffer, homogenized, and applied to RNA purification columns, according to manufacturer's instructions (Rneasy; Qiagen, Mississauga, ON, Canada). After washing the columns, the bound RNA was treated with DNAse I, washed, and eluted.

Real-time Polymerase Chain Reaction
Total RNA (2 µg) was reverse transcribed in a total volume of 50 µl using random hexamers. The TaqMan Universal Master Mix was used according to the manufacturer's protocol (Applied Biosystems, Foster City, CA) in which 50 ng of complementary DNA was amplified for our target genes and 5 ng amplified for 18S using TaqMan primers and probes (Table 1) .


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  		TABLE 1. Taqman primers and probe sequences of rat cytokine genes

 
Relative mRNA Quantitation
For each probe, a dilution series determined the efficiency of amplification of each primer/probe set, allowing the relative quantification method to be employed (34). For relative quantization, polymerase chain reaction signals were compared between groups after normalization using 18S as an internal reference. Fold change was calculated according to Livak and Schmittgen (34).

Statistical Analysis
Experimental and control data were compared using JMP (SAS Institute, Cary, NC) statistical software, by analysis of variance, followed by Student-Neumann-Keuls. Significance was accepted at a p value less than 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Physiologic Data
Dynamic compliance was not altered by 90 minutes of ventilation with 25 ml kg-1 in either adult or newborns (Figure 1) . There was also no statistical difference in PIP (Figure 2A) , PaCO2 (Figure 2B), and pH (Figure 2C) between adult and newborn rats ventilated for 90 minutes with 25 ml kg-1. However, after 90 minutes of ventilation, there was a significant increase in the lung wet to dry weight ratio (Figure 2D) in adult, but not newborn, rats ventilated with 25 ml kg-1 when compared with nonventilated control rats. Histologically, adult lungs showed a greater degree of injury in their airspaces compared with similarly treated newborn rats (Table 2) . Adult, but not newborn, rats ventilated with 25 ml kg-1 for 180 minutes developed a mean decrease in respiratory system compliance of 30% (Figure 1). Newborn rats ventilated with 40 ml kg-1 developed a small decrease in respiratory system compliance by 180 minutes. (Figure 1), whereas adult rats ventilated with 40 ml kg-1 developed significant impairment of respiratory system compliance by 10 minutes (Figure 1) and died within 20 minutes.



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  		Figure 1. Change in respiratory system compliance due to high VT ventilation. Ventilation of newborn rats for 0 to 90 minutes with either 25 ml kg-1 (25 VT) or 40 ml kg-1 (40 VT) did not alter compliance, whereas at 180 minutes, 40 ml kg-1 (40 VT) caused a small decrease in compliance. In the adult rat, ventilation with 25 ml kg-1 (25 VT) for 0–90 minutes did not significantly alter compliance; however, by 180 minutes, compliance was significantly reduced (~ 30%). In the adult rat, ventilation with 40 ml kg-1 (40 VT) for 10 minutes significantly decreased compliance (*p < 0.01 vs. baseline). All graphs are presented as mean change ± SEM (n = 4 individual experiments).

 


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  		Figure 2. Airway pressure and blood gas measurements in newborn and adult animals ventilated for 90 minutes with 25 ml kg-1 (25 VT). After 90 minutes of high VT ventilation, (A) peak inspiratory pressure (PIP), (B) PaCO2, and (C) pH were similar in newborn and adult animals ventilated with 25 ml kg-1. (D) Adult lungs show a significant increase in wet to dry ratio in their lungs after being ventilated for 90 minutes with 25 ml kg-1 (25 VT), which is not seen in newborns (*p < 0.05). All graphs are presented as mean ± SEM (n = 6 individual experiments).

 

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  		TABLE 2. Histologic evaluation demonstrated that lung injury was less severe in newborn animals ventilated for 90 minutes high vT with 25 ml kg- compared with similarly treated adult animals

 
Pulmonary Cytokine mRNA Levels in Nonventilated Adult versus Newborn Rats
Real-time polymerase chain reaction efficiency plots demonstrated that IL-1ß, IL-6, TNF-{alpha}, MIP-2, and IL-10 all amplified with similar efficiencies, allowing comparison of basal cytokine (i.e., nonventilated) mRNA expressions. In adults, mRNA levels of IL-1ß were greater than MIP-2 greater than IL-6 greater than IL-10 relative to TNF-{alpha} (Figure 3A) . In newborns, basal cytokine mRNA expression displayed a similar pattern as in the adult, with the exception that MIP-2 was the most abundant cytokine mRNA followed by IL-1ß (Figure 3B). In contrast to the adult, the basal mRNA levels of each cytokine were significantly lower in the newborns (Figure 3C). In particular, adult versus newborn lungs demonstrated very large differences in basal mRNA levels of IL-1ß, MIP-2, and IL-6 (Figure 3C).



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  		Figure 3. Basal messenger RNA (mRNA) levels of interleukin (IL)-1ß, macrophage inflammatory protein-2 (MIP-2), IL-6, IL-10, and tumor necrosis factor-{alpha} (TNF-{alpha}) in healthy (A) adult and (B) newborn lungs. (C) Compared with adults, newborns had significantly lower levels of IL-1ß, MIP-2, IL-6, IL-10, and TNF-{alpha} mRNA in their lungs (*p < 0.05 vs. neontal basal cytokine mRNA level). All graphs are presented as mean fold change ± SEM (n = 4 individual experiments performed in triplicate).

 
Pulmonary Cytokine mRNA Expression after 30 minutes of Ventilation in Adult versus Newborn Rats
In adult rats ventilated for 30 minutes (with a VT of 25 ml kg-1), the mRNA levels of IL-1ß, IL-6, TNF-{alpha}, MIP-2, and IL-10 were all significantly higher than control values (Figure 4) . In contrast, ventilation (with either 25 or 40 ml kg-1 for 30 minutes) in newborn rats was not associated with a significant increase in IL-6 or TNF-{alpha} expression versus nonventilated control rats (Figure 4). Both MIP-2 and IL-10 mRNA expressions were significantly increased by both 25 and 40 ml kg-1, versus control rats (Figure 4), whereas IL-1ß mRNA expression was increased by tidal volumes of 40 ml kg-1, but not by 25 ml kg-1, versus nonventilated control rats. In addition, we confirmed that mild hypocapnia (PaCO2 = 25–30 mm Hg) did not exacerbate the IL-1ß response in adult rats (Figure E1 in the online supplement).



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  		Figure 4. Alterations in cytokine mRNA expression due to 30 minutes of high VT ventilation. Compared with the adult lung where all five cytokines were significantly elevated by 30 minutes of 25 ml kg-1 (25 VT) ventilation, at 25 ml kg-1 (25 VT), only MIP-2 and IL-10 were significantly increased in the newborn lung. When VT was increased to 40 ml kg-1 (40 VT) in the newborn, IL-1ß was also increased, whereas IL-6 and TNF-{alpha} remained unchanged (*p < 0.05 vs. nonventilated control rats). All graphs are presented as mean fold change ± SEM (n = 4 individual experiments performed in triplicate).

 
Pulmonary Cytokine mRNA Expression after 180 minutes of Ventilation in Newborn Rats
In newborn rats, ventilation with 25 ml kg-1 for 180 minutes was not associated with a significant increase in IL-1ß or TNF-{alpha} expression but was associated with increased levels of IL-6, MIP-2, and IL-10 versus nonventilated control rats (Figure 5) . In contrast, ventilation with tidal volumes of 40 ml kg-1 for 180 minutes was associated with increased expression of mRNA for all the cytokines examined (IL-1ß, IL-6, TNF-{alpha}, MIP-2, and IL-10) versus nonventilated control rats (Figure 5). Furthermore, mRNA levels of IL-6, MIP-2, and IL-10 mRNA at 180 minutes were significantly higher at 40 ml kg-1 than at 25 ml kg-1 (Figure 5).



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  		Figure 5. Alteration in newborn lung cytokine mRNA expression with 180 minutes of high VT ventilation. Compared with 30 minutes of high VT ventilation, when animals were ventilated for 180 minutes, IL-6, MIP-2, and IL-10 were significantly elevated at 25 ml kg-1 (25 VT), whereas at 40 ml kg-1 (40 VT), all five cytokines were significantly elevated (*p < 0.05 vs. nonventilated control rats; #p < 0.05 vs. all groups). All graphs are presented as mean fold change ± SEM (n = 4 individual experiments performed in triplicate).

 
Impact of O2 and VT on Pulmonary Cytokine mRNA Expression in Newborn Rats: 30 minutes of Ventilation
The interactions of VT and inspired O2 content in these experiments were complex. In newborn rats, addition of 100% O2 to VT regimens of 25 or 40 ml kg-1 did not alter the expression of mRNA for IL-6 (Figure 6) . Addition of 100% O2 increased TNF-{alpha} mRNA expression at a VT of 25 ml kg-1 but not when VT was 40 ml kg-1 (Figure 6). Conversely, addition of supplemental O2 decreased the expression of IL-10 when the VT was 25 ml kg-1 but not when it was 40 ml kg-1 (Figure 6). Finally, addition of O2 did not alter the IL-1ß or MIP-2 expression with VT of 25 ml kg-1 but did decrease expression where the VT was 40 ml kg-1 (Figure 6).



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  		Figure 6. Effect of oxygen (O2) and 30 minutes of high VT ventilation on newborn lung cytokine mRNA expression. When newborn rats were ventilated with high VT in the presence of 100% oxygen or room air (21% oxygen) at 25 ml kg-1 (25 VT), there was an additive effect on TNF-{alpha} and a suppressive effect on IL-10. At 40 ml kg-1 (40 VT), oxygen had a suppressive effect on IL-1ß and MIP-2 cytokine mRNA expression (*p < 0.05 vs. paired room air group). All graphs are presented as mean fold change ± SEM (n = 4 individual experiments performed in triplicate).

 
Impact of O2 and VT on Pulmonary Cytokine mRNA Expression in Newborn Rats: 180 minutes of Ventilation
After prolonged mechanical ventilation (i.e., 180 minutes), supplemental O2 had no incremental effect on the mRNA expression of IL-1ß, IL-10, or MIP-2, regardless of whether the VT was 25 or 40 ml kg-1 (Figure 7) . Supplemental O2 increased expression of IL-6 where tital volumes were either 25 or 40 ml kg-1 (Figure 7). Finally, additional O2 increased TNF-{alpha} mRNA expression at 25 ml kg-1 but not when VT was 40 ml kg-1 (Figure 7).



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  		Figure 7. Effect of O2 and 180 minutes of high VT ventilation on newborn lung cytokine mRNA expression. When newborn rats were ventilated with high VT in the presence of 100% oxygen or room air (21% oxygen) at 25 ml kg-1 (25 VT), there was an additive effect on TNF-{alpha} and IL-6. At 40 ml kg-1 (40 VT), oxygen has an additive effect on IL-6 (*p < 0.05 vs. paired room air group). All graphs are presented as mean fold change ± SEM (n = 4 individual experiments performed in triplicate).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this study, we demonstrate that compared with adult animals newborn rats are less susceptible to VALI in terms of overall level of injury and cytokine mRNA responses. Thus, our original hypothesis was wrong. We also demonstrate that in the newborn, the cytokine response depends on the duration of ventilation and the composition of the inspired gas. These data contribute to the ongoing debate about the role played by cytokines in the pathogenesis of VALI (25) and suggest that developmental differences, and in the case of newborns, the use of supplemental O2, may complicate simplistic prediction or extrapolation about the pathogenesis of VALI.

The adult rat has been used in numerous models of VALI (1–13), whereas the newborn rat has been extensively used to study the progression of bronchopulmonary dysplasia or chronic lung disease of the neonate (35). The newborn rat is a particularly useful model for studying newborn lung injury because rats are born with a saccular lung (36), which is structurally similar to the lung of infants most likely to develop chronic lung disease (37).

Previous studies have shown both inverse (38–40) and direct correlations (41) between age and pulmonary barotrauma. However, barotrauma is not the major contributor to VALI but rather volutrauma (42). To our knowledge, we are the first group to compare the effect of age on the evolution of volutrauma-induced VALI and suggest a direct correlation between age and pulmonary injury due to high VT ventilation. We acknowledge that at higher tidal volumes and pressures, conflicting data may arise. Specifically, Adkins and Parker (40) found differences in the filtration coefficient and an increase in the prevalence for pneumothorax in young compared with adult rabbits. However, in their study, PIPs of 30 cm H2O and higher were used, which are well above the PIPs seen in our study. The PIP for our VT = 25 ml kg-1 was less than 15 cm H2O for both our adult and newborn animals, whereas the PIP for our VT = 40 ml kg-1 in the newborns was in the range of 20 to 24 cm H2O. In the adult animals ventilated with VT = 40 ml kg-1, PIP was above 30 cm H2O, but these animals died within 20 minutes and were not analyzed further. Therefore, on the basis of our model of volutrauma, we conclude that there is a positive association between age and pulmonary injury due to high VT ventilation.

Besides VT, the application of PEEP is considered one of the major factors contributing to the development of VALI (42). It is plausible that the application of PEEP would alter the cytokine responses to ventilation. However, Valenza and coworkers (1) recently demonstrated that the application of different levels of PEEP but the same airway pressure will delay but not prevent the emergence of VALI. Consequently, we believe that PEEP may delay the increases in cytokine mRNA levels, seen in both our adult and newborn rat models, but not prevent them.

Cytokines have been shown to contribute to the pathogenesis of numerous diseases through their ability to induce other inflammatory mediators, cause sequestration and accumulation of neutrophils, and enhance vascular permeability (43). By assessing the alterations in mRNA levels of IL-1ß, IL-6, TNF-{alpha}, MIP-2, and IL-10 in adult and newborn rats exposed to high VT ventilation, we provided an alternative means of assessing susceptibility to VALI. Using real-time polymerase chain reaction, we found that the magnitude of cytokine responses to high VT ventilation varied markedly between adult and newborn rats. In the adult rat lung, all five cytokines are induced within 30 minutes of ventilation with 25 ml kg-1. The greatest fold change was seen for IL-10 followed by TNF-{alpha} and then IL-1ß, IL-6, and MIP-2. In newborn rats, MIP-2 and IL-10 were induced at 30 minutes with 25 ml kg-1, whereas increasing the VT from 25 to 40 ml kg-1 induced IL-1ß expression. At 30 minutes, high VT ventilation did not alter either TNF-{alpha} or IL-6 mRNA levels in the newborn. Together, our physiologic and inflammation data demonstrate that high VT ventilation clearly is more injurious to adult rats than to newborn rats.

Although we only present cytokine mRNA data for our newborn and adult models, there is strong evidence to suggest that changes in cytokine proteins would mimic those of their mRNAs but in a temporally delayed pattern. This has been elegantly demonstrated by Quinn and coworkers (44) who ventilated adult rats for 2 hours with a VT = 20 ml kg-1 and did not see any changes in MIP-2 protein expression. However, MIP-2 protein levels were significantly elevated in the VT = 20 ml kg-1 group (compared with nonventilated control rats) when animals were taken off the ventilator after 2 hours and allowed to breath normally for 4 hours before analyzing MIP-2 protein. Because our study involves short-term ventilation, it is unlikely that we will see dramatic changes in protein expression of the cytokines we studied; but, with time, we expect cytokine protein expression to mimic their respective changes in mRNA expression.

There are three major reasons that may explain why the newborn rat is more resistant to VALI than the adult rat. First, the newborn lung is still structurally immature compared with the adult lung, and this is associated with reduced mechanical interdependence between airways and the parenchyma (45). This may result in less mechanical stress and less activation of transcriptional machinery (mechanotransduction) than in the adult lung. This may explain why neither IL-6 nor TNF-{alpha} was induced in the newborn lung by 30 minutes of high VT and why IL-1ß was only induced when VT was extreme (40 ml kg-1). Second, it is well established that the immune system of the preterm lung differs from that of the adult lung (23). The newborn has a lower resident alveolar macrophage population, and those that are present generally have not differentiated from monocytes (46). Compared with adults, monocytes from newborns have a reduced oxidative burst after phagocytosis of bacteria (47), have altered second-messenger responses to exogenous stimuli (48), and differ in their ability to initiate and resolve from an inflammatory stimuli (49). An immature immune system may explain the significantly lower basal expression levels of cytokines in the newborn lung compared with the adult lung, as well as, the differing ability to initiate an inflammatory response due to high VT ventilation. Finally, in terms of body composition, the newborn lung represents a higher proportion of the total body weight than does the adult lung. Thus, the actual volume/lung weight delivered when a VT is determined by body weight changes depending on the age of the animal (20). However, PIPs were similar in adult and newborn when ventilated with 25 ml kg-1, and because at 40 ml kg-1 newborns still produced a less robust inflammatory response than adults, our finding that newborns are more resistant to high VT–induced lung injury holds true.

The current study confirms our previous findings relating to stretch-induced injury in adult lungs (33). However, contrary to our previous study, the current data suggest that the mRNA levels of TNF-{alpha} display a greater fold change than those of IL-1ß due to high VT ventilation in the adult lung (33). This paradox is explained by the sensitivity of detection and expression of the results. First, in the current study, we employed real-time polymerase chain reaction (vs. Northern blotting previously), which is far more sensitive and detects changes even in low-abundance transcripts (50). Second, the current data are expressed as comparisons with basal levels. When we analyzed the basal cytokine mRNA expression levels, we found that MIP-2 and IL-1ß had the highest basal levels in both nonventilated adults and newborns, followed by IL-6 and IL-10, and finally by TNF-{alpha}. In particular, in adult lungs, the basal mRNA level of IL-1ß was 104 times higher than that of TNF-{alpha}. Therefore, the real significance of one cytokine showing a greater mRNA induction compared with another requires knowledge of basal message mRNA levels of each cytokine. Thus, more subtle changes in the mRNA levels of cytokine genes like IL-1ß or MIP-2 after high VT ventilation should maybe be given greater attention than a similar or greater change in TNF-{alpha} mRNA.

In the second part of our study, we determined the temporal mRNA expression of the five cytokines in the newborn lungs ventilated for 30 minutes or 3 hours and showed that the genes considered most important at 3 hours of high VT ventilation were not the same as those considered most important at 30 minutes. Specifically, at 30 minutes, the most prominently upregulated genes were MIP-2 and IL-10, whereas at 3 hours of high VT ventilation, IL-6 and MIP-2 were by far the most strongly induced cytokines. Clearly, if we had only looked at a single time point, very different conclusions would have been drawn. Thus, caution should be exercised when evaluating data regarding ventilation and cytokine expression, particularly when comparing studies where the duration of ventilation is different. This point is substantiated by Naik and coworkers (19), who ventilated premature lambs for 2 or 7 hours and showed that IL-1ß and IL-6 are the most important at 2 hours, whereas at 7 hours, IL-1ß, IL-6, MIP-2, and TNF-{alpha}, all showed similar levels of induction. By studying the temporal pattern of cytokine mRNA levels, we can see that at 30 minutes of high VT ventilation, the newborn lung attempts to minimize proinflammatory stimuli by inducing an antiinflammatory response. However, the dramatic increases in mRNA expression of the proinflammatory cytokines MIP-2, IL-1ß, and IL-6 noted after 180 minutes of high VT ventilation suggest that the proinflammatory response will overwhelm the more modest antiinflammatory response (i.e., IL-10 mRNA), leading to an imbalance. This in turn could have pathologic consequences.

Exposure of newborn rats to high O2 (60%) for 2 weeks results in a lung phenotype that is consistent with the dysplastic lesions observed in chronic human neonatal lung injury (35). It is not known whether ventilation with high O2 has any additive or suppressive effect on VALI in the newborn. From the present study, it appears that high inspired concentrations of O2 at the initiation of ventilation may help suppress the induction of the proinflammatory cytokines MIP-2 and IL-1ß, but if continued for longer periods of time it can help stimulate the production of cytokines like TNF-{alpha} and IL-6. High levels of TNF-{alpha} are clearly detrimental to the lung (51). Whether the additive effect of O2 on IL-6 expression is harmful remains a matter of speculation. IL-6 has long been considered a proinflammatory cytokine (51); yet data over the years have suggested that IL-6 may actually have a protective function. In particular, it has been found that transgenic mice that overexpress IL-6 are more resistant to oxidative injury (52), suggesting that high levels of IL-6 in the lung may actually be beneficial. Further illustrating that high levels of IL-6 may actually be beneficial in the context of ventilator-induced lung injury are data from IL-6 recombinant protein studies (53) and IL-6 knockout mice (54). Both studies demonstrate that IL-6 has the ability to attenuate the synthesis of proinflammatory cytokines like IL-1ß and TNF-{alpha}, although having little effect on antiinflammatory cytokines like IL-10. In the context of ventilator-induced newborn lung injury and the pathogenesis of infant chronic lung disease, the role of IL-6 needs to be further evaluated.

Conclusions
From these experiments, we conclude that the newborn is less susceptible to VALI due to high VT ventilation compared with the adult but when sufficiently challenged can mount a robust inflammatory response whose characteristics are dependent on VT, duration of ventilation, and concentration of inspired O2.


   FOOTNOTES
 
Supported by the Canadian Institutes of Health Research.

This article has an online supplement, which is accessible from this issue's table of contents online at www.atsjournals.org

Conflict of Interest Statement: I.B.C. has no declared conflict of interest; F.M. has no declared conflict of interest; B.P.K. has no declared conflict of interest; D.E. has no declared conflict of interest; C.M. has no declared conflict of interest; J.B. has no declared conflict of interest; M.P. has no declared conflict of interest.

Received in original form October 16, 2003; accepted in final form January 4, 2004


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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