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Published ahead of print on November 25, 2003, doi:10.1164/rccm.200305-660OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 518-524, (2004)
© 2004 American Thoracic Society

Ventilation-induced Neutrophil Infiltration Depends on c-Jun N-Terminal Kinase

Li-Fu Li, Lunyin Yu and Deborah A. Quinn

Pulmonary and Critical Care Units, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts; and Graduate Institute of Clinical Medical Sciences, Chang Gung University, Taoyuan, Taiwan, Republic of China

Correspondence and requests for reprints should be addressed to Deborah A. Quinn, M.D., Pulmonary and Critical Care Unit, Massachusetts General Hospital, 55 Fruit Street, Bulfinch 148, Boston, MA 02114. E-mail: dquinn1{at}partners.org


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Positive pressure ventilation with large VTs has been shown to cause release of cytokines, including macrophage inflammatory protein-2 (MIP-2), a functional equivalent of human interleukin-8. The mechanisms regulating ventilation-induced cytokine production are unclear. Based on our previous in vitro model of lung cell stretch, we hypothesized that high VT ventilation–induced MIP-2 production is dependent on the activation of the c-Jun N-terminal kinase (JNK). We exposed C57BL/6 mice to high VT (30 ml/kg) or low VT (6 ml/kg) mechanical ventilation for 5 hours. High VT ventilation–induced neutrophil migration into the lung, MIP-2 protein production, MIP-2 messenger RNA expression, and JNK activation. Large VT ventilation of JNK knockout mice and pharmacologic JNK inhibition with SP600125 attenuated neutrophil sequestration and blocked MIP-2 messenger RNA expression and MIP-2 production. We conclude that lung cell stretch in vivo results in increased lung neutrophil sequestration and increased MIP-2 production, which was, at least in part, dependent upon the JNK pathway.

Key Words: chemokine • mitogen-activated protein kinase • lung stretch

Mechanical ventilation with high VT can result in overdistention of alveoli in lung regions with the lowest airway resistance and the highest compliance. In acute respiratory distress syndrome, which is an inhomogeneous disease (1), some lung units are more diseased than others are and, therefore, less compliant. Because only a small portion of the lung is compliant and ventilated in acute respiratory distress syndrome, the potential for overdistention of more compliant areas of lung is great, even with the use of moderate-sized VTs. Animal data suggest that ventilation with high VTs leads to ventilator-induced lung injury (VILI). VILI is characterized by noncardiogenic pulmonary edema, release of cytokines, and influx of neutrophils (2–6).

The Acute Respiratory Distress Syndrome Network clinical trial (861 patients) of large volume ventilation versus small volume ventilation was stopped early because 22% fewer deaths were found in the patients ventilated with smaller VTs (7). The use of smaller VTs in humans leads to reduced concentrations of polymorphonuclear cells and cytokines in both plasma and bronchoalveolar lavage fluid (8). Murine macrophage inflammatory protein-2 (MIP-2) is a functional homolog of human interleukin (IL)-8 in rodents and has been shown to be increased in animal models of VILI (9, 10). The exact mechanism of large volume ventilation–induced inflammatory cytokine release is unclear. Understanding these mechanisms may lead to new treatment strategies.

Intracellular messengers that are activated by mechanical cell stretch include the mitogen-activated protein kinases (MAPKs) (11–15). In bronchial epithelial cells, stretch was found to activate c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK)1/2, and p38. ERK1/2 and p38 were found to play a role in stretch-induced IL-8 production, but whether the JNKs mediated IL-8 production could not be evaluated due to the lack of specific inhibitors of the JNK pathway (16). Importantly, in vivo studies of lung stretch found activation of JNK and ERK1/2 but not p38 (17) and increased gene expression of c-Jun, a component of the transcription factor activator protein-1 in the JNK pathway (18).

Based on our previous in vitro study of stretch-induced cytokine production showing that transcriptional regulation of IL-8 messenger RNA (mRNA) and IL-8 production were dependent on JNK activation in human alveolar epithelial A549 cells (19), we generated a murine model system of high- versus low-VT mechanical ventilation to further study the in vivo JNK activation in VILI. We hypothesized that ventilation-induced MIP-2 production would be dependent on the JNK pathway. We compared control nonventilated mice, mice exposed to low VT ventilation, and mice exposed to high VT ventilation as seen in VILI. We used JNK knockout mice and a now available pharmacologic inhibitor to inhibit the JNK. We found that large VT ventilation–induced MIP-2 mRNA expression, MIP-2 protein production, and neutrophil infiltration were dependent, at least in part, on JNK activation.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Laboratory Animals
We used a modification of the established rodent model of VILI in mice as described previously (4). Male C57BL/6, either wild-type JNK+/+ or JNK1-/- and JNK2-/- that have been backcrossed for five generations on a C57BL/6 background, weighing between 20 and 25 g were obtained from Jackson Laboratories (Bar Harbor, ME). The constructs pJNK1KO or pJKN2KO were transfected into D3 ES cells. Chimeras were generated by injecting these ES cells into C57BL/6 (B6) blastocysts. Heterozygotes (+/-) were intercrossed to generate homozygous mutant mice (-/-) (20–22).

Ventilator Protocol
A 20-gauge angiocatheter was introduced into the trachea of the animal under general anesthesia with intraperitoneal ketamine (90 mg/kg) and xylazine (10 mg/kg) while breathing room air. The mice then received 0.9% saline containing maintenance ketamine (0.1 mg/g/hour) and xylazine (0.01 mg/g/hour) at a rate of 0.09 ml/10 g/hour by a continuous intraperitoneal fluid pump and were ventilated at 6 ml/kg at a rate of 135 breaths per minute or 30 ml/kg VT at a rate of 65 breaths per minute in room air.

Histopathologic Grading of Neutrophil Infiltration
The lung tissues from control nonventilated mice, mice exposed to low VT and high VT ventilation for 5 hours with or without pretreatment with SP600125, and JNK knockout mice were paraffin embedded, then sliced at 4 µm and stained with hematoxylin and eosin. For quantification of neutrophil infiltration, histopathology was reviewed in a blinded manner by a pathologist using a modified VILI scoring system as described previously (10, 23). In brief, the number of neutrophils surrounding 10 bronchioles per slide was counted. The infiltration of neutrophils was expressed as the average number of neutrophils per bronchiole.

Pharamacologic Inhibitor
JNK inhibitor II (SP600125, 30 mg/kg subcutaneous injection; Calbiochem, La Jolla, CA), unlike older nonspecific inhibitors, has been shown to be specific for JNK activity at the concentrations used herein (24, 25). The dose was chosen on the basis of previous in vivo studies that showed 30 mg/kg inhibited JNK activity (25). The mice were pretreated with SP600125 in dimethyl sulfoxide (Sigma Chemical, St. Louis, MO) or an equivalent amount of dimethyl sulfoxide without inhibitors for 30 minutes before mechanical ventilation.

Statistical Evaluation
The ribonuclease protection assay and Western blots were quantitated using a National Institutes of Health image analyzer ImageJ 1.27z (National Institutes of Health, Bethesda, MD) and were presented as the ratio of MIP-2 mRNA to the reduced form of glyceraldehyde-3-phosphate dehydrogenase (arbitrary units), phospho-MAPK to MAPK (relative phosphorylation). Values were expressed as the mean ± SEM for at least three experiments. The data of myeloperoxidase (MPO), MIP-2 protein, and average neutrophils per airway were analyzed using Statview 5.0 (Abascus Concepts Inc. and SAS Institute, Inc., Cary, NC). Analysis of variance was used to assess the statistical significance of the differences followed by multiple comparisons with Scheffe's test, and a p value higher than 0.05 was considered statistically significant.

Additional details, including ventilation protocol, harvesting of lung tissue, Evans blue dye analysis, MPO assay, measurement of MIP-2, ribonuclease protection assay, and immunoblot analysis, are listed in the online supplement.


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Physiologic Data
There was no statistical difference in pH, PaO2, PaCO2, mean arterial pressure, and peak inspiratory pressure at the beginning versus the end of mechanical ventilation. Evans blue dye analysis, a quantitative measure of changes of microvascular permeability in VILI, was significantly higher in VT 30 ml/kg mice compared with those of either VT 6 ml/kg or control nonventilated mice (Table 1) .


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  		TABLE 1. Experimental conditions at the beginning and end of ventilation

 
Lung Stretch–induced MIP-2 mRNA Expression and MIP-2 Production
To determine if the increased levels of MPO in high VT mechanical ventilation were associated with upregulation of chemotactic factors for neutrophils, we measured MIP-2 mRNA expression and MIP-2 protein production after 1 and 5 hours of mechanical ventilation, respectively (Figures 1A and 1B) . We found a significant increase in MIP-2 mRNA expression and MIP-2 protein production in the VT 30 ml/kg mice compared with those of either VT 6 ml/kg or control nonventilated mice. There was no significant increase in mRNA expression and MIP-2 protein production in the VT 6 ml/kg mice as compared with control nonventilated mice. This suggested that the increased expression of MIP-2 mRNA and subsequent MIP-2 protein production may have been responsible for increased neutrophil infiltration.



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  		Figure 1. High VT ventilation increased macrophage inflammatory protein-2 (MIP-2) messenger RNA (mRNA) expression, MIP-2 production, and neutrophil sequestration. (A) MIP-2 mRNA (top panel), the reduced form of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA (middle panel), and arbitrary units (bottom panel) from control nonventilated mice and mice ventilated at 6 ml/kg and 30 ml/kg for 1 hour (n = 3/group). Arbitrary units were expressed as the ratio of MIP-2 mRNA to GAPDH. The expression of MIP-2 RNA was performed by ribonuclease (RNase) protection assay (B) MIP-2 production in bronchoalveolar lavage (BAL) fluid from control nonventilated mice and mice ventilated at 6 ml/kg and 30 ml/kg for 5 hours (n = 4/group). (C) Myeloperoxidase (MPO) assay of lung tissue from control nonventilated mice and mice ventilated for 5 hours at VT of 6 ml/kg and 30 ml/kg (n = 5/group). *p Values less than 0.05 versus control nonventilated mice; {dagger}p values less than 0.05 versus mice ventilated at 6 ml/kg.

 
Lung Stretch–induced Infiltration of Neutrophils
MPO assay was used to quantitate total lung neutrophils, i.e., neutrophils marginated in the vasculature, located in the parenchyma and in the alveoli (Figure 1C). The MPO levels were significantly elevated after mechanical ventilation with VT 30 ml/kg for 5 hours compared with control nonventilated and mechanical ventilation with VT 6 ml/kg mice. No significant elevation was found in mice ventilated with VT 6 ml/kg compared with control nonventilated mice.

Lung Stretch–induced MAPK Activation
We measured activity of three members of the MAPK families, JNKs, p38, and ERK1/2, in mice exposed to VT 30 ml/kg mechanical ventilation for 1 hour. There were magnitude-dependent increases in phosphorylation of JNKs and ERK1/2 but no change in the expression of total nonphosphorylated proteins of JNKs and ERK1/2 (Figures 2A and 2C) . JNK2 (arbitrary units pJNK2/JNK2: control 1 ± 0.02, VT 6 ml 1.16 ± 0.04, VT 30 ml 1.65 ± 0.05*; *p < 0.05 vs. control) was more prominently phosphorylated than that of JNK1 (arbitrary units: control 1 ± 0.02, VT 6 ml 0.78 ± 0.04, VT 30 ml 1.29 ± 0.03*; *p < 0.05 vs. control). However, both JNK1 and JNK2 showed increased phosphorylation with high VT ventilation. The phosphorylation of p38 was also increased, but there was no significant difference between the low and high VT–ventilated mice (Figure 2B). In previous in vivo and in vitro studies (16, 17), ERK pathway did not significantly contribute to the ventilation-induced releases of MIP-2 or IL-8 protein despite the increased expression of phopho-ERK1/2. This suggested that an increase in JNK-specific activity may be the mechanism of stretch-induced MIP-2 production and neutrophil infiltration.



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  		Figure 2. High VT ventilation increased c-Jun N-terminal kinase (JNK), p38, extracellular signal-regulated kinase (ERK)1/2 activation. Phosphorylated JNK1, JNK2, p38, or ERK1/2 expressions (A–C, top panel) and total JNK1, JNK2, p38, or ERK1/2 protein expressions (A–C, middle panel), and quantitation by arbitrary units (A–C, bottom panel) (n = 3/group). Arbitrary units were expressed as relative phosphorylation. *p Values less than 0.05 versus control nonventilated mice; {dagger}p values less than 0.05 versus mice ventilated at 6 ml/kg.

 
Inhibition of JNK Activation in JNK Knockout Mice
To determine the role of JNK activation in ventilation-induced MIP-2 production and neutrophil infiltration, we used JNK1 and JNK2 knockout mice. Homozygous JNK1 or JNK2 knockout mice were ventilated at VT 30 ml/kg for 1 hour. Both JNK1-/- and JNK2-/- knockout mice had reduced phosphorylation of JNK2, but there was little effect on phosphorylation of JNK1, as compared with wild-type JNK+/+ mice (Figure 3) . The degree of JNK1 expression and JNK1 phosphorylation was low in wild-type and knockout mice. This suggested that the JNK pathway may be involved in VILI. To further investigate the JNK pathway, we measured MIP-2 production and neutrophil infiltration in the knockout mice and in mice with inhibition of JNK by a specific JNK inhibitor (SP600125).



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  		Figure 3. High VT ventilation in JNK1 and JNK2 knockout mice. Western blot was performed using the antibody that recognizes the phosphorylated JNK expressions (top panel) and the antibody that recognizes total JNK1 and JNK2 protein expressions in lung tissue (middle panel). Arbitrary units were expressed as relative JNK2 phosphorylation (bottom panel) (n = 3/grop). *p Values less than 0.05 versus control nonventilated mice; {dagger}p values less than 0.05 versus mice ventilated at 6 ml/kg.

 
Inhibition of JNK Activation with SP600126 and JNK Knockout Mice Reduced Lung Stretch–induced MIP-2 mRNA Expression and MIP-2 Production
We pretreated mice for 30 minutes with the specific JNK inhibitor (SP600125) to determine whether inhibiting JNK activation would decrease ventilation-induced MIP-2 mRNA expression and MIP-2 production (24, 25). Ventilation-induced expression of MIP-2 mRNA and production of MIP-2 protein were significantly reduced via pharmacologic inhibition or homozygous disruption of the JNK1 or JNK2 locus (Figures 4A and 4B) . This suggested that expression of MIP-2 mRNA and MIP-2 protein production was dependent, at least in part, on the JNK activation.



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  		Figure 4. SP600126 and JNK knockout mice reduced lung stretch–induced infiltration of neutrophils, MIP-2 mRNA expression, and MIP-2 production. The mice ventilated at VT of 30 ml/kg for 1 hour (A) or 5 hours (B and C) were pretreated with 30 mg/kg SP600126 or equivalent amount of vehicle for 30 minutes. (A) MIP-2 mRNA (top panel), GAPDH mRNA (middle panel), and arbitrary units (bottom panel) (n = 3/group). Arbitrary units were expressed as the ratio of MIP-2 mRNA to GAPDH. The expression of MIP-2 RNA was performed by RNase protection assay. (B) MIP-2 production in BAL fluid (n = 4/group for wild-type mice and n = 3/group for either JNK1-/- or JNK2-/- mice). (C) MPO assay of lung tissue (n = 4/group for wild-type mice and n = 3/group for either JNK1-/- or JNK2-/- mice). *p Values less than 0.05 versus ventilation with SP600125; {dagger}p values less than 0.05 versus ventilation in JNK1-/- or JNK2-/- mice.

 
Inhibition of JNK Activation with SP600126 and JNK Knockout Mice Reduced Infiltration of Neutrophils
We found significantly decreased levels of MPO in mice pretreated with SP600125 with VT 30 ml/kg mechanical ventilation as compared with wild-type mice pretreated with vehicle only (Figure 4C). Similar MPO results were found by using either JNK1-/- or JNK2-/- knockout mice receiving VT 30 ml/kg mechanical ventilation compared with wild-type JNK+/+ mice (Figure 4C).

Using histopathology, we confirmed the MPO results. Neutrophil infiltration was increased in mice with VT 30 ml/kg mechanical ventilation as compared with control nonventilated and VT 6 ml/kg mice. The increase in neutrophil infiltration in wild-type mice with VT 30 ml/kg mechanical ventilation was reduced after either pretreatment with SP600125 or by using JNK1-/- or JNK2-/- knockout mice (Figure 5) . We quantified the histopathology by determining the average number of neutrophils per bronchiole. There were also more cells distributed in the loose connective tissue, including lamina propria and submucosa. Neutrophil infiltration was statistically significantly reduced with both SP600125 pretreated mice and JNK knockout mice but still significantly greater than in the control mice (Figure 6) . There was only partial inhibition as compared with controlled mice, which suggested that the JNK pathway was only one of the many pathways contributing to neutrophil accumulation. Because there was no difference between JNK1-/- and JNK2-/-, the data were combined.



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  		Figure 5. Increase of neutrophil sequestration was associated with mechanical ventilation on histopathology. Representative photomicrographs (x400) with hematoxylin and eosin stain of the lung sections after 5 hours of mechanical ventilation. (A) Control wild-type mice; (B) control JNK1-/- or JNK2-/- mice; (C) VT 6 ml/kg mice; (D) VT 30 ml/kg mice; (E) VT 30 ml/kg mice, pretreated with SP600125; (F) VT 30 ml/kg, JNK1-/- or JNK2-/- mice. Neutrophils are pointed out by arrows.

 


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  		Figure 6. Inhibition of JNK activation with SP600126 and JNK knockout mice reduced infiltration of neutrophils. Neutrophil infiltration was quantified as the average number of neutrophils per bronchiole (n = 3/group). Results of JNK1-/- and JNK2-/- were combined because there was no difference between groups. *p Values less than 0.05 versus control nonventilated mice; {dagger}p values less than 0.05 versus mice ventilated at 30 ml/kg.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Large VTs in patients with acute respiratory distress syndrome lead to increased levels of IL-8 in bronchoalveolar lavage fluid (8). Animal studies have shown that simply overdistending lung tissue, in the absence of any other stimuli, causes production of cytokines and chemokines, but the mechanisms are unclear (2).

The reported pattern of stretch-induced MAPK activation differs depending on the cell type (11–14, 16). In some cells, stretch induces activation of all three MAPKs, whereas in others, only a subset is activated. In addition, the regulation of MIP-2, a functional homolog of human IL-8, by MAPs has differed depending on the stimulus (positive or negative pressure, pressure or volume control mode, duration of ventilation with or without waiting period after ventilation, and ex vivo isolated lungs or in vivo lung model) (4, 9, 10, 17). Using high VT ventilation in rats, Uhlig and coworkers (17) found that there were different magnitudes of JNK and ERK1/2 activation between high and low VT–ventilated rats, but p38 was not activated. In isolated mouse lungs, inhibition of ERK1/2 with U0126 MEK inhibitor did not affect lung stretch–induced cytokine production (17). High VT ventilation did not activate p38 kinase, suggesting that it was regulated differently from JNK and ERK1/2 activation. The p38 MAPK pathway may have contributed to induction of IL-8 synthesis by stabilizing its mRNA (26, 27), but this has not been explored in VILI. In our previous in vitro studies of A549 cells, we also found that stretch-induced IL-8 production was not dependent on p38 and ERK1/2 but was dependent on JNKs (19).

The biological roles of JNKs have been extremely difficult to characterize due to lack of specific pharmacologic inhibitors. Though several investigators (16, 17) have found stretch-induced activation of JNK, the role of JNKs has not been defined. To further confirm the results of our previous in vitro study (19), we employed two different strategies to determine whether ventilation-induced MIP-2 production was dependent on JNK activation. We used homozygous JNK knockout mice (20, 21) and the now available specific anthrapyrazolone JNK inhibitor SP600125, a reversible ATP–competitive inhibitor with greater than 20-fold selectivity versus other kinases in inhibiting the action of phospho-JNK but not the expression of phospho-JNK (24, 25). Using SP600125, we found that high VT ventilation–induced neutrophil infiltration, MIP-2 mRNA expression, and MIP production were dependent on JNK activation. We confirmed our findings with JNK1-/- and JNK2-/- knockout mice (Figures 4–6).

We used both JNK1 and JNK2 knockout mice because the combined knockout is nonviable. The JNK1 and the JNK2 knockout mice develop normally, have no known abnormal pathology, are fertile, are of normal size, and have normal lymphocyte development (20, 21). They have normal lung parenchyma and airways (Figure 5B). JNK-deficient mice displayed higher pulmonary MPO activity at baseline than did wild-type mice but did not have difference in the bronchoalveolar lavage neutrophils. When exposed to hyperoxia, both JNK1 and JNK2 knockout mice have a decreased survival in hyperoxia (28). In these mice, hyperoxia caused an increase in bronchoalveolar lavage protein and increased apopotosis in lung parenchyma compared with those in wild-type mice (28). The production of IL-1ß and IL-6 was not different between wild-type, JNK1, and JNK2 knockout mice at baseline. There was no difference in baseline cytokine production of IL-2, IFN-{gamma}, IL-4, IL-5, and IL-10 between JNK1 knockout and wild-type spleen T cells (28, 29). The lack of JNK1 resulted in reduced c-Jun phosphorylation and resistance to UV-induced cell death, but JNK2-deficient cells showed increased sensitivity to UV irradiation (30). Both JNK1 and JNK2 appeared to negatively regulate apoptosis independent of c-Jun phosphorylation (31).

Both JNK1-/- and JNK2-/- mice had decreased MIP-2 production and neutrophil infiltration, with only a small-but-significant increase of pJNK1 with ventilation in wild-type mice. This appeared to be secondary to a large decrease in ventilation-induced JNK2 activation in both JNK1-/- and JNK2-/- knockout mice (Figure 3). This suggested that lung stretch–induced inflammation was dependent on JNK1 and JNK2 phosphorylation. Previous in vitro study of human bronchial epithelial cells (16) showed early JNK phosphorylation in BEAS-2B cells submitted to cell stretching. Phosphorylation of JNK1 (p46) was detected after 5 minutes of cell stretching, whereas JNK2 (p54) was detected later at 10 minutes. It was thus possible the similar reductions in MIP-2 in both JNK1 and JNK2 knockouts were because JNK1 and JNK2 phosphorylation was needed sequentially for full activation of MIP-2 transcription. The levels of total and phosphorylated JNKs in JNK knockout mice were not completely absent because JNK1 knockout mice have an intact JNK2 gene and vice versa because each gene was capable of making the various isoforms of JNK via alternate splicing (28). Thus, the changes in JNK1 and JNK2 in the knockout mice may have also depended on the degree of alternate splicing.

The role of inflammatory mediators involved in VILI is unclear and even discrepant (32). Tremblay and coworkers demonstrated in ex vivo rat lung that high VT ventilation at 40 ml/kg produced an outpouring of cytokines and chemokines such as MIP-2, tumor necrosis factor-{alpha}, IL-1ß, IL-6, IFN-{gamma}, and IL-10 (3, 33). However, Ricard and coworkers (34) found no increase in MIP-2, tumor necrosis factor-{alpha}, and IL-1ß in their isolated nonperfused lungs model. Recently, an in vivo study comparing low peak-pressure (VT 12 ml/kg) versus high peak-pressure (VT 24 ml/kg) mechanical ventilation on C57BL/6 mice supported the fact that increased expression of MIP-2 mRNA and MIP-2 production are important in the pathogenesis of VILI (10). In our study, high VT mechanical ventilation increased neutrophil sequestration and lung injury (Figure 6) and was associated with increased MIP-2 production. In humans, IL-8, a member of the CXC family of cytokines, is a potent chemotactic factor for recruitment of neutrophils in the human lung (35, 36). Interestingly, no exact homolog of IL-8 has been found in rodents. MIP-2, another member of the CXC family of cytokines, appears to play a related role as a chemoattractant for neutrophils in rodent lungs (37). MIP-2 is produced by alveolar macrophages and binds to the CXCR2 receptor in rodents, which is the homolog of the IL-8 receptor ß in humans (38). The MIP-2 promoter, which controls MIP-2 expression, contains the same promoter regions as IL-8, including activator protein-1 and nuclear factor-{kappa}B (39). JNK1 and JNK2 control activator protein-1 binding. We have previously shown that stretch-induced activator protein-1 binding to the IL-8 promoter was dependent on JNK activation in vitro (19). We have now also shown that in vivo pharmacologic inhibition of JNK activation blocked MIP-2 protein production, and mice deficient in JNK 1 or JNK 2 were resistant to lung stretch–induced MIP-2 production and stretch-induced neutrophil infiltration.

As in our rat model with MIP-2 antibody, blocking MIP-2 production with JNK inhibitor or JNK knockout mice only partially blocked neutrophil infiltration (Figure 6). In our rat model of VILI, blocking MIP-2 production in the alveoli and airways with MIP-2–neutralizing antibody blocked influx of neutrophils into the alveoli but did not block total neutrophil infiltration into the lung as measured by MPO assay (4). These data suggested that other mechanisms besides chemoattraction by MIP-2 were present such as stretch-induced upregulation of intracellular adhesion molecule causing sticking of neutrophils in the vasculature and interstitium (40, 41).

In both humans and animals, lung stretch has been shown to be associated with release of chemokines and influx of neutrophils. Though the Acute Respiratory Distress Syndrome Network trial demonstrated that low volume ventilation is safer than high volume ventilation, these findings have been questioned (42, 43). The NHLBI working group on acute lung injury identified examination of the biology of stress-induced injury to the lung in health and disease as a fertile area of future research because ventilation-induced release of cytokines may lead to systemic translocation and multisystem organ failure (44). We have found that lung cell stretch in vivo, resulted in increased lung neutrophil sequestration and increased MIP-2 production, which was, at least in part, dependent, on the JNK pathway. These data have added to the understanding of the effects of mechanical forces in the lung.


   Acknowledgments
 
L-F.L. has no declared conflict of interest; L.Y. has no declared conflict of interest; D.A.Q. has no declared conflict of interest.

The authors thank Susannah Wood for her generous support and encouragement, Kathie Sweeney Laing for her help in preparation of the manuscript, and Piedro Caironi, John Beagle, and Olga Syrkina for their technical support.


   FOOTNOTES
 
Supported by grants HL039020, HL61688, and HL67371 and Susannah Wood's generous financial assistance.

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

Received in original form May 15, 2003; accepted in final form November 22, 2003


   REFERENCES
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 METHODS
 RESULTS
 DISCUSSION
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