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Published ahead of print on October 24, 2003, doi:10.1164/rccm.200303-343OC
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American Journal of Respiratory and Critical Care Medicine Vol 169. pp. 201-208, (2004)
© 2004 American Thoracic Society

Phosphoinositide 3-OH Kinase Inhibition Prevents Ventilation-induced Lung Cell Activation

Ulrike Uhlig, Heinz Fehrenbach, Robert A. Lachmann, Torsten Goldmann, Burkhard Lachmann, Ekkehard Vollmer and Stefan Uhlig

Research Center Borstel, Borstel; Clinical Research Group "Chronic Airway Diseases," Department of Internal Medicine (Respiratory Medicine), Philipps-University, Marburg, Germany; and Department of Anesthesiology, Erasmus MC Faculty, Rotterdam, The Netherlands

Correspondence and requests for reprints should be addressed to Stefan Uhlig, Ph.D., Division of Pulmonary Pharmacology, Research Center Borstel, Parkallee 22, D-23845 Borstel, Germany. E-mail: suhlig{at}fz-borstel.de


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In acute respiratory distress syndrome patients, protective ventilation strategies reduce mortality and proinflammatory mediator levels. It has been suggested that some of the side effects of mechanical ventilation are caused by the excessive release of mediators capable of causing pulmonary inflammation and tissue destruction (biotrauma). Selective inhibition of this process might be used to minimize the side effects of artificial mechanical ventilation. This study was designed to identify the cell types and specific signaling mechanisms that are activated by ventilation with increased pressure/volume (overventilation). In isolated perfused mouse lungs, overventilation caused nuclear translocation of nuclear factor-{kappa}B (NF-{kappa}B) and enhanced expression of interleukin-6 mRNA in alveolar macrophages and alveolar epithelial type II cells. The phosphoinositide 3-OH kinase inhibitor Ly294002 prevented nuclear translocation of NF-{kappa}B and the subsequent release of interleukin-6 and macrophage inflammatory protein–2{alpha} in overventilated but not in endotoxic lungs. Similar results were obtained in rats in vivo, where Ly294002 prevented NF-{kappa}B activation by overventilation but not by endotoxin. These findings show that alveolar macrophages and alveolar epithelial type II cells contribute to the ventilation-induced release of proinflammatory mediators and that selective inhibition of this process is possible without inhibiting the activation of NF-{kappa}B by endotoxin.

Key Words: ventilation-induced lung injury • overventilation • biotrauma nuclear factor-{kappa}B • I-{kappa}Bß

Clinical studies have shown that ventilation with conventionally used high tidal volumes and pressures increases mortality, proinflammatory mediator release, and pulmonary inflammation in acute respiratory distress syndrome patients (1, 2). Although the correlation between mortality, ventilation, and markers of inflammation in these studies does not prove a causal relationship between them, the biotrauma hypothesis is supported by an increasing number of experimental and clinical data (3–5): Stretching alveolar epithelial type II cells or alveolar macrophages in culture triggers mediator release (6, 7). In isolated lungs, ventilation with increased volumes or pressures, termed overventilation (8), elicits local (9) and systemic (10, 11) concentrations of proinflammatory mediators to an extent that is comparable to that achieved by bacterial endotoxins (8). Protective ventilation strategies increased survival and reduced mediator release in animal models of acute lung injury, for example, after lung lavage (12–14) or acid instillation (15–17). It was also demonstrated that the chemokine macrophage inflammatory protein (MIP)-2{alpha} and its receptor (CXCR2) are critical mediators of ventilation-induced lung injury (18). In acute respiratory distress syndrome patients, Ranieri and colleagues noted a close correlation between ventilation-associated release of interleukin (IL)-6 and multiorgan failure (19). Most recently, it was shown that ventilation of patients with increased pressures leads to an elevation in proinflammatory mediators, including IL-6, that was reversible if the ventilation pressures were reduced (20). This last finding establishes a causal link between ventilation and rapid cytokine release in patients.

Key questions in this area concern the cell types and the signaling cascades that become activated by ventilation. Experiments with stretched alveolar macrophages (6), intact mouse lungs (8), and healthy rat lungs (21) have led to the conclusion that a central event in the signaling cascade elicited by overventilation (OV) is activation, that is, nuclear translocation of the transcription factor nuclear factor-{kappa}B (NF-{kappa}B). Overventilation activated NF-{kappa}B also in lipopolysaccharide (LPS)-resistant toll-like receptor 4–deficient mice, demonstrating that different signaling pathways lead to activation of NF-{kappa}B by ventilation and by LPS (8). As pointed out (22), this suggests that it may be possible to target selectively the proinflammatory side effects of mechanical ventilation without interfering with the organisms' ability to respond to bacterial infections.

Phosphoinositide 3-OH kinases (PI3Ks) are a family of ubiquitous heterodimeric lipid-modifying enzymes divided into three classes (23). PI3Ks play an important role in mitogenic signaling and cell survival, cytoskeletal remodeling, metabolic control, and vesicular trafficking (23). Several in vitro studies have established a link between PI3K and activation of NF-{kappa}B. These studies can be divided into those supporting the sequence PI3K -> Akt (protein kinase B) -> NF-{kappa}B (24–30) and those that show activation of NF-{kappa}B by PI3K by pathways that apparently are independent of Akt (31–33).

Here, we investigated the hypothesis that PI3K contributes to the activation of NF-{kappa}B and release of IL-6 and MIP-2{alpha} during ventilation with high-ventilation pressures (overventilation) but not LPS exposure. We focused on IL-6 and MIP-2{alpha} because both mediators are released in great quantities by overventilation (8) and have been shown to be relevant in clinical (1, 19, 20) respectively experimental (18) conditions of ventilator-associated lung injury. In addition, we used immunohistochemistry and in situ hybridization to identify the cell types in which NF-{kappa}B and IL-6 become activated by overventilation. We should emphasize that this study was not designed to study acute lung injury directly, but to characterize the signaling pathways that lead to inflammatory gene activation during ventilation. Although we did not study lung injury itself, there is clear evidence that proinflammatory mediators such as MIP-2{alpha} play an essential role in biotrauma (18). Hence, understanding the molecular mechanisms of ventilation-induced cell activation and mediator release will help to develop strategies to minimize the side effects of mechanical ventilation.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Isolated Perfused Mouse Lung Preparation
The mouse lungs were prepared and perfused as described (8, 10, 11, 34). In this study, we used negative pressure ventilation because the low perfusion pressures that result from negative pressure ventilation reduce vascular shear stress (35) and help to minimize hydrostatic edema formation. Ventilation was always pressure controlled. In all experiments, the lungs were first perfused and ventilated for 60 minutes under baseline conditions with an end-inspiratory pressure of -10 cm H2O and an end-expiratory pressure of -3 cm H2O, resulting in tidal volumes of approximately 200 µL (Figure 1) . Subsequently, the lungs were randomly allocated to one of the following five groups and were perfused and ventilated for another 180 minutes: group 1 with a low end-inspiratory pressure of -10 cm H2O (control); group 2 with a high distending pressure of -25 cm H2O (OV); group 3 with OV (as in group 2) and pretreatment with 50 µM Ly294002 (OV/Ly) from 30 minutes before OV on; group 4 with a low end-inspiratory pressure of -10 cm H2O and 50 µg/ml LPS; group 5 with LPS (as in group 4) and pretreatment with 50 µM Ly294002 (LPS/Ly) from 30 minutes before OV on. Some of the experiments were interrupted after 60 minutes of OV or LPS treatment for electromobility shift assay, Western blots, immunohistochemistry, or in situ hybridization.



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  		Figure 1. Tidal volume of isolated perfused lungs ventilated with an end-inspiratory pressure of either -10 cm H2O (squares, n = 3) or -25 cm H2O (circles, n = 7). Filled circles indicate lungs that were ventilated with -25 cm H2O end-inspiratory pressure and treated with 50 µM of Ly294002 (n = 4). Data are mean ± SEM.

 
In Vivo Studies in Rats
Animals were anesthetized with 65% NO2/ 35% O2 and 2% isoflurane, and a sterile metal cannula was inserted into the trachea as described before (21). After these surgical procedures, gaseous anesthesia was discontinued, and anesthesia was continued with 60 mg/kg of pentobarbital sodium intraperitoneally. Subsequently, muscle relaxation was induced by pancuronium bromide 2 mg/kg intramuscularly, and ventilation was initiated in a pressure-constant time-cycled mode and at an FIO2 of 1.0. Rats were mechanically ventilated with normal (13/3 positive inspiratory pressure/positive end-expiratory pressure in cm H2O) or with high pressure (45/10) for 30 minutes, leading to tidal volumes of approximately 12 and 18 ml/kg (36), respectively. At the end of the study period, the animals were killed with an overdose of pentobarbital. The thorax was opened, and lungs were collected sterile, snap frozen, and stored at -80°C.

The animals were randomly allocated into five experimental groups of six animals each: (1) nonventilated untreated control subjects, (2) injection of the solvent (0.4 mg/kg dimethyl sulfoxide, intravenously) 10 minutes before ventilation with 45/10 cm H2O for 30 minutes, (3) injection of Ly294002 (1.4 mg/kg, intravenously) 10 minutes before ventilation with 45/10 cm H2O for 30 minutes of ventilation, (4) injection of the solvent (0.4 mg/kg dimethyl sulfoxide, intravenously) 10 minutes before injection of LPS (8 mg/kg) and ventilation with 13/3 cm H2O for 30 minutes, and (5) injection of Ly294002 (1.4 mg/kg, intravenously) 10 minutes before injection of LPS (8 mg/kg) and ventilation with 13/3 cm H2O for 30 minutes.

Measurements
Electromobility shift assay for the detection of NF-{kappa}B (8) and Western Blot (21, 37) analysis was performed as described before. Perfusate concentrations of IL-6 and MIP-2{alpha} were determined by ELISA.

Immunohistochemistry and In Situ Hybridization
For immunohistochemistry of NF-{kappa}B and inhibitor of NF-{kappa}B (I-{kappa}Bß) the perfused lungs were fixated with paraformaldehyde in phosphate-buffered saline (38–41). Hepes-glutamic acid buffer–mediated organic solvent protection effect (HOPE)–fixed, paraffin-embedded specimens for the IL-6 in situ hybridization were prepared as described (42–44).

Statistics
The data are shown as mean ± SEM and analyzed by repeated measurement analysis of variance Kruskal-Wallis or the Mann-Whitney test where appropriate. Multiple comparisons were adjusted by the Shaffer procedure (45).


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Tidal Volume
Initially, all lungs were perfused for 60 minutes with 1 ml/minute and ventilated with -10/-3 cm H2O end-inspiratory pressure/end-expiratory pressure (control conditions). To exclude binding of Ly294002 to proteins in the perfusate, subsequently all lungs were perfused with medium devoid of albumin. The lack of serum did not affect tidal volume (Figure 1), pulmonary compliance, vascular resistance, or histologic appearance of the lungs (data not shown). After 60 minutes, either control ventilation was continued or overventilation (-25/-3 cm H2O of end-inspiratory pressure/end-expiratory pressure) was started. Overventilation more than doubled tidal volume, and this response was not affected by the PI3K inhibitor Ly294002 (Figure 1). The tidal volume of lungs perfused with LPS or with Ly294002/LPS was not different from control subjects (data not shown) as shown before. These findings suggest that Ly294002 was well tolerated and had no adverse effects on lung physiology. In line with this, the PO2 or PCO2 levels of the perfusate medium were not different whether animals were overventilated in the presence or absence of Ly294002 (see Figure E1 in the online supplement).

Kinase Activation
The serine kinase Akt is known to be activated by PI3K, and thus, phosphorylation of Akt may serve as an indirect evidence for activation of PI3K. Overventilation caused activation of Akt in the lung homogenate, whereas the effect of LPS was much weaker (Figure 2) . The PI3K inhibitor Ly294002 abolished the effect of overventilation on Akt (Figure 2). To exclude an effect of Ly294002 on other kinases such as mitogen-activated protein kinase, we investigated the effects of Ly294002 on phosphorylation of the mitogen-activated protein kinases Erk 1/2 and p38 (Figure 2). Overventilation for 60 minutes increased phosphorylation of Erk 1/2, which was only slightly affected by Ly294002 (compared with dimethyl sulfoxide control). In contrast, the MEK-inhibitor U0126 reduced Erk 1/2 phosphorylation below baseline levels. Overventilation for 60 minutes had no effect on phosphorylation of p38 kinase. Neither Ly294002 nor U0126 affected the basal phosphorylation of p38.



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  		Figure 2. Effect of Ly294002 (Ly) and U0126 on phosphorylation of Erk 1/2, p38, and Akt in isolated perfused mouse lungs. P-Erk, P-p38 (both upper panel), and P-Akt (lower panel) were analyzed by immunoblot using antibodies specific for the phosphorylated forms. Shown are data from lungs ventilated for 60 minutes with -10/-3 cm H2O end-inspiratory pressure/end-expiratory pressure (C), -25/-3 cm H2O of end-inspiratory pressure/end-expiratory pressure (overventilation [OV]), or 50 µg/ml of lipopolysaccharide (LPS) in the presence of 50 µM of Ly294002 (Ly), 0.05% dimethyl sulfoxide (DMSO), or 20 µM of U0126. Similar results were obtained in three independent experiments. DMSO alone had no effect on the phosphorylation of Akt (data not shown).

 
NF-{kappa}B
We next investigated whether PI3K contributes to activation of NF-{kappa}B. Pretreatment with Ly294002 prevented the nuclear translocation of NF-{kappa}B induced by OV, but not by LPS (Figure 3) . To establish the physiologic relevance of these findings, we examined the effect of Ly294002 on activation of NF-{kappa}B in rats in vivo. Both overventilation and LPS induced NF-{kappa}B in rats in vivo (Figure 4) , although here LPS was clearly more potent, as indicated by the fact that to get a reliable signal in the electromobility shift assay we had to load the gels with 40 µg of protein in the case of OV, but only 10 µg in the case of LPS. Intravenous injection of Ly294002 largely prevented NF-{kappa}B activation induced by overventilation, but not by endotoxin (Figure 4).



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  		Figure 3. Differential inhibition of nuclear translocation of nuclear factor-{kappa}B (NF-{kappa}B) by inhibition of PI3 kinase in mouse lungs. Lungs were ventilated under control conditions for 60 minutes before they were ventilated with -10/-3 cm H2O end-inspiratory pressure/end-expiratory pressure in the absence (Cont) or presence (LPS) of 50 µg/ml of LPS or with -25/-3 cm H2O of end-inspiratory pressure/end-expiratory pressure (overventilation [OV]) for another 60 minutes. Some of the OV- or LPS-treated lungs were treated with 50 µM of Ly294002 (Ly). NF-{kappa}B translocation was determined by electromobility shift assay; each gel slot was loaded with 10 µg of protein. The rightmost band (comp) shows competition of band 4 (LPS) in the presence of unlabeled oligonucleotides. Similar results were obtained in three independent experiments.

 


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  		Figure 4. Differential inhibition of nuclear translocation of NF-{kappa}B by inhibition of PI3 kinase in rats in vivo. A and C show representative electromobility shift assay images. B and D show the densitometric quantification of these electromobility shift assays. A and B show data from rats ventilated for 30 minutes with 45/10 cm H2O (OV) with and without pretreatment with 1.4 mg/kg of Ly294002. Each gel slot was loaded with 40 µg of protein. C and D show data from rats ventilated for 30 minutes with 13/3 cm H2O and treated with 8 mg/kg of LPS with and without pretreatment with 1.4 mg/kg of Ly294002. Each gel slot was loaded with 10 µg of protein. Comp = cold competition of band OV in A; cont = unventilated untreated control subjects. Data are mean ± SEM from five or six animals per group. *p < 0.05 versus cont; §p < 0.05 versus OV.

 
To identify the cell type in which OV activates NF-{kappa}B, we used indirect immunohistochemistry with antibodies against p65 and I-{kappa}Bß . These antibodies were selected because overventilation elicited nuclear translocation of the p65 (also p50) NF-{kappa}B subunit (see Figure E2 in the online supplement) and because PI3K activity is known to lead only to a transient decrease in I-{kappa}B{alpha} but a sustained one in I-{kappa}Bß (46). In control lungs, immunoreactivity for NF-{kappa}B p65 was almost completely restricted to the cytoplasm of parenchymal cells (Figure 5A and see Figure E3 in the online supplement). In all groups, nuclear localization of NF-{kappa}B p65 was regularly seen in endothelial cells of small venules only and in very few bronchiolar epithelial cells (data not shown). In contrast, overventilation resulted in a marked increase in parenchymal cells exhibiting nuclear localization of NF-{kappa}B p65 (Figure 5B and Figures E3B and E3E). Translocation of NF-{kappa}B p65 was mainly but not exclusively seen in alveolar macrophages and alveolar epithelial type II cells. Nuclear localization of NF-{kappa}B p65 was clearly decreased, albeit not to control levels, in overventilated lungs treated with the PI3K inhibitor Ly294002 (Figure 5C and Figures E3C and E3F).



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  		Figure 5. Indirect immunohistochemical localization of NF-{kappa}B p65 (A–C) and the NF-{kappa}B inhibitor I-{kappa}Bß (D–F) in isolated perfused mouse lungs. Shown are high-power micrographs from lungs that were ventilated with either -3/-10 cm H2O end-expiratory pressure/end-inspiratory pressure (control, A and D) or with -3/-25 cm H2O end-expiratory pressure/end-inspiratory pressure (overventilation, B and E) and overventilated lungs, which were treated with the PI3-kinase inhibitor Ly294002 (C and F). Cell types that after overventilation exhibited translocation of NF-{kappa}B p65 into the nucleus (B) and loss of cytoplasmic I-{kappa}Bß (E) were identified as alveolar macro phages (arrows) and alveolar epithelial type II cells (arrowheads). The micrographs are representative of three independent experiments each. Bar = 25µm.

 
Correspondingly, immunoreactivity for the NF-{kappa}B inhibitor I-{kappa}Bß was exclusively seen in the cytoplasm of bronchiolar epithelial cells, alveolar macrophages, and alveolar epithelial cells type II in control lungs. With the exception of bronchiolar epithelial cells, immunoreactivity for I-{kappa}Bß was completely lost in parenchymal cells after overventilation, which was in part prevented by Ly294002 (Figures 5D–5F and Figures E3G–E3I).

IL-6 Expression
To demonstrate that the activation of NF-{kappa}B in alveolar macrophages and alveolar epithelial cells type II corresponded to increased gene expression, we analyzed the expression of IL-6 mRNA in these cells (Figure 6) . After 60 minutes of overventilation, IL-6 mRNA was increased in alveolar macrophages and alveolar epithelial type II cells, and again, this response was prevented by Ly294002.



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  		Figure 6. In situ hybridization for interleukin (IL)-6 mRNA in isolated perfused mouse lungs. Shown are micrographs from lungs that were ventilated with either -3/-10 cm H2O end-expiratory pressure/end-inspiratory pressure (control, A) or -3/-25 cm H2O of end-expiratory pressure/end-inspiratory pressure (B and C) and overventilated lungs, which were treated with the PI3-kinase inhibitor Ly294002 (D). Cell types that after overventilation exhibited activation of IL-6 were identified as alveolar macrophages (arrows) and alveolar epithelial type II cells (arrowheads). The micrographs are representative of three independent experiments. Primary magnification x400, except B (x800).

 
Cytokine Release
In line with its effects on NF-{kappa}B activation and IL-6 mRNA expression, treatment with Ly294002 prevented the ventilation-induced release of IL-6 and attenuated that of MIP-2{alpha}. In contrast, Ly294002 had no effect on the release of these mediators if they were induced by LPS (Figure 7) . Please note that because of the lack of albumin in the perfusate buffer, the mediator levels were lower than previously reported (8).



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  		Figure 7. Differential inhibition of OV- and LPS-induced mediator production by inhibition of PI3K in isolated perfused mouse lungs. OV (overventilation, left) and LPS (right)-induced alterations in IL-6 and macrophage inflammatory protein (MIP)-2{alpha} perfusate levels. Lungs were perfused for 60 minutes under control conditions (open circles, n = 3) before they were exposed to either overventilation (n = 5, closed circles, A and C) or 50 µg/ml of LPS (n = 3, closed circles, B and D); 50 µM of Ly294002 were added 30 minutes before exposure to either OV (n = 3, closed squares, A and C) or LPS (n = 4, closed squares, B and D). IL-6 (A and B) and MIP-2{alpha} (C and D) levels in the perfusate were assessed every 30 minutes. Data are mean ± SEM. (A–D) Mediator release was significantly (p < 0.05) higher in overventilated or LPS-treated lungs compared with control perfused lungs. Pretreatment with Ly294002 significantly (p < 0.05) reduced IL-6 and MIP-2{alpha} concentrations in overventilated but not in LPS-treated lungs.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Our previous finding that overventilation and endotoxin promote proinflammatory responses by different pathways that converge at the level of NF-{kappa}B suggested that it may be possible to block biotrauma without affecting innate immunity (8, 22). This study shows that selective blockade of ventilation-induced mediator-release is indeed feasible, as shown by the differential effect of a PI3 kinase inhibitor on NF-{kappa}B activation and mediator release triggered by OV and LPS, respectively, although for therapeutic application, PI3 kinase inhibitors may have too many side effects (discussed later here). Another key question concerns the cell types that are activated by overventilation. Here, we identify alveolar macrophages and alveolar type II cells as the major cell types in which NF-{kappa}B is activated by overventilation.

Ventilation-induced mediator release can result from stress failure of cell membranes, stress failure of cell contacts, increased vascular shear stress, or mechanotransduction processes triggered by stretch (35). The isolated perfused mouse lung model permits to study relative selectively the latter mechanism in the whole intact organ because increased vascular shear stress and stress failure are largely excluded by ventilation with negative pressure and end-expiratory pressure, respectively (35). It should be noted that in isolated blood-free perfused mouse lungs the mode of overventilation applied does not cause acute lung injury, as none of the typical pathologic changes such as edema or neutrophil influx occurs (10, 11, 47). We therefore have speculated that the decrease in tidal volume is a result of surfactant exhaustion or airway derecruitment (10, 11). The strength of the isolated perfused mouse lung is that it provides a well-defined model to study the signaling events in intact overdistended alveoli, akin to what may happen in the ventilated and healthy parts of lungs with inhomogeneous lung injury (5, 35). Many of the findings in perfused mouse lungs (8, 21) are reproduced in whole animals in vivo, as illustrated by the ventilation-induced release of MIP-2{alpha} and KC in mice (18), the activation of NF-{kappa}B and MAP kinases in rats (21), and the effect of Ly294002 on NF-{kappa}B activation shown in this study (which was measured after 30 minutes and thus too early for inflammation to occur).

Cells respond to external physical forces such as stretch, shear stress, or ultraviolet light by well-defined intracellular responses that include stress activated protein kinases, ion fluxes, NF-{kappa}B, and—as recently recognized—also PI3K. Activation of PI3K was demonstrated in endothelial cells by shear stress (48, 49) and in cardiac myocytes by stretch (50). The present findings together with an accompanying study (37) identify ventilation with high distending volumes/pressures as another physical force that activates PI3K. This was demonstrated by phosphorylation of Akt and by the effects of Ly294002 on activation of Akt, NF-{kappa}B, and mediator release. Ly294002 is a highly specific competitive inhibitor of the PI3K, binding to the ATP-binding site (51). Further evidence for the specificity of Ly294002 in our model is provided by its lack of effect on phosphorylation of other kinases such as Erk 1/2 and p38 and by the fact that it had no effect on LPS-induced NF-{kappa}B activation or mediator release, a process that involves another set of kinases.

A large body of evidence now supports the concept that ventilation with high distending pressures triggers proinflammatory responses in the lung. However, the cell types that become activated to secrete those proinflammatory mediators have not been identified. Recently, we showed that overventilation elicits a PI3K-sensitive activation of Akt in pulmonary endothelial cells leading to production of nitric oxide (37). This pattern of activation is clearly different from the staining pattern for overventilation-induced translocation of the NF-{kappa}B p65 subunit, degradation of I-{kappa}Bß, and enhanced expression of IL-6 mRNA, all of which occurred predominantly in alveolar type II epithelial cells and alveolar macrophages (Figures 5 and 6 and Figure E3). This observation is in line with previous cell culture studies showing stretch-dependent release of IL-8 from both cell types (6, 7). In addition, it was recently shown that ventilation with high pressures activated extracellular matrix metalloproteinase inducer, gelatinase A and gelatinase B in endothelial cells, alveolar type II epithelial cells, and alveolar macrophages (52). All of these findings suggest that in the whole organ both alveolar type II epithelial cells and alveolar macrophages contribute to the release of proinflammatory mediators elicited by overventilation. How these cells sense increased stretch (or more unlikely pressure) is still elusive, and thus, we cannot exclude the possibility that alveolar macrophages and alveolar type II epithelial cells are activated only indirectly by products released from other cells or maybe even from nerve endings (53).

The mammalian Rel/NF-{kappa}B transcription factor family is comprised of five homologous polypeptides: p50, p65, c-Rel, RelB, and p52. These subunits associate in a combinatorial fashion to form transcriptionally active homodimers and heterodimers. The most prevalent and well-characterized species of NF-{kappa}B dimer is the p50/p65 heterodimer. Therefore, it is not surprising that the p50/p65 dimer is also active in overventilated lungs (Figure 5 and Figures E2 and E3). Nuclear translocation of the p50/p65 dimer is prevented by a family of transcription factor inhibitors, most important among them being I-{kappa}B{alpha} and I-{kappa}Bß. Phosphorylation of these inhibitors triggers their degradation and frees NF-{kappa}B to translocate to the nucleus. The present view is that I-{kappa}B{alpha} regulates transient and I-{kappa}Bß persistent NF-{kappa}B activation (54). Therefore, degradation of I-{kappa}Bß may be a critical event for the persistent NF-{kappa}B activation observed in many disease states (54). Unfortunately, the molecular details of the regulation of I-{kappa}Bß are only poorly defined.

Expression of I-{kappa}Bß was diminished in alveolar cells of overventilated lungs and was restored by inhibition of PI3K, suggesting that PI3K leads to phosphorylation and degradation of I-{kappa}B proteins. This is in line with other studies showing either activation of PI3K (50) or degradation of I-{kappa}Bß (55) by stretch in culture. Currently, the mechanism linking PI3K to I-{kappa}Bß/NF-{kappa}B during overventilation is unknown, although a number of recent studies have demonstrated a link between PI3K and I-{kappa}Bß/NF-{kappa}B under a variety of different conditions (discussed previously in this article). The major pathways described involve direct effects on I-{kappa}B or NF-{kappa}B (33) or mediation by Akt or protein kinase C isoenzymes. The different spatial activation of Akt in endothelial cells (37) and NF-{kappa}B in alveolar macrophages and type II cells (Figure 5 and Figure E3 in the online supplement) suggests that Akt is not involved in the activation of NF-{kappa}B by overventilation. However, because immunohistochemistry is a semiquantitative method, we cannot completely exclude that limited activation of Akt may also occur in lung cells other than endothelial cells. An attractive alternative mechanism how PI3K may activate NF-{kappa}B is activation of Ca2+-independent or atypical protein kinase C isoforms by PtdIns(3,4,5)P3 (56–60). For instance, protein kinase C-{zeta}, a target of PI3K (61, 62), can regulate NF-{kappa}B through activation of I-{kappa}B kinases (63, 64) or phosphorylation of the p65 subunit of NF-{kappa}B (60, 62, 65).

Of particular interest is our observation that inhibition of PI3K did not prevent the NF-{kappa}B activation and the cytokine release triggered by LPS. This is in line with previous studies in rat primary astrocytes (66), although controversial findings have been reported (67). Controversial results were also obtained with respect to LPS-induced activation of Akt in macrophages, showing phosphorylation in isolated human alveolar macrophages (68), but not the macrophage cell line RAW 264.7 (69). Thus, currently, the role of PI3K and Akt during LPS stimulation in macrophages and type II cells is not well understood. However, given the redundancy of pathways activated by LPS (70), the failure of PI3K kinase inhibitors to affect LPS-induced NF-{kappa}B activation may not be surprising.

Differing mechanisms of NF-{kappa}B activation by LPS and OV were also suggested in our previous study, where in TLR-4–deficient mice NF-{kappa}B was activated by overventilation, but not by LPS (8). In contrast to steroids, which block both LPS- and ventilation-induced NF-{kappa}B activation and mediator release (8), PI3K inhibition was selective for ventilation, suggesting that it may be possible to reduce some of the side effects of ventilation without causing severe immune suppression. However, the variety of cellular functions that are controlled by the PI3K pathway, including neutrophil activation (71), cell proliferation (72), or insulin receptor signaling (72), let it appear likely that blocking of this pathway might have marked side effects. Therefore, in future studies, it will be important to establish the missing link between PI3K and NF-{kappa}B to define a more selective target. Alternatively, local (tracheal) administration of PI3K inhibitors might be considered as a way to reduce the side effects of PI3K inhibitors.

In summary, we have shown that overventilation triggers activation of NF-{kappa}B in alveolar macrophages and alveolar epithelial type II cells. In line with its effect on NF-{kappa}B and IL-6 mRNA expression, the PI3K inhibitor Ly294002 attenuated the ventilation-induced release of IL-6 and MIP-2{alpha}, suggesting alveolar macrophages and epithelial type II cells as sources of MIP-2{alpha} and IL-6. The failure of Ly294002 to block LPS-induced responses suggests that it may be possible to target selectively the side effects of ventilation related to the release of proinflammatory mediators.


   Acknowledgments
 
The authors thank Dörte Karp and Heike Kühl for excellent technical assistance.


   FOOTNOTES
 
Supported by grants DFG Uh 88/2-4 and Uh 88/4-1 from the Deutsche Forschungsgemeinschaft.

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: U.U. has no declared conflict of interest; H.F. has no declared conflict of interest; R.A.L. has no declared conflict of interest; T.G. has no declared conflict of interest; B.L. has no declared conflict of interest; E.V. has no declared conflict of interest; S.U. has no declared conflict of interest.

Received in original form March 8, 2003; accepted in final form October 20, 2003


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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