| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
Departments of 1Physiology, 2Pathology, 3Microbiology, and 4Pediatrics, College of Medicine, University of South Alabama, Mobile, Alabama 36688
Submitted 25 March 2004 ; accepted in final form 13 July 2004
 |
ABSTRACT |
|---|
 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
, interleukin (IL)-1
, IL-1, macrophage inflammatory protein (MIP)-2, and IL-6 concentrations in perfusate were markedly increased in perfusate at 2 and 4 h, but only MIP-2 was detectable in intact Balb/c mice using the same PIP. In intact wild-type and TNF dual-receptor null mice with ventilation at 45 cmH2O PIP, the MIP-2 and IL-6 levels in BAL were significantly increased after 2 h in both groups, but there were no differences between groups in the BAL albumin and cytokine concentrations or in lung wet-to-dry weight ratios. TNF- was not be detected in BAL fluids in any group of intact mice. These results suggest that the alveolar hyperpermeability induced by high PIP ventilation occurs very rapidly and is initially independent of TNF- participation and unlikely to depend on MIP-2 or IL-6.
ventilator induced lung injury; tumor necrosis factor-; major intrinsic protein-2; interleukin-6; knockout mice
40–50% of cases (50). Recently, ventilator-induced lung injury (VILI) has been recognized as a significant contributing factor to the morbidity and mortality of patients with ARDS because a large-scale clinical trial using a reduced tidal volume improved survival by 22% (4). Numerous investigators have observed that high airway pressures and lung volumes can increase the permeability of pulmonary endothelial and epithelial barriers as well as increase proinflammatory cytokine production by the lung (7, 10, 28, 41). However, the relationship of the increased lung vascular permeability associated with VILI and ARDS to the proinflammatory cytokines remains controversial (9). Tremblay et al. (45) reported a marked increase in lung lavage concentrations of tumor necrosis factor (TNF)-, interleukin (IL)-1, IL-6, macrophage inflammatory protein (MIP)-2, interferon-
, and IL-10 in excised, unperfused rat lungs ventilated with high peak inflation pressures (PIP). This study and others have led some investigators to speculate a causal role for acute response proinflammatory cytokines such as TNF- in the lung permeability lesion (5, 21, 27, 43). Such a role has been disputed based on the rapid increase in lung vascular permeability and often variable cytokine response to VILI (9–11, 38, 39). Still other investigators have suggested separate pathways for mechanical injury and the inflammatory response (19, 47). A major source of difficulty in interpreting the interactions of lung permeability and cytokine production is the wide range of experimental models, species, times, and pressures used to produce VILI.
In the present study, we assessed vascular permeability and cytokine production in both isolated perfused mouse lungs and intact mice using Balb/c and B6/129 mouse strains at high and low PIP. In addition, we also ventilated TNF- dual-receptor (p55 and p75) knockout mice with high PIP to ablate the action of TNF- receptor binding on the measured variables. Acute changes in vascular permeability were evaluated in isolated perfused lungs using the capillary filtration coefficient (Kfc) and the leak of plasma albumin into the air spaces in intact mice. We measured TNF-, IL-1, IL-6, and MIP-2 in the bronchoalveolar lavage (BAL) fluid (BALF) and serum of intact mice and the perfusate of isolated mouse lungs using ELISAs. The rapid onset of increased vascular permeability in different models and species, the delayed, variable, or absent increases in cytokine production and lack of influence of TNF- receptors suggest that the permeability lesion precedes and may contribute to the inflammatory response.
|
METHODS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
7 cmH2O at a respiratory rate of 85 breaths/min, with a positive end-expiratory pressure (PEEP) of 3 cmH2O. The chest was opened by a midline incision, heparin (100 IU) was injected into the right ventricle, and sutures were placed around the pulmonary artery and aorta. Cannulas were placed in the pulmonary artery and left atrium, and pulmonary arterial, venous, and airway pressures were continuously monitored using pressure transducers (Cobe, Lakewood, CO) zeroed at the mid-lung level with pressures recorded on a Grass model 7D polygraph. The initial 1–2 ml of perfusate, which contained residual blood cells and plasma, were discarded and not recirculated. Lungs were then perfused in situ with 4% bovine albumin in RPMI 1640 cell culture medium using a roller pump (Minipuls 2, Gilson, Middleton, WI) at a flow rate of 0.5 ml/min in a recirculating system that had a nominal system volume of 7 ml. Temperature was maintained at 37°C using a heating pad and heat lamp. These animal protocols were approved by the College of Medicine Animal Care and Use Committee and met all National Institutes of Health guidelines for care and handling of animals.
Kfc.
Isolated perfused Balb/c mouse lungs were used to measure Kfc in milliliters per minute per cmH2O per 100 g, which is a sensitive measure of endothelial hydraulic conductivity when capillary surface area is maintained constant (33). After an isogravimetric state was attained, pulmonary venous pressure was increased by 6 cmH2O for 20 min, and the change in capillary pressure (dPpc) was determined by double occlusion before and after the pulmonary venous pressure increase. The rate of lung weight gain (dW/dt) between 18 and 20 min was used to calculate Kfc using
 | (1) |
 | (2) |
Isolated lung protocols. Kfc measurements were performed in two groups of Balb/c mice (n = 5). After a baseline isogravimetric state was obtained during ventilation at 85 breaths/min with a PIP of 10 cmH2O, a Kfc measurement was performed. Mouse lungs were then ventilated for 20 min with either a PIP of 10 cmH2O (low-PIP group) or a PIP of 25–28 cmH2O (high-PIP group). In the high-PIP group, ventilation rate was reduced to 65 breaths/min to prevent automatic PEEP.
The time course for cytokine production was also measured in isolated perfused lungs of Balb/c mice. After a baseline state was attained, the mice were separated into two groups of seven mice each: a high-PIP group where PIP was increased to 25–26 cmH2O and a low-PIP group where PIP was maintained at the baseline level of 7–8 cmH2O. Lungs were perfused for 4 h, and 0.5-ml perfusate samples were obtained from the venous reservoir every hour in the high-PIP group and every 2 h in the low-PIP group. Lungs were weighed at the end of the experiments.
Intact animal preparations.
All intact animal ventilation protocols were also approved by the Institutional Animal Care and Use Committee of University of South Alabama. Specific pathogen-free Balb/c, B6/129J, and TNF- receptor knockout (TNFRKO) mice (p55–/–, p75–/–) were purchased from Jackson Laboratory (Bar Harbor, ME). Mice were anesthetized by intraperitoneal injection of pentobarbital sodium (65 mg/kg). A tracheostomy was performed, and the cannula was inserted into the trachea. Mice were ventilated by use of a rodent ventilator (model 683, Harvard) with various PIP. Anesthesia was maintained by intermittent intraperitoneal injection of pentobarbital sodium. Airway pressure was measured using a Cobe pressure transducer (Lakewood, CO), and electrocardiograph was monitored using a polygraph (model 70, Grass).
Intact animal protocols. The Balb/c mice were divided into three groups (n = 5): 30-min and 2-h high-PIP groups, and a time 0 nonventilated group. The high-PIP groups were ventilated with PIP values between 25 and 26 cmH2O and 2.5 cmH2O PEEP to obtain pressures comparable to the airway pressure used in the isolated perfused lung experiments. The mice in the time 0 group were ventilated at a PIP of 7 cmH2O and 2.5 cmH2O PEEP at a rate of 120 breaths/min for a brief period sufficient for blood sampling.
B6/129 and TNFRKO mice were divided into groups (n = 5) of each strain according to ventilation strategy: a high-PIP group, a low-PIP group, and a nonventilated group. The high-PIP group was ventilated with a PIP of 30 cmH2O and 2.5 cmH2O PEEP at 20 breaths/min for up to 2 h. The low-PIP group was ventilated with PIP of 7 cmH2O and 2.5 cmH2O PEEP at a rate of 120 breaths/min for 2 h. At the end of ventilation, a thoracotomy was performed and blood was sampled by cardiac puncture for gas analysis and measurement of cytokines and albumin. To examine the time course of the effect of high PIP ventilation on cytokine concentrations in BALFs, we ventilated separate groups with high PIP for 30 min and 1 h.
Arterial blood gases. After the ventilation period, blood was sampled by cardiac puncture for gas analysis using a Radiometer ABL 5 blood-gas machine. Respiratory rates were adjusted to maintain blood gases constant at high and low PIP values, as previously described.
BAL.
Immediately after blood samples were collected, the right lung was lavaged three times with 0.5 ml of phosphate-buffered saline. Total cell counts of nucleated cells were performed using a hematocytometer. Albumin concentrations in BALFs and serum were measured with an ELISA kit (Bethyl, Montgomery, TX). The sensitivity of this kit was 20 ng/ml. MIP-2, TNF-, IL-1, and IL-6 were also measured using mouse ELISA kits (R&D Systems, Minneapolis, MN). The sensitivity was 1 pg/ml for MIP-2, 5.1 pg/ml for TNF-, 1.3 pg/ml for IL-1, and 2.5 pg/ml for IL-6.
Lung wet-to-dry weight ratios. The left lung was removed and weighed to obtain the wet weight. The lung was desiccated at 80°C for 1 wk and weighed again to obtain the dry weight. Lung wet-to-dry weight (W/D) ratios were calculated as wet weight divided by dry weight.
Albumin clearance.
To obtain a direct estimation of the time course of actual albumin fluxes as a measure of pulmonary microvascular permeability, albumin concentrations determined in BALFs and serum by ELISA were used to calculate albumin clearance (Calb) where
 | (3) |
Electron microscopy. Lungs from B6/129 wild-type and TNFRKO mice were fixed with 3% glutaraldehyde in cacodylate buffer. After fixation, the tissue was rinsed in 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide in cacodylate buffer for 1 h at room temperature. The specimen was washed again in cacodylate buffer, dehydrated in a graded ethanol series, and placed in propylene oxide. The tissue was infiltrated with Poly/Bed 812 resin (Polysciences, Warrington, PA) for at least 2 h and embedded in capsules using overnight polymerization at 60°C. Thick sections (1 µm) were cut with glass knives using a Leica Ultracut UCT (Leica, Austria), stained, and examined by light microscopy. Thin sections (80 nm) were cut with a diamond knife (Diatome US, Fort Washington, PA) and stained with uranyl acetate and Reynold lead citrate. Thin sections were examined and photographed using a Philips CM 100 transmission electron microscope (FEI, Hillsboro, OR).
Cytokine measurement.
Cytokines were measured using commercially available ELISA kits and plates read on an automated microtiter plate reader from Dynatech Laboratories (Chantilly, VA 22021). The following kits were obtained: mouse ELISA kits for IL-1, IL-6, and TNF- were obtained from Endogen (Woburn, MA); and those for IL-1 and MIP-2 were obtained from R&D Systems. Minimal detection limits for these kits were <6 pg/ml for IL-1, <3 pg/ml for IL-1, <15 pg/ml for IL-6, <10 pg/ml for TNF-, and 1.5 pg/ml for MIP-2. Perfusate cytokine concentrations were expressed as picograms per milliliter.
Statistics. All values are expressed as means ± SE unless otherwise stated. The pressures and resistances were compared using an ANOVA with repeated measures and either a Newman-Keuls or Bonferroni posttest. A least-squares regression analysis was used where indicated. A significant difference was determined where P < 0.05.
|
RESULTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
30 min at a 10 cmH2O PIP was followed by a 20-min ventilation period with either 10 cmH2O PIP (low-PIP group) or 27 cmH2O PIP (high-PIP group). Kfc was unchanged by continued ventilation with 10 cmH2O PIP but increased significantly by threefold after high PIP ventilation for only 20 min. Terminal lung weights increased by 83 ± 9 and 104 ± 7% relative to predicted lung weights for the low-PIP group and high-PIP group, respectively, but the weight increases were not significantly different between the high- and low-PIP groups.
|
|
|
45-fold in both wild-type and TNFRKO mice ventilated for only 30 min at 45 cmH2O PIP and remained elevated over 2 h. However, there were no statistical differences between high PIP-ventilated wild-type and TNFRKO groups. There were also no significant differences between BALF albumin concentrations in the low-PIP group at 2 h compared with unventilated mice.
|
Lung W/D ratios also increased as a function of time in both the wild-type and TNFRKO high-PIP ventilation groups (Table 1). Lung W/D ratios increased by 1.66-fold in wild-type and by 1.50-fold in TNFRKO mice after 2 h of high PIP ventilation. No significant differences were observed in lung W/D ratios between nonventilated mice and the low-PIP group after 2 h of ventilation.
Arterial blood gases. Table 2 summarizes the arterial blood gases obtained at 0 and 2 h at the end of the low-PIP wild-type, high-PIP wild-type, and high-PIP TNFRKO mice. No significant differences were observed since we had previously determined the appropriate ventilation rate to keep total ventilation constant with the different tidal volumes (54).
|
, IL-1, IL-6, TNF-, and MIP-2. Figure 4 shows the perfusate concentrations of IL-1, IL-6, TNF-, and MIP-2 because perfusate IL-1 levels were not increased significantly during the 4-h perfusion period in either the low-PIP or high-PIP groups. Figure 4A shows that TNF-, IL-6, and MIP-2 were significantly greater at 4 h than their baseline values in the low-PIP group. TNF- was increased by 3-fold, IL-6 was increased by 5-fold, and MIP-2 was increased by 17-fold from baseline at 4 h, but IL-1 did not change significantly from baseline over 4 h in the low-PIP group. In the high-PIP group (Fig. 4B), perfusate IL-1 increased 3-fold at 2 h and 12.8-fold at 4 h, TNF- increased 26-fold at 2 h and 38-fold at 4 h, and MIP-2 increased 343-fold at 2 h and 900-fold at 4 h relative to their respective baseline values. These increases were also significantly greater than their time-matched values in the low-PIP ventilation group.
|
in BALF at any time period. BALF MIP-2 increased to detectable levels at 2 h only in 2 of 5 animals in this group (data not shown).
The time course of BALF cytokines measured in intact B6/129 wild-type and TNFRKO mice ventilated at 46 cmH2O PIP are shown in Fig. 5. TNF- was not detected in BALF of low- or high-PIP groups at any time period. In the wild-type high-PIP ventilation group, BALF MIP-2 and IL-6 but not IL-1 levels were increased significantly at 2 h compared with the nonventilated controls and the low-PIP ventilated group. In the TNFRKO high-PIP group, MIP-2 and IL-6 also increased at 2 h relative to time 0 controls, but there were no statistical differences in MIP-2, IL-6, or IL-1 levels between the wild-type and TNFRKO high-PIP groups. In the wild-type mice ventilated at low PIP, there was a small but significant increase in BALF IL-6, but MIP-2 and IL-1 levels did not change during ventilation. The cytokine levels in serum did not change significantly with time in any group, and there were no differences observed between groups.
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
and IL-1 may have a causal role in the vascular permeability increase observed in VILI (7, 46). However, the findings of the present study of a rapid onset of the vascular permeability increase in both isolated ex vivo and in vivo lung preparations, the delayed and variable cytokine responses in the different preparations and mouse strains, the lack of difference in BAL albumin responses between wild-type and TNFRKO mice, the lack of a detectable change in BAL or serum TNF- in the intact animal models, and the negative correlation between lung Calb and BAL MIP-2 and IL-6 concentration data all argue against such a role. The proinflammatory cytokines and chemokines undoubtedly will influence the ultimate degree of injury but do not appear to initiate the permeability increase.
Rapid onset of vascular permeability increases.
In the isolated perfused Balb/c mouse lungs, microvascular permeability (Kfc) increased by threefold in the high-PIP group after only 20 min of ventilation with a PIP of 27 cmH2O compared with both the low-PIP and baseline groups. Lung vascular permeability, as measured by BAL albumin concentration in intact Balb/c mice, also increased after only 30 min of ventilation with a similar PIP. Likewise, ventilation of intact B6/129 wild-type and TNFRKO mice for only 30 min with a somewhat higher PIP resulted in equivalent increases in BAL albumin concentration, which remained elevated for 2 h. Calculated Calb indicates that the albumin flux rate was highest at 30 min and waned significantly at 1 and 2 h of high PIP ventilation. Ventilation of B6/129 mice over a range of PIP for 2 h indicates that significant BAL albumin increases were also observed at lower PIP values (Fig. 3). Thus the presence of TNF- receptors had no measurable effect on the permeability response to high PIP. Quantitative differences in the threshold PIP required for lung injury differ between mouse strains because lung compliance of B6/129 mice is approximately half that of Balb/c mice (44). However, the very high compliance of the chest wall in mice suggests that the susceptibility to injury of isolated mouse lungs and intact mice should be comparable (24, 44), unlike other species where the chest wall significantly limits lung volume and the potential for high PIP injury (20).
In previous studies of high PIP injury in isolated perfused dog lungs, Parker et al. (34) also observed an increase in Kfc after only 20 min of ventilation injurious PIP. Kfc tended to recover toward baseline after moderately severe injury (45 cmH2O PIP) but continued to increase with time after injury with the highest PIP (55 cmH2O). Dreyfuss et al. (11) observed a significant increase in lung extravascular albumin spaces in rats after only 5 min of ventilation with a PIP of 45 cmH2O. The excess interstitial albumin spaces subsequently decreased toward baseline values with time. In other isolated lung studies, Fu et al. (14) exposed lungs to high airway and vascular pressures for only 1 min before fixation and observed epithelial breaks proportional to transmural pressure, and Rippe et al. (41) reported significant increases in Kfc after a transient (3 min) increases in lung vascular pressures, which also returned toward baseline over 2 h. Thus these previous studies as well as the present study indicate a very rapid onset of mechanically induced permeability increases and the capacity for recovery with moderate injury. The initial peak and time-dependent decreases in Calb observed in the present study also suggest a rapid onset of protein leak. The progressive increases in lung W/D ratios in B6/129 wild-type and TNFRKO mice (Fig. 6) suggest that Calb was coupled to transcapillary filtration rather than an active transport process (33).
Although severe mechanical stress can cause rupture of capillary basement membranes and hemorrhage due to stress failure, the bulk of the vascular fluid leak even at high vascular and airway pressures remains modulated by endothelial cell response (14, 30). Increases in vascular permeability involve alterations in the balance of endothelial cell retractile forces and tethering at cell-cell and cell-matrix junctions (42). Mechanical stretch of cultured endothelial monolayers initiates an increase in intracellular calcium as well as phosphorylation of focal adhesion kinase, focal adhesion complex proteins, and proteins of the MAP-kinase pathways (7). Our electron microscopy observations in both wild-type and TNF- receptor knockout mice suggest that injury is initiated by separation of both epithelium and endothelium from their basement membranes (Fig. 7). These changes suggest a failure of the cell-matrix focal adhesion sites and intercellular junctions. Bleb formation in both capillaries and small vessels support our previous observation in isolated rat lung of an increase in regional filtration coefficients in both alveolar and extra-alveolar vessels after high PIP ventilation (32). We previously observed that high PIP-induced increases in Kfc in isolated rat lungs were attenuated by blockade of stretch-activated cation channels and tyrosine kinase activity but was augmented by inhibition of protein tyrosine phosphatase activity (29, 31). Ying and Bhattacharya (53) observed increased calcium transients in epithelial cells in situ at high lung volumes and endothelial cells in situ at high vascular pressures in isolated lungs. However, transient cell wounding, such as observed in stretched alveolar epithelial cells, would initiate a massive calcium entry and also may affect vascular permeability (48). Such studies support the idea that the permeability lesion is a direct mechanical effect involving cation influx and phosphorylation events, which acutely alter cell adhesion, and precedes induction of the proinflammatory cascade of cytokines.
Variable cytokine response to lung overdistention.
In contrast to the consistently rapid increase in lung vascular permeability measured by Kfc and BAL albumin, the cytokine response to lung overdistention was delayed and highly variable. In isolated perfused Balb/c lungs ventilated at 25 cmH2O PIP, a small but significant increase in TNF- was observed in perfusate at 30 min. IL-1, TNF-, and MIP-2 were significantly increased by 1 h, and IL-1, TNF-, IL-6, and MIP-2 were present in high concentrations at 2 h. In the low-PIP group, TNF-, IL-6, and MIP-2 were only increased significantly after 2 h of ventilation and were present at concentrations several times lower than present in perfusate of the high-PIP group of lungs. Our observations confirm those of von Bethmann (49), who also reported significant increases in TNF- and IL-6 in perfusate from isolated perfused Balb/c mouse lungs after 2 h of ventilation with a PIP of 25 cmH2O and who also observed a small increase in cytokines at low PIP, possibly due to tissue manipulation during the isolation procedure. Tremblay et al. (45) previously observed high levels of IL-1, TNF-, IL-6, and MIP-2 in the BALF of isolated unperfused rat lungs ventilated at a PIP of 42–45 cmH2O for 2 h. However, Ricard et al. (39) could not repeat these observations in isolated ischemic rat lungs ventilated under similar conditions and reported elevations of MIP-2 but not IL-1 and TNF-. The reported increase in TNF- after ischemia/reperfusion of isolated perfused rat lungs suggest that an added insult of ischemia may contribute to the cytokine response of isolated lungs (22).
Unlike the isolated lung preparations, the intact mice ventilated with high PIP had no detectable levels of TNF- in BALF and no significant increase in TNF- in serum in either Balb/c or B6/129 mouse strains. Two previous studies in intact rats ventilated at a PIP of 45 cmH2O also failed to detect elevated TNF- in BALF (39, 47). The lack of increases in TNF- and IL-1 levels in BALF and serum in this study is unlikely due to lack of sensitivity in the ELISA because TNF- was detectable in serum and BALF when intraperitoneal lipopolysaccharide was administered (data not shown). In addition, minimal detection limits were <6 pg/ml for IL-1, <3 pg/ml for IL-1, <15 pg/ml for IL-6, <10 pg/ml for TNF-, and <1.5 pg/ml for MIP-2. Ricard et al. (39) found negligible TNF- and IL-1 in BALF of intact rats ventilated at high tidal volumes despite an increase in microvascular permeability that resulted in a 47-fold increase in BALF total protein concentration. We show here that high PIP ventilation of intact mice not only is not associated with lung production of TNF- and IL-1 but that binding of TNF- to both p55 and p75 receptor types is not necessary for the acute vascular permeability increase. The distinction between ventilation-associated cytokine production and receptor-mediated effects is significant because Xavier et al. (52) demonstrated that only minute amounts (<0.5 ng/ml) of TNF- were required to stimulate the release of MIP-2 from alveolar epithelial cells. In the present study, BALF albumin concentration increased 46-fold after 2 h of ventilation with 42 cmH2O PIP with or without the presence of TNF- receptors. Kuebler et al. (23) observed that a Ca2+ signal induced by application of TNF- to the alveolar space was communicated via gap junction between lung epithelial and endothelial cells in intact lungs. Thus the effect of TNF- produced anywhere in the lung would be rapidly spread between lung cells. A lack of receptors would preclude the possibility of low but effective local concentrations of TNF- that could not be detected after dilution in the BALF. The observed increase in MIP-2 in BALF after 2 h of high PIP ventilation also was not affected by the presence or absence of TNF- receptors. In patients, 1 h of high tidal volume ventilation did not cause significant changes of TNF- levels in the systemic circulation, and blockade of TNF- with antibody receptor ligand domains did not increase survival in sepsis (13, 51).
Many of the previous studies implicating TNF- in VILI involve ventilation after preexisting injuries. In rats with preexisting lung injury due to intratracheal hydrochloric acid instillation or cecal ligation with perforation that were ventilated with high volumes, progressive increases in BALF and serum TNF- and MIP-2 levels were reported (6, 26). Application of TNF- to monolayers of endothelial cells from human umbilical vein (12) or bovine pulmonary artery (35) consistently produced increases in monolayer permeability, primarily attributed to binding of the p55 TNF receptor (12). In contrast, infusion of TNF- into intact animals or isolated, perfused lungs without preexisting injury either does (16) or does not produce increases in lung vascular permeability (2, 22), but intratracheal instillation of anti-TNF- antibodies attenuated high PIP injury in surfactant-depleted rabbits (21). Therefore, the mechanical trauma and ischemia during the isolation procedure for isolated lung preparations may also constitute a preexisting injury that primes lung cells to produce the acute response cytokines TNF- and IL-1 and account for the similarities of the cytokine profiles between isolated lung studies and preexisting injury studies subsequently ventilated with high lung volumes. Thus mechanical ventilation and these insults may activate common pathways for priming inflammatory cells and endothelium as suggested in the "two-hit" hypothesis of lung injury (8).
MIP-2, the murine analog of IL-8 in humans, was significantly increased after 2 h of ventilation with 42 cmH2O PIP in our intact wild-type and TNFRKO mouse models but only marginally detected in intact Balb/c mice ventilated at 25 cmH2O PIP. This potent chemoattractant for neutrophils is the most consistently reported cytokine after mechanical stress in isolated and intact lung studies and cultured monolayers of alveolar epithelial cells, fetal lung cells, and lung endothelial cells (17, 36–38). The importance of this cytokine in VILI is emphasized by the attenuated injury in CXC receptor knockout mice and after neutrophil depletion (1). However, neutrophils probably augment the lung injury with inflammation after the initial vascular fluid leak. Yoshikawa et al. (54) reported that increases in BAL albumin and Clara cell secretory protein preceded microscopic evidence of the tissue injury and inflammation in a similar mouse model of VILI. The time course for lung injury and inflammation that we observed was similar to that previously reported by Quinn et al. (37) for rats ventilated for 2 h at 20 ml/kg. Although lung W/D ratios significantly increased, BAL MIP-2 and neutrophil counts did not change significantly at 2 h. Only after waiting an additional 6 h after ventilation were the BAL MIP-2 increased by fourfold and the neutrophil counts increased significantly over the low-volume group. Lung MPO was not elevated after 2 h of ventilation in their study but increased significantly after the additional 6-h wait. Likewise, Belperio et al. (1) only observed attenuation of VILI using CXC receptor knockout mice and CXC receptor antibodies after 6 h of high-volume ventilation after there was significant histological evidence of tissue destruction and edema. We found that there was actually a significant negative correlation of normalized Calb with normalized BAL MIP-2 and IL-6 concentration. We do not infer that these inflammatory cytokines inhibit albumin flux rates but rather that elevated concentrations of these cytokines are unlikely to cause the initial increases in Calb that were measured. Because we did not detect BAL TNF- in any of the intact mouse models and the absence of TNF- receptors did not affect susceptibility to lung injury, we can conclude that these cytokines are unlikely to induce the acute permeability response reported here. The full-blown inflammatory response takes time to develop, and we would expect further increases in permeability and tissue injury with neutrophil infiltration during longer ventilation times.
These studies suggest that the acute proinflammatory cytokines do not play a major role in the initial vascular leak of fluid and protein in the absence of preexisting injury or inflammation. Rather, the initial vascular leak probably results from acute signaling events such as calcium entry and phosphorylation of focal adhesion, and junctional and cyoskeletal proteins that alter endothelial contraction and tethering (15). Protein leak and edema may then cause release of growth factors, proteases, arachadonic acid derivatives, and other mediators that facilitate neutrophil influx and activation. A consequence of such a sequence of events would infer that pharmacological blockade of initial signaling events may be more effective in preventing mechanical injury than immunologic interventions targeted at specific proinflammatory cyokines after inflammation is well established.
|
GRANTS |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
|
FOOTNOTES |
|---|
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
|
REFERENCES |
|---|
|
TOPABSTRACTMETHODSRESULTSDISCUSSIONGRANTSREFERENCES |
|---|
does not cause lung edema in rabbits. J Appl Physiol 73: 173–178, 1992.
-induced vascular permeability. Am J Physiol Cell Physiol 281: C1173–C1179, 2001.
B and is blocked by steroids. Am J Respir Crit Care Med 163: 711–716, 2001.
antibody attenuates ventilator-induced lung injury in rabbits. J Appl Physiol 87: 510–515, 1999.
in ischemic and reperfusion injury in rat lungs. J Appl Physiol 85: 2005–2011, 1998.
and IL-6 levels are consistent and efficient predictors of outcome over time. Chest 107: 1062–1073, 1995.
-induced endothelial cell apoptosis and barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 280: L1168–L1178, 2001.
and IL-1 during high volume mechanical ventilation of ex vivo rat lungs (Abstract). Am J Respir Crit Care Med 161: A211, 2000.
and prostacyclin production in lavaged rat lungs in vivo. Anesthesiology 91: 1834–1843, 1999.
mediates lipopolysaccharide-induced macrophage inflammatory protein-2 release from alveolar epithelial cells. Autoregulation in host defense. Am J Respir Cell Mol Biol 21: 510–520, 1999.
This article has been cited by other articles:
|
|
 |
|
  J. C. Parker and M. I. Townsley Physiological determinants of the pulmonary filtration coefficient Am J Physiol Lung Cell Mol Physiol, August 1, 2008; 295(2): L235 - L237. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Miyahara, K. Hamanaka, D. S. Weber, M. Anghelescu, J. R. Frost, J. A. King, and J. C. Parker Cytosolic phospholipase A2 and arachidonic acid metabolites modulate ventilator-induced permeability increases in isolated mouse lungs J Appl Physiol, February 1, 2008; 104(2): 354 - 362. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. A. Oeckler and R. D. Hubmayr Ventilator-associated lung injury: a search for better therapeutic targets Eur. Respir. J., December 1, 2007; 30(6): 1216 - 1226. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. Hamanaka, M.-Y. Jian, D. S. Weber, D. F. Alvarez, M. I. Townsley, A. B. Al-Mehdi, J. A. King, W. Liedtke, and J. C. Parker TRPV4 initiates the acute calcium-dependent permeability increase during ventilator-induced lung injury in isolated mouse lungs Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L923 - L932. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. Miyahara, K. Hamanaka, D. S. Weber, D. A. Drake, M. Anghelescu, and J. C. Parker Phosphoinositide 3-kinase, Src, and Akt modulate acute ventilation-induced vascular permeability increases in mouse lungs Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L11 - L21. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. R. Wilson, M. E. Goddard, K. P. O'Dea, S. Choudhury, and M. Takata Differential roles of p55 and p75 tumor necrosis factor receptors on stretch-induced pulmonary edema in mice Am J Physiol Lung Cell Mol Physiol, July 1, 2007; 293(1): L60 - L68. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Yoshikawa, T. Miyahara, S. D. Reynolds, B. R. Stripp, M. Anghelescu, F. G. Eyal, and J. C. Parker Clara cell secretory protein and phospholipase A2 activity modulate acute ventilator-induced lung injury in mice J Appl Physiol, April 1, 2005; 98(4): 1264 - 1271. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
P < 0.05 vs. 0- and 0.5-h groups.
P < 0.05 vs. low-PIP group value at same time period.
