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Ann Thorac Surg 2001;71:1825-1832
© 2001 The Society of Thoracic Surgeons
a Department of Biochemistry, The University of Texas Health Center at Tyler, Tyler, Texas, USA
b Vivarium, The University of Texas Health Center at Tyler, Tyler, Texas, USA
c Medical Research Service of the Seattle Veterans Affairs Medical Center, and the Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA
d Department of Thoracic and Cardiovascular Surgery, Saga Medical School, Saga, Japan
Accepted for publication January 24, 2001.
Address reprint requests to Dr Miller, Department of Biochemistry, The University of Texas Health Center at Tyler, 11937 US Highway 271, Tyler, TX 75708–3154
e-mail: ed.miller{at}uthct.edu
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Abstract |
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 Top Abstract IntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Methods. We examined the cell population, epithelial permeability measured by Evans blue dye (EB), ßglucuronidase and cytokine concentrations in bronchoalveolar lavage fluid (BALF) and/or blood using a rabbit RPE model.
Results. We confirmed that RPE is characterized by recruitment of polymorphonuclear leukocytes (PMNs), the release of PMN granular contents into the air spaces, and increased vascular permeability. These findings were highly correlated with increased interleukin-8 (IL-8) and monocyte chemoattractant protein 1 (MCP-1) concentrations in the BALF. Growth related oncogene (GRO) was detected in the BALF from only 2 of the 7 reexpanded lungs while TNF
was not detected in any rabbits. A similar but less severe inflammatory response to the reexpanded lung was found in the contralateral lung.
Conclusions. IL-8 and MCP-1 may play important roles in the development of RPE; the inflammatory response is independent of TNF and unilateral reexpansion of the lung induces an inflammatory response not only in the reexpanded lung but also in the contralateral lung.
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Introduction |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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RPE is characterized by an acute onset lung injury together with a high protein and high osmolality edema fluid with increased lung microvascular permeability [2, 3]. Recently, clinical [4, 5] and animal reports [6] have revealed a strong correlation between polymorphonuclear leukocytes (PMNs) and products of activated PMNs with the development of RPE. Some proinflammatory cytokines such as IL-8, MCP-1 and GRO, which induce leukocyte activation, have been shown to be associated with the development of the lung injury associated with the acute respiratory distress syndrome (ARDS) [7–9]. However, the mechanism of accumulation of PMNs in the air space, the activation of PMNs and the role of cytokines in the development of RPE remains undefined. Also, there have been reports [10] that RPE occurs in the contralateral lung and the mechanism by which this occurs remains unclear. Therefore, the present study was designed to clarify the role of leukocytes and cytokines in the development of the lung injury found in RPE, and to examine the inflammatory effects of unilateral reexpansion on the contralateral lung.
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Material and methods |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Female, New Zealand white rabbits (2.0 to 3.0 kg) were used. Lung collapse was induced by the insertion of a sterile plastic bag filled with 50 ml of saline into the thoracic cavity. The bag was inserted into the right thoracic space through a 3 cm incision at the 4th or 5th intercostal space of the back following anesthesia (intramuscular injection of 35 mg/kg of ketamine, 5.0 mg/kg of xylazine and 0.1 mg/kg of butorphanol). After 7 days, the right carotid artery was cannulated with a 20 ga cannula for blood sampling. The trachea was cut and intubated with a 4.5 Fr tube. Then, the rabbits were randomized into two groups (a) reexpansion, and (b) control groups. In the reexpansion group, the bag was removed under anesthesia through a 3 cm incision at the 4th or 5th intercostal space in the lateral site of the thorax. After removal of the bag, an 8 Fr thoracic tube was inserted into the right thoracic space and the tube was connected to an evacuator set at a negative pressure of -20 cm H2O for 3 hours. To achieve complete reexpansion of the lung, positive pressure ventilation less than 20 cm H2O was applied five times just after the start of the evacuation. In the control group, the bag was not removed and no negative pressure was applied. However, the same incision was made in the same site as the REP group and the thoracic tube was kept subcutaneously. All rabbits were kept under room air and voluntary ventilation for 3 hours before they were sacrificed by exsanguination. Confirmation of the collapse and reexpansion of the right lung was made by Roentgenogram examination before and after removal of the bag followed by evacuation (Fig 1). Any rabbit with lung collapse less than 70% was excluded from this study.
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Lung lavage
A single cycle bronchoalveolar lavage (BAL) [11, 12] was performed in each side of the lung in situ. Briefly, after exsanguination of the rabbit, the chest cavity was opened, and the right main bronchus was clamped to isolate the left lung. A tube (i.d.3-mm; o.d.5-mm) was wedged in the left main bronchus, and tied in place. A single aliquot (20 mL) of sterile saline (0.9% W/V) was instilled into the lung. The fluid was then withdrawn and the final BAL fraction (5 mL), which was from the most peripheral site of the lung, was used for analysis. The same procedure was performed in the right lung. The lung and heart were then removed en bloc for histologic examination. The samples were centrifuged at 800g for 10 minutes and frozen at -70°C immediately for biochemical analysis.
Cell analysis
Total WBC numbers were assessed using a Coulter Counter Model Fn (Coulter Electronics Inc, Hialeah, FL). Slides of each BAL sample were made and stained using a Leukostat stain kit (Fisher Diagnosis, Pittsburgh, PA). Differential cell counts were then performed.
Biochemical analysis
BAL and plasma concentrations of IL-8, GRO, MCP-1, and tumor necrosis factor (TNF) were determined by ELISA as described previously [13, 14]. Briefly, goat antirabbit IL-8, GRO, MCP-1 or TNF antibodies were diluted to 200 µg/mL with 0.1 mol/L bicarbonate buffer (pH 9.6), absorbed to 96-well plates, incubated at 47°C overnight, and then washed with phosphate buffered saline (PBS) twice. Non-specific binding to the plate was blocked by 5% nonfat milk in Dulbecco’s PBS for 1 hour. The diluted samples (100 µL/well) were added and the plates were incubated at 37°C for 2 hours. After washing four times with PBS containing 0.05% Tween 20, a biotinylated goat antirabbit IL-8, MCP-1, TNF or GRO was added to each well and incubated at 37°C for 2 hours. Then the plates were incubated with streptavidin-biotin-peroxdase complex at 37°C for 1 hour. The washed plates were incubated with the peroxidase substrate 3,3,5,5,-tetramethylbenzidine for 1 hour at 37°C, the reaction was stopped with 1.0 mol/L phosphoric acid, and the absorbance at 450-nm was measured.
Pulmonary vascular permeability analysis
Vascular permeability was measured by the extravasation of Evans Blue (EB) dye. The extravasation of EB correlates well with the extravasation of radiolabeled albumin at high rates of plasma leakage [15]. Fifty mg/kg of EB (Sigma, St. Louis, MO) was administered intravascularly at time 0. The absorbance of the BALF and plasma were measured at 620-nm using a spectrophotometer (DU Series 600, Beckman Instruments Inc., Fullerton, CA) and the concentration of EB was calculated by comparison with a standard curve.
Neutrophil enzyme release
The azurophilic neutrophil enzyme, ß-glucuronidase, was measured in BALF to evaluate the activation of PMNs in the lung. Briefly, aliquots (40 µL) of BALF were mixed with 10 µL of 0.01 mol/L phenolphthalein-glucuronic acid (Sigma) and 40 µL of 0.1 mol/L sodium acetate pH 4.6 in 96 well plates for ß-glucuronidase measurement. After 16 hours incubation at 37°C, 0.2 mol/L glycine in 0.2 mol/L NaCl, pH 10.4 (200 µL) was added to each well and the absorbance was measured at 540-nm.
Histologic examination
To exclude animals with complications associated with bag implantation, such as lung necrosis and abscess, we examined the lung tissues from each rabbit microscopically after performing lung lavage. The specimens were fixed with 10% formalin for 18 to 36 hours and embedded in paraffin wax. Sections (7-µm thickness) were made and stained with hematoxylin and eosin (H & E). Microscopic examination was performed on at least 3 sections from the different lobes of each side of the lung.
Statistics
Statistical analysis was performed using the Stat View 4.5J software (Abacus Concepts, Berkeley, CA). Data are expressed as means ±SEM. To determine the effects of reexpansion, we compared the data in each side of the lung between the reexpansion group and the control group using an unpaired t test or the nonparametric Mann-Whitney U test when appropriate. Furthermore, to determine if maintaining the lung in a collapsed state itself lead to inflammatory changes, we also compared the data between the right (collapsed) and left (noncollapsed) lungs in the control group which had been maintained with a lung collapse for 7 days without reexpansion on the 7th day. Wilcoxon’s paired rank test was used to test for within-group differences. The correlation coefficients among the inflammatory cell numbers, EB concentration, ß-glucuronidase concentration and cytokines in BALF were evaluated and p values were calculated using Fisher’s z transformation of r. A value of p less than 0.05 was accepted as statistically significant in all analyses.
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Results |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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Blood gas analysis and WBC counts
All rabbits showed decreased PaO2 at time 0 (the starting point of the evacuation, just after placement of the thoracic tube) due to the anesthesia. The PaO2 levels in the control group recovered gradually, and none of them showed less than 60 Torr after 3 hours (Fig 2). In contrast, 3 out of the 5 reexpansion rabbits showed PaO2 levels less than 35 Torr after 3 hours following reexpansion. There were statistically significant differences between PaO2 levels in the reexpansion (45.2 ± 10.6 Torr) and control groups (76.3 ± 5.3 Torr) 3 hours after surgery (p = 0.04).
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Neutrophil enzyme release
Effect of reexpansion on both sides of the lungs: comparison between reexpansion and control groups
In the right (collapsed) lung, the reexpansion group had higher concentrations of ß-glucuronidase than the control group (1.48 ± 0.31 nmol/L, 0.49 ± 0.07 nmol/L, p = 0.008). However, there were no differences between the 2 groups in the left (noncollapsed) lung (0.64 ± 0.13 nmol/L, 0.40 ± 0.06 nmol/L, p = 0.1)(Fig 3). The increase in ß-glucuronidase was significantly higher in the right lung than in the left lung (p = 0.04).
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Effect of collapse: comparison between the right (collapsed) and left (noncollapsed) lungs in the control group
There were no differences in concentrations of ß-glucuronidase between the right and the left lungs (0.49 ± 0.07 nmol/L, 0.40 ± 0.06 nmol/L).
Pulmonary vascular permeability analysis
Effect of reexpansion on both sides of the lungs: comparison between reexpansion and control groups
In the right (collapsed) lung, EB concentration was much higher (p =0.006) in the reexpansion group (84.9 ± 50.9 ng/µl) than in the control group (2.7 ± 0.3 ng/µL)(Fig 4). In the left (noncollapsed) lung, the concentration was also higher (p = 0.018) in the reexpansion group (13.2 ± 6.5 x 106ng/µL) than in the control group (1.75 ± 0.2 x 106ng/µL). The increase in EB concentration was significantly higher in the right lung than in the left lung (p = 0.04). EB concentration was highly correlated with total PMN numbers (r = 0.993, p < 0.0001), % PMNs (r = 0.905, p < 0.0001) and ß-glucuronidase (r = 0.874, p = 0.0004).On the other hand, there was no correlation between EB concentration and either % macrophage or macrophage numbers in the BALF (p = 0.3, p = 0.3).
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Effect of collapse: comparison between the right (collapsed) and left (noncollapsed) lungs in the control group
There were no significant differences in EB concentrations between right (2.7 ± 0.3 ng/µL) and left lungs (1.8 ± 0.2 ng/µL).
Cytokines in the BALF
Effect of reexpansion on both sides of the lungs: comparison between reexpansion and control groups
In the right (collapsed) lung, IL-8 and MCP-1 concentrations were much higher in the reexpansion group than in the control group (p = 0.007, p = 0.006, respectively). Furthermore, in the left (noncollapsed) lung, IL-8 and MCP-1 concentrations were also higher in the reexpansion group than in the control group (p = 0.008, p = 0.008, respectively)(Fig 5A and B). The increase in IL-8 and MCP-1 were significantly higher in the right lung than in the left lung in the reexpansion group (p = 0.04, p = 0.04, respectively).
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was not detected in any samples. GRO was detected in only two samples of the reexpanded lungs at concentrations less than 1 ng/mL.
Effect of collapse: comparison between the right (collapsed) and left (noncollapsed) lungs in the control group
The right (collapsed) lung had higher MCP-1 concentrations than the left (noncollapsed) lung in the control group (0.78 ± 0.5 ng/mL,0.45 ± 0.3 ng/mL, respectively, p = 0.03). However, there were no differences in IL-8 concentration between right and left lungs (0.13 ± 0.4 ng/mL, 0.15 ± 0.8 ng/mL respectively). TNF and GRO were not detected in any samples.
Cytokines in blood
The reexpansion group had higher MCP-1 concentrations than the control group at both 2 and 3 hours following placement of the chest drainage tube (2 hours: 2.74 ± 0.72 ng/mL versus 0.18 ± 0.18 ng/mL, p = 0.004; 3 hours: 3.70 ± 1.20 ng/mL versus 0.28 ± 0.18 ng/mL, p = 0.005). However, MCP-1 was not detectable before 1 hour after placement of the chest drainage tube. In the reexpansion group, we could not detect any correlation between blood MCP-1 and total PMNs, EB concentration, IL-8, MCP-1 or ß-glucuronidase in the BALF from either the right or left lungs. IL-8, GRO and TNF were not detectable in any samples.
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Comment |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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In spite of the absence of increased numbers of macrophages in the BALF, elevated concentrations of the potent monocyte and basophil chemotactic and activating factor MCP-1 [21], were found in the reexpanded lung. Unexpectedly, the increased MCP-1 level was correlated with PMN rather than macrophage recruitment to the lung, and was also correlated with increased levels of ß-glucuronidase. However, the IL-8 correlation with total PMNs, % PMNs, and ß-glucuronidase in the BALF was stronger than that of MCP-1 levels (Table 2). Although the lack of correlation between macrophages and MCP-1, and the significant positive correlation between PMNs and elevated concentration of MCP-1 in the BALF were surprising, similar results were found in ARDS patients analyzed on days 3,7,14, and/or 21 after the onset of ARDS [8]. Since this increased MCP-1 also had significant correlation with lung injury, it was suggested that MCP-1 may play an important role in the development of ARDS [8]. Though the mechanism of the relationship between MCP-1 and PMNs is unclear, it may be explained in part by the fact that activated PMNs also produce MCP-1 [22].
In a rabbit model of RPE, Jackson et al. demonstrated that hypoxia increases anaerobic glycolysis, decreases mitochondrial oxygen use, pulmonary blood flow (by approximately 45%) and tissue PaO2 to less than 20 mm Hg [6]. The present study showed that lung collapse itself did not induce increased vascular permeability, or increased concentrations of ß-glucuronidase, IL-8, GRO or TNF in the BALF before reexpansion. Increased PMN numbers and MCP-1 were found during collapse, but the levels were much less than those found following reexpansion (PMN: p < 0.01, MCP-1: p < 0.01). The data suggests that the reexpansion (reperfusion or reoxygenation) of the collapsed lung was the main cause of the lung injury associated with RPE. Association of IL-8 or MCP-1 production with ischemic reperfusion injury has been reported in the lung and myocardium [19, 23]. Further, there was a high correlation between IL-8 and MCP-1 in the BALF (r = 0.926, p < 0.0001). Our findings support the idea that RPE may be an analog of the reperfusion injury after ischemia [6]. Although some reports showed the important role of TNF in the development of reperfusion injury in liver or limb [24], we did not detect TNF in the BALF or blood from the RPE rabbits. It has been reported that mononuclear phagocytes and endothelial cells, exposed to hypoxia/hyperoxia mimicking ischemia/reperfusion, produce IL-8 and this production is independent of TNF and IL-1ß [25]. These data are consistent with our findings that the production of IL-8 and the activation of PMNs are independent of TNF in the development of RPE.
GRO peptides (, ß, and 
) may play an important role in lung inflammation in some cases of ARDS and pneumonia [9, 26]. However, we detected GRO in the BALF from only 2 animals at concentrations less than 1 ng/mL in the reexpanded lung from the reexpansion group, and none in the BALF from the control group (data are not shown). Thus, GRO expression is not a prominent feature of RPE.
In the present study, the high correlation (r = 0.993, p < 0.0001) between EB concentration and PMN numbers in the BALF was found, and it is consistent with the theory that leukocyte activation and migration can result in protein leakage into the airspaces [27]. Further, IL-8 is highly correlated with PMNs (r = 0.856, p = 0.0007) and ß-glucuronidase (r = 0.882, p = 0.0004). Sekido and associates have shown that IL-8 mAb prevents both PMNs accumulating in the alveolar space and fibrinous exudation forming in the alveolar lumen after reperfusion lung of rabbits [28]. These findings indicate that IL-8 may play an important role in increasing vascular permeability in RPE by activating PMNs. On the other hand, Lum and associates showed that endothelial injury resulted in increased permeability and was directly induced by oxidants generated by ischemic reoxygenation in the absence of leukocytes in an in vitro model [28]. In the present study, one RPE rabbit died at 30 minutes after reexpansion due to severe respiratory dysfunction, and it showed high EB concentration without increased PMN numbers and cytokines (IL-8, GRO, MCP-1, and TNF) levels in the BALF. Such differences between EB concentration and PMN numbers or cytokines concentration in the BALF were not found in the other 5 rabbits that survived for 3 hours after reexpansion. Therefore, cytokine or PMN independent pathways that induce increased permeability may play an important role in the early phase of the development of RPE.
Contralateral and bilateral RPE
The mechanism by which edema occurs in the contralateral lung remains uncertain. The present study found that in each case, the injury to the reexpanded lung was more severe than in the contralateral lung. However, unilateral reexpansion of the lung induced injury in the contralateral lung that was characterized with PMN accumulation to the air space and increased permeability as in the reexpanded lung (see Table 1, Fig 4). Also, increased levels of IL-8 and MCP-1 concentrations in the BALF were found in the contralateral lung of the reexpansion group (see Fig 5). Our findings suggest that the mechanism of development of bilateral RPE is similar to unilateral RPE. Although, we found increased concentrations of MCP-1 in the blood in the reexpansion group, there were no correlations between blood MCP-1 concentration and IL-8 concentration, EB concentration, PMN numbers, ß-glucuronidase concentration, or MCP-1 concentration in the BALF from the contralateral lung. These findings indicate that MCP-1 is not a primary cause of the inflammatory response in the contralateral lung. However, the increased concentration of MCP-1 in the blood of the reexpansion group suggests that the lung reexpansion results in an increase in proinflammatory mediators in the blood that may induce a systemic inflammatory response, including bilateral lung injury.
In summary, we have shown that RPE, characterized by PMN accumulation in the air space and increased vascular permeability with impaired respiratory function, is an inflammatory response highly associated with IL-8 and MCP-1, but independent of TNF. Furthermore, unilateral reexpansion of the lung induces an inflammatory response both in the reexpanded lung, and to a lesser extent, in the contralateral lung. The data suggest that neutralization of IL-8 and MCP-1 may attenuate to the severity of injury associated with RPE.
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Acknowledgments |
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References |
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TopAbstractIntroductionMaterial and methodsResultsCommentAcknowledgmentsReferences |
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