1 Department of Surgery, Lund University Hospital, SE-221 85 Lund; and 2 Department of Animal Physiology, Lund University, SE-223 62 Lund, Sweden
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ABSTRACT |
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Intestinal ischemia-reperfusion commonly occurs
in critically ill patients and may lead to the development of remote
organ injury, frequently involving the lungs. In the present study, alveolar liquid clearance was studied in ventilated, anesthetized rats
subjected to 45 min of intestinal ischemia followed by 3 h of
reperfusion. An isosmolar 5% albumin solution was instilled into the
lungs, and alveolar liquid clearance was measured from the increase in
alveolar protein concentration as water was reabsorbed over 45 min.
Intestinal ischemia-reperfusion resulted in a 76% increase in
alveolar liquid clearance compared with the control value (P < 0.05). The stimulated alveolar liquid clearance seen after
intestinal ischemia-reperfusion was not inhibited by
propranolol, indicating stimulation through a
noncatecholamine-dependent pathway. Intestinal
ischemia-reperfusion did not result in increased intracellular cAMP levels. Amiloride inhibited similar fractions in animals subjected
to ischemia-reperfusion and control animals. Administration of
a neutralizing polyclonal anti-tumor necrosis factor- antibody before induction of intestinal ischemia completely inhibited
the increased alveolar liquid clearance observed after intestinal ischemia-reperfusion. In conclusion, our results suggest that intestinal ischemia-reperfusion in rats leads to stimulation of alveolar liquid clearance and that this stimulation is mediated, at
least in part, by a tumor necrosis factor-
-dependent mechanism.
tumor necrosis factor-; endothelium; epithelium; lung; lung
injury; pulmonary edema
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INTRODUCTION |
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INTESTINAL ISCHEMIA-REPERFUSION is a potentially
life-threatening condition commonly associated with injury to organs
remote from the initial insult (9, 15, 17). The remote organ injury is
suggested to be caused by release of inflammatory mediators including
arachidonic acid metabolites, reactive oxygen species, complement
factors, and cytokines such as tumor necrosis factor (TNF)- (5, 36,
40). It has previously been demonstrated that TNF-
is an essential
cytokine in the cascade that causes lung endothelial injury after lung
ischemia-reperfusion (13). Also, leukocytes have been suggested
as important mediators of both local intestinal injury (10) and remote
tissue injury (29, 31). Clinical studies as well as animal models have
demonstrated an impairment of pulmonary function after intestinal
ischemia-reperfusion (15, 19, 29). Injury to the intestinal
barrier during ischemia-reperfusion may lead to translocation
of bacteria and bacterial products such as endotoxin, with the systemic
dissemination linked to the development of remote tissue injury (15,
35). Endotoxin can stimulate macrophages to synthesize and secrete
several cytokines and other inflammatory mediators including TNF-
(19).
A previous study (38) has implicated TNF- as a mediator of
hemodynamic dysfunction secondary to intestinal
ischemia-reperfusion in rats. TNF-
has also been
demonstrated to increase pulmonary epithelial permeability secondary to
acute bacterial lung inflammation in rats (18). Furthermore, TNF-
has been reported to increase sodium-coupled amino acid transport in
rat hepatocytes (25), thus suggesting that TNF-
may modulate
vectorial solute transport across other cell membranes. A recent study
by Rezaiguia et al. (28) demonstrated that TNF-
stimulates alveolar
liquid clearance in rats during bacterial pneumonia from
Pseudomonas aeruginosa. Furthermore, several studies have
demonstrated that
-adrenergic stimulation increases alveolar liquid
clearance during pathological conditions (27) as well as under normal
conditions (7, 23).
We hypothesized that lung injury after intestinal
ischemia-reperfusion is caused by an increase in pulmonary
endothelial-epithelial permeability. Furthermore, we hypothesized that
TNF- protects the lungs from a rapid lethal flooding by stimulating
alveolar liquid clearance. Therefore, our first objective was to
investigate alveolar barrier permeability and alveolar liquid clearance
after intestinal ischemia-reperfusion injury. Because
intestinal ischemia-reperfusion stimulated alveolar liquid
clearance and because several authors (27) have shown that endogenous
epinephrine can stimulate alveolar liquid clearance in a variety of
pathological conditions, our second objective was to investigate the
role of endogenous catecholamines and their intracellular second
messenger, cAMP, on alveolar liquid clearance and alveolar epithelial
permeability after intestinal ischemia-reperfusion. Because
-adrenergic stimulation was not responsible for the stimulated
alveolar liquid clearance after intestinal ischemia-reperfusion
and because TNF-
has been demonstrated to increase alveolar liquid
clearance under other pathological conditions (28), our third objective
was to study whether TNF-
may stimulate alveolar liquid clearance
after intestinal ischemia-reperfusion.
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MATERIALS AND METHODS |
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Animals
Adult male Sprague-Dawley rats (n = 49; B&K Universal, Sollentuna, Sweden) weighing 230-550 g were fed standard rat chow (R3, Astra-Ewos, Södertälje, Sweden) and had tap water ad libitum. The rats were maintained in a 12:12-h day-night rhythm at a temperature of 20 ± 2°C and a relative humidity of 50 ± 10%. The Ethical Review Committee on Animal Experiments at Lund University (Lund, Sweden) approved the experimental protocol.Preparation of Instillates and Rhodamine B Isothiocyanate-Conjugated Dextran Injection Solution
A 5% albumin solution was prepared by dissolving 50 mg/ml of bovine serum albumin (Sigma, St. Louis, MO) in 0.9% NaCl. A sample of the instillate was saved for total protein measurement. In some studies (see Specific Experimental Protocols), theRhodamine B isothiocyanate (RITC)-conjugated Dextran 70 (RITC-dextran; mol wt 70,000; Sigma) was dissolved at a concentration of 35 mg/ml in 2.5 ml of 0.9% NaCl and filtered through a PD-10 column (Pharmacia Bioscience, Uppsala, Sweden) to separate free unbound RITC molecules from the RITC-dextran. The filtered RITC-dextran was then diluted with isosmolar 0.9% NaCl to a final concentration of 2.5 mg/ml.
Induction of Intestinal Ischemia-Reperfusion
The rats were anesthetized with an intramuscular injection of ketamine (100 mg/kg body weight; Ketalar, Parke-Davis, Barcelona, Spain) and xylazine (10 mg/kg body weight; Rompun, Bayer, Leverkusen, Germany). Through a midline laparotomy, the superior mesenteric artery was located and isolated from the surrounding tissues, and a vascular clip was placed around the vessel near the aortic origin. The abdominal incision was then temporarily closed, and the rat was placed on a heating pad during the ischemic period. After 45 min, the abdominal incision was opened, and the vascular clip was removed. Reperfusion was noted by visual inspection of the intestine in each animal, after which the abdominal incision was permanently closed. Reperfusion was continued for 3 h. A sham operation on control rats was carried out as above but without placing the vascular clip on the superior mesenteric artery.Surgical Procedures and Ventilation
One hour fifteen minutes after the onset of reperfusion, the animals were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight; Apoteksbolaget, Umeå, Sweden). A 2.0-mm-ID endotracheal tube (PE-240; Clay Adams, Becton Dickinson, Sparks, MD) was inserted into the trachea through a tracheostomy. A 0.58-mm-ID catheter (PE-50; Clay Adams, Becton Dickinson) was inserted into the right carotid artery to monitor systemic blood pressure, administer drugs, and obtain blood samples. Pancuronium bromide (0.3 mg · kg body wtPeak airway pressure, blood pressure, and heart rate were measured with calibrated pressure transducers (UFI model 1050BP; BioPac Systems, Goleta, CA) connected to analog-to-digital converters and amplifiers (MP100 and DA100, respectively; BioPac Systems) and continuously recorded on an IBM-compatible computer with Acknowledge 3.2.4 software (BioPac Systems).
General Experimental Protocol
After the surgical procedures, the animals were placed in the left decubitus position on a slanting board. A heating pad covered the animals during the experiment to control and maintain normal body temperature. A 30-min baseline period with stable blood pressure and heart rate was required before fluid instillation into the lungs. Ten minutes into the baseline period, RITC-dextran was injected intra-arterially (2 ml/kg body weight, resulting in ~ 0.07 mg RITC-dextran/ml blood). A blood sample (1 ml) was taken 10 min after the RITC-dextran injection.After the 30-min baseline period, an instillation catheter (PE-50; Clay Adams, Becton Dickinson) was gently passed through the endotracheal tube and advanced to a wedged position in the distal air spaces of the lungs. Then, 0.8 ml of the 5% albumin solution was instilled over 20 min into the lungs. The instillation catheter was withdrawn after the instillation was completed.
Forty-three minutes after instillation, a 5-ml blood sample was taken,
and at 45 min, the animal was given an overdose (30 mg) of
pentobarbital sodium intra-arterially. The abdomen was opened, and the
rats were exsanguinated by transecting the abdominal aorta. The lungs
and heart were carefully removed en bloc from the thorax through a
midline sternotomy. A PE-50 catheter (Clay Adams, Becton Dickinson) was
gently passed to a wedged position in the instilled lung, and a sample
of the remaining alveolar liquid was aspirated. The lungs were then
homogenized for fluorescence measurements and wet-to-dry weight
determinations. The blood samples were centrifuged at 4,000 g
for 5 min, and the plasma was collected for analysis of fluorescence,
total protein concentration, and TNF- measurements.
Specific Experimental Protocols
Group 1: Control rats. Rats (n = 4) were sham operated as described in Surgical Procedures and Ventilation. After the baseline period, the rats were instilled with the 5% albumin solution, studied for 45 min, and processed as described in General Experimental Protocol. A separate set of control rats (n = 2) was injected with a nonspecific IgG antibody to control for possible nonspecific effects from the anti-TNF-Group 2: Intestinal ischemia-reperfusion. Rats
(n = 5) were subjected to 45 min of intestinal ischemia
followed by 3 h of reperfusion as described in Surgical Procedures
and Ventilation. After the baseline period, the rats were instilled
with the 5% albumin solution and studied for 45 min. The rats were
then treated as described in General Experimental Protocol. A
separate set of rats (n = 4) was injected with a nonspecific
IgG antibody to study possible unspecific antibody effects from the
anti-TNF- MAb injection (see Group 4: Amiloride studies).
The animals were treated and instilled as described in General
Experimental Protocol. Because no effects from the nonspecific IgG
were observed on any of the studied parameters, the rats were combined
with the regular ischemia-reperfusion rats, giving a total of
nine rats, and are referred to as the ischemia-reperfusion
group. Three additional rats were subjected to 45 min of intestinal
ischemia followed by 3 h of reperfusion, and their
lungs were perfused free of blood. cAMP production was determined in
tissue samples over 10 min in 37°C as described in Pulmonary
cAMP Generation.
Group 3: Propranolol studies. Rats (n = 4) were
subjected to 45 min of intestinal ischemia followed by 3 h of
reperfusion as described in Surgical Procedures and
Ventilation. After the baseline period, the rats were instilled
with the 5% albumin solution containing 104 M
propranolol. The rats were studied for 45 min and processed as
described in General Experimental Protocol. A separate set of
rats (n = 5) was sham operated as described in Surgical
Procedures and Ventilation and instilled with the 5% albumin
solution containing 10
4 M propranolol. The rats were
studied for 45 min and processed as described in General
Experimental Protocol.
Group 4: Amiloride studies. Rats (n = 4) were subjected
to 45 min of intestinal ischemia followed by 3 h of reperfusion
as described in Surgical Procedures and Ventilation. After the
baseline period, the rats were instilled with the 5% albumin solution
containing 103 M amiloride. Amiloride was used at
10
3 M because ~50% is bound to the protein in the
instillate, and because of its relatively low molecular weight,
amiloride leaves the air spaces rapidly (24, 39). The rats were studied
for 45 min and processed as described in General Experimental
Protocol. A separate set of rats (n = 4) underwent a sham
operation as described in Surgical Procedures and Ventilation
and instilled with the 5% albumin solution containing
10
3 M amiloride. The rats were studied for 45 min
and processed as described in General Experimental Protocol.
Group 5: TNF- inhibition studies. A neutralizing
anti- TNF-
MAb (0.1 ml; IP-400, Genzyme, Cambridge, MA) originally
directed against mouse TNF-
, which also neutralized rat TNF-
, was
administered intracardially to the rats (n = 6) 30 min before induction of ischemia-reperfusion. After the
baseline period, the rats were instilled with the 5% albumin solution
and studied for 45 min. The rats were then processed as described in
General Experimental Protocol.
Alveolar Liquid Clearance
The increase in alveolar concentration of the instilled protein over 45 min was used to measure the clearance of liquid from the distal air spaces (across the alveolar epithelium and distal airway epithelium) as done before (4, 7, 8, 11, 21, 23). Data on alveolar liquid clearance are shown in two ways. First, alveolar liquid clearance is presented as a ratio of final aspirated alveolar fluid protein concentration to instilled fluid protein concentration. The final-to-instilled protein concentration ratio provides direct evidence for alveolar liquid clearance because liquid must be transported from the air spaces for the final alveolar protein concentration to rise. Because there were small changes in epithelial and endothelial permeabilities to protein (i.e., very little protein left the air spaces in any of the groups; see RESULTS), this method is accurate for measuring liquid clearance from the distal air spaces of the lungs. The second method is based on calculating alveolar liquid clearance (ALC; expressed as a percentage of instilled volume) with Eq. 1
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(1) |
Endothelial Permeability to Protein
To estimate endothelial permeability to protein, the clearance of the vascular tracer RITC-dextran into the extravascular compartments of the lungs (interstitium and air spaces) was measured. The total extravascular RITC-dextran accumulation in the alveolar liquid and the lung homogenate was measured spectrophotofluorometrically (CytoFluor 2300, Millipore, Bedford, MA) and is expressed as extravascular plasma equivalents. Passage of RITC-dextran across the endothelial-epithelial barrier was considered to be equal to that of albumin because they have similar molecular weights (70,000 vs. 67,000). The calculation of endothelial protein passage was done with the RITC-dextran concentration in the different compartments and applying them in Eq. 2
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(2) |
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A ratio between the RITC-dextran concentration in the final alveolar fluid sample and the RITC-dextran plasma concentration provided an index of equilibration of the vascular protein tracer into the alveolar compartment as in an earlier experimental study of epithelial permeability (37).
Pulmonary cAMP Generation
Lungs from six rats were used for the determination of pulmonary cAMP levels after sham operation (n = 3) and after intestinal ischemia followed by reperfusion (n = 3). After 2 h 30 min of reperfusion, the rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight; Apoteksbolaget) and 2,000 IU of heparin (Lövens, Ballerup, Denmark). An endotracheal tube (2.0 mm ID, PE-240) was inserted into the trachea through a tracheostomy. The lungs and heart were exposed through a midline sternotomy, and the lungs were mechanically ventilated with a constant-volume piston pump (Harvard Apparatus, Natick, MA). After removal of the left atrium, the lungs were perfused free of blood with 30 ml of 0.9% NaCl containing 10TNF- Analysis
Plasma levels of TNF- were determined with a commercially available
enzyme-linked immunosorbent assay specific for rat TNF-
(Genzyme).
Intra- and interassay coefficients of variation were 5.15 and 5.15%, respectively.
Statistics
All data are means ± SD. The data were analyzed with Student's t-test or one-way analysis of variance, with Tukey's test post hoc when appropriate. Differences were considered significant when P < 0.05 was reached. ![]() |
RESULTS |
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Alveolar Liquid Clearance Under Basal Conditions and After Intestinal Ischemia-Reperfusion
Anesthetized ventilated rats were instilled with 5% bovine serum albumin in 0.9% NaCl. After 45 min, a sample of the instilled solution was aspirated from the distal air spaces. The increase in total protein concentration during the 45-min period was used as a measurement of the liquid that had been cleared from the distal air spaces of the lungs. Intestinal ischemia followed by reperfusion resulted in a significant increase in alveolar liquid clearance by 76% compared with that in sham-operated control rats (P < 0.05; Fig. 1, Table 1).
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Effect of Propranolol on Alveolar Liquid Clearance
To investigate whether the increased alveolar liquid clearance after intestinal ischemia-reperfusion was mediated by
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Effect of Intestinal Ischemia-Reperfusion on Pulmonary cAMP Generation
Lung tissue samples from three sham-operated rats and three rats subjected to intestinal ischemia-reperfusion were incubated at 37°C for 10 min to determine the generation of intracellular cAMP under normal (unstimulated) conditions and after stimulation with 10There was no increase in intracellular cAMP levels after intestinal
ischemia-reperfusion compared with that in sham-operated control animals (7.74 ± 1.33 vs. 7.77 ± 1.87 pmol/l). Stimulation of adenylate cyclase with forskolin (104 M) raised
the intracellular cAMP levels over 10 min compared with the basal
levels in both the sham-operated and intestinal ischemia-reperfusion animals as expected because forskolin acts directly on adenylate cyclase without receptor stimulation.
Effect of Amiloride on Alveolar Liquid Clearance
To investigate whether the amiloride-sensitive fraction of alveolar liquid clearance was altered after intestinal ischemia-reperfusion, anesthetized ventilated rats (control animals and animals subjected to intestinal ischemia-reperfusion) were instilled with the 5% albumin solution containing amiloride (10
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Effect of Neutralizing TNF- MAbs on Alveolar Liquid
Clearance After Intestinal Ischemia-Reperfusion
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Endothelial Permeability to Protein and Extravascular Lung Water
Extravascular accumulation of RITC-dextran in the lungs, determined as described in Endothelial Permeability to Protein and expressed as extravascular plasma equivalents, was used as a measure of endothelial protein leakage. Extravascular plasma equivalents did not differ between control rats and rats subjected to intestinal ischemia-reperfusion (Fig. 5, Table 2). Furthermore, propranolol did not affect the extravascular plasma equivalent accumulation in either control or intestinal ischemia-reperfusion rats (Table 2).
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Administration of an anti-TNF- MAb before the induction of the
intestinal ischemia led to a significant increase (P < 0.05) in extravascular plasma equivalent accumulation compared with that in rats subjected to intestinal ischemia-reperfusion alone and to sham-operated control rats (Fig. 5, Table 2).
Protein influx (measured by the vascular tracer RITC-dextran) from the
plasma to the air spaces in rats subjected to intestinal ischemia-reperfusion compared with that in sham-operated
control rats was similar in both groups (Table 2). Propranolol did not affect the protein influx in either sham-operated control rats or
intestinal ischemia-reperfusion rats (Table 2). However, the alveolar-to-plasma concentration ratio of RITC-dextran was increased in
rats subjected to intestinal ischemia-reperfusion after
inhibition of TNF- with the MAb compared with that in the
sham-operated rats as well as in the rats subjected to intestinal
ischemia-reperfusion alone, although significance was not
reached (Table 2).
Extravascular lung water was measured in rats subjected to intestinal ischemia-reperfusion and in sham-operated control rats without instillation of the alveolar test solution. Extravascular lung water was similar in the rats subjected to intestinal ischemia-reperfusion (4.5 ± 0.4 g water/g dry lung; n = 4) and in the sham-operated control rats (4.5 ± 0.2 g water/g dry lung; n = 4).
Plasma Levels of TNF- After Intestinal
Ischemia-Reperfusion
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In the samples obtained from the rats at the end of the experiments,
TNF- plasma levels were increased in the intestinal ischemia-reperfusion group (Fig. 6, inset). No TNF-
was seen in the sham-operated control rats. Administration of the
neutralizing anti-TNF-
MAb inhibited the intestinal
ischemia-reperfusion-induced TNF-
generation completely.
Effects on Systemic Blood Pressure and Peak Airway Pressure From Intestinal Ischemia-Reperfusion
There were no significant differences in systemic arterial blood pressure expressed as mean arterial blood pressure, although there was a trend that the rats subjected to intestinal ischemia-reperfusion had a lower systemic blood pressure (control: 79 ± 30 mmHg, n = 11; intestinal ischemia-reperfusion: 59 ± 18 mmHg, n = 11; intestinal ischemia-reperfusion plus TNF- ![]() |
DISCUSSION |
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An intact barrier between the alveolar space and the vascular compartment is of critical importance for normal lung function. Substantial progress has been made in understanding the pathways and mechanisms regulating the clearance of protein and fluid across the alveolar and distal airway epithelia in the uninjured lung (reviewed in Ref. 20). The barrier properties may, however, change during pathological conditions such as acute lung injury and acute respiratory distress syndrome and result in severe pulmonary dysfunction. Increased endothelial permeability to protein has long been a known hallmark of acute lung injury, but it was not until recently that the alveolar and distal air space epithelial barrier has been thoroughly studied. A clinical study has shown that patients who were able to reabsorb alveolar edema fluid after acute lung injury had a better recovery from respiratory failure and a lower mortality compared with patients who were unable to reabsorb alveolar edema fluid (22).
Intestinal ischemia-reperfusion may lead to acute lung injury and acute respiratory distress syndrome characterized by increased pulmonary endothelial leakage and accumulation of inflammatory cells (15, 29). The barrier damage may be followed by an increased transvascular plasma leakage and fluid-filled alveolar spaces. In the present study, we investigated whether the injury to the lung endothelium and epithelium in the early phase of intestinal ischemia-reperfusion would be severe enough for alveolar flooding and/or interstitial pulmonary edema to occur. The study demonstrated that intestinal ischemia-reperfusion did not result in significant extravascular plasma accumulation in the alveolar spaces of the lung. Also, there seemed to be little accumulation of plasma in the interstitial spaces of the lungs. This finding was supported by the fact that there was no difference in extravascular lung water between rats subjected to intestinal ischemia-reperfusion and sham-operated control rats. This study, as well as a study by Khimenko et al. (14), demonstrated that alveolar fluid absorptive mechanisms were operational after both local lung ischemia-reperfusion and injury from remote ischemia-reperfusion such as intestinal ischemia-reperfusion injury. Because fluid is actively absorbed as a result of sodium uptake, our hypothesis was that sodium uptake was stimulated and that this resulted in an increased fluid absorption that counteracted an increased extravasation of fluid into the alveolar spaces. Our results show, in fact, that intestinal ischemia-reperfusion stimulates alveolar fluid clearance. Thus this would suggest that stimulation of alveolar fluid clearance may act as a first line of defense against alveolar flooding.
A variety of endogenous factors may be responsible for the stimulated
alveolar liquid clearance after intestinal
ischemia-reperfusion. Recent studies have suggested endogenous
release of catecholamines, especially epinephrine, as an important
mechanism for the stimulation of alveolar liquid clearance under normal
physiological conditions (7) as well as under pathological conditions
(16, 27). Finley et al. (7) showed that stimulation of alveolar liquid clearance for removal of fetal lung fluid in newborn guinea pigs in
preparation for air breathing depended on elevated plasma epinephrine levels. Under pathological conditions, it has been demonstrated that
the increased plasma epinephrine concentrations seen after septic shock
in rats (27), as well as in a model of neurogenic pulmonary edema in
dogs (16), stimulated alveolar liquid clearance. We therefore
hypothesized that intestinal ischemia-reperfusion would
lead to an increased release of endogenous catecholamines produced
during the ischemic phase, in turn leading to -adrenergic stimulation of alveolar fluid clearance during the reperfusion phase.
We consequently carried out studies using the
-adrenergic antagonist
propranolol to investigate the role of
-adrenergic-receptor stimulation and endogenous catecholamine release. We also investigated whether intracellular cAMP levels in lung tissue increased because studies (7, 8) have shown that
-adrenergic stimulation leads to
elevations in intracellular cAMP that are associated with increased
liquid clearance from the lung. However, contradictory to our
hypothesis, we found that administration of the
-adrenergic antagonist propranolol was without effect on the stimulated alveolar liquid clearance after intestinal ischemia-reperfusion,
indicating that the increased alveolar liquid clearance was not a
result of epinephrine stimulation. This was further confirmed by our cAMP studies in which there was no stimulation of intracellular cAMP
from intestinal ischemia-reperfusion. Therefore, it is unlikely that the stimulation of alveolar liquid clearance was secondary to
increased plasma epinephrine levels and
-adrenergic stimulation.
Because the increase in alveolar liquid clearance was not mediated by
the -adrenergic system and because TNF-
has been demonstrated to
stimulate alveolar liquid clearance in rats after pneumonia (28), we
hypothesized that TNF-
could be involved. It has previously been
shown that intestinal ischemia followed by reperfusion leads to
increased intestinal permeability (34) and that blood levels of
endotoxin increase after intestinal ischemia-reperfusion (33). Endotoxin stimulates monocytes and macrophages to produce and release
TNF-
in several different tissues (19, 33). Attempts have been made
to clarify the role of TNF-
in various pathological conditions,
clinically as well as in experimental animal models (5, 13, 26, 32).
Most studies carried out on the relationship between TNF-
and
intestinal ischemia-reperfusion-induced pulmonary injury have
focused on damage to the endothelial cells and not to the alveolar
epithelium. The increase in plasma TNF-
levels is suggested to be
accompanied by pulmonary neutrophil sequestration and damage to
endothelial beds remote to the original insult in the intestine (5, 32,
38), which may lead to disrupted barrier function in remote organs.
Lately, attempts have also been made to clarify the role of TNF-
in
the pathogenesis of alveolar epithelial injury in animal models of
acute lung injury (18, 28). In a model of bacterially induced acute
lung injury, increased permeability to albumin across the alveolar
epithelium was partly mediated by TNF-
(18). These investigators did
not, however, study the effects of acute lung injury on alveolar liquid clearance. In a study of Pseudomonas aeruginosa-induced
pneumonia in rats, Rezaiguia et al. (28) showed that there was an
increased alveolar liquid clearance after pneumonia in the presence of
alveolar epithelial damage. They also showed that the accelerated
alveolar liquid clearance was mediated by a TNF-
-dependent
mechanism. Therefore, our hypothesis was that intestinal
ischemia would compromise the intestinal barrier function,
leading to an increased translocation of bacterial endotoxin and/or
bacteria, which, in turn, would accumulate in the intestinal blood
vessels during the ischemic phase. On reperfusion of the ischemic
intestine, endotoxin and/or bacteria would subsequently enter the
systemic circulation and activate monocytes and macrophages to generate
and secrete TNF-
, which, in turn, would stimulate alveolar liquid
clearance. The presence of TNF-
in the lung circulation would
initially be beneficial and assist to protect the alveolar and
interstitial spaces from flooding. Blocking of TNF-
would
consequently lead to flooding of the air spaces and the lung
interstitium, with worsening of the initial lung injury as a result. We
therefore used a neutralizing monoclonal anti-TNF-
antibody to inhibit TNF-
activity. Administration of the antibody
30 min before the induction of intestinal ischemia completely
attenuated the stimulation of alveolar liquid clearance seen after
intestinal ischemia-reperfusion. Simultaneously with the
blocking of the stimulated alveolar fluid clearance, there was an
increased endothelial plasma leak and a beginning of alveolar flooding
compared with those in animals subjected to intestinal ischemia-reperfusion without administration of anti-TNF-
antibodies. This implies that the TNF-
-stimulated alveolar liquid
clearance may function as a first line of defense against alveolar
flooding and edema formation by initially accelerating fluid transport out of the alveolar compartments.
We also investigated whether TNF- stimulated alveolar liquid
clearance by increasing intracellular cAMP levels but found no such
effect. This suggests that stimulation of alveolar liquid clearance
during intestinal ischemia-reperfusion is via a pathway different from adenylate cyclase, which has previously been
demonstrated to be one main intracellular pathway for stimulating
liquid removal from the lung (7, 8). Therefore, although not tested in this study, the results suggest a direct action by the TNF-
receptor in stimulating alveolar liquid clearance. In fact, preliminary data
from Jayr et al. (12) suggest that direct binding of TNF-
to its
receptor in A549 pulmonary epithelial cells is directly linked with an
increased capacity to transport sodium. However, in neither that study
nor our investigation has the intracellular signaling pathway been
definitively identified.
This is somewhat in contradiction to findings described by other
authors, although one should bear in mind that anti-TNF- therapy was
shown to be inefficient in clinical trials on septic patients (1) and
that treatment of septic rats with human recombinant TNF-
improved
mortality and other parameters (2). Sorkine et al. (32) reported an
increased leakage of radiolabeled albumin from the blood to the
alveolar spaces after intestinal ischemia-reperfusion and that
blocking of TNF-
reduces the lung injury. Caty et al. (5) also
showed that intestinal ischemia-reperfusion induced a pulmonary
microvascular injury that was prevented by pretreating the rats with
anti-TNF-
antibodies. One explanation may be the different time
periods of ischemia as well as the total study times used in
the different investigations. In the study by Caty et al. (5), the
duration of ischemia was 120 min compared with 45 min used in
our model. At these longer study times, the injury may become too
severe to the endothelium and epithelium and an increased absorption of
fluid is not enough to maintain dry alveolar spaces. Also, if the
epithelium and endothelium become severely injured and large holes open
up in the barrier, the sodium gradient necessary for transepithelial
fluid absorption cannot form. As a result, alveolar and interstitial
flooding may occur.
Alveolar liquid clearance depends, in part, on amiloride-sensitive pathways (7, 23). Intra-alveolar amiloride inhibited 40% of the alveolar liquid clearance after intestinal ischemia-reperfusion as well as in the sham-operated animals in the present study, which is similar to what has previously been reported (11, 14, 23). This indicates that intestinal ischemia-reperfusion increases alveolar liquid clearance by stimulation of both amiloride-sensitive and amiloride-insensitive pathways. In fact, recent data (6, 30) suggest the existence of a rod-type cyclic nucleotide-gated cation channel in the alveolar epithelium that may be involved in fluid movement in the lung.
In conclusion, we found that intestinal ischemia-reperfusion in
rats leads to stimulation of alveolar liquid clearance and that this
stimulation is mediated, at least in part, by a TNF--dependent mechanism. Once TNF-
is eliminated, the alveolar
epithelial-endothelial barrier cannot withstand the pressure from the
circulation and plasma enters the extravascular compartments in the
lung. Our results therefore suggest that TNF-
may act as a first
line of defense against alveolar flooding in rats, at least during the early phase of intestinal ischemia-reperfusion.
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ACKNOWLEDGEMENTS |
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This study was supported by grants from the Swedish Natural Science Research Council, the Crafoord Foundation, the Magnus Bergwall Foundation, the Åke Wiberg Foundation, and the Hierta Retzius Foundation for Scientific Research and by Medical Research Council Grant 11236.
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: H. G. Folkesson, Department of Animal Physiology, Lund University, Helgonavägen 3 B, SE-223 62 Lund, Sweden (E-mail: Hans.Folkesson{at}zoofys.lu.se).
Received 15 March 1999; accepted in final form 20 August 1999.
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REFERENCES |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abraham, E.,
A. Anzueto,
G. Gutierrez,
S. Tessler,
G. San Pedro,
R. Wunderink,
A. Dal Nogare,
S. Nasraway,
S. Berman,
R. Cooney,
H. Levy,
R. Baughman,
M. Rumbak,
R. B. Light,
L. Poole,
R. Allred,
J. Constant,
J. Pennington,
and
S. Porter.
Double-blind randomised controlled trial of monoclonal antibody to human tumour necrosis factor in treatment of septic shock. NORASEPT II Study Group.
Lancet
351:
929-933,
1998[ISI][Medline].
2.
Alexander, H. R.,
B. C. Sheppard,
J. C. Jensen,
H. N. Langstein,
C. M. Buresh,
D. Venzon,
E. C. Walker,
D. L. Fraker,
M. C. Stovroff,
and
J. A. Norton.
Treatment with recombinant human tumor necrosis factor-alpha protects rats against the lethality, hypotension, and hypothermia of gram-negative sepsis.
J. Clin. Invest.
88:
34-39,
1991[ISI][Medline].
3.
Ballard, S. T.,
S. M. Schepens,
J. C. Falcone,
G. A. Meininger,
and
A. E. Taylor.
Regional bioelectric properties of porcine airway epithelium.
J. Appl. Physiol.
73:
2021-2027,
1992
4.
Berthiaume, Y.,
N. C. Staub,
and
M. A. Matthay.
Beta-adrenergic agonists increase lung liquid clearance in anesthetized sheep.
J. Clin. Invest.
79:
335-343,
1987[ISI][Medline].
5.
Caty, M. G.,
K. S. Guice,
K. T. Oldham,
D. G. Remick,
and
S. I. Kunkel.
Evidence for tumor necrosis factor-induced pulmonary microvascular injury after intestinal ischemia-reperfusion injury.
Ann. Surg.
212:
694-700,
1990[ISI][Medline].
6.
Ding, C.,
E. D. Potter,
W. Qiu,
S. L. Coon,
M. A. Levine,
and
S. E. Guggino.
Cloning and widespread distribution of the rat rod-type cyclic nucleotide-gated cation channel.
Am. J. Physiol. Cell Physiol.
272:
C1335-C1344,
1997
7.
Finley, N.,
A. Norlin,
D. L. Baines,
and
H. G. Folkesson.
Alveolar epithelial fluid clearance is mediated by endogenous catecholamines at birth in guinea pigs.
J. Clin. Invest.
101:
972-981,
1998
8.
Folkesson, H. G.,
J.-F. Pittet,
G. Nitenberg,
and
M. A. Matthay.
Transforming growth factor- increases alveolar liquid clearance in anesthetized ventilated rats.
Am. J. Physiol. Lung Cell. Mol. Physiol.
271:
L236-L244,
1996
9.
Harward, T. R. S.,
D. L. Brooks,
T. C. Flynn,
and
J. M Seeger.
Multiple organ dysfunction after mesenteric artery revascularization.
J. Vasc. Surg.
18:
459-469,
1993[ISI][Medline].
10.
Hernandez, L. A.,
M. B. Grisham,
B. Twohig,
K. E. Arfors,
J. M. Harlan,
and
D. N. Granger.
Role of neutrophils in ischemia-reperfusion-induced microvascular injury.
Am. J. Physiol. Heart Circ. Physiol.
253:
H699-H703,
1987
11.
Jayr, C.,
C. Garat,
M. Meignan,
J. F. Pittet,
M. Zelter,
and
M. A. Matthay.
Alveolar liquid and protein clearance in anesthetized ventilated rats.
J. Appl. Physiol.
76:
2636-2642,
1994
12.
Jayr, C.,
Y. Wang,
H. G. Folkesson,
A. Lazrak,
S. Matalon,
R. Lucas,
and
M. A. Matthay.
Mechanisms responsible for TNF--induced increase in alveolar epithelial sodium and fluid transport in rats and A549 cells (Abstract).
Am. J. Respir. Crit. Care Med.
159:
A292,
1999[ISI].
13.
Khimenko, P. L.,
G. J. Bagby,
J. Fuseler,
and
A. E. Taylor.
Tumor necrosis factor- in ischemia and reperfusion injury in rat lungs.
J. Appl. Physiol.
85:
2005-2011,
1998
14.
Khimenko, P. L.,
J. W. Barnard,
T. M. Moore,
P. S. Wilson,
S. T. Ballard,
and
A. E. Taylor.
Vascular permeability and epithelial transport effects on lung edema formation in ischemia and reperfusion.
J. Appl. Physiol.
77:
1116-1121,
1994
15.
Koike, K.,
F. A. Moore,
E. E. Moore,
R. S. Poggetti,
R. M. Tuder,
and
A. Banerjee.
Endotoxin after gut ischemia/reperfusion causes irreversible lung injury.
J. Surg. Res.
52:
656-662,
1992[ISI][Medline].
16.
Lane, S. M.,
K. C. Maender,
E. A. Awender,
and
M. B. Maron.
Adrenal epinephrine increases alveolar liquid clearance in a canine model of neurogenic pulmonary edema.
Am. J. Respir. Crit. Care Med.
158:
760-768,
1998
17.
Levy, P. J.,
M. M. Krausz,
and
J. Manny.
Acute mesenteric ischaemia: improved resultsa retrospective analysis of ninety-two patients.
Surgery
107:
372-380,
1990[ISI][Medline].
18.
Li, X. Y.,
K. Donaldson,
D. Brown,
and
W. MacNee.
The role of tumor necrosis factor in increased airspace epithelial permeability in acute lung inflammation.
Am. J. Respir. Cell Mol. Biol.
13:
185-195,
1995[Abstract].
19.
Maier, R. V.
Endotoxin requirements for alveolar macrophage stimulation.
J. Trauma
30:
S49-S57,
1990[ISI][Medline].
20.
Matthay, M. A.,
H. G. Folkesson,
and
A. S. Verkman.
Salt and water transport across alveolar and distal airway epithelia in the adult lung.
Am. J. Physiol. Lung Cell. Mol. Physiol.
270:
L487-L503,
1996
21.
Matthay, M. A.,
C. C. Landolt,
and
N. C. Staub.
Differential liquid and protein clearance from the alveoli of anesthetized sheep.
J. Appl. Physiol.
53:
96-104,
1982
22.
Matthay, M. A.,
and
J. P. Wiener-Kronish.
Intact epithelial barrier function is critical for the resolution of alveolar edema in humans.
Am. Rev. Respir. Dis.
142:
1250-1257,
1990[ISI][Medline].
23.
Norlin, A.,
N. Finley,
P. Abendinpour,
and
H. G. Folkesson.
Alveolar liquid clearance in the anesthetized ventilated guinea pig.
Am. J. Physiol. Lung Cell. Mol. Physiol.
274:
L235-L243,
1998
24.
O'Brodovich, H.,
V. Hannam,
and
B. Rafii.
Sodium channel but neither Na+-H+ nor Na-glucose symport inhibitors slow neonatal lung water clearance.
Am. J. Respir. Cell Mol. Biol.
5:
377-384,
1991[ISI][Medline].
25.
Pacitti, A. J.,
Y. Inoue,
and
Y. Y. Souba.
Tumor necrosis factor stimulates amino acid transport in plasma membrane vesicles from rat liver.
J. Clin. Invest.
91:
474-483,
1993[ISI][Medline].
26.
Pinsky, M. R.,
J.-L. Vincent,
J. Deviere,
M. Alegre,
R. J. Kahn,
and
E. Dupont.
Serum cytokine levels in human septic shock. Relation to multiple-system organ failure and mortality.
Chest
103:
565-575,
1993[Abstract].
27.
Pittet, J. F.,
J. P. Wiener-Kronish,
M. C. McElroy,
H. G. Folkesson,
and
M. A. Matthay.
Stimulation of lung epithelial liquid clearance by endogenous release of catecholamines in septic shock in anesthetized rats.
J. Clin. Invest.
94:
663-671,
1994[ISI][Medline].
28.
Rezaiguia, S.,
C. Garat,
C. Delclaux,
M. Meignan,
J. Fleury,
P. Legrand,
M. Matthay,
and
C. Jayr.
Acute bacterial pneumonia in rats increases alveolar epithelial fluid clearance by a tumor necrosis factor-alpha-dependent mechanism.
J. Clin. Invest.
99:
325-335,
1997
29.
Schmeling, D. J.,
M. G. Caty,
K. T. Oldham,
K. S. Guice,
and
D. B. Hinshaw.
Evidence for neutrophil-related acute lung injury after intestinal ischemia-reperfusion.
Surgery
106:
195-202,
1989[ISI][Medline].
30.
Schwiebert, E. M.,
E. D. Potter,
T.-H. Hwang,
J. S. Woo,
C. Ding,
W. Qiu,
W. B. Guggino,
M. A. Levine,
and
S. E. Guggino.
cGMP stimulates sodium and chloride currents in rat tracheal airway epithelia.
Am. J. Physiol. Cell Physiol.
272:
C911-C922,
1997
31.
Simpson, R.,
R. Alon,
L. Kobzik,
R. Valeri,
D. Shepro,
and
H. B. Hechtman.
Neutrophil and nonneutrophil-mediated injury in intestinal ischemia-reperfusion.
Ann. Surg.
218:
444-454,
1993[ISI][Medline].
32.
Sorkine, P.,
A. Setton,
P. Halpern,
A. Miller,
V. Rudick,
S. Marmor,
J. M. Klausner,
and
G. Goldman.
Soluble tumor necrosis factor receptors reduce bowel ischemia-induced lung permeability and neutrophil sequestration.
Crit. Care Med.
23:
1377-1381,
1995[ISI][Medline].
33.
Sorkine, P.,
O. Szold,
P. Halpern,
M. Gutman,
M. Greemland,
V. Rudick,
and
G. Goldman.
Gut decontamination reduces bowel ischemia-induced lung injury in rats.
Chest
112:
491-495,
1997
34.
Sun, Z.,
X. Wang,
X. Deng,
Å. Lasson,
R. Wallén,
E. Hallberg,
and
R. Andersson.
The influence of intestinal ischemia and reperfusion on bidirectional intestinal barrier permeability, cellular membrane integrity, proteinase inhibitors, and cell death in rats.
Shock
10:
203-212,
1998[ISI][Medline].
35.
Turnage, R. H.,
K. S. Guice,
and
K. T. Oldham.
Endotoxemia and remote organ injury following intestinal reperfusion.
J. Surg. Res.
56:
571-578,
1994[ISI][Medline].
36.
Turnage, R. H.,
K. M. Kadesky,
L. Bartula,
and
S. I Myers.
Pulmonary thromboxane release following intestinal reperfusion.
J. Surg. Res.
58:
552-557,
1995[ISI][Medline].
37.
Wiener-Kronish, J. P.,
K. H. Albertine,
and
M. A. Matthay.
Differential responses of the endothelial and epithelial barriers of the lung in sheep to Escherichia coli endotoxin.
J. Clin. Invest.
88:
864-875,
1991[ISI][Medline].
38.
Yao, Y. M.,
S. Bahrami,
H. Redl,
and
G. Schlag.
Monoclonal antibody to tumor necrosis factor- attenuates hemodynamic dysfunction secondary to intestinal ischemia/reperfusion in rats.
Crit. Care Med.
24:
1547-1553,
1996[ISI][Medline].
39.
Yue, G.,
and
S. Matalon.
Mechanisms and sequelae of increased alveolar fluid clearance in hyperoxic rats.
Am. J. Physiol. Lung Cell. Mol. Physiol.
272:
L407-L412,
1997
40.
Xiao, F.,
M. J. Eppihimer,
B. H. Willis,
and
D. L. Carden.
Complement-mediated lung injury and neutrophil retention after intestinal ischemia-reperfusion.
J. Appl. Physiol.
82:
1459-1465,
1997