Departments of Anesthesia and Medicine and Cardiovascular Research Institute, University of California, San Francisco, California 94143
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Activation of -adrenergic receptors in the
lung is an important mechanism that can prevent alveolar flooding after
brief but severe hemorrhagic shock. However, a neutrophil-dependent oxidant injury to the alveolar epithelium prevents the normal upregulation of alveolar fluid clearance by catecholamines after prolonged hemorrhagic shock. Because hemorrhage increases
proinflammatory cytokine expression in the lung partly through the
activation of
-adrenergic receptors, the objective of this study was
to determine whether
-adrenergic blockade would restore the normal fluid transport capacity of the alveolar epithelium after hemorrhagic shock. Hemorrhagic shock was associated with a significant increase of
interleukin-1
(IL-1
) concentration in the lung and a failure of
the alveolar epithelium to respond to
-adrenergic agonists, with the
upregulation of vectorial fluid transport despite intra-alveolar administration of exogenous catecholamines. In contrast,
catecholamine-mediated upregulation of alveolar liquid clearance was
restored by pretreatment with phentolamine, an
-adrenergic-receptor
antagonist. Phentolamine pretreatment also significantly attenuated the
shock-mediated increase of IL-1
concentration in the lung.
Additional experiments demonstrated that the inhibition of IL-1
binding to its receptor by the administration of IL-1-receptor
antagonist restored the normal fluid transport capacity of the alveolar
epithelium after hemorrhagic shock. In summary, the results of these
studies indicate that the activation of
-adrenergic receptors after
hemorrhagic shock prevents the
-adrenergic-dependent upregulation of
alveolar fluid clearance by modulating the severity of the pulmonary
inflammatory response.
-adrenergic-receptor antagonist; alveolar liquid clearance; oxidative injury; interleukin-1; interleukin-1-receptor
antagonist
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
endogenous release of catecholamines is a major mechanism that prevents alveolar flooding after the onset of septic or short-term hemorrhagic shock by upregulating vectorial, sodium-dependent fluid transport across the alveolar epithelium (13, 17, 18). However, after prolonged hemorrhagic shock, upregulation of alveolar fluid clearance by catecholamines was prevented by a neutrophil-dependent injury to the alveolar epithelium that occurred in the absence of a major alteration in paracellular permeability to protein (14). Because the expected catecholamine-mediated upregulation of alveolar liquid clearance was restored by normalization of the concentration of reduced glutathione in alveolar epithelial lining fluid by N-acetylcysteine or by inhibition of the conversion of xanthine dehydrogenase to xanthine oxidase, these findings indicated that the decrease in the fluid transport capacity of the alveolar epithelium after hemorrhagic shock was mediated by oxidant stress (14).
Abnormalities in cytokine expression are involved in the development of
the inflammatory response after hemorrhagic shock (24) and depend in
part on the presence of shock-mediated oxidant stress (25). Recently,
Le Tulzo et al. (11) found that the activation of nuclear factor-B
(NF-
B), as well as the increased gene expression of proinflammatory
cytokines in pulmonary mononuclear cell populations after hemorrhagic
shock in mice, was prevented by
-adrenergic blockade. These findings
suggested that the activation of NF-
B through an
-adrenergic
pathway is an important mechanism by which hemorrhagic shock increases
proinflammatory cytokine expression in the lungs. However, the impact
of this mechanism on lung fluid balance and alveolar fluid transport
several hours after fluid resuscitation from hemorrhagic shock has not
been studied.
Therefore, the first objective of these studies was to determine
whether -adrenergic blockade would protect against oxidative injury
and restore the normal fluid transport capacity of the alveolar
epithelium after hemorrhagic shock. Catecholamine-mediated upregulation
of alveolar liquid clearance was restored by pretreatment with
phentolamine, an
-adrenergic-receptor antagonist. Phentolamine pretreatment was also associated with a significant decrease in the
immunoreactive interleukin-1
(IL-1
) content of the lung homogenate of hemorrhaged rats. Thus the second objective of these studies was to determine whether the inhibition of IL-1
binding to
its receptor by the administration of IL-1-receptor antagonist (IL-1ra)
would protect against the shock-mediated oxidative injury to the
alveolar epithelium and thereby restore the normal fluid transport
capacity of the alveolar epithelium after hemorrhagic shock.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The protocol for these studies was approved by the University of California, San Francisco Animal Research Committee.
Lung Barrier Function Studies
Surgical preparation and ventilation.
Male Sprague-Dawley rats (n = 48) weighing
300-350 g were anesthetized with pentobarbital sodium (60 mg/kg
ip), and anesthesia was maintained with 30 mg/kg of pentobarbital
sodium every 2 h. An endotracheal tube (PE-220) was inserted through a
tracheotomy. Pancuronium bromide (0.3 mg · kg1 · h
1
iv) was given for neuromuscular blockade. Catheters (PE-50) were inserted into both carotid arteries to monitor systemic arterial pressure, obtain blood samples, and withdraw blood for induction of
hemorrhagic shock. A catheter was also inserted into the jugular vein
to monitor central venous pressure. The rats were maintained in the
left lateral decubitus position during the experiments and were
ventilated with a constant-volume pump (Harvard, Millis, MA) with an
inspired oxygen fraction of 1.0, peak airway pressures of 8-12
cmH2O supplemented with positive
end-expiratory pressure of 3 cmH2O. The respiratory rate was
adjusted to maintain the arterial
PCO2 between 35 and 40 mmHg during
the baseline period and to correct the metabolic acidosis after the
hemorrhagic shock.
Preparation of instillate. A 5% bovine albumin solution was prepared using Ringer lactate and adjusted with NaCl to be isosmolar with the rat's circulating plasma as previously published (13). Anhydrous Evans blue dye (0.5 mg) was added to the albumin solution to confirm the location of the instillate at the end of the study, and 1 µCi of 125I-labeled human serum albumin (Frosst Laboratories, Quebec, Canada) was also added to the albumin solution. The 125I-albumin served as the alveolar protein tracer in all experiments. A sample of the instilled solution was saved for total protein measurement, radioactivity counts, and water-to-dry weight ratio measurements so that the dry weight of the protein solution could be subtracted from the final lung water calculation. In some studies, epinephrine was added to the instillate.
General protocol.
In all experiments after the surgery, heart rate and systemic blood
pressure were allowed to stabilize for 60 min (Fig.
1). The rat was placed in the left lateral
decubitus position to facilitate liquid deposition into the left lung
120 min after the onset of hemorrhagic shock. Hemorrhagic shock was
induced by withdrawing blood from the carotid artery to maintain a mean
systemic arterial pressure of 30-35 mmHg for 60 min. This
corresponded to the removal of 9-12 ml of blood. After 1 h of
hemorrhagic shock, the rats were resuscitated with intravascular 4%
albumin solution in 0.9% NaCl over 30 min to maintain a central venous
pressure <8 mmHg as we have done before (13, 14). The volume of 4%
albumin solution administered was twice the amount of blood withdrawn.
|
Specific Protocols
Group 1. Effect of hemorrhagic shock and fluid resuscitation on alveolar liquid clearance. Rats (n = 5) were hemorrhaged and resuscitated to determine the effect of hemorrhagic shock and fluid resuscitation on fluid transport across the lung epithelium. Two hours after the onset of hemorrhagic shock, 3 ml/kg of the 5% bovine albumin solution with 1 µCi of 125I-albumin were instilled into the left lung. Then the rats were studied as described in General protocol. Control studies included rats (n = 9) that underwent the same surgical preparation and were studied for the same period of time but were neither hemorrhaged nor fluid resuscitated.
Group 2. Stimulation with exogenous -adrenergic
agonists after hemorrhagic shock.
Because there was no upregulation of alveolar fluid clearance after
hemorrhagic shock, the next series of experiments included rats
(n = 5) that were hemorrhaged and
fluid resuscitated and that had their air spaces instilled with a 5%
bovine albumin solution containing 1 µCi of
125I-albumin and epinephrine
(10
5 M). Control studies
included rats (n = 3) that were
neither hemorrhaged nor fluid resuscitated, but their distal air spaces
were instilled with an albumin solution containing 1 µCi of
125I-albumin and epinephrine
(10
5 M).
Group 3. Pretreatment with phentolamine in rats that underwent
hemorrhagic shock and fluid resuscitation.
Because hemorrhage increases proinflammatory cytokine expression in the
lung through the activation of -adrenergic receptors (11), rats
(n = 7) were pretreated with the
-adrenergic-receptor antagonist phentolamine (Sigma) given (10 mg/kg
ip) 30 min before the onset of hemorrhagic shock. This intraperitoneal
dose of phentolamine has previously been shown to produce an
-adrenergic blockade in mice as determined by its effects on
-adrenergic-induced alterations in intestinal transit (23), insulin
secretion (8), and release of endogenous catecholamines (7). Then rats
were hemorrhaged and fluid resuscitated. Two hours after the onset of
hemorrhagic shock, 3 ml/kg of the 5% bovine albumin solution with 1 µCi of 125I-albumin and
epinephrine (10
5 M) were
instilled into the left lung. Control studies included rats
(n = 3) that were pretreated with
phentolamine (10 mg/kg) and had their air spaces instilled with
epinephrine solution (10
5
M), but did not undergo hemorrhagic shock and fluid resuscitation. In
pilot experiments (n = 5), we found
that pretreatment with phentolamine (10 mg/kg ip) did not affect
alveolar liquid clearance in control rats.
Group 4. Pretreatment with IL-1ra in rats that underwent
hemorrhagic shock and fluid resuscitation.
Because the restoration of the ability of the alveolar epithelium to
upregulate vectorial fluid transport in response to catecholamines after pretreatment with phentolamine was associated with a significant decrease in the immunoreactive IL-1 content of the lung homogenate of hemorrhaged rats, we hypothesized that the inhibition of IL-1
binding to its receptor by the administration of IL-1ra would restore
the normal fluid transport capacity to the alveolar epithelium after
hemorrhagic shock. Therefore, rats (n = 3) were pretreated with IL-1ra (Amgen, Boulder, CO) given as a
continuous intravenous infusion of 25 mg · kg
1 · h
1,
a dose that has been shown to effectively block IL-1
in rats (P. Lee, Amgen, personal communication), started 30 min before the onset of
hemorrhagic shock and continued until the end of the experiment. Then
rats were hemorrhaged and fluid resuscitated. Five hours after the
onset of hemorrhagic shock, 3 ml/kg of the 5% bovine albumin solution
with 1 µCi of 125I-albumin and
epinephrine (10
5 M) were
instilled into the left lung. Control studies included rats
(n = 5) that were pretreated with
IL-1ra and had their air spaces instilled with 3 ml/kg of the 5%
bovine albumin solution with 1 µCi of
125I-albumin and epinephrine
(10
5 M) but did not undergo
hemorrhagic shock and fluid resuscitation. In pilot experiments
(n = 3), we found that pretreatment
with IL-1ra did not affect alveolar liquid clearance in control rats.
IL-1 Measurement in Lung Homogenate
Measurements
Hemodynamics, pulmonary gas exchange, and protein concentration. Systemic arterial, central venous, and airway pressures were continuously measured. Arterial blood gases were measured at 1-h intervals. Samples from the instilled protein solution, from final distal air space fluid, and from initial and final blood were collected to measure total protein concentration with an automated analyzer (AA2 Technicon, Tarrytown, NY).
Albumin flux across endothelial and epithelial barriers. Two different methods were used to measure the flux of albumin across the lung endothelial and epithelial barriers as done before (13, 14). The first method measures residual 125I-albumin (the air space protein tracer) in the lungs as well as accumulation of 125I-albumin in plasma. The second method measures 131I-albumin (the vascular protein tracer) in the extravascular spaces of the lungs.
The total quantity of 125I-albumin (the air space protein tracer) instilled into the lung was determined by measuring duplicate samples of the instilled solution for total radioactivity counts (counts · minAlveolar liquid clearance. Changes in the concentration of the nonlabeled bovine albumin and the instilled 125I-albumin over the study period (1 h) were used to measure liquid clearance from the distal air spaces as we have done before (13, 14). There is a good correlation between the changes in the concentration of instilled nonlabeled bovine albumin and 125I-albumin. Because some reabsorption may have occurred across distal bronchial epithelium, the term alveolar does not imply that all fluid reabsorption occurred at the alveolar level.
Lung liquid clearance (excess lung water measurement). The extravascular water content of the lungs was measured by the gravimetric method as in prior studies (13). In addition, changes in the water-to-dry weight ratio of the noninstilled lung were used as an index of lung endothelial injury since the noninstilled lung did not have the confounding presence of the instilled protein solution in its air spaces.
Tracer binding measurement. To determine 125I binding to albumin, trichloroacetic acid (20%) was added to all tubes, which were then centrifuged to obtain the supernatant for measurement of free 125I radioactivity. The results are expressed as a percentage of the unbound 125I radioactivity to the total amount of 125I-albumin radioactivity instilled. These fluid samples always had <1% of unbound iodine present.
IL-1 ELISA.
After the lung vascular bed had been flushed by injecting 25 ml of
chilled (4°C) PBS into the right ventricle, lung homogenates were
prepared by snap freezing isolated lung in liquid nitrogen. The lungs
were then homogenized (30-s pulse on ice) in lysis buffer containing
1% Nonidet P-40, 50 mM HEPES, 500 mM NaCl, 1 mg/ml leupeptin (Sigma
Chemical) and 1 mg/ml phenylmethylsulfonyl fluoride (Sigma Chemical).
The homogenates were centrifuged at 2,500 rpm at 4°C for 10 min,
and the supernatants were collected. Immunoreactive IL-1
was
quantitated using a commercially available ELISA kit specific for rat
IL-1
(Endogen, Boston, MA) as previously published (11). With this
assay, the threshold of sensitivity for IL-1
is 3 pg/ml.
Statistics
All the data are summarized as means ± SE. One-way ANOVA was used to compare experimental with control groups, followed by Fisher's exact t-test. P < 0.05 was considered statistically significant. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Systemic Hemodynamics and Arterial Blood Gases
To induce hemorrhagic shock and maintain a systemic mean arterial pressure of 30-35 mmHg for 60 min, 42 ± 3% of the blood volume was withdrawn. This corresponded to the removal of 10.6 ± 0.8 ml of blood. The development of hemorrhagic shock resulted in severe systemic arterial hypotension (Table 1) and metabolic acidosis (Table 2). The final calculated base deficit of the arterial blood at the end of the ischemic phase of hemorrhagic shock was 16.5 ± 1.7 in hemorrhaged rats vs. 1.0 ± 0.7 in controls (P < 0.05, Table 2). The hemorrhaged rats were resuscitated over 30 min with twice the volume of shed blood using a 4% bovine albumin solution. At the end of the studies, all physiological parameters in hemorrhaged rats were comparable to those measured in controls (Tables 1 and 2).
|
|
In rats pretreated with intraperitoneal phentolamine, 38 ± 4% of the blood volume was withdrawn (9.6 ± 0.8 ml) to induce hemorrhagic shock and maintain a systemic mean arterial pressure of 30-35 mmHg for 60 min. There was no difference between the severity of hemorrhagic shock (mean arterial pressure, arterial pH, base deficit) in hemorrhaged rats that were or were not pretreated with phentolamine before hemorrhagic shock (Tables 1 and 2).
In rats pretreated with IL-1ra, 42 ± 2% of the blood volume was withdrawn (10.7 ± 0.5 ml) to induce hemorrhagic shock and maintain a systemic mean arterial pressure of 30-35 mmHg for 60 min. There was no difference between the severity of hemorrhagic shock (mean arterial pressure, arterial pH, base deficit) in hemorrhaged rats that were or were not pretreated with IL-1ra prior to hemorrhagic shock (Tables 1 and 2).
Alveolar and Lung Liquid Clearances
In contrast to our results in rats that underwent short-term hemorrhagic shock and fluid resuscitation (13), there was no upregulation of alveolar liquid clearance after hemorrhagic shock. The final-to-initial distal air space unlabeled protein concentration ratio was 1.28 ± 0.02 in control rats and 1.34 ± 0.01 in hemorrhaged rats (Table 3). Alveolar liquid clearance was comparable in control and hemorrhaged rats, measured either with the nonlabeled bovine albumin (29 ± 3 vs. 33 ± 1%, Fig. 2) or with the 125I-albumin (26 ± 0.03 vs. 27 ± 0.02%). Lung liquid clearance was also not different between control and hemorrhaged rats (Fig. 3). When exogenous epinephrine was added to the protein solution instilled into the distal air spaces of the lung, there was a significant increase in the alveolar and lung liquid clearance in control but not in hemorrhaged and fluid-resuscitated rats (Figs. 2 and 3). The inability of the alveolar epithelium to upregulate vectorial fluid transport in response to catecholamines was associated with a significant increase in immunoreactive IL-1
|
|
|
|
In contrast, after pretreatment of hemorrhaged rats with
intraperitoneal phentolamine, there was a restoration of the normal upregulation of alveolar liquid clearance in response to the
instillation of epinephrine
(105 M) in the distal air
spaces (Fig. 2), corresponding to a significant increase in the
final-to-initial distal air space unlabeled protein concentration ratio
(Table 3). Comparable results were obtained for lung liquid clearance
(Fig. 3). The restoration of the ability of the alveolar epithelium to
respond to catecholamines by upregulating vectorial fluid transport in
response to catecholamines after pretreatment with intraperitoneal
phentolamine was associated with a significant decrease in the
immunoreactive IL-1
protein content in the lung homogenate compared
with values measured in non-pretreated but hemorrhaged and
fluid-resuscitated rats (Fig. 4).
After pretreatment of hemorrhaged rats with IL-1ra, there was also a
restoration of the normal upregulation of alveolar liquid clearance in
response to the instillation of epinephrine
(105 M) in the distal air
spaces (Fig. 2), corresponding to a significant increase in
final-to-initial distal air space unlabeled protein concentration ratio
(Table 3). Comparable results were obtained for lung liquid clearance
(Fig. 3).
Lung Endothelial and Alveolar Epithelial Permeability to Protein
Hemorrhagic shock and fluid resuscitation were associated with a modest increase in the endothelial permeability to protein as indicated by the significant increase in extravascular plasma equivalents of noninstilled lung in hemorrhaged rats compared with controls (0.14 ± 0.02 vs. 0.38 ± 0.11 ml, P < 0.05). Interestingly, pretreatment with intraperitoneal phentolamine 30 min before the onset of hemorrhagic shock was associated with normalization of the endothelial permeability to protein. There was a significant decrease in extravascular plasma equivalents of the noninstilled lung in hemorrhaged rats pretreated with phentolamine prior to the onset of shock compared with the values measured in hemorrhaged rats without prior pretreatment (0.11 ± 0.05 vs. 0.38 ± 0.11 ml, P < 0.05). Comparable results were obtained in hemorrhaged rats that were pretreated with IL-1ra compared with the values measured in hemorrhaged rats without prior pretreatment (0.10 ± 0.03 vs. 0.38 ± 0.11 ml, P < 0.05).There was no increase in the protein flux
(125I-albumin) from the air spaces
to the plasma in all experimental groups. The percentages of the
instilled 125I-albumin recovered
in the lungs and in the blood at the end of experiments were comparable
in hemorrhaged and in control rats that were or were not pretreated
with intraperitoneal phentolamine or IL-1ra (Table
4).
|
Finally, arterial PO2 and PCO2 were not statistically different between the experimental groups throughout the study (data not shown).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The overall objective of these studies was to determine whether
-adrenergic blockade would protect against injury to the alveolar
epithelium after hemorrhagic shock. Because the preservation of the
capacity of the alveolar epithelium to actively remove fluid from the
air spaces is critical for the survival of patients with acute lung
injury (12), we developed an experimental animal model to investigate
the effect of hemorrhagic shock on the function of the alveolar
epithelium. We found that the release of endogenous catecholamines
upregulates vectorial, sodium-dependent fluid transport across the
alveolar epithelium during the ischemic phase of hemorrhagic shock as
well as after fluid resuscitation following brief hemorrhagic shock
(13, 17). Interestingly, we have found that this protective mechanism
was inhibited after hemorrhagic shock because of an oxidative stress to
the alveolar epithelium (14). Inasmuch as it has been shown that the
activation of NF-
B through an
-adrenergic pathway is one of the
important mechanisms by which hemorrhagic shock increases
proinflammatory cytokine expression in the lungs (11), we hypothesized
that
-adrenergic blockade would protect the alveolar epithelium
against shock-mediated cytokine-associated injury to the alveolar epithelium.
Therefore, the first objective of these studies was to determine
whether -adrenergic blockade would restore the normal fluid transport capacity of the alveolar epithelium after hemorrhagic shock.
The results indicate that pretreatment with phentolamine restored the
ability of the alveolar epithelium to respond to
-adrenergic
agonists by upregulating alveolar fluid clearance in hemorrhaged and
fluid-resuscitated rats. These results were not explained by a
nonspecific effect of phentolamine on vectorial fluid transport across
the alveolar epithelium. Indeed, pretreatment with phentolamine did not
modify basal alveolar liquid clearance (pilot experiments) or the
normal response of the alveolar epithelium to exogenous catecholamines
in control nonhemorrhaged rats (Fig. 2).
There are several mechanisms that could explain the protective effect
of -adrenergic blockade against the shock-mediated cytokine-associated injury to the alveolar epithelium. First, hemorrhage-induced
-adrenergic stimulation could enhance NF-
B activation and proinflammatory cytokine expression in the lung by
directly increasing the production of reactive oxygen species. Catecholamines cause the formation of oxygen radical species through the degradation of quinones (19) or through the release of iron from
ferritin under anaerobic conditions (1). Activation of NF-
B in the
pulmonary mononuclear cell populations after the onset of hemorrhagic
shock has been shown to be dependent on the presence of
xanthine-derived oxygen radicals (25). Inhibition of the conversion of
xanthine dehydrogenase to reactive oxygen species generating xanthine
oxidase by two different strategies, either by a tungsten-enriched diet
or by pretreatment with allopurinol (14) or inhibition of the
activation of NF-
B by sulfasalazine (15), restored the normal fluid
transport capacity of the alveolar epithelium after hemorrhagic shock.
Second,
-adrenergic stimulation causes the activation of protein
kinase C and thus increases intracellular calcium (2). NF-
B can be
activated either by protein kinase C-mediated phosphorylation of I
B
(4) or by proteolytic degradation of the COOH-terminal protein sequence
of I
B by calcium-dependent intracellular proteases (3). Taken
together, these studies indicate that
-adrenergic stimulation may be
partly responsible for the reactive oxygen species-dependent injury to
the alveolar epithelium after hemorrhagic shock. Third, phentolamine
might have affected alveolar liquid clearance in hemorrhaged rats by mechanisms that are unrelated to its inhibition of the release of
oxygen radicals. For example, phentolamine has been shown to block
ATP-sensitive potassium channels in cardiac ventricular cells by a
mechanism unrelated to its
-adrenergic blockade (28). In addition,
the
Na+/H+
exchanger is known to be activated by
1-adrenergic agonists (6, 20).
If the effect of phentolamine on these two types of ion channels would
have been relevant in our experimental model of hemorrhagic shock, it
would have caused a decrease in the vectorial transport of sodium
across the alveolar epithelium and thus a decrease in the net alveolar
fluid clearance, an effect not observed in the present studies.
Although it has recently been shown that the activation of
-adrenergic receptor causes an increase in the gene expression of
several proinflammatory cytokines (IL-1
, tumor necrosis factor-
, transforming growth factor-
1) in pulmonary mononuclear cell
populations after hemorrhagic shock in mice (11), the authors of that
study also found that IL-1
, but not tumor necrosis factor-
protein, was significantly increased in the lung homogenate of
hemorrhaged mice and that the increase in IL-1
protein was prevented
by
-adrenergic blockade. Based on these results, we hypothesized
that the release of IL-1
within the air spaces by shock-mediated
release of oxygen radicals secondary to
-adrenergic stimulation
might in part mediate the failure of the alveolar epithelium to respond
to
-adrenergic agonists by upregulating alveolar liquid clearance.
Therefore, the second objective of our studies was to determine whether
IL-1
could be an important mediator of the oxidative stress to the alveolar epithelium after hemorrhagic shock. First, we found that hemorrhagic shock was associated with a significant increase in the
immunoreactive IL-1
protein content in the lung homogenate and that
phentolamine pretreatment was associated with a 50% decrease in the
protein content of this proinflammatory cytokine (Fig. 4). Thus the
second series of studies was to determine whether the inhibition of
IL-1
binding to its receptor by the administration of IL-1ra would
protect against the shock-mediated oxidative injury to the alveolar
epithelium and restore the normal fluid transport capacity of the
alveolar epithelium after hemorrhagic shock. The results indicated that
pretreatment with IL-1ra restored the ability of the alveolar
epithelium to respond to
-adrenergic agonists by upregulating
alveolar fluid clearance in hemorrhaged and fluid-resuscitated rats.
These results were not explained by a nonspecific effect of IL-1ra on
vectorial fluid transport across the alveolar epithelium since
pretreatment with IL-1ra modified neither basal alveolar liquid
clearance (pilot experiments) nor the normal response of the
alveolar epithelium to exogenous catecholamines in control nonhemorrhaged rats (Fig. 2).
Interestingly, it has previously been shown that neutrophils are major
contributors to intraparenchymal IL-1 protein expression after
hemorrhage and endotoxemia (16). There was a significant increase in
IL-1
protein expression in neutrophil-rich but not in T or B
lymphocyte-rich fraction of the lung intraparenchymal cells (16). The
same laboratory reported that alveolar macrophages show a different
pattern of cytokine expression after hemorrhage in mice. Indeed,
IL-1
protein expression is elevated in bronchoalveolar fluid only 72 h after hemorrhage (26). Taken together, these results suggest a
central role for neutrophil-derived IL-1
production in the lung
after hemorrhage. In addition, these results are in accordance with the
data presented here as well as with recent results of our laboratory
reporting the first in vivo evidence for a neutrophil-dependent
decrease in alveolar epithelial fluid transport after hemorrhagic shock
and fluid resuscitation (14).
There are several mechanisms that could explain why the inhibition of
IL-1 by pretreatment with IL-1ra would protect against the
shock-mediated oxidative injury to the alveolar epithelium. IL-1 has
been shown to cause
-adrenergic receptor hyporesponsiveness by
different mechanisms that might contribute to the failure of the
alveolar epithelium to upregulate fluid clearance in response to
endogenous and/or exogenous
-adrenergic agonists. First, IL-1
can
disrupt the
-adrenergic receptor-adenylate cyclase-coupled transmembrane signaling mechanism by inducing the expression of inhibitory Gi
protein in rabbit
airway smooth muscle through a cytokine-induced enhanced muscarinic
M2 receptor-Gi
protein coupling (5). Second, IL-1
can cause a defect in adenylyl cyclase activity as shown by an impairment of forskolin-induced cAMP
accumulation in lungs of rats pretreated with IL-1
(9). In that
study, pertussis toxin had no effect on the reduced response to
forskolin, suggesting an abnormality independent of any increase in
Gi
protein or of
-adrenergic
receptor-linked adenylyl cyclase activity (9). Third, IL-1
has been
shown recently to cause cyclooxygenase-2 expression and subsequent
release of prostaglandin E2, causing the activation of
protein kinase A in human airway smooth muscle cells. An increase in
protein kinase A caused the phosphorylation of the
-adrenergic
receptor, resulting in a decreased ability of
-adrenergic agonist to
stimulate its receptor (10). This effect of IL-1
is inhibited by
pretreatment with a specific COX-2 inhibitor (10). Thus these recent
results indicate that prostanoids could have an important role in the
mechanism of action of IL-1
leading to
-adrenergic dysfunction.
In summary, the results of these experimental studies in rats indicate
that the activation of -adrenergic receptors after hemorrhagic shock
prevents the
-adrenergic-dependent upregulation of alveolar fluid
clearance by modulating the severity of the pulmonary inflammatory
response. Because the expected catecholamine-mediated upregulation of
alveolar liquid clearance was restored by preventing the binding of
IL-1
to its receptor by the administration of IL-1ra, these
experiments also indicate that IL-1
is an important mediator that
modulates the oxidant-induced decrease in the fluid transport capacity
of the alveolar epithelium after prolonged hemorrhagic shock. These
results also may have important clinical implications because several
studies have shown that IL-1
is a major proinflammatory cytokine in
bronchoalveolar lavage and pulmonary edema fluid obtained from patients
with acute lung injury (21, 22, 27).
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Ricardo Ciriales for technical assistance in carrying out these studies.
![]() |
FOOTNOTES |
---|
This work was supported primarily by National Heart, Lung, and Blood Institute Grant HL-51854 (M. A. Matthay and J. F. Pittet). It also has been supported in part through a Research Grant from the American Lung Association (J. F. Pittet) and a Fellowship Grant from the American Lung Association-California (K. Modelska).
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: J.-F. Pittet, Dept. of Anesthesia, Rm. 3C-38, San Francisco General Hospital, 1001 Potrero Ave., San Francisco, CA 94110 (E-mail: pittetj{at}anesthesia.ucsf.edu).
Received 1 April 1999; accepted in final form 21 May 1999.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Allen, D. R.,
G. L. Wallis,
and
P. B. McCay.
Catechol adrenergic agents enhance hydroxyl radical generation in xanthine oxidase systems containing ferritin: implication for ischemia-reperfusion.
Arch. Biochem. Biophys.
315:
235-243,
1994[Medline].
2.
Exton, J. H.
Mechanisms involved in -adrenergic phenomena.
Am. J. Physiol.
248 (Endocrinol. Metab. 11):
E633-E647,
1985
3.
Finco, T.,
A. Beg,
and
A. Baldwin.
Inducible phosphorylation of IB
is not sufficient for its dissociation from NF-
B and is inhibited by protease inhibitors.
Proc. Natl. Acad. Sci. USA
91:
884-888,
1994.
4.
Ghosh, S.,
and
D. Baltimore.
Activation in vitro of NF-B by phosphorylation of its inhibitor I
B.
Nature
344:
678-682,
1990[Medline].
5.
Hakonarson, H.,
D. A. Herrick,
and
M. M. Gruenstein.
Mechanism of impaired -adrenoreceptor responsiveness in atopic sensitized airway smooth muscle.
Am. J. Physiol.
269 (Lung Cell. Mol. Physiol. 13):
L645-L652,
1995
6.
Haussinger, D.,
O. Brodde,
and
K. Starke.
Alpha-adrenoceptor antagonistic action of amiloride.
Biochem. Pharmacol.
36:
3509-3515,
1987[Medline].
7.
Heilig, M.,
M. Irwin,
I. Grewal,
and
E. Sercarz.
Sympathetic regulation of T-helper cell function.
Brain Behav. Immun.
7:
154-163,
1993[Medline].
8.
Karlsson, S.,
A. J. Scheurink,
A. B. Steffens,
and
B. Ahren.
Involvement of capsaicin-sensitive nerves in regulation of insulin secretion and glucose tolerance in conscious mice.
Am. J. Physiol.
267 (Regulatory Integrative Comp. Physiol. 36):
R1071-R1077,
1994
9.
Koto, H.,
J. C. W. Mak,
E. B. Haddad,
W. B. Xu,
M. Salmon,
P. J. Barnes,
and
K. J. Chung.
Mechanisms of impaired -adrenoceptor-induced airway relaxation by interleukin-1
in vivo in the rat.
J. Clin. Invest.
98:
1780-1787,
1996
10.
Laporte, J. D.,
P. E. Moore,
R. A. Panettieri,
W. Moeller,
J. Heyder,
and
S. A. Shore.
Prostanoids mediate IL-1-induced
-adrenergic hyporesponsiveness in human airway smooth muscle cells.
Am. J. Physiol.
275 (Lung Cell. Mol. Physiol. 19):
L491-L501,
1998
11.
Le Tulzo, Y.,
R. Shenkar,
D. Kaneko,
P. Moine,
G. Fantuzzi,
C. A. Dinarello,
and
E. Abraham.
Hemorrhage increases cytokine expression in lung mononuclear cells in mice: involvement of catecholamines in nuclear factor kappa B regulation and cytokine expression.
J. Clin. Invest.
99:
1516-1524,
1997
12.
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[Medline].
13.
Modelska, K.,
M. A. Matthay,
M. C. McElroy,
and
J. F. Pittet.
Upregulation of alveolar liquid clearance after fluid resuscitation for hemorrhagic shock in rats.
Am. J. Physiol.
273 (Lung Cell. Mol. Physiol. 17):
L305-L314,
1997
14.
Modelska, K. M.,
M. A. Matthay,
L. A. S. Brown,
E. Deusch,
L. N. Lu,
and
J. F. Pittet.
Inhibition of -adrenergic-dependent alveolar epithelial clearance by oxidant mechanisms after hemorrhagic shock.
Am. J. Physiol.
276 (Lung Cell. Mol. Physiol. 20):
L844-L857,
1999
15.
Morris, D.,
K. Modelska,
L. N. Lu,
D. Sheppard,
M. A. Matthay,
and
J. F. Pittet.
Sulfasalazine restores catecholamine-induced increase in alveolar epithelial fluid transport following prolonged hemorrhagic shock and blocks nitrite production by rat alveolar macrophages (Abstract).
Am. J. Respir. Crit. Care Med.
159:
A291,
1999.
16.
Parsey, M. V.,
R. M. Tuder,
and
E. Abraham.
Neutrophils are major contributors to intraparenchymal lung IL-1 expression after hemorrhage and endotoxemia.
J. Immunol.
160:
1007-1013,
1998
17.
Pittet, J. F.,
T. B. Brenner,
K. Modelska,
and
M. A. Matthay.
Alveolar liquid clearance is increased by endogenous catecholamines in hemorrhagic shock in rats.
J. Appl. Physiol.
81:
830-837,
1996
18.
Pittet, J. F.,
J. P. Wiener-Kronish,
M. C. McElroy,
H. G. Folkesson,
and
M. A. Matthay.
Stimulation of alveolar epithelial liquid clearance by endogenous release of catecholamines in septic shock.
J. Clin. Invest.
94:
663-671,
1994[Medline].
19.
Powis, G.,
and
P. G. Appel.
Relationship of the single-electron reduction potential of quinones to their reduction by flavoproteins.
Biochem. Pharmacol.
29:
2567-2572,
1979.
20.
Puceat, M.,
O. Clement-Chomienne,
A. Terzic,
and
G. Vassort.
Alpha1-adrenoceptor and purinoceptor agonists modulate Na-H antiportin single cardiac cells.
Am. J. Physiol.
264 (Heart Circ. Physiol. 33):
H310-H319,
1993
21.
Pugin, J.,
B. Ricou,
K. P. Steinberg,
P. M. Suter,
and
T. R. Martin.
Proinflammatory activity in bronchoalveolar lavage fluids from patients with ARDS. A prominent role for interleukin-1.
Am. J. Respir. Crit. Care Med.
153:
1850-1856,
1996[Abstract].
22.
Pugin, J.,
G. Verghese,
M. C. Widmer,
and
M. A. Matthay.
The alveolar space is the site of intense inflammatory and profibrotic reactions in the early phase of acute respiratory distress syndrome.
Crit. Care Med.
27:
304-312,
1999[Medline].
23.
Ramaswamy, S.,
D. Srinivasan,
and
J. S. Bapna.
Inhibition of tetrahydroisoxazolo-pyridin-3-ol and muscimol and its mechanism on gastrointestinal transit in mice.
Eur. J. Pharmacol.
220:
147-149,
1992[Medline].
24.
Shenkar, R.,
and
E. Abraham.
Effects of hemorrhage on cytokine gene transcription.
Lymphokine Cytokine Res.
12:
237-247,
1993[Medline].
25.
Shenkar, R.,
and
E. Abraham.
Plasma from hemorrhaged mice activates CREB and increases cytokine expression in lung mononuclear cells through a xanthine oxidase dependent mechanism.
Am. J. Respir. Cell Mol. Biol.
14:
198-206,
1996[Abstract].
26.
Shenkar, R.,
W. F. Coulson,
and
E. Abraham.
Hemorrhage and resuscitation induce alterations in cytokine expression and the development of acute lung injury.
Am. J. Respir. Cell Mol. Biol.
10:
290-297,
1994[Abstract].
27.
Siler, T. M.,
J. E. Swierkosz,
T. M. Hyers,
A. A. Fowler,
and
R. O. Webster.
Immunoreactive interleukin-1 in bronchoalveolar lavage fluid of high risk patients and patients with the adult respiratory distress syndrome.
Exp. Lung Res.
15:
881-884,
1989[Medline].
28.
Wilde, A. A.,
M. W. Veldkamp,
A. C. G. van Ginneken,
and
T. Opthoh.
Phentolamine blocks ATP sensitive potassium channels in cardiac ventricular cells.
Cardiovasc. Res.
28:
847-850,
1994[Medline].