alpha -Adrenergic blockade restores normal fluid transport capacity of alveolar epithelium after hemorrhagic shock

M. Laffon, L. N. Lu, K. Modelska, M. A. Matthay, and J. F. Pittet

Departments of Anesthesia and Medicine and Cardiovascular Research Institute, University of California, San Francisco, California 94143


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of beta -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 alpha -adrenergic receptors, the objective of this study was to determine whether alpha -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-1beta (IL-1beta ) concentration in the lung and a failure of the alveolar epithelium to respond to beta -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 alpha -adrenergic-receptor antagonist. Phentolamine pretreatment also significantly attenuated the shock-mediated increase of IL-1beta concentration in the lung. Additional experiments demonstrated that the inhibition of IL-1beta 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 alpha -adrenergic receptors after hemorrhagic shock prevents the beta -adrenergic-dependent upregulation of alveolar fluid clearance by modulating the severity of the pulmonary inflammatory response.

alpha -adrenergic-receptor antagonist; alveolar liquid clearance; oxidative injury; interleukin-1; interleukin-1-receptor antagonist


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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-kappa B (NF-kappa B), as well as the increased gene expression of proinflammatory cytokines in pulmonary mononuclear cell populations after hemorrhagic shock in mice, was prevented by alpha -adrenergic blockade. These findings suggested that the activation of NF-kappa B through an alpha -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 alpha -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 alpha -adrenergic-receptor antagonist. Phentolamine pretreatment was also associated with a significant decrease in the immunoreactive interleukin-1beta (IL-1beta ) content of the lung homogenate of hemorrhaged rats. Thus the second objective of these studies was to determine whether the inhibition of IL-1beta 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 · kg-1 · 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.


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Fig. 1.   General experimental protocol (see METHODS for further explanation). IL-1ra, interleukin-1-receptor antagonist.

A vascular tracer, 1 µCi of 131I-labeled human albumin, was injected into the blood 90 min after the onset of hemorrhagic shock to calculate the flux of plasma protein into the lung interstitium as previously published (13, 14). An alveolar tracer, 1 µCi of 125I-albumin in 3 ml/kg of the 5% bovine albumin solution, was instilled into the left lower lobe 30 min later to calculate the flux of protein from the air spaces into the circulating plasma. This tracer was instilled by using a 1-ml syringe and a polypropylene tube (0.5 mm ID) over a 20-min period.

At the end of the experiment (1 h after the beginning of the alveolar instillation), the abdomen was opened and the rat was exsanguinated by transecting the abdominal aorta. Urine was obtained for radioactivity counts. The lungs were removed through a median sternotomy. An alveolar fluid sample from the distal air spaces (0.1-0.2 ml) was obtained by gently passing the sampling catheter (PE-50 catheter, 0.5 mm ID) into a wedged position in the instilled area of the left lower lobe. After centrifugation, the total protein concentration and the radioactivity of the liquid sampled were measured. Right and left lungs were homogenized separately for water-to-dry weight ratio measurements and radioactivity counts.

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 beta -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 alpha -adrenergic receptors (11), rats (n = 7) were pretreated with the alpha -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 alpha -adrenergic blockade in mice as determined by its effects on alpha -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-1beta content of the lung homogenate of hemorrhaged rats, we hypothesized that the inhibition of IL-1beta 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-1beta 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-1beta Measurement in Lung Homogenate

Because hemorrhage has been shown to increase levels of immunoreactive IL-1beta in the lungs through the activation of alpha -adrenergic receptors in mice (11), a first series of experiments included rats (n = 3) that were hemorrhaged and fluid resuscitated as described in General protocol. Six hours after onset of hemorrhagic shock, the rats were exsanguinated and the lung vascular bed was flushed by injecting 25 ml of chilled (4°C) PBS into the right ventricle. Then the lungs were snap frozen in liquid nitrogen. A second series of experiments included hemorrhaged and fluid-resuscitated rats that were pretreated with phentolamine (10 mg/kg ip, n = 3). A third series of experiments included control rats (n = 3) that were neither hemorrhaged nor fluid resuscitated. A fourth series of experiments included control rats (n = 3) that were neither hemorrhaged nor fluid resuscitated but were pretreated with phentolamine (10 mg/kg ip).

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 · min-1 · g-1) and multiplying this value by the total volume instilled into the lung. To calculate the residual 125I-albumin in the lungs at the end of the study, the average radioactivity counts of two 0.5-g samples obtained from the lung homogenate was multiplied by the total weight of lung homogenate. The 125I-albumin in the lung homogenate data was added to the recovered counts in the final aspirated distal air space fluid to calculate the quantity of instilled 125I-albumin that remained in the lungs at the end of the study. The 125I-albumin in the circulating plasma was measured from a sample of plasma obtained at the end of the experiment. The plasma fraction was accounted for by multiplying the counts per gram times the plasma volume [body wt × 0.07 (1 - hematocrit)] (13).

The second method requires measurement of the vascular protein tracer 131I-albumin in the extravascular spaces of the lungs. We estimated the quantity of plasma that entered the instilled lungs by measuring the accumulation of the vascular protein tracer 131I-albumin into the extravascular spaces of the instilled lung using the equation of plasma equivalents previously described (13, 14). The extravascular lung plasma equivalents were determined by taking the total counts of 131I-albumin in the lung, subtracting the fraction in the blood in the lung measured by the gravimetric method, and then dividing by the mean counts in the circulating plasma over the duration of the experiment as done previously (13, 14).

Alveolar 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-1beta 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-1beta was quantitated using a commercially available ELISA kit specific for rat IL-1beta (Endogen, Boston, MA) as previously published (11). With this assay, the threshold of sensitivity for IL-1beta 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
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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).

                              
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Table 1.   Effect of pretreatment with phentolamine or IL-1ra on changes in mean systemic arterial pressure in control and hemorrhaged rats


                              
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Table 2.   Effect of pretreatment with phentolamine or IL-1ra on changes in arterial pH and calculated base deficit in control and hemorrhaged rats

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-1beta protein content in the lung homogenate of hemorrhaged and fluid-resuscitated rats (Fig. 4).

                              
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Table 3.   Effect of pretreatment with phentolamine or IL-1ra on change in alveolar fluid protein concentration after hemorrhagic shock (60 min) and fluid resuscitation (30 min)



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Fig. 2.   Alveolar liquid clearance (means ± SE) is shown for control (A) and hemorrhaged (B) rats. Both groups were pretreated with phentolamine (10 mg/kg ip) or interleukin-1-receptor antagonist (IL-1ra, 25 mg · kg-1 · h-1) and instilled with epinephrine (10-5 M) into distal air spaces of the lung. Pretreatment with phentolamine or IL-1ra restored ability of alveolar epithelium to upregulate fluid clearance in hemorrhaged rats that were stimulated with exogenous epinephrine. * P < 0.05 from control rats without air space epinephrine. ** P < 0.05 from hemorrhaged and resuscitated rats that were not pretreated with phentolamine or IL-1ra.



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Fig. 3.   Lung liquid clearance (means ± SE) is shown for control (A) and hemorrhaged (B) rats. Both groups were pretreated with phentolamine (10 mg/kg ip) or IL-1ra (25 mg · kg-1 · h-1) and instilled with epinephrine (10-5 M) into distal air spaces of the lung. Pretreatment with phentolamine or IL-1ra restored ability of alveolar epithelium to upregulate fluid clearance in hemorrhaged rats that were stimulated with exogenous epinephrine. * P < 0.05 from control rats without air space epinephrine. ** P < 0.05 from hemorrhaged and resuscitated rats that were not pretreated with phentolamine or IL-1ra.



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Fig. 4.   Effect of alpha -adrenergic blockade with phentolamine (10 mg/kg ip) on hemorrhage-induced alterations in IL-1beta protein in lungs (means ± SE). Pretreatment with phentolamine significantly decreases level of immunoreactive IL-1beta in lung homogenates from hemorrhaged rats. * P < 0.05 from control rats. ** P < 0.05 from hemorrhaged and resuscitated rats that were not pretreated with phentolamine.

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 (10-5 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-1beta 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 (10-5 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).

                              
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Table 4.   Effect of pretreatment with phentolamine or IL-1ra on recovery of alveolar protein tracer (125I-albumin) from lung and plasma

Finally, arterial PO2 and PCO2 were not statistically different between the experimental groups throughout the study (data not shown).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The overall objective of these studies was to determine whether alpha -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-kappa B through an alpha -adrenergic pathway is one of the important mechanisms by which hemorrhagic shock increases proinflammatory cytokine expression in the lungs (11), we hypothesized that alpha -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 alpha -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 beta -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 alpha -adrenergic blockade against the shock-mediated cytokine-associated injury to the alveolar epithelium. First, hemorrhage-induced alpha -adrenergic stimulation could enhance NF-kappa 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-kappa 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-kappa B by sulfasalazine (15), restored the normal fluid transport capacity of the alveolar epithelium after hemorrhagic shock. Second, alpha -adrenergic stimulation causes the activation of protein kinase C and thus increases intracellular calcium (2). NF-kappa B can be activated either by protein kinase C-mediated phosphorylation of Ikappa B (4) or by proteolytic degradation of the COOH-terminal protein sequence of Ikappa B by calcium-dependent intracellular proteases (3). Taken together, these studies indicate that alpha -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 alpha -adrenergic blockade (28). In addition, the Na+/H+ exchanger is known to be activated by alpha 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 alpha -adrenergic receptor causes an increase in the gene expression of several proinflammatory cytokines (IL-1beta , tumor necrosis factor-alpha , transforming growth factor-beta 1) in pulmonary mononuclear cell populations after hemorrhagic shock in mice (11), the authors of that study also found that IL-1beta , but not tumor necrosis factor-alpha protein, was significantly increased in the lung homogenate of hemorrhaged mice and that the increase in IL-1beta protein was prevented by alpha -adrenergic blockade. Based on these results, we hypothesized that the release of IL-1beta within the air spaces by shock-mediated release of oxygen radicals secondary to alpha -adrenergic stimulation might in part mediate the failure of the alveolar epithelium to respond to beta -adrenergic agonists by upregulating alveolar liquid clearance. Therefore, the second objective of our studies was to determine whether IL-1beta 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-1beta 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-1beta 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 beta -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-1beta protein expression after hemorrhage and endotoxemia (16). There was a significant increase in IL-1beta 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-1beta 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-1beta 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-1beta has been shown to cause beta -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 beta -adrenergic agonists. First, IL-1beta can disrupt the beta -adrenergic receptor-adenylate cyclase-coupled transmembrane signaling mechanism by inducing the expression of inhibitory Gialpha protein in rabbit airway smooth muscle through a cytokine-induced enhanced muscarinic M2 receptor-Gialpha protein coupling (5). Second, IL-1beta 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-1beta (9). In that study, pertussis toxin had no effect on the reduced response to forskolin, suggesting an abnormality independent of any increase in Gialpha protein or of beta -adrenergic receptor-linked adenylyl cyclase activity (9). Third, IL-1beta 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 beta -adrenergic receptor, resulting in a decreased ability of beta -adrenergic agonist to stimulate its receptor (10). This effect of IL-1beta 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-1beta leading to beta -adrenergic dysfunction.

In summary, the results of these experimental studies in rats indicate that the activation of alpha -adrenergic receptors after hemorrhagic shock prevents the beta -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-1beta to its receptor by the administration of IL-1ra, these experiments also indicate that IL-1beta 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-1beta 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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