Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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Endotoxemia produces
elevations in catecholamine levels in the pulmonary and systemic
circulation as well as rapid increases in neutrophil number and
proinflammatory cytokine expression in the lungs. In the present
experiments, we examined the effects of endogenous and exogenous
adrenergic stimulation on endotoxin-induced lung neutrophil
accumulation and activation. Levels of interleukin (IL)-1, tumor necrosis factor (TNF)-
, and macrophage
inflammatory protein (MIP)-2 mRNAs were increased in lung neutrophils
from endotoxemic mice compared with those present in lung neutrophils from control mice or in peripheral blood neutrophils from endotoxemic or control mice. Treatment with the
-adrenergic antagonist
propranolol before endotoxin administration did not affect trafficking
of neutrophils to the lungs or the expression of IL-1
, TNF-
, or MIP-2 by lung neutrophils. Administration of the
-adrenergic antagonist phentolamine before endotoxemia did not alter lung neutrophil accumulation as measured by myeloperoxidase (MPO) levels but
did result in significant increases in IL-1
, TNF-
, and MIP-2 mRNA
expression by lung neutrophils compared with endotoxemia alone.
Administration of the
1-adrenergic agonist
phenylephrine before endotoxin did not affect trafficking of
neutrophils to the lungs but was associated with significantly
increased expression of TNF-
and MIP-2 mRNAs by lung neutrophils
compared with that found after endotoxin alone. In contrast, treatment
with the
2-adrenergic agonist
UK-14304 prevented endotoxin-induced increases in lung MPO and lung
neutrophil cytokine mRNA levels. The suppressive effects of UK-14304 on
endotoxin-induced increases in lung MPO were not affected by
administration of the nitric oxide synthase inhibitor
N-nitro-L-arginine
methyl ester. These data demonstrate that the initial accumulation and
activation of neutrophils in the lungs after endotoxemia can be
significantly diminished by
2-adrenergic stimulation.
Therapy with
2-adrenergic
agents may have a role in modulating inflammatory pulmonary processes
associated with sepsis-induced acute lung injury.
lipopolysaccharide; -adrenergic;
-adrenergic; acute lung
injury; propranolol; phentolamine; UK-14304; phenylephrine; nitric
oxide
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INTRODUCTION |
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ACUTE INFLAMMATORY LUNG INJURY occurs frequently in
patients with severe infections (1, 16). Massive accumulation of neutrophils in the lungs and increased pulmonary immunoregulatory cytokine levels are major characteristics of this condition (7, 16, 19,
46). Proinflammatory cytokines and chemokines, including interleukin (IL)-1, tumor necrosis factor (TNF)-
, IL-8, and macrophage inflammatory protein (MIP)-2, can be produced by resident pulmonary cell populations, including alveolar macrophages and vascular
endothelium (22, 29, 36, 47). However, recent data (32) indicate that
neutrophils are also a source of IL-1
in the lungs after endotoxemia
and other pathophysiological conditions, such as hemorrhage, associated
with the development of acute lung injury. Identification of lung
neutrophils as a significant intrapulmonary source of IL-1
after
endotoxemia may be particularly important because several studies (34,
42) have shown that IL-1
is a major proinflammatory cytokine in
bronchoalveolar lavage fluids obtained from patients with
acute lung injury.
Endotoxemia and sepsis are associated with the release of high levels
of catecholamines into the pulmonary and systemic circulation (40).
Catecholamines, often in large doses, are frequently administered to
critically ill septic and endotoxemic patients to maintain blood
pressure and cardiac function (33). In addition to their vasoactive
effects, catecholamines can affect expression of proinflammatory cytokines (18, 33, 44). For example, TNF- production by lipopolysaccharide (LPS)-stimulated macrophages is augmented by
-adrenergic (43) and decreased by
-adrenergic agonists (37). In
human volunteers, endotoxin-induced increases in serum TNF-
levels
were diminished when pretreatment with epinephrine was provided (48,
49). Even though epinephrine has mixed
- and
-adrenergic agonist
properties, in vitro studies of human peripheral blood mononuclear
cells suggested that the
- but not the
-adrenergic agonist
properties of epinephrine were responsible for the inhibition of
TNF-
release (37). However, no studies have specifically examined
the ability of catecholamines to affect cytokine production by lung neutrophils.
There is evidence that catecholamines can directly affect adhesion of
leukocytes to endothelial surfaces (4, 6). In particular,
-adrenergic stimulation of endothelial cells has been shown to
inhibit neutrophil adhesion (5, 10). Therefore, catecholamines, whether
produced endogenously or administered exogenously, may be capable of
affecting the development and progression of acute lung injury. Such
effects may occur through modulating proinflammatory cytokine
production by neutrophils or other pulmonary cell populations, through
direct effects on neutrophil-endothelial interactions, or through both
mechanisms. To examine these issues, we treated endotoxemic mice with
- and
-adrenergic antagonists or agonists and then determined
whether such adrenergic manipulation affected either the accumulation
of neutrophils in the lungs or the expression of proinflammatory
cytokines by lung neutrophils.
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METHODS |
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Materials.
Escherichia coli 0111:B4 endotoxin,
phenylephrine, UK-14304 (brimonidine), DMSO, collagenase, and
DNase (type I) were obtained from Sigma (St. Louis, MO). RPMI
1640 medium (with 10 mM HEPES and 20 mM
L-glutamine), used in the cell
isolation procedures, was obtained from BioWhittaker Products
(Walkersville, MD). Fetal calf serum (FCS) and penicillin-streptomycin
were purchased from Gemini Bioproducts (Calabasas, CA). Percoll was
obtained from Pharmacia (Uppsala, Sweden). For the RNA extractions,
guanidinium and phenol-chloroform (5:1, pH 4.7) were purchased from
Fisher Scientific (Pittsburgh, PA) and isopropanol was purchased from Sigma. Taq DNA polymerase was obtained
from Perkin-Elmer (Branchburg, NJ), and the cytokine primers for
IL-1 and TNF-
were from Clontech (Palo Alto, CA). Primers for
MIP-2 were synthesized by Operon Technologies (Alameda, CA) with
sequences kindly provided by Dr. David Baltimore (Massachusetts
Institute of Technology, Cambridge, MA). Potassium phosphate,
hexadecyltrimethylammonium bromide, hydrogen peroxide, and
O-dianisidine used in the myeloperoxidase (MPO)
assays were obtained from Sigma.
Animals. Male BALB/c mice (7-8 wk old) were purchased from Harlan Sprague Dawley (Indianapolis, IN). Animals were kept on a 12:12-h light-dark cycle and given free access to food and water. Studies were conducted when the mice were between 8 and 12 wk of age.
Experimental model of endotoxemia. A murine model of endotoxemia was used as previously reported (32). Briefly, mice were given intraperitoneal injections of 25 mg/kg of LPS (E. coli 0111:B4) in a volume of 0.2 ml of phosphate-buffered saline (PBS). This dose of LPS has previously been shown to cause lung injury 24 h postinjection (12, 13).
In designated experiments, mice were treated intraperitoneally with the
-adrenergic antagonist phentolamine (10 mg/kg) or the
-adrenergic
antagonist propranolol (3 mg/kg) 30 min before LPS administration.
These doses of phentolamine and propranolol have been used previously
by our laboratory and result in complete
- and
-adrenergic
blockade (26). To investigate the effects of
-adrenergic-specific
agonists, either phenylephrine
(
1 specific) or UK-14304
(
2 specific) at 1 mg/kg was
administered intraperitoneally 30 min before LPS treatment.
Phenylephrine was resuspended in PBS, whereas UK-14304 was dissolved in
DMSO at 10 mg/ml and then diluted to a 1 mg/kg dose in PBS. All drugs
were administered in a volume of 0.2 ml. The following control groups
were included: normal (unmanipulated), dilute DMSO, dilute DMSO-LPS,
phenylephrine only, UK-14304 only, propranolol only, and phentolamine only.
Isolation of intraparenchymal pulmonary mononuclear cells and neutrophils. Intraparenchymal pulmonary mononuclear cells and neutrophils were isolated with techniques previously described by our laboratory (2). In brief, after the mouse was euthanized by cervical dislocation under methoxyfluorane anesthesia, the chest was opened and the lung vascular bed was flushed by injecting 3-5 ml of cold PBS through the right ventricle of the heart. The lungs were then removed and rinsed two times in iced RPMI 1640 medium. The lungs from each mouse were finely minced and placed in RPMI 1640 medium containing 10 mM HEPES, 20 mM L-glutamine, 5% FCS, 1% penicillin-streptomycin, 20 U/ml of collagenase, and 1 µg/ml of DNase. After incubation for 60 min at 37°C, tissue fragments were forced three times through a 21-gauge needle to disrupt any remaining intact tissue. Tissue fragments and dead cells were removed by rapid filtration on a glass wool column. The resulting cells were collected by centrifugation.
Neutrophil isolation. Neutrophil isolation from peripheral blood as well as from intraparenchymal pulmonary mononuclear cell and neutrophil populations was performed with a modification of the technique described by Sugarawa et al. (45) and previously used in our laboratory (32). To isolate peripheral blood neutrophils, mice were anesthetized with methoxyfluorane and then exsanguinated. Blood was withdrawn by cardiac puncture and collected into 5 U of heparin. The collected blood was then mixed into 2 volumes of chilled PBS and layered onto a gradient of 5 ml each of 1.097 and 1.085 g/ml of Percoll. To isolate lung neutrophils, the intraparenchymal pulmonary mononuclear and neutrophil cell pellet was resuspended in 1 ml of PBS and layered on a gradient of 5 ml each of 1.097 and 1.085 g/ml of Percoll. After centrifugation at 600 g for 25 min at 18°C, the neutrophil-rich fraction was collected from the gradient interface and washed with RPMI 1640 medium. Viability as determined by trypan blue exclusion was consistently >98%. The purity of the isolated neutrophil populations was assessed for each experiment by Wright staining of cytospin preparations and was as great as 98%.
Isolation of alveolar macrophages. Alveolar macrophages were isolated as previously described (38). In brief, bronchoalveolar lavage fluid was collected after 1.0 ml of PBS was injected and then aspirated intratracheally three times. Alveolar macrophages were obtained by centrifugation of bronchoalveolar lavage samples. Macrophage purity and viability, as assessed by trypan blue exclusion and cytospin preparations, were consistently >98%.
MPO assay. MPO assays were performed
essentially as described by Goldblum et al. (15). In brief, red blood
cells were flushed from the lung vascular bed by an injection of 5 ml
of iced PBS through the right ventricle. The lungs were removed, rinsed
two times in iced PBS, blotted dry, and snap-frozen in liquid nitrogen. The frozen lungs were then weighed and stored at 80°C if not used immediately. Frozen tissue was homogenized in 20 mM potassium phosphate buffer (pH 7.4) and then centrifuged for 30 min at 20,000 g, and the resulting pellet was
resuspended in 50 mM potassium phosphate buffer (pH 6.0) containing
0.5% hexadecyltrimethylammonium bromide. Samples were sonicated and
placed at 60°C for 2 h. After incubation, 1 ml of each sample was
centrifuged briefly, and the supernatant was assayed for MPO activity
in a hydrogen peroxide-O-dianisidine buffer via spectrophotometric analysis at 420 nm. Results are given as
units of MPO activity per gram of lung tissue.
Semiquantitative PCR. The basic procedure used for semiquantitative PCR has been described previously by our laboratory (38). Groups of six mice, with PCR results obtained from individual mice, were used for each experimental condition. In brief, after purified neutrophil populations had been lysed in 4 M guanidinium thiocyanate-25 mM sodium citrate-0.5% sarcosyl-0.1 M 2-mercaptoethanol, mRNA was phenol extracted following the method of Chomczynski and Sacchi (8). cDNA was synthesized from the mRNA from 100,000 cells/sample with Moloney murine leukemia virus reverse transcriptase and random hexamer oligonucleotide primers as described by Kawasaki (21). Semiquantitative PCR was performed with the cDNA from 1,000 cells/sample. A single PCR master mix was prepared, and aliquots were used as samples in all treatment groups for each experiment. After an initial 4-min denaturation step at 95°C, between 20 and 40 cycles of PCR were carried out as follows: 1 min, 95°C denaturation; 1 min, 60°C anneal; and 1 min, 72°C extension. Coamplification of the housekeeping genes hypoxanthine phosphoribocil transferase (HPRT) and glyceraldehyde-3-phosphate dehydrogenase was used to standardize the PCR products. PCR products were visualized by electrophoresis on 1.6% agarose gels stained with ethidium bromide. The number of PCR cycles was selected for the cytokine product so that the ethidium bromide-stained amplified DNA products were between barely detectable and below saturation levels. For analysis, the gel image was photographed and scanned with a gel-documentation system (ImageStore 5000 with GelBase Windows Software, Ultraviolet Products, San Gabriel, CA). Results for each cytokine were normalized to those for HPRT or glyceraldehyde-3-phosphate dehydrogenase.
Statistical analysis. Because of inherent variability between groups of mice for each experimental condition, the entire group of animals was prepared and studied at the same time. For each experimental condition, mice in all groups had the same birth date and had been housed together. For semiquantitative PCR, cells were obtained individually from each animal and analyzed individually before group data were calculated. All experiments were repeated two or three times with separate, additional groups of animals. Data are presented as means ± SE for each experimental group. Comparisons between groups were performed by one-way ANOVA and the Student-Newman-Keuls test. A P value of <0.05 was considered significant.
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RESULTS |
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Neutrophil migration and activation in response to
endotoxin. Lung MPO levels increased >16-fold from
baseline within 1 h of endotoxin administration (Fig.
1). The neutrophils that were present in
the lungs after endotoxemia showed significant increases in mRNA levels
of IL-1, TNF-
, and MIP-2 compared with lung
neutrophils from control, unmanipulated mice (Fig.
2). In peripheral blood neutrophils
isolated at the same time point after endotoxemia, levels of mRNA for
TNF-
were increased, but there were no significant changes in the
amounts of mRNA for IL-1
and MIP-2 compared with those in control,
unmanipulated mice (Fig. 2). However, for all three cytokines examined,
mRNA levels after endotoxemia were greater in lung neutrophils than in
peripheral blood neutrophils. No differences in the levels of mRNA for
IL-1
, TNF-
, and MIP-2 were found in alveolar macrophages
collected 1 h after endotoxin administration compared with
those from control animals (Fig. 3).
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Effects of endotoxin-induced catecholamine release on
lung neutrophil accumulation and activation. To examine
the role of endogenous catecholamine release on neutrophil accumulation
in the lungs after endotoxemia, we pretreated mice with either the nonspecific -adrenergic antagonist phentolamine or the nonspecific
-adrenergic antagonist propranolol. Neither propranolol nor
phentolamine pretreatment produced any significant change in lung MPO
concentrations compared with those present in mice given endotoxin
alone (Fig. 4).
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We also examined the effects of - or
-adrenergic blockade on
endotoxin-induced alterations of proinflammatory cytokine
expression in lung neutrophils (Fig. 5).
Pretreatment with the
-adrenergic antagonist propranolol did not
affect endotoxin-induced increases in IL-1
, TNF-
, or MIP-2 mRNA
expression in lung neutrophils. However,
-adrenergic blockade with
phentolamine was associated with up to threefold greater levels of mRNA
for each of the proinflammatory cytokines studied.
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Propranolol or phentolamine given alone without endotoxin administration did not have any effect on lung MPO content. MPO levels with the drug alone were comparable to those seen in control, unmanipulated mice (data not shown). A previous study by our laboratory (26) also showed that these drugs had no effect on baseline levels of cytokine mRNA expression in intraparenchymal pulmonary mononuclear and neutrophil cell populations.
Effects of exogenous -adrenergic stimulation
on lung neutrophil accumulation. To explore more
completely
-adrenergic-mediated effects on endotoxin-induced lung
neutrophil accumulation and activation, we pretreated mice with either
the
1-adrenergic agonist phenylephrine or the
2-adrenergic agonist UK-14304
(Fig. 6). Pretreatment with phenylephrine
had no effect on endotoxin-induced increases in lung MPO
concentrations. However, administration of UK-14304 before endotoxin
decreased lung MPO content to levels not significantly different from
those seen in unmanipulated mice not given endotoxin.
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Administration of vehicle, phenylephrine, or UK-14304 without endotoxin did not alter baseline lung MPO concentrations from levels seen in control, unmanipulated mice (data not shown). Likewise, administration of vehicle did not affect LPS-induced increases in lung MPO content (data not shown).
Increased production of nitric oxide (NO) can attenuate neutrophil
adhesion to endothelial surfaces (3, 24). Because 2-adrenergic stimulation can
enhance NO production by vascular endothelial cells (27, 51), we
examined whether such effects on NO generation contributed to the
observed suppression by UK-14304 of endotoxin-induced neutrophil
accumulation in the lungs.
Administration of the nonspecific NO synthase inhibitor N-nitro-L-arginine methyl ester before endotoxin injection did not significantly affect lung MPO (Fig. 7). Similarly, N-nitro-L-arginine methyl ester administration did not modify the suppressive effects of UK-14304 on lung MPO levels after LPS administration.
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Effects of exogenous -adrenergic stimulation
on lung neutrophil activation. To examine the effects
of
-adrenergic stimulation on endotoxin-induced increases in
proinflammatory cytokine mRNA expression by lung neutrophils, we
pretreated mice with either the
1-adrenergic agonist
phenylephrine or the
2-adrenergic agonist UK-14304
(Fig. 8). Under these conditions, UK-14304
inhibited endotoxin-induced increases in mRNA levels of IL-1
,
TNF-
, and MIP-2 in lung neutrophils. With UK-14304 pretreatment, the
levels of TNF-
and MIP-2 mRNAs in endotoxemic animals were not
different from those present in control, unmanipulated mice. IL-1
mRNA levels in lung neutrophils from UK-14304-pretreated mice were also
decreased compared with those present in animals given endotoxin alone
but were greater than those in lung neutrophils from control, unmanipulated mice. By contrast, administration of phenylephrine before
endotoxin did not reduce LPS-induced elevations in mRNA expression for
any of the three cytokines studied and, in fact, produced increases in
the amounts of mRNA for TNF-
and MIP-2 in lung neutrophils that were
even greater than those found in endotoxemic mice not given other
therapies.
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Administration of vehicle alone or up to fivefold greater
concentrations (i.e., 5 mg/kg) of phenylephrine or UK-14304 without endotoxin did not alter cytokine mRNA expression in lung neutrophils compared with the levels present in control, unmanipulated mice. Similarly, administration of vehicle did not affect LPS-induced elevations in cytokine mRNA expression (data not shown). Treatment with
phenylephrine or UK-14304 in control, unmanipulated animals or before
endotoxemia did not have any effect on IL-1, TNF-
, or MIP-2
expression in peripheral blood neutrophils or alveolar macrophages
(data not shown).
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DISCUSSION |
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In the present study, we found that neutrophils rapidly accumulated in
the lungs after endotoxemia and produced increased amounts of mRNA for
proinflammatory cytokines including IL-1, TNF-
, and MIP-2.
Although blockade of endogenous
- or
-adrenergic stimulation did
not appear to influence the migration of neutrophils to the lungs,
activation of these lung neutrophils to produce proinflammatory
cytokines was affected by
-adrenergic blockade. In particular,
inhibition of endogenous
-adrenergic effects with phentolamine
resulted in further increases in the expression of IL-1
, TNF-
,
and MIP-2 to levels greater than those seen in lung neutrophils
isolated from mice treated with endotoxin alone. The suppressive
effects of
-adrenergic stimulation on lung neutrophil activation
appeared to be due to
2-adrenergic effects because enhanced
2- but not
1-adrenergic stimulation
prevented endotoxin-induced increases in the expression of
proinflammatory cytokines.
Signal transduction through
1-adrenergic receptors involves
G proteins that activate phospholipase C, leading to hydrolysis of
membrane phosphatidylinositol and increases in intracellular calcium
levels (20, 28). Engagement of
2-receptors, through coupling
to inhibitory G proteins, decreases adenylate cyclase-associated cAMP
production (20, 28). Binding sites for the transcriptional regulatory
factors nuclear factor-
B (NF-
B) (17, 41) and cAMP responsive
element binding protein (CREB) (1, 9, 30, 52) are present in the
promoter/enhancer regions of each of the cytokines examined in the
present study. Increases in either intracellular
Ca2+ or cAMP can activate NF-
B
and CREB (14, 25, 35, 39). However, the results from the present
experiments, showing relative dominance of
2-adrenergic effects on lung
neutrophils after endotoxemia, would suggest that cAMP, probably
through affecting protein kinase A-associated
pathways (25, 39), is relatively more important under these conditions
in modulating NF-
B- and/or CREB-associated proinflammatory
cytokine transcription.
Previous studies (37, 43, 44) examined the effects of catecholamines on
the systemic cytokine responses in vivo or the in vitro release of
proinflammatory cytokines by macrophages. However, little information
is available concerning interactions between catecholamines, pulmonary
inflammatory processes, and neutrophils in the setting of endotoxemia.
The addition of -adrenergic agonists to LPS-stimulated macrophages
or peripheral blood mononuclear cells inhibits IL-1, IL-6, and TNF-
production (23, 37, 50). These previous studies (37, 50) did not show
that
-adrenergic stimulation had any significant suppressive effects
on proinflammatory cytokine release by macrophage or mononuclear cell
populations. Treatment of endotoxemic mice with the specific
2-adrenergic antagonist
CH-38083 decreased circulating TNF-
levels, but the mechanism of
this effect appeared to be indirect, resulting from increased
norepinephrine release and enhanced
1-adrenergic stimulation (11,
18).
In previous experiments by our laboratory (26), we found
that -adrenergic-receptor blockade prevented hemorrhage-induced increases in proinflammatory cytokine production by intraparenchymal lung cell populations, which included large numbers of neutrophils. In
contrast,
-adrenergic blockade increased the amounts of mRNA for
IL-1
and TNF-
to levels that were greater than those present in
untreated, hemorrhaged mice (26). The results of the present experiments did not show similar inhibitory effects of
-adrenergic stimulation in lung neutrophils obtained after endotoxemia. Different adrenergic-associated mechanisms would therefore appear to be involved
in lung neutrophil activation after hemorrhage or endotoxemia. In
particular, although the present studies indicate that
2-adrenergic stimulation is
inhibitory after endotoxemia, there is no evidence to suggest that
similar regulatory mechanisms affect lung neutrophils after blood loss.
Although the present study demonstrated that lung neutrophils were
activated to a greater extent after endotoxemia than were peripheral
blood neutrophils, these results do not necessarily imply that
neutrophil activation occurs in the lungs. In previous experiments by
our laboratory (32), immunohistochemical studies found that the
neutrophils present in the pulmonary vasculature as well as in the
pulmonary parenchyma after endotoxemia stained positively for IL-1,
showing that activation could occur within the intravascular space.
Although neutrophil activation may occur as a result of interaction
with vascular endothelial cells in the lungs, it is also possible that
extrapulmonary events may induce activated neutrophils to traffic
rapidly to the lungs. Either of these potential mechanisms for
neutrophil activation could be affected through
2-adrenergic effects. Because
2-adrenergic receptors are
present on neutrophils (31),
2-adrenergic agonists may
directly affect neutrophils in pulmonary or extrapulmonary intravascular sites. Alternatively,
2-adrenergic stimulation may
downregulate expression of neutrophil costimulatory molecules on
vascular endothelium, thereby decreasing neutrophil activation. Mechanisms involving
2-adrenergic-dependent effects
on vascular endothelium would explain the link between the inhibition
of neutrophil accumulation in the lungs and the activation seen with
UK-14304 therapy. However, because inhibition of NO synthase did not
alter the suppressive effects of
2-adrenergic stimulation on
neutrophil accumulation in the lungs, alterations in NO production by
the vascular endothelium do not appear to be involved in this process.
In the present experiments, no increase in the expression of IL-1,
TNF-
, or MIP-2 was present in alveolar macrophages isolated 1 h
after endotoxin administration. These results are consistent with
previous findings by our laboratory (32), where immunohistochemical studies showed increased staining for IL-1
in lung neutrophils and
endothelial cells but not in alveolar macrophages at this same
postendotoxemia time point. Although alveolar macrophages can be
stimulated in vitro by endotoxin to produce proinflammatory cytokines
(46), the present data suggest that such activation does not occur in
vivo in the period immediately after endotoxemia.
Neutrophils, by producing proinflammatory cytokines and expressing
cytotoxic products, appear to play an important role in the development
and progression of acute lung injury (32). Inhibition of accumulation
and/or activation of neutrophils in the lungs may therefore be
important in improving the outcome from this condition. The present
results suggest that administration of 2-adrenergic agonists may have
therapeutic utility in clinical settings in which endotoxemia plays a
major role, such as sepsis-induced acute respiratory distress syndrome.
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ACKNOWLEDGEMENTS |
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We thank Dr. Merdad Parsey for help and careful reading of this manuscript.
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FOOTNOTES |
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This work was supported by National Heart, Lung, and Blood Institute Grant HL-50284.
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: E. Abraham, Univ. of Colorado Health Sciences Center, Division of Pulmonary Sciences and Critical Care Medicine, 4200 E. 9th Ave., Box C-272, Denver, CO 80262.
Received 4 May 1998; accepted in final form 22 September 1998.
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