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(Journal of Leukocyte Biology. 2002;72:571-579.)
© 2002 by Society for Leukocyte Biology

Effects of catecholamines on kinase activation in lung neutrophils after hemorrhage or endotoxemia

John Arcaroli, Kuang-Yao Yang, Ho-Kee Yum, John Kupfner, Todd M. Pitts, Jong Sung Park, Derek Strassheim and Edward Abraham

Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Denver

Correspondence: Edward Abraham, M.D., Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Center, Mail Code C-272, 4200 East Ninth Ave., Denver, CO 80262. E-mail: Edward.Abraham{at}UCHSC.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Catecholamines are released in high levels after hemorrhage or endotoxemia and have been shown to modulate immune function, including cellular release of inflammatory mediators. In the present experiments, we examined the effects of endogenous and exogenous catecholamines on neutrophil accumulation and activation in the lungs using pretreatment with {alpha}- or ß-antagonists or {alpha}-adrenergic agonists before hemorrhage or endotoxemia. These studies showed that {alpha}-, but not ß-adrenergic stimuli, modulated the severity of acute lung injury after hemorrhage or endotoxemia, and {alpha}-adrenergic stimuli was proinflammatory after hemorrhage but anti-inflammatory after endotoxemia. The observed {alpha}-adrenergic effects on lung neutrophil activation appeared to involve primarily the extracellular signal-regulated kinase pathway at the upstream kinase Raf, but not Ras. Although p38 and protein kinase A were activated in lung neutrophils after hemorrhage or endotoxemia, these kinases were not affected by {alpha}- or ß-adrenergic modulation. These results demonstrate that catecholamines have important immunomodulatory effects in vivo that affect intracellular signaling pathways in neutrophils and neutrophil-driven, inflammatory processes such as the development of acute lung injury.

Key Words: intracellular signaling • Ras • Raf • MEK • ERK • p38 • PKA • {alpha}-adrenergic stimulation • ß-adrenergic stimulation


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Acute lung injury (ALI) is characterized by neutrophil accumulation in the lungs, interstitial edema, disruption of epithelial integrity, and leakage of protein into the alveolar space [1 2 3 4 ]. Infection, associated with endotoxemia, and blood loss are frequent predisposing factors to the development of ALI [1 ], and in experimental settings, endotoxemia or hemorrhage produces ALI [5 ]. Neutrophils play a central role in this acute pulmonary inflammatory process, as their elimination can prevent the development of ALI [5 ]. The neutrophils present in the lungs during ALI produce proinflammatory mediators, including cytokines such as tumor necrosis factor {alpha} (TNF-{alpha}) and macrophage inflammatory peptide (MIP)-2, and demonstrate increased activation of transcriptional regulatory factors including cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) and nuclear factor-{kappa}B (NF-{kappa}B) [5 6 7 8 ].

Catecholamines are released in high levels after blood loss or endotoxemia and can modulate inflammatory processes including cytokine release [9 10 11 ]. Exposure of lipopolysaccharide (LPS)-stimulated macrophages to norepinephrine down-regulates TNF-{alpha} and interleukin (IL)-6 expression [12 , 13 ], and infusion of catecholamines into human volunteers diminishes endotoxemia-induced increases in circulating levels of TNF-{alpha}. Both of these effects are primarily a result of ß-adrenergic stimulation [13 , 14 ]. However, {alpha}-adrenergic effects also appear to have immunomodulatory properties. For example, blockade of the {alpha}-adrenergic receptor results in the protection of rats from mortality after lethal doses of LPS, and administration of an {alpha}-adrenergic agonist further increased endotoxemia-induced mortality [15 ]. In previous studies [9 , 10 , 16 ], we found that {alpha}- and ß-adrenergic blockade could modulate proinflammatory cytokine release as well as activation of the transcription factors NF-{kappa}B and CREB in lung cell populations, including neutrophils, after hemorrhage or endotoxemia.

The extracellular signal-regulated (ERK) and p38 mitogen-activated protein kinases play an important role in neutrophil signal transduction pathways activated by diverse extracellular stimuli, including LPS, mitogens, oxidative stress, and cytokines [17 18 19 20 21 ]. For example, p38 activation is associated with increased neutrophil adhesion, chemotaxis, priming, O2- release, synthesis of TNF-{alpha}, and expression of C-X-C chemokines, such as IL-8 and MIP-2 [17 , 18 , 22 ]. Signaling cascades involving ERK1/2 and p38 lead to the activation of transcriptional factors, including NF-{kappa}B and CREB, which are important in regulating transcription of cytokines, adhesion molecules, and other mediators involved in inflammatory responses [17 , 23 24 25 ]. In particular, activation of p38 results in enhanced nuclear translocation of NF-{kappa}B in neutrophils [17 ], and ERK1/2-dependent pathways lead to phosphorylation of serine 133 of CREB [24 ], an essential step for enhancing transcriptional activity of this factor.

Endogenous catecholamines released during endotoxemia or hemorrhage have been shown to modulate NF-{kappa}B and CREB activation among lung cell populations. In previous studies [16 ], we found that activation of CREB and NF-{kappa}B in lung neutrophils was modified when {alpha}- or ß-adrenergic blockade was instituted before hemorrhage or endotoxemia, implying that such interventions might be able to affect the development or severity of ALI. ERK2 appeared to be involved in CREB activation under these pathophysiologic conditions. However, the kinases upstream to ERK2, which may have been responsive to catecholamine effects and responsible for the observed alterations in ERK2 activation, were not examined.

In the present experiments, we examined the effects of endogenous catecholamine release on the development of ALI. These studies showed that {alpha}- but not ß-adrenergic stimuli modulated the severity of ALI after hemorrhage or endotoxemia, and {alpha}-adrenergic stimuli was proinflammatory after hemorrhage, but anti-inflammatory after endotoxemia. The observed {alpha}-adrenergic effects on lung neutrophil activation appeared to involve primarily the ERK pathway at the upstream kinase Raf, but not Ras. Such results demonstrate that catecholamines, released in high levels in stress states such as hemorrhage or endotoxemia, have important immunomodulatory effects in vivo that can modulate intracellular signaling pathways in neutrophils and affect acute neutrophil-driven, inflammatory processes such as ALI.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Mice
Male BALB/c mice, 8 to 12 weeks of age, were purchased from Harlan Sprague-Dawley (Indianapolis, IN). The mice were kept on a 12-h light/dark cycle with free access to food and water. All experiments were conducted in accordance with institutional review board-approved protocols.

Materials
Isoflurane was obtained from Abbott Laboratories (Chicago, IL). Escherichia coli 0111:B4 endotoxin, collagenase, DNase, UK-14304, phenylephrine, propranolol, and phentolamine were purchased from Sigma Chemical Co. (St. Louis, MO). RPMI 1640/25 mM HEPES/L-glutamine was obtained from BioWhittaker Products (Walkersville, MD), and fetal bovine serum (FBS) and penicillin/streptomycin were purchased from Gemini Bioproducts (Calabasas, CA). Percoll was purchased from Amersham-Pharmacia (Piscataway, NJ). Bicinchoninic acid (BCA) protein assay reagent was purchased from Pierce (Rockford, IL). Antibodies specific for p-mitogen-activated protein kinase kinase (MEK)1/2, p-ERK1/ERK2, p-p90rsk, p-p38, and total MEK1/2, ERK1/ERK2, and p38 were purchased from Cell Signaling Technologies (Beverly, MA). Anti-total Raf-1 and anti-total p90rsk were purchased from Transduction Laboratories (Lexington, KY). Ras assay kit and p-Raf-1were purchased from Upstate Biotech (Lake Placid, NY). The colorimetric PKA assay kit was obtained from Pierce. Custom cocktail antibodies and columns for neutrophil isolation were purchased from Stem Cell Technologies (Vancouver, BC).

Models of hemorrhage and endotoxemia
The murine hemorrhage model used in these experiments was reported previously [6 , 9 , 26 ]. With this model, 30% of the calculated blood volume (approximately 0.55 ml for a 20-g mouse) is withdrawn over a 60-s period by cardiac puncture from an isoflurane-anesthetized mouse. The period of isoflurane anesthesia was less than 1 min in all of the cases. The mortality rate with this hemorrhage protocol is approximately 12%.

The model of endotoxemia was used as reported previously [6 , 10 ]. Mice received an intraperitoneal (i.p.) injection of LPS at dose of 1 mg/kg in 0.2 ml phosphate-buffered saline (PBS). This dose has previously been demonstrated to produce acute neutrophilic alveolitis, histologically consistent with acute lung injury in mice [5 , 6 ].

Interventions
In designated experiments, mice were treated i.p. with 0.2 ml PBS (control), the {alpha}-adrenergic antagonist phentolamine (10 mg/kg), or the ß-adrenergic antagonist propranolol (3 mg/kg), 30 min prior to hemorrhage or LPS administration. These doses of phentolamine and propranolol have been used previously by our laboratory and result in complete {alpha}- and ß-adrenergic blockade [9 , 10 ]. To investigate the effects of {alpha}-adrenergic stimulation, phenylephrine ({alpha}1-specific) or UK-14304 ({alpha}2-specific) at 1 mg/kg was administered i.p. 30 min prior to hemorrhage or LPS injection. Phenylephrine was resuspended in PBS, whereas UK-14304 was dissolved in dimethyl sulfoxide at 5 mg/500 µl and then diluted to a 1 mg/kg dose in PBS. All drugs were administered in a volume of 0.2 ml.

Isolation of neutrophils
Lung or peripheral neutrophils were purified from intraparenchymal pulmonary or bone marrow cell suspensions. To obtain the bone marrow cell suspension, the femur and tibia of a mouse were flushed with 5 ml RPMI 1640/penicillin/streptomycin, and the cells were passed through a glass wool column. Intraparenchymal pulmonary cell suspensions were prepared as previously described by our laboratory [6 , 10 , 26 ]. In brief, the chest of the mouse was opened, and the lung vascular bed was flushed with 2–3 ml chilled (4°C) PBS injected into the right ventricle. Lungs were then excised, avoiding the paratracheal lymph nodes and thymus and were washed twice in RPMI-1640/25 mM HEPES/L-glutamine supplemented with penicillin/streptomycin. The excised lungs were minced finely, and the tissue pieces were placed in RPMI-1640 medium containing 5% FBS, 20 U/ml collagenase, and 1 µg/ml DNase. Following incubation for 60 min at 37°C, any remaining intact tissue was disrupted by passage through a 21-gauge needle. Tissue fragments and the majority of dead cells were removed by rapid filtration through a glass wool column, and cells were collected by centrifugation.

The cell pellets from the intraparenchymal pulmonary or bone marrow cell suspensions were resuspended in RPMI 1640/5% FCS and then incubated with 10 µl primary antibodies specific for cell surface markers F4/80, CD4, CD45R, CD5, and TER119 for 15 min at 4°C. This custom cocktail (Stem Cell Technologies) is specific for T and B cells, red blood cells (RBC), monocytes, and macrophages. After 15-min incubation, 100 µl antibiotin tetrameric antibody complexes were added, and the cells were incubated for 15 min at 4°C. Following this, 60 µl colloidal magnetic dextran iron particles were added to the suspension and incubated for 15 min at 4°C. The entire cell suspension was then placed into a column surrounded by a magnet. The T cells, B cells, RBC, monocytes, and macrophages were captured in the column, allowing the neutrophils to pass through by negative selection methods. The neutrophil suspension was then layered on 50% Percoll, centrifuged at 3000 rpm for 15 min, and the neutrophil layer was collected. Viability, as determined by trypan blue exclusion, was consistently greater than 98%. Neutrophil purity, as determined by Wright’s stained cytospin preparations, was greater than 97%.

Myeloperoxidase (MPO) assay
MPO activity was assayed as reported previously [5 ]. Excised lungs from three to four mice per treatment group were frozen in liquid nitrogen, weighed, and stored at -86°C. Lungs were homogenized for 30 s in 1.5 ml 20 mM potassium phosphate, pH 7.4, and centrifuged at 4°C for 30 min at 40,000 g. The pellet was resuspended in 1.5 ml 50 mM potassium phosphate, pH 6.0, containing 0.5% hexadecyltrimethyl ammonium bromide, sonicated for 90 s, incubated at 60°C for 2 h, and centrifuged. The supernatant was assayed for peroxidase activity corrected to lung weight.

Western blot analysis
Whole cell extracts from lung neutrophils were denatured in ice-cold lysis buffer [50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM ethyleneglycol-bis(ß-aminoethylether)-N,N'-tetraacetic acid (EGTA), 1 mM Na2 vanadate, 10 mM Na pyrophosphate, 10 mM NaF, 300 µM p-nitrophenyl phosphate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 µg/ml leupeptin, 10 µg/ml aprotinin, pH 7.3] for 15 min. The protein concentration of each sample was assayed using the BCA protein assay kit standardized to bovine serum albumin (BSA), according to manufacturer’s protocol. Briefly, 50 µg protein was loaded and then run on a 10% Tris-HCl sodium dodecyl sulfate (SDS) polyacrylamide gel. Protein was electrotransferred to a nitrocellulose membrane and then blocked with 5% nonfat dry milk, 20 mM Tris-buffered saline, with 0.1% Tween. After blocking, the membrane was incubated overnight at 4°C with a rabbit polyclonal-specific primary antibody to p-Raf, p-MEK1/2, p-ERK1/ERK2, p-p90rsk, or p-p38 using a dilution of 1:1000 followed by anti-rabbit or anti-rat immunoglobulin (Ig) horseradish peroxidase-coupled secondary antibody at a dilution of 1:2000. After washing five times, bands were detected using enhanced chemiluminescence Western blotting detection reagents (Amersham-Pharmacia). The membranes were then stripped using Immuno Pure IgG elution buffer (Pierce) and were reprobed with antibodies specific for total Raf, MEK1/2, ERK1/ERK2, p90rsk, or p38. Densitometry was performed using a chemiluminescence system and analysis software (BioRad, Hercules, CA) to determine the ratio between phosphorylated and total kinase.

PKA assay
PKA activity was measured by a commericially available PKA assay kit (Pierce) according to the manufacturer’s protocol. In brief, lung neutrophils were resuspended in ice-cold lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2 , 1 mM EGTA, 1 mM Na3 vanadate, 10 mM Na pyrophosphate, 10 mM NaF, 300 µM p-nitrophenyl phosphate, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, pH 7.3) for 15 min. The protein concentration was assayed using the BCA protein assay kit standardized to BSA, according to the manufacturer’s protocol. Equal amounts of protein in each group were used to measure PKA activity. Reaction buffer [10 mM adenosine 5'-triphosphate, 50 mM MgCl2, 0.01% Triton X-100, 100 mM Tris(hydroxymethyl)-amino methane, pH 7.4, PKA substrate (kempeptide) labeled with fluorescent probe, and activator solution (500 mM cAMP)] was added to 50 µg protein and incubated for 30 min at 30°C. After incubation, 20 µl of each sample was directly applied to the SpinZyme unit, and 250 µl phosphopeptide-binding buffer (0.1 M sodium acetate, 0.5 M sodium chloride, 0.02 M sodium azide, pH 5.0) was added and incubated for 3 min. The sample was then centrifuged at 6500 rpm for 1 min. The membrane was transferred to a new receptacle, and 250 µl phosphopeptide-elution buffer (0.1 M ammonium bicarbonate, 0.02% sodium azide, pH 8.0) was added to the membrane and incubated for 3 min. After 3 min, the sample was centrifuged at 6500 rpm for 1 min. This step was repeated for a final elution volume of 500 µl. The sample was mixed, and 300 µl was transferred to an individual well of a flat-bottom 96-well plate. The plate was read at 570 nm. A standard curve was used ranging from 0.2 U/µl to 0.0063 U/µl to determine the kinase activity of each sample. The working range of this assay kit is approximately 0.03–1 unit of PKA activity.

Ras assay
For immunoprecipitation of Raf-associated Ras, lung neutrophils were resuspended in ice-cold lysis buffer (50 mM HEPES, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 1.5 mM MgCl2, 1 mM EGTA, 1 mM Na3 vanadate, 10 mM Na pyrophosphate, 10 mM NaF, 300 µM p-nitrophenyl phosphate, 1 mM PMSF, 10 µg/ml leupeptin, 10 µg/ml aprotinin, pH 7.3) for 15 min. The protein concentration was assayed using the BCA protein assay kit standardized to BSA, according to manufacturer’s protocol. Equal amounts of protein in each group were used for the immunoprecipitation procedures. Anti-Raf monoclonal antibody (mAb; 15 µl; Upstate Biotechnology), immobilized by cross-linkage to agarose-hydrazide beads, was added to each lysate and incubated for 1 h at 4°C. After this, the immune complexes were collected by centrifugation and were then washed three times with lysis buffer. After washing, 25 µl Laemmli buffer (62.5 mM Tris-HCl, pH 6.8, 10% glycerol, 2% SDS, 5% 2-mercaptoethanol) was added to each sample. The samples were boiled for 5 min and loaded onto a 10% Tris-HCl SDS-polyacrylamide gel. A Ras mAb (Upstate Biotechnology) was used for detection at a dilution of 1:1000.

Statistical analysis
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. Separate groups of mice were used for Western blotting, Ras assay, PKA assay, and MPO. Data are presented as mean ± SEM for each experimental group. One-way ANOVA and the Tukey-Kramer Multiple Comparisons test (for multiple groups) or Student’s t-test (for comparisons between two groups) were used. P < 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effects of {alpha}- and ß-adrenergic stimulation on hemorrhage- or endotoxemia-induced lung injury
Although endogenously released catecholamines have previously [16 ] been demonstrated to modulate activation of transcriptional factors, such as NF-{kappa}B, as well as expression of proinflammatory cytokines by neutrophils and other pulmonary cell populations [9 , 10 ], their role in affecting parameters of acute lung injury had not been examined. To explore this issue, we administered the {alpha}-adrenergic inhibitor phentolamine or the ß-adrenergic inhibitor propranolol before hemorrhage or endotoxemia and then determined the severity of lung injury. As shown in Figure 1 , {alpha}-adrenergic blockade before hemorrhage resulted in a significant decrease in the accumulation of neutrophils in the lungs. In contrast, {alpha}-adrenergic blockade before endotoxemia was associated with a significant increase in this parameter. ß-adrenergic blockade did not significantly modify lung neutrophil accumulation induced by hemorrhage or endotoxemia.



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  		Figure 1. Effects of blockade of endogenous {alpha}- or ß-adrenergic stimuli with phentolamine (Phen) or propranolol (Pro) and of pretreatment with the {alpha}2-adrenergic agonist UK-14304 (UK) or the {alpha}1-adrenergic agonist phenylephrine (PE) on hemorrhage (Hem)- or endotoxemia (LPS)-induced neutrophil accumulation. Lung MPO levels were increased after hemorrhage or administration of endotoxin but were modified by {alpha}-adrenergic blockade or by additional {alpha}-adrenergic stimulation given before either of these pathophysiologic events. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus unmanipulated control mice. #, P < 0.05 and ###, P < 0.001 versus untreated hemorrhage. {dagger}{dagger}, P < 0.01 versus untreated LPS. ;N = six mice in each group.

 
The above results with {alpha}-adrenergic blockade suggested that {alpha}-stimulation is anti-inflammatory during endotoxemia, but proinflammatory after hemorrhage. To examine this hypothesis directly, pulmonary neutrophil accumulation was determined in mice in which the {alpha}1 agonist phenylephrine or the {alpha}2 agonist UK-14304 was administered before endotoxin injection or hemorrhage. As shown in Figure 1 , {alpha}2 stimulation significantly decreased endotoxemia, but not hemorrhage-induced neutrophil migration to the lungs. In contrast, pretreatment with the {alpha}1 agonist phenylephrine resulted in enhanced accumulation of neutrophils in the lungs after hemorrhage or endotoxemia.

Activation of the Ras, p38, and PKA pathways in lung neutrophils after hemorrhage or endotoxemia
MEK1/2 and ERK2 were activated in lung neutrophils after hemorrhage or endotoxemia (Fig. 2 ), as shown in our previous results [16 ]. Although we examined ERK1 activation as well, neither hemorrhage nor endotoxemia affected amounts of phosphorylated ERK1 in lung neutrophils, consistent with findings previously reported [16 ]. To determine if there is also activation of upstream and downstream kinases under these conditions, we examined levels of total and phosphorylated Raf and p90rsk. Figure 2 shows that hemorrhage or endotoxemia produces significant increases in Raf and p90rsk activation among lung neutrophils.



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  		Figure 2. Effects of hemorrhage or endotoxemia on activation of ERK-related, p38, and PKA kinases in lung neutrophils. (A) Densitometry results for activation patterns of ERK-related kinases in lung neutrophils after hemorrhage or endotoxemia. Levels of phosphorylated Raf, MEK 1/2, ERK2, and p90rsk were normalized to total amounts of kinase. Ras activation was obtained directly by kinase assay. (B) Hemorrhage- or endotoxemia-induced activation of p38 and PKA in lung neutrophils. The ratio of phosphorylated-to-total p38 is shown as are results from a direct PKA kinase assay. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus peripheral neutrophils from control, unmanipulated mice. N = three to six mice in each group.

 
Ras is considered to be the dominant kinase capable of phosphorylating and activating Raf [27 ]. Because hemorrhage and endotoxemia resulted in Raf activation in lung neutrophils, we expected to find activation of Ras under the same conditions. However, as shown in Figure 2 , no alteration in Ras activity was found in lung neutrophils after hemorrhage or endotoxemia, although Raf, MEK1/2, ERK2, and p90rsk were activated.

In previous studies [7 , 16 ], we found that the transcriptional factors CREB and NF-{kappa}B were activated in lung neutrophils after hemorrhage or endotoxemia. Phosphorylation of serine 133 of CREB is essential for the transcriptional activity of this factor [24 ], and p90rsk has been shown to be capable of activating CREB through participating in this phosphorylation step [28 ]. Because p38 and PKA can also be involved in activation of NF-{kappa}B and CREB [17 , 29 , 30 ], we examined the effects of hemorrhage or endotoxemia on these kinases. As shown in Figure 2B , there was an increase in phosphorylated p38 in lung neutrophils after hemorrhage or endotoxemia. PKA activity was also increased in lung neutrophils after hemorrhage or endotoxemia.

Modification of the Ras, p38, and PKA pathways by {alpha}- and ß-adrenergic effects after hemorrhage or endotoxemia
In previous studies [16 ], we showed that endogenous catecholamines, released after hemorrhage or endotoxemia, could affect NF-{kappa}B and CREB activation in lung neutrophils. In those experiments, alterations in the activation of MEK1/2 and ERK2 associated with inhibition of {alpha}- or ß-adrenergic stimulation paralleled levels of CREB phosphorylation, suggesting that catecholamine-modulated alterations in the activity of MEK1/2 and downstream kinases, such as ERK2, affected CREB phosphorylation in neutrophils. However, ERK has not been shown to be involved in NF-{kappa}B activation, so the catecholamine-modifiable pathways leading to enhanced nuclear translocation of NF-{kappa}B in neutrophils after hemorrhage or endotoxemia remained undefined. To explore interactions between endogenously released catecholamines and kinases known to be involved in NF-{kappa}B and CREB activation, we determined the effects of {alpha}- or ß-blockade on hemorrhage- or endotoxemia-induced activation of Ras, Raf, MEK1/2, ERK2, p90rsk, PKA, and p38 in lung neutrophils.

As shown in Figure 3A and B, there was significantly greater activation of Raf, MEK1/2, ERK2, and p90rsk, but no change in p38, PKA, or Ras in animals treated with the {alpha}-adrenergic inhibitor phentolamine before hemorrhage. In contrast, ß-blockade before hemorrhage resulted in no change in Ras, Raf, MEK1/2, p90rsk, p38, and PKA activation and only a small decrease in ERK2 activation compared with levels present in lung neutrophils from control, hemorrhaged mice treated with PBS alone.



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  		Figure 3. Effects of {alpha}- or ß-adrenergic blockade on hemorrhage-induced activation of ERK-related, p38, and PKA kinases in lung neutrophils. (A) Densitometry results demonstrating patterns in ERK-related kinase activation from lung neutrophils of mice treated with phentolamine or propranolol before hemorrhage. Levels of phosphorylated Raf, MEK 1/2, ERK2, and p90rsk were normalized to total amounts of kinase. Ras activation was obtained directly by kinase assay. (B) Effects of {alpha}- or ß-adrenergic blockade on hemorrhage-induced activation of p38 and PKA in lung neutrophils. The ratio of phosphorylated-to-total p38 is shown as are results from a direct PKA kinase assay. *, P < 0.05; **, P < 0.01; and ***, P < 0.001 versus lung neutrophils from untreated, hemorrhaged mice. N = three to six mice in each group.

 
The effects of {alpha}- or ß-adrenergic blockade on endotoxemia-associated kinase activation in lung neutrophils were different from those found after hemorrhage (Fig. 4A and B). In particular, administration of the {alpha}-adrenergic blocker phentolamine before endotoxemia resulted in a significant decrease in the activation of Raf, MEK1/2, ERK2, and p90rsk but no changes in Ras, p38, or PKA. ß-adrenergic blockade before endotoxemia did not change the activation of any of the kinases examined.



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  		Figure 4. Effects of {alpha}- or ß-adrenergic blockade on endotoxemia-induced activation of ERK-related, p38, and PKA kinases in lung neutrophils. (A) Densitometry results demonstrating patterns in ERK-related kinase activation from lung neutrophils of mice treated with phentolamine or propranolol before endotoxin administration. Levels of phosphorylated Raf, MEK 1/2, ERK2, and p90rsk were normalized to total amounts of kinase. Ras activation was obtained directly by kinase assay. (B) Effects of {alpha}- or ß-adrenergic blockade on endotoxemia-induced activation of p38 and PKA in lung neutrophils. The ratio of phosphorylated-to-total p38 is shown as are results from a direct PKA kinase assay. *, P < 0.05 and **, P < 0.01 versus lung neutrophils from untreated, endotoxemic mice. N = three to six mice in each group.

 
In the above experiments, we demonstrated that {alpha}-adrenergic receptor blockade could affect hemorrhage- or endotoxemia-induced activation of Raf, MEK1/2, ERK2, and p90rsk in lung neutrophils. To examine possible modulatory effects of {alpha}-adrenergic stimulation on the activity of these kinases, we administered the {alpha}1 agonist phenylephrine or the {alpha}2 agonist UK-14304 before hemorrhage or LPS administration. As shown in Figure 5 A , additional {alpha}1 stimulation in the context of hemorrhage resulted in a significant decrease of Raf, MEK1/2, ERK2, and p90rsk activity. Pretreatment with the {alpha}2 agonist UK-14304 did not affect hemorrhage-induced activation of these kinases. In contrast, {alpha}2 stimulation significantly increased LPS-induced activation of Raf, MEK1/2, ERK2, and p90rsk in lung neutrophils, whereas additional {alpha}1 stimulation had no effect (Fig. 5B) .



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  		Figure 5. Effects of pretreatment with the {alpha}1-adrenergic agonist phenylephrine (PE) or the {alpha}2-adrenergic agonist UK-14304 (UK) on hemorrhage- or endotoxemia-induced activation of Raf, MEK1/2, ERK2, and p90rsk in lung neutrophils. (A) Densitometry results for ERK-related kinase activation in lung neutrophils from mice pretreated with phenylephrine or UK-14304 before hemorrhage. (B) Densitometry results demonstrating patterns in ERK-related kinase activation from lung neutrophils of mice pretreated with phenylephrine or UK-14304 before endotoxemia. Levels of phosphorylated Raf, MEK 1/2, ERK2, and p90rsk were normalized to total amounts of kinase. *, P < 0.05 and **, P < 0.01 versus lung neutrophils from untreated hemorrhaged or endotoxemic mice. N = three to six mice in each group.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In previous studies [9 , 10 , 16 ], we found that catecholamines could modulate activation of lung neutrophils and other pulmonary cell populations after hemorrhage or endotoxemia. In particular, proinflammatory cytokine expression in the lungs was decreased by {alpha}-adrenergic blockade before hemorrhage, but exacerbated by such interventions before endotoxemia. Administration of {alpha}2-adrenergic agonists prevented endotoxemia-induced increases in IL-1ß, TNF-{alpha}, and MIP-2 among lung neutrophils [10 ]. Such findings suggested that manipulation of adrenergic stimuli might modulate the development of lung injury after hemorrhage or endotoxemia. This hypothesis was confirmed in the present experiments, where we demonstrated that endogenous {alpha}-adrenergic, but not ß-adrenergic catecholamines are involved in the development of lung injury after hemorrhage or endotoxemia. Inhibition of {alpha}-adrenergic stimuli diminished hemorrhage-induced accumulation of neutrophils in the lungs but worsened this parameter of lung injury after endotoxemia.

In our present experiments, {alpha}1- and {alpha}2-adrenergic agonists exerted differing effects on Raf and downstream kinases after hemorrhage or endotoxemia. In particular, administration of an {alpha}1-adrenergic agonist inhibited hemorrhage-induced activation of Raf but had no effects on Raf activation after endotoxemia. In contrast, pretreatment with an {alpha}2 agonist did not modify hemorrhage-associated Raf activation but increased endotoxemia-induced kinase activation. These findings are consistent with those previously reported by our laboratory, where {alpha}2-, but not {alpha}1-adrenergic agonists, prevented endotoxemia-induced elevations in proinflammatory cytokine expression by lung neutrophils [10 ]. These differing effects of {alpha}1- and {alpha}2-adrenergic stimuli after hemorrhage or endotoxemia suggest that neutrophils are activated by distinct intracellular signaling events in each of these conditions and provide an explanation for the differing effects observed with {alpha}-adrenergic blockade before hemorrhage or endotoxemia (Fig. 6 ).



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  		Figure 6. Proposed mechanisms of {alpha}-adrenergic stimulation on activation of Raf and downstream kinases in lung neutrophils after hemorrhage or endotoxemia.

 
In previous studies, [7 , 16 ], we found that xanthine oxidase-derived reactive oxygen intermediates (ROI) play an important role in neutrophil activation after hemorrhage, but not endotoxemia. ROI are likely generated in response to the intense degree of vasoconstriction associated with blood loss and inhibit MEK1/2 activation in neutrophils under such in vivo conditions [16 ]. Because of their vasoconstrictive properties, further administration of {alpha}1-adrenergic agonists is likely to result in {alpha}1-increased tissue ischemia/reperfusion injury and release of additional ROI in hemorrhage. In contrast, decreased vasoreactivity, particularly in response to {alpha}1 stimulation, is characteristic of endotoxemia [31 ]. In this setting, {alpha}1 agonists would be unlikely to affect xanthine oxidase-derived ROI generation, providing an explanation for the lack of effect of {alpha}1 agonists on neutrophil activation after endotoxemia.

While {alpha}1-adrenergic stimuli, probably as a result of vasoconstriction-derived release of ROI, appear to be dominant in hemorrhage, the decreased endothelial response to {alpha}1 stimuli with endotoxemia is likely to uncover {alpha}2 effects in this setting. Endothelium and neutrophils express {alpha}2 receptors [32 ]. Engagement of {alpha}2 receptors, through coupling to inhibitory G proteins, decreases adenylate cyclase-associated cAMP generation [33 , 34 ]. Because Raf activation is blocked by increased cAMP in neutrophils and other cell types [35 , 36 ], down-regulation of cAMP through {alpha}2-adrenergic-dependent mechanisms would be expected to activate Raf and downstream kinases, as was seen in the present experiments.

In the present studies, we found that Raf, MEK1/2, ERK2, p90rsk, p38, and PKA were activated in lung neutrophils after hemorrhage or endotoxemia. Inhibition of endogenous, {alpha}-adrenergic effects did not produce any changes in the activation of p38 or PKA, indicating that pathways including these kinases were not responsible for the immunomodulatory effects of {alpha}-adrenergic stimulation found in these and previous experiments [9 , 10 ]. In contrast, Raf, MEK1/2, ERK2, and p90rsk were modulated by {alpha}-adrenergic blockade and {alpha}-adrenergic stimulation. Following hemorrhage, activation of these kinases was increased when {alpha}-adrenergic effects were blocked, whereas the opposite effects were found after endotoxemia. Pretreatment with an {alpha}1-, but not an {alpha}2-adrenergic agonist, decreased activation of Raf, MEK1/2, ERK2, and p90rsk after hemorrhage, whereas increased activity of these kinases was found when additional {alpha}2-, but not {alpha}1-adrenergic stimulation, was administered before endotoxemia. Because MEK1/2, ERK2, and p90rsk are all downstream of Raf, these results suggest that Raf has important, anti-inflammatory roles in modulating neutrophil activation and the development of ALI.

There are several mechanisms by which Raf may down-regulate neutrophil activation and exert anti-inflammatory effects in ALI. Many of the cytokines and other proinflammatory mediators associated with the development of ALI are regulated by the transcriptional factor NF-{kappa}B [7 , 37 , 38 ]. The transcriptional coactivator CREB binding protein (CBP) associates with CREB and NF-{kappa}B and is required for optimal activity of these transcriptional regulatory factors [29 , 38 ]. Activation of ERK through the Raf pathway was reported to recruit phosphorylated p90rsk to the third zinc finger domain of CBP in a manner that prevents the binding of essential transcriptional factors such as RNAPII, thereby inhibiting CBP-dependent transcriptional events [39 ]. An additional mechanism by which Raf may inhibit NF-{kappa}B-dependent transcription of proinflammatory cytokines and other mediators involves enhancement of serine 133 phosphorylation of CREB, via activation of MEK, ERK, and p90rsk. Although pathways involving kinases other than Raf, such as PKA, protein kinase C (PKC), and calmodulin kinases II and IV, also can induce CREB phosphorylation at serine 133 [40 41 42 43 44 ], the Raf-MEK pathway appears to have a dominant role, where cellular activation is initiated by cytokines or oxidant stress [45 ], conditions present with hemorrhage or endotoxemia. CBP is required for transcriptional activity of NF-{kappa}B as well as CREB, but is present in limiting quantities in the nucleus [29 , 46 ]. In vitro experiments have found that competition between CREB and NF-{kappa}B for binding to CBP results in alterations in the transcription of CREB- and NF-{kappa}B-dependent genes [29 , 47 , 48 ]. In particular, in situations where phosphorylation of CREB is increased, NF-{kappa}B:CBP association is diminished, and NF-{kappa}B-dependent transcription is decreased, although translocation of NF-{kappa}B to the nucleus continues. We have recently demonstrated that similar inverse relationships between CREB and NF-{kappa}B for association with CBP occur in the lung in vivo [49 ]. In those experiments, interventions that increased CREB phosphorylation, CREB:CBP interactions, and CREB-dependent transcription were associated with parallel decreases in the amount of NF-{kappa}B-associated with CBP- and NF-{kappa}B-dependent transcription, although nuclear translocation of NF-{kappa}B remained unchanged.

Although Ras is upstream to Raf and can phosphorylate as well as activate Raf, we found no change in Ras in lung neutrophils after hemorrhage or endotoxemia. This implies that Ras is not involved in the activation of Raf under such conditions. Similar findings were reported in a study by Buscher and colleagues [50 ], where no activation of Ras occurred after exposure of macrophages to LPS, although Raf, MEK, and ERK were activated. Several kinases, other than Ras, are capable of activating Raf and may play such a role in lung neutrophils after hemorrhage or endotoxemia. It has been shown that phosphatidylinositol-3 kinase (PI-3K) is critical for Raf phosphorylation [51 ]. We recently found that hemorrhage or endotoxemia activates PI-3K in lung neutrophils [52 ]. The PKC family of serine/threonine kinases is activated by LPS and oxidant stress and has also been shown to be capable of directly activating Raf, without the involvement of Ras [53 54 55 56 ]. In particular, PKC{alpha} and PKC{varepsilon} can phosphorylate Raf in vivo [55 ].

In the present experiments, endogenous {alpha}-adrenergic stimuli had differing, modulatory effects on Raf activation after hemorrhage or endotoxemia. In addition, pretreatment with exogenous {alpha}1- or {alpha}2-adrenergic agonists had distinct effects on Raf activity in lung neutrophils isolated after hemorrhage or endotoxemia. Stimulation of heterotrimeric G protein-coupled {alpha}-adrenergic receptors has been demonstrated to activate Raf and downstream kinases, including ERK [57 58 59 ]. This {alpha}-adrenergic-mediated effect occurs via pathways involving phospholipase C, calcium-calmodulin, and tyrosine kinases in a Ras-independent manner [58 ]. In previous studies, we showed that neutrophil signaling pathways leading to NF-{kappa}B and CREB activation and affecting proinflammatory cytokine expression were distinct after hemorrhage or endotoxemia [7 , 16 ]. In those studies, xanthine oxidase-derived ROI had an important role in modulating lung neutrophil activation after hemorrhage, but not endotoxemia. Catecholamines can increase the generation of ROI directly through mechanisms such as their degradation to quinines [60 ] and indirectly by modifying vascular perfusion and contributing to ischemia/reperfusion-induced cellular alterations. However, even if {alpha}-adrenergic-associated alterations in the generation of ROI are involved in modulating Raf activation after hemorrhage or endotoxemia, the signaling event affected by oxidants remains undefined. We are actively investigating this issue.

The present experiments may have implications for the therapy of ALI. Although these studies highlight the involvement of {alpha}-adrenergic stimuli in the development of ALI, a more important finding may be the central role that Raf appears to occupy in this pathophysiologic pulmonary process. Although PKA and p38 were activated in lung neutrophils after hemorrhage or endotoxemia, neither PKA nor p38 was affected by {alpha}-adrenergic modulation, even though this intervention diminished the severity of acute lung injury. Such results do not indicate an important role for PKA or p38 in the genesis of ALI. These findings are consistent with previous reports [18 , 22 ] showing that p38 inhibition did not inhibit endotoxin-induced accumulation of neutrophils into the lungs or the development of ALI. Rather, the present studies suggest that Raf and downstream kinases, including ERK, may be more appropriate therapeutic targets for preventing or diminishing the severity of ALI.


   ACKNOWLEDGEMENTS
 
This work was supported by grant HL 62221 from the National Institutes of Health.

Received October 8, 2001; revised April 17, 2002; accepted April 19, 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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