Surgical Immunology Research Laboratory, Department of Surgery, Division of Trauma, University of California, San Diego, California 92103-8236
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ABSTRACT |
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Hypertonic stress (HS) suppresses neutrophil (PMN) functions. We studied the underlying mechanism and found that HS rapidly (<1 min) increased intracellular cAMP levels by up to sevenfold. cAMP levels correlated with applied hypertonicity and the degree of neutrophil suppression. HS and cAMP-elevating drugs (forskolin and dibutyryl cAMP-acetoxymethyl ester) similarly suppressed extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase activation and superoxide formation in response to N-formylmethionyl-leucyl-phenylalanine (fMLP) stimulation. Inhibition of cAMP-dependent protein kinase A (PKA) with H-89 abrogated the suppressive effects of HS, restoring fMLP-induced ERK and p38 activation and superoxide formation. Inhibition of phosphodiesterase with 3-isobutyl-1-methylxanthine augmented cAMP accumulation and the suppressive effects of HS, while inhibition of adenylyl cyclase with MDL-12330A abolished these effects. These findings suggest that HS-activated cAMP/PKA signaling inhibits superoxide formation by intercepting fMLP-induced activation steps upstream of ERK and p38. In contrast to its effects in the presence of moderate hypertonicity levels (40 mM), H-89 was unable to rescue neutrophil functions from suppression by higher hypertonicity levels (100 mM), indicating that more severe HS suppresses neutrophils via secondary PKA-independent mechanisms.
inflammation; osmotic stimulation; signal transduction
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INTRODUCTION |
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EFFECTIVE NEUTROPHIL (PMN) activation is important for a successful host defense. Rapid activation of neutrophil responses, such as superoxide formation and enzyme release, is critical for the elimination of invading microorganisms. However, under numerous pathological conditions, the cytotoxic mediators released by neutrophils cause host tissue injury. In trauma patients, hemorrhagic shock results in excessive neutrophil activation. The ensuing ischemia-reperfusion injury leads to tissue destruction and serious posttraumatic complications, such as acute respiratory distress syndrome and multiple organ failure syndrome (for review see Ref. 48). In light of the critical role of neutrophils in the development of such posttraumatic complications, therapeutic approaches to control neutrophil activation have become a central focus in trauma research (for review see Ref. 15).
Hypertonic saline resuscitation is a relatively recent addition to the various approaches that are aimed at controlling neutrophil activation after hemorrhagic shock. The finding that hypertonic resuscitation can protect mice and rats from the lethal consequences of hemorrhagic shock, apparently by reducing neutrophil-mediated tissue damage, has triggered a growing interest in hypertonic saline as a potential immunomodulator for the treatment of trauma patients (4, 10, 39). The concept that hypertonic saline could be useful to modulate neutrophil functions in patients is supported by in vitro studies that have demonstrated a rapid suppression of a number of neutrophil functions by physiologically relevant levels of hypertonicity (25, 26, 39).
We have reported that hypertonic stress rapidly activates a number of signaling enzymes in neutrophils that may be part of an osmotic signal transduction pathway (26). This osmotic signal transduction pathway, however, is not well defined in mammalian cells (29). Work from this and other laboratories has clearly shown that hypertonic signals can modulate neutrophil functions by interfering with cell activation events (20, 26, 37, 39, 40). However, to fully grasp the underlying mechanisms, several critical questions remain to be answered. The mechanism whereby neutrophils detect osmotic changes has to be defined, and downstream signaling pathways must be identified. In addition, the regulatory cross talk between osmotic- and activation-signaling pathways needs to be investigated.
Osmotic signaling has been shown to involve protein kinase A (PKA) activation in other cell types (12, 30, 5, 43, 44), and PKA is a known inhibitor of extracellular signal-regulated kinase (ERK) signaling (6). In the present study, we investigated the role of PKA signaling in the suppression of neutrophil superoxide formation by hypertonic stress.
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METHODS |
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Cells. Neutrophils were isolated from peripheral blood of healthy human volunteers. Heparinized blood (40 ml) was mixed with 12 ml of Dextran T500 in normal saline (Pharmacia, Piscataway, NJ) and sedimented for 20 min at room temperature. Cells in the supernatants were centrifuged and separated with Percoll gradient centrifugation according to the manufacturer's recommendations (Pharmacia), and neutrophils were washed twice with Hanks' buffered saline solution (HBSS). Viability was >95% as assessed with trypan blue dye exclusion, and purity exceeded 98%. This isolation procedure was chosen to avoid osmotic shock lysis that is often employed to remove residual red blood cells. Cell isolation and all subsequent experiments were performed under sterile and pyrogen-free conditions.
Cell stimulation. Cells were preincubated at 37°C for 30 min and stimulated with increasing levels of hypertonic saline alone or with N-formylmethionyl-leucyl-phenylalanine (fMLP; both from Sigma-Aldrich, St. Louis, MO) at final concentrations of 100 nM. Cells were stimulated with hypertonic saline by addition to the culture medium of appropriate volumes of HBSS containing additional NaCl (1 mol/l).
Materials. The PKA inhibitor H-89 {N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide · 2HCl}, the phosphodiesterase (PDE) inhibitor 3-isobutyl-1-methylxanthine (IBMX), the adenylyl cyclase (AC) inhibitor MDL-12330A [cis-N-(2-phenylcyclopentyl)azacyclotridec-1-en-1-amine · HCl], and forskolin were obtained from Calbiochem (San Diego, CA), and the cell-permeable cAMP analog N6,O2'-dibutyryl cAMP-acetoxymethyl ester (dibutyryl cAMP-AM) was obtained from Molecular Probes (Eugene, OR).
Assay of neutrophil function.
Superoxide radical (·O
SDS-PAGE and immunoblotting. After stimulation, neutrophils (106 cells) were placed on ice, centrifuged, and lysed by boiling for 5 min in 100 µl of SDS sample buffer containing 100 mM dithiothreitol. Proteins were separated by SDS-PAGE using 8-16% Tris-glycine polyacrylamide gradient gels (Novex, San Diego, CA). Separated proteins were transferred to polyvinylidene difluoride membranes (0.45-µm pore size; Immobilon-P, Millipore, Bedford, MA), and mitogen-activated protein kinase (MAPK) activation was determined by immunoblotting with phosphospecific antibodies that recognize the phosphorylated (and thereby activated) forms of extracellular signal-regulated kinase 1/2 (ERK1/2) and p38 MAPKs (New England Biolabs, Beverly, MA). Secondary antibodies were obtained from Pharmingen (San Diego, CA). Antibody complexes were detected with an enhanced chemiluminescence assay kit (ECL, Amersham, Arlington Heights, IL) according to the manufacturer's instructions.
Intracellular cAMP measurements. Neutrophil cAMP levels were determined with a direct enzyme immunoassay kit (Amersham Pharmacia Biotech, Piscataway, NJ). Neutrophils (106 cells) were plated in 96-well tissue culture plates and incubated at 37°C with IBMX at a final concentration of 80 µM followed by increasing doses of hypertonic saline. After 10 min at 37°C, 20 µl of the lysis reagent supplied with the enzyme immunoassay kit were added, and cAMP levels were determined according to the manufacturer's instructions.
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RESULTS |
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Hypertonic stress causes rapid and dose-dependent accumulation of
cAMP in neutrophils.
In endothelial cells, membrane deformation by cyclic strain has been
reported to cause the accumulation of intracellular cAMP (9). Because cell shrinkage in response to hypertonic
stimulation also causes membrane deformation, we speculated that
hypertonic stress might stimulate cAMP accumulation in neutrophils. We
tested this possibility by exposing neutrophils to increasing levels of
hypertonicity and measuring cAMP accumulation. These assays were
performed in the presence of the PDE inhibitor IBMX to facilitate cAMP
measurements by preventing the rapid turnover of accumulated cAMP. In
the absence of IBMX, hypertonic saline-induced cAMP accumulation was
short lived and less pronounced (data not shown). Hypertonicity caused
rapid cAMP accumulation, increasing baseline cAMP levels by sevenfold
within 1 min after exposure to 100 mM hypertonic saline (Fig.
1A). cAMP levels peaked 10 min
after exposure to hypertonicity and slowly decreased thereafter. The
levels of hypertonicity used to treat the cells correlated with peak
cAMP levels (Fig. 1B) and with the corresponding degree of
suppression of superoxide formation in response to fMLP stimulation
(Fig. 1C). These results demonstrate that hypertonic stress
stimulates cAMP accumulation in neutrophils, and the correlations shown
in Fig. 1C strongly suggest that cAMP accumulation is
involved in the suppression of superoxide formation by hypertonic
stress.
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Hypertonic stress and cAMP-elevating drugs have similar effects on
superoxide formation of fMLP-stimulated neutrophils.
To test the role of cAMP accumulation in the suppression of neutrophil
functions, we compared the suppressive effects of hypertonic stress
with those of cAMP-elevating drugs. Exposure to hypertonic stress for
1 h inhibited fMLP (100 nM)-induced superoxide formation in a
dose-dependent fashion, with half-maximal suppression at a
hypertonicity level of 20 mM (Fig.
2A). For comparison,
neutrophils were exposed to the cell-permeable cAMP analog dibutyryl
cAMP-AM or forskolin for 1 h and stimulated with 100 nM fMLP, and
superoxide formation was measured. Dibutyryl cAMP-AM and forskolin also
caused a dose-dependent inhibition of superoxide formation (Fig. 2,
B and C). These results indicate that hypertonic
stress and cAMP-elevating drugs have similar properties concerning
their suppressive effects on superoxide formation of fMLP-stimulated
neutrophils. Figure 1A shows that hypertonic saline causes
significant cAMP accumulation as early as 1 min after hypertonic
stimulation. If it is assumed that cAMP accumulation is indeed involved
in the suppression of superoxide formation, one would expect similarly
kinetic properties for the suppression of neutrophil responses by
hypertonic saline. To test this theory, we determined the suppressive
effect of hypertonic saline on cells that were simultaneously
stimulated with increasing doses of fMLP (100 nM) and hypertonic saline
(40 mM; Fig. 2D). The suppressive effect of hypertonic
saline under these conditions was similar to that caused by prolonged
exposure to hypertonic saline (Fig. 2A), with 50-100%
suppression of superoxide formation, depending on the fMLP
concentration used to stimulate the cells (Fig. 2D). These
findings suggest that hypertonic saline-induced cAMP accumulation could
indeed account for the suppression of superoxide formation by
hypertonic saline.
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Hypertonic stress and dibutyryl cAMP-AM have similar effects on
MAPK signaling of fMLP-stimulated neutrophils.
Stimulation of neutrophils with fMLP activates the ERK and p38 members
of the MAPK family (26, 49). Both MAPKs have been implicated in the signal transduction pathways leading to several neutrophil responses, including superoxide formation and enzyme release, although their respective roles in these processes are still
controversial (26, 38). We previously reported that hypertonic stress blocks fMLP receptor-initiated signaling pathways upstream of these MAPKs (26). Here we investigated whether
cAMP accumulation in response to hypertonic stress could be responsible for blocking fMLP receptor signaling upstream of these MAPKs. Neutrophils were pretreated with hypertonic saline or dibutyryl cAMP-AM
for 1 h and stimulated with 100 nM fMLP for 3 min, and ERK and p38
MAPK activation was determined by immunoblotting with phosphospecific
MAPK antibodies. Hypertonic saline and the cAMP analog markedly reduced
ERK and p38 MAPK activation (Fig. 3). Dibutyryl cAMP-AM at 0.4 µM suppressed fMLP-induced p38 MAPK
activation by 85%. Similarly, 40 mM hypertonic saline caused a 70%
suppression of this response (Fig. 3A). Half-maximal
suppression of ERK activation required levels of hypertonic saline
twice as high (80 mM) and doses of dibutyryl cAMP-AM four times as high
(1.5 µM) as those needed for 50% suppression of p38 activation. The
similarities between these effects of hypertonic saline and dibutyryl
cAMP-AM on the suppression of MAPK signaling further support the notion that cAMP accumulation may be involved in the suppression of neutrophil functions by hypertonic stress.
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Inhibition of PKA reduces the suppressive effects of moderate but
not high levels of hypertonicity.
Because the cAMP-dependent PKA, which is activated by cAMP, is known to
play an important role in the downregulation of neutrophil functions,
we speculated that hypertonic saline could recruit PKA to suppress
fMLP-induced superoxide formation. To test this possibility, we used
the specific PKA inhibitor H-89. Neutrophils were treated with H-89 for
1 h, exposed to hypertonic saline or forskolin for 5 min, and
stimulated with 100 nM fMLP, and superoxide formation was
determined (Fig. 4). H-89
dose-dependently prevented the suppression of superoxide formation by
40 mM hypertonic saline and forskolin. Interestingly, H-89 was not
effective in preventing the suppression of superoxide production by 100 mM hypertonic saline. These data suggest that low levels of
hypertonicity (40 mM) exert their suppressive effect on neutrophil
function largely by activating PKA, while higher levels of
hypertonicity (100 mM) seem to utilize additional PKA-independent
mechanism(s).
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Suppression of MAPK signaling by hypertonic stress involves PKA.
Although cAMP/PKA signaling is known to suppress fMLP-stimulated
neutrophil responses, the exact sites at which it affects the signal
pathways that lead to superoxide formation have remained unclear
(1, 6). Because we have seen that hypertonic saline blocks
fMLP signaling upstream of ERK and p38 MAPK activation, we speculated
that PKA could be responsible for this effect of hypertonic saline. To
test this concept, cells were pretreated with increasing doses of H-89
for 1 h, exposed to 40 or 100 mM hypertonic saline for 10 min, and
stimulated with 100 nM fMLP for 3 min, and MAPK activation was
determined. Hypertonic stress at 40 mM, and more markedly at 100 mM,
reduced the activation of ERK and p38 MAPK by fMLP. The different
degrees of MAPK suppression shown in Figs. 3 and
5, A and B, are
probably due to the differences in timing of hypertonic saline exposure
(1 h vs. 10 min). Inhibition of PKA with 0.1-1 µM H-89
completely prevented the suppressive effects caused by 40 mM hypertonic
saline and by forskolin (Fig. 5). The data with forskolin clearly
demonstrate that cAMP/PKA signaling interferes with fMLP-induced
signaling at a site(s) upstream of the ERK and p38 MAPKs. Although H-89
completely prevented the suppression of MAPK signaling caused by 40 mM
hypertonic saline (Fig. 5A), H-89 was less effective in
counteracting the suppression caused by high levels of hypertonicity
(100 mM hypertonic saline; Fig. 5B). The different effects
of H-89 in the presence of 40 and 100 mM hypertonic saline correspond
well with the data shown in Fig. 4. These observations provide further
evidence for our conclusion that lower hypertonic saline levels inhibit
fMLP signaling preferentially with a PKA-dependent mechanism, while
higher hypertonic saline levels trigger additional PKA-independent
mechanisms.
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Suppression of superoxide formation by hypertonic stress is
sensitive to manipulations of cAMP levels with PDE and AC inhibitors.
On the basis of the findings described above, cAMP accumulation and PKA
activation appear to be responsible for the inhibition of superoxide
formation by physiologically relevant hypertonicity levels. Thus
manipulation of intracellular cAMP levels with pharmacological agents
would be expected to modulate the suppressive effects of hypertonic
stress. To further examine the role of cAMP/PKA signaling in this
context, neutrophils were treated with IBMX or MDL-12330A, inhibitors
of PDE or AC, respectively, to manipulate intracellular cAMP levels.
The cells were then stimulated with fMLP in the presence or absence of
hypertonic saline, and superoxide formation was determined. Because of
its significant effects on superoxide formation, the IBMX dose used in
this experiment (60 µM) was optimized with pilot studies to permit
clear observations of functional responses to hypertonic saline. IBMX
significantly reduced superoxide formation of fMLP-stimulated cells in
the absence of hypertonic saline, and repressed superoxide formation
was further decreased in the presence of hypertonic saline (Fig.
6A). Figure 6B
shows that IBMX-treated neutrophils were more susceptible to
suppression by hypertonic saline, inasmuch as the hypertonic saline
dose leading to half-maximal suppression decreased from 35 to 25 mM.
This is consistent with the concept that hypertonic saline-induced cAMP accumulation is exaggerated by inhibition of PDE with IBMX and that
exaggerated cAMP accumulation is related to increased neutrophil suppression. On the other hand, when cAMP production was blocked with
the AC inhibitor MDL-12330A, fMLP-stimulated superoxide formation increased and the suppression of superoxide formation by hypertonic saline was completely abrogated (Fig. 6C). These findings
demonstrate the important physiological role of cAMP/PKA signaling in
the regulation of superoxide formation in general and in response to
hypertonic stress. The additive effects of cAMP accumulation by PDE
inhibition and hypertonic saline (Fig. 6, A and
B) and the full restoration of normal cell function by
inhibition of AC (Fig. 6C) clearly demonstrate the important
role of cAMP in the suppression of neutrophil functions by hypertonic
saline.
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DISCUSSION |
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We previously reported that hypertonic stimulation of neutrophils triggers specific signaling processes that involve tyrosine phosphorylation of a number of cellular proteins and the activation of p38 MAPK (26). Although these osmotic signaling processes did not directly elicit functional responses of otherwise unstimulated cells, they clearly are able to modulate responses including superoxide formation, degranulation, and chemotaxis of cells stimulated with agents such as fMLP, complement fragments, and phorbol 12-myristate 13-acetate (20, 26, 28, 37).
An important consequence of hypertonic stimulation is that it renders neutrophils largely incapable of mounting functional responses to subsequent stimulation with chemoattractants. Hypertonic stress elicited by the addition of NaCl, KCl, choline chloride, or sucrose profoundly suppressed neutrophil functions in response to fMLP stimulation, even when the cells were exposed to hypertonic stress and fMLP at the same time (26). This shows that hypertonic stress rapidly elicits an inhibitory signal that is fast enough to intercept activation signals triggered by the fMLP receptor. In addition to this fast response, hypertonic stress suppressed neutrophils for extended periods of time (26). These suppressive properties of hypertonicity have attracted the attention of clinicians who hope to use hypertonic resuscitation fluids to prevent neutrophil-mediated organ damage after hemorrhagic shock. Despite the growing interest in the potential therapeutic use of hypertonic solutions, the underlying molecular mechanisms whereby hypertonic stress inhibits neutrophil activation have remained unclear.
In neutrophils, physiologically relevant hypertonicity levels activate p38 MAPK, but not the c-Jun NH2-terminal kinase 1/2 (JNK1/2) or ERK1/2 members of the MAPK family (26). Stimulation of neutrophils with fMLP strongly activates the ERK1/2 and p38 MAPKs (data not shown; 27, 49). Although p38 MAPK activation in response to hypertonic stress and fMLP differs in timing and signal intensity, both signaling pathways clearly overlap at the level of p38 MAPK activation. Despite this overlap, however, hypertonic stimulation can intercept fMLP-induced signaling upstream of p38 MAPK, particularly when cells are exposed to hypertonic stress before they are stimulated with fMLP. This is evident in Fig. 3, which shows that the fMLP-induced portion of p38 MAPK activation is eliminated when neutrophils are exposed to hypertonicity before stimulation with fMLP. Under these conditions, hypertonicity blocked not only fMLP-induced activation of p38 MAPK but also activation of ERK. This indicates that hypertonic stress triggers a suppressive signal pathway that interferes with fMLP-signaling events at multiple sites upstream of the ERK and p38 MAPK cascades or at a shared site upstream of the point of bifurcation connecting the pathways that lead to ERK and p38 MAPK activation. The suppressive effect of hypertonic stress on the activation of these MAPK cascades closely corresponded with its suppressive effects on superoxide formation (Fig. 2). Although the exact roles of the ERK and p38 MAPKs in the activation of superoxide formation are still controversial, our data suggest that hypertonic stress inhibits superoxide formation by blocking signaling steps upstream of ERK and p38 MAPK signaling. This possibility is in agreement with findings of Detmers et al. (13), who reported that p38 MAPK activation is required for the NADPH oxidase assembly.
In the present study, we were able to show that hypertonic stimulation of neutrophils causes a rapid and dose-dependent accumulation of cAMP, that cAMP-elevating drugs can mimic the suppressive effects of hypertonicity, and that cAMP accumulation and PKA activation seem to play important roles in the suppression of neutrophil functions by hypertonic stress. To our knowledge, this is the first report to show that hypertonic stress results in cAMP accumulation in human neutrophils. Interestingly, osmotic stress has long been known to trigger cAMP accumulation or enhance receptor-stimulated cAMP accumulation in other mammalian cell types, particularly renal cells. For example, hypotonic shock increases the cAMP content of rat renal inner medulla cells by three- to fivefold (12), and hypertonic exposure enhances cAMP accumulation of vasopressin-stimulated renal cells (5, 30, 43, 44). Similarly, hypertonic stress activates cAMP accumulation in Dictyostelium discoideum, where cAMP signaling acts to protect the cells from hypertonic stress and to control spore germination (36, 46). These similarities in the response to hypertonic stress of mammalian cells and their distant relative are intriguing, because they suggest that cAMP accumulation could be another evolutionarily conserved osmotic signaling response, in addition to the better-known response that involves p38 MAPK. Dictyostelium expresses two types of ACs. The aggregation AC (ACA) harbors 12 putative membrane-spanning domains and is highly homologous to mammalian ACs. ACA is activated through G protein-coupled receptors and produces the cAMP signal that regulates cell movement and differentiation. The germination AC (ACG) contains a single transmembrane domain and serves as an osmosensor, producing a cAMP signal that controls germination of Dictyostelium spores under hypertonic conditions. It is unknown whether mammalian cells express an ACG equivalent. Thus the possibility cannot be ruled out that hypertonic stimulation causes cAMP accumulation in neutrophils by direct activation of an osmosensitive AC.
However, considerably more evidence points toward the involvement of G protein-coupled receptors in the cAMP response of mammalian cells to hypertonic stress. In endothelial and smooth muscle cells, mechanical deformation has been shown to cause cAMP accumulation and PKA activation, indicating that mechanical stress may activate Gs protein-coupled mechanoreceptors (9, 14, 24, 33, 35). Because neutrophils shrink in response to hypertonic stress (18, 29), shrinkage-induced membrane deformation could lead to the activation of Gs protein-coupled mechanoreceptors, which may be responsible for the observed cAMP production in response to hypertonic stress (Fig. 1). This model is made more compelling by the known ability of activated G protein-coupled receptors to inhibit other G protein-coupled receptors through an inhibitory cross talk that is referred to as heterologous desensitization (3, 21, 42). Thus the activation of Gs protein-coupled mechanoreceptors in neutrophils could cause heterologous desensitization of the fMLP receptor, which itself is a Gi protein-coupled receptor (41).
PKA plays an important role in the inhibitory cross talk that leads to
the heterologous desensitization of -adrenergic receptors by direct
receptor phosphorylation, which attenuates agonist-stimulated GTPase
activity (21, 42). In neutrophils, heterologous
desensitization of chemoattractant receptors, including the fMLP and
C5a receptors, has been observed (3), although the role of
PKA in this context has not been delineated. It remains to be seen
whether cAMP accumulation and PKA activation in response to hypertonic
stress affect the fMLP receptors directly or whether there are
additional mechanisms whereby PKA inhibits downstream signaling events.
PKA has been shown to inhibit ERK MAPK signaling in a number of cells
by interfering with the activation of Raf-1 by Ras or by directly
targeting Raf-1 (8, 11, 45, 47, 50). In neutrophils, PKA
has been shown to affect fMLP-induced phosphoinositide 3-kinase
activation, which is a critical event in the signaling steps leading to
superoxide formation (1). In addition, PKA may directly
affect components of the NADPH oxidase complex and reduce its ability
to produce superoxide. For example, PKA was reported to downregulate
the activation of the NADPH oxidase component p47phox in
response to fMLP stimulation and to affect Rac2 and Rap1A indirectly
through inhibition of phosphoinositide 3-kinase or through direct
phosphorylation (1, 2, 6, 7, 16).
Our data demonstrate that cAMP/PKA signaling in response to hypertonic stress plays an important role in the suppression of neutrophil function by moderate hypertonicity levels, i.e., levels that could be potentially used in clinical applications aimed at controlling excessive neutrophil activation in trauma patients. However, we observed that more severe hypertonic stress (100 mM beyond isotonicity) suppresses neutrophil responses by additional PKA-independent mechanisms. The nature of these additional suppressive mechanisms that cause inhibition of fMLP-induced signal transduction and superoxide formation remains to be elucidated. Interestingly, a recent report by Rizoli et al. (40) demonstrated that hypertonic stress markedly affected the remodeling of the actin skeleton in neutrophils. These effects were observed at high levels of extracellular hypertonicity (100 mM) and may represent at least one of the PKA-independent suppressive mechanisms whereby severe hypertonic stress blocked fMLP-induced neutrophil responses in our study.
Under normal physiological conditions, plasma tonicity can vary as a
consequence of water loss and salt uptake. In humans, plasma tonicity
was found to vary with age (32), climate and seasonal
changes (23), and such activities as water/salt uptake and
physical exercise (17), which can transiently change
plasma Na+ levels by 10 mM. In addition, neutrophils face
drastic differences in extracellular tonicity when patrolling organs of
the urinary tract system. In trauma patients, hypertonic resuscitation
fluids can be used to restore blood volume, and the infusion of these solutions transiently increases plasma tonicity (31).
Plasma Na+ levels of trauma patients treated with 7.5%
NaCl were found to be elevated by up to 22 mM even when measured as
late as 2 h after infusion of hypertonic saline (31).
These hypertonicity levels suppress neutrophil function by ~50%
(Fig. 2) and can be even more potent when left in contact with
neutrophils for extended periods (34). Hypertonic saline
infusion has been shown to reduce posttraumatic complications in
subgroups of trauma patients (31) and to prevent
neutrophil-mediated organ damage in several animal models of
hemorrhagic shock (4, 10, 25, 39).
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ACKNOWLEDGEMENTS |
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This work was supported in part by National Institute of General Medical Sciences Grants R29-GM-51477 and R01-GM-60475 (W. G. Junger) and Office of Naval Research Grant N00014-00-1-0851 (W. G. Junger).
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FOOTNOTES |
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Address for reprint requests and other correspondence: W. G. Junger, Surgical Immunology Research Laboratory, Dept. of Surgery, Div. of Trauma, University of California San Diego Medical Center, 200 West Arbor Dr., San Diego, CA 92103-8236 (E-mail: wjunger{at}ucsd.edu).
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. Section 1734 solely to indicate this fact.
10.1152/ajpcell.00479.2001
Received 9 October 2001; accepted in final form 4 January 2002.
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