Hypertonicity increases cAMP in PMN and blocks oxidative burst by PKA-dependent and -independent mechanisms

Tatjana Orlic, William H. Loomis, Amy Shreve, Sachiko Namiki, and Wolfgang G. Junger

Surgical Immunology Research Laboratory, Department of Surgery, Division of Trauma, University of California, San Diego, California 92103-8236


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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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


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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.


    METHODS
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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<UP><SUB>2</SUB><SUP>−</SUP></UP>) formation was determined with a slightly modified version of the cytochrome c reduction method described previously (26). Briefly, neutrophils (2 × 106/ml) were incubated with 100 µM cytochrome c (Fe3+; Sigma Chemical) and 100 nM fMLP for 10 min at 37°C, placed on ice, and centrifuged in the cold. The optical density at 550 nm of supernatants was used to calculate ·O<UP><SUB>2</SUB><SUP>−</SUP></UP> formation. As a control, superoxide dismutase (50 µg/ml) was added to verify that cytochrome c reduction was the result of ·O<UP><SUB>2</SUB><SUP>−</SUP></UP> formation.

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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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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Fig. 1.   Hypertonic stress causes cAMP accumulation in neutrophils. A: neutrophils were exposed to hypertonic stress at a hypertonicity level of 100 mM by addition of appropriate volumes of a 1 M NaCl solution in Hanks' balanced saline solution. At the same time, 80 µM IBMX was added to prevent cAMP turnover by inhibition of phosphodiesterase activity. After incubation for the indicated periods, cells were lysed, and cAMP levels were determined with an enzyme-linked immunoassay. B: cells were treated with IBMX as described above, hypertonic saline (HS) was added, and cAMP levels were determined after 10 min. As positive controls, cells were stimulated with 100 µM forskolin for 10 min, which resulted in cAMP levels of 1,110 ± 230 fmol/106 cells. C: peak cAMP levels in response to a 10-min exposure to HS. Cells from the same volunteer were exposed to equivalent HS levels for 10 min, and the resulting superoxide (·O<UP><SUB>2</SUB><SUP>−</SUP></UP>) formation in response to N-formylmethionyl-leucyl-phenylalanine (fMLP) stimulation was determined. Peak cAMP levels in response to HS exposure were correlated with the corresponding degrees of suppression of superoxide formation. Values are means ± SD of 2 experiments performed in triplicate. * P < 0.05; ** P < 0.001 compared with controls at 0 min or 0 mM HS (Student's t-test).

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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Fig. 2.   Hypertonic stress and cAMP inhibit fMLP-induced superoxide formation. A: neutrophils were exposed to the indicated hypertonicity levels by addition of appropriate volumes of 1 M NaCl in Hanks' balanced saline solution. After 1 h, cells were stimulated with 100 nM fMLP, and superoxide formation was measured with the cytochrome c reduction assay. B: neutrophils were pretreated with the indicated concentrations of the cell-permeable cAMP analog dibutyryl cAMP- AM for 1 h and stimulated with 100 nM fMLP, and superoxide formation was measured. C: cells were pretreated with the indicated concentrations of forskolin for 1 h and stimulated with 100 nM fMLP, and superoxide formation was measured. D: neutrophils were simultaneously stimulated with the indicated doses of fMLP and HS at a hypertonicity level of 40 mM, and superoxide formation was determined. Values are means ± SD of 3 experiments with cells from different donors. * P < 0.05; ** P < 0.001, treated vs. untreated in A, B, and C and with vs. without HS in D (Student's t-test).

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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Fig. 3.   Hypertonic stress and cAMP inhibit fMLP-induced activation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinases (MAPKs). Cells were treated for 1 h with HS or dibutyryl cAMP-AM, stimulated with 100 nM fMLP for 3 min, and lysed, and ERK and p38 MAPK activation was determined by immunoblotting with phosphospecific antibodies that recognize the phosphorylated and, thereby, activated forms of the ERK and p38 MAPKs (p-ERK and p-p38, respectively). Membranes were reprobed with antibodies that recognize the active and inactive forms of these MAPKs to control for differences in the amount of total protein in different lanes (bottom panels). Ratio of blot densities was used to calculate increases in MAPK phosphorylation. Experiments were repeated 3 times with cells from different donors.

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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Fig. 4.   Protein kinase A (PKA)-dependent and -independent mechanisms are involved in suppression of fMLP-induced superoxide formation by hypertonic stress. Neutrophils were incubated for 1 h with the PKA inhibitor H-89 and exposed to hypertonic stress by increasing the extracellular tonicity by 40 mM (A) or 100 mM (B). C: 50 µM forskolin was added as a control. After 5 min, cells were stimulated with 100 nM fMLP, and superoxide formation was determined. Values are means ± SD of 3 similar experiments performed in duplicate. * P < 0.05; ** P < 0.001 (Student's t-test).

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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Fig. 5.   PKA is involved in suppression by hypertonic stress of fMLP-induced ERK and p38 MAPK activation. Cells were treated for 1 h with the PKA inhibitor H-89 and exposed for 10 min to 40 mM HS (A), 100 mM HS (B), or 50 µM forskolin (C). After 5 min, cells were stimulated with 100 nM fMLP for 3 min, and ERK and p38 MAPK activation was determined by immunoblotting with phosphospecific antibodies (p-ERK and p-p38). The presence of comparable amounts of protein in different lanes was verified by reprobing membranes with antibodies that recognize active and inactive forms of these MAPKs (bottom blots). Experiments were repeated 3 times with cells from different donors.

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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Fig. 6.   Hypertonic stress-induced neutrophil suppression is subject to modulation of intracellular cAMP levels. A: neutrophils were left unstimulated (no stim) or simultaneously exposed to the phosphodiesterase inhibitor IBMX (60 µM) alone (IBMX), 100 nM fMLP, and/or 20 mM HS, and superoxide formation was determined. B: cells were simultaneously stimulated with HS and 100 nM fMLP in the presence () or absence (open circle ) of the phosphodiesterase inhibitor IBMX (60 µM), and superoxide production was determined. C: neutrophils were incubated for 5 min with increasing doses of the adenylyl cyclase inhibitor MDL-12330A, stimulated with 100 nM fMLP in the absence (open bars) or presence of 40 mM HS (solid bars), and superoxide formation was determined. Values are means ± SD of experiments done in duplicate and repeated 3 times with cells from different donors. ** P < 0.001 (Student's t-test).


    DISCUSSION
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ABSTRACT
INTRODUCTION
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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 beta -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).


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Ahmed, MU, Hazeki K, Hazeki O, Katada T, and Ui M. Cyclic AMP-increasing agents interfere with chemoattractant-induced respiratory burst in neutrophils as a result of the inhibition of phosphatidylinositol 3-kinase rather than receptor-operated Ca2+ influx. J Biol Chem 270: 23816-23822, 1995[Abstract/Free Full Text].

2.   Akasaki, T, Koga H, and Sumimoto H. Phosphoinositide 3-kinase-dependent and -independent activation of the small GTPase Rac2 in human neutrophils. J Biol Chem 274: 18055-18059, 1999[Abstract/Free Full Text].

3.   Ali, H, Richardson RM, Haribabu B, and Snyderman R. Chemoattractant receptor cross-desensitization. J Biol Chem 274: 6027-6030, 1999[Free Full Text].

4.   Angle, N, Hoyt DB, Coimbra R, Liu F, Herdon-Remelius C, Loomis W, and Junger WG. Hypertonic saline resuscitation diminishes lung injury by suppressing neutrophil activation following hemorrhagic shock. Shock 9: 164-170, 1998[ISI][Medline].

5.   Baudouin-Legros, M, Badou A, Paulais M, Hammet M, and Teulon J. Hypertonic NaCl enhances adenosine release and hormonal cAMP production in mouse thick ascending limb. Am J Physiol Renal Fluid Electrolyte Physiol 269: F103-F109, 1995[Abstract/Free Full Text].

6.   Bengis-Garber, C, and Gruener N. Protein kinase A downregulates the phosphorylation of p47phox in human neutrophils: a possible pathway for inhibition of the respiratory burst. Cell Signal 8: 291-296, 1996[ISI][Medline].

7.   Bokoch, GM, Quilliam LA, Bohl BP, Jesaitis AJ, and Quinn MT. Inhibition of rap1A binding to cytochrome b558 of NADPH oxidase by phosphorylation of rap1A. Science 254: 1794-1796, 1991[ISI][Medline].

8.   Burgering, BMT, Pronk GJ, Vanweeren PC, Chardin P, and Bos JL. cAMP antagonizes p21ras-directed activation of extracellular signal-regulated kinase 2 and phosphorylation of mSos nucleotide exchange factor. EMBO J 12: 4211-4220, 1993[Abstract].

9.   Cohen, CR, Mills I, Du W, Kamal K, and Sumpio BE. Activation of the adenylyl cyclase/cyclic AMP/protein kinase A pathway in endothelial cells exposed to cyclic strain. Exp Cell Res 231: 184-189, 1997[ISI][Medline].

10.   Coimbra, R, Hoyt DB, Junger WG, Angle N, Wolf P, Loomis W, and Evers MF. Hypertonic saline resuscitation decreases susceptibility to sepsis following hemorrhagic shock. J Trauma 42: 602-607, 1997[ISI][Medline].

11.   Cook, SJ, and McCormick F. Inhibition by cAMP of Ras-dependent activation of Raf. Science 262: 1069-1072, 1993[ISI][Medline].

12.   Craven, PA, Briggs R, and DeRubertis FR. Calcium-dependent action of osmolality on adenosine 3',5'-monophosphate accumulation in rat renal inner medulla: evidence for a relationship to calcium-responsive arachidonate release and prostaglandin synthesis. J Clin Invest 65: 529-542, 1980[ISI][Medline].

13.   Detmers, PA, Zhou D, Polizzi E, Thieringer R, Hanlon WA, Vaidya S, and Bansal V. Role of stress-activated mitogen-activated protein kinase (p38) in beta 2-integrin-dependent neutrophil adhesion and the adhesion-dependent oxidative burst. J Immunol 161: 1921-1929, 1998[Abstract/Free Full Text].

14.   Du, W, Mills I, and Sumpio BE. Cyclic strain causes heterogeneous induction of transcription factors, AP-1, CRE binding protein and NF-kappa B, in endothelial cells: species and vascular bed diversity. J Biomech 28: 1485-1491, 1995[ISI][Medline].

15.   Fujishima, S, and Aikawa N. Neutrophil-mediated tissue injury and its modulation. Intensive Care Med 21: 277-285, 1995[ISI][Medline].

16.   Gabig, TG, Crean CD, Mantel PL, and Rosli R. Function of wild-type or mutant Rac2 and Rap1a GTPases in differentiated HL60 cell NADPH oxidase activation. Blood 85: 804-811, 1995[Abstract/Free Full Text].

17.   Greenleaf, JE, Jackson CG, and Lawless D. CD4+/CD8+ T-lymphocyte ratio: effects of rehydration before exercise in dehydrated men. Med Sci Sports Exerc 27: 194-199, 1995[ISI][Medline].

18.   Grinstein, S, Woodside M, Sardet C, Pouyssegur J, and Rotin D. Activation of the Na+/H+ antiporter during cell volume regulation: evidence for a phosphorylation-independent mechanism. J Biol Chem 267: 23823-23828, 1992[Abstract/Free Full Text].

19.   Grunewald, JM, Grunewald RW, and Kinne RK. Regulation of ion content and cell volume in isolated rat renal IMCD cells under hypertonic conditions. Am J Physiol Renal Fluid Electrolyte Physiol 267: F13-F19, 1994[Abstract/Free Full Text].

20.   Hampton, MB, Chambers ST, Vissers MCM, and Winterbourn CC. Bacterial killing by neutrophils in hypertonic environments. J Infect Dis 169: 839-846, 1994[ISI][Medline].

21.   Hausdorff, WP, Caron MG, and Lefkowitz RJ. Turning off the signal: desensitization of beta -adrenergic receptor. FASEB J 4: 2881-2889, 1990[Abstract].

22.   Haussinger, D. The role of cellular hydration in the regulation of cell function. Biochem J 313: 697-710, 1996[ISI][Medline].

23.   Henrotte, JG. Variation of plasma potassium and potassium tolerance in man in relation to climatic adaptation. Fed Proc 25: 1375-1379, 1966[ISI][Medline].

24.   Iba, T, Mills I, and Sumpio BE. Intracellular cyclic AMP levels in endothelial cells subjected to cyclic strain in vitro. J Surg Res 52: 625-630, 1992[ISI][Medline].

25.   Junger, WG, Coimbra R, Liu FC, Herdon-Remelius C, Junger W, Junger H, Loomis W, Hoyt DB, and Altman A. Hypertonic saline resuscitation: a tool to modulate immune function in trauma patients? Shock 8: 235-241, 1997[ISI][Medline].

26.   Junger, WG, Hoyt DB, Davis RE, Herdon-Remelius C, Namiki S, Junger H, Loomis W, and Altman A. Hypertonicity regulates the function of human neutrophils by modulating chemoattractant receptor signaling and activating mitogen-activated protein kinase p38. J Clin Invest 101: 2768-2779, 1998[Abstract/Free Full Text].

27.   Krump, E, Nikitas K, and Grinstein S. Induction of tyrosine phosphorylation and Na+/H+ exchanger activation during shrinkage of human neutrophils. J Biol Chem 272: 17303-17311, 1997[Abstract/Free Full Text].

28.   Krump, E, Sanghera JS, Pelech SL, Furuya W, and Grinstein S. Chemotactic peptide N-formyl-Met-Leu-Phe activation of p38 mitogen-activated protein kinase (MAPK) and MAPK-activated protein kinase-2 in human neutrophils. J Biol Chem 272: 937-944, 1997[Abstract/Free Full Text].

29.   Kültz, D, and Burg M. Evolution of osmotic stress signaling via MAP kinases cascades. J Exp Biol 201: 3015-3021, 1998[Abstract/Free Full Text].

30.   Lutz, W, and Kumar R. Hypertonic sucrose treatment enhances second messenger accumulation in vasopressin-sensitive cells. Am J Physiol Renal Fluid Electrolyte Physiol 264: F228-F233, 1993[Abstract/Free Full Text].

31.   Mattox, KL, Maningas PA, Moore EE, Mateer JR, Marx JA, Aprahamian C, Burch JM, and Pepe PE. Prehospital hypertonic saline/dextran infusion for posttraumatic hypotension: the USA multicenter trial. Ann Surg 213: 482-491, 1991[ISI][Medline].

32.   McLean, KA, O'Neill PA, Davies I, and Morris J. Influence of age on plasma osmolality: a community study. Age Aging 21: 56-60, 1992[Abstract].

33.   Mills, I, Letsou G, Rabban J, Sumpio B, and Gewirtz H. Mechanosensitive adenylate cyclase activity in coronary vascular smooth muscle cells. Biochem Biophys Res Commun 171: 143-147, 1990[ISI][Medline].

34.   Murao, Y, Hoyt DB, Loomis W, Namiki S, Patel N, Wolf P, and Junger WG. Does the timing of hypertonic saline resuscitation affect its potential to prevent lung damage? Shock 14: 18-23, 2000[ISI][Medline].

35.   Oluwole, BO, Du W, Mills I, and Sumpio BE. Gene regulation by mechanical forces. Endothelium 5: 85-93, 1997[Medline].

36.   Ott, A, Oehme F, Keller H, and Schuster SC. Osmotic stress response in Dictyostelium is mediated by cAMP. EMBO J 19: 5782-5792, 2000[Abstract/Free Full Text].

37.   Patrick, DA, Moore EE, Offner PJ, Johnson JL, Tamura DY, and Silliman CC. Hypertonic saline activates lipid-primed human neutrophils for enhanced elastase release. J Trauma 44: 592-597, 1998[ISI][Medline].

38.   Rane, MJ, Carrithers SL, Arthur JM, Klein JB, and McLeish KR. Formyl peptide receptors are coupled to multiple mitogen-activated protein kinase cascades by distinct signal transduction pathways: role in activation of reduced nicotinamide adenine dinucleotide oxidase. J Immunol 159: 5070-5078, 1997[Abstract].

39.   Rizoli, SB, Kapus A, Fan J, Li YH, Marshall JC, and Rotstein OD. Immunomodulatory effects of hypertonic resuscitation on the development of lung inflammation following hemorrhagic shock. J Immunol 161: 6288-6296, 1998[Abstract/Free Full Text].

40.   Rizoli, SB, Rotstein OD, Parodo J, Phillips MJ, and Kapus A. Hypertonic inhibition of exocytosis in neutrophils: central role for osmotic actin skeleton remodeling. Am J Physiol Cell Physiol 279: C619-C633, 2000[Abstract/Free Full Text].

41.   Schreiber, RE, Prossnitz ER, Ye RD, Cochrane CG, Jesaitis AJ, and Bokoch GM. Reconstitution of recombinant N-formyl chemotactic peptide receptor with G protein. J Leukoc Biol 53: 470-474, 1993[Abstract].

42.   Sibley, DR, Benovic JL, Caron MG, and Lefkowitz RJ. Regulation of transmembrane signaling by receptor phosphorylation. Cell 48: 913-922, 1987[ISI][Medline].

43.   Skorecki, KL, Conte JM, and Ausiello DA. Effects of hypertonicity on cAMP production in cultured renal epithelial cells (LLC-PK1). Miner Electrolyte Metab 13: 165-172, 1987[ISI][Medline].

44.   Torikai, S, and Imai M. Effects of solute concentration on vasopressin stimulated cyclic AMP generation in the rat medullary thick ascending limbs of Henle's loop. Pflügers Arch 400: 306-308, 1984[ISI][Medline].

45.   Vaillancourt, RR, Gardner AM, and Johnson GL. B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP. Mol Cell Biol 14: 6522-6530, 1994[Abstract].

46.   Van Es, S, Virdy KJ, Pitt GS, Meima M, Sands TW, Devreotes PN, Cotter DA, and Schaap P. Adenylyl cyclase G, an osmosensor controlling germination of Dictyostelium spores. J Biol Chem 271: 23623-23625, 1996[Abstract/Free Full Text].

47.   VanRenterghem, B, Browning MD, and Maller JL. Regulation of mitogen-activated protein kinase activation by protein kinases A and C in a cell-free system. J Biol Chem 269: 24666-24672, 1994[Abstract/Free Full Text].

48.   Weiss, SJ. Tissue destruction by neutrophils. N Engl J Med 320: 365-376, 1989[ISI][Medline].

49.   Worthen, GS, Avdi N, Buhl AM, Suzuki N, and Johnson GL. FMLP activates ras and raf in human neutrophils: potential role in activation of MAP kinase. J Clin Invest 94: 815-823, 1994[ISI][Medline].

50.   Wu, J, Dent P, Jelinek T, Wolfman A, Weber MJ, and Sturgill TW. Inhibition of the EGF-activated MAP kinase signaling pathway by adenosine 3',5'-monophosphate. Science 262: 1065-1069, 1993[ISI][Medline].


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