1 Program in Cell Biology, Department of Pediatrics, National Jewish Medical and Research Center, Denver 80206; and Departments of 4 Immunology, 2 Pediatrics, 3 Medicine, and 5 Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262
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ABSTRACT |
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Inflammation, characterized
by the presence of proinflammatory chemokines and neutrophils, is a
hallmark of early airway disease in infants with cystic fibrosis (CF),
although the underlying mechanisms remain poorly defined. In this
study, we evaluated the role of NaCl and the ensuing hyperosmolar
effect on tumor necrosis factor (TNF)- signaling and
apoptosis in macrophages. Incubation of mouse macrophages with
NaCl activated p38mapk and the p46jnk and
p54jnk c-jun NH2-terminal kinase
isoforms, but not p42mapk/erk2 or Akt. Similar results were
obtained with sorbitol, suggesting a general response to
hyperosmolarity. Strikingly, the activation of p42mapk/erk2
and Akt by TNF-
was also inhibited in the presence of NaCl. Because
the activation of p42mapk/erk2 and Akt has been associated
with survival responses, we investigated the effect of NaCl on
macrophage apoptosis. The results indicated a synergistic
increase in apoptosis when macrophages were exposed to TNF-
in the presence of NaCl compared with stimulation with TNF-
alone or
NaCl alone. Furthermore, pharmacological inhibition of
p42mapk/erk2 and Akt mimicked the effect of NaCl.
Collectively, these findings indicate that modest elevations in NaCl
differentially regulate the activation of mitogen-activated protein
kinases and Akt and potentiate macrophage apoptosis. We
speculate that augmentation of macrophage apoptosis in CF
airways may result in decreased clearance of neutrophils and in
deficiencies in the elimination of common CF pathogens.
mitogen-activated protein kinase; extracellular signal-related kinase
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INTRODUCTION |
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CYSTIC FIBROSIS (CF) is the most common fatal hereditary disorder of the Caucasian population and leads to death due to chronic progressive pulmonary disease. The CF lung is thought to be normal in utero; however, the pulmonary manifestations begin early in infancy with evidence of endobronchial infection (1, 3, 4) and airway inflammation noted as early as 4 wk of age (22). These infants are often found to have increased numbers of neutrophils and interleukin (IL)-8 in their airways in the absence of detectable airway infection. The CF gene encodes a defective cystic fibrosis transmembrane conductance regulator (CFTR) protein that leads to abnormal electrolyte transport in epithelial cells. Although much research has been dedicated to elucidating exactly how the CF electrolyte transport defect leads to persistent lung infection and inflammation, the mechanism is not fully resolved (11).
The electrolyte composition of airway surface liquid (ASL) in healthy
individuals and CF subjects has been difficult to determine and has
been a source of recent controversy (16). Initial studies reported that bronchial chloride concentrations were increased in CF
subjects compared with subjects with chronic bronchitis (170 ± 79 mM and 85 ± 54 mM, respectively) (14). Similar
findings were reported by Joris and colleagues (21) in
their study of ASL [Cl] in CF patients (129 ± 5 mM) and normal subjects (84 ± 9 mM). A similar increase in
[Na+] was found in CF subjects (121 ± 4 mM)
compared with control subjects (82 ± 6 mM). In addition, in a
bronchial epithelial cell xenograft model in nu/nu mice,
Goldman and colleagues (15) found that xenografts of CF
bronchial epithelial cells produced abnormally high [Na+]
and [Cl
] levels ([Na+] = 172 ± 9 mM; [Cl
] = 178 ± 9 mM) compared with normal
grafts ([Na+] = 83 ± 3 mM; [Cl
] = 83 ± 3 mM). However, other studies have failed to detect
significant differences in electrolyte concentrations of ASL obtained
from normal, CF, and chronic bronchitis subjects (20, 23).
Given the findings suggesting that electrolyte concentrations in the
ASL of patients with CF are hypertonic with respect to serum
concentration as well as with respect to the ASL of normal subjects, we
were intrigued by the possible role that differences in Na+
and Cl concentrations may play in the early, exuberant
inflammatory response in CF and, in particular, their effects on
macrophage functions. Previous studies have shown that different cell
types respond to elevated levels of NaCl and the ensuing hyperosmolar effect by activating members of the mitogen-activated protein kinase
(MAPK) family, including p38mapk, p46jnk
(c-jun NH2-terminal kinase), and
p54jnk isoforms (13). Activation of these
serine/threonine kinases has been shown to contribute to protective
stress responses as well as to induce apoptosis in cells
stressed by exposure to hyperosmolarity (12, 25,
39). In addition, Shapiro and Dinarello
(32) have shown that macrophages respond to
hyperosmolarity by expressing and secreting increased levels of
proinflammatory cytokines, including tumor necrosis factor (TNF)-
and IL-8. These findings thus provide important clues that elevated
NaCl or hyperosmolarity in general may contribute to changes in
macrophage function in the airways of patients with CF. To address this
question, we investigated the effect of hyperosmolarity on the
activation of MAPKs and Akt and on the induction of macrophage
apoptosis in primary cultures of mouse macrophages and in the
human monocyte-like cell line THP-1. As we will show, hyperosmolarity
enhanced the activation of p38mapk, p46jnk, and
p54jnk isoforms induced by the proinflammatory cytokine
TNF-
but inhibited the activation of p42mapk/erk2
(extracellular signal-related kinase) and Akt. In addition, these conditions potentiated macrophage apoptosis.
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MATERIALS AND METHODS |
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Materials.
C3H/HeJ mice were bred at the National Jewish Center Biological
Resource Center (Denver, CO) and were used throughout the study to
avoid the possibility of stimulation by trace amounts of endotoxin
contaminants (28). Dulbecco's modified Eagle's medium
(DMEM) was purchased from Whittaker Bioproducts (Walkersville, MD), and
fetal bovine serum (FBS) was obtained from Irvine Scientific (Santa
Ana, CA). The human monocyte-like cell line THP-1 was obtained from
American Type Culture Collection (Rockville, MD). Recombinant c-Jun1-79-GST and anti-p38mapk antiserum
were generously provided by Dr. Gary Johnson (National Jewish Medical
and Research Center, Denver, CO). Protein A-Sepharose beads
were obtained from Sigma (St. Louis, MO). Rabbit anti-phosphospecific p44mapk/erk1 and p42mapk/erk2 antibody was
purchased from Promega (Madison, WI). Rabbit anti-phosphospecific p38mapk and phospho-Ser473 Akt antibodies were
obtained from New England Biolabs (Beverly, MA). Mouse monoclonal
anti-phosphospecific JNK (G-7), rabbit polyclonal anti-JNK1 (C-17),
anti-p38mapk (C-20), and anti-p42mapk/erk2
antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz,
CA) or Upstate Biotechnology (Lake Placid, NY). Donkey anti-rabbit and
sheep anti-mouse IgG F(ab')2 horseradish
peroxidase-conjugated antibodies, [-32P]ATP, and
enhanced chemiluminescence kits were purchased from Amersham Life
Science (Arlington Heights, IL). Recombinant mouse TNF-
and
apoptosis detection kits were obtained from R&D Systems (Minneapolis, MN), and the in situ cell death detection kit was obtained from Roche Molecular Biochemicals (Indianapolis, IN).
Macrophage isolation and culture.
Bone marrow-derived macrophages were cultured from femoral and tibial
bone marrow, as previously described (30). The growth medium was DMEM containing 100 U/ml penicillin, 100 µg/ml
streptomycin, 10% (vol/vol) heat-inactivated FBS, and 10% (vol/vol)
L929 cell-conditioned medium (as a source of colony stimulating
factor-1). Bone marrow cells were cultured at a density of
2.4 × 105 cells/cm2 at 37°C in a 10%
CO2 atmosphere for 5-6 days. One hour before stimulation, one-half of the medium was removed and centrifuged at
1,000 rpm for 10 min to remove any nonadherent cells. NaCl, sorbitol, and/or TNF- were added at twice the concentrations indicated. The conditioned media were then added back to the macrophage monolayers at the time of stimulation. This method of stimulation was
used to avoid the stimulation of MAPKs that occurs upon transfer to
fresh medium. THP-1 cells were cultured in RPMI 1640 containing 10%
(vol/vol) heat-inactivated fetal calf serum and 1% (wt/vol) glutamine,
100 U/ml penicillin, and 100 µg/ml streptomycin and were maintained
at densities between 0.2-1 × 106 cells/ml. The
cells were split every 3-4 days.
In vitro kinase assays.
JNK activity was measured in lysates of mouse macrophages as previously
described (8). GST-c-Jun1-79 was bound to sepharose beads as the affinity matrix and substrate for
phosphorylation in the presence of [-32P]ATP. Kinase
activities of p38mapk, p42mapk/erk2, and
p44mapk/erk1 were measured by immunoprecipitation with the
appropriate antibody followed by in vitro kinase assays using
recombinant activating transcription factor-2 as substrate in
the presence of [
-32P]ATP as previously described
(37). All reactions were terminated by the addition of 2×
Laemmli sample buffer containing 20 mM dithiothreitol (DTT) and were
boiled for 5 min. The proteins were then separated by SDS-PAGE on 12%
gels and transferred to nitrocellulose membranes. The
32P-labeled substrates were detected by autoradiography.
Western blot analysis. Macrophage monolayers were lysed on ice with 300 µl of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 1 mM Na3VO4) at 4°C, the insoluble material was removed by centrifugation at 14,000 rpm for 10 min, and protein was equalized in each sample using the bicinchoninic acid method (29). To each sample, 5× Laemmli sample buffer containing 20 mM DTT was added, and the samples were boiled for 5 min. Samples were separated by SDS-PAGE on 12% gels and were transferred onto nitrocellulose membranes. The blots were washed in Tris-Tween-buffered saline [20 mM Tris, pH 7.6, containing 137 mM NaCl and 0.05% (vol/vol) Tween] and blocked with 5% (wt/vol) dry milk dissolved in Tris-Tween-buffered saline for 1 to 2 h. The membranes were probed overnight at 4°C with phosphospecific p44mapk/erk1, p42mapk/erk2, p38mapk, or JNK antibodies, or nonphosphospecific p42mapk/erk2, p38mapk, or JNK1 antibodies in 5% (wt/vol) bovine serum albumin and 0.02% (wt/vol) sodium azide as described (36). Phosphorylation of Akt at Ser473 was detected by immunoprecipitation of total Akt from lysates of ~2 × 107 bone marrow macrophages or THP-1 cells as described (37), followed by Western blotting with anti-phospho-Ser473 Akt antibody. In some experiments, Ser473-phosphorylated Akt was detected directly by SDS-PAGE of macrophage whole cell lysates, followed by Western blotting as described above. Bound antibody was detected with anti-rabbit or anti-mouse IgG F(ab')2 horseradish peroxidase-conjugated antibody as the secondary antibody for 1 h. The enhanced chemiluminescence method was used to detect bound conjugated secondary antibody.
Quantification of apoptosis. Macrophage monolayers were grown on bacteriological grade petri dishes and stimulated as described in RESULTS for 5 h. Cells were then removed from the culture plates using a gentle jet technique so that cells would not be damaged or unnecessarily disrupted. In all experiments, an unstimulated negative control and an anisomycin-positive control for apoptosis were used. Cells were treated as described in the apoptosis detection kit and as previously reported by Bratton et al. (6). Briefly, the cells were centrifuged at 1,000 rpm for 10 min at 4°C, washed twice with 5 ml of cold 1× Dulbecco's PBS, and suspended in 1× binding buffer at a concentration of 1 × 106 cells/ml. One hundred microliters of cells were transferred to 5-ml culture tubes and stained with 100 ng of fluorescein-conjugated annexin V and 500 ng of propidium iodide reagent. The reactions were gently vortexed and allowed to incubate for 15 min at room temperature. Each sample was given 400 µl of binding buffer, and samples were analyzed by flow cytometry. Analysis was done on a Becton Dickinson (San Jose, CA) FACScalibur flow cytometer, and the results were analyzed with PC Lysis software (Becton Dickinson). Annexin-positive cells were determined as described in the apoptosis detection kit by setting quadrants to separate viable cells from propidium iodide-permeant cells and nonapoptotic cells from those staining highly for the FITC-labeled annexin V probe. Percent apoptosis was determined from the cells staining greater than the control population threshold.
To confirm the apoptosis seen above, cells were also studied using a terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay to detect fragmented DNA. Bone marrow-derived macrophages grown on glass coverslips in 12-well plates were stimulated as described in RESULTS for 24 h. Macrophages were washed with PBS, fixed for 15 min at room temperature in a solution containing 3% (wt/vol) paraformaldehyde and 3% (wt/vol) sucrose in PBS (pH 7.5), washed again, and permeabilized with 0.2% (vol/vol) Triton X-100 for 10 min. Cells were then incubated with terminal transferase reaction solution containing fluorescein-conjugated dUTP for 1 h at 37°C as recommended by the manufacturer (Roche Diagnostics, Indianapolis, IN). The cells were washed three times with 0.03 M sodium citrate, pH 7.4, containing 0.3 M sodium chloride to remove unbound nucleotides and were then washed with PBS. DNA was stained with Hoechst-33342 at 10 µg/ml. After being washed with PBS, the coverslips were incubated overnight in PBS supplemented with 0.02% sodium azide and mounted in a solution containing 90% glycerol, 10% Tris · HCl, pH 8.5, and 20 mg/ml o-phenylenediamine as an antifading agent. Cells were observed with a Leica DMR/XA confocal immunofluorescence microscope using a ×100 plan objective. The percentage of TUNEL-positive cells was determined by counting three times at least 200 cells with a confocal microscope.Osmolality and electrolyte measurement.
Osmolality was measured by freezing-point depression using an Advanced
Micro-Osmometer (model 3MO; Advanced Instruments, Norwood, MA). Sodium
and chloride concentrations were quantitated by ion-selective electrodes using a Beckman CX3 automated chemistry analyzer (Brea, CA).
The osmolality, sodium, and chloride measurements of the media with
NaCl or sorbitol added are shown in Table
1.
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Statistical analysis. The apoptosis data are shown as means ± SE. Results were analyzed using a nonparametric repeated measures analysis of variance to compare different conditions. Statistics were calculated using SAS version 6.12 (Cary, NC). P < 0.05 was considered significant.
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RESULTS |
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Hyperosmolar activation of JNK and p38mapk, but not
ERK, in mouse macrophages.
To investigate the effect of the addition of NaCl on MAPK activation,
we used in vitro kinase assays to quantify the activity of JNK,
p38mapk, p42mapk/erk2, and
p44mapk/erk1. Macrophage monolayers either were left
unstimulated or were stimulated with media containing increasing
concentrations of NaCl (0-200 mM) for 10 min and were then lysed
as described in MATERIALS AND METHODS. We also conducted
experiments in which cell lysates from unstimulated and NaCl-exposed
macrophages were analyzed by Western blotting using phosphospecific
antibodies for JNK, p38mapk, or ERK. The same blots were
then probed with the respective nonphosphospecific antibodies to
control for equal loading. Preliminary experiments established that 10 min was the optimal time point of MAPK activation. As can be seen in
Fig. 1A, after stimulation with NaCl, JNK activity was increased as detected using both the in
vitro kinase assay and Western blot analysis. JNK activity was
stimulated in response to increasing concentrations of NaCl and peaked
at 200 mM. Figure 1B illustrates the results of the in vitro
kinase assay and Western blot analyses for p38mapk, which
also showed increased kinase activity first seen at 50 mM NaCl and
peaking at 200 mM NaCl. In the corresponding Western blot using
phosphospecific p38mapk antibody, maximal stimulation was
seen at 200 mM NaCl. The effect of NaCl on p42mapk/erk2
activity is illustrated in Fig. 1C. In contrast to the
effects of NaCl on JNK and p38mapk, there was no activation
of p42mapk/erk2 or p44mapk/erk1 above baseline
in response to NaCl in either the kinase assay or Western blots using
phosphospecific anti-p42/p44mapk antibody. Thus increasing
concentrations of NaCl stimulate JNK and p38mapk but do not
stimulate p42mapk/erk2 or p44mapk/erk1
activity.
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MAPK activation by hyperosmolarity and TNF-.
In addition to the possibility of exposure to ASL containing increased
amounts of NaCl in CF compared with normal ASL, macrophages and other
cell types are also exposed to a multitude of inflammatory and other
stimuli, including TNF-
, in the airways of patients with CF. We
therefore questioned whether costimulation of macrophages with TNF-
under hyperosmolar conditions would affect MAPK signaling. Macrophage
monolayers were stimulated with a fixed concentration of TNF-
(1 ng/ml) in media containing increasing concentrations of NaCl
(0-200 mM) for 10 min. MAPK activity was quantified by in vitro
kinase assays and Western blot analysis using phosphospecific antibodies for each MAPK. As shown in Fig.
3A and as previously reported
(8, 27), TNF-
alone activated JNK, whereas the addition
of NaCl increased phosphorylation of JNK beginning at 100 mM and
peaking at 200 mM. Figure 3B shows that the TNF-
-induced p38mapk activation was also modestly increased by the
addition of NaCl. p42mapk/erk2 activity is shown in Fig.
3C under the same conditions as described above. TNF-
alone activated p42mapk/erk2; however, unlike the
potentiation of JNK and p38mapk activation, the addition of
NaCl markedly diminished the activity of p42mapk/erk2.
p42mapk/erk2 activity was decreased with as little as 50 mM
NaCl in the in vitro kinase assay and continued to decrease to baseline
with 200 mM NaCl (Fig. 3C). This was demonstrated by both in
vitro kinase assay and Western blot using phosphospecific
p42/p44mapk antibody. Thus costimulation with TNF-
and
increasing concentrations of NaCl resulted in a modest increase in the
activation of JNK and p38mapk and a significant inhibition
of p42mapk/erk2 and p44mapk/erk1 activation.
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Hyperosmolarity inhibits the activation of Akt.
An important function of p42mapk/erk2 is in the control of
cell proliferation and in cell survival responses. In view of the
striking inhibitory effect of hyperosmolarity on the activation of
p42mapk/erk2, we next questioned whether the inhibitory
effect of hyperosmolarity also extends to another prosurvival protein
kinase signaling pathway, namely the activation of Akt
(9). Macrophage monolayers were stimulated with TNF-
(10 ng/ml) in either the presence or absence of 100 mM NaCl for 10 min
before lysis and analysis of Akt activation by Western blotting with a
phosphospecific antibody directed at Ser473. As can be seen
in Fig. 5A, Akt was basally
phosphorylated at low levels in mouse macrophages, consistent with the
presence of growth factors in the culture medium, whereas stimulation
with TNF-
markedly increased phosphorylation of Akt at
Ser473. However, under hyperosmolar conditions induced by
100 mM NaCl, the phosphorylation of Akt was reduced to basal levels
(Fig. 5A). Figure 5B shows a control experiment
confirming the inhibitory effect of hyperosmolarity on the
phosphorylation of p42mapk/erk2. We also observed an
inhibition of Akt phosphorylation in the human embryonic epithelial
cell line HEK-293 (data not shown), suggesting that epithelial cells
may behave similarly to macrophages in this respect. Thus
hyperosmolarity inhibits the activation of the survival pathways
involving both p42mapk/erk2 and Akt while potentiating the
activation of the stress-responsive pathways involving JNK and
p38mapk.
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Effect of hyperosmolarity and TNF- on macrophage
apoptosis.
Based on previous studies associating the activation of JNK and
p38mapk and the inhibition of ERK with apoptosis
(39), we investigated the effect of TNF-
and
hyperosmolarity induced by NaCl on the induction of macrophage
apoptosis. Mouse macrophage monolayers were exposed to 1 ng/ml
of TNF-
and 100 mM NaCl, either alone or combined, for 5 h. The
level of apoptosis was then quantified by flow cytometry after
staining with propidium iodide and annexin V. Figure
6A illustrates the effect of
various conditions on apoptosis. Cells exposed to normal media
had a baseline rate of apoptosis of 12.6 ± 1.2%, and
when exposed to anisomycin, a positive control, apoptosis was
induced in 50.6 ± 2.4% of cells (P < 0.001).
The addition of TNF-
alone or hyperosmolarity alone did not
significantly increase the degree of apoptosis compared with
normal media (TNF-
12.8 ± 1.7%, P = 0.88;
NaCl 18.3 ± 2.3%, P = 0.08). However, the combination of TNF-
and hyperosmolarity induced apoptosis in 23.1 ± 3.3% of macrophages (P = 0.007). These
studies were reproduced using the same conditions with mouse bone
marrow-derived macrophages exposed to 1 ng/ml of TNF-
and
hyperosmolarity induced with 100 mM NaCl alone or together for 24 h when apoptosis was detected using a TUNEL assay. Figure
6B demonstrates the effects of these conditions on
apoptosis using this method. In cells exposed to normal media,
apoptosis was undetectable, but after exposure to anisomycin,
apoptosis was detected in 67.5 ± 16.4% of the cells (P < 0.001). Stimulation with TNF-
alone did not
result in any significant increase in apoptosis above
unstimulated levels (2.4 ± 1.1%, P = 0.13).
However, hyperosmolarity alone resulted in a significant apoptotic
response (8.4 ± 2.7%, P = 0.003), although the
macrophages had an even greater rate of apoptosis when exposed to TNF-
under hyperosmolar conditions (23.9 ± 4.7%,
P = 0.003). Thus exposure of macrophages to TNF-
under hyperosmolar conditions potentiates apoptosis by both
methods of detection.
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Involvement of both p42mapk/erk2 and Akt in protection
against apoptosis.
In view of these findings, we next addressed the role of
p42mapk/erk2 and Akt in the protection against
apoptosis. Because both kinase cascades promote distinct
survival responses in different cell systems, we considered it possible
that either or both kinase cascades may be involved in the protection
against apoptosis after stimulation with TNF-. Thus we
reasoned that pharmacological inhibition of either pathway alone or
both pathways together would mimic the effects of hyperosmolarity
on macrophage apoptosis. Mouse bone marrow-derived macrophages
were incubated with PD-98059, a MEK1 inhibitor, LY-294002, a
phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor, or both
inhibitors to block the activation of ERK, Akt, or both pathways,
respectively. The cells were then left unstimulated or were
stimulated with TNF-
(10 ng/ml) in the presence and absence of the
inhibitors for 18 h before quantifying the degree of
apoptosis by TUNEL assay. As shown in Fig.
7A, in the absence of TNF-
,
incubation in PD-98059 or LY-294002 each modestly augmented the basal
level of apoptosis, whereas the effect of coincubation with
both inhibitors resulted in an additive increase in the basal level of
apoptosis. These findings suggest that both pathways may exert
modest prosurvival effects, likely as a result of the low level of
activation of these pathways seen in unstimulated conditions (Fig.
5). Incubation with PD-98058 or LY-294002 in the presence of TNF-
led to an augmentation in apoptosis compared with the level
seen in the absence of either inhibitor. However, coincubation with
both PD-98059 and LY-294002 in the presence of TNF-
led to a
synergistic increase in the degree of apoptosis to a level that
was comparable with that in cells incubated with TNF-
under
hyperosmolar conditions (Fig. 7A), indicating that both ERK
and Akt combine to exert a protective effect against TNF-
-induced
apoptosis.
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DISCUSSION |
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CF is the most common inherited fatal disease of Caucasians in the United States, with >90% of patients dying from progressive and unrelenting cycles of airway inflammation, infection, mucus plugging, and bronchiectasis. However, while much has been learned about the pathogenesis of pulmonary inflammation and infection, the precise mechanism(s) that connects the genetic mutations in the CFTR to fulminant lung disease is not well understood (11, 19). Until 1995, conventional dogma supported the view that the intense neutrophil recruitment and accumulation in the distal airways in CF arose primarily in response to colonization with common CF pathogens, especially Pseudomonas aeruginosa (24). However, previous studies from this and other laboratories have shown that pulmonary inflammation, characterized by increased levels of IL-8, neutrophils, and neutrophil secretory products, is established early in life and can be detected in bronchoalveolar lavage fluid from infants with CF as young as 4 wk of age (4, 22). These findings have recently been substantiated in a pathogen-free, fetal tracheal xenograft model in scid mice. Tirouvanziam et al. (35) found that approximately eightfold higher levels of IL-8 accumulated in the luminal fluid of xenografts from fetuses bearing mutations in CFTR compared with non-CF fetal tracheal xenografts. In addition, murine neutrophils were found to accumulate in higher numbers in the lamina propria of CF tracheal xenografts compared with non-CF xenografts. Collectively, these studies suggest that abnormalities in the function of CFTR are involved in early airway inflammation in CF and that inflammation can arise in the apparent absence of detectable infection. These observations thus raise the question of how the inflammatory response is both initiated and perpetuated in CF.
Characterization of the composition of ASL has led to two theories
regarding the pathogenesis of lung disease in CF. One hypothesis, originating from Michael Welsh's group in Iowa, proposes that the ASL
in CF has an increased NaCl content compared with normal ASL and has
been referred to as the "high-salt hypothesis" (33). The other hypothesis, proposed by Richard Boucher's group in North Carolina, proposes that CF ASL has a reduced volume compared with normal ASL and has often been referred to as the "low-volume
hypothesis" (23, 26). In the present study, we
investigated the effect of hyperosmolarity induced by NaCl on both the
signaling responses of macrophages to the proinflammatory cytokine
TNF- and on macrophage survival in the presence of TNF-
. The
principal findings of this study are that 1) the pattern of
MAPK and Akt phosphorylation and activation, induced by TNF-
, is
altered by hyperosmolarity and that 2) the changes in MAPK
and Akt activation are accompanied by an increase in macrophage
apoptosis. Previous studies have shown that
p42mapk/erk2, p38mapk,
p46jnk, and p54jnk isoforms are
rapidly and simultaneously activated in mouse macrophages in response
to TNF-
(8, 37, 38). The most striking change in MAPK
and Akt activation observed in the present study was the hyperosmolarity-dependent inhibition of the phosphorylation and activation of both kinases. These changes in kinase activation were
also seen when macrophages were stimulated in the presence of sorbitol,
suggesting that the alteration in activation was not a response to NaCl
per se but represented a more generalized response to hyperosmolarity.
Importantly, while the activation of p38mapk,
p46jnk, and p54jnk was induced only at
relatively high concentrations of NaCl, the inhibition of
p42mapk/erk2 and Akt activation was seen at concentrations
of NaCl similar to those that have been reported in the ASL of patients
with CF.
A growing body of literature supports the conclusion that
hyperosmolarity exerts significant effects on cell function and on the
activity of innate host defense systems. Studies reported by Smith and
colleagues (33) provided the first demonstration that
increases in the concentration of NaCl inhibited the ability of a
-defensin activity to kill P. aeruginosa when exposed
both in vitro and in cultures of airway epithelial cells. Goldman et al. (15) subsequently cloned human
-defensin-1 and
confirmed its exquisite sensitivity to NaCl. In addition, using human
bronchial epithelial cell xenografts in nu/nu mice, they
were able to demonstrate impaired killing of P. aeruginosa
in cells isolated from patients with CF. Other studies have also shown
that cultured human bronchial gland epithelial cells isolated from a CF
patient bearing the
F508 mutation secrete increased amounts of IL-8
compared with non-CF cells in response to NaCl (34). The
results from in vitro studies have also provided insights into how
hyperosmolar conditions are sensed. Based on earlier work on the
yeast-osmosensing gene HOG1, Han et al. (17)
cloned the mammalian homolog, p38mapk and showed it to be
activated in response to exposure of macrophages to hyperosmotic
stress. Similarly, the p46jnk and
p54jnk isoforms have been shown to be activated in response
to hyperosmotic stress in a variety of cell types (13). An
additional study also suggested that the activation of JNK after
exposure to hyperosmolarity was associated with clustering and
internalization of the cell surface receptors for epidermal growth
factor, TNF, and IL-1 (31). Last, pharmacological
inhibitor studies conducted by Hashimoto et al. (18) and
Shapiro and Dinarello (32) have shown that the induction
of IL-8 production in response to hyperosmotic stress is dependent on
the activation of p38mapk. Thus increased concentrations of
NaCl in the ASL of patients with CF compared with non-CF subjects might
be expected to have significant effects on the innate inflammatory
response in both the presence and absence of colonization with common
CF pathogens.
The results of the present study have also provided insight into a
potential mechanism that may also contribute to the high numbers of
neutrophils that are seen in CF pathogen-colonized adolescent patients
and adults with CF as well as in infants, namely decreased neutrophil
clearance as a consequence of the induction of macrophage
apoptosis in the presence of a proinflammatory stimulus
(TNF-) and increased levels of NaCl resulting in conditions of
hyperosmolarity. Hyperosmolarity alone was not sufficient to induce
macrophage apoptosis as has previously been reported in neutrophils (2, 12). Similarly, exposure to TNF-
alone
was found to be ineffective at inducing macrophage apoptosis.
The results of the present study clearly implicate the downregulation in ERK and Akt activation as contributing to the increase in
apoptosis seen when macrophages interact with TNF-
under
hyperosmolar conditions. Previous studies have shown that growth factor
withdrawal from rat PC-12 pheochromocytoma cells is accompanied by a
sustained activation of the JNK and p38mapk pathways and a
concurrent inhibition of p42mapk/erk2 activation
(39). This pattern of divergence in MAPK activation pathways, which is analogous to the results of the present study, also
led to the induction of apoptosis of PC-12 cells
(39). Similar results have also been found in human Jurkat
T lymphocytes in which activation of JNK and p38mapk in the
absence of p42mapk/erk2 activation also resulted in
apoptosis (25). Likewise, inhibition of Akt
activation either by pharmacological inhibition of PI 3-kinase or with
dominant inhibitory Akt mutants has been shown to block growth
factor-mediated survival responses via the phosphorylation-dependent inhibition of the proapoptotic activity of Bad (10)
and by blocking Fas ligand expression via the phosphorylation of the
forkhead transcription family member FKHRL1 (7).
Similarly, Berra and colleagues (5) have shown that
combined inhibition of both p42mapk/erk2 and Akt promotes
apoptosis in HeLa cells through effects on the activation of
p38mapk. Thus since both TNF-
and NaCl are believed to
be present at elevated concentrations in the ASL of the CF airway, it
is tempting to speculate that the excessive burden of neutrophils may
be partially due to apoptosis of airway macrophages, thereby
depriving neutrophils of a major clearance pathway.
In summary, the present study demonstrates that exposure of mouse
macrophages to TNF- in the presence of increasing NaCl leads to an
activation of p46jnk, p54jnk, and
p38mapk isoforms and a concomitant inhibition of the
activation of p42mapk/erk2 and Akt. Under these conditions,
macrophages were also induced to undergo apoptosis via the
inhibitory effects of hyperosmolarity on the activation of
p42mapk/erk2 and Akt. We speculate that this may partially
explain the persistence of inflammation in CF through impairment of an
important mechanism for the clearance of neutrophils.
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ACKNOWLEDGEMENTS |
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The authors thank Linda Remigio and Cheryl Leu for excellent technical assistance and Marci Sontag for statistical support.
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FOOTNOTES |
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This work was supported by a Cystic Fibrosis Foundation Pilot and Feasibility Project grant within the University of Colorado Research and Development Program in Cystic Fibrosis and by National Heart, Lung, and Blood Institute Public Health Service Grant HL-65326. G. S. Kerby was supported by a Harry Shwachman Cystic Fibrosis Clinical Investigator Award from the Cystic Fibrosis Foundation. V. Cottin was supported, in part, by a traveling fellowship from the Société de Pneumologie de Langue Française, the Association pour la Recherche contre le Cancer Fondation Alain Philippe, Fondation Lavoisier du Ministère des Affaires Etrangères, and a Michael and Eleanore Stobin 1999 Pediatric Fellowship from National Jewish Medical and Research Center, Denver, CO. This work was also supported by National Center for Research Resources Grant MO1-RR00069 and the Mike McMorris Cystic Fibrosis Center.
Address for reprint requests and other correspondence: D. W. H. Riches, Program in Cell Biology, Dept. of Pediatrics, National Jewish Medical and Research Center, Neustadt Rm. D405, 1400 Jackson St., Denver, CO 80206 (E-mail: richesd{at}njc.org).
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.
First published March 1, 2002;10.1152/ajplung.00263.2001
Received 16 July 2001; accepted in final form 24 February 2002.
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