 B Activation and Cytokine Release
in Human Alveolar Macrophages Is PKC-independent and
TK- and PC-PLC-dependent
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Abstract |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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A critical feature of sepsis-induced adult respiratory distress syndrome (ARDS) is the release of cytokines
(such as interleukin [IL]-6, IL-8, and tumor necrosis factor [TNF]) from endotoxin (lipopolysaccharide
[LPS])-activated alveolar macrophages (AM). Nuclear factor kappa B (NF-
B) is activated in AM from
patients with ARDS, and it is essential for the transcription of many cytokine genes. In these studies, we
evaluated the regulation of LPS-induced cytokine release and the activation of NF-B in human AM. We
found that the activation of NF-B and the release of IL-6, IL-8, and TNF from AM exposed to LPS was
protein kinase C-independent and tyrosine kinase- and phosphatidylcholine-specific phospholipase C-dependent. We also found that LPS-induced activation of NF-B was enhanced in AM cultured in serum or
in the presence of LPS-binding protein, simulating conditions in the lung that are present in ARDS. In addition, LPS triggered the activation of several different NF-B complexes in AM, and different forms of
NF-B bound to the IL-6, IL-8, and TNF promoter sequences. These observations suggest that physiologic
abnormalities present in the lungs of patients with ARDS facilitate the activation of NF-B and local release of cytokines.
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Introduction |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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The adult respiratory distress syndrome (ARDS) is a form of acute lung injury that often results from sepsis. Many factors are involved in the development of ARDS, but one of the early and ongoing characteristics of this disease is inflammation in the lung. These inflammatory responses are triggered, at least in part, by alveolar macrophage (AM)-derived cytokines. Several studies have shown that various cytokines are increased in the lung in patients with ARDS (1). Further, the persistence of high cytokine concentrations in the lung has been correlated with ongoing inflammatory injury (2). Although many cytokines are present in patients with ARDS, three cytokines, interleukin (IL)-6, IL-8, and tumor necrosis factor (TNF), have been evaluated in many studies, and they are associated with continued inflammation and poor outcome (1).
The regulation of these cytokines in macrophages is
controlled, at least in part, at the level of gene transcription. A crucial transcription factor that regulates expression of the IL-6, IL-8, and TNF genes is nuclear factor
kappa B (NF-B). It regulates expression of these genes
by binding to specific promoter sequences (4). A primary means by which NF-B is activated to bind to the
promoters of these genes is via translocation of the factor from the cytoplasm to the nucleus of the cell. This occurs
following phosphorylation and degradation of an inhibitor
protein, IB, which binds NF-B in the cytoplasm and prevents its translocation to the nucleus (14). It is likely that
the regulation of NF-B in AM after exposure to lipopolysaccharide (LPS) is critical to the inflammatory response that occurs in ARDS. In fact, NF-B is present in
the nucleus of AM in patients with ARDS (15). No prior
studies, however, have evaluated how NF-B is activated in AM after exposure to LPS.
Several studies, using other types of cells, have evaluated second messenger pathways that regulate NF-B after exposure to LPS (16). These studies, as an aggregate, show that NF-B is not regulated by the same
mechanisms in various types of cells. Pathways that have
been linked to NF-B activation include protein kinase C
(PKC) pathways (16, 17), tyrosine kinase (TK) pathways (16, 18), and phosphatidylcholine-specific phospholipase C (PC-PLC) pathways (22). We found that NF-B
activation by LPS in AM is PKC-independent and TK-
and PC-PLC-dependent. Release of IL-6, IL-8, and TNF
was also PKC-independent and TK- and PC-PLC-dependent. In addition, we found that different NF-B complexes bind to specific promoter sequences in the IL-6,
IL-8, and TNF genes. Finally, the ability of LPS to trigger
NF-B activation in AM is enhanced by the presence of
serum and LPS-binding protein (LBP), factors that are increased in the lungs of patients with ARDS.
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Materials and Methods |
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Isolation of AM
The use of normal volunteers to obtain AM by bronchoalveolar lavage was approved by the Human Subjects Review Board of the University of Iowa College of Medicine. AM were obtained from normal volunteers who met the following criteria: (1) age between 18 and 45 yr; (2) no history of cardiopulmonary disease or other chronic disease; (3) no prescription or nonprescription medication except oral contraceptives; (4) no recent or current evidence of infection; and (5) lifetime nonsmoker. The volunteers underwent fiberoptic bronchoscopy and bronchoalveolar lavage in subsegments of the right upper lobe, right middle lobe, and lingula after receiving 0.6 mg atropine delivered intramuscularly and adequate local anesthesia. Each subsegment of the lung was lavaged with five 20-ml aliquots of normal saline, and the first aliquot in each subsegment was discarded. The percentage of AM was determined by Wright-Giemsa stain. The percentage of AM varied from 90 to 98% of the cells.
Expression of NF-B
We initially determined that the maximal expression (nuclear translocation) of NF-B occurred 3 h after stimulation with LPS and that the optimal dose of LPS was 1 µg/
ml. AM were cultured at 37°C for 3 h in Roswell Park Memorial Institute (RPMI)-1640 medium alone, RPMI medium with 100 ng/ml of LBP (a generous gift from Richard
Ulevitch, Scripps Research Institute, La Jolla, CA), or
RPMI medium with 5% fetal calf serum. The cells were
also cultured in each of these three conditions in the presence or absence of 1 µg/ml Escherichia coli serotype 026:
B6 LPS (Sigma Chemical Co., St. Louis, MO). In some instances, various inhibitors (Calbiochem, La Jolla, CA)
were added 15 min prior to the addition of LPS. The PKC
inhibitors were 1 nM staurosporine and 50 nM bisindomaleamide. The TK inhibitors were 40 µM genistein and
10 µM tyrophostin AG 126. The PC-PLC inhibitor was
100 µM D609. After 3 h of exposure to LPS, the cells
were washed in phosphate-buffered saline. They were
then resuspended in a lysis buffer (10 mM Hepes, 10 mM
KCl, 2 mM MgCl2, 2 mM EDTA) for 15 min on ice. Nonidet P-40 (NP-40) (10%) was added to lyse the cells, and
the cells were centrifuged at 4°C at 14,000 rpm. The nuclear pellet was resuspended in an extraction buffer (50 mM Hepes, 50 mM KCl, 300 mM NaCl, 0.1 mM EDTA,
10% glycerol) for 20 min on ice. After centrifuging at 4°C at 14,000 rpm, the supernatant was stored at 
70°C.
NF-B oligonucleotides were labeled with [
-32P]ATP
(New England Nuclear/Dupont, Boston, MA). The consensus oligonucleotide (5'-AGTTGAGGGGACTTTCCCAGGC-3') was obtained from Promega (Madison, WI),
and the IL-6 (5'-AGTTGAGGGGATTTTCCCAGGC-3'), IL-8 (5'-AGTTGAGTGGAATTTCCCAGGC-3'), and
TNF3 (5'-AGTTGAGGGGTTTCTCCCAGGC-3') NF-B oligonucleotides were purchased from Research Genetics (Huntsville, AL). Electrophoretic mobility shift assays (EMSA) were performed at room temperature for 30 min with 5 µg of nuclear extract protein and the 32P-labeled
oligonucleotides in the presence of an incubation buffer (1 M Tris, glycerol, NP-40, 0.1 M ZnSO4, 1 M DTT, 2 M
KCl, and 1 M MgCl2) and 1 µg of poly [d(I-C)] (Boehringer Mannheim, Indianapolis, IN). Supershift assays
were performed with the addition of p50, p52, p65, Rel B,
and c-Rel antibodies (Santa Cruz Biotechnology, Santa
Cruz, CA). The protein-DNA complexes were separated
on 5% polyacrylamide gels at 25 to 45 mA. The gels were
subsequently vacuum-dried and exposed to autoradiographic film (Amersham, Arlington Heights, IL) at 70°C
for 12 to 48 h.
Expression of Mitogen-activated Protein Kinases
AM were cultured for 15 min at 37°C after stimulation
with either 10 ng/ml phorbol myristate acetate (PMA) or
1 µg/ml LPS. PKC inhibitors were used in cells stimulated
with PMA, and TK inhibitors and the PC-PLC inhibitor
were used in cells stimulated with LPS. The cells were harvested, resuspended in a lysis buffer (1% NP-40, 0.15 M
NaCl, 0.05 M Tris [pH 7.4], 100 µg/ml phenylmethylsulfonyl fluoride, 50 µg/ml aprotinin, 10 µg/ml leupeptin, 50 µg/
ml pepstatin, 0.4 M NaVO4, 10 mM NaFl, and 10 mM sodium pyrophosphate), sonicated, and placed on ice for 20 min. After centrifuging at 14,000 rpm at 4°C for 10 min to
remove cellular debris, the lysates were stored at 70°C.
Extracellular signal-regulated kinase-2 (Erk2) was immunoprecipitated from the lysates (500 µg) overnight at 4°C
with Erk2 antibody (Santa Cruz Biotechnology) bound to
Gammabind with sepharose (Pharmacia Biotech, Uppsala,
Sweden). The sepharose pellet was placed in a kinase buffer
(20 mM MgCl2, 25 mM Hepes, 20 mM 
-glycerophosphate,
20 mM P-nitrophenylphosphate, 0.1 mM NaVO4, and 2 mM
dithiothreitol), and kinase activity was assayed by phosphorylation of myelin basic protein (MBP) using 5 µCi/sample [-32P]ATP, 20 µM ATP, and 10 µg/ml MBP (Sigma).
After 15 min, the reaction was stopped with the addition
of SDS sample buffer, and the samples were heated to
95°C for 5 min to separate the protein from the sepharose.
The samples were separated on a 10% SDS-PAGE discontinuous gel at 45 mA. The gels were then vacuum-dried
and exposed to autoradiographic film. Western blots were
performed simultaneously to ensure equal loading of the
samples.
Expression of Cytokines
For these studies, AM were cultured in RPMI medium with 5% fetal calf serum for 24 h in the presence or absence of LPS and with and without inhibitors, as described earlier. The amounts of IL-6, IL-8, and TNF in the supernatant of the cells was measured by enzyme-linked immunosorbent assay (R&D Systems, Minneapolis, MN).
Statistical Analysis
All of the cytokine measurements are shown as means with the standard error. Statistical comparisons were performed using an unpaired t test with a probability value of P < 0.05 considered to be significant.
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Results |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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NF-B DNA Binding Activity
In the absence of LPS, LBP, or serum, a small amount of
NF-B activity was present in AM (Figure 1). Both LBP
and serum alone caused a small increase in NF-B activity.
With each of these conditions, LPS caused a significant increase in NF-B (Figure 1). In absence of LPS, LBP, or serum, only p50 NF-B was detected (data not shown). Both
LBP- and serum-exposed AM had p50/65 NF-B (data not
shown). LPS-stimulated AM contained p50/65 NF-B in all three (serum-free, LBP, and 5% serum) conditions (data
not shown). These initial studies were performed using the
consensus NF-B oligonucleotide. These studies show that
LPS increases NF-B in the nucleus of AM. The studies
further show that both LBP and serum (simulating conditions in the lungs of patients with ARDS) enhance this effect of LPS.
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NF-B Binding to Specific NF-B DNA Sequences
It has previously been shown that specific NF-B complexes exhibit varying affinities for sequences in the promoter regions of cytokine genes (4). Therefore, we
used specific NF-B DNA-binding sequences from the
IL-6, IL-8, and TNF promoters for these studies. In LPS-stimulated AM, both p50 and p65 NF-B proteins bound
to the IL-6 sequence, a p65 NF-B protein bound to the
IL-8 sequence, and a p50 NF-B protein bound to the
TNF sequence (Figures 2a through 2c). We did not detect
p52, Rel B, or c-rel proteins in these assays. These findings
suggest that different NF-B complexes are generated by
LPS in AM and that specific NF-B complexes are likely
used for the transcription of these cytokine genes.
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LPS-induced NF-B Is Independent of PKC Activity
To determine if PKC plays a role in the activation of NF-B
in AM, the cells were cultured in the presence or absence
of staurosporine, a nonspecific PKC inhibitor, and bisindomaleamide, which is a relatively specific PKC inhibitor.
LPS-induced NF-B in AM was not inhibited by bisindomaleamide (Figure 3), and similar results were obtained with
staurosporine (data not shown). To confirm that these PKC
inhibitors were active, we performed Erk2 in vitro kinase
assays in cells stimulated with PMA. We found that PMA-induced Erk2 kinase activity in AM was significantly reduced
by PKC inhibition with staurosporine (data not shown).
These observations strongly suggest that LPS-induced activation of NF-B in AM occurs independently of PKC.
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LPS-induced NF-B Is Partially Dependent on TK Activity
To determine if tyrosine kinases play a role in the generation of NF-B by LPS in AM, the cells were cultured in the
presence of genistein, a specific TK inhibitor with broad-spectrum activity, and tyrophostin AG 126, which is a relatively specific TK inhibitor. LPS-induced NF-B was partially inhibited by both inhibitors (Figures 4a and 4b). To
confirm that other pathways of tyrosine phosphorylation were inhibited with the inhibitors that we used, we performed Erk2 in vitro kinase assays in cells stimulated with
LPS. We found that LPS-induced Erk2 kinase activity in
AM was significantly reduced to near control levels in the
presence of genistein (data not shown). These observations
strongly suggest that tyrosine kinase(s) play an important
role in the activation of NF-B in AM exposed to LPS.
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LPS-induced NF-B Is Dependent on PC-PLC Activity
To determine if activation of a PC-PLC plays a role in the
activation of NF-B in AM exposed to LPS, the cells were
cultured in the presence of D609, a highly specific inhibitor of PC-PLC. D609 significantly inhibited NF-B in AM
exposed to LPS (Figure 5). We also performed Erk2 in
vitro kinase assays in cells exposed to LPS to confirm that
other pathways linked to PC-PLC activity are inhibited by
D609. We found that LPS-induced Erk2 kinase activity in
AM is significantly reduced to control levels in the presence of D609 (data not shown). These observations suggest that the activation of a PC-PLC is necessary for the
activation of NF-B in AM by LPS.
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Effect of Cytokine Release from AM Stimulated with LPS
To determine if the same second messenger pathways that
regulate the activation of NF-B also regulate cytokine release in AM, we measured the amounts of IL-6, IL-8, and
TNF released by AM stimulated by LPS. We found that
IL-6, IL-8, and TNF are all increased in the supernatants
of the cells exposed to LPS (Figure 6). PKC inhibition with
bisindomaleamide did not significantly (IL-6: P = 0.884;
IL-8: P = 0.994; TNF: P = 0.132) affect the release of any
of these cytokines from AM. Similar results were obtained
with staurosporine (data not shown). TK inhibition with genistein caused a significant (IL-6: P = 0.009; IL-8: P = 0.046; TNF: P = 0.005) decrease in the release of all three
cytokines. Similar results were found with tyrophostin AG
126 (data not shown). PC-PLC inhibition with D609 significantly (IL-6: P = 0.002; IL-8: P = 0.025; TNF: P < 0.001)
decreased cytokine release to levels near control values
(Figures 7a through 7c). These observations show that
similar pathways are used for the activation of NF-B and
cytokine release in LPS-stimulated AM.
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Correlation of NF-B Activation and Cytokine
Release in a Dose-dependent Manner
Although we found that similar pathways are used for
both NF-B activation and cytokine release in LPS-stimulated AM, we also evaluated if the inhibitors used had similar effects on each in a dose-dependent manner. Culture
conditions were done as outlined above, but genistein was
used at 10 µM and 40 µM and D609 was used at 25 µM
and 100 µM. We found that genistein and D609 decreased both NF-B activation (Figures 8A and 8B) and cytokine
(IL-6) release (Figure 8C) in a dose-dependent manner.
These observations further suggest that similar pathways
are used for the activation of NF-B and cytokine release
in LPS-stimulated AM.
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Discussion |
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Abstract
Introduction
Materials & Methods
Results
Discussion
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A primary feature of ARDS is a leak of serum proteins
(including LBP) onto the alveolar surface of the lung. This
disorder is also associated with the activation of NF-B
and the release of cytokines from AM. One of the major
causes of ARDS is sepsis. In these studies, we found that
the activation of NF-B in AM by LPS is markedly enhanced by both LBP and serum. LPS-induced NF-B in
AM appeared to be independent of PKC and was dependent on both TK and PC-PLC activity. LPS-induced release of IL-6, IL-8, and TNF from AM also appeared to be
independent of PKC and was significantly reduced with
both TK and PC-PLC inhibition. We also found that LPS
triggered the activation of several different NF-B complexes, and different forms of NF-B bound to the IL-6,
IL-8, and TNF promoter sequences. To our knowledge,
these are the first studies that have evaluated the activation of NF-B in human AM in response to LPS.
The role of PKC in the activation of NF-B and the release of cytokines also has not been previously evaluated
in human AM, but its role in cytokine release has been
evaluated in rat AM (26). In rat AM, LPS-induced TNF
production is reduced by staurosporine. Other studies have
evaluated the consequence of PKC inhibition on NF-B
activation in other types of cells, including human monocytes (18). Human monocytes do not require PKC activity
for NF-B activation by LPS. Interestingly, in both of
these studies, PKC inhibition by staurosporine resulted in
reduced TNF release. As staurosporine can alter expression of other second messenger pathways (27), it is not
clear if the effect on release of TNF was due to inhibition
of PKC. Our studies in human AM strongly suggest that
LPS-induced activation of NF-B and release of IL-6, IL-8, and TNF occurs independently of most PKC isoforms. One
caution in interpreting this data is that some PKC isoforms
(i.e., PKC
) are poorly inhibited by the agents used in this
study.
The role of TK activity in the activation of NF-B has
not been previously evaluated in human AM. Several
studies (16, 18), however, have evaluated the role of
TK in NF-B activation and cytokine release in other
types of cells. The effect of TK inhibition on LPS-induced
NF-B translocation has been controversial. In human
monocytes exposed to LPS, NF-B activation and cytokine release have been shown to be inhibited by both herbimycin A and genistein (18). In Chinese hamster ovary
cells and RAW 264.7 cells, TK inhibitors did not prevent
LPS-induced NF-B translocation (20). A recent study has
also shown that LPS-induced NF-B translocation in
THP-1 cells is not inhibited by genistein (21). Several studies have shown that there is increased TK activity after
LPS binds to the CD14 cell surface receptor in both monocytes and macrophages (28). Tyrosine phosphorylation
has also been reported to be linked to inactivation of IB
(31). Our studies strongly suggest that NF-B translocation and cytokine production in human AM are partially
dependent on TK activity.
The role of PC-PLC activity in LPS-induced generation
of NF-B also has not been previously evaluated in human
AM. A study, using a murine model, however, reported
that the systemic effects of sepsis could be prevented with
D609, a specific and potent inhibitor of PC-PLC (32). The
role of PC-PLC in TNF-induced activation of NF-B has
been evaluated (22). This effect is not due to a diacylglycerol-dependent activation of PKC. Instead, TNF-induced PC-PLC appears to trigger the production of ceramide, which serves as a second messenger downstream
of PC-PLC. Ceramide is produced as a result of the activation of an acid sphingomyelinase. The acid sphingomyelinase, which is activated by diacylglycerol, hydrolyzes
sphingomyelin to produce ceramide. The ceramide then activates ceramide-activated protein kinase, which phosphorylates IB on a serine residue, resulting in its degradation (33, 34). This mechanism would explain why D609
inhibited NF-B and cytokine release in our studies while
inhibitors of PKC had no effect. In our studies, D609 was
the most potent inhibitor of both NF-B and cytokine production in human AM stimulated with LPS.
The NF-B complexes that bind specific cytokine promoters have not been evaluated in human AM. Other
studies have shown, however, that not all NF-B promoter
sequences efficiently bind all forms of NF-B, and the sequence determines which NF-B complex(es) will bind.
Several studies have reported that a p50/65 NF-B binds to both the IL-6 and TNF promoters and that a p65/65
NF-B binds to the IL-8 promoter (4). Our findings in
human AM exposed to LPS are consistent with these previous studies, with one exception: we could detect only the
p50 NF-B protein in the complex that bound the TNF3
promoter.
The enhanced effect of serum and LBP on NF-B activation in AM is relevant to ARDS. The normal lung alveoli have little exposure to serum. In ARDS, however,
there is increased conductance of serum proteins, including LBP, into the lung. We found NF-B activation to be
much greater in AM incubated in serum or with the addition of LBP. NF-B activation has been previously reported to be activated in AM from patients with ARDS
(15). Although NF-B can be activated in AM in serum-free conditions, its increased activity in AM incubated in
serum or LBP simulates conditions present in ARDS, in
which there are increased amounts of plasma and proteins
(including LBP) in the lung. These observations suggest
that the leak of serum proteins into the lung may magnify
the level of production of cytokines and, possibly, accentuate the lung injury.
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Footnotes |
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Address correspondence to: Aaron Brent Carter, M.D., Pulmonary Division, Room C323 GH, University of Iowa Hospitals & Clinics, 200 Hawkins Dr., Iowa City, IA 52242. E-mail: aaron-carter{at}uiowa.edu
(Received in original form March 26, 1997 and in revised form July 22, 1997).
Acknowledgments: These studies were supported by Grants HL 37121 and AI 35018 from the National Institutes of Health and by a VA Merit Review grant. The writers thank Anita Riggan for her outstanding assistance in preparing the manuscript.
Abbreviations
AM, alveolar macrophage(s);
ARDS, adult respiratory distress
syndrome;
EMSA, electrophoretic mobility shift assay(s);
Erk2, mitogen-activated protein kinase;
IL, interleukin;
LBP, lipopolysaccharide-binding protein;
LPS, lipopolysaccharide;
NF-B, nuclear factor kappa B;
NP-40, Nonidet P-40;
PC-PLC, phosphatidylcholine-specific phospholipase C;
PKC, protein kinase C;
PMA, phorbol myristate acetate;
RPMI, Roswell Park Memorial Institute;
TK, tyrosine kinase;
TNF, tumor necrosis factor.
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References |
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Introduction
Materials & Methods
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Discussion
References
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