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B and Activator Protein-21
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Department of Microbiology and Immunology, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642;
The Center for Blood Research and Department of Pathology, Harvard Medical School, Boston, MA 02115;
Department of Molecular Oncology, Cancer Research Institute, Kanazawa University, Kanazawa, Japan; and
Cancer Center, University of Rochester, Rochester, NY 14642
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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B were essential for the maximal induction of IL-8 by
3-O-C12-HSL. This was confirmed by EMSAs, where 3-O-C12-HSL induced a
shift with both AP-2 and NF-B consensus DNA. The activation of
NF-B and subsequent production of IL-8 were found to be regulated by
a mitogen-activated protein kinase pathway. These findings support the
concept that the severe lung damage that accompanies P.
aeruginosa infections is caused by an exuberant neutrophil
response stimulated by 3-O-C12-HSL-induced IL-8. Understanding the
mechanisms of 3-O-C12-HSL activation of lung structural cells may
provide a means to help control lung damage during infections with
P. aeruginosa. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The early stages of P. aeruginosa colonization of the lung are hallmarked by the entry of neutrophils. This neutrophil recruitment is most likely due to the production of the chemokine IL-8. In support of this concept, animal models of P. aeruginosa lung infection demonstrate concomitant increases in IL-8 and neutrophils in the airways (14, 15, 16). When bronchoalveolar lavage fluid from P. aeruginosa-infected humans was examined, IL-8 levels were significantly increased over those of uninfected patients. P. aeruginosa-infected patients also had highly significant increases in the number of lung neutrophils (17, 18, 19). Increases in IL-8 protein leads to massive infiltration of neutrophils, which results in the production of proteolytic enzymes, such as elastase, and subsequently tissue destruction and pulmonary failure. Persistent infection with P. aeruginosa leads to exuberant production of IL-8 and neutrophil migration into the lungs and results in chronic inflammation and tissue destruction.
Supernatants from P. aeruginosa contain a small, nonprotein, non-LPS molecule that stimulates bronchial epithelial cells to produce IL-8 (20). This phenomenon was further examined by DiMango et al. who demonstrated that human epithelial cells incubated with 3-O-C12-HSL in vitro produced significant levels of IL-8 (21). It was also shown that the P. aeruginosa mutant PAOR1 (22), which produces miniscule amounts of the autoinducer 3-O-C12-HSL, had a decreased ability to stimulate IL-8 when compared with the wild-type strain PAO1 (14). The production of Pseudomonas autoinducers have been shown to be essential for the infection and pathology found in both acute and chronic infections (23, 24, 25). In these models it was observed not only that the degree of pathology was decreased but that the infiltration of neutrophils to the site of infection was also reduced with autoinducer mutant strains of Pseudomonas. These data suggest that 3-O-C12-HSL is instrumental in the initial recruitment of neutrophils to the lung.
In this study we examined whether 3-O-C12-HSL could activate human lung cells to produce the neutrophil chemotactic molecule IL-8. We also demonstrate the role of mitogen-activated protein (MAP) kinases and transcription factors in the production of IL-8 with activation 3-O-C12-HSL in structural cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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L828, a nontransformed normal human lung fibroblast strain (26), A549, human type II-like alveolar epithelial cells (ATCC, Manassas, VA), and primary human foreskin fibroblasts (27), were maintained in MEM (Life Technologies, Gaithersburg, MD) supplemented with 10% FBS (HyClone Laboratories, Logan, UT) and 50 U/ml gentamicin (Life Technologies). The 16HBE, human bronchial epithelial cell line, was grown on collagen/ fibronectin-coated culture dishes in MEM with 10% FBS and 50 U/ml gentamicin (28). Cells were passed every 4–5 days using 0.05% trypsin with 0.1% EDTA to dissociate adherent cells. Fibroblasts were used between passages 5 and 20.
3-O-C12-HSL synthesis
3-O-C12-HSL was synthetically produced as previously described (10). This molecule is structurally and functionally identical with the natural 3-O-C12-HSL produced by P. aeruginosa. No detectable levels of endotoxin were found in preparations of 3-O-C12-HSL using a Limulus Amebocyte Lysate assay (Cape Cod Associates, Falmouth, MA).
RNase protection assays
16HBE and L828 cells were grown in 100-mM plates until confluent monolayers formed. Cells were serum-starved for 18 h and then stimulated with 100 µM 3-O-C12-HSL for varying amounts of time. Cells were extracted with Tri Reagent (Molecular Research Center, Cincinnati, OH), and total RNA was quantified by spectrophotometry. DNA templates for human chemokines (PharMingen, San Diego, CA) were used to make 32P-labeled RNA probes. Probes were mixed with 2–8 µg of sample RNA, and a RiboQuant kit (PharMingen) was used to perform RNase protection assays as per the manufacture’s procedures. Protected RNA fragments were resolved on a 5% polyacrylamide gel and quantified by densitometry. GAPDH controls were used to standardize the quantification of RNA samples.
IL-8 measurements
Cell lines were grown in 96-well plates until confluent
(
5 x 104 cells/well) and then serum
starved for 18 h before stimulation. Cells were treated with
25–100 µM 3-O-C12-HSL or C4-HSL for 18 h and the amount of IL-8
in the supernatants determined using an IL-8 ELISA as previously
described (29). In MAP kinase experiments, 50 µM of the
MAP kinase/extracellular signal-regulated kinase (MEK) inhibitor
PD98059 (Calbiochem, La Jolla, CA) was added to cultures along with a
titration of 3-O-C12-HSL. Cells were incubated for 18 h, and IL-8
in the supernatants was measured.
Granulocyte chemotaxis assays
Granulocytes were isolated from healthy human plasma by centrifugation over a Ficoll-Hypaque density gradient. Residual erythrocytes were removed via hypotonic lysis. The resulting population routinely contained 80–90% neutrophils and 1–15% eosinophils and basophils. Using the ChemoTx System (Neuroprobe, Cabin John, MD), 3.25-mm diameter, 3-µm pore polycarbonate membranes were placed over the wells of a corresponding 96-well tissue culture plate. Each well contained 28 µl of MEM or supernatant from 16HBE cultures, which were nonstimulated or induced with 100 µM 3-O-C12-HSL for 18 h. Some wells contained 10 µg/ml mouse anti-human IL-8 mAb (R&D Systems, Minneapolis, MN) or control mouse IgG. As a positive control for migration, a standard curve of 0–5000 ng/ml of recombinant human IL-8 (rhIL-8) (R&D Systems) was added to some wells. Repeatedly, maximal migration was found with 5–500 ng/ml rhIL-8. Granulocytes (2.5 x 105) in 25 µl of DMEM, 10% FBS, 50 µg/ml L-glutamine, 50 U/ml gentamicin were added to the upper side of the membrane, and the plate was incubated at 37°C for 2 h. Granulocytes in the lower chamber were quantified with a FACScan (BD Biosciences, San Jose, CA). Forward vs side scatter was used to identify the granulocytes that were in the migrating population. Chemotactic index was calculated by dividing values for cells migrating to supernatants by values for cells migrating to medium controls (background migration). A chemotactic index of 1 reflects no specific migration. A standard curve was constructed using known numbers of input granulocytes; this was used to calculate the total percentage of the input cell population that migrated to the supernatants.
Transient transfections
Luciferase reporter constructs of the IL-8 promoter consisted of
a full-length 5' unencoded region of the IL-8 promoter (-1481
+44)
and deletions of this construct of (-546+44) and (-133+44).
Site-directed mutations were also made in the binding sites for
NF-B, NF-IL-6, and AP-1 in the -133 construct (see Fig. 5
A) (30). Transfections were performed by
adding 16HBE cells to collagen/fibronectin-coated six-well plates (BD
Labware, Franklin Lakes, NJ) at 2 x 106
cells/well in 1 ml MEM supplemented with 2% FBS and 50 U/ml
gentamicin. DNA was prepared by mixing 5 µg of the IL-8 promoter
constructs in 20 mM HEPES buffer with 20–30 µl DOTAP (Boehringer
Mannheim, Indianapolis, IN) at room temperature for 15 min. The
DNA/DOTAP mixture was added to the 16HBE cells and incubated overnight
at 37°C. Transfection with a vector expressing 
-galactosidase was
used to determine efficiency of transfections. Transfected cells were
serum-starved for 18 h before being stimulated with 100 µM
3-O-C12-HSL or 50 ng/ml hTNF-
+ 200 U/ml hIL-1 (Genzyme,
Cambridge, MA) for an additional 12 h. Cells were assayed for
luciferase activity using a luciferase assay kit (Promega, Madison, WI)
and light intensity determined with a luminometer (Packard, Meriden,
CT). Data were expressed as fold increase over nonstimulated cells
transfected with the same construct.
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16HBE and L828 cells were grown to confluent monolayers in
six-well plates (2 x 106 cells/ml).
Cells were serum-starved for 18 h before stimulation with 100 µM
3-O-C12-HSL, 50 ng/ml hTNF- (Genzyme), or 10 ng/ml PMA (Sigma, St.
Louis, MO) for 30 min or 2 h at 37°C. Cells with MEM only were
used as a control for induction. Nuclear protein extracts were prepared
as previously described (31). Briefly, cells were removed
from plates and washed in cold PBS. Cell pellets were resuspended in
400 µl of an ice-cold hypotonic buffer (10 mM HEPES-KOH pH 7.9, 1.5
mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.5% Nonidet
P-40, and 0.2 mM PMSF) and incubated on ice for 10 min. Lysates were
vortexed for 10 s and centrifuged at 13,000 rpm in a microfuge for
30 s. Supernatants containing cytoplasmic proteins were removed,
and pellets were resuspended in 50 µl cold hypertonic buffer (20 mM
HEPES-KOH pH 7.9, 1.5 mM MgCl2, 25% glycerol,
420 mM NaCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.2 mM PMSF). Samples were
incubated on ice for 20 min then centrifuged for 2 min at 4°C.
Nuclear protein-containing supernatants were removed and quantified by
bicinchoninic acid protein assay (Pierce, Rockford, IL). Consensus
sequences for the AP-1 DNA binding site
(5'-CGCTTGATGAGTCAGCCGGAA-3'), AP-2 DNA binding site
(5'-GATCGAACTGACCGCCCGCGGCCCGT-3'), and NF-B DNA binding site
(5'-AGTTGAGGGGACTTTCCCAGGC-3') (Promega) were labeled with
[
-32P]ATP using T4 polynucleotide kinase for
30 min at 37°C. Labeled DNA was purified over a G-25 column to remove
unbound nucleotides. Nuclear protein extracts, at a concentration of
300–5 µg, were incubated at room temperature for 20 min with
50,000 cpm (0.06 pmol) of the labeled oligonucleotide suspended
in binding buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl, 4% glycerol, 1
mM MgCl2, 0.5 mM EDTA, 0.5 mM DTT, and 0.05 mg/ml
poly(dI:dC)). Samples were resolved on a 4% nondenaturing
polyacrylamide gel at 100 V and exposed to film. Cold competition
assays were performed by adding a 100-fold excess of unlabeled
oligonucleotides to nuclear protein extracts 10 min before the addition
of the labeled oligonucleotides. Supershift experiments were completed
by adding 2 µg of the anti-AP-2 Ab C18 (Santa Cruz Biotechnology,
Santa Cruz, CA) to the binding reaction.
Immunoblots
Cell lysates were obtained from 16HBE cells as previously described for EMSA. Proteins were quantified by bicinchoninic acid protein assay and 20 µg of protein was mixed with an equal volume of 2x SDS sample buffer and boiled for 5 min. Denatured proteins were separated by electrophoresis on a SDS-polyacrylamide gel along with control proteins (nonphosphorylated ERK-2 and phosphorylated ERK-2) (Cell Signaling Technology, Beverly, MA). Proteins were transferred to Hybond-C extra nitrocellulose membrane (Amersham, Piscataway, NJ) in 20 mM Tris base, 150 mM glycine, and 20% v/v methanol overnight at 35 V. Nonspecific binding was blocked by incubating the blot with 10% skim milk in PBS with 0.1% Tween 20 for 2 h at room temperature. Immunoreactive proteins were detected by incubating the blot with specific Abs (phospho-p44/42 mAb E10 or phospho-p38) overnight at 4°C (Cell Signaling Technology). These Abs only recognize the active phosphorylated form of the kinase. Between each step the nitrocellulose was washed four times for 5 min with PBS/0.1% Tween 20. Bound Abs were detected with either an anti-mouse IgG or an anti-rabbit IgG conjugated to HRP. Specific bands were visualized with ECL reagents (Amersham, Piscataway, NJ) then exposed to Kodak X-OMAT film (Kodak, Rochester, NY).
Nuclear mobilization of NF-B
Cells were grown in eight-well Permanox chamber slides (Nalge
Nunc, Naperville, IL) at 1–5 x 104
cells/well in MEM with 10% FBS. Cells were serum-starved for 18 h
before incubation with medium only or with 100 µM 3-O-C12-HSL for
2 h. Some wells were pretreated with 50 µM PD98059 for 1 h
before stimulation. Immunocytochemistry was used to determine the
localization of NF-B in cells. Cells were washed several times with
PBS + 0.05% Tween 20, and endogenous peroxidase activity was quenched
with 3% H2O2 for 20 min.
Cells were blocked with 2% horse serum for 1 h at room
temperature before overnight incubation at 4°C with 2 µg/ml
anti-NF-B p65 Ab (Santa Cruz Biotechnology) or an isotype
control mouse IgG (Caltag, South San Francisco, CA). Cells were washed
and incubated at room temperature for 30 min with 7 µg/ml of a
biotin-labeled anti-mouse IgG secondary Ab (Vector Laboratories,
Burlingame, CA). Cells were washed with PBS/Tween and incubated for
1 h at room temperature with a 1/1000 dilution of streptavidin HRP
(Jackson Immuno- Research Laboratories, West Grove, PA). AEC (Zymed
Laboratories, San Francisco, CA), which reacts with the bound HRP to
form a red precipitate that can be visualized in the cells, was used to
detect anti-NF-B Ab binding.
Statistical tests
A two-tailed Student t test was used to analyze the statistical differences between samples.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Cultures of the 16HBE cells (Fig. 1)
or L828 fibroblasts (Fig. 2) in MEM
without serum induced little IL-8 mRNA or protein. Stimulation of these
cells with an optimal dose of 100 µM 3-O-C12-HSL dramatically
increased the levels of IL-8 mRNA over that of nonstimulated cells
within 2–4 h. 3-O-C12-HSL had no toxic effects on these cells at any
of the concentrations used as assessed by both viability counts with
trypan blue and MTT assays (data not shown). In kinetic studies of
3-O-C12-HSL activation of 16HBE cells, a 15-fold increase in IL-8 mRNA
was stimulated after only 2 h (Fig. 1A). This induction
dissipated after 4 h, and IL-8 mRNA returned to background levels
by 24 h. Supernatants from 3-O-C12-HSL-stimulated 16HBE cells were
also tested in an IL-8 ELISA. Production of IL-8 protein was found
between 4 and 24 h of stimulation, at which point the amount of
IL-8 in the supernatant was starting to decrease. After 8 h of
stimulation with 3-O-C12-HSL, a maximal 14-fold induction in IL-8
protein was detected (Fig. 1B). Although not as dramatic, a
similar induction in IL-8 mRNA and protein was found with
3-O-C12-HSL-stimulated L828 fibroblasts (Fig. 2). Interestingly,
although maximal induction of IL-8 mRNA in L828 cells was found after
4 h of stimulation with 3-O-C12-HSL, levels of IL-8 mRNA persisted
out to at least 24 h.
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). When cells were cultured with IL-1
or TNF-, known potent inducers of IL-8, 7,000–15,000 pg/ml IL-8
were measured in culture supernatants. Interestingly, when A549, a type
II-like alveolar epithelial cell line, and foreskin fibroblasts were
stimulated with 25 or 100 µM 3-O-C12-HSL a similar induction in IL-8
was observed (Fig. 3). This indicates that 3-O-C12-HSL can interact
with multiple cell types, which include epithelial cells and
fibroblasts from different sites in the body, and stimulates a similar
IL-8 response. When 100 µM C4-HSL, a second P. aeruginosa
autoinducer, was added to cultures of epithelial or fibroblast cells,
no IL-8 was detected above that of nonstimulated cells (data not
shown). This indicates that the activation of epithelial cells and
fibroblasts with 3-O-C12-HSL is structurally specific. The structural
specificity of homoserine lactone activation of IL-8 was also
demonstrated by other investigators (21).
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Having shown that 3-O-C12-HSL induced the production of both IL-8
mRNA and protein in multiple cell types, we next wanted to determine
whether this IL-8 was functionally active and whether it could
stimulate the migration of neutrophils. Supernatants from 16HBE cells
that were incubated with medium only or medium containing 100 µM
3-O-C12-HSL were added to a neutrophil chemotaxis assay. When medium
alone, medium containing 3-O-C12-HSL, or supernatant from nonstimulated
16HBE cells was added to the assay, 20% of the neutrophils migrated
though the chamber; this reflects background migration and was assigned
a chemotactic index of 1. When supernatant from 3-O-C12-HSL-stimulated
cells was added to the assay, a chemotactic index of 3.2 was obtained,
which represents the migration of 63% of the cells (Fig. 4). This level of migration was equal to
the maximal migration stimulated by rhIL-8. To demonstrate specificity
of this migration to IL-8, an anti-IL-8 Ab was added to culture
supernatants. When anti-IL-8 was added to rhIL-8 controls, the
migration of neutrophils was inhibited to background levels.
Interestingly, when anti-IL-8 was added to supernatants from
3-O-C12-HSL-stimulated cells, migration was only partially inhibited
(70%), indicating that 3-O-C12-HSL is possibly stimulating an
additional chemotactic factor(s) that is inducing migration. Using
RNase protection assays, no significant increases were found with the
chemotactic factors RANTES, IP-10, macrophage-inflammatory
protein-2, macrophage-inflammatory protein-2, and monocyte
chemoattractant protein-1 with 3-O-C12-HSL stimulation of 16HBE cells
or L828 fibroblasts (data not shown). Similar chemotactic activities
were found with supernatants from 3-O-C12-HSL-stimulated L828 cells
(data not shown).
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Induction of IL-8 synthesis by cytokines usually involves
cis-acting elements that stimulate the transcription of IL-8
(32). Fig. 5
A
shows diagrams of the 5' region of the IL-8 promoter and promoter
deletion constructs. To test whether 3-O-C12-HSL regulation of IL-8 was
due to the activation of transcription factors, luciferase reporter
constructs of the IL-8 promoter were transiently transfected into 16HBE
cells (Fig. 5A). Transfected cells were stimulated with
3-O-C12-HSL or a mixture of TNF- and IL-1 as a control.
Stimulation with TNF- and IL-1 induced the production of
luciferase with all the constructs (2.5- to 4-fold over background)
except for the NF-B-deleted construct, indicating that NF-B is
essential for activation of IL-8 with these cytokines (data not shown).
When transfected cells were stimulated with 3-O-C12-HSL, maximal
induction of luciferase production was seen with the -1481 to +44
full-length construct (construct 1). When a deletion was made at -546
(construct 2), a decrease in activity of 75% was repeatedly found. An
additional deletion at -133 (construct 3) gave no further significant
decrease in luciferase activity (Fig. 5B). This indicates
that the deletion of DNA between -1481 and -546 eliminated sites that
were important for 3-O-C12-HSL induction of maximal IL-8 transcription.
Site-directed mutations in the AP-1, NF-IL-6, and NF-B DNA binding
sites were made in construct 3 and tested in transcription assays. When
compared with construct 3, there was a 60% reduction in luciferase
activity with the construct that contained a mutation in the AP-1 DNA
binding site and a 40% reduction with the NF-IL-6 mutated construct
(Fig. 5C). A mutation in the NF-B binding site resulted
in virtually no luciferase activity with 3-O-C12-HSL activation
(reduction of 99% when compared with construct 3). These data
indicated that NF-B was essential for activation of IL-8 with
3-O-C12-HSL stimulation of 16HBE cells and that the area between -1481
and -546 was needed for maximal IL-8 induction.
EMSA confirms that 3-O-C12-HSL induces NF-B and AP-2 but not
AP-1
Other investigators have shown that AP-1 is important for IL-8
induction in lung cells (32). In transcriptional reporter
assays, when AP-1 was mutated there was a 60% decrease in luciferase
induction, when compared with construct 3 (Fig. 5C). EMSA
was performed to determine whether AP-1 was induced in 16HBE cells with
3-O-C12-HSL stimulation. Using a consensus sequence for the AP-1
binding site revealed constitutive expression of AP-1 even in
nonstimulated cell extracts (data not shown). Therefore, stimulation of
16HBE cells with 3-O-C12-HSL did not induce a significant increase in
binding to the AP-1 DNA probe. A similar constitutive expression of
AP-1 was found by other investigators examining IL-8 expression in A549
epithelial cells (33). These data indicate that the
3-O-C12-HSL stimulation of IL-8 is not dependent on induction of the
transcription factor AP-1. Examination of the genomic DNA sequence of
the 5'-IL-8 promoter region identified many consensus sequences for
transcriptional activator binding sites (Fig. 5A). Based on
transcription reporter assays, interest was focused on the sites
located between -1481 and -546. Using these data we identified a DNA
consensus sequence that could bind the transcription factor AP-2.
Therefore, we examined whether 3-O-C12-HSL could induce the
transcription factor AP-2. Nuclear extracts were made from L828 and
16HBE cells incubated with or without 100 µM 3-O-C12-HSL for 30 min.
Nuclear extracts (5 µg) were added to a
32P-labeled oligonucleotide with a consensus
sequence for the AP-2 DNA binding site. When this hybridized mixture
was run on a nondenaturing gel, a shift from unbound DNA was clearly
seen with extracts from cells that were stimulated with 3-O-C12-HSL
compared with the slight shift observed with extracts from
nonstimulated cells (Fig. 6A).
Nuclear extracts from cells stimulated with PMA, a known inducer of
AP-2, were used as a positive control. When a 100-fold excess of
unlabeled AP-2 oligonucleotide was added to 16HBE extracts, most of the
AP-2 shift was competed away, but 100-fold excess of a nonspecific
oligonucleotide had no effect on the shift (Fig. 6A). To
further prove the specificity of this AP-2 shift, supershifts were
performed using an AP-2 specific Ab. When this Ab was added to 16HBE
extracts along with the labeled AP-2 oligonucleotide, an additional
shift in size was observed (Fig. 6B). These data confirm
that 3-O-C12-HSL induces the transcription factor AP-2. When EMSAs were
performed using a NF-B consensus oligonucleotide, a shift was
observed in extracts from 16HBE cells that were stimulated for 2 h
with 3-O-C12-HSL (Fig. 7A).
This shift could be competed away with a 100-fold excess of unlabeled
NF-B DNA but not a nonspecific DNA sequence (Fig. 7B). We also demonstrated that when 16HBE cells were
stimulated with 100 µM 3-O-C12-HSL, NF-B translocated from the
cytoplasm to the nucleus (Fig. 8C). These data show that
3-O-C12-HSL induces the transcription factor NF-B. Similar shifts
for AP-2 and NF-B were found when L828 fibroblasts were stimulated
with 3-O-C12-HSL (data not shown).
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To better understand the activation of NF-B and the subsequent
production of IL-8 in these cells, we evaluated a potential role for
MAP kinases. When 16HBE cells were stimulated with 3-O-C12-HSL, the
active and phosphorylated forms of the ERK1/2 kinases were induced
(Fig. 8A). Kinetic studies of 3-O-C12-HSL activation
revealed ERK induction as early as 10 min with maximal expression at 15
min. Addition of the MEK inhibitor PD98059, which blocks the activation
of ERK, completely inhibited induction. To determine whether this
induction of MAP kinases was specific to the ERK pathway, samples were
also tested for induction of the p38 kinase. When cellular extracts
from 3-O-C12-HSL-stimulated cells were tested using a p38-specific
Western blot, there was no significant induction over that found with
nonstimulated cells (data not shown). Having shown that ERK was induced
by 3-O-C12-HSL stimulation, we next determined what effect this may
have on IL-8 and NF-B induction. When 16HBE cells were cocultured
with 3-O-C12-HSL and 50 µM of PD98059, the production of IL-8 protein
was reduced by 80–90% (Fig. 8B). The MEK inhibitor was not
found to have toxic effects at this concentration; therefore, the
reduction in IL-8 was not due to cell death (data not shown). To
determine whether the inhibition of IL-8 production was due to the
inhibition of transcription factors, NF-B nuclear mobilization
experiments were performed. In nonactivated cells, NF-B is held in
the cytoplasm by inhibitory proteins called IB. When cells become
stimulated, NF-B is released from IB and translocates into the
nucleus where it activates the transcription of multiple genes. When
16HBE cells were cultured with medium only, NF-B was found mainly in
the cytoplasm, but upon activation with 3-O-C12-HSL, NF-B
accumulated in the nucleus. The addition of a MEK inhibitor with
3-O-C12-HSL stimulation failed to induce nuclear mobilization of
NF-B (Fig. 8C). These data indicate that the induction of
NF-B by 3-O-C12-HSL occurs through the activation of a MAP kinase
pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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). During the initial
colonization of the lung with P. aeruginosa it is thought
that the bacteria adhere to the epithelium in the upper airways and
that some of these bacteria are endocytosed by the epithelial cells
(36). In this environment the epithelial cells would be in
direct contact with P. aeruginosa. Because 3-O-C12-HSL
freely diffuses and is actively pumped out of the bacteria, it would
directly interact with epithelial cells (37). Previous
data have shown that in the squid, where the symbiotic bacteria
V. fisheri produces an autoinducer similar to P.
aeruginosa (3-O-C6-HSL), this molecule was found not only in the
area around the bacteria, but was detected also in the underlying
epithelial cells (38). These observations support the
hypothesis that 3-O-C12-HSL produced by P. aeruginosa
interacts with the surrounding tissue. This production of 3-O-C12-HSL
could initiate inflammation in the lung and start a cascade of IL-8
production and, consequently, neutrophil infiltration. In cystic
fibrosis patients, P. aeruginosa is found at high
concentrations (108 CFU/ml of sputum) and grows
in microcolonies or biofilms, which are clusters of bacteria embedded
in a matrix of polysaccharide (39, 40). Although the
concentration of 3-O-C12-HSL in the lungs of P.
aeruginosa-infected patients remains unknown, mRNA encoding
proteins involved in the regulation of 3-O-C12-HSL were found in the
sputum of cystic fibrosis patients (41). These data
indicate that a functional cell-to-cell signaling mechanism occurs in
vivo and that 3-O-C12-HSL is most likely produced in these patients.
Therefore, in the lung it is likely that P. aeruginosa
attaches to epithelial cells and produces 3-O-C12-HSL, which stimulates
surrounding cells. We also showed that 3-O-C12-HSL stimulates IL-8
production in L828, a primary human lung fibroblast strain (Fig. 2).
During P. aeruginosa lung infections, where tissue
destruction is prominent, the bacteria would also have direct contact
with the underlying matrix of fibroblasts. The activation of L828
fibroblasts with 3-O-C12-HSL persisted longer than that found in 16HBE
cells. This long-lasting activation may be a source of extended IL-8
production and may stimulate a steady infiltration of neutrophils into
the lung, leading to extensive tissue damage.
The concentrations of 3-O-C12-HSL produced in vivo are unknown, just as
the relevant in vivo concentrations for most cytokines are unknown.
Studies with V. fisheri have shown that the concentration of
Vibrio autoinducer isolated from the light organs of squids
was up to 200-fold higher than that produced by the bacteria in culture
(38). There is also evidence that when
Pseudomonas is grown in a biofilm the concentration of
autoinducers produced is significantly higher than that of planktonic
bacteria. This indicates that the in vivo concentration of 3-O-C12-HSL
in the Pseudomonas-infected lung may be significantly higher
than the concentration attained in bacterial cultures (10 µM).
Throughout our studies we found that, in vitro, 3-O-C12-HSL works best
at 25–100 µM, but we are also cognizant of the fact that lower
concentrations of 3-O-C12-HSL may act in concert with other bacterial
products and eukaryotic cytokines to synergistically activate resident
pulmonary cells.
IL-8 production has been investigated in many different experimental
systems and its regulation occurs at both transcriptional and
posttranscriptional levels. Transcriptional regulation occurs through
the differential activation and binding of inducible transcription
factors. Based on the genomic sequence of the IL-8 gene, several
transcription factor binding sites have been identified in the 5'
promoter region (Fig. 5A) (42). Using DNase
footprinting and transcriptional fusion studies, the transcription
factors AP-1, NF-IL-6, and NF-B were found to be the most prominent
activators of IL-8 in most cells (32). We show that
3-O-C12-HSL induction of IL-8 in 16HBE and L828 cells is dependent on
the transcription factors AP-2 and NF-B. When these same cells were
stimulated with TNF- and IL-1, only the transcription factor
NF-B was necessary for maximal IL-8 induction. The transcription
factor AP-2 has never been shown to play a role in the regulation of
IL-8. However, other studies do show that AP-2 may be important in the
regulation of other cytokines such as TNF- and TGF- (43, 44). Based on transcriptional data and EMSA (Figs. 5B
and 6), we show that with 3-O-C12-HSL stimulation, AP-2 is necessary
for maximal induction of IL-8. Inflammatory cytokines, such as IL-6,
have been shown to induce AP-2 (45), but indirect
stimulation of AP-2 by 3-O-C12-HSL, via IL-6 activation, is unlikely
due to the fact that in the EMSA experiments AP-2 induction occurred
within 30 min of activation.
Activation of NF-B is tightly regulated by IB proteins. These
proteins bind to the nuclear localization signal in NF-B and
sequester it in the cytoplasm. When cells are activated, IB is
phosphorylated, which targets it for ubiquitination and subsequent
degradation. NF-B is then released and translocates to the nucleus
where it induces the transcription of multiple genes (46).
It has been shown that in some cases the
Ras-Raf-MEK-MAP-pp90rsk kinase pathway
phosphorylates IB and initiates its degradation (47, 48). It has recently been shown that P. aeruginosa
stimulation of mucin production in epithelial cells uses this pathway
of activation (49). The activation of NF-B and
production of IL-8 found in 3-O-C12-HSL-stimulated cells may also occur
through a similar mechanism. We demonstrate that 3-O-C12-HSL is able to
specifically up-regulate both ERK1 and 2 in the cytoplasm of 16HBE
cells. When a MEK inhibitor was added to these cultures, 3-O-C12-HSL
stimulation of both IL-8 and NF-B were inhibited. Although we have
not shown the involvement of c-Ras and c-Raf in 3-O-C12-HSL activation
of cells, we do know that by inhibiting MEK and the subsequent
activation of MAP kinases we inhibit the nuclear mobilization of
NF-B and the production of IL-8.
3-O-C12-HSL induction of NF-B and AP-2 and the subsequent
up-regulation of IL-8 could directly contribute to the neutrophil
infiltration and inflammation found in P. aeruginosa
infections. Because the transcription factors NF-B and AP-2 are
pivotal in the production of IL-8 during P. aeruginosa
infection, these transcription factors should be attractive targets for
anti-inflammatory therapy. Blocking the activation of these
transcription factors may prevent the early activation of IL-8 and
neutrophil migration, therefore decreasing the inflammation induced by
P. aeruginosa infection. An alternative approach would be to
prevent the initial activation of IL-8 by regulating 3-O-C12-HSL
production in the bacteria. The use of autoinducer analogs to
3-O-C12-HSL is a mechanism that is currently being developed. The use
of analogs to inhibit the activation of R proteins has been
demonstrated in many Gram-negative organisms (50, 51, 52). By
directly inhibiting the production of 3-O-C12-HSL, the inflammation
induced by P. aeruginosa may be greatly reduced and
colonization of the lung repressed. Finally, these autoinducer analogs
may also be useful in blocking the actions of native 3-O-C12-HSL on
lung structural cells. If structural analogs can be found that
antagonize the ability of 3-O-C12-HSL to induce IL-8, they may prove
useful therapeutically in cases where exuberant neutrophil responses
lead to tissue injury.
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Footnotes |
|---|
2 Address correspondence and reprint requests to Dr. Richard P. Phipps, Cancer Center, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Box 704, Rochester, NY 14642. E-mail address: Richard_Phipps{at}urmc.rochester.edu
3 Abbreviations used in this paper: 3-O-C12-HSL, N-3-oxododecanoyl-homoserine lactone; MAP, mitogen-activated protein; ERK, extracellular signal-regulated kinase; MEK, MAP kinase/ERK; C4-HSL, N-butyryl-L-homoserine lactone; h, human.
Received for publication April 5, 2000. Accepted for publication April 18, 2001.
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, Supernatant from nonstimulated cells; 
,
cells stimulated with 100 µM 3-O-C12-HSL. These data are
representative of three independent experiments.
) or 100 µM 3-O-C12-HSL
(
0.005 compared with untreated
cultures.
