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INTRODUCTION |
The pathogenesis of pulmonary infection in cystic fibrosis
(CF),1 the most common lethal
genetic disease in Caucasians, has been a topic of great interest.
Early in the disease process CF patients often have intermittent airway
infection because of Staphylococcus aureus, followed by
Pseudomonas aeruginosa infection, which eventually becomes
chronic. These infections are associated with an excessive inflammatory
response, characterized by the accumulation of large amounts of the
polymorphonuclear leukocyte (PMN) chemokine IL-8, as well as PMNs and
their toxic products in the airways (1). Failure to clear inhaled
pathogens, because of a combination of factors such as diminished
defensin activity (2), obstruction because of inspissated mucin
(3), or other cystic fibrosis transmembrane conductance
regulator-associated defects (4), results in a large bacterial burden.
Cells with cystic fibrosis transmembrane conductance regulator
mutations, as well as damaged or regenerating epithelial cells, express
increased numbers of asialylated glycolipids such as asialoGM1 (5, 6)
with a GalNAc
1-4Gal moiety that acts as a receptor for many
pulmonary pathogens including S. aureus and P. aeruginosa (7, 8). The binding of asialoGM1 on airway epithelial
cells by P. aeruginosa stimulates nuclear translocation of
NF-
B and activates transcription of IL-8 (9), the major airway
chemokine in the lung for PMNs, initiating the epithelial immune
response to this pathogen. This response can be stimulated by intact
P. aeruginosa, the isolated P. aeruginosa adhesin
type IV pilin, or by an antibody to the pilin receptor asialoGM1 (9,
10).
The molecular mechanisms involved in bacterial induction of epithelial
cell signaling have been examined for several mucosal pathogens.
Pathogenic Neisseria, which also express type IV pili, but
not pil mutants, activate cervical epithelial cell lines by increasing [Ca2+]i through an
interaction with CD46 (11). The Ca2+ increase is because of
influx associated with the translocation of a Neisserial
porin into the eukaryotic cells and induces apoptosis, which
accompanies Neisseria invasion of these epithelial cells (12). Invasive pathogens in the gut such as Salmonella also stimulate NF-
B-dependent gene expression through
Ca2+-dependent signaling (13). However, airway
epithelial cells with intact tight junctions are highly resistant to
bacterial invasion (14) and apoptosis (15). In diseases such as CF in which there is prominent stimulation of epithelial cytokine expression (1), the bacterial burden is clearly in the airway lumen and not
intracellular (4), indicating that superficial interactions between
specific bacterial gene products and epithelial receptors are
sufficient to activate epithelial cell signaling. There is ample
evidence that epithelial cells express IL-8 in response to a variety of
pulmonary pathogens through NF-
B-dependent
transcription. However, the more proximal components of the epithelial
cell signaling cascade, the nature of the receptor, and the kinases
involved in signal transduction have not been previously characterized. As perturbation of airway epithelial cells by various stimuli can
initiate Ca2+ oscillations (16) we postulated that
bacterial ligands might activate epithelial responses by stimulating
alterations in [Ca2+]i and causing
activation of Ca2+-dependent kinases.
In the studies presented, we sought to determine whether the common
pulmonary pathogens S. aureus and P. aeruginosa,
which are known to recognize asialylated glycolipid receptors,
stimulate epithelial proinflammatory responses by the activation of a
common pathway and to identify the components of the major signaling cascades involved. We demonstrate that these organisms, but not mutants
lacking specific adhesins that ligate asialoGM1 moieties on the
epithelial surface, generate increases in Ca2+ from
intracellular stores to activate MAPKs, stimulate NF-
B translocation, and initiate IL-8 production.
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MATERIALS AND METHODS |
Cell Lines--
1HAEo
cells, SV40 immortalized
human airway epithelial cells whose properties have been well
characterized (9), obtained from D. Gruenert (University of
California, San Francisco, CA) were grown in 96-well plates in
DMEM-F-12 supplemented with 10% fetal calf serum, 100 units/ml
penicillin, 100 µg/ml streptomycin, 50 µg/ml gentamicin, 4 µg/ml
amphotericin B. 9HTEo
cells were obtained from P. Davis (Case
Western Reserve University) and grown in 6-well plates in DMEM-F-12 as
above. Unless specified, reagents were purchased from Sigma. CHO-K1
cells obtained from ATCC were grown in DMEM + 10% FCS.
Bacterial Strains and Culture Conditions--
PAO1, a motile,
piliated, nonmucoid strain of P. aeruginosa, whose genome
has been sequenced, was grown in M9 or LB media. PAO
algC has a mutation blocking phosphoglucomutase expression that results in defective LPS and alginate biosynthesis (17). PAO/NP (pilA) lacks expression of pilin (10).
S. aureus strains RN6390 and RN6911 (agr) were
grown in CYGP media and diluted as above (18).
Ca2+ Imaging--
Subconfluent monolayers of 1HAEo
airway epithelial cells grown on glass coverslips were loaded with 5 µM Fura-2/AM (Molecular Probes, Eugene, OR) and 0.02%
pluronic acid at room temperature for 45 min and then washed twice with
PBS. Ratiometric imaging with excitation at wavelengths of 340 and 380 nm was done using a Zeiss Axiovert microscope and analyzed using VPROBE
software. Frames were collected at 6-s intervals following the
application of bacteria. Recordings were monitored from 10 cells/field,
and at least five fields were examined for each condition.
Bacterial Binding Studies--
Binding competition assays were
done using 1HAEo
cells grown to confluence in 96-well plates (9).
Wells in quintuplicate were incubated with 2 × 108
cfu/ml 35S-labeled PAO1 plus unlabeled organisms
(P. aeruginosa or S. aureus in 5-fold excess).
After a 60-min incubation the monolayers were washed three times in PBS
and solubilized in 0.2% NaOH, and scintillations were counted. Binding
competition studies in the presence of anti-asialoGM1 were done by
incubating the monolayers with bacteria in DMEM + 0.1% bovine serum
albumin with or without rabbit polyclonal antibody to 270 µg/ml
asialoGM1 (anti-asialoGM1) (Wako, Richmond, VA) or with monoclonal
anti-human epithelial antigen (250 µg/ml) (Dako, Carpinteria, CA) for
1 h at 37 °C, 5% CO2. Assays using S. aureus were done in the presence of 0.1% FCS.
IL-8 Assays--
IL-8 was measured by enzyme-linked
immunosorbent assay (R & D Systems, Minneapolis, MN) following a 60-min
exposure of confluent monolayers of 1HAEo
cells, weaned from serum
for 24 h, in 96-well plates to 1-4 × 108 cfu/ml
of the various bacterial strains preincubated for 60 min with the
reagents indicated (9). The cells were washed and sterilized with
gentamicin, and supernatants were harvested either 4 or 18 h later
for IL-8 enzyme-linked immunosorbent assay. Duplicate wells were
treated with trypan blue to assess epithelial viability during the
assay, which was >75%. Assays using S. aureus were done in
the presence of 0.1% FCS.
Statistical Analysis--
Each IL-8 or luciferase data point was
determined in quintuplicate, a mean and standard deviation was
calculated, and statistical significance was evaluated using a one-way
analysis of variance with Dunnett's post test (GraphPad Instat version
3.00; GraphPad Software, San Diego, CA) to test the null hypothesis
that there was no difference in the amount of the outcome variable
(IL-8 production or luciferase units) under each test condition as
compared with the untreated control. For bacterial binding studies,
means and standard deviations were compared using an unpaired,
two-tailed t test.
Western Hybridizations--
Epithelial cells grown in 6-well
plates to 90% confluence were weaned from serum overnight. The cells
were stimulated with thapsigargin, P. aeruginosa PAO1, or
S. aureus (in DMEM + 0.1% FCS) for the time intervals
indicated, washed, and lysed by the addition of 0.5% Triton X-100 in
PBS for 45 min. Aliquots of the cell lysates (5-20 µg of protein)
were combined with 4× NuPage (Novex, San Diego, CA) sample and
reducing agent and heated to 70 °C for 10 min. Proteins were
separated by electrophoresis on SDS polyacrylamide gels, transferred to
polyvinylidene difluoride membranes and blocked in 5% skim milk
overnight. Immunodetection was done with mouse monoclonal
anti-phosphorylated or anti-nonphosphorylated p44/42 ERK1/2 MAPK
(Thr-202/Tyr-204) (Santa Cruz Biotechnology; Santa Cruz, CA),
anti-phosphorylated p38 MAPK (Tyr-180/Tyr-182), or anti-actin (New
England Biolabs, Beverly, MA). An anti-mouse IgG conjugated to
horseradish peroxidase (Santa Cruz Biotechnology, Santa Cruz, CA) was
used as the secondary antibody and detected with Chemiluminescence
Reagent Plus (PerkinElmer Life Sciences, Boston, MA).
Activation of NF-
B Detected by Luciferase Reporter
Constructs--
1HAEo
cells were grown in 6-well plates to 80-90%
confluence, washed once with PBS, and replated with serum and
antibiotic free DMEM-F-12. Cells were transiently transfected
using LipofectAMINE 2000 reagent (Life Technologies, Inc.,
Gaithersburg, MD), 1 ng of plasmid DNA of pNF-
B-luciferase
(Stratagene, La Jolla, CA), which contains five NF-
B binding sites
upstream of a luciferase reporter gene, and a constitutively active
pRL-TK (Promega, Madison, WI) to control for transfection efficiency
and incubated at 37 °C in 5% CO2 for 18 h. Cells
were washed twice, and selected wells were pretreated with 5 µM BAPTA/AM (Molecular Probes, Eugene, OR) and
resuspended in serum and antibiotic free DMEM-F-12 for 1 h. Cells
were stimulated with antibody to asialoGM1 (100 µg/ml), 15 µM thapsigargin, or 108 cfu/ml of PAO1 in
serum and antibiotic free DMEM-F-12 for 2 h or with S. aureus RN6390 in DMEM-F-12 + 0.1% FCS, washed, lysed, and
harvested using passive lysis buffer (Promega, Madison, WI). Luciferase
assays were performed using the reagents and protocol for the
dual-luciferase reporter assay system (Promega, Madison, WI) and
analyzed with a luminometer. After standardization for transfection
efficiency, data were plotted as the mean of quadruplicate samples and
are representative of at least two independent experiments.
To directly demonstrate that asialoGM1 provides the required receptor
to initiate NF-
B activation, CHO cells, which do not normally
express apically exposed asialoGM1, were incubated with various
concentrations of purified asialoGM1 (Fluka, Milwaukee, WI) for 2 h at 37 °C, and incorporation was demonstrated by flow cytometry
using anti-aGM1 (100 µg/ml) as a primary antibody and goat
anti-rabbit conjugated to fluorescein isothiocyanate
(Zymed Laboratories Inc., South San Francisco, CA) as
a secondary antibody. In subsequent experiments, CHO cells were
incubated with 250 µM aGM1 for 2 h, transiently
transfected with the NF-
B reporter system as above, and exposed to
anti-aGM1 (100 µg/ml), 108 cfu/ml S. aureus,
or 108 cfu/ml P. aeruginosa. Analysis of NF-
B
activation was carried out as above. Experiments using S. aureus were performed in the presence of 0.1% FCS.
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RESULTS |
Pulmonary Pathogens Stimulate Ca2+ Increases in 1HAEo
Cells--
Cellular responses to external stimuli often involve
Ca2+-dependent signaling cascades. We tested
the ability of P. aeruginosa, S. aureus, and
corresponding mutant strains lacking genes expected to be involved in
adherence to stimulate Ca2+ increases in human respiratory
epithelial cells. The addition of P. aeruginosa PAO1 to
Fura-2/AM-loaded 1HAEo
cells prompted a 100 nM rise in
[Ca2+]i with return to baseline
within 5 min (Fig. 1). Single cell
recordings demonstrated that similar 100 nM increases in [Ca2+]i could be elicited by PAO1,
anti-asialoGM1, or purified pilin, but not by the mutant PAO/NP
(pilA), which does not recognize this receptor (5) (Fig.
2). Antibody recognition of human
epithelial antigen on the cells did not elicit Ca2+ fluxes
nor did purified P. aeruginosa LPS alone or in the presence of 0.1% serum. A PAO algC mutant that lacks
phosphomannomutase activity required for both LPS and alginate
synthesis (17) activated a more prolonged Ca2+ response
than the parental strain. P. aeruginosa PAO1 and antibody to
asialoGM1 also stimulated Ca2+ responses in respiratory
epithelial cells derived from nasal polyps in primary culture (data not
shown). S. aureus RN6390 activated Ca2+ fluxes
of similar amplitude as PAO1 (Fig. 2D) and was also able to
initiate Ca2+ oscillations following the initial stimulus.
The agr mutant RN6911 did not activate Ca2+
fluxes. Representative tracings from single cells activated by anti-asialoGM1 demonstrate the cell to cell variations in the Ca2+ response (Fig. 2E).

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Fig. 1.
Pseudocolor images of Fura-2/AM-loaded
1HAEo human airway epithelial cells grown on coverslips at 30-s
intervals following the application of 5 × 108 cfu/ml
P. aeruginosa PAO1 at time 0 (Control). Blue areas represent
basal [Ca2+]i 50-100
nM, and green areas represent a 50-100
nM increase above the baseline.
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Fig. 2.
Spectrophotometric monitoring of
Ca2+ transients in 1HAEo cells loaded with Fura-2/AM and
exposed to the stimuli indicated. A, P. aeruginosa PAO1, anti-asialoGM1, and P. aeruginosa pilA
mutant PAO/NP (10). B, purified PAO1 pilin (5) and
anti-human epithelial antigen (HEA). C, PAO
algC (LPS mutant) and P. aeruginosa LPS (100 µg/ml). D, S. aureus wild type RN6390 and
S. aureus RN6911 (agr). E, composite
tracings of multiple cells stimulated with antibody to asialoGM1.
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Bacterial Recognition of AsialoGM1 Receptors--
The receptor for
P. aeruginosa pilin is the GalNAc
1-4Gal moiety of
asialylated glycolipids such as asialoGM1 (10). Although other adhesins
are also expressed, pilin is the major ligand for this receptor and is
responsible for much of the induction of IL-8 expression (5). A
dose-response relationship of the binding of P. aeruginosa
to cells with increasing amounts of surface asialoGM1 has been
described (19). In addition, some data suggest that S. aureus might compete with P. aeruginosa for receptors
on the surface of epithelial cells (20). We postulated that the
activation of Ca2+ responses by S. aureus,
similarly, was because of ligation of the asialoGM1 receptor. Binding
competition studies were done to see whether antibody to asialoGM1
inhibited bacterial attachment to airway epithelial cells and whether
P. aeruginosa could directly compete with S. aureus for epithelial binding. In the presence of anti-asialoGM1,
60% of PAO1 binding was displaced as compared with 40% of S. aureus RN6390 binding (p < 0.0002 and
p < 0.004, respectively). Control assays done with the
same concentration of antibody to human epithelial antigen resulted in
no inhibition of binding by either species (data not shown). The
S. aureus agr mutant RN6911, which did not activate
Ca2+ fluxes, was not displaced by antibody to asialoGM1.
Radiolabeled P. aeruginosa PAO1 could be displaced by
S. aureus RN6390 as effectively as by unlabeled PAO1 present
in 5-fold excess. An equivalent inoculum of the S. aureus
mutant RN6911 did not compete as efficiently suggesting that it has
less affinity for common receptors. Labeled S. aureus was
also able to compete with P. aeruginosa for binding (data
not shown).
Activation of IL-8 by Adherent Bacteria through
Ca2+-dependent Signaling
Pathways--
Epithelial cells produce the PMN chemokine IL-8 in
response to adherent P. aeruginosa through activation of
NF-
B and transcription of the IL-8 gene (9, 10). S. aureus strains similarly stimulate IL-8 expression (Fig.
3). The wild type S. aureus
strain RN6390 was associated with a greater IL-8 response than the
agr mutant. To determine whether the activation of IL-8
expression is a direct consequence of
[Ca2+]i release, epithelial cells
were pretreated with reagents expected to alter Ca2+
signaling prior to and during P. aeruginosa
stimulation, and IL-8 production was measured (Fig.
4). Although chelation of extracellular Ca2+ with EGTA (21) did not inhibit IL-8 expression,
epithelial cells pretreated with BAPTA/AM (22) to chelate intracellular Ca2+ had significantly decreased IL-8 responses
(p < 0.001 for each). NiCl2, which
functions as an external Ca2+ channel blocker (23), did not
significantly inhibit the induction of IL-8 expression nor did 100 µM verapamil (24) have a major inhibitory effect.
Compounds expected to affect
Ca2+/calcineurin-dependent signaling pathways
such as cyclosporin A (25) or FK506 (25) decreased the IL-8 response
(p < 0.001 for each) although the maximal effect
required overnight incubation with the epithelial cells. TEMPO
(2,2,6,6-tetramethylpiperidine-1-oxyl), an inhibitor of
Fe2+/Zn2+-dependent enzymes
including calcineurin (26), significantly decreased PAO1-induced
expression of IL-8, as well (p < 0.001).

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Fig. 3.
Bacterial stimulation of IL-8
expression. IL-8 was assayed by enzyme-linked immunosorbent assay
18 h after exposure of 1HAEo cells to the bacterial strains
listed (108 cfu/ml) or anti-aGM1 (100 µg/ml). A mean and
standard deviation were calculated from quintuplicate wells. Background
IL-8 expression from unstimulated cells in PBS is indicated.
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Fig. 4.
IL-8 production by 1HAEo cells in the
presence of compounds expected to affect
Ca2+-dependent signaling. A,
P. aeruginosa induction of IL-8 is shown. Individual wells
were preincubated with the compounds indicated for 1 h prior to
the addition of PAO1 5 × 108 cfu/ml suspended in PBS + 1 mM Ca2+ + 1 mM
Mg2+. Background IL-8 production by unstimulated 1HAEo
cells (without PAO1) is indicated. Supernatants harvested 4 h (or
18 h (over night) as indicated) after exposure to PAO1.
B, effects of IL-8 agonists. The effects of reagents
expected to cause the transient increase in
[Ca2+]i were tested for the
ability to stimulate IL-8 expression as compared directly with P. aeruginosa PAO1, indicated as 100% for the purpose of comparison.
Some of the error bars fall within the data bars.
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Although P. aeruginosa may activate epithelial IL-8
expression through a Ca2+-dependent signaling
system, we were interested in establishing whether fluxes in
[Ca2+]i alone, in the absence of a
bacterial stimulus, were sufficient to evoke IL-8 expression. Airway
epithelial cells treated with thapsigargin (27), an inhibitor of the
endoplasmic reticulum Ca2+-dependent ATPase
that transiently increases
[Ca2+]i, or with okadaic acid, a
PP2A phosphatase inhibitor that affects
Ca2+-calmodulin-dependent kinase activity (28),
produced a dose-dependent increase in IL-8 production
(p < 0.01 and p < 0.001, respectively) (Fig. 4B) suggesting that activation of IL-8
expression can be mediated solely through increases in
[Ca2+]i.
P. aeruginosa and S. aureus Stimulate IL-8 Expression through
Ca2+-dependent Activation of MAPKs--
Many
signaling cascades activate the nuclear translocation of NF-
B and
other transcriptional activators involved in IL-8 transcription.
NF-
B translocation can be initiated by
Ca2+-dependent signals through the stimulation
of MAPKs (29). We tested the involvement of two of the major families
of MAPKs in the activation of IL-8 expression induced by ligation of
the asialoGM1 receptor by bacteria or antibody (Fig.
5). Stimulation of epithelial IL-8
expression by anti-asialoGM1 (Fig. 5A), P. aeruginosa (Fig. 5B), or S. aureus (Fig.
5C) was inhibited by either PD98059, a specific inhibitor of
the MAPK/ERK1 kinase (30), or SB202190, which inhibits the p38
kinase (31). Inhibition of S. aureus stimulation of the
cells by PD98059 and SB202190 was significant with p < 0.05 and p < 0.01, respectively, and for all the other stimuli p < 0.001. Moreover, the Ca2+
dependence of the IL-8 response was indicated by the ability of
BAPTA/AM to significantly inhibit the induction of IL-8 by S. aureus, P. aeruginosa, or anti-asialoGM1
(p < 0.001 for each).

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Fig. 5.
IL-8 expression in epithelial cells
stimulated by anti-asialoGM1 or bacteria: effects of MAPK inhibitors
and Ca2+ blockers. Confluent monolayers of 1HAEo
cells in 96-well plates were pretreated with the compounds indicated
for 60 min prior to a 1-h exposure to (A) anti-asialoGM1
(Ab-aGM1), (B) P. aeruginosa PAO1, and
(C) S. aureus RN6390. The epithelial cells were
washed, sterilized with gentamicin, and refed with media containing the
compound being tested, and culture supernatants were assayed for IL-8
18 h later. Control wells were treated with each of the compounds
without bacterial stimulation and were assayed.
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To determine whether the proposed pathway is active in human airway
epithelial cells in general, the kinetics of MAPK activation following
exposure of an unrelated cell line to stimuli that increased [Ca2+]i and caused IL-8 production
was examined. In lysates of 9HTEo
cells exposed to thapsigargin,
phosphorylated ERK (p44/p42) was briefly detectable by Western
hybridization within 15 min of stimulation and was gone by 30 min (Fig.
6). Stimulation of the same cell line
with P. aeruginosa also activated ERK1/2 briskly and
transiently. 1HAEo
cells responded similarly to P. aeruginosa with phosphorylation of the p44/p42 ERK1/2 (Fig.
7A). In the presence of 0.1%
FCS, which is required for staphylococcal binding, phosphorylated ERKs
were detectable in unstimulated cells. There was a rapid response to
S. aureus RN6390 with dephosphorylation of the ERK kinase,
as compared with the total (phosphorylated and nonphosphorylated) p44/p42 kinase in the cell. Addition of anti-asialoGM1 to 1HAEo
cells
resulted in more prolonged ERK1/2 phosphorylation persisting beyond 60 min, and thapsigargin also activated this kinase (Fig. 7B).
Phosphorylated p38 MAPK was induced following either P. aeruginosa or S. aureus exposure and persisted beyond
60 min (Fig. 7C).

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Fig. 6.
Activation of ERK p44/p42 MAPKs following
stimulation of 9HTEo airway epithelial cells detected by Western
hybridization. Cells treated with thapsigargin (15 µM) to acutely increase
[Ca2+]i or P. aeruginosa PAO1 were lysed at timed intervals (min) and screened
for the presence of phosphorylated ERKp44/p42. neg. and
pos. indicate controls for p-ERK.
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Fig. 7.
MAPK activation in 1HAEo cells.
A, activation of ERKp44/p42. Cell lysates harvested at the
intervals (min) indicated following exposure to P. aeruginosa PAO1 (108 cfu/ml) were screened for the
presence of phosphorylated ERK p44/p42 and compared with an actin
control. B, activation of ERKp44/p42 by anti-asialoGM1
( -aGM1) or thapsigargin. Cell lysates were
harvested following incubation with anti-asialoGM1 (100 µg/ml) or 15 µM thapsigargin for the times indicated. C,
activation of p38. Phosphorylated p38 was similarly detected in cell
lysates following the exposure of epithelial cells to P. aeruginosa PAO1 or S. aureus RN6390 (108
cfu/ml).
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Activation of NF-
B following S. aureus or P. aeruginosa
Stimulation of Epithelial Cells--
To demonstrate that stimulation
of epithelial cells by S. aureus, as well as P. aeruginosa, results in the activation of NF-
B as an antecedent
to IL-8 transcription, we used an NF-
B-luciferase reporter construct
to monitor the effects of the adherent bacteria (Fig.
8A). Exposure of 1HAEo
airway epithelial cells to antibody to asialoGM1, thapsigargin,
P. aeruginosa, or S. aureus RN6390 stimulated 4- to 5-fold activation of the reporter construct (p < 0.001 for each) (Fig. 8A). As predicted by the effects of
Ca2+ chelation on IL-8 expression, cells pretreated with
BAPTA/AM had less activation of NF-
B in response to ligation of the
asialoGM1 receptor by anti-asialoGM1 (p < 0.05),
P. aeruginosa, or S. aureus (p < 0.001 for each).

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Fig. 8.
Activation of NF- B
following stimulation of 1HAEo or CHO cells. 1HAEo or CHO
cells were transiently transfected with an NF- B reporter construct
and a renilla luciferase control vector. Following stimulation with
antibody to asialoGM1, thapsigargin, or bacteria luciferase activity
was measured and compared with unstimulated control wells or wells
pretreated with BAPTA/AM. Each condition was done in quadruplicate, and
each experiment was repeated three times. A, exposure of transfected 1HAEo cells to
anti-aGM1, P. aeruginosa PAO1, or S. aureus
RN6390 resulted in activation of the transcription factor NF- B as
measured by the luciferase reporter system. This activation was
consistently inhibited by pretreatment with BAPTA/AM. B,
treatment of CHO cells with increasing concentrations of purified
asialoGM1 (aGM1) leads to incorporation into the membrane as
measured by flow cytometry. Detection was done using anti-aGM1 as the
primary antibody and goat anti-rabbit conjugated to fluorescein
isothiocyanate as the secondary antibody. Percentage of cells
positive for fluorescence is measured on the y axis.
C-E, CHO cells with or without incubation with 250 µM aGM1 for 2 h and exposed to (C)
anti-aGM1, (D) P. aeruginosa, or (E)
S. aureus. Higher endogenous activation of cells in
E is attributed to the presence of 0.1% FCS in the media as
described above.
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To further demonstrate that bacteria activate NF-
B through ligation
of an asialoGM1 receptor, the signaling pathway was reproduced in
CHO-K1 cells, which normally do not have detectable asialoGM1 on their
surface. Exogenous asialoGM1 was incorporated into the surface
membranes of the CHO cells in a dose-dependent fashion as
documented by flow cytometry (Fig. 8B). The CHO cells were transiently transfected with the pNF-
B reporter construct and stimulated with antibody to asialoGM1 (Fig. 8C), P. aeruginosa (Fig. 8D), or S. aureus (Fig.
8E). In response to either anti-asialoGM1 or P. aeruginosa PAO1 there was a 5- to 6-fold increase
(p < 0.001) in reporter activity, as compared with
controls lacking the asialoGM1 receptor. S. aureus increased
reporter activity 2- to 3-fold (p < 0.001). The
endogenous activation of NF-
B was higher in the CHO-K1 cells exposed
to S. aureus, likely as a result of the 0.1% FCS required
for staphylococcal binding.
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DISCUSSION |
Respiratory epithelial cells function as an important component of
the normal host defense against bacterial infection. Pathogens that
elude superficial innate defenses (antimicrobial peptides, mucin, and
cilary activity) and express appropriate adhesins to recognize
GalNAc
1-4Gal binding sites on the epithelial surface, rapidly
stimulate increases in [Ca2+ ]i
that serve to initiate MAPK activation and the transcription of IL-8, a
major PMN chemokine. The presence of this common bacterial receptor was
predicted by Krivan et al. (7), who recognized that many
pulmonary pathogens bind to the GalNAc
1-4Gal moiety present on
asialylated glycolipids including asialoGM1 in vitro. The
association of asialylated glycolipid receptors and cystic fibrosis
transmembrane conductance regulator mutations has been documented by
several investigators working with cell lines (9, 20) and primary
tissues (5). The increased activation of this pathway in CF patients
who have bacterial contamination of the lower airways and greater
numbers of available receptors is likely to be a major contributing
factor to the chronic airway inflammation characteristic of this
disease. However, this pathway is also of significance in the normal
lung, as well, providing a general mechanism for defense of the airway
in response to inhaled pathogens.
Diverse bacterial ligands are recognized by common epithelial
receptors. Incorporation of the asialoGM1 glycolipid into the membranes
of CHO cells, which ordinarily lack superficial asialoGM1 moieties,
provided receptor function and mediated the activation of NF-
B in
response to ligation of the receptor by antibody, P. aeruginosa, or S. aureus. Although bacteria have many
superficial ligands that may interact with epithelial components, there
is clearly some specificity for the recognition of the GalNAc
1-4Gal disaccharide of asialoGM1 and similar glycolipids that provides a
common receptor for diverse respiratory pathogens (7) and initiates
this epithelial proinflammatory response. P. aeruginosa pili
have been shown to bind specifically to the asialylated glycolipids and
stimulate the activation of NF-
B translocation and IL-8 expression (9). Despite expression of multiple virulence factors, nonpiliated P. aeruginosa were unable to activate the Ca2+
fluxes required to stimulate an IL-8 response. Other superficial components of P. aeruginosa, particularly LPS, may activate
epithelial signaling cascades; however, the recognition of the
asialylated glycolipids appears to be a major stimulus for the
expression of IL-8 (9). Although P. aeruginosa LPS can
induce mucin (muc-2) expression through activation of a
c-Src-Ras-MAPK/ERK1/2-MAPK-pp90rsk-NF-
B cascade in colonic
epithelial cells (32), LPS from the same strain of P. aeruginosa did not evoke the
[Ca2+]i changes in respiratory
cells that induced NF-
B and IL-8 expression. Moreover, a P. aeruginosa mutant with defective LPS O-side chains was,
nonetheless, able to activate Ca2+ oscillations and
previously has been shown to stimulate IL-8 expression (9). Thus,
P. aeruginosa activation of IL-8 expression by airway
epithelial cells is independent of LPS.
The staphylococcal ligand, while undefined, appears to be an
agr-dependent cell surface structure like the
S. aureus adhesins that recognize extracellular matrix
components. Unlike wild type S. aureus RN6390, the mutant
RN6911 did not compete with anti-asialoGM1 or with P. aeruginosa PAO1 for binding nor did it activate immediate increases in [Ca2+]i. The binding
of RN6911 did evoke some IL-8 expression indicating that
agr-independent adhesins function through other pathways. As
pathogens often express multiple ligands, independent binding events
are likely, which may result in similar epithelial responses.
The surface components of the Gram-positive staphylococci differ
significantly from those of the Gram-negative P. aeruginosa, and it is unlikely that either the binding affinities or the binding kinetics of the two pathogens are identical. However, evidence for the
activation of a common asialoGM1 pathway is further supported by the
similar kinetics of the host response. Both species, as well as
antibody to asialoGM1, initiated immediate changes in [Ca2+]i followed by
phosphorylation of MAPKs within 15 min and NF-
B activation. This was
too rapid a response to involve epithelial ingestion of organisms, as
hours are required for the internalization of P. aeruginosa
(4). Although bacterial invasion of gut epithelial cells may be
required before some signaling pathways are activated (13, 33), for
airway cells a superficial ligand-receptor was sufficient to initiate
Ca2+ signaling and an IL-8 response.
Stimulation of the 1HAEo
cells initiated immediate increases in
[Ca2+]i that came from
predominantly intracellular stores. The Ca2+ response and
ensuing IL-8 expression were inhibited by BAPTA/AM but not by EGTA at
the level of MAPK and NF-
B activation, and blocking external
Ca2+ channels with NiCl2 did not inhibit the
IL-8 response. The ability of thapsigargin, which causes an immediate
increase in [Ca2+]i, to activate
MAPK phosphorylation and stimulate IL-8 expression is also consistent
with this hypothesis. The Ca2+ response did not appear to
be because of influx from the media, as was the case for
Neisseria (12). Activation of Ca2+ fluxes of
similar magnitude and duration, followed by IL-8 expression, were
observed in an unrelated 9HTEo
cell line, nasal polyp cells in
primary culture, as well as Calu-3 cells (data not shown). The
amplitude of the Ca2+ fluxes, 100-150 nM, is
less than that of excitatory cells but is similar to the responses of
gastrointestinal cells, which activate NF-
B through Ca2+
signaling following Salmonella infection (13), suggesting
that this may represent a common mechanism to initiate protection of mucosal surfaces.
The Ca2+ signal in airway cells, whether initiated by
bacterial ligation of asialoGM1, antibody recognition of the receptor, or thapsigargin, was sufficient to activate p38 and ERK MAPKs. Both
families of MAPKs participate as biochemical inhibition of either was
sufficient to inhibit the IL-8 response.
Ca2+-dependent cytokine signaling is often
mediated by calcineurin-dependent pathways. However, the
relatively modest inhibitory effects of cyclosporin and FK506 on IL-8
expression suggest that calcineurin is not involved to any great
extent. The 6-fold stimulation of IL-8 by the phosphatase inhibitor
okadaic acid (which does not affect calcineurin) is more consistent
with signaling through other Ca2+-dependent
protein kinases and phosphatases (28). This portion of the signaling
cascade of airway epithelial cells may be similar to that of HL-60
cells, a promeylocytic cell line, which responds to okadaic acid by
tyrosine phosphorylation of p44, degradation of I
B-
, activation
of NF-
B, and IL-8 transcription (34). There is likely to be
redundancy within this proinflammatory pathway, as well as cross-talk
with other intracellular signaling mechanisms at a variety of levels.
Inhibition of one component of the pathway, one of the MAPKs, for
example, may not have equivalent consequences on the net production of
IL-8 as interference with other, even downstream, component. This
Ca2+dependent-MAPK-NF-
B signaling system, although not
previously described in respiratory cells, is similar to that
delineated in professional immune cells and neuronal cells (34, 35). However, the more proximal components of the pathway, the glycolipid receptors and possibly associated G-proteins involved in signaling, remain to be identified. The association of glycosphingolipids and
G-protein-coupled receptors has been well documented, and the
participation of glycolipid rafts and inositol 1,4,5-trisphosphate receptor in the spatial organization of several signaling
pathways has been demonstrated (36-38).
The asialoGM1 signaling pathway in the normal respiratory epithelium
provides a mechanism to rapidly elicit a PMN response through IL-8 expression when pathogens contact the surface of epithelial cells. In the normal airway, mucosal glycoconjugates are
fully sialylated, and inhaled organisms rarely encounter
GalNAc
1-4Gal-glycolipid binding sites on the epithelial mucosa,
even if they are able to withstand the antibacterial activities of
airway surface fluid and mucociliary clearance. Damaged epithelial
cells, or those undergoing regeneration, provide asialylated receptors
(6). In cystic fibrosis, defective sialylation (20) results in
increased numbers of epithelial receptor sites and increased IL-8
production induced by adherent bacteria. This is a major contributing
factor to the general proinflammatory milieu in CF airways (39).
Although the airway epithelial response to infection seems appropriate in the normal host, it may be useful to modulate components of this
signaling cascade in specific pathological conditions such as CF, to
prevent undesirable consequences of excessive inflammation in the lung.