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Infection and Immunity, August 2000, p. 4430-4440, Vol. 68, No. 8
Department of Pediatrics and Communicable Diseases,
University of Michigan Medical School,1 and
Department of Epidemiology, University of Michigan School of
Public Health,2 Ann Arbor, Michigan 48109, and
Departments of Pediatrics and Molecular Microbiology,
Washington University School of Medicine, St. Louis, Missouri
631103
Received 16 February 2000/Returned for modification 6 April
2000/Accepted 28 April 2000
Nontypeable Haemophilus influenzae (NTHi) causes
repeated respiratory infections in patients with chronic lung diseases.
These infections are characterized by a brisk inflammatory response which results in the accumulation of polymorphonucleated cells in the
lungs and is dependent on the expression and secretion of
proinflammatory cytokines. We hypothesize that multiple NTHi molecules,
including lipooligosaccharide (LOS), mediate cellular interactions with
respiratory epithelial cells, leading to the production of
proinflammatory cytokines. To address this hypothesis, we exposed
9HTEo Nontypeable Haemophilus
influenzae (NTHi) strains are fastidious gram-negative bacteria
that exist as commensal organisms in the human nasopharynx (42,
48). In addition, NTHi strains cause otitis media, sinusitis, and
conjunctivitis in otherwise healthy individuals (65). NTHi
has also been associated with chronic bronchitis and pneumonia in
patients with chronic pulmonary diseases such as cystic fibrosis and
chronic obstructive pulmonary disease (13, 49, 65). An acute
inflammatory response and the accumulation of polymorphonuclear
leukocytes characterize NTHi respiratory infections. The initial events
leading to respiratory inflammation are thought to involve cellular
interactions between infecting bacteria and respiratory epithelial
cells; these bacterial-epithelial cell interactions stimulate the
release of inflammatory mediators (i.e., cytokines and chemokines)
(26, 35).
Several respiratory pathogens such as Pseudomonas
aeruginosa, Burkholderia cepacia, Streptococcus
pneumoniae, and Bordetella pertussis produce a variety
of molecules that interact with epithelial cells and cause a secretion
of proinflammatory cytokines (10, 19, 25, 26, 29, 39, 54, 58, 63,
74, 76). These interactions involve bacterial cell-associated and
extracellular products that include cell wall fragments, outer membrane
proteins, autoinducers, pili and flagella, and secreted molecules. The
bacterial molecules that are capable of perturbing cytokine networks
are called modulins (26, 74). In response to bacterial
modulins, airway epithelial cells produce inflammatory mediators such
as interleukin-6 (IL-6), IL-1 Preliminary studies have begun to characterize the modulin activity of
NTHi and its ability to perturb the inflammatory response. These
studies have focused on H. influenzae lipooligosaccharide (LOS) as a major stimulator of proinflammatory cytokines. Recent in
vitro studies showed that H. influenzae LOS stimulated the release of IL-6, IL-8, and TNF- Bacteria.
Table 1 describes
the bacterial strains used in this study. All bacteria were cultured on
chocolate agar (Becton Dickinson, Cockeysville, Md.) in 5%
CO2 at 37°C overnight and transferred to 10 ml of
Levinthal broth (brain heart infusion broth [Difco Laboratories,
Detroit, Mich.] supplemented with hemin [100 µg/ml] and NAD [20
µg/ml]) and grown in 5% CO2 at 37°C overnight to
stationary phase. Adhesin-deficient NTHi strains carrying the kanamycin
resistance (Kanr) gene were cultured in medium containing
25 µg of kanamycin (Sigma-Aldrich Co., St. Louis, Mo.) per ml of
medium. Long-term storage of bacteria was in sterile skim milk at
0019-9567/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Induction of Proinflammatory Cytokines from Human Respiratory
Epithelial Cells after Stimulation by Nontypeable
Haemophilus influenzae
ABSTRACT
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
human tracheal epithelial cells to NTHi and compared the
resulting profiles of cytokine gene expression and secretion using
multiprobe RNase protection assays and enzyme-linked immunosorbent
assays (ELISA), respectively. Dose-response experiments demonstrated a
maximum stimulation of most cytokines tested, using a ratio of 100 NTHi
bacterial cells to 1 9HTEo tracheal epithelial cell. Compared with
purified LOS, NTHi bacterial cells stimulated 3.6- and 4.5-fold
increases in epithelial cell expression of interleukin-8 (IL-8) and
IL-6 genes, respectively. Similar results were seen with epithelial
cell macrophage chemotactic protein 1, IL-1
, IL-1
, and tumor
necrosis factor alpha expression. Polymyxin B completely inhibited LOS
stimulation but only partially reduced NTHi whole cell stimulation.
Taken together, these results suggest that multiple bacterial molecules
including LOS contribute to the NTHi stimulation of respiratory
epithelial cell cytokine production. Moreover, no correlation was seen
between NTHi adherence to epithelial cells mediated by hemagglutinating
pili, Hia, HMW1, HMW2, and Hap and epithelial cytokine secretion. These
data suggest that bacterial molecules beyond previously described NTHi
cell surface adhesins and LOS play a role in the induction of
proinflammatory cytokines from respiratory epithelial cells.
INTRODUCTION
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
, IL-1, and tumor necrosis factor alpha (TNF-), along with the chemokines IL-8 and macrophage
chemotactic protein 1 (MCP-1), which stimulate the activation and
influx of neutrophils, monocytes, and macrophages (2, 5-7,
10, 19, 39-41, 50, 53, 57, 62, 64).
from cultured, primary human bronchial epithelial cells and TNF-, IL-1, and IL-6 from human monocytes (18, 37, 38, 51). Adherent, intact H. influenzae bacterial cells have also been shown to stimulate IL-6
secretion from human macrophages and IL-6 and IL-8 from respiratory
syncytial virus-infected human respiratory epithelial cells
(31; C. Maguire, and G. Noel, Abstr. 36th Intersci.
Conf. Antimicrob. Agents Chemother., abstr. B16, p. 24. 1996). These
studies, however, have not comprehensively analyzed all of the
proinflammatory mediators involved in H. influenzae epithelial cell infection or characterized completely the bacterial factors that influence this host response. The aim of the present study
was to examine the relative ability of intact H. influenzae, purified H. influenzae LOS, and specific adhesin-deficient
H. influenzae to stimulate respiratory epithelial cytokine
production in vitro.
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
70°C.
TABLE 1.
Strains used in this study
used is this study is a nonpiliated
phase variant and has been described previously (14).
H. influenzae strain N187hap was
constructed using plasmid pJS104, a pT7-7 derivative that contains a
6.5-kb PstI fragment harboring a full-length hap
gene (66). pJS104 was partially digested with
BglII, and linearized DNA was purified and then ligated with
the 1.3-kb BamHI fragment harboring the Kanr
cassette from pUC4K (72). Recombinant plasmids were analyzed by restriction mapping, and a plasmid with the Kanr
cassette inserted at the BglII site in the middle of the
hap coding sequence was designated pJS107. Subsequently
pJS107 was linearized with BglII and transformed into NTHi
strain N187 made competent by the M-IV method (27).
Transformants were selected by growth on agar containing kanamycin.
Southern analysis and Western immunoblotting were used to confirm that
the mutants contained an interrupted hap locus and failed to
produce Hap. One mutant was saved for further study and was designated
N187hap.
Cell culture.
The human tracheal epithelial cell line
9HTEo was obtained from Dieter C. Gruenert (University of California,
San Francisco) (17). Cells were cultured at 37°C and 5%
CO2 in 75-cm2 tissue culture flasks (Costar
Corp., Cambridge, Mass.) in 20 ml of Eagle's minimum essential medium
supplemented with 10% fetal bovine serum, 1% L-glutamine,
1% penicillin, and 1% streptomycin (all from Gibco BRL, Gaithersburg,
Md.). The confluent, adherent monolayers were released from the plastic
surface after treatment with trypsin-EDTA (Gibco BRL) and split 1:4
three times a week.
Stimulation of 9HTEo cells.
Six-well tissue culture plates
(Costar) were seeded with 106 9HTEo cells per well in 3 ml of fresh supplemented Eagle's minimal essential medium and allowed
to grow overnight at 37°C in 5% CO2. On the second day,
the medium was replaced with 3 ml of fresh serum-free medium (SAGM from
Clonetics, San Diego, Calif.) and allowed to grow overnight. On the
third day, cells were 95 to 100% confluent. The growth medium was
replaced with 6 ml of fresh serum-free medium. All experiments included
an unstimulated negative control well(s), a positive control well(s)
containing IL-1 (20 ng/ml; Pharmingen, San Diego, Calif.)
(7), and experimental wells containing various stimuli. Cell
culture supernatant fluid and host cell RNA were isolated at various
times after the start of the experiment.
epithelial cells were analyzed using regression analysis.
Correlation coefficients were used to determine the linearity of
epithelial cytokine responses to increasing amounts of
AAr176p+ whole cells and AAr176p+ LOS.
Control experiments using the lipopolysaccharide (LPS) inhibitor
polymyxin B (Sigma) were performed to determine the contribution of
NTHi LOS to the induction of epithelial cytokines. In these experiments, IL-1 (20 ng/ml), purified LOS from NTHi strain
AAr176p+ (0.21 µg/ml), NTHi strain AAr176p+
whole cells (5 × 107 CFU/ml), and sterile tissue
culture medium were incubated with increasing amounts of polymyxin B
(0.05, 0.5, 5.0, and 50 µg/ml) at room temperature for 30 min. The
levels of polymyxin B used for these experiments were based on studies
by Hedlund et al. (23). Confluent layers of 9HTEo cells
were then exposed to these stimuli for 16 h at 37°C in 5%
CO2. After exposure, the tissue culture fluid from the
9HTEo cells was removed and assayed for the presence of chemokine
IL-8, using commercially available enzyme-linked immunosorbent assay
(ELISA) kits (R&D Systems, Minneapolis, Minn.).
LOS purification and analysis. LOS was isolated from NTHi strain AAr176p+ after growth overnight at 37°C in 5% CO2 on 150-mm-diameter Levinthal agar plates (18, 20). The bacteria were harvested from 18 plates into 180 ml of buffer containing 10 mM Tris-HCl (pH 8.0)-5 mM EDTA buffer. S1 nuclease (20,000 U; Gibco BRL), micrococcal nuclease (50 U; Sigma) and RNase T1 (40,000 U; Gibco BRL) were added, and the digestion mixture was incubated at 37°C for 1 h. Proteinase K (5 mg; Gibco BRL) was added, and the mixture was incubated at 65°C for 2 h. The sample was incubated on ice for 1 h, sonicated briefly on ice to reduce viscosity, and centrifuged at 3,000 × g for 30 min at 4°C. The supernatant fluid was transferred to dialysis tubing (Spectrum Medical, Laguna Hills, Calif.) and electrodialyzed against Milli-Q water (Millipore, Bedford, Mass.) at 500 V for 2 h with frequent changes of water and then overnight at 100 V. The precipitated LOS was removed from the dialysis bags, transferred to centrifuge tubes, and centrifuged at 23,000 × g for 30 min at 4°C. The pellet was washed once with HPLC (high-pressure liquid chromatography)-grade water, resuspended in HPLC-grade water, and further purified by the classical phenol-water extraction method (73). LOS was precipitated with sodium acetate (0.3 M, final concentration) and 2 volumes of ice-cold 95% ethanol. Pellets were washed twice with 70% ethanol and then resuspended in HPLC-grade water. The LOS was lyophilized, weighed, and resuspended to a final concentration of 1 mg/ml in 0.85% saline and stored at 4°C.
Purified and cell-bound LOS from AAr176p+ was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 16% Tris-glycine polyacrylamide gels (NOVEX, San Diego, Calif.) according to the procedure of Gu et al. (18). Purified LOS (0.375, 0.75, 1.5, and 3.0 µg) was mixed with SDS-PAGE digestion buffer, boiled for 10 min, and then loaded on an SDS-polyacrylamide gel. For cell-bound LOS, 3 × 108 CFU of strain AAr176p+ cells was resuspended and boiled in SDS-PAGE digestion buffer and incubated with 100 mg of proteinase K (final concentration, 0.5 mg/ml; Gibco BRL) at 60°C for 2 h before being loaded (28). After electrophoresis, the gels were silver stained and scanned with a Quickscan R&D laser densitometer (Helena Laboratories, Beaumont, Tex.). The amount of cell-bound LOS was compared to a standard curve generated with increasing amounts of the purified LOS (18). LOS data are means of four different experiments. Benchmark (Gibco BRL) prestained molecular weight markers were run to estimate size of bands.Total RNA isolation.
Total RNA was isolated from the 9HTEo
tracheal epithelial cells using Trizol Reagent (Gibco BRL) according to
the manufacturer's directions.
RNase protection assay.
The expression of cytokine and
chemokine mRNA was determined and quantified by the RiboQuant RNase
protection assay system (Pharmingen) according to the manufacturer's
directions. The 32P-labeled antisense HL-14 probe set
simultaneously detected expression of the human cytokine IL-6, IL-1,
IL-1, and TNF- genes (61). The 32P-labeled
antisense hCK-5 multiprobe (Pharmingen) detected simultaneous expression of IL-8 and MCP-1 genes. Both probe sets contain the housekeeping gene L32 as an internal standard for normalization of
cytokine and chemokine gene expression. A PhosphorImager (Storm 860; Molecular Dynamics, Sunnyvale, Calif.) with ImageQuaNT version 4.2 software was used to measure and analyze the expression intensity of
the cytokine and chemokine mRNA species; the data were expressed as
fold increase over the unstimulated control (61).
ELISA.
Secreted cytokines IL-1, IL-6, and chemokine MCP-1
were determined by ELISA kits (CYTImmune Sciences, Inc. College Park, Md.). Biosource International (Camarillo, Calif.) ELISA kits were used
to assay for TNF- and IL-1. R&D Systems ELISA kits were used to
assay IL-8. Comparisons of cytokine secretion were made using
Student's two-tailed paired t tests and were calculated using Microsoft Excel 97 software.
Adherence assay. Adherence assays were performed according to the procedure of Gilsdorf et al. (16). Briefly, epithelial monolayers in 96-well tissue culture plates were fixed with 1% glutaraldehyde, washed with 0.2% Triton X-100, and blocked with phosphate-buffered saline containing 2% bovine serum albumin. The epithelial monolayers were then incubated with 107 CFU of biotinylated bacteria (Sulfo-NHS-Biotin; Pierce Chemical Company, Rockford, Ill.) for 1 h, washed, and incubated further with ExtrAvidin-peroxidase conjugate (Sigma). The wells were developed with the enzyme substrate o-phenylenediamine dihydrochloride (Sigma), and the intensities of the reactions were determined at A490 on a Dynatech microplate reader (Dynatech Laboratories, Inc., Alexandria, Va.). Control wells contained epithelial cells with no bacteria. Comparisons of adherence, as measured by A490 of the immunoassays, were made using Student's two-tailed paired t test, calculated using Microsoft Excel 97 software.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Time course of epithelial cell cytokine production using different
stimuli in the normal tracheal cell line 9HTEo.
To determine the
kinetics of cytokine gene expression and secretion, extended time
courses were performed using the normal human tracheal epithelial cell
line 9HTEo coincubated with different H. influenzae
stimuli. Representative time courses for IL-6 and IL-8 gene expression
and secretion are shown in Fig. 1.
Stimulation of 9HTEo cells with IL-1 (the positive control),
purified LOS, and NTHi bacterial stimuli showed an initial peak mRNA
response with cytokines IL-6 and IL-8 between 2 and 4 h of
coincubation. A second peak of IL-6 and IL-8 gene expression was
observed after 16 h of costimulation with the various stimuli
(Fig. 1A and B). The stimulation of epithelial cells with the stimuli
showed a peak mRNA response with cytokines IL-1, IL-1, MCP-1, and
TNF- between 2 and 4 h of coincubation that decreased over the
rest of the time course with no secondary peak (data not shown). The stimulation of epithelial cytokine gene expression with intact NTHi
cells generated a response greater than that seen with purified LOS.
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), 20 ng of IL-1
), 1 µg of
LOS per ml (
), and 5 × 107 CFU of NTHi strains
AAr176p+ (×), 11 (
), and AA238 (
) per ml. An NTHi
cell density of 5 × 107 CFU/ml corresponds to a
100::1 ratio of bacteria to epithelial cells. Based on the
calculations of Gu et al. ( cells coincubated with different
stimuli were assayed from culture supernatant fluids using commercially
available ELISAs (Fig. 1C and D). Peak secretion of IL-6 and IL-8 was
seen between 16 and 24 h after coincubation with different
stimuli. Similar secretion was seen for cytokine MCP-1; no IL-1 or
IL-1 secretion was seen under any stimulation conditions (data not
shown). TNF- secretion, however, rose faster than that of IL-6 and
IL-8, showing a range of 39 to 75% maximal response, compared to a
range of 1 to 17% for both IL-6 and IL-8 after 4 h of
coincubation of 9HTEo cells with various stimuli. Similar to the mRNA
expression data, intact bacterial cells stimulated greater cytokine
secretion than the purified LOS.
As a negative control, monolayers of unstimulated control 9HTEo cells
demonstrated low levels of cytokine gene expression and secretion of
IL-6 and IL-8 (Fig. 1), as well as with cytokines IL-1, IL-1,
TNF-, and MCP-1 (data not shown). Control experiments with
epithelial cells coincubated with sterile bacterial growth medium or
0.85% saline demonstrated no cytokine expression or secretion (data
not shown).
Dose response of 9HTEo tracheal epithelial cytokine production
with NTHi strain AAr176p+ whole cells and purified
LOS.
To assess the contribution of H. influenzae LOS to
cytokine gene expression by H. influenzae whole cells,
subsequent experiments assayed RNA expression at the 4-h peak
expression time. Dose-response experiments were performed with 9HTEo
cells coincubated with increasing levels of LOS purified from strain
AAr176p+ and with increasing amounts of
AAr176p+ bacterial cells. The levels of purified LOS used
in these experiments were chosen based on the levels used by Khair et
al. (38) (10 and 100 µg/ml) with human bronchial
epithelial cells and the levels used by Gu et al. (18) (0.01 ng/ml to 10 µg/ml) with human monocytes. The NTHi levels used in
these experiments represent bacterium-to-epithelial cell ratios of 1:1
to 10,000:1 and were chosen based on those used by Bresser et al.
(4). Representative graphs for cytokine gene expression are
shown in Fig. 2 and
3.
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), 0.61 (IL-1), 0.78 (IL-6), 0.93 (TNF-), 0.88 (IL-8), and 0.92 (MCP-1). The magnitude of incremental steps over the 5-log range of doses of
LOS, however, was small. The difference in fold increase of cytokine
expression between the lowest and highest levels of LOS used (0.01 and
100 µg/ml, respectively) ranged between 1.3 and 12.
All levels of LOS stimulated a 0.4- to 2.5-fold increase in IL-1 and
IL-6 gene expression over that of unstimulated controls. In addition, a
1.7- to 5.8-fold increase was observed with TNF- and IL-1, and a
7.6- to 17.1-fold increase was seen with IL-8 and MCP-1 over
unstimulated controls (Fig. 2).
Strain AAr176p+ bacterial cells also showed a dose response
with respect to cytokine gene expression (Fig. 3); regression analysis of this response showed correlation coefficients of 0.78 (IL-1), 0.89 (IL-1), 0.61 (IL-6), 0.71 (TNF-), 0.88 (IL-8), and 0.84 (MCP-1). The peak response of all cytokines was seen with either 5 × 107 CFU/ml (3 × 108 CFU/well) or
5 × 108 CFU/ml (3 × 109 CFU/well)
bacteria, which represented a approximate 100:1 and 1,000:1,
respectively, ratios of bacteria to epithelial cells.
Quantitation of cell-bound LOS on NTHi strain
AAr176p+.
The dose-response studies presented above
indicated that peak cytokine expression was seen with a
bacterium/epithelial cell ratio of approximately 100:1, or 3 × 108 CFU of strain AAr176p+ per well (5 × 107 CFU/ml). To better assess the relative contributions of
NTHi whole cells and LOS to epithelial cytokine gene expression, the amount of cell-bound LOS on 3 × 108 CFU of bacterial
whole cells (the peak stimulating inoculum) was determined. For this,
the LOS of 3 × 108 bacterial cells and increasing
amounts of purified LOS were isolated, fractionated by SDS-PAGE, and
quantitated by scanning laser densitometry (Fig.
4). The results showed that 3 × 108 CFU of AAr176p+ bacterial cells contained
1.25 µg of LOS. Standardizing for the amount of LOS in the inoculum,
AAr176p+ bacterial cells showed significant levels of
cytokine gene expression over that of the purified LOS (Table
2).
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cellular
response to AAr176p+ cells, polymyxin B, which binds to LOS
and neutralizes the endotoxic activity, was used as a control (Fig.
5) (23, 37). Polymyxin B at
all levels (0.05 to 50 µg/ml) did not affect IL-8 secretion when
added to 9HTEo cells with and without IL-1. All levels of
polymyxin B blocked the secretion of IL-8 from 9HTEo cells in the
presence of purified LOS from NTHi strain AAr176p+.
Incubation of AAr176p+ cells with increasing amounts of
polymyxin B (0.05 µg/ml to 50 µg/ml) showed a 55.8 to 73.7%
reduction in 9HTEo IL-8 secretion compared to AAr176p+
cells without polymyxin B (Fig. 5).
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Stimulation of epithelial cell cytokine production by adherent
NTHi.
Adhesive interactions between bacterial pathogens and
mucosal epithelial cells have been shown to play a key role in the
stimulation of epithelial cytokine production (9, 10, 26, 35,
74). Several NTHi adhesins that mediate adherence to epithelial
cells have been described (15, 59). To assess the role of
NTHi adherence in the stimulation of respiratory epithelial cytokine
production, sets of wild-type and adhesin-deficient NTHi strains,
representing five different adhesins, were first tested for their
adherence to respiratory epithelial cells. H. influenzae
strain Rd, which lacks the genes for pili, HMW1, and HMW2 and contains
a truncated, nonfunctional gene for Hia and a mutated, nonfunctional
gene for Hap, was used as a minimal adherence control (Table 1)
(12). In this assay, adhesin-proficient strains adhered
significantly better to 9HTEo cells than adhesin-deficient strains
for all adhesins tested (P < 0.0001) (data not shown).
cells for
16 h before the culture supernatant fluids were harvested and
assayed for secreted cytokines. Secreted IL-8 and IL-6 were chosen as
models for cytokine secretion and were assayed from the epithelial
culture supernatant fluid; the data for IL-8 secretion are presented in
Fig. 6.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
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Mucosal epithelial cells play an active role in the host response to microbial pathogens and act not only as a mechanical barrier to infection but also as environmental sensors that modulate the host immune response through the secretion of cytokines (35, 39, 69, 74). These cytokine responses occur through microbial attachment to specific receptors and transmembrane signaling, microbial invasion of epithelial cells followed by intracellular activation, or microbial activation of nonepithelial cytokines (69, 74). This study sought to identify those interactions between respiratory epithelial cells and NTHi that resulted in epithelial cytokine production and focused on epithelial cell interactions mediated by intact NTHi bacterial cells, purified NTHi LOS, and known NTHi adhesins.
Our results showed that epithelial cell interactions with intact NTHi
bacterial cells and purified NTHi LOS stimulated the early expression
of the proinflammatory cytokines IL-1, IL-1, TNF-, IL-6, IL-8,
and MCP-1. This early gene expression led to the secretion of the
cytokines TNF-, IL-6, IL-8, and MCP-1; no secretion of cytokines
IL-1 and IL-1 was seen. This result is not surprising since
IL-1 tends to be cell associated, and IL-1 transcription does not
always correlate with translation (11).
The secretion of cytokines TNF-, IL-6, IL-8, and MCP-1 in response
to NTHi stimulation correlates well with the cytokines found in the
lungs of patients with cystic fibrosis and chronic obstructive
pulmonary disease and in the middle ear effusions of patients with
otitis media (2, 34, 45, 47, 49, 52, 53, 55, 57, 68).
Further, these data correlate with studies showing the secretion of
cytokines from primary bronchial epithelial cells in response to
stimulation with NTHi LOS (38, 51).
The expression of IL-6 and IL-8 declined after the 4-h peak to a low
level of expression by 8 h followed by a secondary peak at 16 h (Fig. 1A and B). This secondary peak of IL-6 and IL-8 gene expression
was more pronounced with 9HTEo cells stimulated with intact NTHi
bacterial cells; LOS-stimulated gene expression fell off by 8 h
and showed either no increase or a small increase at 16 h. The
secondary peak of IL-6 and IL-8 expression is probably due to the
autocrine effects elicited by the initial secretion of TNF- (7,
35, 39, 43, 64). A recent study by Khair et al. (39)
demonstrated a similar biphasic IL-8 gene expression pattern with human
bronchial epithelial cells stimulated with H. influenzae
LOS. They also showed that antibodies directed against IL-1 and
TNF- abrogated the secondary peak of IL-8 expression (39). This secondary peak of IL-6 and IL-8 expression may
play a role in sustaining the bacterium-induced inflammation and
recruitment of polymorphonuclear leukocytes into the respiratory tract
(7, 35, 39, 43, 64).
TNF- secretion by 9HTEo cells in response to NTHi whole cell and
LOS stimulation rose faster than that of IL-6 and IL-8 secretion (data
not shown). This, in part, may represent a selective early response by
epithelial cells to bacterial insult resulting in sustained production
of other cytokines like IL-8 and IL-6 and activation of other cells of
the immune system (7, 35, 39, 43, 64, 69). Hence, epithelial
cells may act as sensors and play a key role in the modulation of the
immune response to microbial infections (22, 35, 39).
Further, IL-1 and TNF- have been shown to damage respiratory
epithelial cells (25, 36), and H. influenzae has
been shown to preferentially adhere to damaged epithelial cells
(60, 75). Thus, cytokine-induced respiratory epithelial cell
damage may provide a means for H. influenzae to adhere to
and invade host cells.
The time courses of epithelial cytokine production suggested that NTHi bacterial cells stimulated a response greater than that of purified LOS. The dose-response (Fig. 2 and 3), LOS quantitation (Table 2), and polymyxin B inhibition (Fig. 5) experiments corroborated that observation, demonstrating that NTHi cells elicited an epithelial cytokine response greater than that of purified LOS. These experiments suggest that NTHi factors, beyond LOS, contribute to the stimulation of respiratory epithelial cytokine production. Henderson et al. (26) have suggested that bacterial factors (i.e., modulins) beyond LPS play a role in the induction of cytokine synthesis by host cells. For example, cell surface adhesins (pilin and flagellin), secreted products (autoinducer and pyocyanin), and periplasmic components (nitrite reductase) of P. aeruginosa stimulated IL-8 production from a variety of human respiratory epithelial cells (8, 10, 54, 63). P. aeruginosa LPS, however, minimally stimulated IL-8 production from the respiratory epithelial cells (10, 54). Similar results have been described for cytokine production from human epithelial cells stimulated with B. cepacia, uropathogenic Escherichia coli, and Helicobacter pylori (21, 24, 30, 56). The present study demonstrated that NTHi bacterial adherence mediated by hemagglutinating pili, HMW1, HMW2, Hap, or Hia to the tracheal epithelial cells did not correlate with IL-8 and IL-6 secretion (Fig. 6). In fact, the least adherent H. influenzae strain (Rd) elicited strong IL-6 and IL-8 responses from the epithelial cells, suggesting that other bacterial modulins contribute to epithelial cytokine stimulation. Jiang and Patel (31) showed that P5 fimbriae enhanced the IL-6 and IL-8 secretion from respiratory syncytial virus-infected and uninfected type II alveolar epithelial cells (A549). None of the H. influenzae strains tested in this study were known P5 fimbriae mutants. The strong epithelial cytokine response stimulated by strain H. influenzae strain Rd could in part be due to adherence by P5 fimbriae. Bresser et al. (4) recently identified a heat-stable soluble factor from NTHi culture fluid that stimulated IL-6 and IL-8 production from H292 human lung epithelial cells. Approximately 70% of this cytokine stimulating activity, however, was due to LOS (4).
The role of NTHi LOS in respiratory epithelial cell cytokine production is unclear. Khair et al. (38) and Nichols et al. (51) have shown that purified NTHi LOS stimulate the production of proinflammatory cytokines from primary bronchial epithelial cells. No comparative analyses, however, between NTHi bacterial cells and LOS were performed in these studies to determine the relative contribution of each to epithelial cytokine production (38, 51). The present study suggests that NTHi molecules, beyond LOS, play a role in respiratory epithelial cytokine synthesis.
The stimulation of respiratory epithelial cell cytokine production by
NTHi LOS appears to be somewhat of a paradox. Several reports have
suggested that epithelial cells lack the membrane bound CD14 receptor
for LPS (22, 24, 39, 44). Preliminary fluorescence-activated
cell sorting analysis in our laboratory of a variety of human
respiratory epithelial cells (e.g., A549, HEp-2, 16HBE14o, and
CFTE29o) demonstrated no cell-associated CD14 (data not shown).
LPS-binding protein (LBP), a serum-derived protein that binds LPS and
delivers it to CD14, is not present in this system since serum-free
culture media was used for all epithelial cytokine stimulation assays.
Further, LBP alone cannot mediate cellular responses to LPS independent
of CD14 (71). Levels of respiratory epithelial cytokine
stimulation by LPS/LOS vary greatly; this variation may depend on the
bacterial source and structure of the LPS/LOS (10, 18, 51, 54, 59,
63, 64, 70).
The actual signaling pathway for H. influenzae LOS-mediated stimulation of respiratory epithelial cytokine production remains to be determined. Three separate mechanisms, however, have been proposed in the literature. First, Li et al. (44) suggested that as yet unidentified receptors are responsible for LPS-dependent expression in human epithelial cells. Khair et al. (39) postulated that the alternate receptors could include the platelet-activating factor (PAF) receptors since PAF receptor antagonists block LPS-induced Ca2+ increase in platelets. Alternatively, NTHi LOS may act as an epithelial cell cytotoxin and stimulate a stress response resulting in epithelial cytokine synthesis. LOS has been shown to act as an epithelial cytotoxin in organ cultures of rat trachea (32). Further cytokine production by the epithelial cells could then lead to further epithelial cell damage (25, 36). Finally, Joseph et al. (33) suggested that LPS could act as a structural analog of ceramide and hence could stimulate host cells by mimicking the second messenger function of ceramide.
In summary, the data presented show that NTHi possess other modulins,
beyond LOS, that stimulate the expression of proinflammatory cytokines
from respiratory epithelial cells. Further, our studies showed that
NTHi adherence to epithelial cells mediated by hemagglutinating pili,
Hia, HMW1, HMW2, or Hap did not play a role in epithelial cytokine
stimulation. Epithelial cells derived from a variety of locations in
the human respiratory tract have been shown to respond differentially
to bacterial stimuli (4, 7, 8, 10, 31, 39, 51, 54, 63, 64).
Here we have presented results using only one human respiratory
epithelial cell line (9HTEo). Additional studies incorporating
different cell lines need to be done to more fully understand the
cytokine response to NTHi of the entire human respiratory tract. Future
studies will focus on the identification and characterization of the
modulins from NTHi responsible for the stimulation of a variety of
respiratory epithelial cells to secrete cytokines.
| |
ACKNOWLEDGMENTS |
|---|
We are very grateful to R. Rochford for providing the human riboprobe template set HL-14 and to Mayurika Patel for excellent technical assistance.
This work was supported in part by grants from the American Lung Association of Michigan (to D.L.C.), the University of Michigan Office of the Vice-President for Research and Biomedical Research Council (to D.L.C.), and the National Institutes of Health (RO1-AI25630 to J.R.G.).
| |
FOOTNOTES |
|---|
* Corresponding author. Mailing address: Department of Pediatrics and Communicable Diseases, The University of Michigan, 109 South Observatory Street, SPH 1/Rm. 2059, Ann Arbor, MI 48109-2029. Phone: (734) 647-3943. Fax: (734) 764-3192. E-mail: dclemans{at}umich.edu.
Deceased.
Editor: J. D. Clements
| |
REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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| 1. | Barenkamp, S. J., and J. W. St. Geme, III. 1996. Identification of a second family of high-molecular-weight adhesion proteins expressed by non-typable Haemophilus influenzae. Mol. Microbiol. 19:1215-1223[Medline]. |
| 2. | Bonfield, T., J. Panuska, M. Konstan, K. Hilliard, J. Hilliard, H. Ghnaim, and M. Berger. 1995. Inflammatory cytokines in cystic fibrosis lungs. Am. J. Respir. Crit. Care Med. 152:2111-2118[Abstract]. |
| 3. | Bonfield, T., M. Konstan, P. Burfeind, J. Panuska, J. Hilliard, and M. Berger. 1995. Normal bronchial epithelial cells constitutively produce the anti-inflammatory cytokine interleukin-10, which is downregulated in cystic fibrosis. Am. J. Respir. Cell Mol. Biol. 13:257-261[Abstract]. |
| 4. | Bresser, P., L. van Alphen, F. J. M. Habets, A. A. M. Hart, J. Dankert, H. M. Jansen, and R. Lutter. 1997. Persisting Haemophilus influenzae strains induce lower levels of interleukin-6 and interleukin-8 in H292 lung epithelial cells than nonpersisting strains. Eur. Respir. J. 10:2319-2326[Abstract]. |
| 5. | Broug-Holub, E., G. B. Toews, J. F. Van Iwaarden, R. M. Strieter, S. L. Kunkel, R. Paine III, and T. J. Standiford. 1997. Alveolar macrophages are required for protective pulmonary defenses in murine Klebsiella pneumonia: elimination of alveolar macrophages increases neutrophil recruitment but decreases bacterial clearance and survival. Infect. Immun. 65:1139-1146[Abstract]. |
| 6. | Crestani, B., P. Cornillet, M. Dehoux, C. Rolland, M. Guenounou, and M. Aubier. 1994. Alveolar type II epithelial cells produce interleukin-6 in vitro and in vivo. J. Clin. Investig. 94:731-740. |
| 7. |
Cromwell, O.,
Q. Hamid,
C. J. Corrigan,
J. Barkans,
Q. Meng, and P. Collins.
1992.
Expression and generation of interleukin-8, IL-6 and granulocyte-macrophage colony-stimulating factor by bronchial epithelial cells and enhancement by IL-1 and tumour necrosis factor-.
Immunology
77:330-337[Medline].
|
| 8. |
Denning, G. M.,
L. A. Wollenweber,
M. A. Railsback,
C. D. Cox,
L. L. Stoll, and B. E. Britigan.
1998.
Pseudomonas pyocyanin increases interleukin-8 expression by human airway epithelial cells.
Infect. Immun.
66:5777-5784 |
| 9. |
DiMango, E.,
A. Ratner,
R. Bryan,
S. Tabibi, and A. Prince.
1998.
Activation of NF- B by adherent Pseudomonas aeruginosa in normal and cystic fibrosis respiratory epithelial cells.
J. Clin. Investig.
101:2598-2606[Medline].
|
| 10. | DiMango, E., H. J. Zar, and A. P. R. Bryan. 1995. Diverse Pseudomonas aeruginosa gene products stimulate respiratory epithelial cells to produce interleukin-8. J. Clin. Investig. 96:2204-2210. |
| 11. | Dinarello, C. A. 1998. Interleukin-1, p. 35-72. In A. W. Thompson (ed.), The cytokine handbook, 3rd ed. Academic Press, San Diego, Calif. |
| 12. |
Fleischmann, R. D.,
M. D. Adams,
O. White,
R. A. Clayton,
E. F. Kirkness,
A. R. Kerlavage,
C. J. Bult,
J.-F. Tomb,
B. A. Dougherty,
J. M. Merrick,
K. McKenney,
G. Sutton,
W. FitzHugh,
C. Fields,
J. D. Gocayne,
J. Scott,
R. Shirley,
L.-I. Liu,
A. Glodek,
J. M. Kelley,
J. F. Weidman,
C. A. Phillips,
T. Spriggs,
E. Hedblom,
M. D. Cotton,
T. R. Utterback,
M. C. Hanna,
D. T. Nguyen,
D. M. Saudek,
R. C. Brandon,
L. D. Fine,
J. L. Fritch,
J. L. Fuhrmann,
N. S. M. Geoghagen,
C. L. Gnehm,
L. A. McDonald,
K. V. Small,
C. M. Fraser,
H. O. Smith, and J. C. Venter.
1995.
Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.
Science
269:496-512 |
| 13. |
Foxwell, A.,
J. Kyd, and A. Cripps.
1998.
Nontypeable Haemophilus influenzae: pathogenesis and prevention.
Microbiol. Mol. Biol. Rev.
62:294-308 |
| 14. |
Gilsdorf, J. R.,
H. Y. Chang,
K. W. McCrea, and L. O. Bakaletz.
1992.
Comparison of hemagglutinating pili of Haemophilus influenzae type b with similar structures of nontypeable H. influenzae.
Infect. Immun.
60:374-379 |
| 15. | Gilsdorf, J. R., K. W. McCrea, and C. F. Marrs. 1997. Role of pili in Haemophilus influenzae adherence and colonization. Infect. Immun. 65:2997-3002[Medline]. |
| 16. | Gilsdorf, J. R., M. Tucci, and C. F. Marrs. 1996. Role of pili in Haemophilus influenzae adherence to, and internalization by, respiratory cells. Pediatr. Res. 39:343-348[Medline]. |
| 17. |
Gruenert, D. C.,
C. B. Basbaum,
M. J. Welsh,
M. Li,
W. E. Finkbeiner, and J. A. Nadel.
1988.
Characterization of human tracheal epithelial cells transformed by an origin-defective simian virus 40.
Proc. Natl. Acad. Sci. USA
85:5951-5955 |
| 18. | Gu, X.-X., C. M. Tsai, M. A. Apicella, and D. J. Lim. 1995. Quantitation and biological properties of released and cell-bound lipooligosaccharides from nontypeable Haemophilus influenzae. Infect. Immun. 63:4115-4120[Abstract]. |
| 19. | Hakansson, A., I. Carlstedt, J. Davies, A.-K. Mossberg, H. Sabharwal, and C. Svanborg. 1996. Aspects on the interaction of Streptococcus pneumoniae and Haemophilus influenzae with human respiratory tract mucosa. Am. J. Respir. Crit. Care Med. 154:S187-S191. |
| 20. | Hancock, I., and I. Poxton. 1988. Preparation of lipopolysaccharide and enterobacterial common antigen, p. 93-95. In I. Hancock, and I. Poxton (ed.), Bacterial cell surface techniques. John Wiley & Sons Ltd., Chichester, England. |
| 21. |
Hedges, S. R.,
M. Svensson, and C. Svanborg.
1992.
Interleukin-6 response of epithelial cell lines to bacterial stimulation in vitro.
Infect. Immun.
60:1295-1301 |
| 22. | Hedges, S. R., W. W. Agace, and C. Svanborg. 1995. Epithelial cytokine responses and mucosal cytokine networks. Trends Microbiol. 3:266-270[CrossRef][Medline]. |
| 23. | Hedlund, M., C. Wachtler, E. Johansson, L. Hang, J. E. Somerville, R. P. Darveau, and C. Svanborg. 1999. P fimbriae-dependent, lipopolysaccharide-independent activation of epithelial cytokine responses. Mol. Microbiol. 33:693-703[CrossRef][Medline]. |
| 24. | Hedlund, M., W. Agace, G. Godaly, M. Haraoka, and C. Svanborg. 1998. Fimbriae-mediated attachment, transmembrane signaling and epithelial cytokine responses. Bacterial Protein Toxins 29:200-207. |
| 25. |
Heiss, L. N.,
S. A. Moser,
E. R. Unanue, and W. E. Goldman.
1993.
Interleukin-1 is linked to the respiratory epithelial cytopathology of pertussis.
Infect. Immun.
61:3123-3128 |
| 26. |
Henderson, B.,
S. Poole, and M. Wilson.
1996.
Bacterial modulins: a novel class of virulence factors which cause host tissue pathology by inducing cytokine synthesis.
Microbiol. Rev.
60:316-341 |
| 27. |
Herriott, R. M.,
E. M. Meyer, and M. Vogt.
1970.
Defined nongrowth media for stage II development of competence in Haemophilus influenzae.
J. Bacteriol.
101:517-524 |
| 28. |
Hitchcock, P. J., and T. M. Brown.
1983.
Morphological heterogeneity among Salmonella lipopolysaccharide chemotypes in silver-stained polyacrylamide gels.
J. Bacteriol.
154:269-277 |
| 29. |
Houldsworth, S.,
P. Andrew, and T. Mitchell.
1994.
Pneumolysin stimulates production of tumor necrosis factor alpha and interleukin-1B by human mononuclear phagocytes.
Infect. Immun.
62:1501-1503 |
| 30. | Huang, J., W. O'Toole, P. Doig, and T. Trust. 1995. Stimulation of interleukin-8 production in epithelial cell lines by Helicobacter pylori. Infect. Immun. 63:1732-1738[Abstract]. |
| 31. | Jiang, Z., and J. Patel. 1998. Fimbriae-mediated attachment of and cytokine potentiation by nontypable Haemophilus influenzae (NTHI) in respiratory syncytial virus (RSV)-infected epithelial cell cultures. Pediatr. Res. 43:148A. |
| 32. |
Johnson, A. P., and T. J. Inzana.
1986.
Loss of ciliary activity in organ cultures of rat trachea treated with lipo-oligosaccharide from Haemophilus influenzae.
J. Med. Microbiol.
22:265-268 |
| 33. |
Joseph, C. K.,
S. D. Wright,
W. G. Bornmann,
J. T. Randolph,
E. R. Kumar,
R. Bittman,
J. Liu, and R. N. Kolesnick.
1994.
Bacterial lipopolysaccharide has structural similarity to ceramide and stimulates ceramide-activated protein kinase in myeloid cells.
J. Biol. Chem.
269:17606-17610 |
| 34. | Juhn, S. K., C. Lees, R. Amesara, Y. Kim, C. T. Lee, and G. S. Giebink. 1993. Role of cytokines in the pathogenesis of otitis media, p. 431-434. In D. Lim, C. Bluestone, J. Klein, J. Nelson, and P. Ogra (ed.), Recent advances in otitis media. B. C. Decker, Philadelphia, Pa. |
| 35. | Kagnoff, M. F., and L. Eckmann. 1997. Epithelial cells as sensors for microbial infection. J. Clin. Investig. 100:6-10[Medline]. |
| 36. |
Kampf, C.,
A. J. Relova,
S. Sandler, and G. M. Roomans.
1999.
Effects of TNF-, IFN- , and IL-1 on normal human bronchial epithelial cells.
Eur. Respir. J.
14:84-91[Abstract].
|
| 37. | Khair, O. A., J. L. Devalia, M. M. Abdelaziz, R. J. Sapsford, and R. J. Davies. 1995. Effect of erythromycin on Haemophilus influenzae endotoxin-induced release of IL-6, IL-8 and sICAM-1 by cultured human bronchial epithelial cells. Eur. Respir. J. 8:1451-1457[Abstract]. |
| 38. |
Khair, O. A.,
J. L. Devalia,
M. M. Abdelaziz,
R. J. Sapsford,
H. Tarraf, and R. J. Davies.
1994.
Effect of Haemophilus influenzae endotoxin on the synthesis of IL-6, IL-8, TNF-, and expression of ICAM-1 in cultured human bronchial epithelial cells.
Eur. Respir. J.
7:2109-2116[Abstract].
|
| 39. | Khair, O. A., R. J. Davies, and J. L. Devalia. 1996. Bacterial-induced release of inflammatory mediators by bronchial epithelial cells. Eur. Respir. J. 9:1913-1922[Abstract]. |
| 40. | Khan, T. Z., J. S. Wagener, T. Bost, J. Martinez, F. J. Accurso, and W. Riches. 1995. Early pulmonary inflammation in infants with cystic fibrosis. Am. J. Respir. Crit. Care Med. 151:1075-1082[Abstract]. |
| 41. | Konstan, M., and M. Berger. 1997. Current understanding of the inflammatory process in cystic fibrosis: onset and etiology. Pediatr. Pulmonol. 24:137-142[CrossRef][Medline]. |
| 42. | Kuklinska, D., and M. Kilian. 1984. Relative proportions of Haemophilus species in the throat of healthy children and adults. Eur. J. Clin. Microbiol. 3:249-252[CrossRef][Medline]. |
| 43. |
LaFleur, R. L.,
M. S. Abrahamsen, and S. K. Maheswaran.
1998.
The biphasic mRNA expression pattern of bovine interleukin-8 in Pasteurella haemolytica lipopolysaccharide-stimulated alveolar macrophages is primarily due to tumor necrosis factor alpha.
Infect. Immun.
66:4087-4092 |
| 44. |
Li, J.-D.,
W. Feng,
M. Gallup,
J.-H. Kim,
J. Gum,
Y. Kim, and C. Basbaum.
1998.
Activation of NF-B via a Src-dependent Ras-MAPK-pp90rsk pathway is required for Pseudomonas aeruginosa-induced mucin overproduction in epithelial cells.
Proc. Natl. Acad. Sci. USA
95:5718-5723 |
| 45. |
Lindberg, K.,
B. Rynnel-Dagoo, and K.-G. Sundqvist.
1994.
Cytokines in nasopharyngeal secretions; evidence for defective IL-1 production in children with recurrent episodes of acute otitis media.
Clin. Exp. Immunol.
97:396-402[Medline].
|
| 46. | Malefyt, R. W., and K. W. Moore. 1998. Interleukin-10, p. 333-364. In A. W. Thomson (ed.), The cytokine handbook, 3rd ed. Academic Press, San Diego, Calif. |
| 47. |
Mills, P. R.,
R. J. Davies, and J. L. Devalia.
1999.
Airway epithelial cells, cytokines, and pollutants.
Am. J. Respir. Crit. Care Med.
160:S38-S43 |
| 48. |
Moxon, E.
1986.
The carrier state: Haemophilus influenzae.
J. Antimicrob. Chemother.
18(Suppl. A):17-24 |
| 49. | Murphy, T., and S. Sethi. 1992. Bacterial infection in chronic obstructive pulmonary disease. Am. Rev. Respir. Dis. 146:1067-1083[Medline]. |
| 50. | Nakamura, H., K. Yoshimura, N. G. McElvaney, and R. G. Crystal. 1992. Neutrophil elastase in respiratory epithelial lining fluid of individuals with cystic fibrosis induces interleukin-8 gene expression in a human bronchial epithelial cell line. J. Clin. Investig. 89:1478-1484. |
| 51. |
Nichols, W.,
C. R. H. Raetz,
T. Clementz,
A. L. Smith,
J. A. Hanson,
M. R. Ketterer,
M. Sunshine, and M. A. Apicella.
1997.
htrB of Haemophilus influenzae: determination of biochemical activity and effects on virulence and lipooligosaccharide toxicity.
J. Endotoxin Res.
4:163-172 |
| 52. |
Nixon, L. S.,
B. Yung,
S. C. Bell,
J. S. Elborn, and D. J. Shale.
1998.
Circulating immunoreactive interleukin-6 in cystic fibrosis.
Am. J. Respir. Crit. Care Med.
157:1764-1769 |
| 53. | Noah, T. L., H. R. Black, P.-W. Cheng, R. E. Wood, and M. W. Leigh. 1997. Nasal and bronchoalveolar lavage fluid cytokines in early cystic fibrosis. J. Infect. Dis. 175:638-647[Medline]. |
| 54. | Oishi, K., B. Sar, A. Wada, Y. Hidaka, S. Matsumoto, H. Amano, F. Sonoda, S. Kobayashi, T. Hirayama, T. Nagatake, and K. Matsushima. 1997. Nitrite reductase from Pseudomonas aeruginosa induces inflammatory cytokines in cultured respiratory cells. Infect. Immun. 65:2648-2655[Abstract]. |
| 55. | Ophir, D., T. Hahn, A. Schattner, D. Wallach, and A. Aviel. 1988. Tumor necrosis factor in middle ear effusions. Arch. Otolaryngol. Head Neck Surg. 114:1256-1258. |
| 56. | Palfreyman, R. W., M. L. Watson, C. Eden, and A. W. Smith. 1997. Induction of biologically active interleukin-8 from lung epithelial cells by Burkholderia (Pseudomonas) cepacia products. Infect. Immun. 65:617-622[Abstract]. |
| 57. | Pastore, F. D., S. E. Schlegel-Haueter, D. C. Belli, T. Rochat, T. S. Dudez, and S. Suter. 1998. Chemotactic factors in bronchial secretions of cystic fibrosis patients. J. Infect. Dis. 177:1413-1417[Medline]. |
| 58. | Pier, G. 1998. Pseudomonas aeruginosa: a key problem in cystic fibrosis. ASM News 64:339-347. |
| 59. | Rao, V. K., G. P. Krasan, D. R. Hendrixson, S. Dawid, and J. W. St. Geme, III. 1999. Molecular determinants of the pathogenesis of disease due to non-typable Haemophilus influenzae. FEMS Microbiol. Rev. 23:99-129[CrossRef][Medline]. |
| 60. | Read, R., R. Wilson, A. Rutman, V. Lund, H. Todd, and P. Brain. 1991. Interaction of nontypable Haemophilus influenzae with human respiratory mucosa in vitro. J. Infect. Dis. 163:549-558[Medline]. |
| 61. | Rochford, R., M. J. Cannon, R. E. Sabbe, K. Adusumilli, G. Picchio, J. M. Glynn, D. J. Noonan, D. E. Mosier, and M. V. Hobbs. 1997. Common and idiosyncratic patterns of cytokine gene expression by Epstein-Barr virus transformed human B cell lines. Viral Immunol. 4:183-195. |
| 62. | Ruef, C., D. Jefferson, S. Schlegel-Haueter, and S. Suter. 1993. Regulation of cytokine secretion by cystic fibrosis airway epithelial cells. Eur. Respir. J. 6:1429-1436[Abstract]. |
| 63. | Sar, B., K. Oishi, K. Matsushima, and T. Nagatake. 1999. Induction of interleukin 8 (IL-8) production by Pseudomonas nitrite reductase in human alveolar macrophages and epithelial cells. Microbiol. Immunol. 43:409-417[Medline]. |
| 64. | Standiford, T. J., S. L. Kunkel, M. A. Basha, S. W. Chensue, J. P. Lynch III, G. B. Toews, J. Westwick, and R. M. Strieter. 1990. Interleukin-8 gene expression by a pulmonary epithelial cell line. J. Clin. Investig. 86:1945-1953. |
| 65. | St. Geme, J. W., III. 1993. Nontypeable Haemophilus influenzae disease: epidemiology, pathogenesis, and prospects for prevention. Infect. Agents Dis. 2:1-16[Medline]. |
| 66. | St. Geme, J. W., III, M. L. de la Morena, and S. Falkow. 1994. A Haemophilus influenzae IgA protease-like protein promotes intimate interaction with human epithelial cells. Mol. Microbiol. 14:217-233[CrossRef][Medline]. |
| 67. |
St. Geme, J. W., III,
S. Falkow, and S. J. Barenkamp.
1993.
High-molecular-weight proteins of nontypable Haemophilus influenzae mediate attachment to human epithelial cells.
Proc. Natl. Acad. Sci. USA
90:2875-2879 |
| 68. | Storgaard, M., K. Larsen, S. Blegvad, H. Nodgaard, T. Ovesen, P. L. Andersen, and N. Obel. 1997. Interleukin-8 and chemotactic activity of middle ear effusions. J. Infect. Dis. 175:474-477[Medline]. |
| 69. | Svanborg, C., G. Godaly, and M. Hedlund. 1999. Cytokine responses during mucosal infections: role in disease pathogenesis and host defence. Curr. Opin. Microbiol. 2:99-105[CrossRef][Medline]. |
| 70. | Tang, H. B., E. DiMango, R. Bryan, M. Gambello, B. H. Iglewski, J. B. Goldberg, and A. Prince. 1996. Contribution of specific Pseudomonas aeruginosa virulence factors to pathogenesis of pneumonia in a neonatal mouse model of infection. Infect. Immun. 64:37-43[Abstract]. |
| 71. |
Tapping, R. I.,
S. L. Orr,
E. M. Lawson,
K. Soldau, and P. S. Tobias.
1999.
Membrane-anchored forms of lipopolysaccharide (LPS)-binding protein do not mediate cellular responses to LPS independently of CD14.
J. Immunol.
162:5483-5489 |
| 72. | Vieira, J., and J. Messing. 1982. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene 19:259-268[CrossRef][Medline]. |
| 73. | Westphal, O., and K. Jann. 1965. Bacterial lipopolysaccharide extraction with phenol:water and further application of the procedure. Methods Carbohydr. Chem. 5:83-91. |
| 74. |
Wilson, M.,
R. Seymour, and B. Henderson.
1998.
Bacterial perturbation of cytokine networks.
Infect. Immun.
66:2401-2409 |
| 75. | Wilson, R., R. B. Dowling, and A. D. Jackson. 1996. The biology of bacterial colonization and invasion of the respiratory mucosa. Eur. Respir. J. 9:1523-1530[Abstract]. |
| 76. | Wilson, R., R. B. Dowling, and A. D. Jackson. 1996. The effects of bacterial products on airway cells and their function. Am. J. Respir. Crit. Care Med. 154:S197-S201. |
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Tong, H. H., Chen, Y., James, M., Van Deusen, J., Welling, D. B., DeMaria, T. F.
(2001). Expression of Cytokine and Chemokine Genes by Human Middle Ear Epithelial Cells Induced by Formalin-Killed Haemophilus influenzae or Its Lipooligosaccharide htrB and rfaD Mutants. Infect. Immun.
69: 3678-3684
[Abstract] [Full Text] -
Wang, B., Lim, D. J., Han, J., Kim, Y. S., Basbaum, C. B., Li, J.-D.
(2002). Novel Cytoplasmic Proteins of Nontypeable Haemophilus influenzae Up-regulate Human MUC5AC Mucin Transcription via a Positive p38 Mitogen-activated Protein Kinase Pathway and a Negative Phosphoinositide 3-Kinase-Akt Pathway. J. Biol. Chem.
277: 949-957
[Abstract] [Full Text]
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