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B-Dependent Network1
Department of Infectious Diseases, Imperial College of Science Technology and Medicine, Hammersmith Campus, London, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-induced transcripts. CoMTB stimulated nuclear binding of p65,
p50, and c-Rel subunits of NF-
B to IL-8 promoter sequences.
Transient transfections with IL-8 promoter reporter constructs showed
NF-B binding-site mutations abolished IL-8 promoter activity while
NF-IL-6 binding-site mutations decreased promoter activity to 50.2
± 6.3% of wild-type activity. IL-1R antagonist but not neutralizing
anti-TNF- inhibited epithelial cell IL-8 secretion, mRNA
accumulation, and NF-B binding. Recombinant IL-1ß (2 ng/ml)
induced similar levels of IL-8 secretion to CoMTB in both A549 cells
and NHBE. Pulmonary epithelial cells are a major source of IL-8 in the
initial host response to pulmonary tuberculosis. Such IL-8 secretion is
NF-B dependent, NF-IL-6 requiring, and activated by an IL-1-mediated
pathway as a consequence of phagocytosis of M.
tuberculosis by monocytes. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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3 million deaths annually worldwide (1). The
histological hallmark of the immune response to pulmonary tuberculosis
is the formation of lung granulomas consisting of monocyte-derived
cells and Ag-specific T lymphocytes. A prerequisite for granuloma
formation is the recruitment of such cells to areas of infection.
Chemokines have a pivotal role in controlling cellular influx into
sites of infection (2). IL-8, first isolated from
monocytes as a neutrophil attractant (3, 4, 5), is a CXC
chemokine that is also chemotactic for T lymphocytes (6)
and possibly monocytes (7). IL-8 recruits T lymphocytes
stimulated with the purified protein derivative of M.
tuberculosis (8) and critically was absolutely
required for granuloma formation during in vivo mycobacterial challenge
in rabbits (9). IL-8 has been found during human infection
with M. tuberculosis in bronchoalveolar lavage fluid
(BALF)3
(10, 11, 12), tuberculous lymph nodes (13), and
in plasma, where IL-8 concentrations were higher in patients who died
from tuberculosis than in survivors (14).
The pulmonary epithelium, covering a vast surface area of 70
m2 (15), is a first line of defense
to inhaled pathogens. The type II alveolar epithelial cell is known to
contribute indirectly to immune responses during tuberculosis by its
product surfactant protein A, which enhances macrophage phagocytosis of
M. tuberculosis (16). Epithelial cells may also
contribute directly to immune responses by secreting chemokines.
Epithelial cells have been shown to secrete IL-8 and other chemokines
following infection by pathogenic respiratory viruses (17, 18), bacteria (19, 20), and after stimulation with
proinflammatory cytokines (21, 22, 23, 24). The cellular sources
of the high IL-8 concentrations found in BALF of patients with
pulmonary tuberculosis, and the mechanisms regulating this IL-8
release, have not been dissected. Alveolar macrophages phagocytosing
M. tuberculosis are one source of IL-8 (11, 12, 25), but there are only few macrophages per alveolus. Virulent
M. tuberculosis, strain H37-Rv, invades and replicates
within alveolar epithelial cells at a low multiplicity of infection
(MOI) of 1–10 (1–10 organisms per epithelial cell) (26, 27) and at a higher MOI of 10–20 after culture periods of over
4 days may be cytotoxic for epithelium (28). In addition,
M. tuberculosis has recently been shown to directly
stimulate pulmonary epithelial A549 cells to secrete a low level of
IL-8 over 1–6 days (29), suggesting pulmonary epithelial
cells are a source of IL-8 detected in BALF during disease.
The consequences of high-level IL-8 secretion during pulmonary tuberculosis will include neutrophil and T cell influx (30). BALF from patients with tuberculosis show dramatic increases in neutrophils, which are proportional to BALF IL-8 protein content (12). In addition, the extent of expression of IL-8 mRNA in tuberculous lymph node granulomas correlates with neutrophil infiltration (13). Neutrophils chemoattracted into the alveolus by IL-8 during pulmonary tuberculosis may be involved in host defense by themselves secreting chemokines (31), by releasing mediators such cathepsin G and azurocidin, which stimulate secondary T cell influx (32), and may also have a direct role in mycobacterial killing (33). Furthermore, IL-8, with its angiogenic properties (34), may be involved in tissue repair. However, an excess of neutrophils may contribute to the extensive lung damage often seen in pulmonary tuberculosis by generation of damaging free radicals (35), proteases, elastases (36), and matrix metalloproteinases (37, 38).
IL-8 secretion has been shown to be dependent on transcriptional
activation of the IL-8 gene in a number of cell types
(39, 40, 41). The IL-8 promoter region contains binding sites
for a number of important transcription factors (42)
including NF-B, which comprises a family of Rel-related proteins
that are normally retained in the cytoplasm bound to the inhibitor
IB (43). Following cellular activation, IB is
phosphorylated (44) on specific serine residues,
ubiquinated, and degraded (45), allowing NF-B to pass
into the nucleus where it can bind to the IL-8 B-binding site. This
binding site is at position -80 to -71 in the IL-8 genome and
adjacent to the NF-IL-6 binding site (42). Cooperative
binding between NF-B and NF-IL-6 has been demonstrated in alveolar
epithelial cells infected with respiratory syncitial virus
(46) and in TNF--stimulated HeLa cells
(40), but did not occur in TNF--stimulated alveolar
epithelial cells (47), suggesting that IL-8 gene
activation is regulated in a cell type- and stimulus-specific
manner.
The first aim of the present study was to determine whether pulmonary
epithelial cells are involved in the secretion of IL-8 as a consequence
of cytokine networks activated by monocyte phagocytosis of M.
tuberculosis. The second aim was to analyze the role of NF-B
and NF-IL-6 in the control of IL-8 secretion by pulmonary epithelial
cells. We demonstrate for the first time that pulmonary epithelial
cells are a major source of IL-8 during the immune response to M.
tuberculosis infection. Pulmonary epithelial cell IL-8 secretion
is NF-B dependent, NF-IL-6 requiring, and dependent on IL-1 secreted
by M. tuberculosis-infected monocytes. In view of the large
number of epithelial cells in the lung, such mechanisms will result in
greater IL-8-dependent cell recruitment than either from phagocytosis
of M. tuberculosis by monocytes or from direct infection of
epithelial cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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DMEM and RPMI 1640 media were obtained from Life Technologies
(Paisley, U.K) and Dubos’ media from Difco (Detroit, MI). Normal human
primary bronchial epithelial cells and bronchial epithelial growth
medium were purchased from Clonetics (San Diego, CA). Recombinant human
TNF-, IL-1R antagonist (IL-1Ra), and neutralizing polyclonal
anti-human TNF- were obtained from Peprotech (Rocky Hill, NJ).
Matched Ab pairs for the IL-8 ELISA were obtained from R&D Systems
(Minneapolis, MN). The protease inhibitors leupeptin, chymostatin,
pepstatin, antipain, bestatin, and Pefabloc SC and the transfection
reagent FuGene were obtained from Boehringer Mannheim (East Sussex,
U.K). Rabbit polyclonal Abs to p52, p65, Rel B, c-fos, and
goat polyclonal Ab to p50 were obtained from Santa Cruz Biotechnology
(Santa Cruz, CA). Serotec (Oxford, U.K.) supplied c-rel
rabbit polyclonal Ab. Rabbit Ig fraction (normal) for negative control
(X0903) was purchased from Dako (Glostrup, Denmark). Ficoll-Paque was
purchased from Pharmacia Biotech (Uppsala, Sweden). Hybond-N was
purchased from Amersham (Little Chalfont, U.K.). Oligonucleotides were
synthesized by Genosys Biotechnologies (Cambridge, U.K) and Oswell
(Southampton, U.K.). Renilla luciferase control reporter vector pRL-TK
and the reporter gene assay kit, Dual-Luciferase Reporter Assay System,
were obtained from Promega (Madison, WI). All other chemical reagents
were obtained from Sigma (Poole, U.K.).
Human cell culture
The human pulmonary type II alveolar epithelial A549 cell line (48) was purchased from European Collection of Animal Cell Cultures (no. 86012804) and maintained in DMEM supplemented with 10% FCS (containing endotoxin levels <0.02ng/ml), 2 mM glutamine, and 10 µg/ml ampicillin, an antibiotic with no significant antimycobacterial activity at this concentration, in a humidified 5% CO2 atmosphere. Epithelial cells were seeded in tissue culture plates 48 h before use. Immediately before experiments, medium was replaced with serum-free DMEM supplemented with 2 mM glutamine and 10 µg/ml ampicillin. As a primary cell correlate, normal human bronchial epithelial cells (NHBE) were used. These primary cells originate from the upper airway epithelium, in contrast to the alveolar epithelial cell line A549, which is derived from type II pneumocytes present in the alveoli. NHBE were maintained and seeded in bronchial epithelial growth medium (according to suppliers instructions).
Human peripheral blood monocytes were prepared from pooled buffy coats obtained from North Thames Blood Transfusion Service (Colindale, U.K). PBMCs were separated out by density gradient centrifugation on Ficoll-Paque, and monocytes were purified by adhesion to tissue culture plastic for 2 h. Monocytes were maintained in RPMI 1640 supplemented with 10% FCS, 2 mM glutamine, and 10 µg/ml ampicillin.
Mycobacterial culture
The virulent strain of M. tuberculosis H37-Rv (obtained from the National Collection of Type Cultures, Colindale, U.K) was used in all experiments. Stocks of these organisms were maintained in Dubos’ medium enriched with albumin Cohn fraction V plus dextrose and sodium chloride. Before use in experiments, 1 ml of a mid-logarithmic-phase mycobacterial suspension was briefly sonicated (20 s) to break up clumps, left to stand for 10 min, and the top 750 µl used. This method was shown to generate single-cell mycobacterial suspensions by using a modified Kinyoun method to stain suspensions. The amount of viable mycobacteria present was quantitated for each experiment by plating in triplicate serial dilutions of the prepared mycobacterial suspension on Middlebrook (7H10) plates (Royal Brompton Hospital, London, UK) and colony counting 4 wk later.
Epithelial cell and monocyte stimulation
A549 cells were grown to confluence on Thermanox cover slips (ICN Biomedicals, Cleveland, OH) in 35-mm petri dishes and exposed to M. tuberculosis (MOI = 1). After 1, 2, 4, 8, and 24 h, monolayers were washed and the coverslips air dried, heat and formaldehyde fixed, and stained using a modified Kinyoun stain. Microscopy was performed to determine the percentage of cells infected with M. tuberculosis by counting the number of epithelial cells associated with mycobacteria. Counts were performed in three high-power fields per slide for each time point, and the experiment performed in triplicate. We confirmed previously published data (26) demonstrating that M. tuberculosis was able to invade A549 cells over 24 h by invasion assays using amikacin to kill extracellular organisms, washing, and then lysing cell monolayers before culturing lysates on 7H10 plates, and colony counting mycobacterium 4 wk later.
Human peripheral blood monocytes were infected with M.
tuberculosis (strain H37-Rv; MOI = 10) for 24 h, and the
conditioned media (CoMTB) were harvested and stored at -70°C.
Conditioned media from unstimulated human monocytes cultured for
24 h (CoMControl) was used as a negative control. Epithelial cells
were exposed to M. tuberculosis (MOI = 1–10), CoMTB,
CoMControl, or to the positive control TNF- (20 ng/ml). The CoMTB
was used at the dilutions 1:10, 1:100, and 1:1000, diluted in
serum-free DMEM. In experiments where IL-1Ra was used, epithelial cells
were preincubated for 2 h at 37°C before stimulation, and in
those using neutralizing anti-TNF- Ab, CoMTB was preincubated
with the Ab for 1 h at 37°C before use. Anti-TNF- (50
µg/ml) neutralizes 10 ng/ml of TNF- (data supplied by
manufacturer). We confirmed that 50 µg/ml anti-TNF- abolished
the ability of 10 ng/ml TNF- to stimulate IL-8 release from A549
cells and to induce nuclear translocation of NF-B in A549 cells.
RNA extraction and Northern blotting
Following aspiration of supernatant at specific time points, epithelial cell monolayers were washed in ice-cold PBS, homogenized in RNA extraction buffer (4 mM guanidine thiocyanate, 25 mM Tris, pH 7.0, 0.5% N-lauroylsarcosine, and 0.1 M 2-ME), and frozen at -70°C. RNA extraction was performed by a guanidium thiocyanate-phenol-chloroform extraction method modified from previously described protocols (25, 49). Aliquots (20 µg) of RNA were run on a denaturing formaldehyde 1% agarose gels, transferred by capillary blotting to Hybond-N, and fixed by exposure to UV light using a UV Stratalinker 1800 (Stratagene, La Jolla, CA).
Blots were prehybridized and then hybridized with
[
-32P]-end-labeled oligonucleotide probes:
IL-8 with a 33-mer probe and ß-actin with a 42-mer probe
(50). ß-actin probing and assessment of 18S/28S
ribosomal RNA (rRNA) bands on ethidium-stained gels were used to ensure
uniform RNA loading. Blots were autoradiographed with intensifying
screens at -70°C for 24–48 h. Autoradiograph images were digitized
(Umax, Power Look II) and analyzed with NIH Image 1.52 (from National
Institutes of Health Research Services Branch, Bethesda, MD). IL-8
signal densitometry was normalized for total RNA loading using
ß-actin mRNA densitometry, except for mRNA decay studies when 18S/28S
rRNA were used. Between probings, blots were stripped by heating for
1 h at 65°C in a solution of 0.005 M Tris-HCl, pH 8.0, 0.0002 M
EDTA, and 0.1x Denhardt’s solution.
Cytokine assay
IL-8 was assayed in cell-culture supernatants by ELISA using matched Ab pairs according to the manufacturer’s instructions. The lower limit of sensitivity of the IL-8 ELISA was 15 pg/ml. IL-8 concentrations are expressed in pg/106 cells as mean ± SEM of three independent experiments.
Preparation of nuclear extracts
At the indicated time points, nuclear extracts from stimulated and control cells were prepared from 107 A549 cells using a modified version of a previously published protocol (51). In brief, cell monolayers were washed with PBS and then cytoplasmic extraction buffer (10 mM Tris-HCl, 60 mM KCL, 1 mM EDTA, 1 mM DTT) containing a mixture of protease inhibitors (leupeptin, E64, chymostatin, pepstatin, antipain, bestatin, and Pefabloc SC) applied for 10 min. Then, 0.15% Nonidet P-40 was added and the cell lysate was centrifuged at 500 x g for 5 min, and the resultant cell nuclei pellet was washed in cytoplasmic extraction buffer, pelleted, and then resuspended in nuclear extract buffer (20 mM Tris-HCl, pH 8.0, 400 mM NaCl, 1.5 mM MgCl2, 1.5 mM EDTA, 25% glycerol, 1 mM DTT, and the above protease inhibitors) and incubated on ice for 10 min. After centrifugation at 500 x g, the soluble nuclear extract was aspirated and stored at -70°C. Protein quantitation was performed spectrophotometrically at 590 mm using the Bradford assay (52) before storage.
EMSA
Nuclear extract (5 µg) was mixed with 0.7 ng
[-32P]-end-labeled double-stranded DNA probe
with sp. act. >1 x 108 cpm/µg in DNA
binding buffer containing 10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1
mM DTT, 0.25 mg/ml BSA, 3 µg poly(dI-dC), and 5% glycerol. The
oligonucleotides used contained either the sequence specific for the
IL-8 NF-B binding site
(-82GTGGAATTTCC-72)
(42) or mutated NF-B binding sequence
(-82GaatAATTTCC-72).
Bound and free probe were resolved on a 4% polyacrylamide gel
containing 50 mM Tris, 0.38 M glycine, and 2 mM EDTA. Gels were dried
and autoradiographed at -70°C overnight with intensifying screens.
Probe binding specificity was demonstrated by competition assays using
up to a 25-fold excess of cold, unlabeled probe specific for the IL-8
NF-B-binding region and by failure to compete out the signal with a
25-fold excess of unlabeled mutated probe. Supershift assays were
performed using 1 µg of Abs to the NF-B subunits p50, p52, p65,
Rel B, and c-rel or to c-fos (a subunit of the
unrelated transcription factor, AP-1). These were added to the binding
mix and incubated for 15 min at room temperature before running on a
4% polyacrylamide gel.
Transfections and luciferase assays
Three promoter-reporter constructs of the 5' flanking region
(-1370 to +82) of the IL-8 gene (a generous gift of Dr. W. Reed,
Environmental Protection Agency, Chapel Hill, NC) were used. One
construct contained wild-type NF-B and NF-IL-6 binding sites (WT),
one a mutated NF-B binding site (
NF-B), and one a mutated
NF-IL-6 binding site (NF-IL-6). These constructs had been inserted
into the firefly luciferase expression plasmid PGL2-basic. Plasmid DNA
was purified from the Escherichia coli host using Qiagen
plasmid kits according to manufacturers instructions (Qiagen,
Chatsworth, CA).
For transfection studies, A549 cells were grown to 50% confluence in 35-mm petri dishes. Cells were then transfected using FuGene 6, a lipid formulation. Then, 5 µg of the required IL-8 promoter-reporter gene was cotransfected with 0.25 µg of a control reporter plasmid PRL-TK, which constitutively expresses low-level Renilla luciferase, and left overnight (16 h). Following stimulation with CoMTB for 2 h, cell extracts were harvested by first washing the monolayer in PBS and then manually scraping the cells from the culture dishes in the presence of passive lysis buffer (Promega, Madison, WI). Cell extracts were stored at -70°C. Firefly and Renilla luciferase activity was later measured in cell extracts using the Dual-Luciferase Reporter Assay System using a luminometer Bio-Orbit 1253 (Labtech International, East Sussex, U.K.). Measurement of Renilla luciferase activity was used to normalize experimental firefly luciferase values and control for variability in transfection efficiency. The WT IL-8 promoter-reporter gene luciferase activity in response to CoMTB stimulation was expressed as maximal luciferase activity (100%). WT IL-8 promoter-reporter gene activity in response to CoMControl, and mutated IL-8 promoter-reporter gene activity in response to CoMTB, were expressed as a percentage of this maximum.
Analysis and presentation of data
Results of ELISAs are expressed as pg/106 cells, means ± SEM of at least three independent experiments. Luciferase assays are expressed as percentage maximal luciferase activity, means ± SEM of at least three independent experiments, each of which were performed in triplicate. Northern blots (together with a computerized, densitometric analysis taking into account total RNA loaded) and EMSA are shown, representative of at least three independent experiments.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The percentage of A549 cells infected with M. tuberculosis (strain H37-Rv, MOI 1), assessed by microscopy of Kinyoun-stained cell preparations, showed that 15.8 ± 1.3% (mean ± SEM) of A549 cells were infected at 1 h, 33.4 ± 1.5% at 2 h, 31.3 ± 0.9% at 4 h, and 60.1 ± 5.4% at 8 h with no significant increase by 24 h. This is consistent with a study demonstrating by invasion assays that 41 ± 4% of A549 cells were invaded by M. tuberculosis strain H37-Rv (MOI = 1) after 2 h incubation (26).
A549 cells were exposed to M. tuberculosis (strain H37-Rv;
MOI = 10), and IL-8 was measured in cell-culture supernatants by
ELISA over a 24-h period. At this MOI, no significant A549 cell
cytotoxicity occurs over a 24-h incubation (28). Increased
IL-8 secretion was detected in cell-culture supernatants after 4 h
and rose to a maximal level of 7720 ± 930
pg/106epithelial cells by 24 h (Fig. 1
A). The positive control
TNF- (20 ng/ml) stimulated IL-8 secretion within 1 h, which
rose to 17,700 ± 5,200 pg/106 cells by
24 h, levels consistent with previous reports (22, 47). M. tuberculosis at a lower, more
pathophysiological MOI also stimulated IL-8 secretion from A549 cells
in a dose-dependent manner. At an MOI of 1 and 0.1, IL-8 concentrations
were 1600 ± 367 pg/106cells and 943 ±
207 pg/106cells, respectively, at 24 h (Fig. 1B). In contrast, the levels of IL-8 secretion from M.
tuberculosis-infected human monocytes were much greater and
reached 184,100 ± 3,100 pg/106 monocytes
after 24 h, levels similar to previous studies (12, 25) (Fig. 1C). Such levels were 25-fold more than
those observed from pulmonary epithelial cells directly infected with
M. tuberculosis.
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To determine whether phagocytosis of M. tuberculosis
may have indirect effects on pulmonary epithelial cell IL-8 secretion,
we used conditioned media, harvested from human peripheral
blood-derived monocytes infected for 24 h with M.
tuberculosis, to stimulate A549 cells. This CoMTB was used to
stimulate A549 cells over a 24-h period at dilutions of 1:10, 1:100,
and 1:1000 in serum-free DMEM and IL-8 measured in cell-culture
supernatants by ELISA. CoMControl was used as the negative control at a
1:10 dilution. Monocyte-derived IL-8 present in the CoMTB is
represented by the baseline IL-8 measured at t = 0.
Taking this baseline into account, A549 cells stimulated with CoMTB, at
a dilution of 1:10, secreted 23,050 ± 1,220
pg/106 cells of IL-8 within 4 h, and
92,635 ± 13,180 pg/106 cells after 24
h incubation (Fig. 2). The CoMTB-induced
IL-8 secretion at 24 h was 12-fold greater than that following
direct exposure of epithelial cells to M. tuberculosis
(92,635 ± 13,180 pg/106 cells vs 7,720
± 930 pg/106 cells) and 5-fold higher than
following stimulation with TNF- (20 ng/ml) (Fig. 1
A).
CoMTB was a potent stimulus, as even a dilution of 1:100 induced
14,630 ± 3,865 pg/106 cells of IL-8 after
8 h, and low level secretion was evident with a 1:1000 dilution of
CoMTB. These data indicate that IL-8 secretion from CoMTB-stimulated
pulmonary epithelial cells is much greater than that following direct
infection of cells with M. tuberculosis and of a similar
order of magnitude to that secreted by M.
tuberculosis-infected human monocytes.
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IL-8 mRNA, assessed by Northern analysis, was not detected in A549
cells stimulated by CoMControl but appeared within 1 h of
stimulation with CoMTB, peaked between 2–4 h, and persisted 24 h
poststimulation (Fig. 3). These kinetics
of IL-8 gene expression are consistent with the IL-8 protein secretion.
There was a clear dose-response effect of CoMTB on IL-8 mRNA
accumulation over the 3 log-fold dilutions investigated. Strong
expression of IL-8 mRNA expression was evident at 24 h and
suggested either continued IL-8 gene transcription or
posttranscriptional stabilization of IL-8 mRNA. Because evidence exists
that a number of cytokines are posttranscriptionally modified to
increase mRNA stability (53), this possibility was
investigated in transcriptional blockade studies. The RNA polymerase II
inhibitor actinomycin D (10 µg/ml) was added to A549 cell cultures
2 h after stimulation by either CoMTB, CoMControl, or TNF- 20
ng/ml, at which time significant IL-8 mRNA accumulation had occurred.
Cells were harvested for RNA extraction and Northern analysis 1, 2, 4,
8, and 24 h later. Nearly 70% of the baseline IL-8 mRNA signal
from CoMTB-stimulated cells was still detectable 24 h after
inhibition of new gene transcription (Fig. 4A). In contrast, in
TNF--stimulated cells IL-8 mRNA decayed rapidly, falling to 30% of
baseline signal within 1 h and was barely detectable 8 h
after the addition of actinomycin D (Fig. 4B).
CoMControl-stimulated cells showed no IL-8 mRNA accumulation both
before or after actinomycin D (Fig. 4C). These results
indicate that CoMTB induces in a dose-dependent manner an early,
specific up-regulation of IL-8 mRNA, which is posttranscriptionally
regulated to cause mRNA stabilization.
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B in CoMTB-stimulated A549 cells
To investigate the mechanisms underlying IL-8 gene activation in
response to CoMTB, we analyzed the role of the transcriptional
regulator NF-B in the control of IL-8 gene expression in pulmonary
epithelial cells. Nuclear extracts prepared from A549 cells stimulated
by CoMTB or CoMControl for 1, 2, 4, 8, and 24 h were analyzed by
EMSA for the presence of NF-B using double-stranded oligonucleotides
corresponding to the B binding site in the IL-8 promoter region.
CoMTB stimulated a high level of NF-B nuclear binding, which was
clearly present at 1 h and maintained at 4 h (Fig. 5A). Such a time course of
NF-B binding was consistent with the kinetics of IL-8 gene
expression. No NF-B activation was seen following exposure to
CoMControl, but NF-B translocation (without NF-IL-6 activation) was
observed in the TNF--stimulated control cells (data not shown and
consistent with published literature (47)). The NF-B
binding due to CoMTB was a specific effect, competed out in a
dose-dependent manner by a 25-fold excess of cold oligonucleotide
probe, but not competed out with a 25-fold cold excess of an
oligonucleotide containing a mutated NF-B binding site (Fig. 5B).
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B subunits involved were p65, p50,
and c-rel (Fig. 5C). There was no supershift
related to either p52 or to Rel B subunits of NF-B, and an
irrelevant Ab to c-fos did not affect binding. Thus CoMTB
activated nuclear translocation of three distinct subunits of NF-B,
including p50 and p65, which form the activating p50/p65
heterodimer.
CoMTB-activation of the IL-8 promoter is dependent on NF-B and
NF-IL-6 binding
Both NF-B and NF-IL-6 have been shown to coregulate IL-8 gene
expression (39, 40, 46), and therefore a promoter-reporter
analysis was undertaken to investigate the effect of mutations of
either of these transcription factor binding sites on the function of
the IL-8 promoter in response to CoMTB stimulation. The three
constructs used included most of the 5' flanking region of the IL-8
gene and contained either the WT NF-B and NF-IL-6 binding sites or a
mutated NF-B with a WT NF-IL-6 binding site or a WT NF-B with a
mutated NF-IL-6 binding site inserted into a firefly luciferase
expression plasmid (Fig. 6A).
A549 cells were transfected with each of the IL-8 promoter-reporter
genes, stimulated for 2 h by CoMControl or CoMTB, and luciferase
activity was measured in cell extracts. NF-B nuclear translocation
had previously been demonstrated by EMSA at 2 h.
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B) that mutations of the NF-B
binding site in the IL-8 promoter caused luciferase activity to fall to
11.0 ± 1.4% of WT promoter activity, levels lower than that
measured from CoMControl-stimulated cells (15.2 ± 3.7%). This
demonstrated that NF-B binding was absolutely necessary for
activation of the IL-8 promoter in response to CoMTB. NF-IL-6 binding
site mutations resulted in a reduction of luciferase activity to
50.2 ± 6.3% of WT promoter activity and suggest that, in
contrast to NF-B, NF-IL-6 is involved in but is not absolutely
required for IL-8 activation in response to CoMTB. Monocyte-derived IL-1 is essential for IL-8 production by CoMTB-stimulated A549 cells
To determine which specific mediators may mediate the effects of
CoMTB on pulmonary epithelial cells, we investigated the role of two
proinflammatory cytokines, TNF- and IL-1, by using neutralizing
polyclonal anti-human TNF- Ab and IL-1Ra, the naturally
occurring competitive inhibitor of IL-1 bioactivity (54).
Both TNF- and IL-1 are known to activate pulmonary epithelial cell
IL-8 secretion (22) and are released by M.
tuberculosis-infected monocytes (55, 56).
TNF- 10 ng/ml stimulated IL-8 secretion from A549 cells, and we
demonstrated that preincubation for 1 h of 10 ng/ml TNF- with
50 µg/ml of human neutralizing anti-TNF-, but not with an
isotype-matched negative control Ig, completely inhibited IL-8
secretion from A549 cells at 24 h (Table I). This confirmed the neutralizing
capability of the Ab and that its bioactivity was in accordance with
the manufacturers data. The initial concentrations of anti-TNF-
of 1, 2, 5, and 10 µg/ml used to neutralize the TNF- bioactivity
in CoMTB did not inhibit IL-8 mRNA accumulation in CoMTB-stimulated
A549 cells (Fig. 7A). The
monocytes that had phagocytosed M. tuberculosis for 24
h secreted 2.9 ng/ml TNF- into CoMTB, levels consistent with
previous reports (25, 57). However, CoMTB was used after
10-fold or greater dilutions, making its final TNF- concentration
<300 pg/ml. This TNF- concentration is much lower than that used as
a positive control, which stimulated 6-fold less IL-8 secretion after
24 h (Fig. 1A) and 60% less IL-8 mRNA accumulation
after 2 h (Fig. 4, A and B) than did CoMTB.
This indicated that the TNF- present in CoMTB was insufficient to
account for the IL-8 secretion.
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B). Following this
observation, the effect of anti-TNF- (50 µg/ml, capable of
neutralizing 10 ng/ml of TNF-) and IL-1Ra (200 ng/ml), both alone
and in combination on IL-8 secretion, mRNA accumulation, and NF-B
nuclear binding to IL-8 B-binding sites, was determined (Fig. 8). IL-1Ra completely blocked IL-8
secretion from CoMTB-stimulated A549 cells to background levels, while
anti-TNF- had no effect (Fig. 8A). IL-1Ra treatment
of A549 cells decreased IL-8 mRNA accumulation significantly from
93.7 ± 5.5% to 22.8 ± 2.1% (mean normalized
densitometry ± SEM), while anti-TNF- alone had no effect.
In combination (Fig. 8B, lane 5),
anti-TNF- and IL-1Ra decreased IL-8 gene expression to 8.7
± 2.1%, down to levels similar to those observed with CoMControl
(6.3 ± 1.2%). The effect of IL-1Ra and anti-TNF- on
NF-B nuclear binding was similar, with IL-1Ra almost completely
inhibiting binding activity (Fig. 8C). These data
demonstrate that IL-1 is the essential constituent of CoMTB, which
mediates NF-B-dependent IL-8 gene expression and secretion in
pulmonary epithelial cells.
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It was important to confirm that the IL-8 secretion from the cell
line A549 was reproducible in primary lung epithelial cells. NHBE are a
readily available source of such cells and are widely used as a primary
cell correlate (54, 58, 59). NHBE originate from the upper
airway epithelium, in contrast to the type II alveolar epithelial cell
line A549, which is derived from type II pneumocytes found in the
alveoli. Direct infection of NHBE by M. tuberculosis
(MOI = 10) did not stimulate IL-8 secretion (Fig. 9A), in contrast to direct
infection of the A549 cell line. However, CoMTB at a dilution of 1:10
stimulated significant IL-8 secretion from the primary epithelial cells
(Fig. 9B). CoMControl resulted only in low-level
constitutive IL-8 secretion from NHBE. Taking baseline monocyte-derived
IL-8 secretion into account, NHBE secreted 2798 ± 796
pg/106 cells of IL-8 at 8 h and 13,416
± 3,529 pg/106 cells after 24 h stimulation
by CoMTB. We also confirmed in primary cells that IL-1Ra blocked the
ability of CoMTB to stimulate IL-8 secretion, while anti-TNF-
had no inhibitory effect (Fig. 9C). These results show that
CoMTB but not direct infection stimulates IL-8 secretion from pulmonary
epithelial cells, an effect that is IL-1 mediated.
|
Because IL-1Ra inhibited IL-8 secretion from epithelial cells
stimulated by CoMTB, we investigated whether recombinant IL-1ß
stimulated IL-8 secretion from A549 cells and NHBE. IL-1ß 2 ng/ml
stimulated A549 cells to secrete 75,917 ± 3,444 pg
IL-8/106 cells after 24 h, which was
inhibited by preincubation with a 100-fold excess IL-1Ra (Fig. 10A). Similar findings were
observed in IL-1ß-stimulated NHBE, which secreted 15,354 ±
4,564 pg IL-8/106 cells at 24 h (Fig. 10B), concentrations similar to those secreted by NHBE
stimulated with CoMTB. Effects of IL-1ß on NHBE were inhibited by
IL-1Ra. These data confirm a central role for IL-1 as the active
constituent of CoMTB. However, blocking the activity of any autologous
IL-1 by IL-1Ra did not alter IL-8 secretion from A549 cells directly
infected by M. tuberculosis, indicating that IL-1 is not
implicated in this aspect of IL-8 secretion (Fig. 10C).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Alveolar macrophages and monocytes, recruited early in the course of pulmonary infection with M. tuberculosis, have previously been considered the major cellular source of immunoregulatory and proinflammatory mediators, including chemokines, in the lung (12, 25, 61, 62). However, our data suggest that although phagocytosis of M. tuberculosis by monocytes and macrophages may initiate host immune defenses by cytokine production, pulmonary epithelial cells subsequently have a pivotal role. Such epithelial IL-8 secretion is likely to be an early feature of the immune response to M. tuberculosis because IL-8 mRNA accumulation was detectable within 1 h. Furthermore, CoMTB caused significant IL-8 gene expression and secretion from pulmonary epithelial cells even at large dilutions. Thus even in the earliest stages of infection, when relatively few monocytic cells will have phagocytosed M. tuberculosis, there will be a significant release of IL-8 from the alveolar epithelium, stimulating early recruitment of neutrophils and T lymphocytes. The vast surface area of the pulmonary epithelium suggests that this is likely to be a major cellular source of IL-8 in host defense in pulmonary tuberculosis.
We observed that direct infection of A549 cells with M.
tuberculosis stimulated A549 cells to secrete low IL-8
concentrations within 8 h. These kinetics of IL-8 secretion were
consistent with the time required for M. tuberculosis
infection to become established within A549 cells, which we and others
have found to occur within 4–8 h (26). IL-8 secretion
from infected A549 cells varied with MOI, and was detected even at a
low pathophysiological MOI. IL-8 secretion by epithelial cells is
pathogen specific; for example, respiratory syncytial virus-stimulated
IL-8 release from alveolar epithelial cells occurs rapidly, within
2 h of infection, and in high concentrations (17).
Our results with A549 cells are in keeping with a recent study that
reported that M. tuberculosis was able to directly stimulate
low-level secretion of both IL-8 and monocyte chemoattractant protein-1
but not of macrophage inflammatory protein-1 ß or RANTES in
pulmonary epithelial cells over a period of 1–6 days
(29). However, when we investigated IL-8 secretion from
primary pulmonary epithelial cells directly infected with M.
tuberculosis, we found no release of this chemokine. Therefore,
direct exposure of the alveolar epithelium to virulent mycobacteria
does not appear to be a significant source of IL-8 in pulmonary
tuberculosis.
Epithelial cell IL-8 mRNA accumulation activated by CoMTB was not only
rapid but stable over a 24-h period. Almost 70% of IL-8 mRNA persisted
24 h after inhibition of new gene transcription by actinomycin D.
This was a specific response to CoMTB because we and others have found
that <25% of IL-8 mRNA accumulated following TNF- stimulation of
A549 cells remains detectable by Northern analysis 4 h after
blocking transcription with actinomycin D (60).
Posttranscriptional regulatory mechanisms that increase mRNA stability
are critical in regulating gene expression of a number of cytokines
(53). Mechanisms mediating mRNA stability involve
conserved AU-rich sequences within the 3' untranslated region of the
gene (63, 64) and such AU-rich regions are found in IL-8
mRNA. It is possible that persistent mRNA is a characteristic finding
of chronic infection such as tuberculosis. CoMTB-stimulated pulmonary
epithelial cells are probably not the only source of persistent IL-8
secretion because human monocytic cells phagocytosing M.
tuberculosis secreted high IL-8 concentrations for 5 days
(25).
IL-8 gene activation in response to CoMTB stimulation of alveolar
epithelial cells was regulated by NF-B. This transcription factor
exists in the cytoplasm as a family of at least five
rel-related subunits; p65, p50, p52, c-rel, and
Rel B, which have varied stimulatory and inhibitory effects on promoter
regions of different genes. The commonest activating heterodimer of
NF-B is comprised of p50/p65 (43), and we have
previously demonstrated that it is this heterodimer that is activated
in pulmonary epithelial cells after infection with replicating
respiratory syncytial virus (59). In the present study,
the kinetics of NF-B activation in CoMTB-stimulated epithelial cells
showed nuclear translocation over a 4-h period, consistent with the
kinetics of IL-8 mRNA accumulation. Studies of the transcriptional
control of the IL-8 gene suggest that gene activation is differentially
regulated by various combinations of NF-B subunits in a cell type-
and stimulus-specific fashion (41, 47, 65) and often
involves cooperative interactions with other transcription factors
(40, 66). In PMA-treated Jurkat T cells, p65 was the major
subunit involved in regulation of the IL-8 promoter, and the subunit
p50 was shown not to be involved (41). This may reflect an
isolated interaction of the p65 subunit with B-binding sites, or
that p65 may be able to interact with other transcription factors. In
contrast, in TNF--stimulated HeLa cells, p65 and to a lesser extent
c-Rel, p50, and p52 subunits were all activated to regulate the IL-8
promoter (40). Recently, TNF--stimulated alveolar
epithelial cells were shown to induce p65 binding by supershift assay,
and p50 and c-Rel binding were only demonstrable by a highly sensitive
two-step microaffinity isolation/Western immunoblot DNA binding assay
(47). Our study showed a similar pattern of NF-B
subunit translocation to the nucleus, but p50, c-Rel, and p65 subunit
binding were all readily detectable on supershift analysis. The
transient transfection assays we performed confirmed NF-B to be
essential for activation of the IL-8 promoter in response to CoMTB as
mutation of the NF-B binding site abolished promoter-reporter gene
activation. NF-IL-6 and NF-B have been shown to exhibit synergistic
and cooperative binding during regulation of the IL-8 gene (40, 46). We show that NF-IL-6 binding site mutation in the IL-8
promoter-reporter gene caused luciferase activity to fall to 50.2
± 6.3% of WT in response to CoMTB. Therefore, NF-IL-6 contributes to
but is not essential for IL-8 gene activation in this setting. These
results are in contrast to TNF--stimulated A549 cells, where no role
for NF-IL-6 was found (47), and demonstrate stimulus
specificity in the mechanism of activation of IL-8 gene expression in
pulmonary epithelial cells.
Monocytes phagocytosing M. tuberculosis secrete both IL-1ß
and TNF- (55, 56, 57), and both these cytokines are
increased in BALF of patients with tuberculosis (67). We
demonstrated that in CoMTB-mediated IL-8 secretion from epithelial
cells, monocyte-derived IL-1 was absolutely necessary, whereas TNF-
was not. In contrast, in pulmonary networks activated by conditioned
media from LPS-stimulated macrophages, IL-8 gene expression from A549
cells was reduced by 28 ± 7% with anti-TNF- Ab, by
44 ± 8% with anti IL-1ß Ab, and by 83 ± 4% using both
Abs together (22). Thus LPS and M. tuberculosis
stimulate differing profiles of proinflammatory cytokine secretion from
monocytic cells resulting in stimulus specificity for the effects of
conditioned media on IL-8 secretion from epithelial cells. IL-1ß has
been shown to induce IL-8 secretion from A549 cells within 4 h,
increasing over 24 h of stimulation (22). We
confirmed IL-1 to be the critical mediator of CoMTB by demonstrating
that IL-1Ra abolished IL-8 secretion from A549 cells and NHBE, and
inhibited IL-8 mRNA accumulation and nuclear binding of NF-B. We
also showed that recombinant IL-1ß could induce the same levels of
IL-8 from both A549 and NHBE as CoMTB. NHBE secreted less IL-8
following the CoMTB stimulus than did A549 cells. Such reduced
responses of primary epithelial cells compared with cell lines have
been noted previously in response to TNF- (60). In
summary, IL-1 was absolutely required for the NF-B-mediated IL-8
secretion from epithelial cells exposed to CoMTB, although blocking
autologous IL-1 secretion did not decrease IL-8 secretion from directly
infected A549 cells. The upstream events leading to NF-B induction
by IL-1ß are poorly understood, but may involve distinct signal
transduction pathways in epithelial cells compared with other cell
types (68).
This study has defined an important early role for pulmonary epithelial
cells (which are present in large numbers in vivo) in the amplification
of NF-B-dependent, NF-IL-6-requiring IL-8 gene expression and
secretion in response to cytokine networks established during infection
with M. tuberculosis. Similar amplification cascades may
also be important for other chemokines, such as RANTES, that have a
central role in host defense to tuberculosis (69).
Specifically, increased IL-8 secretion due to intercellular networks
will result in neutrophil and T lymphocyte influx. Excess neutrophilic
infiltration, a feature of many destructive lung diseases
(70), may contribute to the debilitating loss of lung
architecture often seen in pulmonary tuberculosis. This study
implicates IL-1 as a critical mediator in immune response to pulmonary
tuberculosis by its ability to induce IL-8 release from alveolar
epithelial cells. Inhibiting the bioactivity of IL-1 effectively
reduced IL-8 protein release, steady-state IL-8 mRNA accumulation, and
nuclear translocation and binding to B sites in the promoter of the
IL-8 gene, suggesting potential future areas to target
immunotherapeutic interventions.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Jon S. Friedland, Department of Infectious Diseases, Imperial College, Hammersmith Hospital, Du Cane Road, London, W12 0NN, United Kingdom. E-mail address:
3 Abbreviations used in this paper: AP-1, activator protein 1; CoMControl, conditioned media from unstimulated human monocytes; CoMTB, conditioned media from M. tuberculosis-infected human monocytes; IL-1Ra, IL-1 receptor antagonist; MOI, multiplicity of infection; NHBE normal human bronchial epithelial cell; rRNA, ribosomal RNA; WT, wild type; BALF, bronchoalveolar lavage fluid.
Received for publication January 12, 1999. Accepted for publication July 21, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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B and NF-IL-6. J. Infect. Dis. 174:262.[Medline]
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