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Infectious Disease Unit, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA 02129;
Meakins Christie Laboratories, Montreal Chest Institute Research Centre, McGill University, Montreal, Quebec, Canada; and
Department of Molecular Sciences, Pfizer Inc., Groton, CT 06340
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(Mig), and IFN-inducible T-cell 
-chemoattractant (I-TAC),
which specifically chemoattract activated T cells by signaling through
the chemokine receptor CXCR3, were inducible in respiratory epithelial
cells. The effects of proinflammatory cytokines, including IFN-
(Th1-type cytokine), Th2-type cytokines (IL-4, IL-10, and IL-13), and
dexamethasone were studied in normal human bronchial epithelial cells
(NHBEC) and in two human respiratory epithelial cell lines, A549 and
BEAS-2B. We found that IFN-, but not TNF- or IL-1ß, strongly
induced IP-10, Mig, and I-TAC mRNA accumulation mainly in NHBEC and
that TNF- and IL-1ß synergized with IFN- induction in all three
cell types. High levels of IP-10 protein (>800 ng/ml) were detected in
supernatants of IFN-/TNF--stimulated NHBEC. Neither dexamethasone
nor Th2 cytokines modulated IP-10, Mig, or I-TAC expression. Since
IFN- is up-regulated in tuberculosis (TB), using in situ
hybridization we studied the expression of IP-10 in the airways of TB
patients and found that IP-10 mRNA was expressed in the bronchial
epithelium. In addition, IP-10-positive cells obtained by
bronchoalveolar lavage were significantly increased in TB patients
compared with normal controls. These results show that activated
bronchial epithelium is an important source of IP-10, Mig, and I-TAC,
which may, in pulmonary diseases such as TB (in which IFN- is highly
expressed) play an important role in the recruitment of
activated T cells. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, while Th2 cells principally produce IL-4, IL-5,
IL-10, and IL-13 1 . Th1 cells and IFN- expression are increased in
several pulmonary diseases, including pulmonary tuberculosis
(TB)3, 2 . In TB,
CD4+ T cells are required to provide a state of
delayed-type hypersensitivity to Mycobacterium tuberculosis
3 , and lack of CD4+ T cells, as found in AIDS patients,
favors the development of TB as well as other mycobacteria diseases 4, 5 . Recruitment of T cells into sites of airway inflammation is a multistep process involving adherence to and Migration across the pulmonary endothelium, trafficking through the interstitium, and finally moving into and through the airway epithelium 6, 7 . Airway epithelial cells are likely to play an important role in this process 8, 9 . They can express adhesion molecules, such as ICAM-1 and VCAM-1 10 , facilitating the adherence of lymphocytes to the epithelial surface. Airway epithelial cells can also be induced to express HLA-DR 11 and therefore may present Ag to T cells. In addition, activated bronchial epithelial cells secrete a variety of mediators, including prostaglandins, platelet-activating factor, and proinflammatory cytokines, as well as chemokines important for the process of inflammatory cell recruitment into the lung interstitium and alveolar spaces 8 .
Chemokines are a superfamily of 8- to 10-kDa secreted proteins that
direct the recruitment of leukocytes to sites of inflammation. All but
two of the chemokines belong to the CC (ß-chemokine) or CXC
(-chemokine) family, defined by the primary sequence of the first
two of four invariant cysteine residues 12 . In general, CC chemokines
chemoattract monocytes, eosinophils, basophils, and T cells and signal
through the chemokine receptors CCR1 to CCR9 (reviewed in 12 . The
CXC chemokine family can be divided in two classes based on the
presence or absence of an NH2-terminal ELR sequence
(Glu-Leu-Arg). The ELR-containing CXC chemokines (e.g., IL-8)
chemoattract neutrophils 13 , while the non-ELR CXC chemokines
chemoattract lymphocytes. For example, IFN-inducible protein of 10 kDa
(IP-10), monokine induced by -IFN (Mig), and IFN-inducible T-cell
-chemoattractant (I-TAC) are potent chemoattractants for activated T
lymphocytes 14, 15, 16, 17, 18 , while stromal cell-derived factor-1 is active on
resting T 19 and immature B cells 20 , and B cell-attracting
chemokine-1 is specific for mature B cells 21 . The CXC chemokines
signal through the chemokine receptors CXCR1 to CXCR5.
Several members of the CC chemokine family, including monocyte chemoattractant protein (MCP)-1 22 , MCP-3 23 , MCP-4 24, 25 , RANTES 23 , and eotaxin 26, 27 are expressed by activated bronchial epithelial cells. Likewise, the ELR-containing CXC chemokines IL-8 28 and epithelial cell-derived neutrophil-activating peptide-78 29 have been shown to be inducible in bronchial epithelium. In contrast, non-ELR chemokines have not been demonstrated to be expressed in bronchial epithelium. Since bronchial epithelium is likely to regulate T cell trafficking into the lung, we asked whether human bronchial epithelial cells could express IP-10, Mig, and I-TAC.
IP-10 was initially identified as an abundantly induced mRNA in U937
cells upon IFN- stimulation 30 , and its expression is
predominantly induced by IFN- in endothelial cells, monocytes,
fibroblasts 30 , astrocytes 31 , keratinocytes 32 , and neutrophils
33 . Human Mig, which is 37% identical to IP-10 at the amino acid
level, is expressed in IFN--induced THP-1 cells, PBMCs,
endothelial cells, keratinocytes, and fibroblasts 17, 34 .
I-TAC, a newly identified non-ELR chemokine, is 
40%
identical at the amino acid level to IP-10 and Mig and is expressed by
activated monocytes and astrocytes 18 . Both IP-10, Mig, and I-TAC
chemoattract activated T cells and NK cells, but not resting T cells, B
cells, or neutrophils 14, 15, 16, 17 . IP-10 expression has been found in
various clinical conditions such as psoriasis 35 , tuberculoid leprosy
36 , sarcoidosis 37 , and viral meningitis 38 , as well as in
experimental animal models of autoimmune encephalomyelitis 31 and
nephrosis 39 . Mig was also found in psoriatic lesions by in situ
hybridization 17 . All of these diseases are associated with an
increased expression of IFN- (Th1-type diseases) 1 , which may
induce IP-10 Mig and I-TAC expression in involved tissues.
IP-10, Mig, and I-TAC share a common receptor, CXCR3, which is specific
for these three chemokines 15 . CXCR3 is expressed on peripheral blood
T cells activated in vitro and on a significant fraction of circulating
CD4+ and CD8+ T cells, B cells, and NK cells
40 , but not on monocytes or neutrophils. In addition, most peripheral
CXCR3+ T cells expressed CD45RO+ (memory T
cells) and ß1 integrins 40 , which are implicated in the
binding of lymphocytes to endothelial cells, epithelial cells, and
extracellular matrix 6 . Recent studies revealed that
CXCR3+ T cell clones were predominantly of the Th1 type
41, 42, 43 . In addition, it was recently reported that virtually all
synovial fluid T cells isolated from patients with rheumatoid arthritis
expressed CXCR3 40, 42 . Furthermore, the vast majority of T
lymphocytes found in colonic tissue obtained from patients with
ulcerative colitis were CXCR3+ 40 . These data suggest
that in Th1-type diseases, in which IFN- is up-regulated, IP-10,
Mig, and I-TAC play an important role in the recruitment of activated
CXCR3+ T cells into inflamed tissue.
In this study, we demonstrate that human bronchial epithelial cells can
be induced to express high levels of IP-10, Mig, and I-TAC mRNA and
secrete high levels of IP-10 protein in response to IFN-
stimulation. In addition, we show that IP-10 is expressed in bronchial
epithelial cells and in cells recovered from bronchoalveolar lavage
(BAL) in patients with active pulmonary TB.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BEAS-2B cells and A549 cells were obtained from the American
Type Culture Collection (Manassas, VA) and cell culture reagents
from Cellgro (Mediatech, Herndon, VA). Recombinant human IFN-,
TNF-, IL-4, IL-10, IL-13, IP-10, and Mig were obtained from
PeproTech (Rocky Hill, NJ), and IL-1ß from R&D Systems (Minneapolis,
MN). Normal human bronchial epithelial cells (NHBEC) and their specific
medium were purchased from Clonetics (Walkersville, MD).
Chemicals were obtained from Sigma (St. Louis, MO).
Cell culture
BEAS-2B, an SV-40-transformed human bronchial epithelial
cell line, was cultured in F12/DMEM supplemented with 2 mM
L-glutamine, 100 IU/ml penicillin, 100 µg/ml
streptomycin, 10 mM HEPES, and 10% heat-inactivated FCS (Mediatech)
(referred to as complete F12/DMEM). A549, a human lung adenocarcinoma
cell line with the alveolar type II phenotype, was maintained in
complete F12K in a 5% CO2 atmosphere at 37°C.
NHBEC were maintained in serum-free bronchial epithelial cell
basal medium (Clonetics) supplemented with 50 µg/ml bovine
pituitary extract, 50 ng/ml human epidermal growth factor, 0.5 µg/ml
hydrocortisone, 0.5 µg/ml epinephrine, 10 µg/ml transferrin, 5
µg/ml insulin, 0.1 ng/ml retinoic acid, 6.5 ng/ml triiodothyronine,
50 µg/ml gentamicin, and 50 ng/ml amphotericin-B. BEAS-2B and A549
cells were grown to confluence in 100-mm cell culture petri dishes, and
24 h before activation they were serum starved in their respective
media without FCS. Twelve to 16 h before activation, medium from
90% confluent NHBEC was replaced by BEBM alone. Cells were then
stimulated with IFN-, TNF-, or IL-1ß alone or in combination at
indicated doses and times. In experiments involving cycloheximide (10
µg/ml) 26 ; dexamethasone; and IL-4, IL-13, and IL-10 (20 ng/ml),
the agents were added 1 h before cell stimulation by cytokines.
RNA analysis
Total RNA was extracted from samples using Stat-60 (Tel-Test,
Friendswood, TX). For Northern analysis, 20 µg of total RNA was
electrophoresed on a 1.2% agarose-formaldehyde gel and then capillary
transferred to a GeneScreen membrane (NEN Life Science Products,
Boston, MA). Following overnight prehybridization (50% formamide, 1%
SDS, 4x SSC, 4x Denhardt’s solution, 0.8% glycine, and 0.17 mg/ml
denatured salmon sperm DNA) at 42°C, blots were hybridized at 42°C
in 50% formamide, 10% dextran, 1% SDS, 5x SSC, 1x Denhardt’s
solution, and 0.17 mg/ml denatured salmon sperm DNA with 1 x
106 cpm/ml [-32P]dCTP-radiolabeled
cDNA probe prepared by nick translation. The following fragments were
used as probes: a 1-kb PstI fragment from IP-10 cDNA, a 3-kb
NotI fragment from human Mig cDNA (kindly provided by J.
Farber, National Institutes of Health, Bethesda, MD), a
300-bp BamHI/AvaI fragment from human I-TAC cDNA,
and a 346-bp EcoRI fragment from human IL-8 cDNA (a kind
gift from H. Oettgen, Children’s Hospital, Boston, MA). A
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA probe was
used as a control for RNA loading and to normalize signals obtained
with the different probes. Signal quantitation was determined using a
PhosphorImager (Molecular Imager System, Bio-Rad, Hercules, Ca). The
data shown in all of the figures are representative of at least two
independent experiments.
Generation of anti-human IP-10 (hIP-10) mAbs
Fifty micrograms of histidine-tagged hIP-10 44 in CFA was
injected i.p. into 6-wk-old female BALB/c mice. Mice were injected i.p.
with 25 µg of His-tagged hIP-10 in IFA on days 14 and 21. On
day 25, spleen cells were harvested and fused with P3X63 myeloma cells
using a standard PEG 1500 protocol. Hybridoma cells were cloned by two
rounds of single-cell cloning using medium supplemented with 10%
IL-6-conditioned supernatant from Sp2/IL-6 cells. Direct ELISA, using
recombinant non-histidine-tagged hIP-10 (PeproTech) containing only
amino acids found in the mature IP-10 protein, was used to screen 750
colonies. Ten of 18 initial positive clones survived two rounds of
single-cell cloning. All clones were found to be specific for rhIP-10
when tested by ELISA and dot blot analysis using the following CXC
chemokines: human growth-regulated oncogene (GRO), IL-8, Mig,
neutrophil-activating peptide-2(NAP-2), platelet factor 4 (PF4),
stromal cell-derived factor-1, and murine IP-10. One clone,
D1D2, was chosen for use in Western blots and ELISA. D1D2 mAb was
purified from serum-free supernatants using protein G chromatography
(Pharmacia, Piscataway, NJ).
Western blot analysis of cell supernatants
Unconcentrated cell supernatants and different amounts of rhIP-10 were separated by SDS-PAGE using 12.5% Tris-tricine gel. Proteins were then transferred to a polyvinylidene difluoride membrane (NEN Life Science Products). The membrane was blocked 1 h in 5% dry milk diluted in PBS-0.1% Tween (PBST) at room temperature and incubated with D1D2 mAb (2 µg/ml in 1% BSA) overnight at 4°C. After the membrane was washed in TBST, 1/10,000 dilution of peroxidase-conjugated goat anti-mouse Ig Ab (Kirkegaard & Perry Laboratories, Gaithersburg, MD) was added for 1 h, followed by additional washes and detection solution for chemiluminescence (Amersham Life Science, Arlington Heights, IL).
Sandwich ELISA
D1D2 mAb (100 µl) at 10 µg/ml in PBS was added to wells of a
high binding-efficiency 96-well ELISA plate (Costar, Cambridge, MA) and
incubated overnight at 4°C. The plate was then incubated with
blocking buffer (3% BSA and 3% goat serum in PBS) overnight at 4°C.
After the plate was washed with PBS, 100 µl of standards or
unconcentrated cell supernatants (in triplicates) were added to each
well and incubated for 2 h at 37°C. The plate was washed again
three times with PBST and three times with PBS and incubated with 15
ng/well of affinity-purified rabbit anti-hIP-10 44 for 1 h
at 37°C. Following similar washes, 100 µl of peroxidase-conjugated
goat anti-rabbit Ig Ab (Kirkegaard & Perry Laboratories) diluted
1/10,000 in blocking buffer was added for 1 h at 37°C, followed
by detection solution (Kirkegaard & Perry Laboratories). The plate was
then read at 650 nm for 15 min. The limit of detection of this assay
was 500 pg/ml.
BALs and bronchial biopsies
Seven control individuals (nonsmokers, nonasthmatics, and nonatopics) and seven patients with active TB (prior antituberculous chemotherapy) underwent bronchofibroscopy and BAL as previously described 2 . Bronchial biopsies of the lower bronchus were performed during bronchoscopy.
In situ hybridization
BAL cells were processed as described 2 . Labeled riboprobe was prepared from IP-10 cDNA using 35S-labeled UTP or digoxigenin-UTP. The prehybridization, hybridization, and posthybridization protocols were as previously described 45 . Hybridization signal was visualized using autoradiography or antidigoxigenin. For negative controls, preparations were hybridized with sense probes. The specificity of the hybridization signal was confirmed by treating preparations with ribonuclease (RNase) before hybridization.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Preliminary experiments using A549 and BEAS-2B cells showed that
IP-10 mRNA was not found in unstimulated cells, but was detectable
2 h after and peaked 8 h after stimulation with IFN- (100
ng/ml) or TNF- (10 ng/ml) (data not shown). We therefore used the
8-h time point for studying the effect of different doses and
combinations of IFN-, TNF-, and IL-1ß on IP-10, Mig, and I-TAC
mRNA accumulation (Fig. 1
, A
and B) and compared it with the expression of the
ELR-containing CXC chemokine IL-8. We found that IFN- alone induced
IP-10 in BEAS-2B but not in A549 cells and that the maximal effect was
observed at 100 ng/ml. IL-1ß weakly induced IP-10 in A549 but not in
BEAS-2B cells. Both cell lines expressed IP-10 in response to TNF-
in a dose-dependent manner. Mig and I-TAC expression was barely
detected in BEAS-2B cells treated with IFN- and was not seen in
cells treated with TNF- or IL-1ß. TNF- and IL-1ß strongly
synergized in a dose-dependent manner with IFN- (100 ng/ml) in
inducing the accumulation of IP-10, Mig, and I-TAC mRNA. IP-10 was
induced to higher levels than Mig and I-TAC in both cell lines; this
can be appreciated in Fig. 1A, which shows blots for IP-10
and Mig that were exposed for similar times (25 and 26 h,
respectively) and a blot for I-TAC exposed for 7 days. In contrast,
IL-8 was strongly induced in A549 cells by TNF- or IL-1ß, with
IFN- being only weakly synergistic. We also studied the effect of
the proinflammatory cytokines IL-6 and granulocyte-macrophage CSF,
alone or combined with IFN-. These two cytokines had no effect on
IP-10 or Mig expression (data not shown).
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B). IFN- induced IP-10, Mig, and I-TAC expression
to higher levels than those seen in A549 and BEAS-2B cells. In
addition, only 10 ng/ml of IFN- induced maximal IP-10, Mig, and
I-TAC expression, indicating that NHBEC were more sensitive to IFN-
than the two cell lines. TNF- and IL-1ß, alone or together, did
not induce IP-10, Mig, and I-TAC expression in NHBEC, while
they were potent inducers of IL-8 expression in these cells. Moreover,
TNF- and IL-1ß only slightly synergized with IFN- in the
induction of IP-10, Mig, and I-TAC, suggesting that IFN- alone was
maximally inducing IP-10, Mig, and I-TAC in NHBEC. To determine whether
TNF- could be induced in response to IFN- and therefore have an
autocrine effect, NHBEC were treated with IFN- in the presence of a
neutralizing anti-hTNF- goat polyclonal Ab (5 µg/ml) or goat
IgG control (5 µg/ml). We found that these treatments had no effect
on IP-10 or I-TAC mRNA expression (data not shown). However, as a
control, we found that this concentration of anti-TNF- Ab
completely inhibited the effects of 10 ng/ml TNF- (data not shown).
These data suggest that TNF- did not contribute to the effect of
IFN- on IP-10 or I-TAC induction in NHBEC. When the level of
expression of the four chemokines was compared (Fig. 1B)
according to their respective times of Northern blot exposure (IP-10,
2 h; Mig, 2 h; I-TAC, 10 h; and IL-8, 3 days), the data
show that IP-10 was induced to significantly higher levels than Mig,
I-TAC, or IL-8 in NHBEC. Kinetic studies
The kinetic response of IP-10, Mig, I-TAC, and IL-8 mRNA
accumulation after stimulation with combinations of IFN-, TNF-,
and IL-1ß was also examined. Northern blot analysis of NHBEC showed
that the pattern of IP-10, Mig, and I-TAC expression was very similar
(Fig. 2A). IP-10, Mig, and
I-TAC mRNA accumulation was detected between 1 and 2 h after
cytokines were added, peaked at 8 h, and slowly decreased
thereafter. In contrast, IL-8 expression was bimodal with an early peak
at 1 h followed by a second peak at 16 h (Fig. 2A).
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B. These data revealed minor differences in IP-10 and Mig
expression in BEAS-2B and A549 cells compared with NHBEC. There was a
suggestion of a bimodal expression of IL-8 in BEAS-2B cells; however,
this was not seen in A549 cells. Effect of protein synthesis inhibition
To examine the effect of protein synthesis inhibition on chemokine
mRNA accumulation, cells were pretreated 1 h before cytokine
stimulation with cycloheximide at a concentration (10 µg/ml)
previously shown to inhibit >90% of protein synthesis in A549 cells
26 . As shown in Fig. 3A, in
NHBEC, Mig and I-TAC expression induced by IFN-, IFN-/TNF-,
and IFN-/IL-1ß was significantly inhibited by
cycloheximide. The effect of cycloheximide on IP-10 expression
was weak compared with the effects of Mig and I-TAC and was even absent
in IFN-/TNF--stimulated NHBEC. IP-10, Mig, and I-TAC mRNA was not
detected in untreated or in TNF-- and in IL-1ß-stimulated cells
and was not affected by cycloheximide treatment. The effect of
cycloheximide on IP-10, Mig, and I-TAC induction in BEAS-2B cells was
less pronounced than in NHBEC. However, in A549 cells we observed a
superinduction of IP-10, Mig, and I-TAC mRNA accumulation in response
to IFN-/TNF- stimulation (Fig. 3B). IL-8 expression
was strongly superinduced by cycloheximide in the three cell lines and
in all situations studied (Fig. 3, A and B). As
an example, in A549 cells treated with TNF-, cycloheximide increased
IL-8 expression 6.6-fold.
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Glucocorticoids are potent inhibitors of chemokine expression.
However, treatment of NHBEC stimulated by IFN-, IFN-/TNF-, and
IFN-/IL-1ß with 10 µM dexamethasone (Fig. 4) was associated with no or only a weak
inhibition of IP-10, Mig, and I-TAC expression. This is in contrast to
the clear inhibition of IL-8 mRNA expression (Fig. 4). Data obtained
with A549 and BEAS-2B cells were similar to those obtained with NHBEC
(data not shown), although the effect of dexamethasone on IL-8
expression by these cells was significantly less marked.
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Since IL-4 has been reported to down-regulate IP-10 in monocytes
46 and macrophages 47 , we asked whether IL-4, IL-13, and IL-10
(three Th2-type cytokines) had the capacity to down-regulate IP-10,
Mig, and I-TAC expression in epithelial cells. We found that IP-10,
Mig, I-TAC, and IL-8 expression by NHBEC treated with IFN- or
IFN-/TNF- was not affected by pretreatment with IL-4, IL-13, or
IL-10 (all at 20 ng/ml) (Fig. 5).
However, NHBEC were responsive to IL-4 and IL-13, since IL-8 expression
increased by 156% and 93% in TNF--stimulated cells pretreated with
IL-4 and IL-13, respectively. Experiments performed on IFN-- or
IFN-/TNF--stimulated A549 and BEAS-2B cells pretreated
with IL-4, IL-13, and IL-10 gave results comparable with those
described for NHBEC (data not shown).
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To assess whether IP-10 protein was secreted by human bronchial
epithelial cells, we performed Western blot analysis on unconcentrated
supernatants from A549 cells, BEAS-2B cells, and NHBEC using a newly
generated murine mAb (D1D2) specific for IP-10. We found a significant
amount of IP-10 secreted in cell supernatants, as demonstrated by the
unique band of 10 kDa that coMigrated with rIP-10 (Fig. 6A). Using a sandwich ELISA
developed in our laboratory, we detected high levels of IP-10 in
unconcentrated supernatants. IP-10 levels were detected between 4 and
8 h after activation with IFN-/TNF- (Fig. 6B). At
32 h, the amount of IP-10 in supernatants of A549 cells, BEAS-2B
cells, and NHBEC was 253.1 ± 36.6 (SEM), 558.7 ±
62.9, and 843.8 ± 73.2 ng/ml, respectively. IP-10 levels at 8,
16, and 32 h were significantly higher than at 4 h in all
three cell type supernatants. Moreover, IP-10 levels detected by ELISA
were positively correlated with mRNA levels in experiments in which
stimulated cells were treated with dexamethasone or with IL-4, IL-13,
and IL-10 (data not shown).
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To determine whether IP-10 was expressed in the airways of
patients with pulmonary TB, a Th1-type disease in which IFN- has
been shown to be up-regulated, we compared IP-10 mRNA expression by in
situ hybridization using an IP-10 cRNA probe on BAL cells obtained from
seven patients with active TB and seven normal controls. Quantitation
of BAL data is shown in Table I and
demonstrates a statistically significant increase in the percentage of
IP-10 mRNA-positive cells in BAL from TB patients compared with
controls (p < 0.001). Fig. 7, A and B, shows a
representative in situ hybridization of BAL cells from one TB patient
and one control. Although the type of the positive cells was not
determined, morphologically they appeared to be macrophages. In
situ hybridization was also performed on bronchial biopsies obtained
from patients with active TB and normal controls. The bronchial
epithelium was strongly positive for IP-10 mRNA (Fig. 7D).
No signal was detected using a sense probe as a control (Fig. 7C), confirming the specificity of the hybridization.
Bronchial epithelium obtained from normal volunteers had little or no
IP-10 signal (data not shown).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study, we demonstrate that primary human bronchial
epithelial cells highly express IP-10, and to a lesser extent Mig and
I-TAC, in response to IFN-, but not in response to TNF- or
IL-1ß. In addition, as has been seen in other cell types 33, 49 ,
IL-1ß and TNF- synergized with IFN- in inducing IP-10 and Mig
mRNA expression. Similar synergism was found with I-TAC expression.
However, using the human epithelial cell lines A549 (alveolar type II
phenotype) and BEAS-2B (bronchial phenotype), we found that IFN-, as
well as TNF- and IL-1ß alone, weakly induced IP-10 but not Mig or
I-TAC. This finding may reflect differences between transformed or
tumor cell lines and primary cells.
We also found that significant amounts of IP-10 protein were secreted
from activated bronchial epithelial cells. Greater than 800 and 500
ng/ml of IP-10 protein was detected in unconcentrated supernatants
collected from NHBEC and BEAS-2B cells, respectively, activated for
32 h with IFN- and TNF-. These are the highest levels
reported to date for chemokine secretion from bronchial epithelial
cells. In comparison, in a 24-h period, BEAS-2B treated with IFN-
and TNF- released 20-fold less RANTES 48 , and A549 cells maximally
stimulated with IL-1ß secreted 10-fold less IL-8 28 and 10,000-fold
less eotaxin 26 . IP-10 mRNA accumulation in bronchial epithelial
cells closely reflected levels of secreted IP-10 protein, as previously
shown in endothelial cells 32 , suggesting that in these cells the
IP-10 protein is not stored.
In contrast with IP-10, Mig, and I-TAC, IL-8 expression was weakly
induced by IFN- but was strongly induced by TNF- or IL-1ß,
confirming a previous study 28 . These results show that in bronchial
epithelial cells, IP-10, Mig, and I-TAC are regulated differently from
IL-8. This pattern of differential regulation was also seen when we
explored the effects of cycloheximide, glucocorticoids, and Th2
cytokines on the expression of these four chemokines in bronchial
epithelial cells.
In NHBEC, cycloheximide reduced IP-10, Mig, and I-TAC mRNA
accumulation; however, in BEAS-2B and A549 cells, the effect on IP-10,
Mig, and I-TAC expression was less marked. It was previously found that
cycloheximide did not affect IP-10 expression on IFN--treated U937
cells 30 but increased its expression in IFN--induced
keratinocytes 49 . Therefore, the effect of cycloheximide on IP-10
expression appears to differ according to cell type. Interestingly, the
effect of cycloheximide was more pronounced on I-TAC mRNA accumulation
in NHBEC, suggesting that different pathways Might be involved in
IP-10, Mig, and I-TAC expression, even though they are all induced by
IFN-. In contrast, cycloheximide increased IL-8 mRNA accumulation in
all three cell types, suggesting again that IP-10, Mig, and I-TAC are
regulated differently from IL-8.
Glucocorticoids are used in many inflammatory processes involving the lung, such as asthma and sarcoidosis. Steroids modulate cytokine gene expression and lead to an impairment of T cell function in vivo (reviewed in 50 . They also down-regulate the expression of chemokines, including IL-8 51 , MCP-1 52 , RANTES 48 , eotaxin 26 , and MCP-4 25 . In contrast to IL-8, we found that IP-10, Mig, and I-TAC expression was not diminished by pretreatment of epithelial cells with dexamethasone.
Th2 cells secrete IL-4, IL-5, IL-10, and IL-13 and are associated with
allergic and humoral-type immune responses 1 . Since IL-4
down-regulates IP-10 expression in human monocytes 46 and murine
macrophages 47 , we asked whether IL-4, IL-13, and IL-10 would
similarly modulate IP-10 and Mig expression in bronchial epithelial
cells. We found that none of these cytokines affected IP-10, Mig, or
I-TAC expression induced by IFN- or IFN-/TNF-. Again
in contrast, IL-8 expression was increased by IL-4 and IL-13 in
TNF--treated NHBEC.
In addition to their differences in leukocyte selectivity and cytokine
regulation, IP-10/Mig and IL-8 have opposing effects on endothelial
cells. IP-10 and Mig are antiangiogenic 44, 53, 54, 55, 56 , whereas IL-8 is
proangiogenic 57 . This has led to speculation that the relative
levels of IP-10/Mig and IL-8 may affect neovascularization in the lung
following a given inflammatory process 58 . For example, in idiopathic
pulmonary fibrosis in which neovascularization is prevalent, pulmonary
levels of IP-10 were decreased, while those of IL-8 increased compared
with the levels in normal lung 58 . It is interesting to note that
high levels of TNF- have been demonstrated in the lungs of patients
with idiopathic pulmonary fibrosis 59, 60, 61 . Our finding in bronchial
epithelial cells that TNF- was a much better inducer of IL-8 than
IP-10 or Mig may explain the up-regulation of IL-8 compared with IP-10
and resultant angiogenic environment found in idiopathic pulmonary
fibrosis.
Activated epithelial cells secrete high levels of IP-10 in vitro and
appear to be a major source of IP-10 in vivo. IFN--treated human
keratinocytes secreted more IP-10 than IFN--treated endothelial
cells, monocytes, or fibroblasts 32 . Furthermore, IP-10 was expressed
in keratinocytes in psoriatic lesions, while it was not detected in
normal keratinocytes 35 . Similar observations were reported in fixed
drug eruptions 62 and in cutaneous T-cell lymphomas 63 . IFN- is
up-regulated in these skin disorders 1, 64 and Might be responsible
for keratinocyte activation and IP-10 expression. Our findings extend
these observations to bronchial epithelial cells, which secreted very
high levels of IP-10 following IFN- stimulation. Furthermore, IP-10
was expressed in the bronchial epithelium in patients with active TB,
in which IFN- and IL-12 have been shown to be up-regulated 2 .
IP-10 secretion from epithelial cells may play an important role in
recruiting activated T cells into the epithelium, which is necessary
for protective immunity at these surfaces in contact with the
environment.
IP-10 is expressed in inflamed tissues in which the Th1-type cytokine
IFN- is up-regulated. This has been demonstrated for psoriasis 35 ,
tuberculoid leprosy 36 , ulcerative colitis 65 , sarcoidosis 37 ,
and atherosclerosis (A. Sauty and F. Mach, unpublished observations),
and we now present data for active TB. Interestingly, CXCR3 is found
expressed predominantly on Th1 clones and on a significant fraction of
memory circulating T cells 41, 42 . Recent studies have implicated
CXCR3+ T cells in the pathogenesis of rheumatoid arthritis
42 , ulcerative colitis 40 , and atherosclerosis (A. Sauty and F.
Mach, unpublished observations), since the vast majority of
infiltrating T cells were CXCR3+. In addition, it has
recently been shown that IP-10 selectively activated and enhanced Ag
and mitogen-driven IFN- (but not IL-4) cytokine gene expression in T
cells 66 . Therefore, it is tempting to speculate that in Th1-type
disease, IP-10, Mig, and I-TAC are induced by IFN- and chemoattract
CXCR3+ T cells, which in turn are induced to express more
IFN-, establishing a mechanism for amplifying a Th1-type immune
response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Andrew D. Luster, Infectious Disease Unit, Massachusetts General Hospital East, Building 149, 13th Street, Charlestown, MA 02129. E-mail address:
3 Abbreviations used in this paper: TB, tuberculosis; IP-10, IFN-induced protein of 10 kDa; Mig, monokine induced by IFN-; I-TAC, IFN-inducible T-cell chemoattractant; MCP, monocyte chemoattractant protein; BAL, bronchoalveolar lavage; NHBEC, normal human bronchial epithelial cell; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; h, human.
Received for publication June 17, 1998. Accepted for publication December 7, 1998.
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C. Agostini, F. Calabrese, F. Rea, M. Facco, A. Tosoni, M. Loy, G. Binotto, M. Valente, L. Trentin, and G. Semenzato CXCR3 and Its Ligand CXCL10 Are Expressed by Inflammatory Cells Infiltrating Lung Allografts and Mediate Chemotaxis of T Cells at Sites of Rejection Am. J. Pathol., May 1, 2001; 158(5): 1703 - 1711. [Abstract] [Full Text] [PDF]
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M. Cecilia Berin, L. Eckmann, D. H. Broide, and M. F. Kagnoff Regulated Production of the T Helper 2-Type T-Cell Chemoattractant TARC by Human Bronchial Epithelial Cells In Vitro and in Human Lung Xenografts Am. J. Respir. Cell Mol. Biol., April 1, 2001; 24(4): 382 - 389. [Abstract] [Full Text]
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B. Qiu, K. A. Frait, F. Reich, E. Komuniecki, and S. W. Chensue Chemokine Expression Dynamics in Mycobacterial (Type-1) and Schistosomal (Type-2) Antigen-Elicited Pulmonary Granuloma Formation Am. J. Pathol., April 1, 2001; 158(4): 1503 - 1515. [Abstract] [Full Text] [PDF]
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R. E. Wiley, K. Palmer, B. U. Gajewska, M. R. Stampfli, D. Alvarez, A. J. Coyle, J.-C. Gutierrez-Ramos, and M. Jordana Expression of the Th1 Chemokine IFN-{{gamma}}-Inducible Protein 10 in the Airway Alters Mucosal Allergic Sensitization in Mice J. Immunol., February 15, 2001; 166(4): 2750 - 2759. [Abstract] [Full Text] [PDF]
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J. J. Campbell, C. E. Brightling, F. A. Symon, S. Qin, K. E. Murphy, M. Hodge, D. P. Andrew, L. Wu, E. C. Butcher, and A. J. Wardlaw Expression of Chemokine Receptors by Lung T Cells from Normal and Asthmatic Subjects J. Immunol., February 15, 2001; 166(4): 2842 - 2848. [Abstract] [Full Text] [PDF]
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C. AGOSTINI, M. FACCO, M. SIVIERO, D. CAROLLO, S. GALVAN, A. M. CATTELAN, R. ZAMBELLO, L. TRENTIN, and G. SEMENZATO CXC Chemokines IP-10 and Mig Expression and Direct Migration of Pulmonary CD8+/CXCR3+ T Cells in the Lungs of Patients with HIV Infection and T-Cell Alveolitis Am. J. Respir. Crit. Care Med., October 1, 2000; 162(4): 1466 - 1473. [Abstract] [Full Text] [PDF]
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A. E. Dixon, J. B. Mandac, D. K. Madtes, P. J. Martin, and J. G. Clark Chemokine expression in Th1 cell-induced lung injury: prominence of IFN-gamma -inducible chemokines Am J Physiol Lung Cell Mol Physiol, September 1, 2000; 279(3): L592 - L599. [Abstract] [Full Text] [PDF]
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M. T. Liu, B. P. Chen, P. Oertel, M. J. Buchmeier, D. Armstrong, T. A. Hamilton, and T. E. Lane Cutting Edge: The T Cell Chemoattractant IFN-Inducible Protein 10 Is Essential in Host Defense Against Viral-Induced Neurologic Disease J. Immunol., September 1, 2000; 165(5): 2327 - 2330. [Abstract] [Full Text] [PDF]
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C. Albanesi, C. Scarponi, S. Sebastiani, A. Cavani, M. Federici, O. De Pita, P. Puddu, and G. Girolomoni IL-4 Enhances Keratinocyte Expression of CXCR3 Agonistic Chemokines J. Immunol., August 1, 2000; 165(3): 1395 - 1402. [Abstract] [Full Text] [PDF]
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F. N. Lauw, A. J. H. Simpson, J. M. Prins, S. J. H. van Deventer, W. Chaowagul, N. J. White, and T. van der Poll The CXC Chemokines Gamma Interferon (IFN-gamma )-Inducible Protein 10 and Monokine Induced by IFN-gamma Are Released during Severe Melioidosis Infect. Immun., July 1, 2000; 68(7): 3888 - 3893. [Abstract] [Full Text] [PDF]
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D. P. Widney, Y.-R. Xia, A. J. Lusis, and J. B. Smith The Murine Chemokine CXCL11 (IFN-Inducible T Cell {alpha} Chemoattractant) Is an IFN-{gamma}- and Lipopolysaccharide- Inducible Glucocorticoid-Attenuated Response Gene Expressed in Lung and Other Tissues During Endotoxemia J. Immunol., June 15, 2000; 164(12): 6322 - 6331. [Abstract] [Full Text] [PDF]
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N. Marx, F. Mach, A. Sauty, J. H. Leung, M. N. Sarafi, R. M. Ransohoff, P. Libby, J. Plutzky, and A. D. Luster Peroxisome Proliferator-Activated Receptor-{gamma} Activators Inhibit IFN-{gamma}-Induced Expression of the T Cell-Active CXC Chemokines IP-10, Mig, and I-TAC in Human Endothelial Cells J. Immunol., June 15, 2000; 164(12): 6503 - 6508. [Abstract] [Full Text] [PDF]
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K. Kinoshita, G. Tesch, A. Schwarting, R. Maron, A. H. Sharpe, and V. R. Kelley Costimulation by B7-1 and B7-2 Is Required for Autoimmune Disease in MRL-Faslpr Mice J. Immunol., June 1, 2000; 164(11): 6046 - 6056. [Abstract] [Full Text] [PDF]
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M. M. Mazanet, K. Neote, and C. C. W. Hughes Expression of IFN-Inducible T Cell {alpha} Chemoattractant by Human Endothelial Cells Is Cyclosporin A-Resistant and Promotes T Cell Adhesion: Implications for Cyclosporin A-Resistant Immune Inflammation J. Immunol., May 15, 2000; 164(10): 5383 - 5388. [Abstract] [Full Text] [PDF]
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D. Jones, R. J. Benjamin, A. Shahsafaei, and D. M. Dorfman The chemokine receptor CXCR3 is expressed in a subset of B-cell lymphomas and is a marker of B-cell chronic lymphocytic leukemia Blood, January 15, 2000; 95(2): 627 - 632. [Abstract] [Full Text] [PDF]
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N. A. Lee and J. J. Lee The Macroimportance of the Pulmonary Immune Microenvironment Am. J. Respir. Cell Mol. Biol., September 1, 1999; 21(3): 298 - 302. [Full Text]
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P. Loetscher, A. Pellegrino, J.-H. Gong, I. Mattioli, M. Loetscher, G. Bardi, M. Baggiolini, and I. Clark-Lewis The Ligands of CXC Chemokine Receptor 3, I-TAC, Mig, and IP10, Are Natural Antagonists for CCR3 J. Biol. Chem., January 26, 2001; 276(5): 2986 - 2991. [Abstract] [Full Text] [PDF]
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