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-Inducible Protein 10 (CXCL10) Contributes to Airway Hyperreactivity and Airway Inflammation in a Mouse Model of Asthma1
* Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology,
Pulmonary and Critical Care Unit, and
Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114; and
Division of Allergology and Clinical Immunology, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein 10 (IP-10; CXCL10) is an IFN--inducible
chemokine that preferentially attracts activated Th1 lymphocytes. IP-10
is up-regulated in the airways of asthmatics, but its function in
asthma is unclear. To investigate the role of IP-10 in allergic airway
disease, we examined the expression of IP-10 in a murine model of
asthma and the effects of overexpression and deletion of IP-10 in this
model using IP-10-transgenic and IP-10-deficient mice. Our experiments
demonstrate that IP-10 is up-regulated in the lung after allergen
challenge. Mice that overexpress IP-10 in the lung exhibited
significantly increased airway hyperreactivity, eosinophilia, IL-4
levels, and CD8+ lymphocyte recruitment compared with
wild-type controls. In addition, there was an increase in the
percentage of IL-4-secreting T lymphocytes in the lungs of
IP-10-transgenic mice. In contrast, mice deficient in IP-10
demonstrated the opposite results compared with wild-type controls,
with a significant reduction in these measures of Th2-type allergic
airway inflammation. Our results demonstrate that IP-10, a Th1-type
chemokine, is up-regulated in allergic pulmonary inflammation and that
this contributes to the airway hyperreactivity and Th2-type
inflammation seen in this model of asthma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of chemokines have been shown to be up-regulated in allergic
asthma, and they are believed to be important in the pathogenesis of
this disease (2). Studies in murine models of allergic
airways disease have shown that chemokines are essential for the
initiation and progression of airways inflammation (14).
Chemokines expressed in these models are predominantly those that
attract eosinophils and Th2-type lymphocytes (14). Th2
lymphocytes are preferentially recruited into the lung in murine models
of asthma and are crucial for the development of airway inflammation
(4). The role of chemokines preferentially active on Th1
lymphocytes has not been thoroughly examined in these models.
IFN--inducible protein 10 (IP-10; CXCL10) is a chemokine that
preferentially attracts Th1 lymphocytes through its receptor CXCR3,
which is expressed at high levels on these cells (15, 16, 17, 18).
IP-10 is induced in a variety of cells in response to the Th1 cytokine
IFN- (19, 20). IP-10 expression is most often
associated with Th1-type inflammatory diseases, where it is thought to
play an important role in the recruitment of Th1 lymphocytes into
tissues. However, IP-10 expression has also been shown to be increased
in the airways of asthmatics (21), but its role in
Th2-type inflammatory diseases such as asthma remains unknown.
In our study we used a murine model of allergic airway inflammation to evaluate the role of IP-10 in a Th2-type inflammatory response characteristic of asthma. The experiments performed demonstrate that IP-10 is up-regulated during the inflammatory response seen in this model. We used genetically modified mice that overexpress IP-10 in lung epithelial cells or mice that are deficient in IP-10 expression to show that IP-10 contributes to the inflammatory response in the airways as well as to AHR.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wild-type FVB mice used in these experiments were purchased from
Charles River Breeding Laboratories (Wilmington, MA).
IFN--/- mice (on the BALB/c background) and
BALB/c control mice were purchased from The Jackson Laboratory (Bar
Harbor, ME). IP-10-transgenic mice (in FVB background) and
IP-10-/- mice (in Sv129 background) were
generated in our laboratory (22, 23, 24). Sv129 wild-type mice
were used from our own colony of mice and were the same Sv129 strain
used to generate the IP-10-/- mice. Mice were
used at 6–8 wk of age and were housed in a pathogen-free animal
facility. All animals were fed sterile food and autoclaved water ad
libitum. For each experiment, age- and sex-matched groups of mice
were used.
Immunization and challenge protocol
Mice were injected i.p. with 10 µg OVA (Sigma-Aldrich, St. Louis, MO) and 1 mg aluminum hydroxide suspended in 0.5 ml normal saline on days 0 and 7. Sham-immunized mice received aluminum hydroxide alone. Mice underwent aerosol challenge with OVA (50 mg/ml in normal saline) or normal saline alone on days 14, 14–17, or 14–18. OVA challenge was performed by placing mice in a Plexiglas box (22 x 23 x 14 cm) and aerosolizing OVA using a nebulizer (DeVilbiss, Somerset, PA), driven by compressed air for 20 min. Mice were sacrificed 18–24 h after the last aerosol challenge, except in the kinetics studies where they were sacrificed at the indicated times postchallenge.
Airway responsiveness
AHR was measured noninvasively using a whole-body plethysmograph (Buxco, Sharon, CT). AHR was expressed as the enhanced pause (Penh), a calculated number based on inspiratory and expiratory times and pressures. Penh has been shown to correlate with measurements of airway resistance (25). The average Penh over 3 min was determined after 2-min exposure to aerosolized normal saline as a baseline. The average Penh over 3 min was then determined after exposing the mice for 2 min to aerosolized methacholine (Sigma-Aldrich) at increasing concentrations (3.125, 6.25, 12.5, and 25 mg/ml in normal saline) and was expressed as the percentage of change from the baseline.
Bronchoalveolar lavage
Bronchoalveolar lavage (BAL) was performed at 18–24 h after the last aerosol challenge. Mice were anesthetized with chloral hydrate (400 µg/g). The trachea was exposed and cannulated with polyethylene tubing. The lungs were lavaged with six 0.5-ml aliquots of PBS (Mediatech, Herndon, VA) containing 0.6 mM EDTA. Lavage fluid recovered from the first 1 ml of instilled PBS/EDTA was collected separately from the rest of the BAL. Both BAL fractions were centrifuged at 540 x g at 4°C, and the pelleted cells from both fractions were pooled for analysis. The supernatant of the BAL recovered from the first 1 ml instilled was kept frozen at -80°C for subsequent analysis. The cells from both BAL fractions were exposed for 30 s to Tris (0.014 M)/NH4Cl (0.14 M) to lyse RBC, and the remaining live cells, as determined by trypan blue exclusion, were washed in PBS and enumerated in a hemocytometer. Cell differential counts were determined by enumerating macrophages, neutrophils, eosinophils, and lymphocytes on cytocentrifuge preparations of the cells stained with a combination of Wright stain (EM Sciences, Gibbstown, NJ) and Diff-Quick (Dade Behring, Newark, DE).
Flow cytometry
Cells recovered from the BAL were resuspended in PBS with 1%
BSA (Intergen, Purchase, NY) and incubated for 30 min with 25 µg/ml
2.4G2 anti-FcRIII/II (CD16/CD32) at 4°C. Samples of
106 cells were then stained with
FITC-conjugated anti-murine CD3 mAb and PE-conjugated
anti-murine CD19 mAb in one reaction and with FITC-conjugated
anti-murine CD8 mAb, PE-conjugated anti-murine CD4 mAb, and
allophycocyanin-conjugated anti-murine CD25 mAb in a separate
reaction. All Abs were obtained from BD PharMingen (San Diego, CA).
Cells were washed with PBS and then fixed in 2% paraformaldehyde. Flow
cytometry was performed after gating on the lymphocyte population using
a FACSCalibur analytical flow cytometer (BD Biosciences, Mountain View,
CA) and were analyzed using CellQuest software (BD Biosciences).
Limulus amebocyte lysate assay for LPS
Five percent OVA in normal saline was serially diluted in water. The samples were added to the tubes provided in the Limulus assay kit (Charles River Endosafe, Charleston, SC). The solution was mixed and incubated at 37°C for 1 h and then assessed for coagulation.
Cytokine assays
Levels of IL-4, IL-5, and IFN- in BAL were measured by ELISA
(Endogen, Woburn, MA).
IP-10 ELISA
The level of IP-10 in the serum of IP-10-transgenic and control animals was determined by ELISA. Serial dilutions of a known quantity of IP-10 (standard), serum samples, and serum samples with added IP-10 (to a final concentration of 10 ng/ml) were coated onto 96-well plates overnight. After washing and blocking the plate with 1% BSA, 0.3 µg/ml biotin-labeled Ab to murine IP-10 (Cedarlane Laboratories, Hornby, Canada) was added to each sample. After a 2-h incubation, avidin (Sigma-Aldrich) was added to the samples for 30 min. Finally, ABTS (Sigma-Aldrich) was added, and the samples were incubated for another 20 min. The absorbance was measured at 405 nm, and the concentration of IP-10 in the samples was determined by comparison to the standards. As a positive control for this assay, we were able to detect 10 ng/ml recombinant IP-10 added to serum samples.
Histopathologic examination
Lungs were flushed free of blood by slowly injecting 10 ml PBS into the right ventricle before excision for all studies. The left lung was inflated with 10% buffered formalin to 25 cm H2O pressure and transferred into vials containing 10% buffered formalin. Multiple paraffin-embedded, 5-µm sections of the entire mouse lung were prepared and stained with H&E and periodic acid-Schiff. The slides were evaluated by light microscopy, and the amounts of inflammation and mucous were assessed by a pathologist blinded to the genotype of the animals.
Whole-lung digests and flow cytometry on lung lymphocytes
The right lung was excised and minced into small pieces with a
scissors. The pieces were digested for 1 h in a lysis solution
containing PBS, 10% FCS, 150 U/ml collagenase III (Worthington
Biochemical, Lakewood, NJ), and 850 U/ml hyaluronidase (Sigma-Aldrich).
The digested lungs were then extruded through a mesh strainer, and the
collected cells were washed once with PBS. Live cells were enumerated
by a hemocytometer, as determined by trypan blue exclusion. Cells were
then resuspended in RPMI with 10% FCS to a concentration of
1 x 106 cells/ml. Five 1-ml aliquots per
lung were put into 24-well plates and incubated for 1 h at 37°C
after adding PMA (50 ng/ml; Sigma-Aldrich) and ionomycin (250 ng/ml;
Sigma-Aldrich). Monensin (1 µg; Golgi stop; BD PharMingen) was
added to three of the five wells per lung (Th2 samples), and 1 µg
brefeldin A (Golgi plug; BD PharMingen) was added to the other two
wells (Th1 samples). The cells were incubated for 4 h at 37°C.
The cells were then placed at 4°C overnight. The cells were pelleted
and washed with PBS/1% BSA. The cells were incubated for 30 min with
2.4G2 anti-FcRIII/II at 4°C. The Th2 samples were stained with
FITC-conjugated anti-murine CD3 mAb, and the Th1 samples were
stained with PE-conjugated CD4 mAb for 20 min at 4°C. The samples
were washed with PBS, and 100 µl reagent A (Fix and Perm kit; Caltag
Laboratories, Burlingame, CA) was added to each sample. The cells were
incubated for 20 min at room temperature and were washed twice with
PBS. Reagent B (100 µl; Fix and Perm kit; Caltag Laboratories) was
added to each sample. The Th2 samples were then stained with
PE-conjugated IgG1, PE-conjugated anti-IL-4 mAb, or PE-conjugated
anti-IL-5 mAb. The Th1 samples were stained with either FITC-conjugated
IgG1 or FITC-conjugated anti-IFN- mAb. All Abs were obtained from BD
PharMingen. Samples were incubated for 20 min at 4°C and washed twice
with PBS/1% BSA. The samples were resuspended in 200 µl PBS/1% BSA.
Flow cytometry was performed after gating on the lymphocyte population
using a FACSCalibur analytical flow cytometer and analyzed using
CellQuest software.
Serum IgE and OVA-specific IgE determination
Blood was obtained by right ventricular puncture. The blood was allowed to clot, and serum was removed after centrifuging the sample at the highest speed in a microcentrifuge for 5 min. Total IgE levels were measured by ELISA (BD PharMingen). OVA-specific IgE levels were measured by plating 100 µl 10 µg/ml OVA onto 96-well plates. Samples were incubated on the plates for 2 h, and bound IgE Abs were determined using an ELISA.
Northern blot analysis
Total cellular RNA was isolated from the lungs by homogenizing
the tissue with a Polytron (Brinkmann Instruments, West Orange, NY) in
4 M guanidine hydrochloride and pelleting the RNA through a 5.7 M
CsCl2 cushion. Northern blotting was performed by
fractionating 10 µg total RNA/lane on a 1.2% agarose gel containing
0.7% formaldehyde, transferring the RNA to GeneScreen (DuPont,
Wilmington, DE), and then hybridizing the membrane with
[32P]dCTP Klenow-labeled random-primed mouse
cDNA probes. The following fragments were used as probes: a cDNA from
the murine IP-10 gene (26), a cDNA from the murine
monokine induced by IFN- (Mig; CXCL9) gene (provided by J. Farber,
National Institutes of Health, Bethesda, MD), a cDNA from the murine
IFN-inducible T cell 
-chemoattractant (I-TAC; CXCL11) gene (provided
by G. Werner-Felmayer, University of Innsbruck, Innsbruck, Austria), a
cDNA from the murine eotaxin gene (27), and a 2-kb
ClaI-KpmI fragment of the bovine keratin 5 (BK5)
gene that contains the 3' intron, splice site, and polyadenylation
signal (BK5-I/pa) (22). 
-Actin cDNA was used as a
control for RNA loading. Signal quantitation was determined using a
phosphor imager (Molecular Imager System; Bio-Rad, Hercules,
CA).
Western blot analysis
Total cellular protein was isolated from the lungs by homogenizing the tissue with a Polytron in lysis buffer consisting of 50 mM Tris (pH 7.5), 1 mM EDTA, 1 mM DTT, 150 mM NaCl, 1% Nonidet P-40, 0.5% deoxycholic acid, 0.1% SDS, and 1% Triton X-100 supplemented with anti-proteases (0.1 mg/ml pepstatin A, 0.03 mM leupeptin, 145 mM benzamidine, 0.37 mg/ml aprotinin, and 1 mM PMSF). Cell lysates were incubated on ice for 30 min, and residual tissue was removed by two centrifugations at 16,000 x g at 4°C for 15 min. Total protein concentrations of the samples were determined by bicinchoninic acid assay (Endogen). Two hundred micrograms of total protein was taken, and the final volume was adjusted to 200 µl with 10 mM Tris. The samples were pelleted with a 1-h centrifugation at 55,000 rpm at 4°C. The pellets were resuspended in PBS and sample buffer and then boiled for 5 min. The samples were fractionated on a 12.5% Tris-Tricene gel. Proteins were transferred onto a polyvinylidene difluoride membrane (NEN, Boston, MA) and blocked in PBS containing 0.1% Tween 20 and 5% nonfat dry milk. IP-10 was identified by sequentially incubating the membrane at room temperature with a 1/2,000 dilution of an affinity-purified polyclonal rabbit anti-murine IP-10 Ab (28), followed by a 1/3,000 dilution of an HRP-conjugated goat anti-rabbit Ig Ab (Bio-Rad). The membrane was washed repeatedly in PBS containing 0.1% Tween 20 between incubations and was developed using an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ).
Data analysis
Data are expressed as the mean ± SEM, unless otherwise indicated. Results were interpreted using two-tailed Student’s t test or two-way ANOVA. High and low data values were discarded from each group in the BAL cell counts and flow cytometric analysis from IP-10-/- mice and their wild-type controls to eliminate the effects of extreme outliers. Differences were considered statistically significant at p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are induced in the lung following
OVA sensitization and challenge
We determined the expression of IP-10, Mig, I-TAC, IFN-, and
TNF- mRNA in the lung at baseline and 3, 6, 12, 24, and 48 h
after a single OVA challenge in BALB/c mice (Fig. 1
A). IP-10 expression was
induced at 3, 6, and 12 h after OVA challenge in both mice
immunized with OVA and sham-immunized mice. This induction was greater
and more sustained in OVA-immunized and -challenged mice. Mig, I-TAC,
and IFN- were induced to a greater extent in OVA-immunized and
-challenged mice, but to a lesser degree than IP-10 (almost 10-fold
less expression). TNF- was induced to the same extent after OVA
challenge in both sham- and OVA-immunized animals. The induction of
TNF-, IP-10, Mig, I-TAC, and IFN- in sham-immunized,
OVA-challenged animals is believed to be secondary to LPS contamination
of the OVA solution. A representative Northern blot of lung RNA
harvested 24 h after the last of four OVA challenges demonstrates
increased expression of IP-10, Mig, and I-TAC in mice immunized with
OVA compared with sham-immunized animals (Fig. 1B).
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Northern blot analysis of lung mRNA from wild-type and
IFN--/- mice after four OVA challenges
demonstrates attenuated up-regulation of IP-10 expression in
OVA-immunized IFN--/- compared with
wild-type animals (Fig. 2). Mig
expression was undetectable in IFN--/- mice
compared with wild-type mice 24 h after OVA challenge.
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Transgenic mice were engineered by our laboratory to overexpress
IP-10 in epithelial cells, including cells in the lung, using the BK5
promoter (22). IP-10 protein was not detected in the serum
of transgenic and wild-type mice using an ELISA that was sensitive to 3
ng/ml. However, Western blot analysis revealed the presence of IP-10 in
the serum at 1 ng/ml in the transgenic mice. Northern blot analysis
was performed on lung mRNA from wild-type and IP-10-transgenic mice
after four OVA challenges (Fig. 3A). IP-10 expression was
up-regulated in the OVA-immunized wild-type mice as well as the sham-
and OVA-immunized IP-10-transgenic mice. All IP-10-transgenic mice had
greater overall expression of IP-10 than wild-type mice. In contrast,
Mig and eotaxin expression was up-regulated only in OVA-immunized mice.
Expression of the IP-10 transgene, which contains a portion of the BK5
gene, was detected with a BK5-specific probe only in
IP-10-transgenic mice.
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B). IP-10 expression was seen in wild-type OVA-immunized
mice and in OVA-immunized IP-10-transgenic mice, as well as
IP-10-transgenic mice that were not exposed to OVA (naive). IP-10
protein expression was greater in IP-10-transgenic mice than in
wild-type mice. AHR is enhanced by IP-10 expression
AHR is a prominent component of this murine model of asthma.
OVA-immunized and -challenged IP-10-transgenic mice had increased AHR
compared with OVA-immunized and -challenged wild-type mice, as measured
by Penh, a noninvasive correlate of airway resistance (Fig. 4A). All OVA-immunized and
-challenged mice were more reactive than OVA-immunized and
saline-challenged mice. Two-way ANOVA demonstrated a significant
positive interaction between genetic strain (IP-10 transgenic vs wild
type) and dose of methacholine. Thus, the rate of change in Penh with
increasing dose of methacholine was significantly greater for the
IP-10-transgenic mice than for wild-type animals. This implies
significantly greater AHR in the OVA-immunized and -challenged
IP-10-transgenic animals.
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B) as determined
by two-way ANOVA. IP-10 expression modulates BAL total cell counts and eosinophils
Following OVA immunization and four OVA challenges, total cell
counts in the BAL were significantly increased in IP-10-transgenic mice
compared with wild-type mice. Differential counts of the BAL cells
demonstrated significantly increased eosinophils and macrophages in the
IP-10-transgenic mice (Fig. 5A) compared with wild-type
controls. Neutrophil counts were not significantly different (data not
shown). BAL cellular analysis in IP-10-/- mice
demonstrated the opposite findings, with significantly fewer total
cells and eosinophils compared with wild-type mice (Fig. 5B). Again, there was no difference in neutrophil levels
(data not shown).
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Analysis of lymphocyte subsets by flow cytometry demonstrated a
significant increase in the number of CD8+
lymphocytes in BAL in IP-10-transgenic mice compared with wild-type
mice. In addition, there was a nonsignificant trend toward increased
CD3+ lymphocytes and CD4+
lymphocytes in BAL of IP-10-transgenic mice compared with
wild-type mice (Fig. 6A).
Similar analysis in IP-10-/- mice revealed the
opposite finding, with significantly reduced T cells, B cells
(CD19+ lymphocytes), CD4+
lymphocytes, and CD8+ lymphocytes compared with
wild-type controls (Fig. 6B).
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Analysis of cytokine levels in BAL fluid was performed by ELISA
following OVA immunization and five OVA challenges. IP-10-transgenic
animals had significantly increased IL-4 levels and a nonsignificant
increase in IL-5 compared with wild-type mice (Fig. 7A). In contrast,
IP-10-/- mice had significantly decreased IL-4
and IL-5 levels compared with wild-type controls (Fig. 7B).
IFN- levels were not different in wild-type and IP-10-transgenic or
IP-10-/- mice. The differences in the levels of
IL-4/IL-5 and IFN- in the two wild-type groups probably relate to
the different strains used for the experiments (FVB strain for the
IP-10-transgenics and Sv129 for the IP-10-/-
mice).
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Serum levels of total IgE and OVA-specific IgE were determined in wild-type mice and IP-10-transgenic mice after five OVA challenges, and no difference was seen (data not shown). Serum total IgE and OVA-specific IgE were also not different in wild-type and IP-10-/- mice after five OVA challenges (data not shown).
Qualitative analysis of inflammation in the lung by histopathology is unaffected by changes in IP-10 expression
The histopathology of lungs from IP-10-transgenic mice, IP-10-/- mice, and their controls after OVA immunization and five OVA challenges was reviewed by a pathologist blinded to the origin of the tissue. The pathologist assessed the tissue for peribronchial inflammation and mucous gland hypertrophy. There were no apparent differences in the degree of peribronchial inflammation or in the number of mucous-secreting cells (data not shown).
IP-10 expression modulates lymphocyte trafficking into the lung
To better quantitate the effect of IP-10 expression on type 2 and
type 1 lymphocyte recruitment into the lung, we isolated lymphocytes
from the lungs of OVA-immunized and OVA-challenged animals and analyzed
them for surface marker expression as well as intracellular cytokine
expression by flow cytometry. IP-10-transgenic mice had a greater
percentage of IL-4- and IL-5-positive T cells
(CD3+) compared with wild-type mice (Fig. 8A). Only the percentage of
IL-4-positive T cells was significantly different. In contrast,
IP-10-/- mice had a significantly lower
percentage of IL-4-positive T cells compared with wild-type animals
(Fig. 8B). The percentage of IFN--positive T cells
recruited into the lung was not statistically different between the
genetically modified mice and their wild-type controls.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression or Th1 lymphocyte
recruitment). These data suggest that IP-10 may directly influence
Th2-type inflammatory responses.
Few studies to date have examined the effect of alterations in the
expression of Th1-type chemokines, such as IP-10, in animal models of
asthma. IP-10 is a chemokine that has been associated with Th1-type
responses based on its induction by IFN- expression
(30) and the ability of IP-10 to preferentially attract
Th1 lymphocytes (15, 16, 17, 18). In this work we show that IP-10
expression is enhanced in a murine model of asthma. This is consistent
with data demonstrating elevated levels of IP-10 protein in the BAL
fluid of asthma patients compared with healthy controls
(21). We found that the increase in IP-10 expression in
the OVA model of asthma is only partially dependent on IFN-
expression, suggesting that additional pathways contribute to IP-10
up-regulation in this model. In contrast, the expression of another
CXCR3 chemokine ligand, Mig, was undetectable in the absence of
IFN-.
The role of IP-10 in allergic inflammation has not been well defined.
In vitro studies suggest that IP-10 could antagonize allergic
inflammation by blocking the chemokine receptor CCR3 and inhibiting
eosinophil and Th2 lymphocyte recruitment (31). The data
we present here suggest that this effect is not significant in vivo, at
least in the robust model of Th2-type inflammation used in our study.
Another recent study has examined the consequences of overexpression of
IP-10 in a murine model of asthma using an adenovirally mediated gene
delivery system (32). This study demonstrated that
increased IP-10 expression reduced airway eosinophilia and IL-4 levels
and increased IFN- levels. Our results differ from these findings in
that we show that IP-10 directly enhances Th2-type inflammation. The
divergent results seen between these two studies may be related to the
different models used. The previous study used an adenovirus to deliver
the GM-CSF gene to the airways during OVA challenge. This technique
creates a Th2-type inflammatory response in the lung without prior OVA
immunization. They then coexpressed IP-10 with GM-CSF in the airways,
again using adenoviral vectors, and this reduced allergic airway
inflammation compared with a control viral infection. The model in this
study is clearly different from the model used in our study. The
response to IP-10 in the airways may be modified by GM-CSF expression
and the lack of an immunization step before OVA challenge. In addition,
coexistent viral infection in the setting of allergen challenge may
affect the response to IP-10.
The data presented in our paper suggest a pathogenic role for IP-10 in
a model of allergic airways disease. There are several potential
mechanisms for the effects of IP-10 on allergic inflammation. Some
prior studies have suggested that augmentation of Th1-type inflammatory
disease can increase allergic airway disease (3). However,
the fact that IFN- expression or Th1 lymphocyte recruitment was not
altered when the OVA model of allergic airways disease was induced in
IP-10-transgenic or IP-10-deficient mice suggests that IP-10
contributes to Th2-type inflammation directly rather than by increasing
Th1-type inflammation.
In our study we demonstrated an increase in type 2 lymphocyte recruitment into the lungs. Because we used CD3 as a surface marker to identify T lymphocytes for the intracellular cytokine staining experiments, we are unable to distinguish whether the IL-4+ T cells recruited into the lung are Th2 lymphocytes and/or Tc2 lymphocytes (type 2 cytotoxic cells; IL-4+CD8+ lymphocytes). Flow cytometry of lymphocytes from the BAL revealed that both CD4+ and CD8+ lymphocyte recruitment is affected by alterations in IP-10 expression, suggesting that IP-10 may be directly increasing both Th2 and Tc2 recruitment. The receptor for IP-10, CXCR3, has been reported to be expressed on Th2 lymphocytes in humans and mice, albeit at lower levels than on Th1 lymphocytes (15, 18). Other studies have suggested that CXCR3 is found on both Th1 and Th2 lymphocytes recruited into inflammatory sites in vivo (33, 34). Thus, IP-10 could enhance allergic inflammation in the OVA model of asthma by direct recruitment of Th2 lymphocytes into the lung. While increased Th2 lymphocyte recruitment could clearly increase allergic inflammation, the potential implications of alterations in the number of Tc2 lymphocytes are unclear. Some studies have suggested that CD8+ lymphocytes have minimal effects on OVA-induced AHR and inflammation (4, 35), while others have indicated that CD8+ lymphocytes are necessary for the allergic response in the airways (36) and may be a marker of more severe disease in humans (37). Taken together, the data presented above suggest that IP-10 can increase allergic inflammation by enhancing type 2 lymphocyte recruitment into the lung.
We also observed that changes in the level of IP-10 could modulate eosinophil recruitment to the airways in the OVA model of allergic pulmonary inflammation used in our studies. This effect on eosinophil recruitment could be secondary to the effects of IP-10 on the modulation of type 2 lymphocyte recruitment into the lung or could be a direct effect of IP-10 on eosinophils. In this regard there is a single study that reported CXCR3 expression on eosinophils (38). In that paper IP-10 was shown to mediate eosinophil chemotaxis and activation (38). If similar findings can be demonstrated in vivo, this would suggest that IP-10 could be modulating the eosinophilic inflammation seen in the OVA model of asthma by directly affecting the recruitment of eosinophils into the airways.
AHR is a primary feature of asthma and the OVA-induced model of allergic airway disease. In our study we found that transgenic mice (on an FVB background) that overexpress IP-10 in the lung had significantly increased AHR, as assessed by Penh. AHR appeared to be unchanged in IP-10-/- mice; however, these mice were on an Sv129 background, which develops less AHR in the OVA model of allergic airway disease and thus may not demonstrate subtle differences. The change in AHR seen with increased IP-10 could occur indirectly from the effects on type 2 lymphocyte and eosinophil recruitment seen in our experiments. Alternatively, IP-10 could enhance AHR by directly modulating smooth muscle cell function. There is a single study demonstrating that IP-10 directly stimulates vascular smooth muscle cells to proliferate and migrate in vitro (39). If this activity is relevant in the OVA model of allergic pulmonary inflammation, then IP-10 could enhance AHR by directly modulating the density of muscle cells around the airway and/or their contractile properties.
In our murine model of asthma we found that IP-10 modulates Th2-type
airway inflammation. We hypothesize that IP-10 contributes to AHR and
airway inflammation by increasing the recruitment of Th2 lymphocytes
and eosinophils. The fact that the receptor for IP-10 is expressed on
Th2 lymphocytes and possibly on eosinophils and smooth muscle cells
provides a potential mechanism for this effect. In broader terms, our
study suggests that chemokines and complex inflammatory diseases, such
as asthma, cannot be completely compartmentalized into Th1-type or
Th2-type inflammation. Factors from both types of inflammation may
indeed be involved in establishing the allergic inflammatory state in
the airways. Furthermore, attempts to modify the polarization of the
inflammatory response by altering chemokine expression may prove
complicated. Indeed, asthma therapies that up-regulate IP-10 (such as
IFN-) may actually lead to enhancement of airway inflammation.
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Acknowledgments |
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Footnotes |
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2 B.D.M. and A.S. contributed equally to this work.
3 Address correspondence and reprint requests to Dr. Andrew D. Luster, Center for Immunology and Inflammatory Diseases, Division of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, Building 149-8301, 13th Street, Charlestown, MA 02129. E-mail address: luster{at}helix.mgh.harvard.edu
4 Abbreviations used in this paper: AHR, airway hyperreactivity; BAL, bronchoalveolar lavage; BK5, bovine keratin 5; IP-10 (CXCL10), IFN--inducible protein 10; I-TAC, IFN-inducible T cell -chemoattractant; Mig (CXCL9), monokine induced by IFN-; Penh (CXCL11), enhanced pause.
Received for publication November 6, 2001. Accepted for publication March 6, 2002.
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inhibits the development of secondary allergic responses in mice. J. Immunol. 157:1432.[Abstract]
regulate allergic airway inflammation and mucus production. J. Exp. Med. 190:1309.. J. Immunol. 166:207.-Interferon transcriptionally regulates an early-response gene containing homology to platelet proteins. Nature 315:672.[Medline]
interferon-inducible cytokine (IP-10). J. Exp. Med. 166:1084.-inducible protein; monocyte chemotactic proteins 1, 3, and 4; and eotaxin in Th1- and Th2-mediated lung diseases. J. Allergy Clin. Immunol. 107:664.[Medline]
-inducible protein 10 (IP-10, CXCL10)-deficient mice reveal a role for IP-10 in effector T cell generation and trafficking. J. Immunol. 168:3195.-inducible protein 10 in the airway alters mucosal allergic sensitization in mice. J. Immunol. 166:2750.-inducible protein-10 and monokine induced by IFN-. J. Immunol. 165:1548.This article has been cited by other articles:
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  S. Ying, B. O'Connor, J. Ratoff, Q. Meng, C. Fang, D. Cousins, G. Zhang, S. Gu, Z. Gao, B. Shamji, et al. Expression and Cellular Provenance of Thymic Stromal Lymphopoietin and Chemokines in Patients with Severe Asthma and Chronic Obstructive Pulmonary Disease J. Immunol., August 15, 2008; 181(4): 2790 - 2798. [Abstract] [Full Text] [PDF]
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 C-H. Hung, Y-T. Chu, Y-M. Hua, S-H. Hsu, C-S. Lin, H-C. Chang, M-S. Lee, and Y-J. Jong Effects of formoterol and salmeterol on the production of Th1- and Th2-related chemokines by monocytes and bronchial epithelial cells Eur. Respir. J., June 1, 2008; 31(6): 1313 - 1321. [Abstract] [Full Text] [PDF]
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L. Y. Liu, M. E. Bates, N. N. Jarjour, W. W. Busse, P. J. Bertics, and E. A. B. Kelly Generation of Th1 and Th2 Chemokines by Human Eosinophils: Evidence for a Critical Role of TNF-{alpha} J. Immunol., October 1, 2007; 179(7): 4840 - 4848. [Abstract] [Full Text] [PDF]
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S. Y. Thomas, A. Banerji, B. D. Medoff, C. M. Lilly, and A. D. Luster Multiple Chemokine Receptors, Including CCR6 and CXCR3, Regulate Antigen-Induced T Cell Homing to the Human Asthmatic Airway J. Immunol., August 1, 2007; 179(3): 1901 - 1912. [Abstract] [Full Text] [PDF]
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 N. L. Korpi-Steiner, M. E. Bates, W.-M. Lee, D. J. Hall, and P. J. Bertics Human rhinovirus induces robust IP-10 release by monocytic cells, which is independent of viral replication but linked to type I interferon receptor ligation and STAT1 activation J. Leukoc. Biol., December 1, 2006; 80(6): 1364 - 1374. [Abstract] [Full Text] [PDF]
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 N. Wang, M. Campo, L. Ting, C. Fleming, C. Terhorst, and P. W. Finn The Costimulatory Molecule SLAM Is Critical for Pulmonary Allergic Responses Am. J. Respir. Cell Mol. Biol., August 1, 2006; 35(2): 206 - 210. [Abstract] [Full Text] [PDF]
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M. Koch, M. Witzenrath, C. Reuter, M. Herma, H. Schutte, N. Suttorp, H. Collins, and S. H. E. Kaufmann Role of Local Pulmonary IFN-{gamma} Expression in Murine Allergic Airway Inflammation Am. J. Respir. Cell Mol. Biol., August 1, 2006; 35(2): 211 - 219. [Abstract] [Full Text] [PDF]
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P. J. Dunford, N. O'Donnell, J. P. Riley, K. N. Williams, L. Karlsson, and R. L. Thurmond The Histamine H4 Receptor Mediates Allergic Airway Inflammation by Regulating the Activation of CD4+ T Cells. J. Immunol., June 1, 2006; 176(11): 7062 - 7070. [Abstract] [Full Text] [PDF]
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G. Boulday, Z. Haskova, M. E. J. Reinders, S. Pal, and D. M. Briscoe Vascular Endothelial Growth Factor-Induced Signaling Pathways in Endothelial Cells That Mediate Overexpression of the Chemokine IFN-{gamma}-Inducible Protein of 10 kDa In Vitro and In Vivo. J. Immunol., March 1, 2006; 176(5): 3098 - 3107. [Abstract] [Full Text] [PDF]
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G. F. Debes, M. E. Dahl, A. J. Mahiny, K. Bonhagen, D. J. Campbell, K. Siegmund, K. J. Erb, D. B. Lewis, T. Kamradt, and A. Hamann Chemotactic Responses of IL-4-, IL-10-, and IFN-{gamma}-Producing CD4+ T Cells Depend on Tissue Origin and Microbial Stimulus J. Immunol., January 1, 2006; 176(1): 557 - 566. [Abstract] [Full Text] [PDF]
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