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-Inducible Protein 10 kDa in a Cockroach Antigen-Induced Model of Allergic Airway Hyperreactivity: Systemic Versus Local Effects1
Department of Pathology, University of Michigan, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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to antagonize established Th2 type allergic
responses is well documented. To investigate the role of
IFN--inducible protein 10 kDa (IP10) in the allergic
response, we chose to investigate the effect of IP10 neutralization on
an established Th2 response. Systemic neutralization of IP10 at the
time of allergen challenge increased airway hyperreactivity as well as
airway eosinophil accumulation. Interestingly, IFN- levels were
markedly reduced in both the lung and peripheral lymph node following
IP10 neutralization. Furthermore, the number of
CXCR3+CD4+ T cells was decreased in the
peripheral lymph node following neutralization of IP10. Introduction of
exogenous IP10 into the airway at the time of allergen challenge also
dramatically increased eosinophil accumulation in the airway. Protein
levels of IL-4, IL-5, and IL-13 were significantly increased in the
lung following exogenous airway administration of IP10 with allergen.
Interestingly, airway hyperreactivity was significantly decreased at
early time points following concurrent IP10 and allergen challenge but
rebounded at 24 and 48 h post allergen challenge. Although IP10
may initially be acting locally to dampen the allergic response, its
ability to recruit eosinophils may ultimately supersede any
immunomodulatory effect it may have in an established allergic
response. These results suggest that while systemic levels of IP10 are
beneficial in controlling the allergic response, possibly by regulating
cellular trafficking in the lymph node, local administration of
exogenous IP10 into an established allergic response may be
detrimental. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Chemokines are chemotactic cytokines that function in the recruitment
of specific cellular populations to sites of injury and inflammation.
Chemokines are divided into two main families, CC and CXC, according to
the positioning of two conserved cysteine residues in the N terminus.
The CXC family of chemokines can be further divided into families based
on the presence of a glutamate-leucine+ arginine amino acid
motif (10). IFN--inducible protein 10 kDa
(IP10),3 monokine
induced by IFN- (MIG), and IFN-inducible T cell 
chemoattractant
are members of the CXC/ELR- family of
chemokines. These proteins are induced by IFN- in a variety of cells
including monocytes and epithelial cells (13). IP10, MIG,
and IFN-inducible T cell chemoattractant are ligands for the
chemokine receptor CXCR3 (14). CXCR3 is predominantly
expressed on CD4+ and CD8+
T cells that are CD45RO+ and express high levels
of 
1 integrins along with NK cells and
monocytes (15, 16, 17). In addition, it is widely reported
that T cells expressing the receptor CXCR3 are specifically recruited
to sites of inflammation that exhibit a "Th1" type phenotype such
as rheumatoid arthritis and inflammatory bowel disease
(18, 19, 20, 21).
It has been established that Th1 and Th2 responses are antagonistic to
each other (22, 23, 24, 25, 26). Administration of IFN- into the
airways of allergic mice has been demonstrated to reduce the severity
of the Th2 type response and attenuate asthma-like responses (27, 28). However, it is unclear whether IFN- acts directly or
indirectly by altering local and/or systemic inflammatory immune
responses. Recently, it has been shown that overexpression of IP10
during sensitization to OVA decreased levels of IL-4 and eosinophil
accumulation in the airway following OVA challenge (29).
To further investigate the role of IP10 in the allergic response, the
effect of neutralization of IP10 at the time of allergen challenge was
investigated in these studies. Unlike previous studies, the role of
IP10 was examined during an established allergic response that is
characterized by eosinophilia and airway hyperreactivity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BALB/c mice 6–8 wk of age were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained under standard pathogen-free conditions. Our institutional animal use and care committee approved all experiments described.
Sensitization and induction of the allergic airway response
Normal BALB/c mice were sensitized i.p. and s.c. with 1,000
protein nitrogen units (PNUs) of cockroach Ag (CRA) (Holliser Stier,
Toronto, Canada) in IFA (Sigma-Aldrich, St. Louis, MO). The mice were
challenged intranasally (IN) with 150 PNU CRA on day 14 following
initial sensitization to localize the response to the lung. Previous
studies in our lab have demonstrated that IN CRA challenge on day 14
following sensitization induced little inflammation in the lung. To
systemically neutralize IP10, mice were injected i.p. with 0.5 ml of
either control rabbit serum or serum containing Ab against murine IP10
(generated in our lab as previously described) (30) on day
20 following initial sensitization. Briefly, New Zealand white rabbits
were purchased from The Jackson Laboratory and housed under standard
conditions. Rabbits were immunized using 20 µg of recombinant murine
IP10 (PeproTech, Rocky Hill, NJ) in CFA, 1.5 ml was injected
intradermally in multiple sites, and 0.5 ml was injected i.p. Two and 4
wk following the initial immunization, the rabbits were boosted with 10
µg of IP10 in IFA intradermally and i.p. The rabbits were injected
with 10 µg of IP10 protein in IFA every 2–4 mo thereafter. Blood was
collected from an ear artery and the serum was extracted. The rabbit
serum was titered by a direct ELISA and only serum with an Ab titer
>1 x 105 was used in in vivo experiments.
The serum was also tested for cross-reactivity against murine C10,
eotaxin, IL-1, IL-4, IL-12, IL-13, IL-18, JE, KC, monocyte
chemoattractant protein 3, monocyte chemoattractant protein 5,
macrophage-derived chemokine, MIG, macrophage-inflammatory
protein 1, macrophage inflammatory protein 2, RANTES, stromal
cell-derived factor, thymus and activation-regulated chemokine, TCA3,
TNF-, IFN-, six-cysteine chemokine, and fractalkine using a
direct ELISA. Forty-five min following Ab administration, the mice were
anesthetized with 150 µg/kg ketamine and 45 µg/kg xylazine in
normal saline and the trachea aseptically exposed and prebored using a
21-gauge needle. CRA (320 PNU) in sterile PBS in a total volume
of 40 µl was instilled into the trachea using a Hamilton syringe and
wounds were closed using surgical staples. In separate experiments, 320
PNU of CRA in PBS or 80 ng of recombinant murine IP10 (PeproTech) in
CRA was instilled directly into the lung via an intratrachial (IT)
challenge on day 20 following initial sensitization. In additional
experiments, the mice were also challenged IT with 90 µg of purified
anti-IP10 Ab and CRA on day 20. Anti-IP10 Ab was purified from
serum using protein A Sepharose columns and buffers developed by Pierce
(Rockford, IL). The eluted Ab was concentrated by repeatedly
spinning the eluate through a 30,000 m.w. centricon (Millipore,
Bedford, MA) and the Ab concentration determined by dividing its
absorbance at 280 nm by the Ab extinction coefficient (1.35). All
described time points were measured after IT challenge on day
20.
Measurement of airway hyperreactivity
Airway hyperreactivity was measured using a Buxco mouse
plethysmograph (Buxco Electronics, Troy, NY). Mice were
anesthetized with 3.3 mg of pentobarbital (Vortech Pharmaceuticals,
Dearborn, MI). The trachea was cannulated using an 18-gauge needle and
mice were ventilated using a Harvard pump ventilator (tidal volume, 0.4
ml; frequency, 120 breaths/min; positive end-respiratory pressure,
2.5–3.0 cm H2O; Harvard Apparatus, Holliston,
MA). Changes in the lung volume, represented by the change in box
pressure were measured by a differential transducer. A second
transducer was used to measure pressure swings at the opening of the
trachea tube, referenced to the body box (i.e., pleural pressure) and
to provide a measure of transpulmonary pressure
((Ptp) = trachea tube - box
pressure)). The trachea transducer was calibrated at a constant
pressure of 20 cm of H20. Resistance
(R) was calculated by Buxco software (Buxco Electronics) by
dividing the change in pressure (Ptp) by the
change in flow (F)(
Ptp/F; units =
centimeters of H2O per milliliter per second) at
two time points from the volume curve based on a percentage of
inspiratory the volume. Baseline hyperreactivity was recorded and
change in hyperreactivity (
R) was measured after i.v. injection if
12.5 µg methacholine (Sigma-Aldrich) as previously described
(31).
Morphometric analysis of airway and peribronchial eosinophil accumulation
Lungs were lavaged with 1.0 ml 0.9% saline (Abbott Laboratories, Abbott Park, IL). Bronchioalveolar cells were cytospun and stained with Diff-Quik (Dade International, Miami, FL) and the total number of airway cellular populations (monocytes, lymphocytes, neutrophils, and eosinophils) was determined. The left lobe of lung was excised, inflated with 10% formalin, and embedded in paraffin. Five-micron sections were cut and stained with H&E. The number of peribronchial eosinophils was determined by examining 50 consecutive x100 high power fields around the largest bronchioles in each lung section.
Cytokine quantitation using specific ELISA
The lower right lobe of lung and mediastinal lymph nodes was
excised and snap-frozen in liquid nitrogen. Lungs and lymph nodes were
homogenized on ice using a Tissue Tearor (Biospec Products, Racine, WI)
for 30 s in 1.0 ml of PBS (BioWhittaker, Walkersville, MD)
containing 0.05% Triton X-100 (Sigma-Aldrich) and one tablet of
complete protease inhibitor (Roche, Basel, Switzerland). Cytokine
protein concentrations were quantified using basic sandwich ELISAs
developed in our lab. Briefly, flat-bottom 96-well microtiter plates
(Nunc Immunoplate I 96-F; Nunc, Roskilde, Denmark) were coated with 50
µl/well of polyclonal Abs specific for the chemokine or cytokine in
question for 16 h at 4°C and then washed with PBS and 0.05%
Tween 20. All Abs used for ELISA were purchased from R&D Systems
(Minneapolis, MN) with the exception of IFN- in which Abs generated
in our lab (as described above) were used. Nonspecific binding sites
were blocked with 2% BSA in PBS an incubated for 90 min at 37°C.
Plates were washed four times with wash buffer and cell-free
supernatants were added (1:2 and 1:10) followed by incubation for
1 h at 37°C. Plates were washed four times before their
incubation with the appropriate biotinylated secondary Ab for 1 h
at 37°C. The plates were again washed four times,
strepavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was
added, and the plates were incubated at room temperature to the desired
extinction. The reaction was terminated with 50 µl/well of 3 M
H2SO4. Standards were 0.5
log dilutions of recombinant protein from 1 pg/ml to 100 ng/ml.
Absorbance at 490 nm was measured using an ELISA reader.
Immunohistochemical detection of IP10 protein expression
The left lobe of lung from mice immunized and subsequently challenged with CRA were excised 24 h post IT CRA challenge, inflated with formalin, and embedded with paraffin. Five-micrometer sections were cut and mounted onto glass slides. Lung sections were deparaffinized, rinsed in fresh xylene, and rehydrated through graded alcohol: 100, 95, 70, 50%, PBS for 3 min each. Endogenous peroxidase activity was quenched by incubating the rehydrated section in 1:1 methanol and 3% hydrogen peroxide for 3 min. IP10 protein expression was visualized using an HRP-3-amino-9-ethylcarbazole cell and tissue staining kit (R&D Systems) along with primary rabbit anti-mouse Ab against IP10 generated and purified in our lab (5.0 µg/ml) or rabbit IgG (Jackson ImmunoResearch Laboratories, West Grove, PA) (5.0 µg/ml) as an isotype control.
Analysis of mRNA expression
The upper right lobe of lung from mice in which IP10 was systemically neutralized or which had been challenged with IP10 was excised and snap-frozen in liquid nitrogen. The lungs were homogenized in TRIzol (Life Technologies, Rockville, MD) and the RNA purified. The purified RNA was subjected to a DNAase treatment using a DNA-free kit developed by Ambion (Austin, TX) to remove any genomic DNA. A total of 1.0 µg of RNA was then reverse transcribed using an 18-mer Oligo(dT) (Sigma-Aldrich) and Moloney murine leukemia virus reverse transcriptase at 38°C for 60'; the reaction was stopped by incubating the cDNA at 90°C for 5'. One microgram of cDNA was then amplified using predeveloped chemokine receptor primer and probe pairs from Applied Biosystems (Foster City, CA) in a TaqMan 7700 thermocycler for 40 cycles of 50°C for 2', 95°C for 10', 95°C for 15 s, and 60°C for 1 h. The fold difference in mRNA expression between treatment groups was determined using software developed by Applied Biosystems.
Flow cytometric analysis
Lungs were dispersed in 0.2% collagenase (Sigma-Aldrich) in RPMI (BioWhittaker) + 15% FCS (Atlas, Fort Collins, CO) at 37°C for 45 min. Lymph nodes were dispersed over a 4-µm mesh filter into RPMI. After lysing RBCs with ammonium chloride buffer (4.01 g NH4Cl, 0.42 g NaHCO3, and 0.185 g tetrasodium EDTA in 500 ml dH2O), the cells were stained with either 0.5 mg/ml anti murine CD4-FITC (BD PharMingen, San Diego, CA) and anti-murine CXCR3 (Y-16) (Santa Cruz Biotechnology, Santa Cruz, CA) along with an anti-goat IgG-PE secondary Ab (Jackson ImmunoResearch Laboratories, West Grove, PA) (5 µg/ml) or anti-murine DX5 (Research Diagnostics, Flanders, NJ) (0.5 mg/ml) along with an anti-rat IgG-PE secondary Ab (Jackson ImmunoResearch Laboratories)(5 µg/ml) and anti-murine CD3-FITC (BD PharMingen) (0.5 mg/ml) in Dulbecco’s PBS + 5% FCS + 0.05% NaN3 for 20 min on ice protected from light. Cells were fixed in 4% paraformaldehyde and kept in the dark at 4°C until analysis. Cell populations were analyzed using a Coulter XL flow cytometer (Beckman Coulter, Fullerton, CA).
Isolation of murine eosinophils from Schistosome mansoni-infected mice
Swiss albino mice infected with the parasite S. mansoni were obtained from Dr. F. Lewis (Biomedical Research Institute, Bethesda, MD). At 7–8 wk postinfection the mice were injected i.p. with 3,000 S. mansoni eggs and 50 ng of rIL-5 (R&D Systems) in 1 ml of 1.0% thioglycolate medium (Difco, Detroit, MI). Eosinophils were isolated via a peritoneal wash with 25 mM EDTA 48 h following i.p. egg injection. The isolated eosinophils were incubated in MC-9 media (DMEM + 15% PMSF, 15% FCS, 10% T-Stim culture supplement without Con A; Collaborative Biomedical Products, Bedford, MA), and 1 x 10-3% 2-ME) for 1 h at 37°C before further purification. The isolated cells were then resuspended in 90 µl of PBS + 0.5% BSA per 107 cells and incubated with 10 µl per 107 cells of anti-CD90 (Thy 1.2), anti-CD45RO (B220), and anti-MHC class II Abs coupled to magnetic beads (Miltenyi Biotec, Auburn, CA) and purified via negative selection by running the cell suspension through a magnetic column. The purified population was consistently >98% eosinophils and these eosinophils were consistently >95% viable by trypan blue exclusion. The purified eosinophils were analyzed flow cytometrically (as described above) for the expression of CCR3 and CXCR3 using anti-murine CCR3 (R&D Systems) (0.2 mg/ml) along with anti-rat IgG-PE (Jackson ImmunoResearch Laboratories) and anti-murine CXCR3 (Y-16) (0.2 mg/ml) (Santa Cruz Biotechnology) along with anti-goat IgG-FITC (Jackson ImmunoResearch Laboratories).
CXCR3 expression on airway eosinophils
Airway cells were isolated from the lungs of mice challenged with IP10 and CRA on day 20 following initial sensitization via bronchioalveolar lavage (BAL) were cytospun and fixed in 4% paraformaldehyde for 10 min. The cytospins were stained using and anti-goat HRP-3-amino-9-ethylcarbazole immunostaining kit (R&D Systems) along with anti-murine CXCR3 Ab (Y-16; Santa Cruz Biotechnology) and goat IgG (0.2 mg/ml; Jackson ImmunoResearch Laboratories). Cytospins were counterstained with hematoxylin and photographed at x100. In additional experiments, cells isolated from the BAL of mice 24 h following IT challenge with CRA or IP10 and CRA were stained with anti-murine CXCR3 (Y-16; Santa Cruz Biotechnology) along with anti-goat IgG-FITC (Jackson ImmunoResearch Laboratories) and anti-murine CCR3 (R&D Systems) along with anti-rat IgG-PE (Jackson ImmunoResearch Laboratories) and examined flow cytometrically, as described above.
Statistics
Statistical significance was determined by an ANOVA with p < 0.05, error is representative of the SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Analysis of IP10 protein expression in the lymph nodes of allergic
mice challenged IT with CRA revealed an increase in IP10 expression at
24 h (0.82 ± 0.25 ng/ml) in comparison to sensitized but
unchallenged animals (0.47 ± 0.05 ng/ml). IP10 protein expression
in the lungs was also increased over 48 h following allergen
challenge (Fig. 1
A).
Immunohistochemical analysis of paraffin-embedded lung sections from
sensitized and challenged mice illustrate that bronchial epithelial
cells and alveolar macrophages express IP10 (Fig. 1B). To
better elucidate the role of IP10 in the allergic response, IP10 was
systemically neutralized before allergen challenge and the phenotype of
the ensuing inflammatory response in the lung determined. The ability
of the Ab to neutralize IP10 was confirmed by the reduction of IP10
protein levels in both the lung (anti-IP-10 0.01 ± 0.001
ng/ml vs control Ab (cnt Ab) 1.54 ± 0.6 ng/ml) and mediastinal
lymph node (anti-IP-10 0.56 ng/ml vs cnt Ab 2.4 ng/ml) at 24 h
post IT challenge. Systemic neutralization of IP10 at the time of
allergen challenge resulted in a significant increase in airway
hyperreactivity which peaked at 24 h post IT challenge and
returned to control levels by 48 h (Fig. 2A). Eosinophil accumulation
in the airspace was significantly increased following IP10
neutralization. This increased accumulation was observed as early as
8 h post IT allergen challenge and continued for 24 h
following IT challenge (Fig. 2B). Histological analysis of
lung sections taken from treated mice revealed a trend toward an
increase in the amount of eosinophilic inflammation around the airway
24 h following neutralization of IP10 (Figs. 2C and
3). Analysis of mRNA expression in these
lungs 24 h post allergen challenge revealed a marked increase in
CCR3 and CXCR3 expression that paralleled the observed eosinophil
influx (6.4 ± 0.65- and 4.8 ± 1.0-fold, respectively).
Changes in bronchioalveolar populations of neutrophils and lymphocytes
were not statistically different following systemic neutralization.
Interestingly, the number of bronchioalveolar macrophages was
significantly increased following IP10 neutralization (Table I). Neutralization of IP10 in the airway
at the time of allergen challenge had no effect on the allergic
response in the lungs of these mice (data not shown). Thus, systemic,
but not local, neutralization of IP-10 significantly altered the
allergen-induced responses.
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To determine the influence of IP10 on cytokine and chemokine
expression, peripheral lymph nodes and lungs were excised and
homogenized at various time points following allergen challenge.
Systemic neutralization of IP10 at the time of allergen challenge
reproducibly decreased IFN- levels in the lymph node at 4, 12, and
24 h following allergen challenge (Fig. 4). Furthermore, IP10 neutralization also
significantly decreased protein levels of IFN- in the lung at
12 and 24 h post allergen challenge (Fig. 4). No significant
changes in protein levels of IL-4, IL-5, IL-10, IL-12, or IL-13 were
observed following IP10 neutralization (data not shown). These data
suggest that systemic neutralization of IP10 may modulate the allergic
immune response by decreasing the levels of the Th1-associated cytokine
IFN- in the lung and lymph node.
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Peripheral lymph nodes and lungs were excised and dispersed at
various time points following neutralization and IT allergen challenge,
stained for the presence of
CXCR3+CD4+ and
DX5+CD3+ double positive
cells, and analyzed using a Coulter flow cytometer. We found that the
number of CXCR3+CD4+ cells
was decreased in the lymph node following IP10 neutralization (Fig. 5). In addition, the number of NKT cells
(DX5+CD3+) were
significantly increased in the mediastinal lymph nodes of mice in which
IP10 was neutralized (Fig. 5). NKT cell and
CXCR3+CD4+ cell populations
in the lung were not significantly affected by systemic IP10
neutralization (data not shown). Our results suggest that
neutralization of IP10 may alter the trafficking and/or expansion of
lymphocyte subsets within the lymph node and thereby permit an
exacerbated allergic response to develop in the lung.
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Given our above findings on the ability of IP-10 to regulate the
allergic response, the effect of exogenous airway IP10 administration
was investigated. Sensitized mice were challenged IT with either
recombinant murine IP10 (80 ng/mouse) in CRA or PBS in CRA. Airway
hyperresponsiveness was significantly decreased 8 h following
local instillation of IP10 and allergen challenge (Fig. 6A). Subsequently, airway
hyperresponsiveness rebounded and increased substantially at 24 h,
although it was not significantly different from CRA control animals.
Eosinophil accumulation in the lung (Figs. 3 and 6B) and in
the airway (Fig. 6C) was also increased following
administration of exogenous IP10. Analysis of mRNA expression in these
lungs 24 h post IT challenge revealed a significant increase in
both CCR3 and CXCR3 expression (14.3 ± 2.8- and 3.0 ±
1.0-fold, respectively) that paralleled the observed leukocyte influx
(see Fig. 3). Immunohistochemical analysis of airway cells isolated
from the BAL fluid of mice challenged with IP10 and CRA reveal that the
recruited airway eosinophils and alveolar macrophages express CXCR3
(Fig. 7A). Analysis of
eosinophils isolated from mice infected with S. mansoni
using flow cytometry reveal that >98% (98 ± 0.12%) of this
cell population express both CCR3 and CXCR3 (Fig. 7B). Flow
cytometric analysis of BAL cells demonstrated a significant increase in
the number of both
CCR3+CXCR3+ and
CCR3+CXCR3- eosinophils in
the airways of mice 24 h post IT challenge with IP10 and CRA (Fig. 7C). There was no significant difference in the number of
CCR3-CXCR3+ granulocytic
eosinophils in the airways of these mice. Local administration of IP10
into the lungs resulted in a significant increase in total lymphocyte
recruitment as well as a significant increase in the number of
CXCR3+, CD4+, and
DX5+ cells in the lung (Fig. 8A).
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B). As
IFN- levels are already quite low in this Th2 type model, no
additional change was observed (data not shown). Thus, the exogenous
instillation of IP-10 into the airway during allergen challenge
resulted in the increased accumulation of eosinophils and lymphocytes
with a corresponding increase in Th2 type cytokines. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. Interestingly, the number of IFN--expressing cells was
increased while CD4+ T cells expressing the
putative Th2 marker TS1/2 were decreased in the lung by IP10
overexpression. The present studies are patterned after the concept
that IFN--inducible proteins, such as IP10, may antagonize the
development of Th2 responses (27, 29). In addition, the
observation that introduction of exogenous IFN- into the airway can
initially down-regulate the Th2-induced response may suggest that
IP10 can have an immunomodulatory role during allergic responses
(23, 26). A recent study using
IP10-/- and systemic IP10 transgenic mice
suggested that IP10 may play a contributory role in the allergic
response (32). The data in the present study suggest that
the role of IP10 would depend on the location and timing of its
production.
To further characterize the role of IP10 in the allergic response, we
investigated the effect of IP10 neutralization on an established
allergic response induced by CRA. Systemic neutralization of IP10
dramatically decreased protein levels of IFN- in the lymph node as
well as in the lung. Together with the reduction of
CXCR3+CD4+ T cells in the
lymph node, the reduced levels of IFN- may allow for the development
of an exaggerated Th2 response in the lungs (27). Airway
hyperreactivity along with eosinophil accumulation in the lung and
airway were increased following IP10 neutralization illustrating an
enhanced allergic response. Our findings are in agreement with those of
Wiley et al. (29), suggesting a crucial role for IP10 in
not only directing the developing immune response to allergen but also
in influencing an established allergic response. The effect of IP10 may
be important in the lymph node as well as the lung and play a role in
the trafficking of lymphocyte populations. IFN- is involved in the
regulation of the prototypical Th1-mediated immune response through its
ability to inhibit IL-4 and thereby the development of a Th2 type
response (23, 33). Therefore, one possible effect of the
reduction of IFN- following systemic neutralization of IP10 may be a
reduction in the number of Th1 type cells that are primed and/or
recruited in the lymph node that subsequently traffic as effector cells
to the lung. Recent studies by Dufour et al. (34) using
IP10-/- mice lends support to this concept as
these mice demonstrate defective generation and trafficking of effector
T cells in the lymph node. The observed decrease in IFN- in the
present studies after neutralization of IP10 may have allowed for the
development of an exaggerated allergic response in the lung as IFN-
can inhibit IL-13-induced goblet cell hyperplasia and airway eosinophil
accumulation (27).
Interestingly, increased numbers of NK (DX5+CD3-) and NKT (DX5+CD3+) cells were observed in the lymph node following systemic neutralization of IP10. NKT cells are capable of rapidly secreting significant amounts of IL-4 upon repeated exposure to Ag and thereby polarize T cells toward a Th2 phenotype (35). Likewise, depletion of NK cells before initial immunization has been shown to inhibit airway eosinophil and CD3+ T cell accumulation as well as IL-4 and IL-5 levels in the BAL fluid (36). The absence of IP10 in the lymph node may allow for the trafficking of NK cells into the lymph node, leading to their subsequent activation. NK cells and certain subsets of activated CD4+ and CD8+ T cells have also been reported to express CXCR3 (37, 38). It has been established that CXCR3 is preferentially expressed on activated memory CD4+ T cells (CD45RA+ CD45RO+ CD69+ CD25+) but not on resting or naive CD4+ T cells (16, 37, 39). Although several reports have suggested that CXCR3 expression by CD4+ T cells is indicative of a Th1 phenotype in those cells, studies by Nanki et al. (40) using single-cell RT-PCR methodology have demonstrated that CXCR3 is not a definitive marker for Th1 cells in vivo. Additionally, Butcher and colleagues (37) have identified a subset of IL-4-producing CD4+ T cells that also express CXCR3. Therefore, the administration of exogenous IP10 into the airway of mice with an established allergic response may have resulted in an increase in the gradient for recruitment of CXCR3 expressing Th2 type cells and eosinophils.
Previous studies have linked IP10 and its receptor CXCR3 to Th1 type immune responses (19, 20, 21). However, local administration of exogenous IP10 in the present studies in sensitized and challenged mice increased the allergic response in the lung and airway along with increased IL-4, IL-5, and IL-13 levels. Together, these data suggest an increase in the activation or recruitment of Th2 type cells into the lung (41). Interestingly, airway hyperreactivity was significantly reduced at early time points following instillation of IP10 with allergen, but subsequently resulted in increased airway hyperreactivity and eosinophil accumulation. Human peripheral eosinophils were originally reported to migrate to CXCR3 ligands MIG and IP10, confirming that CXCR3 was functionally expressed on these cells (42). Additionally, we have shown that murine eosinophils recruited into the airway during an allergic response also express CXCR3. Our results also suggest that exogenous administration of IP10 altered the local response by increasing recruitment of specific lymphocyte population and increased Th2 type cytokines. The latter effects resulted in an exacerbated allergic response that may be due to an increased chemotactic gradient for CXCR3 expressing cells.
Taken together our findings demonstrate differential roles of IP10 characterized by the systemic vs local modulation of the allergic response. Our results suggest that systemically IP10 may be acting at the level of the lymph node, influencing the recruitment and/or activation of T cells. However, locally, the ability of IP10 to recruit eosinophils and possibly CXCR3 expressing Th2 type cells overrides any ability it may have to dampen the allergic response.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Nicholas W. Lukacs, Department of Pathology, University of Michigan, 5214 Medical Sciences Building I, 1301 Catherine, Ann Arbor, MI 48109. E-mail address: nlukacs{at}med.umich.edu
3 Abbreviations used in this paper: IP10, IFN--inducible protein 10 kDa; MIG, monokine induced by IFN-; PNU, protein nitrogen unit; CRA, cockroach Ag; IN, intranasally; IT, intratrachial; Ptp, transpulmonary pressure; BAL, bronchioalveolar lavage; cnt Ab, control Ab.
Received for publication April 12, 2002. Accepted for publication October 9, 2002.
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References |
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|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
chemoattractant (CXCL11). J. Immunol. 167:7084. gene transfer inhibits pulmonary allergic responses in mice. J. Immunol. 157:3216.[Abstract]
regulate allergic airway inflammation and mucus production. J. Exp. Med. 190:1309. production: defining a novel role for IFN- in the regulation of allergic airway inflammation. J. Immunol. 166:2760.: interactions in lung inflammation. J. Immunol. 167:1769.-inducible protein 10 in the airway alters mucosal allergic sensitization in mice. J. Immunol. 166:2750.-inducible protein 10 (CXCL10) contributes to airway hyperractivity and airway inflammation in a mouse model of asthma. J. Immunol. 168:5278.-dependent mechanism. J. Immunol. 166:1471.-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.-galactosylceramide polarizes CD1-reactive NK T cells towards Th2 cytokine synthesis. Eur. J. Immunol. 29:2014.[Medline]
-inducible protein-10 and monokine induced by IFN-. J. Immunol. 165:1548.This article has been cited by other articles:
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  K. B. Walsh, R. A. Edwards, K. M. Romero, M. V. Kotlajich, S. A. Stohlman, and T. E. Lane Expression of CXC Chemokine Ligand 10 from the Mouse Hepatitis Virus Genome Results in Protection from Viral-Induced Neurological and Liver Disease J. Immunol., July 15, 2007; 179(2): 1155 - 1165. [Abstract] [Full Text] [PDF]
|
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L. N. Stiles, J. L. Hardison, C. S. Schaumburg, L. M. Whitman, and T. E. Lane T Cell Antiviral Effector Function Is Not Dependent on CXCL10 Following Murine Coronavirus Infection J. Immunol., December 15, 2006; 177(12): 8372 - 8380. [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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 X. Zeng, T. A. Moore, M. W. Newstead, J. C. Deng, S. L. Kunkel, A. D. Luster, and T. J. Standiford Interferon-Inducible Protein 10, but Not Monokine Induced by Gamma Interferon, Promotes Protective Type 1 Immunity in Murine Klebsiella pneumoniae Pneumonia Infect. Immun., December 1, 2005; 73(12): 8226 - 8236. [Abstract] [Full Text] [PDF]
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M. S. Thomas, S. L. Kunkel, and N. W. Lukacs Regulation of Cockroach Antigen-Induced Allergic Airway Hyperreactivity by the CXCR3 Ligand CXCL9 J. Immunol., July 1, 2004; 173(1): 615 - 623. [Abstract] [Full Text] [PDF]
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 C. Pilette, J.N. Francis, S.J. Till, and S.R. Durham CCR4 ligands are up-regulated in the airways of atopic asthmatics after segmental allergen challenge Eur. Respir. J., June 1, 2004; 23(6): 876 - 884. [Abstract] [Full Text] [PDF]
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 P. C. Fulkerson, N. Zimmermann, E. B. Brandt, E. E. Muntel, M. P. Doepker, J. L. Kavanaugh, A. Mishra, D. P. Witte, H. Zhang, J. M. Farber, et al. Negative regulation of eosinophil recruitment to the lung by the chemokine monokine induced by IFN-{gamma} (Mig, CXCL9) PNAS, February 17, 2004; 101(7): 1987 - 1992. [Abstract] [Full Text] [PDF]
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, positively stained
macrophage.
