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-Inducible Protein 10 in the Airway Alters Mucosal Allergic Sensitization in Mice1
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Department of Pathology and Molecular Medicine and Division of Respiratory Diseases and Allergy, Centre for Gene Therapeutics, McMaster University, Hamilton, Ontario, Canada; and
Millennium Pharmaceuticals Inc., Cambridge, MA 02139
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein (IP) 10 in the airways of mice
undergoing a mucosal sensitization regimen known to result in a
Th2-polarized allergic response. This resulted in a 
60–75%
inhibition of eosinophils in the bronchoalveolar lavage (BAL); these
inflammatory changes were accompanied by enhanced IFN-, ablated
IL-4, and, peculiarly, unaltered IL-5 and eotaxin levels in the BAL.
The effect of IP-10 expression was shown to be dependent on IFN-, as
there was no statistically significant reduction in BAL eosinophilia in
IFN- knockout mice subjected to the IP-10 intervention. Flow
cytometric analysis of mononuclear cells in the lung revealed a 60%
reduction in the fraction of CD4+ cells expressing T1/ST2,
a putative Th2 marker, and a parallel increase in the proportion
expressing intracellular IFN- following IP-10 treatment. The effect
of IP-10 expression at the time of initial Ag encounter is persistent,
as mice rechallenged with OVA following the resolution of acute
inflammation exhibited reduced eosinophilia and IL-4 in the BAL.
Collectively, these data illustrate that local expression of the
chemokine IP-10 can introduce Th1 phenomena to a Th2-predisposed
context and subvert the development of a Th2
response. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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-inducible protein
(IP)3 10, and
therefore the accumulation of granuloma-promoting mononuclear cells, is
initiated by T cell-independent mechanisms but is ultimately amplified
and sustained by activated T cells at the site of inflammation
(6). Likewise, Lloyd et al. (7) have
documented temporal regulation of chemokines in a murine model of
allergic airways disease; they have shown that as the immune response
evolves in a repeatedly challenged airway, eotaxin, which is initially
dominant in the recruitment of CCR3+ Th2 cells,
is ultimately supplanted by the CCR4 ligand macrophage-derived
chemokine. Collectively, these observations strongly suggest that the
elaboration of an immune response may in part hinge on the coordinated
expression of chemokines.
It has also been proposed that the acquisition of a chemokine and
chemokine receptor repertoire is an integral part of Th
differentiation; indeed, although Th2 cells are associated with the
chemokines eotaxin, thymus and activation-regulated chemokine,
macrophage-derived chemokine, and the chemokine receptors CCR3, CCR4,
and CCR8, Th1 cells correlate with the chemokines IP-10, monokine
induced by IFN-, and macrophage-inflammatory protein (MIP) 1
and
the chemokine receptors CXC chemokine receptor (CXCR) 3 and CCR5
(8, 9, 10, 11, 12, 13). The association between chemokines, chemokine
receptors, and Th phenotype is convincingly illustrated by IP-10. IP-10
is chemoattractant for T cells, monocytes (14, 15, 16), and NK
cells (17) but not for neutrophils (18, 19),
and has been shown to facilitate selective recruitment of Th1 cells
that preferentially express the receptor CXCR3 both in vitro
(9) and in vivo (20). Indeed, abundant CXCR3
expression has been reported on T cells infiltrating Th1-associated
multiple sclerosis lesions (21) and rheumatoid arthritis
synovial fluid (22), and sustained, protective expression
of IP-10 has been described in murine models of Th1-polarized
leishmaniasis (12, 23). IP-10 has also been implicated in
the recruitment of lymphocytes to sites of atheroma formation
(24) and has been detected in the bronchoalveolar lavage
(BAL) fluid of patients with pulmonary sarcoidosis (25).
The relationship between IP-10 and IFN- is unequivocal:
stimulation with IFN- elicits IP-10 expression by activated human
bronchial epithelial cells (26) and neutrophils
(27), whereas levels of IP-10 mRNA are markedly reduced in
lung interstitial macrophages in IFN- receptor knockout (KO) mice
(28). On the other hand, IP-10 has been documented to
induce IFN- expression in cultured human PBMC (29).
IP-10, therefore, may serve not only to mobilize differentiated Th1
cells but also to reinforce the evolution of a Th1 response.
We speculate that the preferential recruitment of a particular immune
cell population by chemokines has implications for the short- and
long-term features of an adaptive immune response. To test this
hypothesis, we used an adenovirus (Ad)-mediated gene transfer approach
to express IP-10, a prototype Th1 chemokine, in the airways of mice
subjected to a mucosal sensitization regimen that results in a
Th2-polarized allergic response. We have previously shown that
intranasal administration of Ad/GM-CSF to mice followed by 10 daily
exposures to aerosolized OVA results in cardinal Th2 events
(30). In this study, we demonstrate that concurrent
expression of IP-10 in the airway microenvironment significantly
attenuates eosinophilia and elaborates Th1 phenomena in an
IFN--dependent fashion. Moreover, OVA rechallenge long after the
clearance of IP-10 and resolution of acute inflammation elicits an
inflammatory response that is primarily mononuclear and not
eosinophilic, indicating that airway expression of IP-10 affects
sensitization and the maturation of T cell memory. Collectively, these
data suggest that IP-10, through preferential recruitment of
Th1-privileging immune-inflammatory cells, can subvert a Th2-polarized
response in vivo and attribute to this chemokine an immunoregulatory
function that transcends chemotaxis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Female BALB/c mice (6–8 wk old) were purchased from Charles
River Laboratories (Montreal, Quebec, Canada). Female IFN- KO mice
on a BALB/c background were purchased from The Jackson Laboratory (Bar
Harbor, ME). The mice were housed under specific pathogen-free
conditions following a 12-h light-dark cycle. A total of 623 mice was
sacrificed during the course of these experiments. All experiments
described in this study were approved by the Animal Research Ethics
Board of McMaster University.
Aerosolization protocol
Over a period of 10 consecutive days (days 0–9), mice were placed in a Plexiglas chamber (10 cm x 15 cm x 25 cm) and exposed for 20 min daily to aerosolized OVA (1% w/v in 0.9% saline; Sigma-Aldrich, Oakville, Ontario, Canada). The OVA aerosol was generated by a Bennett (Kansas City, MO) nebulizer at a flow rate of 10 L/min. For the rechallenge experiment, mice were exposed to a 1% OVA aerosol for two 1-h periods separated by 4 h on day 50 of the protocol.
Administration of adenoviral constructs
To elicit local expression of GM-CSF, IP-10, or IFN-, a
replication-deficient human type 5 Ad construct carrying murine GM-CSF,
human IP-10, or human IFN- cDNA in the E1 region of the viral genome
was delivered intranasally (i.n.) to anesthetized animals on day -1,
24 h before the first exposure to OVA; the human IP-10 sequence
was selected since it has been characterized extensively and is the
standard genetic instrument in murine systems (31).
Ad/GM-CSF, Ad/IP-10, and Ad/IFN- were administered at doses of
3x107, 1x108, and
1x108 PFU, respectively, in 30 µl of PBS
vehicle; an appropriate dose of an E1-deleted replication-deficient
adenovirus (RDA) was used to control for the higher viral burden in the
Ad/IP-10 and Ad/IFN- groups. The Ad/IFN- vector was a kind gift
from J. Kolls (Louisiana State University Medical Center, New Orleans,
LA), and the Ad/IP-10 (31) and Ad/GM-CSF (32)
vectors were engineered and characterized previously by our
laboratory.
Collection and measurement of specimens
Two days after the last OVA exposure (day 11) and at various time points during the aerosolization regimen, mice were sacrificed and BAL was obtained as previously described (33). In brief, the lungs were dissected and the trachea cannulated with a polyethylene tube (Becton Dickinson, Sparks, MD). The lungs were lavaged twice with PBS (0.25 ml followed by 0.2 ml). Approximately 0.3 ml of the instilled fluid was consistently recovered. Total cell counts were determined using a hemocytometer. After centrifugation, supernatants were stored at -20°C for cytokine measurements by ELISA; cell pellets were resuspended in PBS and smears were prepared by cytocentrifugation (Shandon, Pittsburgh, PA) at 300 rpm for 2 min. Diff-Quik (Baxter, McGraw Park, IL) was used to stain all smears. Differential counts of BAL cells were determined from at least 500 leukocytes using standard hemocytological criteria to classify the cells as neutrophils, eosinophils, lymphocytes, or macrophages/monocytes. Additionally, blood was collected by retro-orbital bleeding. Serum was obtained by centrifugation after incubating whole blood for 30 min at 37°C. Finally, lung tissue was fixed in 10% Formalin and embedded in paraffin. Sections (3-µm thick) were stained with hematoxylin and eosin or with periodic acid-Schiff (PAS) to distinguish mucous production in goblet cells.
Cytokine and Ig measurement
ELISA kits for IL-4, IFN-, eotaxin, monocyte chemoattractant
protein (MCP) 1, MIP-1, and RANTES were purchased from R&D Systems
(Minneapolis, MN), while the kit for IL-5 was obtained from Amersham
(Buckinghamshire, U.K.). IP-10 was measured by ELISA according to a
standard alkaline phosphatase/streptavidin assay protocol (R&D
Systems). Briefly, IP-10 was captured with an anti-human IP-10 Ab
in the solid phase and developed with biotinylated anti-human IP-10
(R&D Systems); recombinant human IP-10 (PeproTech, Rocky Hill, NJ) was
used as a standard. Levels of OVA-specific IgE were detected using an
Ag-capture (biotinylated OVA) ELISA method that has been described
previously (33); the ELISA was standardized with serum
obtained from mice sensitized to OVA according to a conventional i.p.
sensitization model (34) and bled 24 h after the
second sensitization. OVA-specific IgG2a was measured by sandwich ELISA
with OVA in the solid phase. Ninety-six-well plates were coated with 5
µg/ml OVA in borate buffer (100 µl/well) for 1 h at 37°C,
3 h at room temperature, and then overnight at 4°C. Plates were
blocked for 2 h at room temperature with 150 µl/well 1% BSA in
PBS before loading samples (50 µl/well). Plates were then incubated
overnight at 4°C and washed before adding 50 µl of 0.25 µg/ml
biotinylated anti-mouse IgG2a Ab (Southern Biotechnology
Associates, Birmingham, AL) to each well. Following a 2-h incubation at
room temperature, plates were washed, incubated with alkaline
phosphatase/streptavidin for 1 h at room temperature (50 µl/well
at a concentration of 1:1000), and developed with
p-nitrophenyl phosphate substrate dissolved in
diethanolamine buffer. This ELISA was standardized with serum obtained
from mice sensitized to OVA according to our Th1-polarized mucosal
sensitization model (33) and bled at day 11 of the
protocol. Ig levels are expressed in units per milliliter relative to
standard sera.
Lung cell isolation and flow cytometric analysis of lung cell subsets
Lungs were perfused with 10 ml of prepared buffer (10% FBS, 1%
penicillin/streptomycin in HBSS) through the right ventricle, cut into
small (2-mm diameter) pieces, and agitated at 37°C for 1 h in
15 ml of collagenase III (Life Technologies, Rockville, MD) at a
concentration of 150 U/ml in HBSS. Using the plunger from a 5-ml
syringe, the lung pieces were triturated through a metal screen into
HBSS, and the resulting cell suspension was filtered through nylon
mesh. Mononuclear cells were isolated at the interphase between layers
of 30 and 60% Percoll following density gradient centrifugation. Cells
were washed twice and stained for flow cytometric analysis. For each Ab
combination, 0.5 x 106 cells were incubated
with mAbs at 0–4°C for 30 min; the cells were then washed and
treated with second-stage reagents. For detection of intracellular
IFN-, cells were first polyclonally stimulated with PMA/ionomycin in
4-h cultures and then permeabilized with saponin according to a
standard protocol (BD PharMingen catalogue, PharMingen, San Diego, CA).
Data were collected using a FACScan (Becton Dickinson, Sunnyvale, CA)
and analyzed using WinMDI software (The Scripps Research Institute, La
Jolla, CA). The following Abs and reagents were used: anti-CD3, PE
conjugated (145-2C11); anti-CD3, CyChrome conjugated (145-2C11);
anti-CD4, FITC conjugated (RM4-5); anti-CD4, CyChrome
conjugated (RM4-5); anti-CD8, FITC conjugated (53-6.7);
anti-CD69, PE conjugated (H1.2F3); anti-IFN-, PE conjugated
(XMG1.2) (all purchased from BD PharMingen); anti-T1/ST2, PE
conjugated (Millennium Pharmaceuticals, Cambridge, MA); and
streptavidin PerCP (BD PharMingen). The Abs were titrated to determine
optimal concentration.
Data analysis
Data are expressed as mean ± SEM, unless otherwise indicated. Results were interpreted using ANOVA followed by the Student-Newman-Keuls post hoc test. Differences were considered to be statistically significant when p < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To investigate the impact of expressing a Th1-affiliated chemokine
on a system that is otherwise predisposed to the development of a
Th2-polarized response, we used a mucosal model of allergic airways
inflammation in which mice are exposed daily to aerosolized OVA over a
period of 10 consecutive days in the context of GM-CSF expression in
the airway. In this study, 1x108 PFU Ad/IP-10 or
control virus (RDA) were coadministered i.n. with Ad/GM-CSF to BALB/c
mice, which were then subjected to the OVA exposure regimen.
Administration of Ad/IP-10 to naive mice resulted in sustained
expression of IP-10 in the airways for 7–10 days, with peak
expression of 2100 pg/ml in the BAL 1 day after vector
administration, followed by resolution at day 4 and a secondary peak of
IP-10 (600 pg/ml) in the BAL at day 7. Two days after the last OVA
exposure, mice were sacrificed and the cellular profile in the BAL was
assessed (Fig. 1
). Mice exposed to OVA in
the presence of IP-10 but in the absence of GM-CSF had modestly
elevated mononuclear cells in the BAL. As expected, exposure to OVA in
the context of GM-CSF expression resulted in marked mononuclear and
eosinophilic inflammation in the BAL; concurrent administration of RDA
did not significantly alter this inflammatory response. Although
concurrent IP-10 expression did not appreciably affect the quantity of
inflammation in mice treated with GM-CSF and OVA, it did change the
phenotype of the infiltrate; in particular, IP-10 reduced the number of
eosinophils in the BAL by 60–75%. To verify that this reduction in
airways eosinophilia was not the consequence of eosinophil retention in
the circulation, peripheral blood leukocytes were assessed; IP-10
expression in the airway resulted in a >50% decrease in eosinophil
counts in the circulation (Table I).
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We conducted a detailed histological analysis that demonstrates a
correlation between processes occurring in lung tissue and the findings
in BAL at day 11 of the aerosolization protocol. Exposure to OVA in the
context of GM-CSF led to marked peribronchial and perivascular
inflammation that was distinctly eosinophilic in nature (Fig. 2A). Evidence of goblet cell
hyperplasia was confirmed in PAS-stained tissue (Fig. 2E),
which documents pervasive expansion of mucous-producing cells. Analysis
of lung tissue from RDA-treated mice revealed similar histopathological
phenomena (data not shown). In contrast, concurrent expression of IP-10
reduced the accumulation of eosinophils in lung tissue, although robust
mononuclear inflammation persisted (Fig. 2, B and
D); moreover, there was significant, if somewhat attenuated,
goblet cell hyperplasia along the airway epithelium of these mice (Fig. 2F). Exposure to OVA in the context of IP-10 expression
alone resulted in a comparably modest mononuclear cell infiltrate (Fig. 2C), with no evidence of mucous-secreting cells (data not
shown).
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To evaluate IP-10-mediated changes in the Th1/Th2 balance of the
immune response to OVA, we assayed a number of definitive mediators
(Table II); we report only peak levels of
expression, which occurred on day 7 of the protocol for cytokines and
chemokines in the BAL and on day 11 for Igs in serum. As shown in Table II, delivery of RDA did not significantly alter the levels of IFN-,
IL-4, and IL-5 detected in mice exposed to OVA in the context of
GM-CSF. In contrast, IP-10 intervention resulted in a 4- to 10-fold
increase in the level of IFN- in the BAL, while IL-4 was virtually
undetectable and IL-13 was significantly attenuated; IL-5 content in
the BAL was also reduced by about 40%, although this change did not
reach statistical significance. Moreover, mice treated with IP-10
presented 2- to 3-fold higher levels of the proinflammatory chemokines
MCP-1, MIP-1, and RANTES in the BAL and produced three to four times
more OVA-specific IgG2a, a Th1-affiliated Ig, than mice treated with
GM-CSF alone or concurrently infected with GM-CSF and RDA control. It
is noteworthy that IP-10 treatment did not result in a statistically
significant reduction in levels of OVA-specific IgE, nor did it alter
expression of eotaxin, hallmarks of the eosinophilic response, an
intriguing observation given the dramatic reduction in BAL, tissue, and
peripheral blood eosinophilia in IP-10-treated animals.
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in IP-10-mediated suppression of eosinophilia
Given the demonstrated association between IP-10 and IFN-
(26, 27, 28, 29), and the dramatic up-regulation of IFN- in the
BAL elicited by IP-10 expression in the airway, we proceeded to explore
mechanistically the role of this prototype Th1 mediator in
IP-10-mediated inhibition of airways eosinophilia. Therefore, we
executed the exposure regimen in IFN- KO mice. In this setting,
airway expression of IP-10 did not attenuate BAL eosinophilia,
indicating that the modulatory effect of IP-10 is dependent on IFN-
(Fig. 3A). To expand this
observation and understand more comprehensively the role of IFN- in
this system, we substituted Ad/IP-10 with an adenoviral construct
expressing the transgene for IFN-; this construct results in
sustained expression of IFN- in the BAL for 10 days and reaches
levels of 2000 pg/ml in the BAL 7 days after i.n. delivery. In mice
exposed to OVA in the context of GM-CSF, concurrent expression of
IFN- completely abrogated BAL eosinophilia, indicating that IFN-
per se, when expressed at levels comparable to those elicited by
Ad/IP-10, is sufficient to prevent the airways eosinophilia to which
this protocol is predisposed (Fig. 3B).
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We used flow cytometric analysis of dispersed lung mononuclear
cells to evaluate the effect of IP-10 expression on the pattern of T
lymphocyte recruitment to the airway. In particular, we examined
CD3+CD4+ and
CD3+CD8+ T cells at day 7
of the aerosolization protocol for expression of the early activation
marker CD69. Table III indicates that
approximately two to three times as many CD8+ T
cells were isolated from the lungs of mice treated with IP-10 and
exposed to OVA in a GM-CSF milieu compared with RDA-treated control
mice; compared with GM-CSF alone (data not shown), delivery of RDA had
no distinguishable effect on any of the T cell parameters we examined.
These differences are particularly pronounced when expression of the
early activation marker CD69 is examined (Fig. 4), as IP-10 treatment resulted in an
increase in the fraction of both
CD4+CD69+ and
CD8+CD69+ T cells in the
airway compared with RDA-treated control mice. Moreover, these T cells
expressed a phenotype consistent with the alleged Th1 bias of IP-10; as
shown in Table IV, while the proportion
of CD3+CD4+ cells
expressing the putative Th2 marker T1/ST2 (35, 36, 37)
declined by 40–50% in IP-10-treated mice, there was a concomitant
50% increase in the frequency of CD4+ cells
expressing intracellular IFN-. These data indicate that the
Th1-biased immune phenomena observed in both the inflammatory and
cytokine responses of IP-10-treated animals may have practical
implications for T cell phenotype.
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To determine whether expression of IP-10 in the airway at the time
of the initial mucosal sensitization affected the development of T cell
memory, we investigated the long-term in vivo response of treated mice
to aerosolized OVA. On day 50, several weeks following the resolution
of airways inflammation, mice were re-exposed to aerosolized OVA for
1 h on two occasions 4 h apart. Seventy-two hours later, mice
were sacrificed and the cellular profile of the BAL was assessed (Fig. 5). Although there was a reconstitution
of airways eosinophilia in mice treated with GM-CSF alone or with
GM-CSF and RDA, IP-10 expression at the time of initial Ag exposure
resulted in markedly attenuated eosinophilia upon OVA rechallenge,
indicating that the OVA-specific immune response was substantively
altered by IP-10. Flow cytometric analysis of T cell subsets isolated
from homogenized lungs of mice sacrificed 72 h after in vivo OVA
recall indicates that while IP-10 intervention did not appreciably
alter the recruitment of CD4+ and
CD8+ T cells to the airways of mice exposed to
OVA in the context of GM-CSF and RDA, the ratio of activated
(i.e., CD69+) CD4 to CD8 T cells
changed considerably, from 3 in control mice to 0.9 in mice exposed
to OVA in the presence of both GM-CSF and IP-10. This change was the
consequence of a 50% increase in the number of activated
CD8+ T cells and a parallel 50% reduction in
activated CD4+ T cells in the airways of
IP-10-treated mice compared with mice initially exposed to OVA in the
context of GM-CSF and RDA (Fig. 6A); RDA intervention had no
distinguishable effect on the lymphocyte profile following OVA
rechallenge compared with mice receiving GM-CSF alone (data not shown).
Cytokine content in the BAL was assessed 24 h following OVA
rechallenge (Fig. 6B); interestingly, although similar
levels of IFN- and IL-5 were detected in all groups, mice initially
treated with IP-10 produced considerably less IL-4 than mice sensitized
to OVA in the context of GM-CSF alone (data not shown) or GM-CSF
and RDA.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), which results in changes in the molecular
and cellular profile both locally and systemically. In particular,
airway expression of IP-10 attenuates IL-4 and IL-13 production,
induces vigorous IFN- and OVA-specific IgG2a responses, and elicits
a cascade of proinflammatory chemokines, including MCP-1, RANTES, and
MIP-1. These molecular changes correlate with a 60–75% inhibition
of BAL eosinophilia and the recruitment of mononuclear cells and
neutrophils, inflammatory changes that were confirmed by
histopathological analysis of lung sections.
Several observations, however, are not satisfactorily explained by the
conclusion that IP-10 expression in the context of a GM-CSF milieu
merely elicits a Th1-polarized response. Our data at early time points
show that significant levels of OVA-specific IgE, the cardinal humoral
feature of the Th2 phenotype, are produced in animals exposed to OVA in
the context of IP-10. In the effector organ, i.e., the airway, the two
prototypic Th2 cytokines IL-5 and IL-13 are readily detected, whereas
histopathological features associated with a Th2 response, such as
goblet cell hyperplasia and mucous production, persist. On the other
hand, that IP-10 simultaneously induces a robust IFN- response and
substantially elevated levels of OVA-specific IgG2a in serum suggests
that IP-10 expression, rather than deviate the system away from Th2
polarization, superimposes a competing Th1 response upon an assiduous
Th2 background.
However, although IL-5 and IL-13 are detected in the BAL of
IP-10-treated mice, the levels of these cytokines are
diminished—significantly in the case of IL-13—compared with control
mice exposed to OVA following concurrent administration of RDA and
GM-CSF. We argue that these attenuated Th2 phenomena are the
consequence of the diminished influx of Th2 cells into the tissue; our
data, which demonstrate a 40% reduction in the proportion of
CD4+ T cells expressing the bonafide Th2 marker
T1/ST2, support this hypothesis. Moreover, that both the level of IL-13
and the pervasiveness of goblet cell hyperplasia in the airway are
reduced, but persistent, in IP-10-treated mice agrees with the
demonstrated role of IL-13 in promoting epithelial remodeling and
mucous production (38); the presence of IL-13 may also
account for the significant levels of OVA-specific IgE detected in
IP-10-treated animals (39). Finally, the comparative
reduction in IL-5 in mice exposed to OVA in the context of IP-10 and
GM-CSF is consistent with reduced, but not absent, peripheral blood
eosinophilia in these animals. Indeed, it may be that dramatically
reduced levels of IL-4 in BAL prevent the up-regulation of VCAM-1 on
pulmonary endothelium, resulting in the compartmentalization of
eosinophils in the circulation (40). As a future
consideration, the competing Th1 phenomena elicited by IP-10 may
provide a prolific experimental setting to explore the role of
eosinophils, IgE, and Th2 mediators in the development of bronchial
hyperreactivity.
The dramatically elevated levels of IFN- in the BAL of IP-10-treated
animals prompted us to explore the role of this cytokine in the
subversion of the Th2-polarized, GM-CSF-driven response to aerosolized
OVA. We therefore executed the same treatment regimen in IFN- KO
mice and found that IP-10 expression did not significantly inhibit BAL
eosinophilia, demonstrating that the effect of IP-10 on this outcome is
dependent on IFN-. To evaluate more comprehensively the role of
IFN- in immune deviation in this experimental setting, we directly
investigated the effect of IFN- airway expression in wild-type
(i.e., IFN- sufficient) mice. We observed complete abrogation of
eosinophilia, and a reduction in mononuclear cell inflammation
generally, in the BAL of Ad/IFN--treated animals, indicating
that overexpression of IFN- is sufficient to subvert the
eosinophilic character of the GM-CSF-driven inflammatory response to
OVA. These findings inform a comparison with the apparently parallel
effect induced by airway expression of IL-12 in this model of mucosal
allergic sensitization (33). Expression of IL-12 in this
setting results in an Ag-specific, Th1-polarized immune response that
is IFN- independent, whereas the ultimately similar effect elicited
by IP-10 is dependent on IFN-. This suggests different pathways
capable of Th2 subversion: although IL-12 can directly mediate Th1
polarization at the level of the APC (33), we hypothesize
that IP-10 initiates a cascade of events, such as preferential
recruitment and activation of IFN--producing cells, that introduce
Th1 phenomena to an existing Th2 context.
The levels of IP-10 protein in BAL exhibit a curious time course: there
is an initial burst of IP-10 in BAL 1 day after i.n. administration of
the vector, followed by a secondary, less robust peak on the seventh
day. We suggest that this second peak is mediated by IFN-. In this
regard, Gangur et al. (29, 41) have shown that rIP-10
selectively enhances polyclonally driven and Ag-specific IFN-
production by cultured PBMC, and a variety of leukocyte subsets and
structural cells have been reported to express IP-10 upon stimulation
with IFN- (26, 27, 28). Additionally, Xie et al.
(20) have shown that IP-10 injected into the peritoneum of
mice preferentially recruits adoptively transferred, Th1-polarized
CD4+ T cells, which in turn may serve as a
cellular source of IFN-. Collectively, these observations support
the hypothesis that the secondary peak of IP-10 expression 7 days after
vector administration (and 3 days after resolution of the initial burst
of vector-derived IP-10) may in fact reflect IFN- production by
resident cells or by IFN--producing cells preferentially recruited
by exogenous IP-10.
To explore the viability of this hypothesis, we used flow cytometry to
examine T cell subsets in the lung at day 7 of the aerosolization
protocol, the point at which we observe peak expression of IFN- in
the BAL of IP-10-treated mice. We found that mice exposed to OVA in the
context of GM-CSF and IP-10 had elevated CD8+ T
cell populations in the lung compared with control mice. These
differences were particularly pronounced when the activation status of
these lymphocytes was considered: IP-10 intervention resulted in
markedly enhanced expression of CD69 on both CD4+
and CD8+ T cells, an observation consistent with
the finding that IP-10 preferentially recruits activated T cells
(15, 19). To characterize more comprehensively the
phenotype of T lymphocytes in the lung, we studied T cells for surface
expression of T1/ST2, a putative marker of Th2 differentiation
(35, 36, 37), and for intracellular IFN-, the prototypic
Th1 effector cytokine. Although CD8+ T cells,
irrespective of treatment regimen, expressed only basal levels of
T1/ST2 and exhibited no change in intracellular IFN- (data not
shown), there was a reduction in the proportion of
CD4+ T cells displaying T1/ST2 and a
corresponding increase in the fraction of CD4+
cells expressing intracellular IFN-. Thus, it is tempting to
envision a model in which IP-10, through the preferential recruitment
of activated CD8+ and Th1 cells, establishes an
airway microenvironment conducive to the differentiation and
amplification of a Th1-polarized response to OVA.
To elucidate these Th1-biased phenomena and to investigate whether
IP-10 intervention altered the memory response to OVA, we subjected
mice to an OVA aerosol challenge well after resolution of acute
inflammation in the airway and clearance of the vector-encoded IP-10
and GM-CSF transgenes. We observed that OVA-rechallenged mice whose
initial Ag encounter occurred in the context of an IP-10/GM-CSF milieu
exhibited significantly less eosinophilia, though comparable total
inflammation, in the BAL compared with mice initially treated with
GM-CSF alone. This observation argues strongly for the persistence of a
distinct, Ag-specific memory lymphocyte population and, concomitantly,
suggests that the airway microenvironment established by IP-10 during
primary OVA exposure influenced the evolution of the adaptive response
to OVA. Examination of cytokine production in the BAL and of lymphocyte
populations in the lung following OVA rechallenge complement findings
during primary Ag exposure. Indeed, concurrent IP-10 expression in the
airway at the time of initial OVA delivery privileges the recruitment
of activated CD8+ T cells to the lung upon in
vivo OVA recall. Moreover, although IFN- and IL-5 content in the BAL
of IP-10-treated mice is unaltered—observations that speak to the
heterogeneity of the Th1/Th2 balance and the persistence of Th2
phenomena following IP-10 intervention—IL-4 production is ablated,
indicating that the underlying Th2 character of GM-CSF-mediated
sensitization to OVA has been permanently impoverished by concurrent
expression of IP-10.
In summary, we have shown that expression of the chemokine IP-10 can introduce a competing Th1 phenotype to an immunological setting otherwise predisposed to the development of allergic airways inflammation and can thereby subvert Th2 features of the ensuing response. Extrapolated to a broader immunological and clinical context, these data illustrate the plasticity of T helper responses and reinforce the importance of the microenvironment in elaborating and maintaining such responses. That an IP-10 immunological milieu has the capacity to alter the nature of an Ag-specific, Th2-polarized immune-inflammatory response attributes to this chemokine an immunomodulatory function that transcends chemotaxis.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Manel Jordana, Department of Pathology and Molecular Medicine, Room 4H21, Health Sciences Center, McMaster University, 1200 Main Street West, Hamilton, Ontario, Canada L8N 3Z5.
3 Abbreviations used in this paper: IP, IFN-inducible protein; MIP, macrophage-inflammatory protein; CXCR, CXC chemokine receptor; BAL, bronchoalveolar lavage; KO, knockout; Ad, adenovirus; i.n., intranasal; RDA, replication-deficient Ad; PAS, periodic acid-Schiff; MCP, monocyte chemoattractant protein; Rx, treatment.
Received for publication September 5, 2000. Accepted for publication November 30, 2000.
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