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University of New Mexico, Departments of
* Pathology and
Internal Medicine, Albuquerque, New Mexico; and
Lovelace Respiratory Research Institute, Albuquerque, New Mexico
Correspondence: Julie A. Wilder, Ph.D., University of New Mexico, Department of Pathology, Albuquerque, NM 87131. E-mail: jwilder{at}salud.unm.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: rodent • lung • allergy • T lymphocytes • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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R1 on mast cells and eosinophils, which stimulate
the release of compounds capable of mediating airway smooth-muscle
contraction and airway lumen narrowing (reviewed in
[2
]). Murine models of allergic asthma have been developed to study the pathobiology of the disease (reviewed in [3 ]). Most of these models involve systemic priming with antigen, often in adjuvant, followed by pulmonary exposure to the antigen. The results are varied, in part because of the strain of mouse being tested and immunization protocol used, but generally include pulmonary inflammation, often eosinophilic in nature, high serum levels of allergen-specific IgE, and airway hyperreactivity (AHR) in response to a nonspecific bronchoconstrictive agent such as methacholine or acetylcholine. In contrast, few models have been described that use aerosol exposures exclusively for the induction of the disease state [4 ]. Indeed, antigen delivery via multiple aerosol exposures has been shown to induce tolerance in rodents as measured by their subsequent inability to respond to the same antigen when administered with adjuvant via an immunogenic route such as the intraperitoneal (i.p.) one [5 , 6 ].
We developed a murine model of asthma initiated by immunization solely via the pulmonary route to more closely mimic the pathobiology of the human disease. Mice hemizygous for the DO11.10 ovalbumin (OVA)-T cell receptor transgene (DO11.10 +/-) [7 ] were exposed to an OVA or saline aerosol once/week for 3 consecutive weeks. These mice bear the transgene on the background of a BALB/c mouse, a strain that, once immune, requires only limited exposures to OVA aerosol to exhibit AHR [8 ]. Additionally, DO11.10 mice are resistant to tolerance induction by OVA given via a normally tolerogenic route [i.p. without adjuvant or intravenous (i.v.)] [9 ]. A colony of hemizygous mice was established (DO11.10 +/-), in which 40% of peripheral T cells stain with the clonotypic monoclonal antibody (mAb) KJ1-26. In mice from this colony, the accumulation and activation status of OVA-T cell receptor (TCR) bearing T cells in the lung and lung-associated lymph nodes (LALNs) could be monitored in response to OVA- or saline-aerosol exposure.
We show here that DO11.10 +/- but not DO11.10 transgene-negative mice developed AHR after limited exposure to OVA aerosols. AHR was accompanied by a mild peribronchiolar inflammatory response and accumulation of OVA-TCR+ T cells in the lung, which expressed decreased levels of CD62L and CD45RB and increased levels of CD69, indicating that they were recently activated. We failed to observe significant increases in total serum IgE or OVA-specific IgG. Nor were we able to demonstrate an increase in eosinophils in the bronchoalveolar lavage fluid or in the lung parenchyma, even when focusing on peribronchiolar areas. These data suggest that AHR can be mediated by a very small number of antigen-specific T cells that have an activated phenotype.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Aerosol exposures
Mice were exposed in a nose-only aerosolization chamber (Intox,
Albuquerque, NM) to pH neutral saline or 0.5% OVA (Grade V, Sigma
Chemical Co., St. Louis, MO) in pH neutral saline. All mice received
three aerosol exposures, 1 h in duration, delivered 7 days apart.
Aerosol particles were generated using a Lovelace Nebulizer such that
the size was <1 µ in diameter (6 L air/min; 42 psi).
Pulmonary physiology
Changes in total lung resistance (RL) were measured
in anesthetized, tracheotomized, ventilated mice, which were placed in
a volume-displacement plethysmograph as described previously
[8
]. Mice were anesthetized with an i.p. injection of a
solution of xylazine and ketamine in sterile saline at a dose of 16
µg xylazine and 80 µg ketamine/g bodyweight. A tail-vein catheter
[10 cm polyethylene (PE)-10 tubing attached to a 30-gauge
needle and filled initially with heparinized saline] and a tracheal
catheter (20-gauge needle hub) were inserted, sealed, and secured with
cyanoacrylate adhesive. Once placed in the plethysmograph, mice were
ventilated at a rate of 150 breaths/min and a 0.25 ml tidal vol. An
opening was made in either side of the caudal chest wall by removing a
portion of a rib to equilibrate pleural-surface pressure to
body-surface (box) pressure and facilitate the measurement of
transpulmonary pressure. The resistance of the tracheal cannula was
determined by ventilation of the plethysmograph in the absence of a
mouse and the value subtracted from all resistance measurements.
Custom-designed computer software (LabView 3.0.1, National Instruments,
Austin, TX) was used to facilitate integration of the flow signal to
yield volume and to derive pulmonary resistance using the method of
least-squares linear regression. Baseline measurements of
RL were recorded prior to delivery of saline and half-log
increasing doses of methacholine (.014–3.7 mg/kg) administered via the
tail-vein catheter. Peak responses were recorded and values allowed to
return to within 10% of baseline before delivery of the next dose.
Recovery was facilitated by 2–3 forced vital-capacity maneuvers. Data
are presented as the actual change in RL from baseline
values.
Lung histology
Lungs were inflated with 10% buffered neutral formalin fixative
injected through PE-50 tubing inserted into the trachea. The lungs,
once excised, were fixed for at least 24 h before the left lobes
were sectioned longitudinally along the major airway and submitted to
the UNM Hospital Pathology Laboratory (Albuquerque, NM) where they were
embedded, sectioned, and stained with hematoxylin and eosin.
Three 10x fields per lung, generally covering the whole left lobe in
total, were examined and scored separately for the percent inflammatory
cell infiltration around bronchioles, peribronchial arteries, and
veins, according to the following scheme as previously described
[8
]: Less than 1% of the circumferential or
longitudinal area involved with any type of inflammation was scored as
a 0; 1–5% involvement = 0.5; 5–10% involvement = 1;
10–25% involvement = 2; 25–50% involvement = 3; and
>50% involvement = 4. The scores of each of the three sections
were averaged to obtain a peribronchiolar, periarterial, and
perivenular score for each lung. The total lung score represents the
sum of the scores of these three areas. Two independent observers
(J.A.W. and M.F.L.) scored each lung lobe in a blinded fashion. The
average of the two observations is shown for each mouse examined. Data
presented are the average (±SE) of individual mice from
several experiments. In addition, all inflamed areas of the lungs of
DO11.10 +/- mice exposed to OVA aerosols were examined under oil
immersion (100x) to distinguish the types of inflammatory cells
present. Monocytoid cells (a combination of lymphocytes and monocytes),
macrophages, polymorphonuclear cells, and eosinophils were enumerated
in inflamed peribronchiolar, periarterial, and perivenular areas.
Harvest of tissue and fluid
All mice were sacrificed by inhalation of CO2 1 day
following the last aerosol exposure. Serum and tracheobronchial lavage
(TBL; 0.3 ml/mouse) were collected as previously described
[8
]. Cells recovered from the TBL were counted and used
for cytospin preparations (25,000 or less/slide). These cytospins were
stained with the Baxter Diff-Quick kit (VWR Scientific Products, San
Francisco, CA), and differentials were calculated. LALN cells were
dispersed into single-cell suspensions by gently rubbing the tissue
between the frosted ends of two glass slides followed by red blood cell
(RBC) lysis with ammonium chloride. Single-cell suspensions of lung
cells were prepared by mincing saline-perfused lungs followed by a
90-min incubation with collagenase (0.7 mg/ml) and DNAse (30 µg/ml)
at 37°C. Lung cells were then gently pushed through a wire mesh and
passed over a loose, nylon wool plug quickly to remove connective
tissue and debris. RBCs were lysed, and the remaining cells were spun
through a layer of 30% Percoll to remove cell debris and enrich for
viable cells.
Cell phenotyping
Lung and LALN cells (0.5–1x106) were stained with
antibodies of interest in a volume of 0.06 ml staining buffer
[phosphate-buffered saline (PBS)+1% fetal calf serum (FCS)] for 30
min on ice. Antibodies included KJ1-26-biotin [10
]
(prepared from hybridoma supernatants by sodium ammonium sulfate
precipitation and biotinylated), CD4-fluorescein isothiocyanate (FITC)
or CD4-allophycocyanin (APC), CD62L-PE, CD45RB-PE, and
CD69-FITC (all from Pharmingen, San Diego, CA). Incubation with these
primary antibodies was followed by three washes in staining buffer and
fixation with 0.5% paraformaldehyde at 4°C or in the case of
KJ1-26-biotin-stained cells, incubation with Streptavidin-PerCP
(Becton-Dickinson, San Jose, CA) for 30 min on ice and subsequent
washing and fixation. All cells were analyzed on a Becton-Dickinson
FACScan or FACSCalibur and analyzed using PCLYSIS or CELLQUEST software
(both from Becton-Dickinson), respectively.
Immunoglobulin enzyme-linked immunosorbent assays (ELISAs)
OVA-specific IgG in the sera was measured as previously
described [8
] using OVA-coated polyvinylchloride (PVC)
plates (Falcon brand, Fisher Scientific, Pittsburgh, PA; 0.1 mg/ml
OVA). Sera was diluted in PBS containing 0.25% bovine serum albumin
(BSA) and 0.05% Tween 20 (blocking buffer). Three dilutions of
sera/mouse were added to the plates in duplicate, and incubation
proceeded overnight at 4°C. OVA-specific IgG was detected using
horseradish peroxidase (HRP)-coupled goat anti-mouse IgG1 or IgG2a
(Southern Biotechnologies, Birmingham, AL) diluted in blocking buffer,
incubated 2 h at room temperature (RT), followed by addition of
HRP substrate (ABTS, Sigma). Color development proceeded at RT until
the least dilute-serum sample approached an OD405 of
1.0–2.0. Plates were read on a Dynatech ELISA reader (Bio-Tek
Instruments, Inc., Winooski, VT). The OD405 of each serum
dilution was multiplied by its dilution factor to give an arbitrary
unit. For each mouse, the units of IgG anti-OVA are calculated from the
three serum dilutions and an average presented. Total IgE levels in the
serum were determined by a sandwich ELISA using PVC plates coated with
rat anti-mouse IgE (clone R35, Pharmingen; 2 µg/ml) and detected
using rat anti-mouse IgE-HRP (Southern Biotechnologies). Coating, serum
dilutions, washing, and development procedures were carried out as
described for the OVA-specific IgG ELISA. A known amount of monoclonal
IgE was used to construct a standard curve in each assay, and values
are shown as ng/ml.
Production and analysis of cytokines
Lung cell cultures and cytokine analysis were performed as
described previously with minor modifications [11
]. Lung
cells were incubated on plastic tissue culture dishes for 2 h at
37°C, and nonadherent cells were harvested, washed, and resuspended
at 5 x 106/ml in culture medium [RPMI 1640
supplemented with 10% fetal bovine serum (FBS), 100 U/ml
penicillin/streptomycin, 2 mM L-glutamine, 1 mM sodium pyruvate, 1 mM
nonessential amino acids (all from Gibco BRL Life Technologies, Grand
Island, NY), 5 x 10-5 M
2-mercaptoethanol (Eastman Kodak, Rochester, NY), 1 µg/ml
indomethacin (Sigma), and 250 units/ml catalase (Worthington Biochem,
Freehoff, NJ)]. Anti-IL-4 receptor (1 µg/ml; Genzyme, Cambridge, MA)
was included in all cultures to prevent uptake of secreted IL-4 and
facilitate its assessment by ELISA. Cells were left unstimulated or
stimulated with OVA protein (100 µM), OVA peptide (323–339, 4 µM;
Research Genetics, Huntsville, AL), or keyhole limpet hemocyanin (KLH;
5 µg/ml; Calbiochem Novabiochem, La Jolla, CA) in duplicate or
triplicate for 48 h, after which supernatants were collected and
analyzed for cytokine content by sandwich ELISA. ELISA plates (Nunc
Maxisorb Immunoassay, VWR) were coated with capture antibodies diluted
in 0.1 M Na2HPO4 overnight at 4°C, washed,
and blocked with 1% BSA in PBS. Samples were added to the plates after
subsequent washes and incubated overnight at 4°C. Detection proceeded
the following day by adding biotinylated mAbs, streptavidin-HRP (1
mg/ml), and ABTS substrate, and the OD405 was read as
described above. mAb pairs for the cytokines were purchased from
Pharmingen in the case of IL-4 (11B11 and biotin-BVD6-24G2), IL-5
(TRFK5 and biotin-TRFK4), and interferon (IFN)-
(R46A2 and
biotin-XMG1.2). Antibodies specific for IL-13 (38213.11 and
biotin-BAF413) were purchased from R&D Systems, Minneapolis, MN. All
cytokines were quantified by comparison to standard curves generated
using recombinant cytokines (Phamingen). Detection limits for each
cytokine assay were assigned as the lowest concentration in the linear
portion of the standard curve, generally between 16 and 250 pg/ml.
Statistics
Differences in RL responses to
methacholine between groups were analyzed by repeated measures analysis
of variance (ANOVA). RL responses between groups at
individual doses were compared using unpaired t-tests to
determine significance. Differences in all other measured variables
were analyzed using ANOVA statistics using the Bonferroni-Dunn post-hoc
test when four groups were being compared or unpaired two-tailed
t-tests when two groups were being compared. Values of
P < .05 were considered significant for all
comparisons.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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secretion above that seen in cultures of
saline-exposed DO11.10 lung cells could be demonstrated. For reasons
that are unclear to us, secretion of IL-5 by naïve DO11.10 +/-
lung cells in response to OVA protein and OVA peptide was also
increased significantly compared with that secreted by similarly
treated lung cells from saline-exposed mice. This was not the case for
any other cytokine measured. It is important that cytokine secretion in
response to KLH, a nonspecific antigenic stimulus, was never
significantly greater than that observed in media alone, regardless of
aerosol exposure.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The fact that DO11.10 +/- mice responded to a limited number of OVA-aerosol exposures by developing AHR suggests that an expanded repertoire of OVA-specific T cells is sufficient for mice to manifest AHR in response to this minimal pulmonary stimulus. These data help explain why most murine models of allergic asthma, which include AHR as a manifestation of antigen exposure, require a systemic priming step with antigen prior to pulmonary-antigenic challenge [15 16 17 18 19 20 21 ]. Systemic priming serves to expand the pool of antigen-specific T cells. In naïve DO11.10 +/- mice, however, 40% of the cells in the T cell repertoire bear the OVA-TCR receptor by virtue of the transgene expression, which eliminates a systemic priming requirement. However, DO11.10 -/- littermates, which have a normal T cell repertoire and numbers of OVA-TCR+ cells similar to normal BALB/c mice, were unable to respond to limited pulmonary exposure to OVA by exhibiting AHR. The only other study of normal mice developing AHR in response to pulmonary antigen exposure in the absence of systemic priming or adoptive transfer of previously primed cells required a more prolonged aerosol exposure protocol (daily for 10 days) [4 ]. These latter mice did not develop pulmonary inflammation but developed high levels of circulating OVA-specific IgE and IgG.
It was recently shown that homozygous, transgenic DO11.10 mice exposed
to a single whole-body OVA aerosol (20 min in duration) or four OVA
aerosol exposures delivered on consecutive days failed to develop AHR,
as measured by increases in enhanced pause (PenH) to aerosolized
methacholine [22
]. These data suggest that the timing of
OVA aerosol exposure (acute vs. more chronic), the mode of OVA aerosol
delivery (whole-body or nose-only), the number of transgenic T cells
(presumably more in homozygous vs. hemizygous DO11.10 mice), or the
methodology used to measure AHR (Buxco box vs. whole-body
volume-displacement plethysmography) is critical in inducing AHR in
DO11.10 transgene-positive mice. It is interesting that, in agreement
with our current studies, these authors also described TBL
neutrophilia, little eosinophilia, and a failure to mount a humoral
immune response after a single exposure of DO11.10 +/+ mice to OVA by
the aerosol route. However, in contrast to our data, Knott et
al. [22
] describe the appearance of mucins in the
bronchial epithelium in response to OVA aerosol exposure, whereas we
never found evidence of mucous-cell metaplasia (unpublished results).
They also describe that a single OVA aerosol exposure induced a skewing
toward a Type 1 cytokine milieu in the lung as evidenced by the
appearance of IFN- but no IL-4 or IL-13 in the BAL. Conversely, we
show a clear skewing toward a Type 2 immune response in the lung, as
evidenced by increases in IL-4, IL-5, and IL-13 cytokine secretion by
OVA-exposed DO11.10 +/- lung cells ex vivo. Again, the
differences in these results may be because of many factors, such as
the strain of mice, the mode of OVA exposure, and the protocols used to
measure secreted cytokines.
In DO11.10 Tg +/- mice, AHR developed in the absence of eosinophilia and IgE. Eosinophils have been shown to play important roles in the manifestation of AHR in some murine models of allergic asthma [23 24 25 26 ], whereas other studies have suggested that they are neither required nor sufficient for AHR [15 , 17 18 19 ]. Similarly, IgE has been shown to be essential for the development of AHR [27 ] in some studies while appearing unnecessary in others [18 , 25 , 28 , 29 ]. Recently, we showed that neither eosinophilia nor elevated serum levels of IgE were good predictors of AHR [8 ]. In these studies, we used three different inbred strains of mice and a more classical, systemic priming event with OVA-alum followed by a limited pulmonary challenge with aerosolized OVA (two aerosol exposures delivered in a single day). In these studies, BALB/c mice exhibited AHR in the absence of eosinophilia, whereas C57BL/6 and BDF1 mice had eosinophilia and elevated IgE levels and failed to develop AHR.
Although controversy remains about the precise immune mechanisms that lead to AHR in murine asthma models, a role for T cells is undisputed. If CD4+ T cells are depleted [20 ] or their activation is inhibited [21 ], AHR fails to develop. Furthermore, exogenous OVA-specific Th2 cells, when transferred to naïve mice, mediate AHR in response to pulmonary challenge with OVA [30 ]. Our data support the critical role that activated T cells play in manifesting AHR in that we can show accumulation of OVA-specific T cells in the lungs of DO11.10 +/- mice after OVA-aerosol exposure, which has decreased L-selectin and CD45RB expression and increased CD69 expression, a phenotype indicating that the T cells were activated recently or are of the memory phenotype [12 13 14 ]. Further, we have shown that the accumulation of these cells in the lungs of OVA-exposed DO11.10 +/- mice results in increased IL-4, IL-13, and IL-5 secretion by OVA-stimulated, nonadherent lung cells in vitro.
The data presented here do not identify the mechanism by which
CD62Llo/med OVA-TCR+ cells increase in numbers in the lungs
of OVA-exposed DO11.10 mice. The possibilities are that OVA-aerosol
exposure induced 1) resting, resident lung OVA-TCR+ T cells to
proliferate in situ and become activated to secrete Th2
cytokines, 2) activation and proliferation of naïve OVA-TCR+
cells in the LALNs, which were then recruited to the lung, or 3)
enhanced recruitment of OVA-TCR+ T cells from the periphery to the
lung. The first possibility is unlikely given recent data, suggesting
that antigen delivery to the lung induces inflammation that is
primarily a result of T cell recruitment rather than in situ
proliferation [31
]. Indeed, although OVA-aerosol
exposure of DO11.10 +/- mice results in accumulation of OVA-TCR+ cells
in the lung, which express an activated phenotype, these cells may
arrive in the lung already expressing this activated phenotype and need
not necessarily become activated in direct response to the OVA
aerosols. These memory T cells could be recruited to the lung from the
LALNs where they were initially activated or from the periphery.
Regarding the latter possibility, it is interesting to note that many
OVA-TCR+ cells in DO11.10 +/- mice co-express a second T cell receptor
through which the cells could be activated (by recombining endogenous
TCR-
chains with transgenic ß chains). Thus, these cells may
arrive in the lung having already been activated via an environmental
antigen, not OVA [32
, 33
]. However,
activation via their alternate TCR has been shown not to preclude them
from acting as OVA-specific memory cells on exposure to OVA
[34
, 35
]. Thus, upon restimulation in the
lung through their OVA-TCR, OVA-specific memory T cells may have
mediated AHR. In addition, it is possible that OVA aerosols recruited
naïve and memory OVA-specific T cells to the lung and that both
types of T cells activated in situ caused AHR.
The precise mechanisms by which the activated or memory T cells induce AHR in OVA aerosol-exposed DO11.10 +/- mice are also not known. However, it has been shown recently that inoculation of naïve mice with IL-4 or IL-13 can cause AHR in the absence of any antigen exposure [36 , 37 ]. Therefore, the increase in OVA-stimulated IL-4 and IL-13 secretion by OVA-exposed DO11.10 lung cells may be playing a role in mediating the AHR observed in our model, although it is clear from other studies that IL-4 is not required for AHR manifestation [18 , 30 ].
Whether these cytokines cause AHR directly or cause it indirectly by
influencing other cells to make the primary mediators is unclear.
Grunig et al. [36
] showed that intranasal
administration of IL-4 or IL-13 to naïve BALB/c mice induced
significant pulmonary eosinophilia and goblet-cell metaplasia with
mucous-cell overproduction, leaving open the possibility that AHR was
mediated by eosinophils and their products. Neutralization of IL-13
activity by intranasal delivery of sIL-13R2-Fc protein also
significantly reduced eosinophilia and goblet-cell metaplasia, normally
induced by intranasal OVA challenge of OVA-immune mice, and these
reductions were accompanied by a loss of AHR. In contrast, Wills-Karp
et al. [37
] showed that whereas IL-13 given
to naïve mice induced early pulmonary eosinophilia (after 1 day
of intratracheal delivery), it had resolved by the time AHR was
detected (after 3 days of delivery). In addition, although IL-13
induced trends toward increased IgE and mucous-containing cells, these
values were not significantly different than PBS controls. Finally,
these authors demonstrated that blockade of IL-13 action by i.p
injections of sIL-13R2-Fc protein failed to alter the increased
pulmonary eosinophilia and OVA-specific IgE induced by pulmonary
challenge of OVA-immune A/J mice with OVA but did abolish AHR,
suggesting that eosinophilia and IgE were incapable of mediating AHR in
the absence of IL-13.
It is interesting that the levels of IL-4 and IL-5 produced by lung cells from DO11.10 +/- mice after three OVA aerosol exposures are not sufficient to induce elevated serum IgE or IgG1 levels or pulmonary eosinophilia. Our results showing that AHR can develop in the absence of increased IgE or eosinophilia support recent studies conducted in OVA-immune IL4 -/- mice treated with anti-IL-5 antibodies that manifest AHR but fail to display eosinophilia or increases in OVA-specific serum immunoglobulins [18 ].
We did note marked increases in neutrophils in the TBL, although monocytoid cells and macrophages were the predominant cell types observed in peribronchial and perivascular areas of inflammation, suggesting that the TBL compartment does not always reflect the peribronchiolar inflammatory compartment accurately and that certain cell types may be recruited selectively to the TBL over the more abundant cell types present in the lung. The presence of neutrophils in the TBL of OVA-exposed DO11.10 +/- mice is interesting, however, given recent evidence that significant airway neutrophilia is often a characteristic of severe asthma [38 ] and has also been observed in allergic asthmatics within hours of segmental allergen challenge [39 ]. BAL neutrophilia was also observed by Knott et al. [22 ] after exposure of DO11.10 +/+ mice to a single OVA aerosol.
Previously, it has been shown that repeated OVA exposure via the
pulmonary route in the absence of systemic priming causes tolerance
[5
] or is an immunologically null event in rodents
[16
]. In DO11.10 +/- mice exposed to three OVA
aerosols, however, we observed evidence of OVA-specific T cell
activation and/or recruitment of memory cells to the lungs instead. In
addition, we showed that after three OVA aerosol exposures, T cells
isolated from the lungs of DO11.10 +/- mice retain the ability to
secrete IL-4, IL-13, IL-5, and IFN- in response to OVA protein or
specific peptide (323–339) stimulation in vitro. The
failure of OVA aerosol exposure to induce tolerance in our studies is
consistent with the observation that DO11.10 mice appear somewhat
resistant to the induction of tolerance when OVA is given via normally
tolerogenic routes (i.e., i.v. or i.p. in the absence of adjuvant)
[9
]. Conversely, Lee et al.
[40
] demonstrated recently that lung cells from DO11.10
mice, which had received repeated OVA-aerosol exposures, failed to
proliferate or secrete IL-2 in vitro in response to
restimulation with OVA, specific peptide, or anti-CD3
[40
]. The suppression of these activities was not a
result of intrinsic anergy or tolerance, however, because it was
alleviated by removal of F4/80+ macrophages from the lung-cell
population before culture. It is interesting that repeated aerosol
exposures did not suppress IFN-, IL-4, or IL-5 production by OVA
peptide-stimulated DO11.10 lung cells in their hands. These data
suggest that although proliferation is inhibited in the lung by
repeated OVA aerosols, OVA-TCR+ cells can retain effector function.
This inhibition of proliferation in DO11.10 lungs may also keep
pulmonary inflammation in check, as reflected by only small increases
in total lung cell numbers in their study and our own.
In summary, our data show that small numbers of activated or memory T cells are present in the lung after limited antigen exposure by the aerosol route. Once in the lung, these T cells or their products induce AHR in the absence of significant pulmonary eosinophilia or antigen-specific immunoglobulin. These data are in agreement with recent studies showing that T cells or their products can mediate AHR [36 , 37 , 41 ]. The observation that very limited exposures to antigen can initiate a pulmonary immune response driven by antigen-specific T cells suggests AHR may be detected before the development of clinical symptoms of allergic asthma (episodes of reversible airway obstruction, pulmonary eosinophilia, and high levels of IgE). Preliminary observations indicate that upon more chronic OVA-aerosol exposure (6 h/day, 5 days/week for up to 6 weeks), DO11.10 +/- mice do develop pulmonary eosinophilia, mucous cell hyperplasia, and high-serum OVA-specific IgG1 and IgE (unpublished results, J. A. W. and D. E. B.). These data indicate that the development of AHR may be an important, early predictor of asthma development in genetically predisposed patients who are chronically exposed to allergens.
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ACKNOWLEDGEMENTS |
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FOOTNOTES |
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Received July 10, 2000; revised November 30, 2000; accepted December 1, 2000.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ß-T cell receptor transgenic mice Am. J. Respir. Crit. Care Med. 161,1340-1348This article has been cited by other articles:
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 T. H. March, J. A. Wilder, D. C. Esparza, P. Y. Cossey, L. F. Blair, L. K. Herrera, J. D. McDonald, M. J. Campen, J. L. Mauderly, and J. Seagrave Modulators of Cigarette Smoke-Induced Pulmonary Emphysema in A/J Mice Toxicol. Sci., August 1, 2006; 92(2): 545 - 559. [Abstract] [Full Text] [PDF]
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