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* Division of Immunology, Allergy and Infectious Diseases, Department of Dermatology, Vienna International Research Cooperation Center, and
Institute of Clinical Pathology, University of Vienna Medical School, Vienna, Austria
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Immunological memory is characterized by a more effective response to repeated Ag exposure (reviewed in Refs. 6, 7, 8). Although this is a mechanism important for protecting the host against pathogens, it is not obvious what an effective immune response is to an allergen that subsequently leads to asthma. However, long-lived, vigorous immune responses against allergens can be potentially life threatening, and remarkably little is known about CD4 Th2 memory cells in allergic diseases.
Immunological memory has been classically studied in animals using replicating micro-organisms such as viruses, Ag coinjected with adjuvant, and Ag-specific TCR transgenic mice (9). However, the study of memory has been difficult for many reasons. In a normal immune response there is a low precursor frequency of memory cells often below the level of detection. Additionally, it is difficult to study the survival of memory cells in the absence of Ag due to immunization protocols including adjuvants, which leave large Ag depots, or replicating organisms, which also result in a constant and available Ag load. To circumvent these problems, many laboratories use adoptive transfers into syngeneic recipients (10), but there may be transferred Ag, and this may not reflect the immune response under normal circumstances. Studies exploiting the large precursor frequency in TCR transgenic mice do not take into account the lack of nonspecific bystander cells that may play a role in disease. However, adoptive transfer of TCR transgenic T cells provides an opportunity to have a sufficient number of Ag-specific cells within the context of a heterogeneous repertoire. We sought to induce experimental allergic asthma in mice by immunizing with soluble and aerosolized Ag and to subsequently explore the generation and maintenance of Th2 immunological memory upon rechallenge in the same host. The advantages of this approach are that we avoid Ag depots and adoptive transfers and can evaluateTh2 immunological memory by disease and by characterizing Th2 immune responses in lymphoid organs and the respiratory tract.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Six- to 8-wk-old female BALB/c mice maintained in the VIRCC facility and provided OVA-free food and water ad libitum were used in all experiments. All experimental protocols complied with the requirements of the Animal Care Committee of the University of Vienna Medical School.
Immunization
To establish acute allergic asthma, we immunized large groups of
mice i.p. with 10 µg of OVA (grade V; Sigma-Aldrich, St. Louis, MO)
in 200 µl of PBS or with PBS on days 0 and 21. One week later, on
days 28 and 29, mice were nebulized with 1% OVA in PBS or with PBS
alone using an ultrasonic nebulizer for 60 min twice daily. Groups of
mice following acute disease induction are referred to as OVA-OVA
(OVAipOVAaerosol), OVA-PBS
(OVAipPBSaerosol), and
PBS-PBS (PBSipPBSaerosol).
To determine whether immunological memory was generated and maintained,
we allowed mice with acute disease to recover, and then
challenged them a second time with aerosolized 1% OVA or PBS for 60
min twice daily on 2 consecutive days. Memory and control groups are
referred to as OVA-OVA-OVA
(OVAipOVAaerosolOVAaerosol),
OVA-OVA-PBS (OVAipOVAaerosol
PBSaerosol), OVA-PBS-OVA
(OVAipPBSaerosolOVAaerosol), and
PBS-PBS-PBS (PBSip
PBSaerosolPBSaerosol). More than six
experimental groups were immunized at different time points over
4 years. Evaluation for disease was performed in two or three separate
experiments on days 31, 60, 90, 120, 143, 170, 220, 260, and 433. Mice
at later time points (days 675, 708, and 807) were used for
investigating lung T cells and Ig. Evaluations for disease on indicated
days are representative. See Table I
for
protocol details.
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Forty-eight hours following the last aerosol challenge, mice were evaluated for inflammation and mucus hypersecretion. The tracheas of lethally anesthetized mice were cannulated and lavaged with 1 ml of PBS. Total leukocytes in the bronchoalveolar lavage fluid (BAL)3 were counted with a hemocytometer, and cytospin slides were prepared and stained with May-Grünwald-Giemsa to determine the cell differential. Following BAL, tracheas were perfused with PBS and then with 4% formalin. Paraffin-embedded lung sections of 4 µm were stained with H&E for morphological evaluation and with periodic acid-Schiff (PAS) for mucopolysaccharide staining, which reflects mucus production. Stained lung sections were evaluated in all experiments.
Airway responsiveness
Twenty-four hours following the last aerosol challenge, mice were evaluated for airway hyperresponsiveness (AHR). We measured AHR in conscious, unrestrained mice by whole-body plethysmography (Buxco Electronics, Sharon, CT) using a method previously reported (11). Briefly, mice were placed into the main chamber, and pressure differences between the main and a reference chamber were recorded. Readings were taken at 5-min intervals at baseline and then with nebulized PBS and titrated doses of methacholine (Sigma-Aldrich) for 5 min each. Pressure readings were averaged for each nebulization dose in a progressive fashion. Results are expressed as a calculated, dimensionless value termed the enhanced pause (Penh) that correlates with pulmonary resistance and airway responsiveness.
Serum Ig
Sera for the measurement of OVA-specific Ig were obtained at 48 h and in some experiments at 96 h after the last aerosol challenge. For the measurement of OVA-specific IgG1, ELISA plates were coated with OVA at 10 µg/ml overnight at 4°C. The plates were washed and blocked with 2% BSA/0.05% Tween 20 for 2 h at 37°C. Titrated sera were incubated for 2 h at room temperature. After washing, biotinylated anti-IgG1 detection mAb (BD PharMingen, San Diego, CA) was added, and plates were incubated for 1 h. Europium (Eu3+)-streptavidin (Delfia; Wallac, Turku, Finland) was added to each well after the plates were washed. Enhancement solution (100 µl) was added (Delfia), and Eu3+ release was measured by fluorometry at 340 nm excitation and 614 nm emission. The assay used to measure OVA-specific IgE was the same as that described above, except that the plates were coated with anti-mouse IgE (LO-ME-3; Serotec, Oxford, U.K.) and detected with biotinylated OVA.
Cytokine analysis
Pooled spleen or mediastinal lymph node cells from mice 48 h after the last aerosol challenge were incubated at 5 x 105/well with 1000 µg/ml OVA, Con A/IL-2, or medium alone (IMDM with 10% FCS and additives) for 48 h on anti-IL-4- and anti-IL-5-coated Immunospot ELISPOT plates (Cellular Technology, Cleveland, OH). The mAb pairs used for capture/biotinylated detection were as follows: IL-4, BVD4-1D11/BVD6-24G2; IL-5, TRFK-4/TRFK-5 (BD PharMingen). For color development, we added streptavidin POD (Roche, Mannheim, Germany), diaminobenzidene/NiCl2 substrate (Sigma-Aldrich), and then water. Spots were then counted per well and subtracted from wells containing medium alone. For spleen cell cytokine analysis, cells (5 x 105/well) were incubated with OVA (1 mg/ml) or medium alone for 96 h. Cell supernatants were tested for IL-4 and IL-5 using ELISAs (Endogen, Woburn, MA) according to the manufacturer’s instructions.
To prepare lung cells for cytokine analysis, lungs were cut into small pieces and were then incubated at 37°C for 1 h with 150 U/ml collagenase D (Sigma-Aldrich) and 50 U/ml DNase (Sigma-Aldrich). Tissue fragments were gently homogenized with a glass homogenizer (Kontes, Vineland, NJ). The resulting cell suspension was flushed through 70-µm pore size mesh filter and centrifuged over a Lympholyte-M (Cedarlane Laboratories, Hornby, Ontario, Canada) gradient. To further purify CD4+ T cells from lung cells, we incubated total lung cells with CD4+ Dynabeads (Dynal, Oslo, Norway) for 20 min at 4°C. The cell suspension was placed on the Dynal Magnetic Particle Concentrator and washed with medium. Beads were removed by incubating the cell suspension with DETACHaBEAD for 45 min at 4°C, followed by bead removal using the Dynal Magnetic Particle Concentrator. Lung cells were incubated with equal numbers of irradiated T depleted spleen APC (5 x 105/well) in the presence of graded doses of OVA in IMDM with 10% FCS and additives or in medium alone on ELISPOT plates for 48 h (as above). Purified populations of lung CD4 cells (2 x 105/well) were incubated with equal numbers of irradiated T cell-depleted spleen APC in the presence of graded doses of OVA in IMDM with 10% FCS and additives or in medium alone in 96-well microtiter plates for 96 h. Supernatants were frozen at -20°C until use. Supernatants were tested for IL-4 and IL-5 levels using ELISA assays (Endogen, Woburn, MA) following the manufacturer’s instructions.
Lungs removed 3 h after a 1-h secondary aerosol challenge in mice
on day 475 for IFN-
, TNF-
, IL-2, IL-4, IL-5, IL-6, IL-10, and
IL-13 RNA expression were immediately frozen in liquid nitrogen. Total
RNA from lungs was extracted with TRIzol reagent (Life Technologies,
Gaithersburg, MD) according to the manufacturer’s instructions. Total
RNA (20 µg) was incubated with RQ1 DNase buffer, DTT, RNasin RNase
inhibitor, and RQ1 RNase-free DNase (Promega, Madison, WI) for 25 min
at 37°C. RNA was further purified by phenol/chloroform/isoamyl
(25/24/1) extraction. First-strand cDNA was synthesized from all the
digested RNA with avian myeloblastosis virus reverse transcriptase
(Roche) in a final volume of 50 µl. cDNA was diluted to a total
volume of 500 µl with Advantage buffer (Clontech, Palo Alto, CA) and
was frozen at -20°C until use. Amplification protocols for cytokines
consisted of 35 repetitive cycles of denaturing at 95°C (0.5 min),
annealing at 60–64°C (0.5 min), and extension at 72°C (1.30 min).
cDNA (5 µl) was added to a reaction mixture containing Advantage
polymerase according to the manufacturer’s protocol. Primers for the
housekeeping gene hypoxanthine-guanine phosphoribosyl- transferase
(HPRT) were used as internal controls. The PCR primers used to detect
all cytokines and HPRT were purchased from Clontech. Amplified cDNA was
separated by agarose gel electrophoresis and visualized with ethidium
bromide.
FACS analysis
Total lung cells were stained with mAb directed against CD4 (GK1.5-FITC), CD8 (53-6.7-PE), B cells (B220-PE), and MHC class II (M5/114-FITC). Purified CD4 T cells were stained with anti-CD4-PE and counterstained with mAb directed against CD45RB (16A-FITC), CD44 (Pgp-1-FITC), CD62L (Mel-14-FITC), and CD25 (7D4-FITC). All mAb were purchased from BD PharMingen.
Statistical analysis
Groups were compared by unpaired Student’s t test. Values of p < 0.05 were considered significant. For AHR, mean Penh values at each dose for each group were summed, and then the sums were compared using unpaired Student’s t test. All data are presented as the mean ± SEM.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Inflammation of the respiratory tract in acute allergic asthma is
characterized by Ag-specific eosinophilic inflammatory infiltrates. To
test whether mice developed allergic lung inflammation, we evaluated
mice by BAL and H&E-stained sections 48 h following the last
aerosol challenge. We assessed airway inflammation using BAL cell
counts and leukocyte differentials. Total BAL cell counts (TBC)
indicates the intensity of airway inflammation. Upon the induction of
acute allergic asthma, we observed a 20-fold Ag-specific increase in
TBC in OVA-OVA compared with OVA-PBS mice on day 31 (Fig. 1). However, when we rechallenged mice a
second time with aerosol-OVA, airway inflammation was higher than
during acute disease (p < 0.05), illustrating
Ag-specific memory responses.
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). We observed a
consistent, statistically significant, OVA-specific increase in
absolute counts per milliliter of BAL for each cell type
(p < 0.05). Airway inflammatory infiltrates in
OVA-OVA-OVA mice consisted predominantly of eosinophils and
lymphocytes, but neutrophils and macrophages were also increased
compared with OVA-OVA-PBS control mice (p <
0.05). Although in the absence of further Ag exposure eosinophils
disappeared within 30 days, OVA-OVA-PBS mice on day 90 had increased
numbers of BAL neutrophils, lymphocytes, and macrophages, suggesting
that mice had not yet remitted from acute airway inflammation. Aerosol
challenge with BSA, an irrelevant Ag, did not induce inflammation (data
not shown). Taken together, these data show an OVA-specific Th2-type
inflammatory response in the airways >400 days after disease
induction.
Further evaluation of lung inflammation in H&E-stained sections
demonstrated that OVA challenge-induced inflammation that was similar
to human asthma. We observed both peribronchial and perivascular
infiltrates containing 
30% eosinophils, numerous lymphocytes,
macrophages, scattered neutrophils, and few plasma cells in tight foci
in the periphery of lungs from OVA-OVA mice during acute disease on day
31 (Fig. 2). Lung tissue sections from
OVA-OVA-OVA mice during relapses revealed 30–50% eosinophils, with
many macrophages, lymphocytes, and plasma cells within the infiltrates
up until day 433. In the absence of further Ag aerosol exposure,
infiltrating eosinophils disappeared within 30 days of acute disease
(data not shown). However, lungs from recuperated OVA-OVA-PBS mice
contained focal peribronchial and perivascular lymphocytic infiltrates
for >1 year after acute disease. These infiltrates were not present in
PBS-PBS-PBS or naive age-matched control mice (data not shown). These
results demonstrate eosinophilic lung inflammation closely resembling
human lesions, memory responses >400 days after acute disease, and
persistent lung inflammatory infiltrates in the absence of Ag
re-exposure.
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A characteristic feature of allergic asthma is excessive
production of viscous mucus. To test Ag-specific mucus hypersecretion,
we stained paraffin-embedded lung sections with PAS. Upon OVA aerosol
challenge a large percentage of goblet cells produced mucus in the
respiratory mucosa of the large airways compared with a very low level
in naive, PBS-PBS-PBS, and OVA-OVA-PBS mice (Fig. 3). Peak mucus hypersecretion appeared on
day 90, as evaluated by high numbers of mucus-producing cells, goblet
cell hyperplasia, and mucus within the airways. Mucus hypersecretion
did not follow BSA aerosolization and remitted 3 wk after the last Ag
aerosol exposure (data not shown). These results demonstrate
Ag-dependent mucus hypersecretion in response to aerosol-OVA.
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Wheezing, difficulty breathing, and coughing are cardinal features
of asthma and are a consequence of airway obstruction and AHR. To
determine whether mice developed Ag-specific AHR, we tested the
response of mice to a methacholine aerosol challenge. We nebulized mice
with methacholine and measured airway resistance using total body
plethysmography 24 h after the last OVA aerosol challenge. OVA
aerosol was necessary for the induction of AHR in response to graded
doses of nebulized methacholine at all time points tested (Fig. 4). Naive, OVA-PBS, OVA-OVA-PBS, PBS-PBS,
and PBS-PBS-PBS mice also respond to methacholine, but the responses
were significantly less than those of OVA-OVA and OVA-OVA-OVA mice.
Experiments were internally controlled and were performed on different
days; thus, relative differences between groups were not assessed.
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To test whether Th2-type Abs were produced in acute disease and
whether they were boosted following aerosol rechallenge, we measured
serum OVA-specific IgG1 and IgE at 48 and/or 96 h after aerosol
challenge. We observed that serum OVA-specific IgG1 and IgE were
elevated on day 31, 48 h after the last aerosol challenge, in mice
immunized with i.p. OVA (OVA-OVA and OVA-PBS) regardless of aerosol
challenge compared with PBS-PBS mice (p <
0.05, Fig. 5A), indicating
that systemic immunization was effective at generating Ab. We then
tested serum OVA-specific IgG1 and IgE at 48 h after secondary
aerosol challenge and observed that Ab levels were not significantly
different for OVA-OVA-OVA and OVA-OVA-PBS mice (data not shown),
suggesting that aerosol challenge did not boost Ab responses. However,
we reasoned that aerosol challenge might boost Ab production later than
48 h. When we tested sera 96 h after challenge with OVA
aerosol, both OVA-specific IgG1 and IgE were significantly elevated
compared with levels in the PBS aerosol controls
(p < 0.05; Fig. 5B), indicating
that aerosol OVA boosted Ab production. BAL Ab levels mirrored serum
levels for both isotypes at all time points (data not shown).
OVA-specific IgG2a was consistently low compared with Th2-type isotypes
(data not shown). IgG1, but not IgE, titers were persistently elevated
in OVA-OVA-PBS mice, indicating the maintenance of IgG1 production
regardless of Ag re-exposure. In one experiment OVA-specific IgG1
levels continued to be elevated at 675 days (data not shown).
Remarkably, serum and BAL IgE 48 h after secondary aerosol
challenge were undetectable at the later time points, demonstrating
that serum IgE levels do not correlate with the onset of
OVA-specific inflammation, AHR, and mucus hypersecretion (data not
shown). Taken together, these data show that Th2 Ig isotype production
increases following aerosol Ag exposure long after initial Ag priming
and long-lived production of IgG1 in recuperated
mice.
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To prove that memory responses that induce disease depend upon
aerosol lung challenge, we tested the OVA-PBS-OVA groups for recall
disease and cytokine production. Animals sensitized with OVA i.p.,
nebulized on days 28 and 29 with PBS, and subsequently nebulized with
OVA (OVA-PBS-OVA) developed less BAL and parenchymal inflammation than
OVA-OVA-OVA mice on days 60 and 90 and had no inflammation after day
120 (data not shown). This indicates that lung challenge is required
<4 mo after sensitization for lung memory responses. A representative
experiment from day 260 demonstrated diminished BAL infiltrates upon
OVA aerosol challenge in OVA-PBS-OVA mice compared with OVA-OVA-OVA
mice (Fig. 6A). Nebulized
methacholine did not induce AHR in OVA-PBS-OVA mice compared with
OVA-OVA-OVA mice (Fig. 6B). After 120 days mucus
hypersecretion and inflammation, as evaluated by histology, were absent
in OVA-PBS-OVA mice, and by day 90 serum OVA-specific IgE was not
detected (data not shown). Additionally, spleen cell production of IL-4
and IL-5 diminished rapidly and was not detected on day 260 in
OVA-PBS-OVA compared with OVA-OVA-OVA mice (Fig. 6C),
indicating that aerosol allergen challenge plays a role in maintaining
spleen Th2 memory. Taken together, these data demonstrate that aerosol
allergen challenge soon after sensitization is important for the
maintenance of Th2 memory responses.
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To test for the presence of Ag-specific Th2 cells in lymphoid
tissue, we tested spleen and draining mediastinal lymph node cytokine
secretion from OVA-OVA-OVA and OVA-OVA-PBS mice. In vitro stimulation
with OVA induced Ag-specific IL-4 and IL-5 from OVA-OVA-OVA and
OVA-OVA-PBS spleen cells (Fig. 7). These
data indicate the persistence of Th2 memory cells in spleen and an in
vivo aerosol Ag memory response. In contrast, we detected OVA-specific
IL-4 and IL-5 secretion from OVA-OVA-OVA, but not OVA-OVA-PBS, draining
lung lymph nodes (Fig. 7), suggesting that long-lived Th2 cells are not
present in draining mediastinal lymph nodes. Taken together, these data
provide evidence for long-lived Th2 memory cells in mice long recovered
from acute disease in spleen, but not in draining lymph nodes unless
mice receive in vivo aerosol Ag.
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Mice recuperated from acute allergic asthma had persistent
lymphocytic lung infiltrates as seen in H&E-stained lung sections from
OVA-OVA-PBS mice (Fig. 2). To determine whether these infiltrates
contained Ag-specific memory Th2 cells, we stained the total lung cell
suspensions and observed that the population contained 25%
CD4+, 40% B220+, 45% MHC
class II+, and no CD8+
cells (Fig. 8A). To
demonstrate the phenotype of CD4 T cells present in the lungs, we
purified CD4+ cells from total lung cells. A
>90% enriched population of lung CD4 T cells expressed high CD44 and
low CD45RB, CD62L, and CD25, indicating a memory cell phenotype (Fig. 8A). Additionally, we stained frozen lung sections with
anti-CD4 and observed many CD4 T lymphocytes within the infiltrates
in OVA-OVA-PBS mice, with little difference between OVA-OVA-PBS and
OVA-OVA-OVA stained sections (data not shown).
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, B and C), indicating the
presence of long-lived, Ag-specific memory CD4 T cells in the lungs of
mice recuperated from acute allergic asthma. To further investigate the
Ag specificity of persistent lung memory cells, we evaluated in vivo Ag
responsiveness by measuring total lung cytokine RNA expression 3 h
after a 1-h aerosol challenge from recovered mice. We reasoned that
early evaluation after a brief aerosol exposure was necessary to avoid
the contribution of immigrant systemic memory cells, particularly from
the spleen. While IFN- and TNF- remained at background levels, a
1-h OVA aerosol challenge induced increases in RNA expression of IL-2,
IL-4, IL-5, IL-6, IL-10, and IL-13 on day 475 (Fig. 8D).
Taken together, these data demonstrate that Ag-specific Th2 memory
cells persist in the lungs of mice >1 year after recuperating from
acute disease and suggest that they play a role in the initiation of
allergic asthma relapses. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Long-lived Th2 memory cells persist in the spleen and lungs of mice long recovered from acute allergic asthma. They have a memory phenotype and respond in vitro by secreting IL-4 and IL-5 upon Ag stimulation. Additionally, they respond rapidly to in vivo aerosol challenge, as detected by the expression of Th2 cytokine RNA in the lungs as early as 3 h. This suggests rapid activation of lung Th2 cells, which then initiates the subsequent cascade of events leading to disease. Moreover, our observation that OVA-PBS-OVA mice did not sustain memory responses in the absence of early aeroallergen encounter illustrates that rapid immune responses depend on previous lung encounter with allergen, which results in sentinel tissue-specific memory cells. The evidence that nonlymphoid tissue harbors resting memory cells that upon activation become effector-memory cells that are capable of initiating allergic asthma is supported by an emerging paradigm suggesting that memory T cells in nonlymphoid tissues contribute to systemic immunity (12). In several studies functionally distinct memory T cell subsets were distinguished by surface receptors such as CCR7, CD62L, and CD45RB; effector function; and capacity to home to lymphoid organs and sites of tissue inflammation (12, 13, 14, 15, 16, 17). Furthermore, memory CD8 T cells have been found in the lungs of mice recovered from viral diseases (18, 19, 20, 21, 22). Taken together, these data implicate a role for tissue-specific memory T cells.
Although memory Th2 cells were present in the spleen and lungs of recovered mice, we were unable to detect OVA-specific IL-4 and IL-5 in draining lung lymph nodes unless the mice were OVA aerosol challenged. This may be due to the sensitivity of our assay or the absence of memory Th2 cells. We speculate that memory cells are not present in the draining lymph nodes during remission, and that following aerosol challenge Th2 cells migrate to draining lymph nodes. Data demonstrating that memory cells may not be in peripheral lymph nodes (23) and that memory, naive, and effector cells have distinct circulation patterns (24, 25, 26) support this hypothesis. Moreover, in a recent study draining lymph node and nonlymphoid tissue T cells were distinguished based on their response to Ag (27). Interestingly, they found that Ag-specific T cells in the lungs did not clonally expand, but produced effector cytokines, while those in draining lymph nodes did the opposite (27). Similarly, cells from resting lungs (OVA-OVA-PBS mice) produced effector cytokines, although draining lung lymph node cells did not. However, upon aerosol exposure Th2 cells were present in the draining lymph nodes. In addition, the numbers of CD4 T cells in lung infiltrates in OVA-OVA-PBS and OVA-OVA-OVA groups were not different. Thus, it is possible that distinct subsets of nonlymphoid/lung and lymphoid memory T cells stimulated upon Ag challenge develop into memory effector cells capable of either producing Th2 cytokines or proliferating, respectively.
A fundamental question regarding the maintenance of memory T cells that remains unanswered is whether persisting Ag is a requirement. Many experiments focusing on this question have resulted in disparate results, particularly when addressing the mechanisms underlying CD4 vs CD8 T cell memory (28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38), and little is known about the role of persisting Ag in the maintenance of Th2 memory cells, especially during allergic disease. It has been shown that persistent Ag is necessary for Th cell survival (10, 38). Ag epitopes in the form of immune complexes reside in vivo on the surface of follicular dendritic cells and are thought to be a reservoir of Ag for circulating memory T cells (39). Often the study of immunological memory is hindered by the presence of Ag depots when animals are immunized with Ag precipitated in alum, emulsified in Freund’s adjuvant, and replicating micro-organisms. Although we avoided Ag deposits in this model, we cannot exclude the role of Ag persistence in the maintenance of memory.
T cell memory has also been shown to depend on B cells and Ig (39, 40). While serum and BAL OVA-specific IgE were boosted after OVA aerosol challenge, they were not detectable in OVA-OVA-PBS mice after 143 days. However, elevated levels of serum and BAL OVA-specific IgG1 persisted up until day 675 in OVA-OVA-PBS mice without further Ag challenge (data not shown). While this demonstrates that an allergic response can occur with low levels of serum and BAL IgE, it does not exclude a role for IgE during disease relapses or in the maintenance of Th2 cell memory, because IgE may be tissue bound. Alternatively, IgG1 may play an important role in the maintenance of memory Th2 cells.
The administration of soluble OVA, followed by aerosol challenge, resulted in focal areas of eosinophilic inflammation that tended to be near the periphery of the lung rather than centrally located and to resemble human lesions (41, 42). This pattern of inflammatory cell infiltration differs from well-established protocols inducing disease in <2 wk using OVA precipitated in alum. Immunization with 25 µg of OVA precipitated in alum on days 0 and 5, followed by aerosol OVA 1 wk later, leads to eosinophilic inflammation characterized by diffuse, centrally located lesions (43). The mechanism underlying the differences remains unknown.
Although persistent lymphocytic infiltrates remained in the lungs of recuperated OVA-OVA-PBS mice, other physiologic changes, including, mucus hypersecretion, eosinophilia, and AHR, were not apparent. Patients with seasonal or intermittent allergic asthma are asymptomatic unless exposed to their inciting allergen. However, asymptomatic asthmatics may exhibit methacholine-induced AHR, which has been correlated with persistent subclinical inflammation (44, 45, 46). We observed that methacholine-induced AHR was transient and was evident in OVA-OVA-OVA mice for up to 5 days after OVA aerosol challenge (data not shown). It is not clear why AHR is short-lived in mice.
In summary, we established a model of experimental allergic asthma that more closely resembles human asthma and demonstrated the maintenance of long-lived Th2 memory cells in spleen and lungs. Whether the long-lived local and systemic memory we observed depends on Ag persistence remains to be tested. However, the immunization protocol we used reduces continuous Ag exposure in the form of Ag depots. This model offers an opportunity to further study peripheral and systemic Th2 immunological memory and may assist in the development of novel strategies for the treatment of allergic asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Michelle Epstein, Department of Dermatology, University of Vienna Medical School, Division of Immunology, Allergy and Infectious Diseases, Vienna International Research Cooperation Center, Brunner Strasse 59, A-1235, Vienna, Austria. E-mail address: michelle.epstein{at}univie.ac.at
3 Abbreviations used in this paper: BAL, bronchoalveolar lavage fluid; AHR, airway hyperresponsiveness; HPRT, hypoxanthine-guanine phosphoribosyltransferase; PAS, periodic acid-Schiff; Penh, enhanced pause; TBC, total BAL cell counts.
Received for publication May 22, 2002. Accepted for publication August 27, 2002.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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),
OVA-PBS (
), OVA-OVA-OVA (
), and OVA-OVA-PBS (
).
), and PBS-PBS (
, OVA-OVA-PBS; 
,
OVA-OVA-OVA) (B). PBS-immunized mice have no detectable Ig
at all indicated days. Data shown are from three independent
experiments. These data were generated from testing sera from different
time points in the same assay. Each line represents the mean ±
SEM Eu3+ release for each serum dilution for each group of
mice (n = 4–6).
