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of June 16, 2017.
T Cells from Human Allergen-Induced Late
Asthmatic Responses Express IL-12 Receptor
β2 Subunit mRNA and Respond to IL-12 In
Vitro
Eva Maria Varga, Petra Wachholz, Kayhan T. Nouri-Aria,
Adrienne Verhoef, Christopher J. Corrigan, Stephen J. Till
and Stephen R. Durham
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The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 2000 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
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J Immunol 2000; 165:2877-2885; ;
doi: 10.4049/jimmunol.165.5.2877
http://www.jimmunol.org/content/165/5/2877
T Cells from Human Allergen-Induced Late Asthmatic
Responses Express IL-12 Receptor ␤2 Subunit mRNA and
Respond to IL-12 In Vitro1
Eva Maria Varga,* Petra Wachholz,* Kayhan T. Nouri-Aria,* Adrienne Verhoef,*
Christopher J. Corrigan,† Stephen J. Till,* and Stephen R. Durham2*
A
llergen-specific T cells from the peripheral blood of patients with allergic asthma or rhinitis are characterized
by elevated expression of the Th2-type cytokines, IL-5,
IL-4, and IL-13 (1–5), and mRNA encoding these cytokines is
up-regulated within the target organ on allergen exposure (6 – 8).
Of the Th2-type cytokines, IL-5 is considered to be of particular
relevance to asthma pathogenesis by virtue of its multiple proeosinophilic properties. For example, IL-5 promotes eosinophil
maturation, activation, survival (9 –11), release from bone marrow
(12), and enhanced responsiveness to chemokines such as RANTES (13) and eotaxin (14). In murine models, the allergen-induced
asthmatic late response is dependent on IL-5 and eosinophils (15).
In vitro studies suggest that the ability of allergen-specific T
cells to produce Th2-type or Th1-type cytokines can be regulated
by exposure to cytokines such as IL-12, IFN-␥, and IL-4. Specifically, addition of IL-12 (and to a lesser extent IFN-␥) to freshly
isolated peripheral blood T cells obtained from atopic subjects
shifts the expression of cytokines away from a Th2-type profile
and toward a Th1-type profile (16, 17). Conversely, addition of
IL-4 results in a shift from Th1- to Th2-type cytokine production
in vitro (18). The effects of IL-12 on Th2 cytokine-producing T
cells that have undergone recent stimulation by Ag are more con-
*Upper Respiratory Medicine, Imperial College School of Medicine at National Heart
and Lung Institute, London, United Kingdom; and †Department of Allergy and Respiratory Medicine, Guy’s Hospital, London, United Kingdom
Received for publication September 8, 1999. Accepted for publication June 12, 2000.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This study was supported by National Asthma Campaign and Medical Research
Council (U.K.).
2
Address correspondence and reprint requests to Dr. Stephen R. Durham, Upper
Respiratory Medicine, National Heart and Lung Institute, Imperial College School of
Medicine, Dovehouse Street, London, SW3 6LY, U.K. E-mail address: s.durham@
rbh.nthames.nhs.uk
Copyright © 2000 by The American Association of Immunologists
troversial: it has been reported that certain Th2-type T cells have
the capacity, when activated and expanded in vitro (i.e., as T cell
lines or clones), to become resistant to the cytokine-modulating
effects of IL-12 when restimulated (17, 19, 20). This has been
linked to loss of function in the IL-12 signal transduction pathway,
as evidenced by a failure to phosphorylate STAT4 on tyrosine
residues (19, 20), and more recently, through failure to express the
␤2 subunit of the IL-12R (IL-12R␤2) (21, 22).
These observations raise the question of whether T cells present
in human tissues exposed to allergen become refractory to IL-12.
There is indirect evidence to suggest that IL-12 and IFN-␥ may be
able to suppress allergic inflammation locally. First, numbers of
IL-12 mRNA-expressing cells are elevated in bronchial biopsies
from nonasthmatics as compared with asthmatic subjects, and
treatment of the latter with corticosteroids is associated with a rise
in the numbers of IL-12 mRNA-expressing cells (23). Second,
following allergen immunotherapy, a reduction in the cutaneous
late phase response to allergen is associated with elevated local
expression of IL-12 and IFN-␥ mRNA (24, 25). However, recent
data suggest that fewer lung T cells from patients with asthma are
recognized by an anti-IL-12R␤2 mAb than in patients with sarcoidosis (a putative Th1 disease) (26), consistent with down-regulation in asthma, up-regulation in sarcoidosis, or both. We therefore examined the effects of IL-12 (and IFN-␥) on cytokine
production by bronchoalveolar lavage (BAL)3 T cells isolated
from the bronchial lumen during an allergen-induced late response,
and compared these with the responses of PBMC obtained from
the same patients. We hypothesized that IL-5 production by both
BAL T cells and T cells in PBMC would be inhibited by IL-12.
Furthermore, we have used in situ hybridization to examine
whether mRNA transcripts encoding the IL-12R␤2 subunit can be
3
Abbreviations used in this paper: BAL, bronchoalveolar lavage; FEV1, forced expiratory volume in 1 s; PC20, that concentration of inhaled methacholine resulting in
a 20% reduction in baseline FEV1.
0022-1767/00/$02.00
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IL-12 suppresses proallergic Th2-type cytokine production and induces Th1-type cytokine production by peripheral blood T cells
from subjects with allergic disease. The objective of the present study was to examine the relevance of these findings to target organ
T cell responses in human asthma. Bronchoalveolar lavage (BAL) and PBMC were collected from atopic asthmatics 24 h after
fiberoptic allergen challenge of a segmental bronchus. BAL T cells and PBMC were cultured with allergen in the presence of
recombinant IL-12 or IFN-␥, and cytokines were measured in culture supernatants after 6 days. IL-5 production by BAL T cells
and PBMC was inhibited by IL-12 and, to a lesser extent, by IFN-␥. IL-12 also induced IFN-␥ production by BAL T cells and
PBMC. The effects of IL-12 nor IFN-␥ on IL-5 production could not be reversed by neutralizing anti-IFN-␥ or anti-IL-12 mAbs,
respectively. Thus, the effect of neither IL-12 nor IFN-␥ appeared to be mediated through induction of the other cytokine. In situ
hybridization revealed that approximately one-third of BAL T cells expressed mRNA transcripts encoding the IL-12R ␤2 subunit
following allergen challenge. Thus, human T cells obtained from BAL during asthmatic late responses, like T cells in the peripheral
circulation, remain susceptible to immunomodulation by IL-12. These findings raise the possibility that IL-12 may hold therapeutic potential in allergic diseases such as asthma. The Journal of Immunology, 2000, 165: 2877–2885.
2878
EFFECTS OF IL-12 ON LUNG T CELL CYTOKINE RESPONSES IN ASTHMA
detected in enriched BAL T cells collected from asthmatic patients
during the allergen-induced late response.
Materials and Methods
Subject populations
All subjects were required to have a forced expiratory volume in 1 s (FEV1)
⬎80% predicted and a methacholine PC20 (that concentration of inhaled
methacholine resulting in a 20% reduction in baseline FEV1) of ⬎1 mg/ml,
but ⬍32 mg/ml. All of these subjects had ⱖ5-mm diameter wheal at 15
min following skin-prick testing with Phleum pratense (timothy grass pollen) or Dermatophagoides pteronyssinus (house dust mite) extract in the
presence of negative diluent and positive histamine controls. These subjects were also required to have elevated concentrations of serum IgE Abs
specific for P. pratense or D. pteronyssinus (radioallergosorbent test,
RAST ⬎0.70 IU/ml; CAP system; Pharmacia Diagnostics, Uppsala, Sweden). All subjects participating in this study were nonsmokers and, where
appropriate, inhaled corticosteroid therapy was withheld 2 wk before bronchoscopy. The study was approved by the Ethics Committee of the Royal
Brompton Hospital (London, U.K.) and all subjects gave written informed
consent.
Fiberoptic bronchoscopy and allergen challenge
Cell preparation
PBMC were isolated from heparinized blood samples by density-gradient
centrifugation over Histopaque (Sigma, Poole, U.K.), washed twice in
HEPES-buffered RPMI (Sigma), and resuspended in RPMI supplemented
with 5% human AB serum, 100 U/ml penicillin, 100 ␮g/ml streptomycin,
and 2 mM L-glutamine (all Sigma). This supplemented medium (complete
medium) was used for all cell culture experiments. BAL cells were isolated
by passing BAL fluid through two layers of sterile gauze to remove mucus,
washed twice in HEPES-buffered RPMI, and resuspended in complete medium. A differential cell count was performed on a cytospin of BAL cells
using May-Grünwald-Giemsa stain. Mononuclear cells were enriched from
BAL cells by density-gradient centrifugation over Histopaque, washed
twice, and resuspended in HEPES-buffered RPMI. T cells were then enriched from BAL mononuclear cells by passage of cells through a T cell
enrichment column containing anti-human Ig-coated glass beads (R&D
Systems, Abingdon, U.K.). Lymphocytes constituted ⬎90% of such preparations, as judged by morphology, and showed good viability (⬎90%), as
determined by trypan blue exclusion. Purity of BAL T cells enriched over
these columns is 85–90%, as judged by flow cytometry with anti-CD3 mAb
(27). The BAL T cell enrichment protocol typically yielded about 0.15–
0.5 ⫻ 106 cells.
BAL T cell cultures
BAL T cell concentrations in culture were restricted by the relatively low
numbers of enriched BAL T cells that could be obtained from human
volunteers. Therefore, BAL T cells were resuspended at 8 ⫻ 104 cells/ml
in 96-well round-bottom plates (Nunc, Roskilde, Denmark) with irradiated
(3000 rad) autologous PBMC at 4 ⫻ 105 cells/ml as APCs, in the presence
and absence of 10 ␮g/ml allergen extract (Aquagen extract; ALK Abelló).
All cultures were performed in 250 ␮l vol. In all cases, control cultures
PBMC cultures
Detection of IL-5 production by peripheral blood T cells necessitated different culture conditions from those that could be used to detect IL-5 production by enriched BAL T cells. To elicit allergen-induced IL-5 production by peripheral blood T cells, PBMC were resuspended at 5 ⫻ 106
cell/ml in triplicate 250 ␮l vol in 96-well flat-bottom cell culture plates
(Nunc) in the presence or absence of 10 ␮g/ml of allergen extract. We
previously showed that allergen-induced IL-5 production is optimal at this
cell density, is dependent on CD4⫹ T cells, and is specifically elevated in
patients with atopic asthma or allergic rhinitis, but not asymptomatic atopic
or nonatopic controls (2). PBMC cultures were supplemented with 0.5, 5,
and 50 U/ml human rIL-12 (R&D Systems), and 5, 50, and 500 U/ml
human rIFN-␥ (R&D Systems) or vehicle control. In some experiments,
cultures were supplemented with 10 ␮g/ml of neutralizing anti-IFN-␥ mAb
(R&D Systems) or 50 ␮g/ml of neutralizing anti-IL-12 mAb (R&D Systems) to examine whether the effects of IL-12 were IFN-␥ dependent and
whether the effects of IFN-␥ were IL-12 dependent. Optimal concentrations of anti-IFN-␥ and anti-IL-12 mAbs were determined by titration of
the concentration of Ab required to reverse the inhibition of allergen-induced IL-5 production by PBMC by IFN-␥ and IL-12, respectively. Culture supernatants were removed from each microculture well on day 6 for
ELISA measurements.
T cell clone culture
The AC1 T cell clone specific for house dust mite (Der p 2) was isolated
as previously described (29). To elicit IL-5 production, T cell clones (105
cells/ml) were stimulated with and without a synthetic peptide (homologous for a Der p 2 epitope) in the presence of irradiated autologous EBVtransformed B cells and human rIL-12 (R&D Systems). Culture supernatants were collected after 48 h for the determination of IL-5 concentrations
by ELISA.
Cytokine assays
IL-5 concentrations in BAL T cell culture supernatants were measured in
duplicate using a commercial assay sensitive above 1 pg/ml (R&D Systems). IL-5 in PBMC culture supernatants were determined in duplicate
using commercially available Ab pairs (PharMingen, Cowley, U.K.), sensitive to 10 pg/ml. The same human rIL-5 standard (R&D Systems) was
used in both assays. IFN-␥ concentrations were measured by ELISA, also
in duplicate, using commercially available Ab pairs (PharMingen), sensitive to 10 pg/ml.
In situ hybridization
Riboprobes, antisense and sense, were prepared from cDNA encoding IL12R␤2 (generous gift from Dr. Gubler, Hoffmann-LaRoche, Nutley, NJ).
IL-12R␤2 cDNA was subcloned into the bluescript SKII⫺ vector. cDNA
was linearized with appropriate enzymes before transcription. Transcription was performed in the presence of [35S]UTP) and the appropriate T7 or
T3 RNA polymerases. In situ hybridization was performed on paraformaldehyde-fixed cytospin preparations of enriched BAL T cells, isolated as
above. Briefly, cytospins were permeabilized with Triton X-100 in PBS,
followed by proteinase K digestion. To inhibit nonspecific binding of 35S,
sections were treated with iodoacetamide and N-ethylmaleimide and then
in acetic anhydride/triethanolamine before hybridization with 35S-labeled
riboprobes. As a negative control, sections were hybridized with the sense
probe or treated with RNase A solution before the prehybridization step
with antisense probes. Specific hybridization was recognized as clear dense
deposits of silver grains in the photographic emulsion overlying cytospins.
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All subjects were premedicated with 2.5 mg nebulized albuterol, and 0.6
mg atropine and 5–10 mg midazolam administered i.v. Local anesthesia of
the vocal cords and trachea was induced with 2– 4% lidocaine. After inspection of the bronchial tree, the tip of the bronchoscope (an Olympus
BFP20; Olympus, London, U.K.) was wedged at random in a segmental
bronchus of the lingula or middle lobe, and BAL was performed by sequentially instilling two 60-ml aliquots of sterile warmed saline, followed
by gentle aspiration into a sterile glass bottle (baseline BAL). Allergen
challenge was then performed in a segmental bronchus of the lingula or
middle lobe (contralateral to that which had been lavaged) by instilling 100
biological units (BU) of P. pratense or D. pteronyssinus (Aquagen extract,
kindly provided by ALK Abelló, Horshølm, Denmark) made up in 5 ml of
sterile saline. The challenge site was observed for an additional 5 min, and
in the absence of excessive local bronchoconstriction, a further 400 BU of
allergen was introduced in 5 ml of sterile saline. All subjects were subsequently detained in hospital overnight for observation. During this period,
nebulized bronchodilator (5 mg albuterol) was administered as necessary to
the asthmatics to maintain FEV1 ⬎80% of the predicted value. A second
bronchoscopy was repeated after 24 h. Just before premedication for the
second bronchoscopy, a sample of peripheral venous blood was collected
in a sterile heparinized syringe. BAL (two 60-ml aliquots of saline) was
then performed in the allergen-challenged segment, as described above.
were performed with APC with allergen (i.e., without BAL T cells) to
confirm the absence of a background response in the irradiated PBMC
population. We previously showed that under these conditions, allergeninduced IL-5 production is detectable in cultures of enriched BAL T cells
from atopic asthmatics, but not peripheral blood T cells from the same
patients, or BAL T cells from nonatopic normal subjects. Moreover, we
reported that allergen-induced IL-5 production by BAL T cells correlated
with allergen-induced eosinophilia in the bronchi (28).
Allergen-stimulated BAL T cell cultures were supplemented with 0.5, 5,
and 50 U/ml human rIL-12 (R&D Systems), and 5, 50, and 500 U/ml
human rIFN-␥ (R&D Systems) or vehicle control. All cultures were performed in a minimum of triplicate. In some experiments, cultures were
supplemented with 10 ␮g/ml of neutralizing anti-IFN-␥ mAb (R&D Systems) to examine whether the effects of IL-12 were IFN-␥ dependent. Culture supernatants were removed from each microculture well on day 6 for
ELISA measurements.
The Journal of Immunology
2879
Statistics
Results are expressed as mean ⫾ SEM. Statistical analysis of data was
performed using a one-way ANOVA or paired Student’s t test, as described
in Fig. 1. All analyses were performed using a commercially available
statistical package (Minitab, State College, PA), and p ⬍ 0.05 was considered as significant.
Results
Effects of IL-12/IFN-␥ on IL-5 production by peripheral blood
T cells
FIGURE 2. Lack of effect of exogenous IL-12 on IL-5 production by an
allergen-specific T cell clone stimulated with specific peptide derived from
Der p 2. Data shown represent mean ⫾ SEM of duplicate cultures.
centrations of IFN-␥ chosen for use in subsequent cultures with
BAL T cells were 5, 50, and 500 U/ml.
Effects of IL-12 on IL-5 production by an established
allergen-specific T cell clone
To confirm previous reports that production of Th2 cytokines by
allergen-specific T cell lines and clones can be refractory to inhibition by IL-12 (17, 20), the effect of IL-12 on IL-5 production by
a T cell clone specific for Der p 2 (derived from house dust mite)
was also examined. T cell clones were stimulated with Der p 2-derived peptides and APCs (EBV-transformed B cells) in the presence of 0.005, 0.05, 0.5, 5, and 50 U/ml IL-12. In contrast to
PBMC, IL-5 production by the T cell clone appeared to be resistant
to the effects of IL-12 at these concentrations (Fig. 2).
Effects of IL-12 and IFN-␥ on cytokine production by BAL
T cells: comparison with effects on peripheral blood T cells
BAL T cells were enriched and cultured from four atopic asthmatics 24 h after segmental challenge with grass pollen allergen. The
characteristics of the subjects and the cell populations recovered in
BAL fluid are described in Table I and Table II, respectively (subjects 1 to 4). Two of the four subjects had current bronchial hyperreactivity (histamine PC20 ⬍ 8 mg/ml), whereas two patients
with pollen-induced asthma were asymptomatic out of season,
with histamine PC20 values ⬎8 mg/ml. All four subjects were
highly sensitized to P. pratense grass pollen, as evidenced by
markedly raised allergen-specific IgE levels. Both the total numbers of BAL cells and the percentages of eosinophils were increased following segmental allergen challenge in all subjects
(Table II).
Table I. Characteristics of subjects undergoing segmental allergen
challenge
FIGURE 1. Effect of exogenous recombinant IL-12 (a) and IFN-␥ (b)
on allergen-induced IL-5 production by PBMC from patients with allergen
asthma and/or rhinitis (mean ⫾ SEM, n ⫽ 4). Cells were cultured with
allergen in the absence/presence of recombinant cytokines and IL-5 measured by ELISA in supernatants collected after 6 days. In allergen-stimulated cultures, the statistically significant effects of IL-12 (p ⫽ 0.0005) and
IFN-␥ (p ⫽ 0.001) were determined by ANOVA. Comparison of individual IL-12 and IFN-␥ concentrations with baseline (i.e., allergen stimulation
without exogenous IL-12 or IFN-␥) was performed using the paired Student t test (ⴱ, p ⬍ 0.05 and ⴱⴱ, p ⬍ 0.005, vs baseline).
Patient
Age
(yr)
Sex
Methacholine
PC20 (mg/ml)
RAST (IU/ml)
PEFR % Falla
1
2
3
4
5
6
7
43
22
30
34
24
41
33
F
M
F
M
F
F
M
15
25
4.0
0.9
1.8
1.2
2.0
⬎100 (GPb)
45.9 (GP)
25.6 (GP)
⬎100 (GP)
54.4 (HDM)
33 (GP)
44.2 (GP)
33
12
41
39
22
21
12
a
b
Maximal fall in peak expiratory flow rate within 12 h of allergen provocation.
GP, grass pollen; HDM, house dust mite.
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Initial experiments were performed to determine the optimal concentration ranges of exogenous IL-12 and IFN-␥ required to downregulate IL-5 production by PBMC stimulated with allergen.
PBMC were isolated from four patients with allergic asthma
and/or rhinitis (not the same patients who later underwent segmental allergen challenge) and stimulated under conditions that were
previously shown to result in optimal CD4⫹ T cell-dependent IL-5
production (2). Addition of human rIL-12 significantly affected
allergen-induced IL-5 production (Fig. 1a; p ⫽ 0.0005 by
ANOVA). Comparison of individual IL-12 concentrations with
baseline showed that IL-12 significantly inhibited allergen-induced
IL-5 production at 0.5, 5, and 50 U/ml. Based on these results, the
concentrations of IL-12 chosen for use in subsequent cultures with
BAL T cells were 0.5, 5, and 50 U/ml. rIFN-␥ also had a significant effect on allergen-induced IL-5 production by PBMC (Fig.
1b; p ⫽ 0.001 by ANOVA), and this was statistically significant
compared with baseline at 5, 50, and 500 U/ml IFN-␥. The con-
2880
EFFECTS OF IL-12 ON LUNG T CELL CYTOKINE RESPONSES IN ASTHMA
Table II. Cell populations in BAL fluid of asthmatic patients before and after segmental allergen challenge
Patient
1
2
3
4
5
6
7
Sample
Total cells
(⫻ 106)
Lymphocytes
(%)
Eosinophils
(%)
Neutrophils
(%)
Macrophages
(%)
Baseline
Allergen
Baseline
Allergen
Baseline
Allergen
Baseline
Allergen
Baseline
Allergen
Baseline
Allergen
Baseline
Allergen
1.3
32.0
22.5
69.8
7.3
75.0
6.6
381.0
7.1
66.5
1.2
12
6
19.8
8
11
5
10
3
1
9
3
3
11
5
10
ND
13
1
9
2
9
1
6
0
21
2
20
0
24
ND
12
0
18
1
1
4
44
0
45
3
28
0
10
ND
1
91
62
92
81
92
49
89
28
92
41
95
56
ND
73
FIGURE 3. Effects of IL-12 and
IFN-␥ on IL-5 production by enriched
BAL T cells collected from the airways
of four atopic asthmatic subjects 24 h after segmental allergen challenge. BAL T
cells were cultured with P. pratense allergen and irradiated autologous APCs
(PBMC) in the absence/presence of recombinant cytokines. IL-5 was measured
in supernatants collected after 6 days by
ELISA. Bars represent mean of duplicate
ELISA measurements. NT, Not tested.
sponse, respectively ( p ⫽ 0.88 for BAL vs PBMC, by paired t
test). At 500 U/ml IFN-␥, IL-5 production by allergen-stimulated
BAL T cells and PBMC was 48.1 ⫾ 4.1% and 37.7 ⫾ 12.5% of the
maximal observed response, respectively ( p ⫽ 0.32 for BAL vs
PBMC, by paired t test). Similarly, for the purposes of comparing
responses of BAL T cell and PBMC cultures with IL-12, IFN-␥
concentrations in cultures were also expressed as a percentage of
the maximal response (Fig. 5). For enriched BAL T cells and
PBMC, maximal IFN-␥ production was observed at 50 U/ml IL12, and the percentage of this maximal response observed without
IL-12 was 11 ⫾ 6.6% and 11 ⫾ 8.9%, respectively ( p ⫽ 1 for
BAL vs PBMC, by paired t test). Thus, both IL-12 and IFN-␥
inhibited IL-5 production by enriched BAL T cells and PBMC to
a similar degree. Similarly, IL-12-induced IFN-␥ production,
though not clearly up-regulated by the presence of allergen alone,
was up-regulated in BAL T cells and PBMC by IL-12 to similar
degrees.
Inhibition of IL-5 responses by IL-12 is not affected by
neutralizing endogenous IFN-␥ production
To address the possibility that IL-12 indirectly suppresses allergen-induced IL-5 production through its ability to up-regulate
IFN-␥ expression, cultures from three asthmatics (PBMC in two
cases; enriched BAL T cells in one case) were supplemented with
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In each of the four patients tested, culture of enriched BAL T
cells with allergen and autologous irradiated PBMC (as APCs) was
accompanied by detectable IL-5 production. In three of the four
patients tested, IL-5 production appeared to be clearly induced by
the allergen, whereas in the other patient, allergen had only a minor effect in up-regulating IL-5 secretion (Fig. 3). In all four patients, addition of exogenous recombinant IL-12 or IFN-␥ resulted
in inhibition of IL-5 production (Fig. 3). In contrast to IL-5, production of IFN-␥ by enriched BAL T cells was not clearly induced
by allergen in any of the four patients examined, but increased in
the presence of IL-12 in all cases (Fig. 4). IL-5 and IFN-␥ could
not be detected in culture supernatants of irradiated PBMC alone
(either with or without allergen).
To facilitate direct comparison of the responsiveness of T cells
in BAL and PBMC to IL-12 and IFN-␥, data have also been expressed as a percentage of the maximal response (Fig. 5). IL-12
had significant effects on both IL-5 and IFN-␥ production by enriched BAL T cells ( p ⫽ 0.002 and p ⫽ 0.0005 by ANOVA,
respectively) and PBMC ( p ⫽ 0.001 and p ⫽ 0.004 by ANOVA,
respectively). Similarly, IFN-␥ also significantly affected IL-5 production by enriched BAL T cells and PBMC (both p ⫽ 0.02, by
ANOVA). The degree of inhibition of the IL-5 response was comparable in enriched BAL T cells and PBMC. At 50 U/ml IL-12,
IL-5 production by allergen-stimulated BAL T cells and PBMC
was 27.6 ⫾ 6.6% and 30.1 ⫾ 9.7% of the maximal observed re-
The Journal of Immunology
2881
FIGURE 4. Effects of IL-12 on IFN-␥ production by enriched BAL T cells collected from the
airways of four atopic asthmatic subjects 24 h after segmental allergen challenge. BAL T cells
were cultured with P. pratense allergen and irradiated autologous APCs (PBMC) in the absence/
presence of recombinant cytokines. IFN-␥ was
measured in supernatants collected after 6 days by
ELISA. Bars represent mean of duplicate ELISA
measurements.
that the effects of IFN-␥ on allergen-induced IL-5 production are
mediated through up-regulation of IL-12 production, PBMC cultures from two patients were supplemented with neutralizing antiIL-12 mAb. Although anti-IL-12 mAb reversed the effects of
IL-12 on allergen-induced IL-5 production, neutralization of IL-12
FIGURE 5. Comparison of the effects of IL-12 and IFN-␥ on IL-5 and IFN-␥ production by enriched BAL T cells harvested 24 h after segmental allergen
challenge and PBMC from the same patients (n ⫽ 4 atopic asthmatics). BAL T cells (8 ⫻ 104 cells/ml) were cultured with irradiated autologous APCs
(PBMC) and P. pratense allergen (as in Fig. 3), and PBMC (5 ⫻ 106 cells/ml) were cultured with P. pratense allergen only (as in Fig. 1). IL-5 and IFN-␥
were measured in supernatants after 6 days of culture. The statistically significant effects of IL-12 on enriched BAL T cell IL-5 production (p ⫽ 0.002)
and IFN-␥ production (p ⫽ 0.0005), and PBMC IL-5 production (p ⫽ 0.001) and IFN-␥ production (p ⫽ 0.004) were determined by ANOVA. Similarly,
the significance of the effects of IFN-␥ on BAL T cell IL-5 production (p ⫽ 0.02) and PBMC IL-5 production (p ⫽ 0.02) was also determined by ANOVA.
Comparison of individual IL-12 and IFN-␥ concentrations with baseline (i.e., allergen stimulation without exogenous IL-12 or IFN-␥) was performed using
the paired Student’s t test. Mean (⫾SEM) maximal IL-5 production by BAL T cells in cultures supplemented with IL-12 was 78.9 pg/ml (⫾47.6), and in
cultures supplemented with IFN-␥ was 77 pg/ml (⫾43.3). Mean (⫾SEM) maximal IL-5 production by PBMC in cultures supplemented with IL-12 was
2024 pg/ml (⫾965), and in cultures supplemented with IFN-␥ was 2005 pg/ml (⫾945). Mean (⫾SEM) maximal IFN-␥ production by BAL T cells in
cultures was 2201 pg/ml (⫾1053), and in PBMC in cultures supplemented with IL-12 was 1923 pg/ml (⫾517). (ⴱ, p ⬍ 0.05 and ⴱⴱ, p ⬍ 0.005, vs baseline).
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neutralizing anti-IFN-␥ mAb. The Ab reversed the inhibition of
IL-5 production caused by addition of exogenous rIFN-␥ (Fig. 6),
confirming the validity of the Ab. In contrast, the neutralizing antiIFN-␥ mAb failed to reverse the inhibitory effects of IL-12 in any
of the three experiments. Furthermore, to address the possibility
2882
EFFECTS OF IL-12 ON LUNG T CELL CYTOKINE RESPONSES IN ASTHMA
FIGURE 6. Effects of neutralizing anti-IFN-␥
mAb (10 ␮g/ml) and anti-IL-12 mAb (50 ␮g/ml) on
inhibition of T cell allergen-induced IL-5 production by IL-12 (5 U/ml) and IFN-␥ (50 U/ml), respectively. BAL T cells (8 ⫻ 104 cells/ml) were
cultured with irradiated autologous APCs (PBMC)
and P. pratense allergen (as in Fig. 2), and PBMC
(5 ⫻ 106 cells/ml) were cultured with P. pratense
allergen only (as in Fig. 1). IL-5 was measured in
supernatants after 6 days of culture. Bars represent
mean (⫹SEM) of duplicate ELISA measurements.
Expression of IL-12R␤2 subunit mRNA by BAL T cells
BAL T cells were isolated from five asthmatics (patients 3 to 7;
Tables I and II) before and after segmental allergen challenge using T cell enrichment columns. Expression of IL-12R␤2 subunit
mRNA transcripts was examined in cytospin preparations of these
cells by in situ hybridization (Fig. 7). The percentages of cells
expressing IL-12R␤2 mRNA were 68 (⫾12)% and 33 (⫾15)%,
respectively. Comparison of samples obtained before and after
segmental challenge revealed that there was a trend for the percentage of enriched BAL T cells expressing mRNA for the IL12R␤2 subunit to decrease following allergen provocation ( p ⫽
0.047 for baseline vs postallergen challenge, by paired t test).
Discussion
Allergen-specific T cells freshly isolated from the peripheral blood
of subjects with active atopic disease are characterized by production of Th2-type cytokines, indicating some degree of in vivo differentiation. In these T cells, production of IL-5, IL-13 (4), IL-4,
and IFN-␥ (16, 17) is consistently sensitive to the effects of IL-12.
According to several reports, allergen-specific T cell clones and
lines, activated and expanded in vitro from peripheral blood T
cells, can undergo commitment to the Th2-type lineage through
loss of sensitivity to the regulatory cytokine IL-12 (17, 19, 20).
Inthe present study, we tested the hypothesis that IL-5 production
by T cells present in the lungs of asthmatics during the allergeninduced late response, thought to be at least partly driven by T
cell-derived IL-5 (15, 30), is sensitive to inhibition by IL-12. There
is good reason that the functional status of such cells might be
ambiguous in relation to freshly isolated peripheral blood T cells
and peripheral blood T cells that have been activated and expanded
with Ag in vitro. First, the allergen-induced late asthmatic response is characterized by an increase in expression of IL-5, IL-4,
and IL-13 by bronchial mucosal or luminal T cells (6, 7, 31) and
an increased frequency of T cells expressing surface activation
markers, such as CD25 (7). This suggests some degree of allergendriven T cell activation and differentiation. Second, bronchial T
cells are phenotypically and functionally distinct from freshly iso-
lated peripheral blood T cells: for example, BAL T cells are almost
exclusively CD45RO⫹ memory T cells (32), show an increased
tendency to undergo apoptosis (33), and, when collected 24 h aftersegmental allergen challenge, BAL T cells produce significantly
greater amounts of IL-5 than equivalent numbers of freshly isolated peripheral blood T cells when stimulated with allergen in
vitro (28).
In the present study, we chose to study T cells in BAL fluid
rather than biopsy tissue specimens because it was our aim to
investigate the responses of T cells that had been freshly isolated
in an ex vivo culture model: the number of enriched T cells in free
suspension that can be obtained from bronchial biopsy tissue is
much lower than with BAL, necessitating prolonged expansion in
vitro (34). We previously reported that IL-5 production by BAL,
but not peripheral blood T cells stimulated with allergen and irradiated PBMC (as APC) correlates with the degree of airway eosinophilia and is higher in asthmatics than control subjects (28).
Although the APC types in PBMC are unlikely to reflect the exact
composition of APC in the lung, a previous report did not find
differences in IL-12 responsiveness by allergen-specific peripheralblood T cells stimulated by different APC types (17). We conclude, therefore, that the conditions adopted for this study are of
physiological relevance and are preferable to alternative means,
such as use of nonspecific mitogens or expanded T cell clones or
lines. The relationship between the properties of BAL T cells and
bronchial mucosal tissue T cells does, however, remain to be established conclusively. Nevertheless, there is strong evidence from
human studies to suggest that the properties of BAL T cells are
highly relevant to asthma: first, CD3⫹ BAL T cells, like CD3⫹ T
cells in the bronchial mucosa, are characterized by elevated expression of IL-5 and IL-4 mRNA (35). Second, the proportion of
BAL T cells that are activated and express Th2-type cytokine
mRNA has been correlated with asthma symptoms (36). Third,
following inhalational allergen challenge, the numbers of BAL T
cells expressing Th2-type cytokine mRNA are increased, correlating with the numbers of recruited eosinophils (7). Finally, improvement in asthma symptoms and decreased BAL eosinophilia
following steroid therapy is accompanied by decreased expression
of Th2-type cytokine mRNA by BAL T cells (37).
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did not reduce the inhibitory effect of IFN-␥ on IL-5 production
(Fig. 6).
The Journal of Immunology
To our knowledge, this is the first study to directly examine the
effects of IL-12 and IFN-␥ on cytokine responses by T cells isolated from human asthmatic airways. BAL T cells collected 24 h
after a segmental allergen challenge produced IL-5 in response to
allergen stimulation and, as with IL-5 production by T cells in
PBMC, this was inhibited by IL-12 in a concentration-dependent
manner. Furthermore, the degree of inhibition at particular IL-12
concentrations, expressed in percentage terms, was similar for T
cells from both sources (Fig. 5). Addition of IFN-␥ to cultures also
resulted in similar patterns of inhibition of IL-5 production by
enriched BAL T cells and PBMC (Fig. 5). Furthermore, in addition
to inhibiting IL-5 production, IL-12 also up-regulated IFN-␥ production in BAL and PBMC cultures.
Collectively, these data suggest that in terms of their ability to
respond to IL-12 and IFN-␥, cultured T cells derived from asth-
matic late responses closely resemble T cells in the peripheral
circulation. Thus, T cells present in the airways of asthmatics following allergen exposure, although predisposed to produce cytokines such as IL-5, appear not to have undergone commitment to
loss of IL-12 sensitivity. It was previously reported that human
naive cord blood T cells or CD45RA⫹ peripheral blood T cells
show induction of IL-12R␤2 mRNA and surface protein on stimulation with anti-CD3 mAb, and that this is potentiated by IL-12
(22, 26). It is therefore possible, in theory, that the responsiveness
of BAL T cells to IL-12 in our study was due to up-regulation of
the IL-12R␤2 subunit on in vitro restimulation with allergen and
IL-12, and that the same cells would have been unresponsive to
IL-12 in vivo. There are a number of observations, however, that
suggest that this is unlikely to be the case. First, Marshall et al. (17)
restimulated allergen-specific T cells from atopic patients with
specific allergen through a number of expansion cycles in the presence or absence of IL-12 before analyzing cytokine production.
The effects of adding IL-12 at the time of restimulation with allergen through the TCR progressively diminished through each
cycle until there was no effect on IL-4 and IFN-␥ production. Second, Hilkens et al. (20) stimulated allergen-specific Th2 clones
with anti-CD3 and anti-CD28 mAb in the presence of IL-12 and
also reported no effect on production of cytokines. These findings
are consistent with our own data obtained with an allergen-specific
T cell clone (Fig. 2). Thus, IL-12 responsiveness does not appear
to be a global feature of all human memory allergen-specific T
cells restimulated through the TCR in vitro.
Current evidence strongly suggests that failure to express IL12R␤2 mRNA correlates with IL-12 insensitivity in Th2 cells (21,
22), although recent studies suggest, at least in mice, that restoration of IL-12R␤2/STAT4 signaling in committed Th2 cells may
not reverse IL-12 insensitivity (38, 39). In the light of this correlation, our aim was to seek support for our hypothesis that airway
T cells in asthmatics are IL-12 responsive by determining whether
IL-12R␤2 mRNA transcripts are detectable in enriched BAL
CD3⫹ T cells after segmental allergen provocation. In the event,
significant numbers of BAL T cells could be demonstrated to contain IL-12R␤2 mRNA transcripts (mean ⫽ 33.1%) following segmental allergen challenge (Fig. 7). Although we are unable to determine whether or not these cells were allergen specific, these data
are consistent with the observed IL-12 responsiveness of these T
cells when stimulated with allergen in vitro. Although the proportion of BAL T cells expressing IL-12R␤2 mRNA was higher before provocation with allergen, we cannot be certain whether this
reflects in situ down-regulation of IL-12R␤2 mRNA expression in
Th2-type cells, or a dilution of the IL-12R␤2-expressing population by T cells recruited during the asthmatic late response. As
with the in vitro experiments, we elected to perform these measurements on the entire CD3⫹ population since studies of human
cord blood CD8⫹ and CD4⫹ T cell lines induced to produce type
1 and type 2 cytokines suggest that the proposed restricted patterns
of IL-12R␤2 are applicable to both CD8⫹ and CD4⫹ T cells (26).
Furthermore, studies on BAL and bronchial biopsies from asthmatics have indicated that CD8⫹ cells producing type 2 cytokines
(Tc2 cells), as well as CD4⫹ T cells, are present in the airways of
these subjects (40, 41). Thus, studying the entire CD3⫹ population
may be more relevant than studying CD4⫹ cells in isolation.
Although IL-5 production by BAL T cells was up-regulated by
culture with allergen, this was not the case with IFN-␥. It was not
apparent whether the increased IFN-␥ production in response to
IL-12 was spontaneous or allergen induced in specific T cells because the small numbers of cells that could be isolated precluded
setting up cultures in the absence of allergen. Nevertheless, it
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FIGURE 7. a, Percentages of enriched BAL T cells isolated before
(baseline) and after segmental allergen challenge (allergen) expressing
mRNA encoding the IL-12R␤2 subunit. ⴱ, p ⬍ 0.05, by paired Student’s
t test. b, Photomicrograph of a cytospin preparation of BAL T cells isolated
after segmental allergen challenge and hybridized with a radiolabeled antisense IL-12R␤2 riboprobe (arrows indicate examples of positive cells).
c, Photomicrograph of a cytospin preparation of BAL T cells isolated after
segmental allergen challenge and hybridized with a radiolabeled sense IL12R␤2 riboprobe (negative control).
2883
2884
EFFECTS OF IL-12 ON LUNG T CELL CYTOKINE RESPONSES IN ASTHMA
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
Acknowledgement
We thank Fiona O’Brien and Wendy Noble for their expert technical assistance.
24.
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of IL-12 on IFN-␥ production by all T cells that would be biologically important. A further issue relates to whether the effects of
IL-12 on IL-5 production are independent of endogenously produced IFN-␥, and similarly, whether the effects of IFN-␥ are IL-12
independent. In experiments performed with allergen-stimulated
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other cytokine.
One question raised by our findings concerns the role of extinction of IL-12 sensitivity in Th2 immune responses if this is not a
property of effector T cells at the site of inflammation. Although
entirely speculative, one possibility is that extinction plays a role
in long-term memory of Th2-type immune responses and occurs
only in a subpopulation of long-lived T cells that are selected by
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and lines.
This study, which suggests that the presence of IL-12 and IFN-␥
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even during an ongoing asthmatic late response, is consistent with
previous reports suggesting that the bronchial mucosa (23) and
peripheral blood (42) of asthmatics are characterized by abnormally low IL-12 expression. Furthermore, clinical improvement
following specific allergen immunotherapy (24) or corticosteroid
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IFN-␥. We believe that these findings provide a valuable insight
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human allergic disease.
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