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ß, But Not 
, T Cells1
University Medicine, Southampton General Hospital, Southampton, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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, but
secreted the chemokine RANTES without any overt stimulation. In
addition to allergen, stimulation of asthmatic explants with mAbs to
CD3 and TCR-
ß but not TCR-
induced IL-5 secretion.
Allergen-induced IL-5 and IL-13 production by the asthmatic tissue was
inhibited by anti-CD80 and, to a lesser extent, by anti-CD86
mAbs. In contrast, the production of these cytokines by PBMCs
was not affected by mAbs to CD80, was inhibited by anti-CD86, and
was strongly attenuated in the presence of both Abs. FACS analysis
revealed that stimulated asthmatic bronchial tissue was comprised of
CD4+ T cells that expressed surface CD28 (75.3%) but
little CTLA-4 (4.0%). Neutralizing mAbs to CD40 ligand had no effect
on the cytokine levels produced by asthmatic tissue or PBMCs.
Collectively, these findings suggest that allergen-specific ß T
cells are resident in asthmatic bronchial tissue and demonstrate that
costimulation by both CD80 and CD86 is essential for allergen-induced
cytokine production. In contrast, CD86 appears to be the principal
costimulatory molecule required in PBMC responses. Attenuation of type
2 ß T cell responses in the bronchial mucosa by blocking these
costimulatory molecules may be of therapeutic potential in
asthma. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CD4+ T cell activation and cytokine production require two distinct signals from the APC. The first signal is triggered by interaction of the Ag-specific TCR with the MHC-peptide complex. The second is a costimulatory signal, of which the most widely studied are CD28 and CTLA-4 molecules expressed on T cells (for review, see Ref. 3). Their ligands CD80 (B7-1) and CD86 (B7-2) are expressed on several types of APCs, including monocytes/macrophages, dendritic cells (DCs)3 (3), activated B cells, keratinocytes, and some activated T cells (4, 5, 6, 7). Inhibition of costimulation prevents T cell activation and can lead to long term T cell unresponsiveness or anergy (8). A costimulatory signal mediated by CD28, which is expressed constitutively on CD4+ and most CD8+ T cells, is required for the activation and the production of various cytokines, including IL-2 (9). In contrast, CTLA-4, which is up-regulated upon T cell activation, has been reported to inhibit T cell proliferation (10), promote Ag-specific apoptosis (11), and suppress the production of cytokines by both Th1 and Th2 cells (12).
Murine models of Ag-provoked airway inflammation revealed that blockade of CD80 and/or CD86, using CTLA-4Ig fusion protein, inhibited T cell activation in vivo (13, 14). Krinzman et al. (13) demonstrated the attenuation of airway hyper-responsiveness and pulmonary inflammation in mice treated with CTLA-4Ig during aerosolized Ag challenge. Using a mutant form of CTLA-4Ig that bound to CD80 but not CD86, it was found that CD80 costimulation was not necessary for the induction of Th2 responses but was required for the maintenance or amplification of lung inflammatory responses in mice (15). Airway administration of an anti-CD86 mAb inhibited Ag-induced airway hyper-responsiveness in vivo and attenuated eosinophil infiltration, IgE production, and Th2 cytokine production (16). These experiments in animals are important because they demonstrate the requirement of T cell costimulation in lung inflammatory responses and implicate components of allergic inflammation that influence airway function.
More recent data suggest that CD86-mediated costimulation may favor IL-4 production and Th2-type immune responses. Kuchroo et al. (17) demonstrated that CD28 ligation with CD80 was required for the generation of a Th1 response, while engagement of CD86 promoted the development of a Th2 response in mice. Other studies also support an important role for CD86 in the signaling of IL-4 production and the development of Th2 cells (18). However, notable exceptions exist. Greenwald et al. have demonstrated that either CD80 or CD86 ligand interactions can provide the required costimulatory signals that lead to T cell effector function during a type 2 mucosal immune response in mice following nematode infection (19). The elucidation of Ag presentation and T cell costimulatory requirements in human bronchial asthma has been difficult, partly because DCs juxtaposed to the airway epithelium or tissue macrophages are likely to be the most effective APCs, but these cells are poorly represented in bronchoalveolar lavage (BAL) or PBMC samples from asthmatic subjects. Nevertheless, there is accumulating evidence to suggest that CD86, rather than CD80, is involved in allergen-induced T cell proliferation and cytokine production from asthmatic BAL or PBMCs (20). However, to date, the relative importance of CD80 and CD86 or other T cell/APC interactions in driving the allergic T cell response in the diseased asthmatic bronchial mucosa has not been determined. Such studies are crucial for the development of useful therapeutic interventions in asthma.
We have previously demonstrated that ex vivo allergen stimulation of
bronchial biopsies from mild atopic asthmatics induced the secretion of
IL-5 and IL-13, and this was inhibited by CTLA-4Ig (21).
These observations identify B7 costimulation as being a prerequisite
for the production of Th2 cytokines in human bronchial asthma. In the
present study we have extended our investigation into the role of CD80
and CD86 individually and the involvement of CD40/CD40L interactions in
the allergen-driven Th2 cytokine production from asthmatic bronchial
tissue and PBMCs. Our results demonstrate an important role
for resident ß T cells in IL-5 production by asthmatic bronchial
mucosa and an essential requirement for both CD80 and CD86
costimulation, but not CD40/CD40L interactions, in allergen-induced Th2
cytokine expression.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sixteen mild atopic asthmatic forced respiratory volume in
1 s (FEV1; >80% predicted) and 10 atopic
nonasthmatic control subjects participated in the study (some of the
asthmatic volunteers participated on more than one occasion). We
compared atopic asthmatic with atopic, nonasthmatic volunteers to be
certain that the observed allergen responses in the airways were
indicative of events characteristic of this type of asthma rather than
atopy. Both groups of subjects were selected on the basis of having
positive skin-prick tests (
3 x 3 mm wheal and flare reaction)
to house dust mite extract, Dermatophagoides pteronyssinus
(Der p). In addition, the asthmatics were selected on the basis of an
increased airway responsiveness to methacholine, i.e., cumulative
concentration producing a fall in FEV1 of 20%
from baseline (PC20) < 16 mg/ml. The
asthmatic patients (6 females and 10 males; mean age, 29.8 ± 2.9
yr) had not experienced an exacerbation of their asthma or upper
respiratory tract infection at least 6 wk before participation in the
study and were using only inhaled short-acting
ß2-agonist medication as required (less than
three or four times a day) for relief of symptoms (National Institutes
of Health guidelines (22)). Their mean serum IgE was
303 ± 159 IU/ml, methacholine PC20 was
3.6 ± 1.0 mg/ml, and FEV1 was 90.7 ±
3.6% (predicted). The atopic, nonasthmatic control volunteers (five
females and five males; mean age, 22.3 ± 1.1 yr) had no history
of asthma, normal FEV1 values, a mean
methacholine PC20 >32 mg/ml, and serum IgE of
86.4 ± 41.1 IU/ml. The control subjects were asymptomatic, with
the exception of one who suffered from rhinitis. All volunteers were
nonsmokers. Informed written consent was obtained from the subjects
before participation, and the study was approved by the joint ethics
committee of Southampton University and General Hospital.
Endobronchial biopsy and peripheral blood samples
Peripheral venous blood was obtained from the subjects for analysis, and fiberoptic bronchoscopy was performed using a standard technique conforming to published guidelines (23). Briefly, subjects were premedicated with nebulized salbutamol (2.5 mg), ipratropium bromide (0.5 mg), and i.v. midazolam (5–10 mg). Topical lidocaine 2% (upper airways) or 1% (lower airways) was used to obtain local anesthesia. Using alligator forceps, endobronchial mucosal biopsies were obtained from subcarinae separating the basal segmental bronchi of the right lower lobe and placed in culture medium.
Culture protocol
Eight separate bronchial biopsies (each 1–2 mm in diameter)
were obtained from each subject, and to minimize effects due to tissue
heterogeneity or variability in composition, two biopsies were used per
culture condition in a given experiment. Thus, using eight biopsies
from each patient, a total of four culture conditions were set up in a
particular experiment. Bronchial biopsies (two biopsies per culture
condition) were cultured for 24 h in serum-free medium alone (500
µl; AIM V, Life Technologies, Paisley, U.K.), in the presence of Der
p allergen (5000 U/ml or 0.35 µg/ml; ALK, Horsholm, Denmark), or Der
p and 10 µg/ml blocking Abs (CD80 or CD86, and in other experiments
CD40 or CD40L, azide-free Abs purchased from Alexis Corp., Nottingham,
U.K.). The effect of stimulation of biopsies with immobilized
anti-CD3, anti-TCR-ß, and anti-TCR- mAbs on
cytokine production was also examined in separate experiments. The
tissue was cultured for 24 h in wells precoated with immobilized
anti-CD3, anti-TCR-ß, or anti-TCR- (2 µg/ml;
PharMingen, Oxford, U.K.). Appropriate isotype control Abs (azide free)
were used. Twenty-four-well, flat-bottom culture plates were used, and
culture supernatants were harvested and stored at -80°C pending
ELISA analysis. A defined culture medium (AIM V) was used throughout
this study, as described previously (21), to preclude the
possibility of stimulation of the tissue with serum components. The ALK
allergen extract was tested using an E-Toxate kit (Sigma, Poole, U.K.)
and was found to be free of endotoxins.
PBMCs (3 x 106 cells/ml) from same subjects were cultured for 6 days using the same conditions as those for the lung biopsies described above. For isolation of PBMCs, heparinized venous blood (20 ml) was layered onto Ficoll-Isopaque (20 ml Lymphoprep, Nycomed, Oslo, Norway) in sterile tubes. After centrifugation at 1000 x g for 25 min at 20 °C, PBMCs were gently aspirated from the plasma/Ficoll interface, transferred to sterile universal tubes, and washed twice (centrifugation at 250 x g for 10 min) with AIM V medium.
Cytokine protein measurement
The level of cytokine proteins in the culture supernatants of
biopsy and blood samples was determined by commercially available
Quantikine ELISA kits for IL-4 (ultrasensitive), IL-5 and IFN- (R&D
Systems, Abingdon, U.K.), and by Cytoscreen kits for IL-13, RANTES, and
GM-CSF (Biosource International, Lifescreen, Watford, U.K.), according
to the manufacturer’s instruction. The sensitivity of most of these
kits is <5 pg/ml, except for IL-4, which is <0.05 pg/ml. In general,
samples and standards were diluted with assay diluent and added to a
96-well microtiter plate precoated with Ab against the appropriate
cytokine. The plate was sealed and incubated at room temperature for
1–2 h. After washing the plate four times, the appropriate conjugated
Ab was added and incubated for an additional 1–2 h, followed by four
washings. Finally, substrate solution was added to the wells, and color
development was stopped after 20- to 30-min incubation. Plates were
read by an ELISA plate reader at 450 nm. A standard curve was plotted,
and the cytokine concentration (picograms per milliliter) of the
samples was calculated. Cytokine levels in biopsy supernatants were
normalized by expressing them as picograms per milligram wet weight of
tissue. Tissue weight was determined after culture and careful removal
of excess medium. It is important to note that an average of 93.3 pg/ml
of IL-5 was detected in supernatants of allergen-stimulated asthmatic
biopsies, and this value was divided by the tissue weight (typically
6.1 mg for two biopsies).
Flow cytometric analysis
FACS analysis was used to determine the expression of CD28 or
CTLA-4 on CD4+ T cells and of CD80 or CD86 on
APCs in allergen-stimulated and unstimulated bronchial tissue and
PBMCs (from same asthmatic subjects). PBMCs were
prepared and cultured for 6 days as described above. For FACS analysis
bronchial biopsies were cultured in medium alone (four biopsies) or
stimulated with Der p allergen (four biopsies) for 24 h. Following
culture the tissue was digested in 1 ml of medium containing
collagenase (0.1%; Sigma) and hyaluronidase (0.01%; Sigma) for 1
h in a shaking water bath at 37 °C. After filtering using a 70-µm
pore size strainer, cells were washed with medium and counted (average
yield was 1 x 105 cells/biopsy).
Nonspecific binding to Fc receptors was blocked by incubating
dispersed bronchial cells or PBMCs for 30 min with human
Fc Ig (5 µg/106 cells; gift from Prof. M.
Glennie, Tenovus Institute, Southampton, U.K.). Cells were stained for
30 min on ice with FITC mAbs to CD4 (Becton Dickinson, Oxford, U.K.),
CD80 or CD86 (Serotec, Oxford, U.K.), PE CTLA-4 (PharMingen, Oxford,
U.K.), and CD28, CD19, or CD14 (Becton Dickinson), appropriately
diluted in PBS. Appropriate PE or FITC isotype controls (IgG1 and
IgG2a from Becton Dickinson; IgG2b and IgM from Serotec) were also
used. After staining, cells were washed in PBS and analyzed by FACScan
(Becton Dickinson) using Consort 30 software, and 10,000 events were
acquired.
Statistical analysis
Cytokine protein levels were compared between study groups using the Mann-Whitney U test. The Wilcoxon signed rank test for paired data was used for within-group comparisons. Analysis was performed using StatView (BrainPower, Calabasa, CA) for Macintosh. Values of p < 0.05 were accepted as statistically significant.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Our previous analysis had demonstrated that stimulation of
bronchial tissue from atopic asthmatic, but not normal control,
subjects with allergen induced secretion of IL-5 and IL-13
(21). We extended the study and monitored the production
of various cytokines by airway tissue from atopic asthmatic compared
with atopic nonasthmatic subjects. Bronchial biopsies were stimulated
ex vivo with Der p allergen, and the production of cytokine proteins
was determined by ELISA. Initially, a time course for the production of
IL-5 by asthmatic biopsies was performed. Secretion of this cytokine by
the explants commenced after 12 h of allergen stimulation and
peaked at 24 and 48 h (Fig. 1
). A
24-h culture period was used for monitoring cytokine production by
bronchial explants throughout this study.
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show that bronchial
tissue from asthmatic subjects clearly secreted higher levels of IL-5,
IL-13, and IL-4 after 24-h allergen stimulation than explants from
atopic, nonasthmatic control subjects. Consistent with our previous
findings (21), there was no production of the Th1 cytokine
IFN- from either asthmatic or nonasthmatic explants stimulated with
allergen. Fig. 2 represents a compendium of cytokine responses of
medium alone and Der p cultures measured during the course of the
study. To ascertain that T cells resident in the asthmatic bronchial
tissue produce IL-5, biopsies were stimulated with immobilized mAbs to
CD3, TCR-ß, or TCR-. In addition to allergen, significant
(p < 0.05) IL-5 production was induced by
tissue stimulated with immobilized anti-CD3 or anti-TCR-ß
(p < 0.05), but not with anti-TCR-
(Fig. 3).
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PBMCs from the asthmatic subjects stimulated with allergen for 6 days also secreted both IL-5 (5.3 pg/ml with medium alone vs 377.4 pg/ml with allergen; n = 8) and IL-13 (16.4 pg/ml with medium vs 140.6 pg/ml with allergen), but not IL-4 (0.7 pg/ml with medium vs 1.2 pg/ml with allergen). PBMCs from atopic nonasthmatic control subjects also secreted IL-5 and IL-13 after prolonged allergen stimulation, but the levels were one-third of those produced by PBMCs from atopic asthmatic subjects (data not shown). Cytokine levels produced by PBMCs from both asthmatic or control subjects when stimulated with allergen for 1–4 days only were low or essentially undetectable (data not shown).
It is important to note that a pool of two asthmatic biopsies was found
to typically contain 2 x 105 cells and
secrete 
93.3 pg of IL-5 in response to allergen. Thus,
106 bronchial cells typically produce 466.5 pg of
IL-5 after 24-h allergen stimulation (3-fold more than
106 PBMCs after 6-day stimulation).
Effects of anti-CD80 and anti-CD86 Abs on allergen-induced cytokine production
We have shown in this study that ß T cells resident in the
asthmatic bronchial tissue can be activated to secrete IL-5. Moreover,
we have previously described a requirement for B7 costimulation for
cytokine production (21). To further extend this work, we
sought to determine whether this response involved both CD80 and CD86
costimulation, or whether one accessory molecule predominated over the
other. Our results show that allergen-induced secretion of IL-5 and
IL-13 by asthmatic airway tissue was inhibited by anti-CD80 and, to
a lesser extent, by anti-CD86 mAbs (Fig. 4). In contrast, allergen-induced
secretion of these cytokines from PBMCs of the same subjects
were not affected by blocking Abs to CD80 alone, were inhibited by
anti-CD86 (p < 0.05), and were strongly
attenuated in the presence of both anti-CD80 and anti-CD86 mAbs
(p < 0.05; Fig. 5). The inhibition in the presence of
both Abs was significantly stronger compared with that induced by
anti-CD86 mAb alone (p < 0.05).
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Expression of costimulatory molecules in asthmatic bronchial tissue
Flow cytometry was used to determine the cellular expression of
CD80 and CD86 in the airways. FACS analysis revealed that enzymatically
dispersed asthmatic bronchial explants were comprised of
CD4+ (44.8 ± 1.0% gated live mononuclear
cells; n = 3) and CD8+ T cells
(23.2 ± 5.2%; n = 3), and few B cells (4.7
± 1.1% CD19+ cells; n = 3) and
macrophages (6.9 ± 2.6% CD14+ cells;
n = 3). Fig. 6 shows that
CD4+ T cells in the bronchial tissue expressed
CD28 (75.3 ± 9.4%; n = 3), but little or
undetectable surface CTLA-4 (4.0 ± 0.5%; n = 3).
Very few APCs expressing CD86 and CD80 were detected in the explants
(data not shown).
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), but not in that of CD80, was observed
after allergen stimulation.
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It has been suggested that ligation of CD40 up-regulates the
expression of CD80 and CD86 molecules on APC (24). We
therefore examined the effects of blocking CD40/CD40L interactions on
the allergen-induced production of cytokines by asthmatic biopsies and
PBMCs. However, neutralizing mAbs to CD40L or CD40 did not
have any effect on the levels of Th2 cytokines produced by either the
tissue or blood (Fig. 8). The
anti-CD40L mAb (10 µg/ml) used in this study did block a mixed
lymphocyte reaction in which PBMCs (3 x
105 responders) were cultured with irradiated
allogeneic PBMCs (1 x 105
stimulators). To measure proliferation, cultures were pulsed with
[3H]thymidine after 6 days and harvested
18 h later.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In the present study we demonstrate that ex vivo allergen stimulation
of bronchial mucosal tissue from mild atopic asthmatic, but not atopic
nonasthmatic control, volunteers elicited an increase in the production
of IL-5, IL-4, and IL-13. There was no production of IFN- or GM-CSF
by the bronchial explants from both asthmatic and control subjects.
Moreover, RANTES was secreted by bronchial explants from both groups of
subjects without allergen stimulation. The constitutive production of
RANTES suggests that this chemokine plays a role in the homeostasis of
the lung, possibly by promoting the migration of cells across the
bronchial epithelium (31). Interestingly, we found that
neutralizing mAbs to CD80 blocked allergen-induced cytokine secretion
by the asthmatic tissue, and mAbs to CD86 were only marginally less
effective at inhibiting the response. Because both these CD28 ligands
are required for Th2 cytokine production, it seems likely that T cells
resident in the asthmatic bronchial tissue are the major source of the
allergen-induced cytokines. This is supported by the observation that
stimulation of the biopsies with mAbs to CD3 and TCR-ß, but not
TCR-, induced IL-5 secretion. Our findings are in contrast with
the recent suggestion that T cells may play an important role in
allergen recognition and inflammation in the airways (32).
Although our data imply that T cells are the major cellular providence
of allergen-induced cytokines in the asthmatic bronchial tissue, other
cell types that are thought to be an important source of IL-5, IL-13,
and IL-4 in this disease include eosinophils, mast cells, and basophils
(33, 34, 35, 36). However, it is unlikely that they are the main
source of cytokines in our system because of the requirement for B7
costimulation, which has not been demonstrated to be essential for
allergen-driven cytokine production by these cells. Moreover, IL-5
production could not be elicited from bronchial tissue following IgE
receptor cross-linking (21).
We have demonstrated that allergen stimulation of bronchial tissue from
atopic nonasthmatic volunteers failed to induce the production of Th1
or Th2 cytokines. This suggests that either there is a low frequency of
allergen-specific T cells in the airway tissue from these subjects or
the Ag was not presented efficiently. However, we have shown previously
that the polyclonal T cell activator, PHA, did induce higher levels of
IFN- production from nonasthmatic compared with asthmatic bronchial
tissue, and therefore, Ag presentation does not limit the response
(21). Conversely, allergen-stimulated bronchial tissue
from atopic asthmatics produced significant levels of IL-5 and IL-13,
but not IFN-, demonstrating not only that a higher frequency of
allergen-specific T cells is resident in the asthmatic tissue, but that
they are predominantly of a Th2 phenotype.
In the present study PBMCs from asthmatic subjects secreted IL-5 and IL-13, but not IL-4, following protracted (6 days) allergen stimulation. Furthermore, in contrast to the airway tissue, allergen-induced PBMC cytokine production was not affected by Abs to CD80, but was inhibited by anti-CD86. The PBMC response was strongly attenuated in the presence of both anti-CD80 and anti-CD86 Abs. The latter findings are consistent with those of other studies (29, 30), demonstrating that CD86 is an important costimulatory molecule for human PBMC responses to allergens. Thus, allergen responses in the airway mucosal tissue and PBMCs differ in the relative contributions of CD80 and CD86. Specifically, both CD80 and CD86 stimulations are essential for cytokine expression by the asthmatic tissue, whereas CD86 is the principal costimulatory molecule required in PBMC responses. It is likely that these differences reflect the availability of the costimulatory molecules at these sites. Consistent with this view is the finding that in blood, CD86, but not CD80, is expressed by resting B cells and monocytes (5, 37). The reason for the requirement for both CD80 and CD86 costimulation for cytokine production by the asthmatic airway tissue is unclear, since monitoring their expression in the tissue during culture proved difficult. Possibly, both CD80 and CD86 are expressed at low levels, and either alone is insufficient to elicit a costimulatory response. However, collectively they may provide sufficient signal to costimulate cytokine expression by T cells. Another explanation could be that CD80 and CD86 may interact with receptors other than CD28 or CTLA-4 in ways that have not been characterized. Interestingly, Chambers et al. have reported that NK cells express receptors other than CD28 and CTLA-4 that interact with CD80 (38). Moreover, a novel inducible T cell costimulator that is structurally and functionally related to CD28 has recently been identified (39). It seems possible that complex ligand-receptor interactions, involving multiple cell types, take place in the asthmatic airways, making it difficult to prioritize which specific cellular interaction is important in this disease.
We used flow cytometry to examine the expression of CD28 and CTLA-4 on CD4+ T cells or CD80 and CD86 on APCs present in bronchial tissue from asthmatic volunteers after culture for 24 h in the presence or the absence of allergen. FACS analysis revealed that CD4+ T cells (44.8 ± 1.0% live mononuclear cells) were present in asthmatic airway tissue, and they expressed cell surface CD28 (75.3 ± 9.4%), but little CTLA-4 (4.0 ± 0.5%). CTLA-4 function requires cell surface expression, but unlike CD28 it is predominantly localized in intracellular vesicles (40). However, during T cell activation, intracellular stores relocate to the cell surface and become focused at the sites of TCR ligation (40). Thus, although we observed very little surface expression of CTLA-4 in asthmatic bronchial tissue, it is possible that this molecule is primarily expressed intracellularly. The small numbers of APCs present and therefore limiting amounts of CD80 and CD86 expression in the tissue has made it difficult to resolve which APC type(s) resident in the asthmatic airways is involved in the presentation of the allergen. In general, PBMCs from asthmatic subjects expressed higher levels of CD86 than CD80 on both B cells and monocytes. Moreover, a significant increase in the expression of CD86 on B cells was observed after prolonged allergen stimulation. CD80 and CD86 are expressed on professional APCs that include monocytes/macrophages, B cells, and DCs. Resting monocytes and B cells have been shown to express CD86 but no detectable CD80 (5, 37, 41), and both molecules are up-regulated after activation (5, 37, 41, 42) but display different expression kinetics (43).
In this study an accelerated T cell response was observed in the asthmatic bronchial tissue (24 h) compared with those in PBMCs (6 days). This is probably due to an increased frequency of Ag-specific memory T cells in the airways, resulting in a more rapid and efficient response. A number of studies have suggested that memory and effector T cells are less dependent than naive T cells on costimulatory signals (44, 45). Gause et al. demonstrated that memory T cells do not require B7/CD28 interactions for their development into effector cells that can mediate a host protective type 2 response (46). We have shown that B7 costimulation is required for driving effector T cell responses in bronchial tissue from mild asthmatic subjects. Whether T cell responses in the airways of patients with more severe asthma are less dependent on costimulation is currently under investigation.
Animal models of airway inflammation have been useful in identifying specific immune processes required for driving type 2 responses and the development of pulmonary eosinophilia (14, 15, 16). The relevance of these observations to human asthma is unclear, since we (47) and others (48, 49, 50) have found that lung T cell responses to inhaled Ags in mice are typically transient and show evidence for strong immune regulation. We reported that following OVA inhalation, lung parenchymal T cell proliferative responses were prevented by the action of interstitial macrophages in BALB/c mice that have been given OVA-specific DO11.10 Th cells (47). This form of regulation, which appears as a selective defect in IL-2-driven proliferation, may serve to prevent the development of chronic pulmonary lymphoproliferative responses (47). In the present study we have demonstrated that there is a critical role for both CD80 and CD86 costimulation in Th2 responses in human asthma. These observations, which differ from those seen in murine models of "asthma," are the first to resolve the exact costimulatory requirements for activation of allergen-specific T cells in the diseased bronchial tissue. The contribution of CD80 to the induction of airway hyper-responsiveness and inflammation using murine models has been controversial. Tsuyuki et al. (16) found no role for CD80 in airway responsiveness, whereas Harris et al. (15) observed that blocking CD80 did inhibit eosinophil and lymphocyte infiltration into the lung, although systemic Th2 responses were unaffected. These inconsistencies highlight the importance of using human airway tissue in resolving T cell costimulatory requirements in asthma. Studies in this disease have been hindered by poor accessibility of bronchial tissue and by ethical concerns associated with the administration of allergens or other agents to patients. We have circumvented this issue by inhibiting allergen-induced responses in bronchial tissue ex vivo, thus enabling us for the first time to probe mucosal T cell costimulatory requirements in human asthma.
Because signaling through CD40 has been shown to up-regulate the expression of CD80 and CD86 on APCs (24, 51), we examined the effects of blocking CD40/CD40L interactions on the allergen-induced cytokine production. We found that the levels of Th2 cytokines produced by the bronchial tissue or PBMCs from asthmatics in response to the Ag were not affected by addition of neutralizing anti-CD40L mAbs, indicating that these responses are independent of CD40/CD40L interactions. Monoclonal Abs against CD40L have been shown to prevent the activation of Ag-specific T cells (51). In mice, CD40-CD40L interactions have been implicated in the recruitment of eosinophils to the airways, but not in Th2 cytokine production (52). These authors demonstrated that in CD40L knockout mice, the magnitude of airway eosinophilic inflammation developing in response to inhaled Ag was dramatically reduced compared with that in control mice. However, levels of IL-5 present in the BAL fluid remained unchanged (52). Pu et al. have shown that during an in vivo type 2 response CD40/CD40L interactions were required for lymphocyte proliferation, Ab production, and eosinophilia, but not for activating T cells to produce IL-4 (53). Our finding that Th2 cytokine production in the airway mucosa is independent of CD40/CD40L interaction is consistent with these observations. Collectively, these results imply that CD40/CD40L interactions are not required to up-regulate CD80 and CD86 in the airways, possibly because the latter are already expressed in vivo at the site of inflammation or are induced ex vivo by events other than CD40 ligation, e.g., cytokines (54). Similarly, it is likely that the difference in CD80 involvement observed between the bronchial tissue and PBMC allergen responses arises because CD80 is expressed at the site of mucosal inflammation. The expression of CD80 and CD86 on B cells, T cells, macrophages, and DCs varies depending on their state of activation (for review, see Ref. 3). However, both DCs and pulmonary macrophages constitutively express CD80 (47, 55, 56). In the human lung, interstitial macrophages and DCs are the principal resident APCs. DCs in the airways, which are found closely associated with the bronchial epithelium, are ideally located to sample inhaled allergens (57, 58). Thus, the differential roles of CD80 and CD86 signaling observed in bronchial tissue and blood may reflect differences between the two sites in the availability or expression of CD80 by APCs such as DCs. The identification of different costimulatory requirements in the lung compared with PBMCs emphasizes the importance of studying bronchial tissue in this disease. The use of bronchial explants provided an opportunity to monitor T cell responses taking place in the mucosal environment and those associated with the disease process. Our data suggest that agents that target CD80 rather than CD86 may be useful in the development of specific therapy for bronchial asthma.
In conclusion, our results demonstrate an important role for ß T
cells in IL-5 production by asthmatic bronchial mucosa and an essential
requirement for both CD80 and CD86 costimulation in allergen-induced
cytokine expression. The attenuation of type 2 responses in the airways
by blockade of B7 costimulation of ß T cells may provide a useful
approach in the development of effective treatment for allergic
asthma.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Zeina Jaffar, University Medicine, Level D, Centre Block, Southampton General Hospital, Southampton, United Kingdom SO16 6YD. E-mail address:
3 Abbreviations used in this paper: DC, dendritic cell; BAL, bronchoalveolar lavage; CD40L, CD40 ligand; Der p, Dermatophagoides pteronyssinus; FEV1, forced respiratory volume in 1 s; PC20, concentration producing a 20% fall from baseline.
Received for publication May 10, 1999. Accepted for publication September 14, 1999.
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plus TNF induce regulated expression of CD80 (B7-1) but not CD86 (B7-2) on murine fibroblasts. J. Immunol. 158:4921.[Abstract]
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