CD80 and CD86 interact with CD28 and deliver costimulatory signals required for T cell activation. We demonstrate that ex vivo allergen stimulation of bronchial biopsy tissue from mild atopic asthmatic, but not atopic nonasthmatic, subjects induced production of IL-5, IL-4, and IL-13. Explants from both study groups did not produce IFN-γ, but secreted the chemokine RANTES without any overt stimulation. In addition to allergen, stimulation of asthmatic explants with mAbs to CD3 and TCR-αβ but not TCR-γδ 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 αβ 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 αβ T cell responses in the bronchial mucosa by blocking these costimulatory molecules may be of therapeutic potential in asthma.

Human allergic asthma is characterized by airway hyper-responsiveness and inflammation. CD4+ T lymphocytes play a central role in orchestrating the bronchial inflammation by producing Th2-type cytokines such as IL-4, IL-13, and IL-5, which cause the activation and recruitment of eosinophils into the airways (1, 2).

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 αβ 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.

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 × 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 β2-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.

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.

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-αβ, and anti-TCR-γδ 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-αβ, or anti-TCR-γδ (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 × 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 × 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 × g for 10 min) with AIM V medium.

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-γ (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).

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 × 105 cells/biopsy). Nonspecific binding to Fcγ receptors was blocked by incubating dispersed bronchial cells or PBMCs for 30 min with human Fcγ 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.

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.

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.

FIGURE 1.

Time course for the production of IL-5 by asthmatic bronchial explants. Biopsies were cultured in medium alone or stimulated ex vivo with Der p allergen (5000 U/ml) for up to 7 days. Interleukin-5 production in supernatants taken at various time points (see Materials and Methods) was determined by ELISA. Values were normalized for tissue weight and expressed as picograms per milligrams of tissue. Data are the mean ± SEM (n = 3).

FIGURE 1.

Time course for the production of IL-5 by asthmatic bronchial explants. Biopsies were cultured in medium alone or stimulated ex vivo with Der p allergen (5000 U/ml) for up to 7 days. Interleukin-5 production in supernatants taken at various time points (see Materials and Methods) was determined by ELISA. Values were normalized for tissue weight and expressed as picograms per milligrams of tissue. Data are the mean ± SEM (n = 3).

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The results in Fig. 2 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-γ 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-αβ, or TCR-γδ. In addition to allergen, significant (p < 0.05) IL-5 production was induced by tissue stimulated with immobilized anti-CD3 or anti-TCR-αβ (p < 0.05), but not with anti-TCR-γδ (Fig. 3).

FIGURE 2.

Cytokine production by bronchial explants from atopic asthmatic and atopic nonasthmatic control subjects. The tissue was cultured in medium alone (M) or stimulated ex vivo with allergen (Dp; 5000 U/ml) for 24 h. The production of IL-5 (A), IL-13 (B), IL-4 (C), and IFN-γ (D) was determined by ELISA. Values were normalized for tissue weight and expressed as picograms per milligram of tissue. Solid lines denote means of 10 (IL-13, IL-4, and IFN-γ) or 12 (IL-5) asthmatic and 10 nonasthmatic explants. ∗, Statistically different from control explants (p < 0.05, by Mann Whitney U test).

FIGURE 2.

Cytokine production by bronchial explants from atopic asthmatic and atopic nonasthmatic control subjects. The tissue was cultured in medium alone (M) or stimulated ex vivo with allergen (Dp; 5000 U/ml) for 24 h. The production of IL-5 (A), IL-13 (B), IL-4 (C), and IFN-γ (D) was determined by ELISA. Values were normalized for tissue weight and expressed as picograms per milligram of tissue. Solid lines denote means of 10 (IL-13, IL-4, and IFN-γ) or 12 (IL-5) asthmatic and 10 nonasthmatic explants. ∗, Statistically different from control explants (p < 0.05, by Mann Whitney U test).

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FIGURE 3.

Effect of stimulation of asthmatic bronchial biopsies with immobilized anti-CD3, anti-TCR-αβ, and anti-TCR-γδ mAbs on IL-5 production. The tissue was cultured for 24 h in wells precoated with immobilized anti-CD3, anti-TCR-αβ, or anti-TCR-γδ (2 μg/ml). Cytokine production was determined by ELISA. Values were normalized for tissue weight and expressed as picograms per milligram of tissue. Data are the mean ± SEM (n = 3). ∗, Statistically different from media (p < 0.05, by Wilcoxon’s test).

FIGURE 3.

Effect of stimulation of asthmatic bronchial biopsies with immobilized anti-CD3, anti-TCR-αβ, and anti-TCR-γδ mAbs on IL-5 production. The tissue was cultured for 24 h in wells precoated with immobilized anti-CD3, anti-TCR-αβ, or anti-TCR-γδ (2 μg/ml). Cytokine production was determined by ELISA. Values were normalized for tissue weight and expressed as picograms per milligram of tissue. Data are the mean ± SEM (n = 3). ∗, Statistically different from media (p < 0.05, by Wilcoxon’s test).

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We then investigated the production of other cytokines thought to be important in the pathogenesis of asthma. We found that GM-CSF was not produced by either asthmatic or nonasthmatic bronchial tissue with or without allergen stimulation (data not shown). RANTES was secreted by the explants during culture without any overt stimulation (control biopsies secreted 26.7 pg/mg tissue, and asthmatic biopsies secreted 15.3 pg/mg tissue; n = 5). These levels were not changed by allergen stimulation (control biopsies secreted 20.4 pg/mg tissue and asthmatic biopsies secreted 19.6 pg/mg tissue). There was no statistically significant differences in the level of RANTES secretion between the two study groups.

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 × 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).

We have shown in this study that αβ 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).

FIGURE 4.

Anti-CD80 and anti-CD86 Abs inhibit allergen-induced IL-5 and IL-13 production from asthmatic bronchial biopsies. Bronchial biopsies were cultured for 24 h in the presence of Der p allergen (5000 U/ml) or Der p plus blocking Abs to CD80 or CD86 mAbs (10 μg/ml). Cytokine production in the culture supernatants was quantified by ELISA. Values were normalized for tissue weight and expressed as picograms per milligram of tissue. Data are the mean ± SEM (n = 7). ∗, Statistically different from Der p alone (p < 0.05).

FIGURE 4.

Anti-CD80 and anti-CD86 Abs inhibit allergen-induced IL-5 and IL-13 production from asthmatic bronchial biopsies. Bronchial biopsies were cultured for 24 h in the presence of Der p allergen (5000 U/ml) or Der p plus blocking Abs to CD80 or CD86 mAbs (10 μg/ml). Cytokine production in the culture supernatants was quantified by ELISA. Values were normalized for tissue weight and expressed as picograms per milligram of tissue. Data are the mean ± SEM (n = 7). ∗, Statistically different from Der p alone (p < 0.05).

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FIGURE 5.

Effects of anti-CD80 and anti-CD86 Abs on allergen-induced IL-5 and IL-13 production from PBMCs of asthmatic subjects. PBMCs were cultured for 6 days in the presence of Der p allergen (5000 U/ml) or Der p plus blocking Abs (10 μg/ml) to CD80 or CD86 alone or to CD80 and CD86 together. Cytokine production in the culture supernatants was quantified by ELISA. Allergen cultured with control IgM and IgG1 (isotype-matched for anti-CD80 and anti-CD86 Abs) secreted 390.1 ± 88.7 and 387.9 ± 79.9 pg/mg of IL-5, respectively, and 198.7 ± 65.3 and 192.3 ± 69.5 pg/mg of IL-13, respectively. Data are the mean ± SEM (n = 4). ∗, Statistically different from Der p alone (p < 0.05).

FIGURE 5.

Effects of anti-CD80 and anti-CD86 Abs on allergen-induced IL-5 and IL-13 production from PBMCs of asthmatic subjects. PBMCs were cultured for 6 days in the presence of Der p allergen (5000 U/ml) or Der p plus blocking Abs (10 μg/ml) to CD80 or CD86 alone or to CD80 and CD86 together. Cytokine production in the culture supernatants was quantified by ELISA. Allergen cultured with control IgM and IgG1 (isotype-matched for anti-CD80 and anti-CD86 Abs) secreted 390.1 ± 88.7 and 387.9 ± 79.9 pg/mg of IL-5, respectively, and 198.7 ± 65.3 and 192.3 ± 69.5 pg/mg of IL-13, respectively. Data are the mean ± SEM (n = 4). ∗, Statistically different from Der p alone (p < 0.05).

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Because a maximum of only four bronchial explant cultures per experiment is possible, in some instances a second set of biopsies was taken from the same asthmatic subjects on a separate occasion to ascertain the effects of control IgM and IgG1 (isotype matched for anti-CD80 and anti-CD86 Abs, respectively). Isotype control Abs had no effect on allergen-induced cytokine production. Allergen cultured with control IgM and IgG1 secreted 11.2 ± 6.1 and 14.5 ± 5.9 pg/mg of IL-5, respectively (compared with 12.9 ± 6.1 pg/mg IL-5 with allergen alone) and 6.3 ± 1.5 and 5.4 ± 0.4 pg/mg of IL-13, respectively (compared with 6.9 ± 1.1 pg/mg IL-13 with allergen alone; n = 3). Thus, these control Abs had no effect on allergen-induced cytokine production by the 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).

FIGURE 6.

Expression of CD28 and CTLA-4 on CD4+ T cells in asthmatic bronchial tissue by FACS analysis. Bronchial biopsies were stimulated with Der p allergen for 24 h. After culture the tissue was digested in collagenase, and cells were then stained with FITC mAbs to CD4 and PE CTLA-4 or CD28. Isotype-matched Ig were used as a control. Representative data from three experiments are shown. CD4+ T cells in the tissue expressed CD28 (75.3 ± 9.4%; n = 3), but little or undetectable CTLA-4 (4.0 ± 0.5%; n = 3).

FIGURE 6.

Expression of CD28 and CTLA-4 on CD4+ T cells in asthmatic bronchial tissue by FACS analysis. Bronchial biopsies were stimulated with Der p allergen for 24 h. After culture the tissue was digested in collagenase, and cells were then stained with FITC mAbs to CD4 and PE CTLA-4 or CD28. Isotype-matched Ig were used as a control. Representative data from three experiments are shown. CD4+ T cells in the tissue expressed CD28 (75.3 ± 9.4%; n = 3), but little or undetectable CTLA-4 (4.0 ± 0.5%; n = 3).

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Our analysis also demonstrated that PBMCs from asthmatic subjects expressed higher levels of CD86 than CD80 on CD19+ and CD14+ APCs (data not shown), and, importantly, an increase in the expression of CD86 on CD19+ cells (from 14.5 to 27.0%; Fig. 7), but not in that of CD80, was observed after allergen stimulation.

FIGURE 7.

Expression of CD80 and CD86 by B cells in PBMCs from asthmatic subjects by FACS analysis. PBMCs from asthmatic subjects were stimulated with allergen for 6 days and stained with FITC mAbs to CD80 or CD86 and PE CD19. Isotype-matched Ig were used as a control. Representative data from three experiments are shown.

FIGURE 7.

Expression of CD80 and CD86 by B cells in PBMCs from asthmatic subjects by FACS analysis. PBMCs from asthmatic subjects were stimulated with allergen for 6 days and stained with FITC mAbs to CD80 or CD86 and PE CD19. Isotype-matched Ig were used as a control. Representative data from three experiments are shown.

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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 × 105 responders) were cultured with irradiated allogeneic PBMCs (1 × 105 stimulators). To measure proliferation, cultures were pulsed with [3H]thymidine after 6 days and harvested 18 h later.

FIGURE 8.

Effects of anti-CD40 and anti-CD40L Abs on allergen-induced IL-5 production from asthmatic bronchial tissue and PBMCs. Bronchial biopsies (A) and PBMCs (B) were cultured in the presence of Der p allergen (5000 U/ml) or Der p plus blocking Abs to CD40 or CD40L (10 μg/ml). Cytokine production in the culture supernatants was quantified by ELISA. In A, values are expressed as picograms per mg tissue. Data are the mean ± SEM (n = 4).

FIGURE 8.

Effects of anti-CD40 and anti-CD40L Abs on allergen-induced IL-5 production from asthmatic bronchial tissue and PBMCs. Bronchial biopsies (A) and PBMCs (B) were cultured in the presence of Der p allergen (5000 U/ml) or Der p plus blocking Abs to CD40 or CD40L (10 μg/ml). Cytokine production in the culture supernatants was quantified by ELISA. In A, values are expressed as picograms per mg tissue. Data are the mean ± SEM (n = 4).

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There is overwhelming evidence to suggest that activation of Ag-specific type 2 CD4+ T cells in the airways of allergic asthmatic subjects plays a central role in the pathogenesis of bronchial asthma. These T cells are known to produce IL-4 and IL-13, which promote Ab isotype switching to IgE (25, 26), and IL-5, which is involved in eosinophil recruitment and activation (27). RANTES is also thought to be involved in promoting eosinophil accumulation in the airways, and mRNA expression of this chemokine has been demonstrated on bronchial mucosal biopsies of asthmatics (28). In addition to the well-characterized interaction of the TCR with Ag complexed with MHC class II on APCs, T cell activation requires a second costimulatory signal. We have previously shown that the production of Th2 cytokines by asthmatic bronchial tissue stimulated with allergen was inhibited by CTLA-4Ig fusion protein (21). These observations identified B7 costimulation as being a prerequisite for allergen-driven T cell activation and cytokine expression in the airways in atopic asthma. However, it was unclear whether this response involved both CD80 and CD86 costimulation or whether the contribution of one molecule predominated over that of the other. Analysis of murine airway responses has revealed that CD86 is an important costimulatory molecule for lung Th2 responses and airway hyper-reactivity (16). Moreover, 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, 29, 30).

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-γ 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-αβ, but not TCR-γδ, induced IL-5 secretion. Our findings are in contrast with the recent suggestion that γδ 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-γ 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-γ, 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 αβ 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 αβ T cells may provide a useful approach in the development of effective treatment for allergic asthma.

We thank Sue Martin and Kathy Bodey for their technical assistance, and Andrea Corkhill for her assistance with the clinical screening and bronchoscopy of volunteers.

1

This work was supported in part by the Wessex Medical Trust (Southampton, U.K.) and the Medical Research Council (Grant G8604034, U.K.).

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.

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