Abstract
T cells are thought to play an important regulatory role in atopic asthma. We hypothesized that human blood and BAL T cell subsets bearing various TCR-Vβ genes might show selective differences in their cytokine profile. Peripheral blood (PB) and bronchoalveolar lavage (BAL) T cells from seven atopic asthmatic and six non-atopic non-asthmatic subjects were stimulated with PMA and ionomycin in the presence of monensin and analysed for TCR-Vβ expression and production of cytokines at the single cell level. The percentage of IFN-γ- and IL-2-producing BAL T cells was elevated compared with PB T cells from both the asthmatic subjects and the non-atopic, non-asthmatic controls. A small percentage of PB and BAL T cells produced IL-4 and IL-5, in asthmatic and normal subjects. In peripheral blood, the percentage of T cells expressing each cytokine was similar in the various TCR-Vβ subsets and in total CD3+ T cells in all normal and six of seven asthmatic subjects. However, there was a substantial degree of heterogeneity in the cytokine profile of BAL TCR-Vβ subsets compared with the total CD3+ T cells. This was more obvious in the asthmatic subjects with a reduction in the percentage of IFN-γ- and IL-2-expressing T cells (five of seven asthmatic subjects) and an increase in the percentage of IL-4- and IL-5-expressing T cells (two of seven asthmatic subjects). These data confirm previous findings of an elevated proportion of IFN-γ- and IL-2-producing BAL T cells while only a small proportion of PB and BAL T cells produce IL-4 and IL-5. Moreover, subsets of BAL T cells, defined by their TCR-Vβ usage, may differ in their cytokine profile compared with the total CD3+ T cells, implying that T cells expressing different Vβ elements may play different roles in regulating the airway inflammation in asthma.
Keywords: asthma, cytokines, flow cytometry, TCR
INTRODUCTION
Atopic asthma is characterized by airway inflammation in which many cells participate, including mast cells, eosinophils and T lymphocytes [1]. T lymphocytes have been suggested to play an important role in regulating the inflammatory and immunological processes thought to underlie asthma [2], by manufacturing and releasing an array of cytokines and chemokines [2,3]. In particular, interferon-gamma (IFN-γ) has many proinflammatory activities on endothelium and eosinophils [4,5], IL-4 and IL-13 promote isotype switching of B-lymphocytes towards IgE production and IL-5 is the main cytokine regulating the differentiation and activation of eosinophils [6,7].
T cell cytokines are synthesized transiently, stored in the Golgi apparatus and released rapidly by exocytosis. This process can be interrupted by monensin or brefeldin A, allowing cytokine protein to accumulate in the Golgi apparatus, where it can be detected by flow cytometry [8,9]. We have applied this technique previously to address cytokine production by bronchoalveolar lavage (BAL) and blood T cells in asthma and allergic disorders [10].
The T cell receptor (TCR) is a disulphide-linked heterodimer composed either of an α and a β or of a γ and a δ chain. β-chain diversity is crucial for the ability of the T cell population to recognize and respond to the vast number of different antigen-MHC ligands that may be encountered [11]. The β chain is encoded by V (variable), D (diversity), J (joining) and C (constant) gene segments and extensive diversity is generated not only from the simple combinations of V, D, J and C regions but also from N-region diversity and joining-site variation. Vβ gene segments have been assigned to families, based upon nucleotide and amino acid sequences similarities.
At present, 25 different Vβ families have been described in man [12,13]. The formation of the T cell repertoire is shaped by the genetic background of the individual and the response to environmental antigens [14]. Antigen-driven stimulation leads to oligoclonal expansion of T cells with common VDJ segments, reflecting the influence of the antigen, while superantigenic stimulation leads to polyclonal expansion of a particular TCR Vβ family with different D and J segments and hence random antigen specificity [15]. There is evidence from murine models of asthma that T cell subsets bearing different Vβ chains show differences in their cytokine production, which in turn may lead to functional differences of the T cell subpopulations [16,17]. A similar observation has been made in experimental allergic encephalomyelitis in the mouse, where the pathological response can be modulated by using monoclonal antibodies to eliminate particular T cell subpopulations [18]. We hypothesized that human blood and BAL T cell subsets bearing various TCR-Vβ genes might show selective differences in their cytokine profile compared to one another and to the overall T cell population. We have compared this in patients with asthma and in a group of healthy normal subjects.
MATERIALS AND METHODS
Subjects
Seven atopic asthmatics and six non-atopic non-asthmatic subjects were recruited by advertising. All the asthmatic subjects met the American Thoracic Society criteria for diagnosis of asthma. They were atopic as confirmed by skin prick tests to a panel of common aeroallergens and had increased airway responsiveness to methacholine (PC20 < 16 mg/ml). All asthmatic subjects were using inhaled salbutamol, when needed for relief of symptoms, but none had received inhaled or oral corticosteroid therapy for at least 6 weeks before the study. The normal controls had no history of asthma or other allergic disease, negative skin-prick tests and a methacholine PC20 > 32 mg/ml. All subjects were non-smokers and none had ever smoked regularly. All subjects were volunteers and gave their written consent after being fully informed about the nature and purpose of the study. The study was approved by the Southampton Joint University and Hospital Ethics Committee.
Fibreoptic bronchoscopy
Bronchoscopy and BAL were performed by a standard technique conforming to current National Heart, Lung and Blood Institute guidelines [19]. Briefly, all subjects were premedicated with nebulized albuterol (2·5 mg), intravenous atropine (0·6 mg) and intravenous midazolam (5–8 mg). Topical upper airway anaesthesia was achieved with 4% lidocaine spray. Supplementary oxygen was administered by nasal cannulae, while oxygen saturation and pulse rate were monitored throughout. The fibreoptic bronchoscope (Olympus BF-XT20 or BF-IT20) was introduced by the oral route and additional lidocaine 2% was given in order to achieve lower airway anaesthesia. BAL was performed by instilling six 20 ml aliquots of warmed isotonic saline into a subsegment of the right upper lobe. The fluid was then aspirated via the suction channel into plastic tubes and kept at 4°C until transfer to the laboratory for processing. Heparin-anticoagulated blood (10 ml) was collected under sterile conditions prior to bronchoscopy, and kept on ice until processed.
Antibodies and reagents
A panel of 18 fluorescein-isothiocyanate (FITC)-labelled monoclonal antibodies (MoAbs) against TCR-Vβ family products 1, 2, 3, 5·1, 5·2/3, 6·7, 8, 11, 12, 12·1, 13·1, 13·1/3, 14, 16, 17, 20, 21·3, 22 and three phycoerythrin (PE)-labelled MoAbs against TCRBV 9, 18, 23, were obtained from Serotec and Immunotech. Anti-CD3-peridin chlorophyll protein conjugate (PerCP), anti-CD3-PE and anti-CD3-FITC were purchased from Becton-Dickinson (Oxford, UK). Anti-IFN-γ and anti-IL-4 MoAbs labelled with PE were purchased from Becton-Dickinson (Oxford UK),while anti-IL-2 and anti-IL-5 MoAbs labelled with PE were obtained from Pharmingen USA. Phorbol 12-myristate 13-acetate (PMA), ionomycin, monensin, paraformaldehyde, saponin and AB serum were obtained from Sigma (Poole, UK). Glutamine, sodium pyruvate, penicillin, streptomycin, mercaptoethanol and RPMI-1640 were purchased from Gibco (Paisley, UK).
Processing of cells and staining procedure
100 μl whole blood was incubated in polypropylene tubes with saturating concentrations of anti-CD3-PE and each one of the anti-TCR-Vβ-FITC MoAbs or anti-CD3-FITC and each of the three anti-TCRVβ-PE MoAbs, in order to determine the percentage of CD3+ cells bearing each particular TCR-Vβ chain.
The recovered BAL fluid was filtered through a 100-nm nylon filter and centrifuged at 1500 g for 10 min. The cell pellet was resuspended in RPMI-1640 medium and adjusted to 1 × 106 cells/ml by counting the cells with a Neubauer haemocytometer. For intracellular cytokine detection, aliquots of 1 × 106 BAL cells, in RPMI-1640 supplemented with 2 mML-glutamine, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 0·5 μg/ml amphotericin B, 20 μM mercaptoethanol and 5% AB serum were cultured in 24-well flat-bottomed plates (Falcon, Becton-Dickinson) for 5h at 37°C in a humidified atmosphere containing 5% CO2. Cells were stimulated with PMA (50 ng/ml) and ionomycin (1 μM) in the presence of monensin (2·5 μM).
Heparin-anticoagulated blood was cultured in 200 μl aliquots under the same conditions in 24-well flat-bottomed plates. Cultured cells (from blood and BAL) were washed in PBS and aliquotted in polypropylene tubes containing 10 μl of anti-CD3-PerCP and 5–10 μl of anti-TCR-Vβ-FITC MoAbs. After incubation for 20 min, the cells were fixed in ice-cold PBS containing paraformaldehyde for 10 min and then permeabilized by resuspension in saponin buffer (PBS containing 0·1% saponin). After a further wash in saponin-buffer, 2–8 μl of PE-labelled anticytokine MoAb was added to each tube and cells were incubated for 20 min. After a final wash in saponin buffer, cells were resuspended in PBS and analysed in a FACScan flow cytometer.
Due to the limited yield of cells that can be obtained with BAL we were able to stain only for intracellular cytokines (IFN-γ, IL-2, IL-4, IL-5) in three different TCR-Vβ subpopulations of the BAL CD3+ cells. These subpopulations were chosen on the basis of the surface marker staining in the peripheral blood samples, as there was no limitation in the number of blood cells available. We performed TCR-Vβ usage analysis in peripheral blood in all subjects studied. Subsequently, we picked three subpopulations with a high percentage of expression in peripheral blood and stained for intracellular cytokines in both blood and BAL, but this had to be carried out before we were aware of the percentage of expression of these TCR-Vβ subsets in the BAL samples. The relative number of T cells expressing the various Vβ regions in the surface marker analysis did not change following culture and intracellular cytokine staining.
Samples were analysed on a FACScan flow cytometer (Becton-Dickinson) equipped with a 15-mW argon ion laser and appropriate filters for FITC (530 nm), PE (585 nm) and PerCP (>650 nm). For surface marker analysis of peripheral blood lymphocytes, 10 000 cells were computed and analysed using appropriate software. For intracellular cytokine detection in blood and BAL lymphocytes, 30 000 cells were collected and analysed with the same software. An electronic gate was set on the lymphocytes on the forward and side scatter plot, and CD3+ (PerCP+) cells falling within the gated area were then identified and selected into a second gate set on the forward scatter and CD3+ (PerCP+) plot. Cytokine production in each of the three TCR-Vβ subpopulations was analysed by detection of FITC and PE staining. The number of TCR-Vβ+ cells staining for each cytokine was expressed as a percentage of the total Vβ+ cells and was compared to the percentage of all CD3+ cells staining for the same cytokine in that sample. A difference greater than 10% for IFN-γ- and IL-2-producing cells or greater than 5% for IL-4- and IL-5-producing cells was considered significant when TCR-Vβ subsets were compared to the total CD3+ cells as regards cytokine production. Three sets of negative controls were used: cell samples from which all the antibodies had been omitted; samples stained with PerCP-CD3 only; samples stained with PerCP-CD3 and the relevant FITC-conjugated TCR-Vβ MoAb.
Statistical analysis
Statistical analysis was carried out using SPSS for Windows on a PC platform. The Mann–Whitney U-test was used for comparisons between different groups and the Wilcoxon signed rank test for paired data for within-group comparisons. Fisher’s exact test was used for categorical analysis. Values of P < 0·05 were accepted as statistically significant.
RESULTS
In this study T cell cytokine expression was studied in blood and BAL in seven asthmatic and six normal non-asthmatic subjects on selected TCR-Vβ families that were found to be over-represented in the blood. The expression of these selected families in the blood ranged from between 2% and 8% of the total CD3+ cells in the asthmatic patients and between 3% and 9% in the control group. Initial analysis revealed that in the control group nine of the selected families (total 18) in the six individuals were found to be expressed in BAL at levels equal to, or greater than, that which had been observed in the blood. In the asthmatic patients much more variation was observed, with five of the selected families (total 21) in the seven individuals demonstrating expression in BAL equal to or greater than that observed in blood (Tables 1 and 2).
Table 1.
Percentage of CD3+ cells and TCRVβ T cell subsets expressing cytokines in peripheral blood and BAL of normal non-asthamatic subjects
| % total | CD3+ | Blood | Bal | Blood | Bal | Blood | Bal | Blood | Bal | ||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Subject | Vβ | blood | Bal | IFN-γ | IFN-γ | Il-2 | IL-2 | IL-4 | IL-4 | IL-5 | IL-5 |
| N6 | CD3+ | 28·5 | 62·1 | 28·0 | 58·6 | 2·0 | 2·0 | 0·5 | 0·5 | ||
| Vβ2 | 7·4 | 7·3 | 25·1 | 62·1 | 29·0 | 63·2 | 3·0 | 0·0 | 1·0 | 0·0 | |
| Vβ6·7 | 4·5 | 3·0 | 25·5 | 57·5 | 35·7 | 63·8 | 3·1 | 2·5 | 1·0 | 0·0 | |
| Vβ22 | 3·2 | 2·0 | 29·6 | 68·0 | 32·7 | 64·0 | 2·0 | 2·7 | 1·0 | 0·0 | |
| N7 | CD3+ | 23·1 | 83·1 | 15·6 | 64·1 | 0·5 | 1·0 | 0·5 | 2·1 | ||
| Vβ1 | 4·3 | 4·4 | 24·0 | 86·6 | 15·6 | 65·7 | 0·0 | 6·0 | 0·0 | 6·0 | |
| Vβ2 | 7·6 | 6·2 | 17·5 | 81·0 | 16·5 | 73·4 | 1·0 | 3·8 | 0·0 | 6·3 | |
| Vβ5·1 | 4·6 | 3·6 | 28·3 | 76·6 | 15·2 | 57·8 | 1·1 | 0·0 | 0·0 | 6·3 | |
| N8 | CD3+ | 28·6 | 75·4 | 27·6 | 62·6 | 1·0 | 0·5 | 0·5 | 0·0 | ||
| Vβ6·7 | 5·6 | 5·3 | 26·3 | 75·0 | 28·4 | 57·9 | 2·1 | 0·0 | 4·2 | 0·0 | |
| Vβ8 | 4·4 | 3·2 | 25·0 | 56·5 | 27·1 | 24·2 | 2·1 | 0·0 | 0·0 | 0·0 | |
| Vβ13·1/3 | 5·7 | 12·7 | 26·8 | 51·3 | 28·9 | 29·5 | 0·0 | 0·0 | 0·0 | 0·0 | |
| N9 | CD3+ | 25·0 | 83·2 | 20·0 | 64·0 | 1·0 | 0·0 | 0·5 | 0·0 | ||
| Vβ2 | 9·2 | 1·1 | 28·0 | 74·1 | 23·0 | 60·3 | 1·0 | 0·0 | 1·0 | 0·0 | |
| Vβ13·1/3 | 5·3 | 4·3 | 30·6 | 80·3 | 19·4 | 56·3 | 1·0 | 1·4 | 0·0 | 0·0 | |
| Vβ17 | 5·2 | 6·9 | 25·5 | 91·5 | 24·5 | 65·9 | 0·0 | 0·0 | 0·0 | 0·0 | |
| N10 | CD3+ | 4·0 | 53·5 | 2·5 | 29·3 | 0·0 | 0·5 | 0·5 | 0·5 | ||
| Vβ1 | 3·8 | 3·5 | 2·0 | 57·1 | 3·0 | 35·7 | 0·0 | 4·8 | 0·0 | 1·2 | |
| Vβ5·2/3 | 3·4 | 8·3 | 4·0 | 60·2 | 3·0 | 30·7 | 0·0 | 1·1 | 0·0 | 1·1 | |
| Vβ13·1/3 | 6·3 | 9·4 | 4·0 | 54·9 | 2·0 | 36·3 | 0·0 | 4·4 | 0·0 | 2·2 | |
| N11 | CD3+ | 12·0 | 40·2 | 4·5 | 29·1 | 0·5 | 0·5 | 0·5 | 0·0 | ||
| Vβ2 | 6·4 | 4·6 | 9·1 | 37·4 | 6·1 | 27·5 | 0·0 | 0·0 | 1·0 | 0·0 | |
| Vβ5·1 | 3·2 | 1·1 | 5·2 | 21·5 | 5·2 | 15·2 | 0·0 | 0·0 | 0·0 | 0·0 | |
| Vβ13·1/3 | 3·3 | 7·6 | 10·2 | 37·5 | 8·2 | 25·0 | 0·0 | 0·0 | 0·0 | 0·0 |
Table 2.
Percentage of CD3+ cells and TCRVβ T cell subsets expressing cytokines in peripheral blood and BAL of asthamatic subjects
| % total | CD3+ | Blood | Bal | Blood | Bal | Blood | Bal | Blood | Bal | ||
|---|---|---|---|---|---|---|---|---|---|---|---|
| Subject | Vβ | blood | Bal | IFN-γ | IFN-γ | Il-2 | IL-2 | IL-4 | IL-4 | IL-5 | IL-5 |
| A5 | CD3+ | 26·6 | 60·3 | 6·5 | 21·1 | 0·0 | 0·5 | 0·0 | 0·5 | ||
| Vβ2 | 7·2 | 4·8 | 22·7 | 38·5 | 9·3 | 20·8 | 0·0 | 0·0 | 0·0 | 1·1 | |
| Vβ13·1/3 | 5·6 | 10·1 | 14·5 | 47·3 | 7·3 | 14·0 | 0·0 | 0·0 | 0·0 | 0·0 | |
| Vβ17 | 3·8 | 0·5 | 10·0 | 62·5 | 8·3 | 26·3 | 2·1 | 1·3 | 0·0 | 0·0 | |
| A6 | CD3+ | 23·0 | 75·4 | 19·0 | 52·3 | 0·5 | 0·5 | 0·5 | 0·0 | ||
| Vβ2 | 8·2 | 2·1 | 20·0 | 63·3 | 21·0 | 53·3 | 2·0 | 5·5 | 1·0 | 1·1 | |
| Vβ13·1/3 | 5·5 | 2·4 | 20·2 | 73·3 | 22·2 | 55·8 | 1·0 | 0·0 | 2·0 | 0·0 | |
| Vβ17 | 3·8 | 5·9 | 25·5 | 88·0 | 21·4 | 53·3 | 0·0 | 0·0 | 1·0 | 0·0 | |
| A7 | CD3+ | 34·2 | 81·8 | 21·6 | 63·1 | 0·5 | 1·5 | 0·0 | 0·5 | ||
| Vβ2 | 6·8 | 4·1 | 24·5 | 81·9 | 26·5 | 41·0 | 0·0 | 1·2 | 0·0 | 0·0 | |
| Vβ8 | 2·4 | 0·9 | 24·7 | 77·0 | 23·7 | 63·9 | 1·1 | 3·3 | 0·0 | 0·0 | |
| Vβ13·1/3 | 4·8 | 3·7 | 24·5 | 73·9 | 21·3 | 49·3 | 0·0 | 0·0 | 0·0 | 0·0 | |
| A8 | CD3+ | 15·0 | 78·8 | 10·0 | 61·6 | 0·5 | 1·5 | 0·5 | 0·0 | ||
| Vβ1 | 4·0 | 1·5 | 15·2 | 87·3 | 10·1 | 75·9 | 1·0 | 0·0 | 1·0 | 3·8 | |
| Vβ5·1 | 4·6 | 2·4 | 16·3 | 75·3 | 13·3 | 53·4 | 0·0 | 1·4 | 0·0 | 13·7 | |
| Vβ8 | 3·5 | 0·6 | 14·6 | 86·4 | 10·4 | 57·6 | 0·0 | 0·0 | 0·0 | 0·0 | |
| A9 | CD3+ | 48·5 | 76·9 | 23·0 | 56·8 | 1·0 | 1·0 | 0·5 | 0·5 | ||
| Vβ2 | 8·0 | 5·5 | 45·5 | 72·0 | 24·2 | 55·9 | 0·0 | 0·0 | 0·0 | 1·1 | |
| Vβ5·1 | 3·0 | 2·1 | 38·8 | 67·1 | 28·6 | 40·5 | 0·0 | 0·0 | 1·0 | 0·0 | |
| Vβ22 | 3·2 | 1·1 | 42·9 | 84·3 | 32·7 | 51·4 | 0·0 | 5·7 | 0·0 | 0·0 | |
| A10 | CD3+ | 8·5 | 44·0 | 3·5 | 30·9 | 0·0 | 1·6 | 0·0 | 7·3 | ||
| Vβ5·1 | 4·1 | 4·8 | 6·3 | 48·1 | 2·1 | 32·7 | 0·0 | 9·6 | 0·0 | 15·4 | |
| Vβ5·2/3 | 2·3 | 7·7 | 14·1 | 53·3 | 0·0 | 38·3 | 0·0 | 11·7 | 0·0 | 8·3 | |
| Vβ13·1/3 | 4·5 | 11·6 | 6·4 | 53·0 | 2·1 | 37·9 | 1·1 | 6·1 | 0·0 | 10·6 | |
| A11 | CD3+ | 10·0 | 29·0 | 2·5 | 15·0 | 0·5 | 0·5 | 0·5 | 0·5 | ||
| Vβ1 | 3·8 | 1·2 | 11·1 | 20·6 | 3·0 | 14·4 | 0·0 | 0·0 | 0·0 | 1·0 | |
| Vβ2 | 8·0 | 2·6 | 8·0 | 23·5 | 3·0 | 14·3 | 1·0 | 0·0 | 0·0 | 1·0 | |
| Vβ3 | 5·7 | 0·8 | 11·0 | 10·3 | 3·0 | 11·3 | 1·0 | 0·0 | 1·0 | 0·0 |
No differences were found between the two groups in the ability of their T cells to produce any particular cytokine, either in blood or in BAL. There were relatively higher proportions of IFN-γ+ T cells and IL-2+ T cells in BAL compared to blood, in both groups (Fig. 1, Tables 1 and 2). IL-4 and IL-5 were found in only a small percentage of T cells in either compartment, and there was no systematic difference between blood and BAL. In blood the percentage of T cells expressing each cytokine in the various TCR-Vβ subsets was similar to that in the total CD3+ T cell population in all normal and asthmatic subjects, with the exception of one asthmatic subject (A5), in whom there was a reduced proportion of IFN-γ+ cells in the Vβ13·1/3 and Vβ17 subsets. However, some heterogeneity was found in BAL with regard to the percentage of T cells expressing each cytokine in the various TCR-Vβ subsets, seen more often in asthmatic subjects as compared to normal subjects. This was seen mainly as a reduction in the percentage of IFN-γ+ or IL-2+ cells in one or more TCR-Vβ subsets. This was found in five of seven asthmatic subjects, while one asthmatic subject (A6) had also an increase in IFN-γ expression in the Vβ17 subset and another (A8) had an increased percentage of IL-2+ cells in the Vβ1 subset. Only one asthmatic subject (A10) did not show any difference in the percentage of T cells expressing IFN-γ and IL-2 in the TCR-Vβ subsets compared to the total CD3+ T cell population. This heterogeneity in the cytokine profile of the various TCR-Vβ subsets in BAL was not confined to the asthmatic group, also being noted in two of six normal subjects (N8 and N11). The subject N8 showed a reduced percentage both in IFN-γ and IL-2 expression in the Vβ8 and the Vβ13·1/3 subsets, while the N11 subject had a decreased percentage in IFN-γ and IL-2 expression in the Vβ5·1 subset. The Th2-cytokines IL-4 and IL-5 were demonstrated in only a small percentage of T cells. Just one asthmatic subject (A10) had a clearly elevated percentage of IL-5+ CD3+ T cells in BAL (7·3% of total CD3+ cells). In the subset analysis, in all normal subjects the percentage of IL-4+ and IL-5+ cells in the TCR-Vβ subsets was similar to that in the total CD3+ cells. However, two of the asthmatic subjects (A8, A10) showed some TCR-Vβ subsets which expressed IL-4 or IL-5 at > 5% above the proportion seen in corresponding CD3+ total T cell population. The subject A8 showed an increase in IL-5 expression in the Vβ5·1 subset while the subject A10 had an increased percentage of IL-4+ cells in all three Vβ subsets studied (Vβ5·1, Vβ5·2/3 and Vβ13·1/3) and an increased percentage of IL-5+ cells in the Vβ5·1 and Vβ13·1/3 subsets. It was interesting to note that the only asthmatic subject who did not show any heterogeneity in the percentage of T cells expressing Th1 cytokines (IFN-γ and IL-2) in the TCR-Vβ subsets compared to the total CD3+ population was the subject with an increased proportion of IL-4+ and IL-5+ T cells in some subsets (A10).
Fig. 1.
Dot plots of cytokine staining versus TCR-Vβ13·1/3 staining in BAL T cells (gated on CD3+ cells) from an asthmatic patient (A5)
DISCUSSION
We have investigated the cytokine profile of T cell subpopulations, isolated from both peripheral blood and BAL, from seven atopic asthmatic and six non-atopic asthmatic subjects. In each individual three TCR-Vβ families, which had demonstrated elevated expression in peripheral blood by flow cytometry, were selected and examined in BAL T cells with regard to their cytokine profile. We felt justified in using this selection process as previous studies have demonstrated that the pattern of Vβ expression is broadly similar in BAL and peripheral blood of asthmatics and normal individuals [20,21]. Thus, it can be assumed that those families found to be elevated in the peripheral blood of the patients studied will be elevated in BAL.
We employed a flow cytometric method of intracellular cytokine detection that permits the simultaneous detection of cytokine production and T cell surface markers at the single cell level. This method is based on the stimulation of T cells in the presence of a pharmacological inhibitor of secretion, followed by cell fixation and permeabilization and then intracytoplasmic staining of accumulated cytokines [8,9]. Unfortunately, BAL T cells do not appear to produce cytokines spontaneously, so in vitro stimulation was required using PMA and ionomycin, providing a polyclonal stimulus to all viable T cells. Previous studies have shown that PMA and ionomycin stimulation of BAL T cells readily induces both IFN-γ and IL-2 production and results in IL-4 and IL-5 production similar to that observed in cultures using more physiological stimulants such anti-CD3 [22]. Monensin does not appear to alter the profile of cytokine production and has no effect on viability of T cells at the concentration and culture duration used in our method.
We have previously analysed BAL T cell function by cloning T cells from human airways [23]. Although we were able to demonstrate functional heterogeneity in our T cell clones, it now appears that the cytokine profile of T cells may be altered during T cell cloning by both selection (only cells capable of extensive cell division are evaluated) and the long-term in vitro culture required [24,25]. In the present study, both the short culture period and the inhibition of secretion by monensin greatly diminish the possibility that T cell or accessory-cell cytokines could modulate the T cell and TCR-Vβ-subset cytokine responses. Other studies have shown that mitogen stimulation of peripheral blood (PB) T cells leads to the rapid and synchronized production of IL-2, IL-4 and IFN-γ, peaking 4–6 h after stimulation [26]. We therefore believe that the results obtained here are a fair reflection of the cytokine profile of the T cells at the time that they were sampled.
We did not find any clear differences between atopic asthmatics and normal subjects in the ability of their T cells to produce a particular cytokine, either in blood or in BAL. Previous studies have reported that BAL concentrations of IL-4 and IL-5 protein were similar in asthmatic and healthy subjects [27], while others have reported increased IL-4 and IL-5 protein concentrations in BAL fluid and in culture supernatants from PB and BAL T cells of allergic asthmatics as compared to normal subjects [3]. This is not necessarily inconsistent with our findings, but could reflect increased production of Th2 cytokines by small numbers of cells in each compartment, rather than an increased number of Th2-cytokine producing T cells. Both atopic asthmatics and normal subjects showed relatively higher proportions of IFN-γ+ and IL-2+ T cells in BAL compared to PB, whereas our previous studies demonstrated this only for IFN-γ+ T cells [10].
The TCR repertoire of mature circulating T cells is determined both by the genetic background of an individual and by clonal selection of T cells in response to environmental antigens [28,29]. Specific interactions between T cells and antigens may increase the frequency of T cells using selected TCR V gene segments, while superantigens stimulate T cells in a Vβ-specific manner [15]. In certain inflammatory and autoimmune conditions, samples of affected sites show restricted or preferential expression of distinct Vβ or Vα families [30–32]. Moreover, T cells obtained from patients with various different disease states have different relative ratios of cytokine gene expression and/or secretion [33,34].
Our study suggests that T cell subset heterogeneity in cytokine expression also occurs in asthmatic airways. Interestingly, a recent study has reported selective expansion of TCR-Vβ T cells in the BAL CD8+ subpopulation following allergen provocation [35]. In another study it was found that subjects with poorly controlled asthma had increased TCR-Vβ8+ T cells in BAL and suggested that this expansion might be driven by superantigenic stimulation [36].
The selection of families found to be expanded in the blood for study in BAL is not ideal. Identification of oligoclonality within TCR-Vβ families in the periphery, using molecular techniques, may provide a more appropriate means of selecting families for further study of cytokine profiles at disease sites. We have recently used this method to identify oligoclonality in TCR-Vβ families in the periphery of patients with rheumatoid arthritis, and found these families to demonstrate cytokine profiles in the synovium that differed from total CD3+ cells.
Although we did not assess clonality in PB or BAL in this study, we have previously shown that the PB repertoire is polyclonal in both normal and asthmatic subjects, with a few clonal expansions in BAL before and after allergen challenge [20]. In the present study, we were particularly careful to study normal subjects and mild asthmatics, who had not had any recent respiratory infection and were stable as regards their disease activity status. Therefore, their lung T cell repertoire should not have been modified by recruitment of T cells from PB or local expansion due to viral or antigenic stimulation. However, it cannot be precluded that the differences in cytokine expression between TCR-Vβ subsets and total CD3+ T cells observed in the asthmatic group are related to atopy. It would be very important to examine an atopic non-asthmatic group in order to elucidate whether any occurring differences in the cytokine profile of TCR-Vβ subsets are related to atopy or asthma.
In ovalbumin-sensitized mice, T cells expressing different TCR-Vβ elements were found to differ in their ability to regulate IgE production and other immune responses [16]. In this murine study ovalbumin-reactive Vβ8+ T cells were able to stimulate specific IgE synthesis, whereas ovalbumin-reactive Vβ2+ T cells did not. In fact, Vβ2+ T cells inhibited IgE synthesis induced by the Vβ8+ T cells. Transfer of Vβ8·1/8·2+ T cells from sensitized mice to naive recipients passively transferred the capacity to develop allergen-specific IgE, immediate cutaneous hypersensitivity and airway hyperresponsiveness, whereas co-transfer of Vβ2+ T cells prevented these responses. These differences reflect differential cytokine production, with Vβ8·1/8·2+ cells producing relatively more IL-4 and less IFN-γ than Vβ2+ cells [16]. In another murine model of allergic asthma Vβ8+ T cells were essential for the development of airway eosinophilia and airway hyperresponsiveness and treatment with an anti-Vβ8 monoclonal antibody prior to sensitization prevented the development of airway hyperresponsiveness and almost completely abolished BAL eosinophilia after ovalbumin challenge [37]. These data indicate the potential for small subsets of T cells to modify the allergic response.
Although the present data are necessarily incomplete, they provide evidence for differences in the cytokine profile of TCR-Vβ subsets in BAL of asthmatic subjects. This is interesting especially in view of recent findings that classic antigens such as birch pollen and cat allergen have the capacity to selectively expand T cells bearing particular TCR-Vβ genes [38]. Thus, it would be instructive to study Vβ usage and cytokine profile of Vβ subsets in the lung following stimulation with specific antigens. Ideally such studies should be performed both in atopic asthmatic and in atopic non-asthmatic subjects to assess the respective contributions of asthma and allergy to the response. If combined with in vitro studies of T cell proliferation to different epitopes, this approach should help identify critical epitopes and T cell subsets with relevant functional differences and thus provide a better insight into the potential for modulating the immune response in allergic patients.
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