Published ahead of print on April 21, 2005, doi:10.1165/rcmb.2005-0100OC
© 2005 American Thoracic Society DOI: 10.1165/rcmb.2005-0100OC Interleukin-10–Secreting "Regulatory" T Cells Induced by Glucocorticoids and ß2-AgonistsDepartment of Asthma, Allergy and Respiratory Science, GKT School of Medicine, King's College London, Guy's Hospital, London, United Kingdom Correspondence and requests for reprints should be addressed to Dr. Catherine M. Hawrylowicz, Department of Asthma, Allergy and Respiratory Science, 5th Floor Thomas Guy House, GKT School of Medicine, King's College London, Guy's Hospital, London SE1 9RT, UK. E-mail: catherine.hawrylowicz{at}kcl.ac.uk
Greater clinical benefit in controlling the symptoms of asthma is frequently observed through combining moderate doses of inhaled glucocorticoids together with long-acting ß2-agonists, as compared with increasing glucocorticoid dosage alone. To address in vitro whether glucocorticoids plus ß2-agonists, compared with glucocorticoids alone, have greater inhibitory activity on CD4+ T cell responses to allergen, peripheral blood CD4+ T cell responses to allergen were compared in the presence or absence of the glucocorticoid fluticasone proprionate and the short- and long-acting ß2-agonists salbutamol and salmeterol, respectively. Fluticasone proprionate inhibited interleukin (IL)-5 and IL-13 and enhanced IL-10 synthesis in allergen-stimulated cultures in a concentration-dependent manner. Salmeterol, but not salbutamol, inhibited IL-5 and IL-13 and enhanced IL-10 synthesis in these cultures. When used in combination the two drugs demonstrated an additive effect on this pattern of cytokine production. Allergen-specific T cell lines induced in the presence of salmeterol and fluticasone proprionate inhibited IL-5 and IL-13 production by allergen-specific Th2 cell lines in an IL-10–dependent manner. Thus fluticasone proprionate and salmeterol increased IL-10 and reduced Th2 cytokine synthesis additively in allergen stimulated human CD4+ T cells.
Key Words: interleukin-10 • glucocorticoids • ß2-agonists • regulatory T cells
The Th2 cell–derived cytokines interleukin (IL)-4, IL-5 and IL-13 play a central role in asthma and allergy by regulating IgE synthesis, eosinophil accumulation and activation, mast cell activation, and airway remodeling (1, 2). Inhaled aeroallergens are an important stimulus for the production of these cytokines, and allergen-specific Th2 cells are present in the bronchial lumen of individuals with atopic asthma (3). IL-5 production by these cells correlates with the presence and severity of atopic disease (4). Glucocorticoids and ß2-agonists form the cornerstone of current asthma therapy. Glucocorticoids have well-documented anti-inflammatory effects (5). In particular, they directly inhibit T cell activation and Th2 cytokine production, which may contribute to their therapeutic effects (6). Short-acting ß2-agonists such as salbutamol produce a bronchodilatory response, which may last for up to an hour, whereas long-acting ß2-agonists, such as salmeterol, exhort a more sustained effect (8–12 h) (7). Unlike glucocorticoids, there is debate whether ß2-agonists possess significant anti-inflammatory activity. Nevertheless, ß2-receptors are widely distributed on inflammatory leukocytes, and there is increasing evidence that ß2-agonists are able to affect functions of T cells and other leukocytes, at least in vitro (8). Previous clinical studies have shown that, in patients whose asthma symptoms cannot be controlled by moderate dosages of inhaled glucocorticoid, further clinical benefit can be achieved by additional treatment with a long-acting ß2-agonist, rather than by solely continuing to increase the glucocorticoid dosage (9–11). The mechanism of this phenomenon remains unexplained (12). One mechanism by which glucocorticoids may inhibit T cell function is through the induction of the anti-inflammatory cytokine IL-10 (13, 14). We previously documented the capacity of glucocorticoids to induce regulatory T cells expressing relatively large quantities of the anti-inflammatory cytokine IL-10, but relatively small quantities of Th1 and Th2 cytokines. These cells were capable of inhibiting, or regulating, autologous T cell responses in an IL-10–dependent manner (15). We recently also demonstrated that glucocorticoid-induced IL-10 synthesis is deficient in peripheral blood T cells from patients clinically resistant to glucocorticoid therapy (16), supporting the hypothesis that induction of IL-10 may contribute to the clinical efficacy of glucocorticoid therapy. Based on these observations, we hypothesized that the combination of a long-acting ß2-agonist with a glucocorticoid results in greater inhibition of allergen-driven Th2 effector responses and enhanced production of IL-10 by human CD4+ T cells when compared with glucocorticoids alone.
Patients Atopy was defined as a positive wheal at 15 min after skin prick testing to  1 of a panel of aeroallergens (cat, grass, and house dust mite, Soluprick; ALK, Horsholm, Denmark) performed with diluent and histamine controls in individuals with a clinical history of allergy. All subjects (9 male, 15 female atopic; 1 male, 6 female nonatopic) were between 20 and 48 yr old. The study was approved by Guy's Hospital Ethics Committee and full written informed consent obtained from all subjects before study.
Cell Purification
Allergen Stimulation Cultures
Generation of Allergen-Specific T Cell Lines
Cytokine Measurement
Statistical Analysis
Both Long-Acting ß2-Agonists and Glucocorticoids Inhibit Allergen-Induced Th2 Responses CD8+ T cell–depleted peripheral blood mononuclear cells from atopic and nonatopic donors were cultured with allergen alone or in the absence or presence of serial dilutions of fluticasone proprionate (10–8–10–11M) or salmeterol (10–6–10–8M). After 7 d of culture, the concentrations of the Th2 cytokine IL-13 was determined in culture supernatants by ELISA. Cells cultured with allergens secreted significantly more IL-13 compared with cells cultured in medium alone (Figure 1, left-hand panels) and atopic donors synthesized much higher levels of cytokine compared with nonatopic donors, as predicted from earlier published reports (1, 2, 18).
As the magnitude of the allergen-induced response varied between individuals, the effects of drugs on IL-13 production were expressed as percentages of the positive control response (Figure 1, right-hand panels). In atopic individuals, fluticasone proprionate alone caused a concentration-dependent inhibition of IL-13 production, which was significant at a concentration 10–9 M or greater (IL-13 10–8 M, P  0.01; IL-13 10–9 M, P 0.05). This inhibition was more profound in nonatopic individuals, where the magnitude of the response to allergen was > 6-fold lower than that seen in the atopic individuals (10–8 M and 10–9 M, P 0.01; 10–10 M, P 0.05) Similar data were observed with IL-5 (data not shown).
Salmeterol alone (10–6 M) also significantly inhibited production of IL-13 at 10–6 M (P The effects of fluticasone proprionate and salmeterol on Th2 cytokine responses were tested in combination in atopic individuals, as this group demonstrated robust Th2 responses to allergen in culture. The two drugs showed an approximately additive effect in inhibiting both IL-13 and IL-5 production (Figure 2). Thus, at concentrations of fluticasone proprionate significantly effective alone (10–8 M, 10–9 M), the addition of salmeterol (10–6 M) virtually abolished cytokine production, whereas with lower concentrations of fluticasone proprionate the combined effect approximated to that of salmeterol alone.
Long-Acting but Not Short-Acting ß2-Agonist Inhibits Allergen-Induced Th2 Responses: Dependence on ß2-Adrenoreceptor Stimulation In contrast to salmeterol, the short-acting ß2-agonist salbutamol (albuterol) at an equivalent concentration (10–6 M) showed no significant inhibitory affect on allergen-induced production of IL-13 and IL-5. Furthermore, it did not add to the inhibitory effect of fluticasone proprionate (Figure 3A). Nevertheless, the inhibitory affect of salmeterol was exerted through the ß2-adrenoreceptor, because addition of the selective ß2-adrenoreceptor antagonist ICI 118.551 (10–6 M) fully reversed the inhibitory effect of salmeterol on allergen-induced IL-13 and IL-5 production (Figure 3B, left-hand panel, and data not shown).
Both Long-Acting ß2-Agonists and Glucocorticoids Enhance Allergen-Induced IL-10 Production Allergen stimulation alone induced only low levels of IL-10 synthesis by CD8+ T cell–depleted peripheral blood mononuclear cells above medium control (medium control 56 ± 16 pg/ml versus with allergen 168 ± 42 pg/ml; n = 10; P 0.05). Addition of fluticasone proprionate to the allergen-stimulated cultures induced a concentration-dependent enhancement of IL-10 production, which was significant at a concentration of 10–10 M or greater (10–8 M and 10–9 M, P 0.01; 10–10 M, P 0.05).
Salmeterol alone also enhanced IL-10 production, which reached statistical significance at 10–6 M (P
Allergen-specific T cells are present at a very low frequency in these cultures. In order, therefore, to directly visualize the effects of fluticasone proprionate and salmeterol on the number of IL-10–secreting cells, purified CD4+ T cells were stimulated by the polyclonal T cell activator anti-CD3 together with anti-CD28 in the absence of APC. Figure 4B shows a typical experiment representative of three. Both salmeterol (10–6 M) and fluticasone proprionate (10–9 M) independently increased the frequency of IL-10–immunoreactive cells, and the combined effect of the two drugs was approximately additive. The pattern of inhibition of IL-13 expression by salmeterol and fluticasone proprionate, singly and in combination, was similar to that observed by ELISA.
Generation of Allergen-Specific, Functional T Regulatory Cell Lines in the Presence of Long-Acting ß2-Agonists and Glucocorticoids
The inhibitory properties of these putative T regulatory lines was investigated by co-culturing them together with cells from autologous Th2 effector lines, irradiated APC, and allergen for 3 d. When cultured alone, the Th2 line produced IL-5 and IL-13 in response to allergen as before, and the regulatory T line produced little or none (Figure 6A). The two lines were co-cultured at a ratio of four Th2 cells to one regulatory T cell, a ratio calculated to be optimal based on earlier studies in an anti-CD3–driven system (data not shown). In this co-culture the production of IL-5 and IL-13 by the Th2 cells was abrogated (Figure 6A). Furthermore, culture in the presence of a blocking anti–IL-10 receptor antibody not only increased IL-13 production by the Th2 effector line alone, as predicted from earlier reports, but also reversed the inhibition of IL-13 production by the T regulatory cells in the co-culture experiments (Figure 6B).
We have shown that the long-acting ß2-agonist salmeterol, as well as the glucocorticoid fluticasone proprionate, can inhibit allergen-driven Th2 cytokine production by CD4+ T cells from atopic subjects. The effect of salmeterol was dependent on ß2-adrenoreceptor stimulation as demonstrated by its abrogation by a selective ß2-receptor antagonist. In contrast, the short-acting ß2-agonist salbutamol had no significant effect on cytokine synthesis under identical conditions. In addition, we have shown that high concentrations of salmeterol, as well as fluticasone proprionate, skew the cytokine profile of allergen-driven CD4+ T cells in atopic subjects from a Th2 phenotype toward a T regulatory phenotype, with elevated production of IL-10. Again, this phenomenon was abolished by ß2-receptor blockade. Allergen-specific T cell lines raised in the presence of fluticasone proprionate and salmeterol, in addition to producing elevated IL-10, were functionally active in suppressing syngeneic Th2 effector responses to allergen. Independent studies implicate IL-10 as an important anti-inflammatory cytokine in asthma and the lung (19–21). The present study extends the beneficial mechanisms via which long-acting ß2-agonists and glucocorticoids may co-operate to greater therapeutic benefit in asthma by causing additive inhibition of Th2 cytokine production and enhancing synthesis of the anti-inflammatory cytokine IL-10. The T cell–inhibitory effects of salmeterol documented in the present study are consistent with previous studies showing a range of inhibitory effects of this drug on cytokine production by T cells activated by mitogen or specific antigen (22–26). Inhibition of Th2 cytokine secretion by basophils and mast cells by salmeterol has also been reported (reviewed in Ref. 8). One very recent report (27), however, failed to observe an inhibition of Th2 cytokine production by salmeterol alone using a slightly lower concentration of the drug. One additional difference between that and the present study is the use of mitogen versus allergen to stimulate a T cell response. The authors did, however, observe that combining salmeterol and fluticasone proprionate led to greater inhibition of both proliferation and Th2 cytokine production than either drug alone in healthy donors. The capacity of salmeterol to inhibit cytokine production by Th1 cells has been reported to reflect actions on APC (28). In the current study, enhancement of IL-10 and inhibition of Th2 cytokine production may reflect both a direct effect on the T cell, since experiments to demonstrate changes in intracellular cytokine staining were performed using purified CD4+ T cell populations, and indirect effects at the level of the APC population which were present in all allergen-stimulated cultures. Our findings suggest that the functional interaction between salmeterol and fluticasone proprionate, at least in terms of suppression of Th2 effector responses and the promotion of IL-10 synthesizing T cells, is seen at comparatively high concentrations of salmeterol but lower concentrations of fluticasone proprionate. This reflects the clinical situation in which combined treatment with inhaled, long-acting ß2-agonist and glucocorticoid at low dosage has clearly been shown to be advantageous in comparison with high dosages of glucocorticoid used alone (9, 10, 29). It is tempting to speculate that this clinical effect reflects the collaborative inhibitory effect of long-acting ß2-agonist and glucocorticoid, which we have demonstrated in the present study. Studies on the anti-inflammatory affects of inhaled long acting ß2-agonist alone in asthma have produced variable results, with some studies reporting reduction in mucosal mast cells (10), eosinophils (26, 30), or neutrophils (31), and others reporting no effect at all (32). These discrepancies most likely reflect the variable power of these studies to detect significant differences and systematic studies of possible collaborative effects of these two drugs on asthmatic bronchial inflammation in vivo are required to clarify this issue. Our data suggest an additive, but not a synergistic effect of long-acting ß2-agonist and glucocorticoid on T cell effector and regulatory functions. Recent studies in the literature suggest the possibility of direct interactions between these drugs at the molecular level. Glucocorticoids increase the expression of ß2-adrenoreceptors by increasing transcription of their gene. This may theoretically counteract tachyphylaxis to ß2-adrenoreceptor stimulation caused by receptor internalization (10). In addition, it has been suggested that ß2-agonists and glucocorticoids act on common transcriptional regulatory proteins (33), and that ß2-agonist affects ligand-independent activation (possibly by phosphorylation) of the glucocorticoid receptor that facilitates nuclear translocation (reviewed in Ref. 10). Experimental evidence is currently discordant as to whether or not this effect is confined to long-acting ß2-agonist or seen with both short- and long-acting ß2-agonist (34, 35). The present study was not designed to address these mechanistic questions, but does clearly demonstrate the potential for a collaborative anti-inflammatory effect of long-acting ß2-agonists and glucocorticoids on T cell–mediated processes thought to be critical to their therapeutic profile.
C.H. and T.H.L. gratefully acknowledge the support of Asthma-UK.
This study was funded in part by an unrestricted research grant from GlaxoSmithKline, UK (E.J.P.). Conflict of Interest Statement: E.J.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; D.F.R. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; A.F. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; P.L. received £195,000 in an unrestricted research grant from GlaxoSmithKline; T.H.L. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript; C.J.C. received $400 in lecture fees and £60,000 in industry-sponsored grant support from GlaxoSmithKline, up to the 3 years ending 2004; C.M.H. received £200,000 in an unrestricted research grant from GlaxoSmithKline, to study "Possible synergistic effect of fluticasone proprionate and salmeterol in a number of in vitro systems." Received in original form March 10, 2005 Received in final form March 10, 2005
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Abstract
Introduction
and IL-12 (BD-Pharmingen, Abingdon, UK). For regulatory T cell lines, CD4+ T cells plus APC were cultured with 10 µg/ml allergen, 10 ng/ml IL-4, 10–9 M fluticasone proprionate and 10–6 M salmeterol (TFlut/Sal). Both lines were cultured for 7 d, then restimulated at a 1:1 ratio with irradiated autologous APC under identical conditions as the original culture. The lines underwent two rounds of stimulation and then supernatants were harvested for cytokine analysis. To assay regulatory/suppressive activity, cells were harvested at 14 d. Cultures contained the indicated combinations of 105 Th2 cells, 2.5 x 104 TFlut/Sal line cells, 10 µg/ml specific allergen, 5 µg/ml anti–IL-10 receptor (3F9–2, DNAX, CA) or 5 µg/ml control antibody (R35–9S; BD-Pharmingen). All wells contained 105 irradiated autologous APC. On Day 3, 100 µl supernatant was removed for cytokine measurement. 
