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Ciclesonide modulates in vitro allergen-driven activation of blood mononuclear cells and allergen-specific T-cell blasts
Immunology Letters 141 (2011) 190–196
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Ciclesonide modulates in vitro allergen-driven activation of blood mononuclear cells and allergen-specific T-cell blasts Michela Silvestri a,∗,1 , Fabio Morandi b,1 , Vito Pistoia b , Ignazia Prigione b , Giovanni A. Rossi a a b
Pediatric Pulmonary and Allergy Unit, G. Gaslini Institute, Largo G. Gaslini 5, 16147 Genoa, Italy Laboratory of Oncology, G. Gaslini Institute, Largo G. Gaslini 5, 16147 Genoa, Italy
a r t i c l e
i n f o
Article history: Received 22 March 2011 Received in revised form 5 October 2011 Accepted 5 October 2011 Available online 12 October 2011 Keywords: Allergen-specific T-cell blasts Antigen Atopy Corticosteroid Peripheral blood mononuclear cells
a b s t r a c t Background: Ciclesonide, an inhaled corticosteroid with almost no affinity for the glucocorticoid receptor, is highly effective in downregulating in vitro pro-inflammatory activities of airway parenchymal cells when converted into the active metabolite desisobutyryl-ciclesonide. Objective: We evaluate whether ciclesonide could effectively downregulate also antigen- or allergeninduced activation of peripheral blood mononuclear cell and of allergen-specific T-cell blasts. Methods: Peripheral blood mononuclear cells were isolated from non atopic and atopic asthmatic children sensitized to Phleum pratense (PhlP5). Proliferation toward Candida albicans or PhlP5 in the presence of ciclesonide or desisobutyryl-ciclesonide (0.003–3.0 M) was evaluated as [3 H]thymidine incorporation. Modulation of PhlP5-specific T-cell blasts proliferation and PhlP5-induced interleukin 4 expression by ciclesonide and desisobutyryl-ciclesonide were measured. Results: Peripheral blood mononuclear cell proliferation to C. albicans was dose-dependently inhibited by 0.3–3.0 M ciclesonide and desisobutyryl-ciclesonide but inhibition by desisobutyryl-ciclesonide was higher. A significant proliferation to PhlP5 was observed only in cultures from atopic subjects: an effective downregulation was already detected at 0.03 M ciclesonide and 0.003 M desisobutyryl-ciclesonide (complete inhibition at 3 M ciclesonide and 0.03 M desisobutyryl-ciclesonide). 3 M ciclesonide and desisobutyryl-ciclesonide reduced the PhlP5-specific T-cell blast proliferation and interleukin 4producing cell proportion. Conclusions and clinical relevance: These in vitro data, obtained at concentrations similar to those reached in vivo at bronchial level, are in favor of an efficient inhibition of ciclesonide on the T-cell mediated response toward allergens. Additional studies are required to confirm these preliminary data on the reduced activity of the drug on allergen-specific T-cell blast activation that may have clinical relevance. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Inhaled corticosteroids (ICs) are the standard-of-care controller medications for asthma [1]. The clinical effectiveness of this class of drugs in asthma, as in many other inflammatory diseases, is related
Abbreviations: ICs, inhaled corticosteroids; PBMC, peripheral blood mononuclear cell; CIC, ciclesonide; des-CIC, desisobutyryl-ciclesonide; Ca, Candida albicans; PhlP, Phleum pratense; yrs, years; DMSO, dimethyl sulfoxide; FCS, foetal calf serum; MNC, mononuclear cells; APC, antigen presenting cells; PBS, phosphate buffered saline; mAb, monoclonal antibody; SE, standard error; GCR, glucocorticoid receptor; IL-2R, interleukin 2 receptor. ∗ Corresponding author. Tel.: +39 010563547; fax: +39 010383953. E-mail addresses: [email protected] (M. Silvestri), [email protected] (F. Morandi), [email protected] (V. Pistoia), [email protected] (I. Prigione), [email protected] (G.A. Rossi). 1 These authors contributed equally to this work. 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.10.003
to their ability to block multiple pathways involved in the inflammatory processes that characterize this disease [2–4]. Treatment with ICs reduces the number of inflammatory cells in the bronchi, such as mast cells and eosinophils but also decreases the proportion of alveolar macrophages and of type 2 helper T-lymphocytes and their related proinflammatory cytokines in asthmatic airways [5,6]. These observations support the concept that a key role of ICs may be the downregulation in vivo of T-cell activation by allergen-presenting cells, inhibiting T-cell differentiation toward the Th2 phenotype, an early key event leading to stimulation of IgE production and, in general, to inflammation promotion [6,7]. This inhibitory activity has been extensively demonstrated in vitro testing a variety of ICs on allergen-stimulated peripheral blood mononuclear cell (PBMC) [8–11] and allergen-specific Th2 T-cell lines [12]. All these effects at cellular levels, but also the therapeutic benefits in patients with asthma, are usually achieved at relatively low ICs doses, with a high benefit-to-risk ratio also for long term treatments [13]. However, despite the improved safety profile of
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ICs, local and systemic side effects are still a concern, some (like oropharyngeal candidiasis) being common also with low-dose and short-term use [13,14]. Ciclesonide (CIC), a new-generation nonhalogenated IC, possesses many key physicochemical properties resulting in pharmacokinetic characteristics associated with high efficacy, favorable tolerability, and low risk for systemic side effects [14,15]. Administered as a parent compound with almost no binding affinity for the glucocorticoid receptor, CIC is rapidly converted in the airway mucosa into the active metabolite, desisobutyryl-ciclesonide (des-CIC) known to have a remarkable anti-inflammatory activity [14,15]. In in vitro cellular models CIC is highly effective in downregulating within 3 h the pro-inflammatory activities of airway epithelial cells and fibroblasts when converted by these cells into the active metabolite desisobutyryl (des)-CIC [16,17]. However it is not known whether: (a) conversion of CIC into des-CIC may occur also at human mononuclear cell level, resulting in an effective downregulation of allergeninduced T-lymphocyte activation and (b) the inhibitory activity of CIC could be higher for a Th2 response than for a Th1 response. A study was therefore designed to compare the ability of CIC and its active metabolite des-CIC to modulate the proliferative response to Candida albicans (Ca) (as a prototype of “Th1 response”) and to Phleum pratense (PhlP5) (as a prototype of “Th2 response”) of PBMCs isolated from non atopic subjects and atopic asthmatic individuals sensitized to PhlP. PhlP5-specific T-cell blasts were then generated and cell proliferation and IL-4 expression in response to PhlP5, in the presence of CIC or des-CIC, were studied. Ca was chosen as antigen because of the high frequency of oropharyngeal candidiasis as side effects of ICs treatment in asthma, while PhlP5, a major grass pollen allergen from P. pratense, because of its clinical relevance as inhalant allergen in our patient population.
2. Materials and methods 2.1. Study population Ten atopic Caucasian patients (mean age: 11.59 ± 0.84 yrs old, 2 females and 8 males) and 8 healthy non atopic individuals (mean age: 10.08 ± 1.05 yrs old, 2 females and 6 males), as controls, entered the study. Demographic and clinical characteristics of the atopic individuals are reported in Table 1. For atopic patients, the inclusion criteria were: (i) rhino-conjunctivitis with or without mild intermittent bronchial asthma and (ii) allergic sensitization to P. pratense (PhlP), demonstrated by skin prick test positivity to PhlP5 antigen and by the detection of allergen-specific serum IgE (see below); (iii) being asymptomatic at the time of the study. None of the patients was currently under anti-asthmatic treatment other than short-acting 2 -stimulants on an ‘as necessary’ basis, had received any drug known to interfere with T-cell mediated immune response in the last 3 months or any medication in the 24 h preced-
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ing the study entry. All subjects and parents/tutors were informed of the nature and the purpose of the study and gave written consent. Access to health records complied with the Italian legislation. Institutional Review Board approval was obtained for this study. 2.2. Diagnosis of allergic sensitization Sensitization to the most common aeroallergens was evaluated by skin-prick test (Lofarma, Milan, Italy) as previously described [8]. Allergen-specific serum IgE levels were determined by solid-phase, chemiluminescent immunometric assays (Immulite, Medical System; Genoa, Italy). Allergen-specific IgE levels ≥3.5 kU/L were considered consistent with sensitization to that allergen. 2.3. Isolation of peripheral blood mononuclear cells (PBMCs) and evaluation of allergen- and antigen-induced proliferation Isolation of PBMCs was performed as previously described [8]. Cell viability was determined by Trypan blue dye exclusion test [Euroclone, Siziano–Pavia, Italy) and found >90% in all the experimental conditions. PBMCs (105 cells/well) in 10% autologous heat-inactived serum were incubated in a 96-well plate for 6 days at 37 ◦ C in 5% CO2 with or without Ca (5 × 104 autoclaved bodies/ml), prepared as previously reported [18] or PhlP5 extract (20 g/ml) (gently provided by ALK Abellò, Spain). We used heat-inactived serum to prevent possible conversion of CIC into des-CIC by serum esterases. In preliminary sets of experiments, 20 g/ml PhlP5 was found to be the optimal dose promoting PBMC proliferation (not shown). To evaluate the effect of CIC and des-CIC on PBMC proliferation, cells were stimulated with PhlP5 or Ca in the presence of different concentrations (0.003–3 M) of the steroids. At day 6, tritiated [3 H]thymidine (0.5 Ci/well) (Amersham International, Bucks, UK) was added and the cells collected 18 h later by a cell harvester. The [3 H]thymidine incorporation in cell cultures, evaluated by a -counter (Wallac beta counter, PerkinElmer, Waltham, MA, USA), was used to assess PBMC proliferation and expressed as count per min (cpm). The vehicle used for corticosteroids dilution was dimethyl sulfoxide (DMSO, Euroclone), tested at a final concentration of 0.15%. At this concentration, DMSO did not affect cell viability, as assessed by Trypan blue dye exclusion test (not shown). 2.4. In vitro expansions of PhlP5-specific T-cell blasts and evaluation of allergen-induced proliferation To generate PhlP5-specific T-cell blasts, PBMCs from 5 sensitized individuals were stimulated with PhlP5 for 6 days as previously described. Low density T-cell blasts were then isolated using Percoll gradient (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and cultured for an additional 4–6 days in complete medium in the presence of 10% foetal calf serum (FCS, Euroclone) and 100 U/ml rIL-2 (Chiron Corp., Emeryville, CA, USA).
Table 1 Demographic and clinical characteristics of the atopic population. Pt Id
Gender
Age (yrs)
Total serum IgE (ku/l)
PhlP5-specific serum IgE levels (ku/L)
C.S. M.F. B.M. F.S. S.L. V.F. O.N. C.L.L. G.M. S.F.
F M M M M M F M M M
11.9 14.0 12.5 14.0 15.5 9.4 10.4 10.1 11.8 6.3
492 285 649 187 288 >2000 1492 481 100 128
23.2 3.6 >100 3.7 8.3 8.5 6.3 >100 >100 5.5
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Fig. 1. Proliferative response to Candida albicans (Ca) by peripheral blood mononuclear cells (PBMCs) isolated from non atopic and atopic subjects and effect of different concentrations of ciclesonide (CIC) and des-CIC on this response. (A) Cell proliferation, evaluated as tritiated thymidine ([3 H]TdR) incorporation and expressed as count per minute (cpm) is shown on the ordinate, whereas the different experimental conditions [without stimulus (medium) and with Ca] are reported on the abscissa. (B and C) The percentage of inhibition for non-atopic (B) and atopic (C) individuals is shown on the ordinate whereas the different concentrations (0.003–3 M) of the drugs are reported on the abscissa. The data are expressed as mean ± standard error of the mean. ¶¶¶: p < 0.001, vs. unstimulated cells; *: p < 0.05, **: p < 0.01, vs. cells grown in the absence of the drug; §: p < 0.05, CIC vs. des-CIC.
To test allergen-specificity of T-cell blasts, cells were collected, washed three times and resuspended in complete medium plus 10% AB serum and cultured (3 × 104 cells/well) with PhlP5 (20 g/ml) in the presence of autologous irradiated (45 Gy) mononuclear cells (MNCs), as antigen presenting cells (APCs) (8 × 104 cells/well) in 96well flat-bottom plates in a total volume of 0.2 ml for 48–72 h [19]. To evaluate the effects of CIC and des-CIC on cell proliferation, blasts and autologous irradiated MNCs were also cultured in the presence of PhlP5 and different concentrations of the two molecules. The incubation conditions with CIC, des-CIC as well as the addition of the APC was performed as described above. Cells were pulsed with 0.5 Ci/well [3 H]thymidine for the last 18 h of culture and harvested. The cell-associated radioactivity was determined by liquid scintillation counting.
2.5. Intracellular cytokine staining on PhlP5-specific T-cell blasts by flow cytometry For intracellular cytokine staining, allergen-specific T-cell blasts (106 cells/ml) were collected after 48–72 h of culture with PhlP5 and in the presence or not of CIC and des-CIC different concentrations, as described in detail in the previous section. Briefly, cells were incubated for 4 h in brefeldin-A (5 g/ml; Sigma–Aldrich, St. Louis, MO, USA). After washing in phosphate buffered saline (PBS) with 1% FCS (staining buffer), cells were stained with anti CD4-PE Cy5.5 monoclonal antibody (mAb) (BD Biosciences, Mountain View, CA, USA) for 30 min at 4 ◦ C in the dark and then washed again in staining buffer and fixed in Cytofix/CytopermTM solution (BD Biosciences) for 20 min at 4 ◦ C in the dark. Subsequently cells were washed twice with Perm/Wash buffer (BD Biosciences) and stained with FITC-conjugated anti-human IFN-␥ mAb and PE-conjugated
anti-human IL-4 mAb (Caltag Laboratories, Burlingame, CA, USA) and for 30 min at 4 ◦ C in the dark. After washing in staining buffer, cells were analyzed by flow cytometry (FACScan, BD Biosciences) gating on the CD4+ T-cell subsets. PE- and FITC-conjugated mouse Ig control mAb (Caltag Laboratories) were used as negative controls in all these experiments. CellQuest software (BD Biosciences) was used for analyses. The results of flow cytometry experiments were expressed as percentage positive cells and as mean relative fluorescence intensity (MRFI). MRFI represents the ratio between the mean fluorescence intensity of cells stained with the selected mAb and the mean fluorescence intensity of cells stained with the isotype-matched mouse Ig. 2.6. Data analysis and statistical evaluation Data were reported as mean and standard error (SE). Statistical analysis (GraphPad Prism Software Inc, San Diego, CA, USA) of results was carried out by Student’s t-test or Mann Whitney U-test as appropriate. Multiple comparisons were made by means of the Analysis of Variance test; post-hoc comparisons after the ANOVA were performed applying the Dunnett’s Multiple Comparison Test. All tests used were two sided and a p values less than 0.05 were considered statistically significant. 3. Results 3.1. Modulation of Ca-induced PBMC proliferation in non atopic and in atopic subjects by CIC and des-CIC In PBMC cultures from atopic and non atopic subjects, a similar 40-fold significant increase in [3 H]thymidine incorporation was
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Fig. 2. Proliferative response to Phleum pratense (PhlP5) by peripheral blood mononuclear cells (PBMCs) isolated from atopic and non atopic subjects and effect of different concentrations of ciclesonide (CIC) and des-CIC on this response. (A) Cell proliferation, evaluated as tritiated thymidine ([3 H]TdR) incorporation and expressed as count per minute (cpm) is shown on the ordinate, whereas the different experimental conditions [without stimulus (medium) and with PhlP5] are reported on the abscissa. (B) The percentage of inhibition is shown on the ordinate whereas the different concentrations (0.003–3 M) of the drugs are reported on the abscissa. The data are expressed as mean ± standard error of the mean. ¶¶¶: p < 0.001, vs. unstimulated cells; ***: p < 0.001, vs. cells grown in the absence of the drug; §: p < 0.05, §§§: p < 0.001, CIC vs. des-CIC.
observed in the presence of Ca, as compared with unstimulated cells (Fig. 1A). A dose-dependent inhibition of Ca-induced PBMC proliferation was observed in the presence of CIC and des-CIC, similar in atopic and non atopic subjects, but significantly higher for des-CIC than for CIC at 0.3 M and 3 M (Fig. 1B and C). Also the inhibitory effect induced by lowest concentration of des-Cic in atopic subjects was not statistically different from that observed in cultures by non atopic subjects (p = 0.17).
des-CIC (p < 0.001, each comparison), with a nearly complete inhibition observed at 3 M CIC and at 0.03 M des-CIC (Fig. 2B). The inhibitory activity toward PhlP5-induced PBMC proliferation was higher for des-CIC than for CIC at 0.003 M (p < 0.05), 0.03 M (p < 0.001) and 0.3 M (p < 0.05).
3.2. Modulation of PhlP5-induced PBMC proliferation by CIC and des-CIC
In allergen-specific T-cell blast cultures, a significant 5-fold increase in [3 H]thymidine incorporation was observed in the presence of PhlP5 (Fig. 3A). Proliferation was similarly downregulated by CIC and des-CIC, but the inhibitory effect was significant only at 3.0 M, at the highest concentration tested amounting to about 37% and 54% reduction (Fig. 3B). Stimulation with PhlP5 of the T-cell blasts also significantly increased the proportion of IL-4-producing cells (p < 0.05) but not that of IFN-␥-producing cells (Fig. 4A). The increased proportion of IL-4-producing cells was partially reduced to comparable extent
As expected, a significant 8-fold increase in [3 H]thymidine incorporation was observed in the presence of PhlP5 in PBMCs cultures from all atopic patients (p < 0.001), but not in PBMC cultures from non atopic subjects not sensitized to the allergen (Fig. 2A). In PBMCs cultures from atopic patients, both CIC and des-CIC induced a dose-dependent downregulation of PhlP5-induced proliferation. The effect was already significant at 0.03 M CIC and at 0.003 M
3.3. Modulation of PhlP5-induced T-cell blast proliferation and cytokine production by CIC and des-CIC
Fig. 3. Proliferative response to Phleum pratense (PhlP5) by PhlP5-specific T-cell blast proliferation and inhibitory effect of different concentrations of ciclesonide (CIC) and des-CIC. (A) T-cell blast proliferation, evaluated as tritiated thymidine ([3 H]TdR) incorporation and expressed as count per minute (cpm) is shown on the ordinate, whereas the different experimental conditions [without stimulus (medium) and with PhlP5] are reported on the abscissa. (B) The percentage of inhibition is shown on the ordinate whereas the different concentrations (0.003–3 M) of the drugs are reported on the abscissa. The data are expressed as mean ± standard error of the mean. ¶¶: p < 0.01, vs. T-cell blasts not re-stimulated; *: p < 0.05, vs. cells grown in the absence of the drug.
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Fig. 4. Proportion of interleukin (IL)-4-producing and interferon (IFN)-␥-producing T-cell blasts following re-stimulation with Phleum pratense (PhlP5) in the presence of ciclesonide (CIC) and des-CIC. The proportion of IL-4-producing (panels A and B) and interferon (IFN)-␥-producing (panels A and C) T-cell blasts is shown on the ordinate, whereas the different experimental conditions [without re-stimulation (Ctr), with re-stimulation with PhlP5 (PhlP5) and in the presence of CIC or des-CIC 3 M] are reported on the abscissa. The percentage of positive cells was calculated on the basis of fluorescence intensity obtained with an irrelevant isotype-matched antibody control. The data are expressed as mean ± standard error of the mean. ¶: p < 0.05, vs. T-cell blasts not re-stimulated; *: p < 0.05, vs. cells grown in the absence of the drug.
(40–50%) by 3 M CIC and 3 M des-CIC (Fig. 4B and C). No significant modification in proportion of IFN-␥-producing cells was observed (Fig. 4A–C). Representative flow-cytometry histograms of IL-4 and IFN-␥ production by PhlP5-specific T-cell blasts and of the inhibitory effect of CIC or des-CIC (3 M) are reported in Fig. 5A and B. 4. Discussion Using PBMCs from non atopic and atopic subjects, we demonstrated that CIC and des-CIC, are effective in downregulating the proliferative response to C. albicans (an antigen stimulating predominantly a “Th1 response”) and to P. pratense 5 (an allergen inducing a “Th2 response”). CIC is a “pro-drug” with almost no affinity for the glucocorticoid receptor, and consequently was added to cell cultures as an inactive compound. Therefore the results here reported demonstrate that, as already shown for airway epithelial cells [16,20] and lung fibroblasts [17], also blood mononuclear cells are able to convert the parent compound in the active principle desCIC, to exert anti-inflammatory functions on the same cells. This is a relevant information considering both the key role that mononuclear cells, i.e. antigen-presenting cells and T-lymphocytes, have in asthma pathogenesis and the pharmacokinetic and pharmacodynamic characteristic of CIC. Indeed, carboxylesterases, one of the enzymes involved in isobutyrate cleaving at the CIC C21 position [21], appear to be abundant in human monocytes and to be present in T-lymphocytes [22]. It is very unlike that over the long period of incubation (6 days at 37 ◦ C) CIC could have been converted into desCIC by esterases in the serum. Indeed: (a) carboxylesterase is rare in human blood [21] and (b) cells were cultured in heat-inactivated serum. Des-CIC was more efficient than CIC in inhibiting PBMC proliferative response but only at the highest concentrations, when the cells were stimulated with Ca antigen, and at mid-to-low
concentrations, when the cells were stimulated with PhlP5 allergen. These results on cell proliferation that indicate a partial conversion of CIC to des-CIC by lymphocytes are in agreement with findings recently reported by Stoeck et al. [23]. Using highly purified human CD4 T-cells stimulated with anti CD3/anti CD28, or PBMCs stimulated with ConA, they obtained a similar efficacy on the inhibition of cell proliferation but at lower des-CIC than CIC concentrations. We observed a higher inhibitory effect on PhlP5-stimulated than on Ca-stimulated PBMCs, being remarkable (50–60%) for PBMC stimulated with Ca and nearly complete for PBMC stimulated with PhlP5 when the highest concentration of CIC and des-CIC was used. One may hypothesize therefore, that the Th2 -driven response (PhlP5) is more sensitive to inhibitory effects of the glucocorticoid than that driven by Th1 cells (Ca). One explanation for these results could be related to differences between antigen (Ca)- and allergen (PhlP5)-specific T-cells in glucocorticoid receptor sensitivity and/or in the balance between glucocorticoid receptor (GCR) ␣ and  expression, respectively the active and the dominant negative GCR isoforms [24]. Another observation was that Ca-induced proliferation of Tcells from atopic subjects was more efficaciously suppressed by the active metabolite compared to ciclesonide. Although carboxylesterases appear to be equally present in T-lymphocyte subsets [22], we may speculate that under different stimulations, they may show different rates of enzymatic activities. Indeed, even though they are ubiquitous in human tissues [25], their relative concentration may be dissimilar and contribute differently to the conversion to des-CIC in the different organs [21,26,27]. We also found a reduced activity of CIC on the allergen-induced activation of T-cell blasts, as compared with PBMCs. This may be due to a different sensitivity to the drug, related to the presence of IL-2 in the medium used to obtain T-cell blasts in vitro [28]. In addition, it has been suggested that high IL-2 concentrations, when related to endogenous overproduction, may be associated
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Fig. 5. Representative flow-cytometry histograms of IL-4 (panel A) and IFN-␥ (panel B) production by PhlP5-specific T-cell blasts and of the inhibitory effect of CIC or des-CIC (3 M). x-axis is SSC and y-axis is specific fluorescence intensity (PE or FITC).
with glucocorticoid resistance [29]. Finally, Haczku et al. [28] also demonstrated that dexamethasone was ineffective in inhibiting lymphoblasts i.e. cells already committed to proliferation, suggesting that this drug may inhibit only early events in the T-lymphocyte proliferative cycle but not the proliferative events subsequent to the synthesis of IL-2. The exact mechanism by which IL-2 promotes steroid resistance is not entirely clear but several possible pathways have been described. For example Goleva et al. [30] and Irusen et al. [31] showed that treatment of PBMCs with a combination of IL2 and IL-4 abrogated dexamethasone-induced GCR-alpha nuclear translocation by activating p38 MAPK that may impair GCR function. Further, Ito and Mercado [32] found that hyperacetylation of the glucocorticoid receptor may also account for steroid resistance following IL-2 and IL-4 exposure of PBMC. The role of IL-2 in response to steroid treatment is also supported by the demonstration that the blockade of IL-2 signaling with an anti-CD25 mAb increased steroid sensitivity in cells from steroid resistant patients [33]. In addition, it has been shown that during the pollen season there was an increase in T lymphocyte activation in the BAL fluid as identified by increased expression of interleukin 2 receptor (IL-2R) as compared to that found out of the pollen season (before the pollen season) [34]. Therefore, the addition of IL-2 to favor blast growth in vitro may, at least in part, mimic what happens in vivo during the pollen season. Taken together these in vitro results are in favor of the effective conversion of CIC into its active metabolite by human PBMCs and prove the ability of this pro-drug to downregulate the immune response toward allergens at concentrations similar to those
obtained in vivo at bronchial level [35]. In the present study we did not evaluate the concentrations of ciclesonide and des-ciclesonide in cell culture supernatants. However, we previously demonstrated both in human bronchial epithelial cell and in human lung fibroblast cultures that after 24 h incubation with the drug, CIC was below the levels of quantification in all the samples whereas only its active compound des-ciclesonide was present, confirming an effective and rapid biotransformation of CIC into the active metabolite. Apparently, the glucocorticoid revealed somewhat more effective to reduce proliferation to PhlP5, as a Th2 response compared to Ca, as a Th1 response in PBMC from atopic subjects. These in vitro results may at least partially explain the low susceptibility to oral candidiasis observed in patients treated with CIC [14]. These preliminary data on the reduced activity of the drug on allergen-specific T-cell blast activation need to be confirmed by additional studies to establish a possible clinical relevance. Funding This study was supported by Grant “Ricerca Finalizzata” from Italian Ministry of Health, Rome, Italy and by a grant from Nycomed S.p.A., Italy. Competing interest MS, FM, VP and IP have no declared conflict of interest. GAR has been reimbursed by ALTANA PHARMA (Nycomed) for conference attendance and served as a consultant to ALTANAPHARMA
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Immunology Letters journal homepage: www.elsevier.com/locate/immlet
Ciclesonide modulates in vitro allergen-driven activation of blood mononuclear cells and allergen-specific T-cell blasts Michela Silvestri a,∗,1 , Fabio Morandi b,1 , Vito Pistoia b , Ignazia Prigione b , Giovanni A. Rossi a a b
Pediatric Pulmonary and Allergy Unit, G. Gaslini Institute, Largo G. Gaslini 5, 16147 Genoa, Italy Laboratory of Oncology, G. Gaslini Institute, Largo G. Gaslini 5, 16147 Genoa, Italy
a r t i c l e
i n f o
Article history: Received 22 March 2011 Received in revised form 5 October 2011 Accepted 5 October 2011 Available online 12 October 2011 Keywords: Allergen-specific T-cell blasts Antigen Atopy Corticosteroid Peripheral blood mononuclear cells
a b s t r a c t Background: Ciclesonide, an inhaled corticosteroid with almost no affinity for the glucocorticoid receptor, is highly effective in downregulating in vitro pro-inflammatory activities of airway parenchymal cells when converted into the active metabolite desisobutyryl-ciclesonide. Objective: We evaluate whether ciclesonide could effectively downregulate also antigen- or allergeninduced activation of peripheral blood mononuclear cell and of allergen-specific T-cell blasts. Methods: Peripheral blood mononuclear cells were isolated from non atopic and atopic asthmatic children sensitized to Phleum pratense (PhlP5). Proliferation toward Candida albicans or PhlP5 in the presence of ciclesonide or desisobutyryl-ciclesonide (0.003–3.0 M) was evaluated as [3 H]thymidine incorporation. Modulation of PhlP5-specific T-cell blasts proliferation and PhlP5-induced interleukin 4 expression by ciclesonide and desisobutyryl-ciclesonide were measured. Results: Peripheral blood mononuclear cell proliferation to C. albicans was dose-dependently inhibited by 0.3–3.0 M ciclesonide and desisobutyryl-ciclesonide but inhibition by desisobutyryl-ciclesonide was higher. A significant proliferation to PhlP5 was observed only in cultures from atopic subjects: an effective downregulation was already detected at 0.03 M ciclesonide and 0.003 M desisobutyryl-ciclesonide (complete inhibition at 3 M ciclesonide and 0.03 M desisobutyryl-ciclesonide). 3 M ciclesonide and desisobutyryl-ciclesonide reduced the PhlP5-specific T-cell blast proliferation and interleukin 4producing cell proportion. Conclusions and clinical relevance: These in vitro data, obtained at concentrations similar to those reached in vivo at bronchial level, are in favor of an efficient inhibition of ciclesonide on the T-cell mediated response toward allergens. Additional studies are required to confirm these preliminary data on the reduced activity of the drug on allergen-specific T-cell blast activation that may have clinical relevance. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Inhaled corticosteroids (ICs) are the standard-of-care controller medications for asthma [1]. The clinical effectiveness of this class of drugs in asthma, as in many other inflammatory diseases, is related
Abbreviations: ICs, inhaled corticosteroids; PBMC, peripheral blood mononuclear cell; CIC, ciclesonide; des-CIC, desisobutyryl-ciclesonide; Ca, Candida albicans; PhlP, Phleum pratense; yrs, years; DMSO, dimethyl sulfoxide; FCS, foetal calf serum; MNC, mononuclear cells; APC, antigen presenting cells; PBS, phosphate buffered saline; mAb, monoclonal antibody; SE, standard error; GCR, glucocorticoid receptor; IL-2R, interleukin 2 receptor. ∗ Corresponding author. Tel.: +39 010563547; fax: +39 010383953. E-mail addresses: [email protected] (M. Silvestri), [email protected] (F. Morandi), [email protected] (V. Pistoia), [email protected] (I. Prigione), [email protected] (G.A. Rossi). 1 These authors contributed equally to this work. 0165-2478/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.imlet.2011.10.003
to their ability to block multiple pathways involved in the inflammatory processes that characterize this disease [2–4]. Treatment with ICs reduces the number of inflammatory cells in the bronchi, such as mast cells and eosinophils but also decreases the proportion of alveolar macrophages and of type 2 helper T-lymphocytes and their related proinflammatory cytokines in asthmatic airways [5,6]. These observations support the concept that a key role of ICs may be the downregulation in vivo of T-cell activation by allergen-presenting cells, inhibiting T-cell differentiation toward the Th2 phenotype, an early key event leading to stimulation of IgE production and, in general, to inflammation promotion [6,7]. This inhibitory activity has been extensively demonstrated in vitro testing a variety of ICs on allergen-stimulated peripheral blood mononuclear cell (PBMC) [8–11] and allergen-specific Th2 T-cell lines [12]. All these effects at cellular levels, but also the therapeutic benefits in patients with asthma, are usually achieved at relatively low ICs doses, with a high benefit-to-risk ratio also for long term treatments [13]. However, despite the improved safety profile of
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ICs, local and systemic side effects are still a concern, some (like oropharyngeal candidiasis) being common also with low-dose and short-term use [13,14]. Ciclesonide (CIC), a new-generation nonhalogenated IC, possesses many key physicochemical properties resulting in pharmacokinetic characteristics associated with high efficacy, favorable tolerability, and low risk for systemic side effects [14,15]. Administered as a parent compound with almost no binding affinity for the glucocorticoid receptor, CIC is rapidly converted in the airway mucosa into the active metabolite, desisobutyryl-ciclesonide (des-CIC) known to have a remarkable anti-inflammatory activity [14,15]. In in vitro cellular models CIC is highly effective in downregulating within 3 h the pro-inflammatory activities of airway epithelial cells and fibroblasts when converted by these cells into the active metabolite desisobutyryl (des)-CIC [16,17]. However it is not known whether: (a) conversion of CIC into des-CIC may occur also at human mononuclear cell level, resulting in an effective downregulation of allergeninduced T-lymphocyte activation and (b) the inhibitory activity of CIC could be higher for a Th2 response than for a Th1 response. A study was therefore designed to compare the ability of CIC and its active metabolite des-CIC to modulate the proliferative response to Candida albicans (Ca) (as a prototype of “Th1 response”) and to Phleum pratense (PhlP5) (as a prototype of “Th2 response”) of PBMCs isolated from non atopic subjects and atopic asthmatic individuals sensitized to PhlP. PhlP5-specific T-cell blasts were then generated and cell proliferation and IL-4 expression in response to PhlP5, in the presence of CIC or des-CIC, were studied. Ca was chosen as antigen because of the high frequency of oropharyngeal candidiasis as side effects of ICs treatment in asthma, while PhlP5, a major grass pollen allergen from P. pratense, because of its clinical relevance as inhalant allergen in our patient population.
2. Materials and methods 2.1. Study population Ten atopic Caucasian patients (mean age: 11.59 ± 0.84 yrs old, 2 females and 8 males) and 8 healthy non atopic individuals (mean age: 10.08 ± 1.05 yrs old, 2 females and 6 males), as controls, entered the study. Demographic and clinical characteristics of the atopic individuals are reported in Table 1. For atopic patients, the inclusion criteria were: (i) rhino-conjunctivitis with or without mild intermittent bronchial asthma and (ii) allergic sensitization to P. pratense (PhlP), demonstrated by skin prick test positivity to PhlP5 antigen and by the detection of allergen-specific serum IgE (see below); (iii) being asymptomatic at the time of the study. None of the patients was currently under anti-asthmatic treatment other than short-acting 2 -stimulants on an ‘as necessary’ basis, had received any drug known to interfere with T-cell mediated immune response in the last 3 months or any medication in the 24 h preced-
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ing the study entry. All subjects and parents/tutors were informed of the nature and the purpose of the study and gave written consent. Access to health records complied with the Italian legislation. Institutional Review Board approval was obtained for this study. 2.2. Diagnosis of allergic sensitization Sensitization to the most common aeroallergens was evaluated by skin-prick test (Lofarma, Milan, Italy) as previously described [8]. Allergen-specific serum IgE levels were determined by solid-phase, chemiluminescent immunometric assays (Immulite, Medical System; Genoa, Italy). Allergen-specific IgE levels ≥3.5 kU/L were considered consistent with sensitization to that allergen. 2.3. Isolation of peripheral blood mononuclear cells (PBMCs) and evaluation of allergen- and antigen-induced proliferation Isolation of PBMCs was performed as previously described [8]. Cell viability was determined by Trypan blue dye exclusion test [Euroclone, Siziano–Pavia, Italy) and found >90% in all the experimental conditions. PBMCs (105 cells/well) in 10% autologous heat-inactived serum were incubated in a 96-well plate for 6 days at 37 ◦ C in 5% CO2 with or without Ca (5 × 104 autoclaved bodies/ml), prepared as previously reported [18] or PhlP5 extract (20 g/ml) (gently provided by ALK Abellò, Spain). We used heat-inactived serum to prevent possible conversion of CIC into des-CIC by serum esterases. In preliminary sets of experiments, 20 g/ml PhlP5 was found to be the optimal dose promoting PBMC proliferation (not shown). To evaluate the effect of CIC and des-CIC on PBMC proliferation, cells were stimulated with PhlP5 or Ca in the presence of different concentrations (0.003–3 M) of the steroids. At day 6, tritiated [3 H]thymidine (0.5 Ci/well) (Amersham International, Bucks, UK) was added and the cells collected 18 h later by a cell harvester. The [3 H]thymidine incorporation in cell cultures, evaluated by a -counter (Wallac beta counter, PerkinElmer, Waltham, MA, USA), was used to assess PBMC proliferation and expressed as count per min (cpm). The vehicle used for corticosteroids dilution was dimethyl sulfoxide (DMSO, Euroclone), tested at a final concentration of 0.15%. At this concentration, DMSO did not affect cell viability, as assessed by Trypan blue dye exclusion test (not shown). 2.4. In vitro expansions of PhlP5-specific T-cell blasts and evaluation of allergen-induced proliferation To generate PhlP5-specific T-cell blasts, PBMCs from 5 sensitized individuals were stimulated with PhlP5 for 6 days as previously described. Low density T-cell blasts were then isolated using Percoll gradient (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) and cultured for an additional 4–6 days in complete medium in the presence of 10% foetal calf serum (FCS, Euroclone) and 100 U/ml rIL-2 (Chiron Corp., Emeryville, CA, USA).
Table 1 Demographic and clinical characteristics of the atopic population. Pt Id
Gender
Age (yrs)
Total serum IgE (ku/l)
PhlP5-specific serum IgE levels (ku/L)
C.S. M.F. B.M. F.S. S.L. V.F. O.N. C.L.L. G.M. S.F.
F M M M M M F M M M
11.9 14.0 12.5 14.0 15.5 9.4 10.4 10.1 11.8 6.3
492 285 649 187 288 >2000 1492 481 100 128
23.2 3.6 >100 3.7 8.3 8.5 6.3 >100 >100 5.5
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Fig. 1. Proliferative response to Candida albicans (Ca) by peripheral blood mononuclear cells (PBMCs) isolated from non atopic and atopic subjects and effect of different concentrations of ciclesonide (CIC) and des-CIC on this response. (A) Cell proliferation, evaluated as tritiated thymidine ([3 H]TdR) incorporation and expressed as count per minute (cpm) is shown on the ordinate, whereas the different experimental conditions [without stimulus (medium) and with Ca] are reported on the abscissa. (B and C) The percentage of inhibition for non-atopic (B) and atopic (C) individuals is shown on the ordinate whereas the different concentrations (0.003–3 M) of the drugs are reported on the abscissa. The data are expressed as mean ± standard error of the mean. ¶¶¶: p < 0.001, vs. unstimulated cells; *: p < 0.05, **: p < 0.01, vs. cells grown in the absence of the drug; §: p < 0.05, CIC vs. des-CIC.
To test allergen-specificity of T-cell blasts, cells were collected, washed three times and resuspended in complete medium plus 10% AB serum and cultured (3 × 104 cells/well) with PhlP5 (20 g/ml) in the presence of autologous irradiated (45 Gy) mononuclear cells (MNCs), as antigen presenting cells (APCs) (8 × 104 cells/well) in 96well flat-bottom plates in a total volume of 0.2 ml for 48–72 h [19]. To evaluate the effects of CIC and des-CIC on cell proliferation, blasts and autologous irradiated MNCs were also cultured in the presence of PhlP5 and different concentrations of the two molecules. The incubation conditions with CIC, des-CIC as well as the addition of the APC was performed as described above. Cells were pulsed with 0.5 Ci/well [3 H]thymidine for the last 18 h of culture and harvested. The cell-associated radioactivity was determined by liquid scintillation counting.
2.5. Intracellular cytokine staining on PhlP5-specific T-cell blasts by flow cytometry For intracellular cytokine staining, allergen-specific T-cell blasts (106 cells/ml) were collected after 48–72 h of culture with PhlP5 and in the presence or not of CIC and des-CIC different concentrations, as described in detail in the previous section. Briefly, cells were incubated for 4 h in brefeldin-A (5 g/ml; Sigma–Aldrich, St. Louis, MO, USA). After washing in phosphate buffered saline (PBS) with 1% FCS (staining buffer), cells were stained with anti CD4-PE Cy5.5 monoclonal antibody (mAb) (BD Biosciences, Mountain View, CA, USA) for 30 min at 4 ◦ C in the dark and then washed again in staining buffer and fixed in Cytofix/CytopermTM solution (BD Biosciences) for 20 min at 4 ◦ C in the dark. Subsequently cells were washed twice with Perm/Wash buffer (BD Biosciences) and stained with FITC-conjugated anti-human IFN-␥ mAb and PE-conjugated
anti-human IL-4 mAb (Caltag Laboratories, Burlingame, CA, USA) and for 30 min at 4 ◦ C in the dark. After washing in staining buffer, cells were analyzed by flow cytometry (FACScan, BD Biosciences) gating on the CD4+ T-cell subsets. PE- and FITC-conjugated mouse Ig control mAb (Caltag Laboratories) were used as negative controls in all these experiments. CellQuest software (BD Biosciences) was used for analyses. The results of flow cytometry experiments were expressed as percentage positive cells and as mean relative fluorescence intensity (MRFI). MRFI represents the ratio between the mean fluorescence intensity of cells stained with the selected mAb and the mean fluorescence intensity of cells stained with the isotype-matched mouse Ig. 2.6. Data analysis and statistical evaluation Data were reported as mean and standard error (SE). Statistical analysis (GraphPad Prism Software Inc, San Diego, CA, USA) of results was carried out by Student’s t-test or Mann Whitney U-test as appropriate. Multiple comparisons were made by means of the Analysis of Variance test; post-hoc comparisons after the ANOVA were performed applying the Dunnett’s Multiple Comparison Test. All tests used were two sided and a p values less than 0.05 were considered statistically significant. 3. Results 3.1. Modulation of Ca-induced PBMC proliferation in non atopic and in atopic subjects by CIC and des-CIC In PBMC cultures from atopic and non atopic subjects, a similar 40-fold significant increase in [3 H]thymidine incorporation was
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Fig. 2. Proliferative response to Phleum pratense (PhlP5) by peripheral blood mononuclear cells (PBMCs) isolated from atopic and non atopic subjects and effect of different concentrations of ciclesonide (CIC) and des-CIC on this response. (A) Cell proliferation, evaluated as tritiated thymidine ([3 H]TdR) incorporation and expressed as count per minute (cpm) is shown on the ordinate, whereas the different experimental conditions [without stimulus (medium) and with PhlP5] are reported on the abscissa. (B) The percentage of inhibition is shown on the ordinate whereas the different concentrations (0.003–3 M) of the drugs are reported on the abscissa. The data are expressed as mean ± standard error of the mean. ¶¶¶: p < 0.001, vs. unstimulated cells; ***: p < 0.001, vs. cells grown in the absence of the drug; §: p < 0.05, §§§: p < 0.001, CIC vs. des-CIC.
observed in the presence of Ca, as compared with unstimulated cells (Fig. 1A). A dose-dependent inhibition of Ca-induced PBMC proliferation was observed in the presence of CIC and des-CIC, similar in atopic and non atopic subjects, but significantly higher for des-CIC than for CIC at 0.3 M and 3 M (Fig. 1B and C). Also the inhibitory effect induced by lowest concentration of des-Cic in atopic subjects was not statistically different from that observed in cultures by non atopic subjects (p = 0.17).
des-CIC (p < 0.001, each comparison), with a nearly complete inhibition observed at 3 M CIC and at 0.03 M des-CIC (Fig. 2B). The inhibitory activity toward PhlP5-induced PBMC proliferation was higher for des-CIC than for CIC at 0.003 M (p < 0.05), 0.03 M (p < 0.001) and 0.3 M (p < 0.05).
3.2. Modulation of PhlP5-induced PBMC proliferation by CIC and des-CIC
In allergen-specific T-cell blast cultures, a significant 5-fold increase in [3 H]thymidine incorporation was observed in the presence of PhlP5 (Fig. 3A). Proliferation was similarly downregulated by CIC and des-CIC, but the inhibitory effect was significant only at 3.0 M, at the highest concentration tested amounting to about 37% and 54% reduction (Fig. 3B). Stimulation with PhlP5 of the T-cell blasts also significantly increased the proportion of IL-4-producing cells (p < 0.05) but not that of IFN-␥-producing cells (Fig. 4A). The increased proportion of IL-4-producing cells was partially reduced to comparable extent
As expected, a significant 8-fold increase in [3 H]thymidine incorporation was observed in the presence of PhlP5 in PBMCs cultures from all atopic patients (p < 0.001), but not in PBMC cultures from non atopic subjects not sensitized to the allergen (Fig. 2A). In PBMCs cultures from atopic patients, both CIC and des-CIC induced a dose-dependent downregulation of PhlP5-induced proliferation. The effect was already significant at 0.03 M CIC and at 0.003 M
3.3. Modulation of PhlP5-induced T-cell blast proliferation and cytokine production by CIC and des-CIC
Fig. 3. Proliferative response to Phleum pratense (PhlP5) by PhlP5-specific T-cell blast proliferation and inhibitory effect of different concentrations of ciclesonide (CIC) and des-CIC. (A) T-cell blast proliferation, evaluated as tritiated thymidine ([3 H]TdR) incorporation and expressed as count per minute (cpm) is shown on the ordinate, whereas the different experimental conditions [without stimulus (medium) and with PhlP5] are reported on the abscissa. (B) The percentage of inhibition is shown on the ordinate whereas the different concentrations (0.003–3 M) of the drugs are reported on the abscissa. The data are expressed as mean ± standard error of the mean. ¶¶: p < 0.01, vs. T-cell blasts not re-stimulated; *: p < 0.05, vs. cells grown in the absence of the drug.
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Fig. 4. Proportion of interleukin (IL)-4-producing and interferon (IFN)-␥-producing T-cell blasts following re-stimulation with Phleum pratense (PhlP5) in the presence of ciclesonide (CIC) and des-CIC. The proportion of IL-4-producing (panels A and B) and interferon (IFN)-␥-producing (panels A and C) T-cell blasts is shown on the ordinate, whereas the different experimental conditions [without re-stimulation (Ctr), with re-stimulation with PhlP5 (PhlP5) and in the presence of CIC or des-CIC 3 M] are reported on the abscissa. The percentage of positive cells was calculated on the basis of fluorescence intensity obtained with an irrelevant isotype-matched antibody control. The data are expressed as mean ± standard error of the mean. ¶: p < 0.05, vs. T-cell blasts not re-stimulated; *: p < 0.05, vs. cells grown in the absence of the drug.
(40–50%) by 3 M CIC and 3 M des-CIC (Fig. 4B and C). No significant modification in proportion of IFN-␥-producing cells was observed (Fig. 4A–C). Representative flow-cytometry histograms of IL-4 and IFN-␥ production by PhlP5-specific T-cell blasts and of the inhibitory effect of CIC or des-CIC (3 M) are reported in Fig. 5A and B. 4. Discussion Using PBMCs from non atopic and atopic subjects, we demonstrated that CIC and des-CIC, are effective in downregulating the proliferative response to C. albicans (an antigen stimulating predominantly a “Th1 response”) and to P. pratense 5 (an allergen inducing a “Th2 response”). CIC is a “pro-drug” with almost no affinity for the glucocorticoid receptor, and consequently was added to cell cultures as an inactive compound. Therefore the results here reported demonstrate that, as already shown for airway epithelial cells [16,20] and lung fibroblasts [17], also blood mononuclear cells are able to convert the parent compound in the active principle desCIC, to exert anti-inflammatory functions on the same cells. This is a relevant information considering both the key role that mononuclear cells, i.e. antigen-presenting cells and T-lymphocytes, have in asthma pathogenesis and the pharmacokinetic and pharmacodynamic characteristic of CIC. Indeed, carboxylesterases, one of the enzymes involved in isobutyrate cleaving at the CIC C21 position [21], appear to be abundant in human monocytes and to be present in T-lymphocytes [22]. It is very unlike that over the long period of incubation (6 days at 37 ◦ C) CIC could have been converted into desCIC by esterases in the serum. Indeed: (a) carboxylesterase is rare in human blood [21] and (b) cells were cultured in heat-inactivated serum. Des-CIC was more efficient than CIC in inhibiting PBMC proliferative response but only at the highest concentrations, when the cells were stimulated with Ca antigen, and at mid-to-low
concentrations, when the cells were stimulated with PhlP5 allergen. These results on cell proliferation that indicate a partial conversion of CIC to des-CIC by lymphocytes are in agreement with findings recently reported by Stoeck et al. [23]. Using highly purified human CD4 T-cells stimulated with anti CD3/anti CD28, or PBMCs stimulated with ConA, they obtained a similar efficacy on the inhibition of cell proliferation but at lower des-CIC than CIC concentrations. We observed a higher inhibitory effect on PhlP5-stimulated than on Ca-stimulated PBMCs, being remarkable (50–60%) for PBMC stimulated with Ca and nearly complete for PBMC stimulated with PhlP5 when the highest concentration of CIC and des-CIC was used. One may hypothesize therefore, that the Th2 -driven response (PhlP5) is more sensitive to inhibitory effects of the glucocorticoid than that driven by Th1 cells (Ca). One explanation for these results could be related to differences between antigen (Ca)- and allergen (PhlP5)-specific T-cells in glucocorticoid receptor sensitivity and/or in the balance between glucocorticoid receptor (GCR) ␣ and  expression, respectively the active and the dominant negative GCR isoforms [24]. Another observation was that Ca-induced proliferation of Tcells from atopic subjects was more efficaciously suppressed by the active metabolite compared to ciclesonide. Although carboxylesterases appear to be equally present in T-lymphocyte subsets [22], we may speculate that under different stimulations, they may show different rates of enzymatic activities. Indeed, even though they are ubiquitous in human tissues [25], their relative concentration may be dissimilar and contribute differently to the conversion to des-CIC in the different organs [21,26,27]. We also found a reduced activity of CIC on the allergen-induced activation of T-cell blasts, as compared with PBMCs. This may be due to a different sensitivity to the drug, related to the presence of IL-2 in the medium used to obtain T-cell blasts in vitro [28]. In addition, it has been suggested that high IL-2 concentrations, when related to endogenous overproduction, may be associated
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Fig. 5. Representative flow-cytometry histograms of IL-4 (panel A) and IFN-␥ (panel B) production by PhlP5-specific T-cell blasts and of the inhibitory effect of CIC or des-CIC (3 M). x-axis is SSC and y-axis is specific fluorescence intensity (PE or FITC).
with glucocorticoid resistance [29]. Finally, Haczku et al. [28] also demonstrated that dexamethasone was ineffective in inhibiting lymphoblasts i.e. cells already committed to proliferation, suggesting that this drug may inhibit only early events in the T-lymphocyte proliferative cycle but not the proliferative events subsequent to the synthesis of IL-2. The exact mechanism by which IL-2 promotes steroid resistance is not entirely clear but several possible pathways have been described. For example Goleva et al. [30] and Irusen et al. [31] showed that treatment of PBMCs with a combination of IL2 and IL-4 abrogated dexamethasone-induced GCR-alpha nuclear translocation by activating p38 MAPK that may impair GCR function. Further, Ito and Mercado [32] found that hyperacetylation of the glucocorticoid receptor may also account for steroid resistance following IL-2 and IL-4 exposure of PBMC. The role of IL-2 in response to steroid treatment is also supported by the demonstration that the blockade of IL-2 signaling with an anti-CD25 mAb increased steroid sensitivity in cells from steroid resistant patients [33]. In addition, it has been shown that during the pollen season there was an increase in T lymphocyte activation in the BAL fluid as identified by increased expression of interleukin 2 receptor (IL-2R) as compared to that found out of the pollen season (before the pollen season) [34]. Therefore, the addition of IL-2 to favor blast growth in vitro may, at least in part, mimic what happens in vivo during the pollen season. Taken together these in vitro results are in favor of the effective conversion of CIC into its active metabolite by human PBMCs and prove the ability of this pro-drug to downregulate the immune response toward allergens at concentrations similar to those
obtained in vivo at bronchial level [35]. In the present study we did not evaluate the concentrations of ciclesonide and des-ciclesonide in cell culture supernatants. However, we previously demonstrated both in human bronchial epithelial cell and in human lung fibroblast cultures that after 24 h incubation with the drug, CIC was below the levels of quantification in all the samples whereas only its active compound des-ciclesonide was present, confirming an effective and rapid biotransformation of CIC into the active metabolite. Apparently, the glucocorticoid revealed somewhat more effective to reduce proliferation to PhlP5, as a Th2 response compared to Ca, as a Th1 response in PBMC from atopic subjects. These in vitro results may at least partially explain the low susceptibility to oral candidiasis observed in patients treated with CIC [14]. These preliminary data on the reduced activity of the drug on allergen-specific T-cell blast activation need to be confirmed by additional studies to establish a possible clinical relevance. Funding This study was supported by Grant “Ricerca Finalizzata” from Italian Ministry of Health, Rome, Italy and by a grant from Nycomed S.p.A., Italy. Competing interest MS, FM, VP and IP have no declared conflict of interest. GAR has been reimbursed by ALTANA PHARMA (Nycomed) for conference attendance and served as a consultant to ALTANAPHARMA
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