An in vitro comparison of commonly
`used topical glucocorticoid preparations
`
`Cristiana Stellato, MD, PhD,a Jun Atsuta, MD, PhD,b Carol A. Bickel, MS,a and
`Robert P. Schleimer, PhDa Baltimore, Md, and Tsu Mie, Japan
`
`Background: Glucocorticoids (GC) are potent inhibitors of
`peripheral blood eosinophil, basophil, and airway epithelial
`cell function.
`Objectives: We compared in vitro the inhibitory activity of
`synthetic GC used for topical treatment in asthma and rhinitis
`on basophil histamine release (HR), eosinophil viability, and
`expression of vascular cell adhesion molecule-1 (VCAM-1) in
`the human bronchial epithelial cell line BEAS-2B.
`Methods: Cells were treated for 24 hours with increasing con-
`centrations (range 10–13 to 10–6 mol/L) of fluticasone propi-
`onate (FP), mometasone furoate (MF), budesonide (BUD),
`beclomethasone dipropionate (BDP), triamcinolone acetonide
`(TAA), hydrocortisone (HC), or dimethyl sulfoxide diluent
`before challenge. HR was measured by a fluorometric assay,
`viability of purified eosinophils was assessed by erythrosin B
`dye exclusion, and expression of VCAM-1 was measured by
`flow cytometry.
`Results: GC induced a concentration-dependent inhibition of
`anti-IgE-induced HR. Maximum inhibition ranged from
`59.7% to 81%, with a rank order of GC potency of FP > MF >
`BUD > BDP ≅ TAA >> HC. Three-day treatment of eosinophils
`with GC concentration-dependently inhibited IL-5–induced
`eosinophil viability, with a rank of potency almost identical to
`that observed with basophil HR. The rank order of potency of
`GC for inhibition of the expression of VCAM-1 in BEAS-2B
`cells was MF ≅ FP >> BUD > TAA > HC ≅ BDP. Inhibitory
`concentration of 50% values revealed that epithelial cells were
`the most sensitive and eosinophils were the least sensitive.
`Conclusions: These data, combined with information on phar-
`macodynamics of these drugs in vivo, may be useful in estimat-
`ing GC local anti-inflammatory effects. (J Allergy Clin
`Immunol 1999;104:623-9.)
`
`Key words: Glucocorticoids, basophil, eosinophil, epithelial cells,
`allergy
`
`Glucocorticoids (GC) have been the most potent and
`effective drugs for the treatment of severe asthma since
`their discovery and synthesis in the late 1940s. Over the
`last 2 decades the use of potent inhaled GC preparations,
`which are metabolized rapidly once absorbed, has
`expanded widely. For many physicians treating asthma,
`
`From the Johns Hopkins Asthma and Allergy Center, Baltimore, Md,a and the
`Mie National Hospital, Tsu Mie, Japan.b
`Supported by grants from the National Institutes of Health and by gifts from
`Astra, Glaxo, and Schering-Plough.
`Received for publication Dec 21, 1998; revised June 7, 1999; accepted for
`publication June 7, 1999.
`Reprint requests: Cristiana Stellato, MD, PhD, Johns Hopkins Asthma and
`Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224-
`6801.
`Copyright © 1999 by Mosby, Inc.
`0091-6749/99 $8.00 + 0 1/1/100691
`
`Abbreviations used
`BDP: Beclomethasone dipropionate
`17-BMP: Beclomethasone-17-monopropionate
`BUD: Budesonide
`DMEM: Dulbecco’s modified Eagle’s medium
`DMSO: Dimethyl sulfoxide
`β-E: β-Estradiol
`FP: Fluticasone propionate
`GC: Glucocorticoid
`HC: Hydrocortisone
`HR: Histamine release
`IC50: Concentration at which each GC produced 50%
`inhibition
`MF: Mometasone furoate
`PIPES: Piperazine- N,N´-bis-(2-ethanesulfonic acid)
`TAA: Triamcinolone acetonide
`VCAM-1: Vascular cell adhesion molecule-1
`
`inhaled GCs have now become a drug of first choice for
`even mild to moderate asthma because of the relative
`safety and clear efficacy of these compounds. Although a
`considerable amount is known about the mechanism of
`GC actions as anti-inflammatory drugs in general and for
`the treatment of asthma, relatively few studies have been
`performed to compare the available inhaled GC prepara-
`tions that are being widely used.1,2 It is not a certainty
`that all GC will behave similarly, either in vivo or in dif-
`ferent in vitro cellular systems. Variation in lipid solubil-
`ity, receptor affinity, binding to lung tissue at sites other
`than the glucocorticoid receptor, and other factors can
`dramatically influence the potency and duration of action
`of GC in the lungs. We have recently shown that human
`lung tissue is active in metabolizing the endogenous GC
`hydrocortisone (HC) because of the presence of the
`enzyme 11β-hydroxysteroid dehydrogenase in epithelial
`cells and perhaps other cells in the lung.3,4 Thus local
`metabolism may also influence the effectiveness of
`inhaled or endogenous GC in the airways. The purpose
`of this study was to compare commonly used inhaled
`GCs for efficacy in cellular assays with several important
`targets of GC action in the airway. The goal was to estab-
`lish a rank order of potency of the following GCs in
`assays of steroid inhibition of basophil histamine release,
`eosinophil survival, and vascular cell adhesion molecule-
`1 (VCAM-1) expression in airway epithelial cells:
`mometasone furoate (MF), fluticasone propionate (FP),
`beclomethasone dipropionate
`(BDP),
`budesonide
`(BUD), triamcinolone acetonide (TAA), and HC. We set
`out to determine whether there are differences in their
`rank order of potency and concentration at which each
`
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`
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`SEPTEMBER 1999
`
`GC produced 50% inhibition (IC50) in these systems. We
`have observed that basophils are slightly more sensitive
`to GCs than eosinophils in the assays used and that air-
`way epithelial cells are particularly sensitive to these
`inhaled GCs. The rank order of potency was relatively
`similar with all 3 assays.
`
`MATERIAL AND METHODS
`The following materials were purchased: piperazine- N,N´-bis-
`(2-ethanesulfonic acid) (PIPES), D-glucose, BSA, heparin, dimethyl
`sulfoxide (DMSO), erythrosin B (Sigma, St Louis, Mo), Percoll
`(Pharmacia, Uppsala, Sweden), RPMI containing 25 mmol/L
`HEPES, penicillin-streptomycin solution, FBS, L-glutamine
`(Gibco, Grand Island, NY), rhu-IL-5 (R&D Systems, Minneapolis,
`Minn), goat antihuman IgE (Kirkegaard and Perry Laboratories,
`Gaithersburg, Md), alcian blue dye (Fisher Scientific, Pittsburgh,
`Pa), Dynabeads (Dynal, Great Neck, NY), CD16 mAB
`(Immunotech, Westbrook, Me), Dulbecco’s modified Eagle’s medi-
`um (DMEM), HAM’s F-12 medium, Ca++- and Mg++-free HBSS,
`Versene (Ca++- and Mg++-free HBSS with 0.02% EDTA), trace ele-
`ments, phosphoethanolamine-ethanolamine, retinoic acid (Bioflu-
`ids, Rockville, Md), insulin, HC, epidermal growth factor, and
`endothelial cell growth supplement (Collaborative Research, Bed-
`ford, Mass), cholera toxin, tri-iodothyronine (Sigma); heat-inacti-
`vated FCS, penicillin-streptomycin solution (Life Technologies,
`Gaithersburg, Md), recombinant human TNF-α (R&D Systems).
`The following GCs were provided or purchased from the indi-
`cated sources: FP, BDP, and beclomethasone-17-monopropionate
`(17-BMP) (Glaxo); MF and TAA (Schering-Plough); BUD (Astra
`Draco); and HC and β-estradiol (β-E) (Sigma). Stock solutions of
`the GCs were prepared in DMSO at 0.1 mol/L concentration and
`stored at –20°C. Buffers used for basophil histamine release were
`PAG (PIPES buffer [25 mmol/L] containing 110 mmol/L sodium
`chloride, 5 mmol/L potassium chloride, 0.003% BSA, 0.1% D-glu-
`cose), PAGCM (PAG containing calcium chloride 1 mmol/L and
`magnesium chloride 1 mmol/L).
`
`Preparation, culture, and histamine release
`from basophil-enriched leukocyte
`suspensions
`Venous blood was obtained from adult donors who had given
`informed consent and basophils were isolated with double-step Per-
`coll gradients as previously described.5 Briefly, whole blood was
`anticoagulated with 0.1 mol/L EDTA and diluted with an equal vol-
`ume of PIPES buffer, and the density was adjusted to 1.065 g/mL
`with 100% isotonic Percoll. The blood-Percoll mixture was layered
`over a cushion of Percoll of density 1.079 g/cm3 and then cen-
`trifuged at 400g for 15 minutes at 22°C. The cells were collected
`from the supernatant and from the plasma-Percoll interface, washed
`once in EDTA–sodium chloride solution to remove the Percoll and
`washed twice in PAG-EDTA, and the number and percentage of
`basophils in each fraction was determined. The mean percentage of
`basophils found in these fractions ranged from 5% to 12%, as deter-
`mined by cell counts in Spiers-Levy chambers with use of Alcian
`blue.6 Cells were then washed and cultured in replicates of 1 mL in
`24-well plates (Costar, Cambridge, Mass) at cell densities of 1 to 10
`× 106 cells, depending on basophil purity, as described.7 Briefly,
`cells resuspended in RPMI 1640 media supplemented with 1% glu-
`tamine, 1% penicillin-streptomycin, and 10% FBS were cultured in
`various concentrations of GC or DMSO diluent for 24 hours at
`37°C. Previous studies have shown that GCs have no effect on
`basophil viability during a 24-hour culture (not shown). Cells were
`then harvested, washed, and challenged with 0.1 µg/mL of anti-IgE
`
`(45 minutes, 37°C). At the end of the incubation, cell-free super-
`natants were assayed for histamine release with the automated flu-
`orometric technique of Siraganian.8 Basophil histamine release was
`calculated as the percent of total cellular histamine after subtraction
`of spontaneous release (5%-8%).
`
`Preparation, culture, and viability
`assessment of peripheral blood eosinophils
`Human granulocytes were isolated from EDTA-anticoagulated
`venous blood of normal donors or patients with asymptomatic aller-
`gic rhinitis or asthma by Percoll (1.090 g/mL) gradient centrifuga-
`tion at room temperature. After centrifugation, all procedures were
`carried out at 4°C to minimize cell activation. Red blood cells were
`removed by hypotonic lysis followed by removal of CD16-positive
`cells (neutrophils) with an immunomagnetic bead technique.9
`Eosinophil purity (on the basis of examination of Diff-Quik–stained
`cytocentrifugation preparations) was 99% ± 1% and viability (on
`the basis of erythrosin B dye exclusion) was 99% ± 1%. Cells were
`resuspended in RPMI 1640 medium supplemented with 1% gluta-
`mine, 1% penicillin-streptomycin, 10% FBS, and IL-5 (0.01
`ng/mL) and placed (in replicates of 100 µL) in 96-well flat-bottom
`plates (Costar) at a cell density of 2.5 × 105 eosinophils per milli-
`liter in the presence of various concentrations of GC, DMSO dilu-
`ent, or medium alone for 3 days at 37°C. After the incubation peri-
`od 70 µL of supernatant was carefully removed, without disturbing
`the eosinophils settled at the bottom of the wells. Erythrosin B (2-3
`µL) was added to wells with gentle pipette mixing, and staining was
`allowed for precisely 5 minutes. A cell sample was then counted at
`×40 magnification. Viability of cells was expressed as percent of
`total cells counted.
`
`Culture and challenge of BEAS-2B epithelial
`cells
`BEAS-2B cells were a generous gift from Dr Curtis Harris
`(National Cancer Institute, Bethesda, Md). This cell line was
`derived from human bronchial epithelium transformed by an aden-
`ovirus 12-SV40 hybrid virus.10 These cells retain electron micro-
`scopic features of epithelial cells and show positive staining with
`antibodies to cytokeratin but do not form tight junctions11 (data not
`shown). BEAS-2B cells were cultured in 25-cm2 tissue culture
`flasks and maintained in F12/10× medium consisting of Ham’s F12
`nutrient medium containing penicillin (100 U/mL) and strepto-
`mycin (100 U/mL) and supplemented with insulin (5 µg/mL), HC
`(10–7 mol/L), tri-iodothyronine (3.1 × 10–9 mol/L), cholera toxin
`(10 ng/mL), endothelial cell growth supplement (3.75 µg/mL), epi-
`dermal growth factor (12.5 ng/mL), phosphoethanolamine-
`ethanolamine (5 × 10–6 mol/L), trace elements (1×), and retinoic
`acid (0.1 µg/mL). Cells were used between passages 35 and 47 and
`were plated on 6-well cluster plates (Costar) and cultured for at least
`48 hours in F12/DMEM medium (which lacks added HC) contain-
`ing 5% heat-inactivated FCS, penicillin (100 U/mL), and strepto-
`mycin (100 U/mL) before harvesting.
`Flow cytometric analysis
`In the experiments assessing the inhibitory effects of glucocorti-
`coids on VCAM-1 expression on BEAS-2B cells, cells were prein-
`cubated with increasing concentrations of GCs or an equivalent
`concentration of DMSO diluent for 24 hours and then incubated
`with TNF-α (10 ng/mL) for 24 hours. These methods are described
`in detail elsewhere.12,13 Cells were then washed 3 times with Ca++-
`and Mg++-free HBSS and treated for 10 minutes with Versene (Ca++
`and Mg++-free HBSS containing 0.02% EDTA) without trypsin and
`then removed from plates by repeated pipetting. For each analysis 1
`× 106 cells were incubated in 30 µL of PBS/0.2% BSA containing
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`
`Stellato et al 625
`
`TABLE I. Concentration of glucocorticoids required for
`inhibition of basophil HR, eosinophil survival, and
`epithelial VCAM-1 expression
`Eosinophil
`survival
`(mol/L)
`2.5 × 10–10†
`7 × 10–10
`5.9 × 10–9
`3 × 10–8
`1 × 10–8
`2.5 × 10–6
`
`GC
`
`FP
`MF
`BUD
`BDP
`TAA
`HC
`
`Basophil HR
`(mol/L)
`7 × 10–11*
`2 × 10–10
`5.9 × 10–10
`1 × 10–9
`3 × 10–9
`1.5 × 10–7
`
`BEAS-2B VCAM-1
`expression
`(mol/L)
`4.6 × 10–12‡
`1.8 × 10–12
`3 × 10–10
`3 × 10–8
`6.3 × 10–10
`1.1 × 10–8
`
`*IC50 of HR.
`†Concentration for half maximal reduction in eosinophil survival.
`‡IC50 of TNF-α–induced VCAM-1 expression.
`
`saturating concentrations of each mAb and 4 mg/mL of human IgG
`(to reduce nonspecific binding) on ice for 30 minutes, as previous-
`ly described.14,15 mAb for detection of intercellular adhesion mole-
`cule-1 was RR1 (AMAC, Westbrook, Me) and for VCAM-1 was
`BBIG-V1 (R&D Systems). The cells were washed, resuspended in
`saturating amounts of fluorescein-conjugated goat F(ab´)2 anti-
`mouse IgG antibody (Bio Source, Camarillo, Calif) for another 30
`minutes, and then washed again. Negative staining with propidium
`iodide (2 µg/mL) and a combination of scatter characteristics were
`used to identify a uniform population of viable cells. Fluorescence
`was measured with an EPICS Profile II flow cytometer (Coulter
`Electronics, Hialeah, Fla) and was expressed as percent of control
`IgG mean fluorescence intensity by comparison to control staining
`with an irrelevant isotype-matched mouse mAb. For each sample, at
`least 5000 events were collected.
`
`Statistical analysis
`Analysis of data was performed with use of Statview II software
`(Abacus Concepts, Berkeley, Calif) on a Macintosh IIsi computer.
`Data are expressed as mean ± SEM. Statistical analysis between
`groups was performed with the ANOVA test with a post-hoc analy-
`sis (Fisher PLSD test). A P value <.05 was considered significant.
`
`RESULTS
`Glucocorticoids are first-line drugs in the therapy of
`airway allergic diseases such as asthma and rhinitis.2 The
`inflammatory cells infiltrating the site of allergic reac-
`tions from peripheral blood, such as eosinophils and
`basophils, are all targets of the anti-inflammatory activi-
`ty of GCs.16 These compounds are effective inhibitors of
`IgE-mediated mediator release by basophils.7,16 It has
`been shown that several GCs used by oral administration
`exert their inhibitory effect on basophils with a rank of
`potency that parallels that found in vivo.14 GCs also exert
`profound inhibitory effects on the recruitment, activa-
`tion, and survival of eosinophils.17 In the current in vitro
`study we compared the effect of several synthetic GCs
`currently used for topical treatment in asthma and rhini-
`tis on basophil mediator release, eosinophil viability, and
`epithelial cell activation. Among the resident cells in the
`airways, epithelial cells are a particularly important tar-
`get of GC action.18
`Fig 1 shows the results of a series of experiments (n =
`2-6) in which basophil-enriched cell suspensions were
`
`FIG 1. Inhibition of basophil histamine release with GC. Basophils
`were cultured for 24 hours in presence of indicated GC or DMSO
`diluent and subsequently challenged with 0.1 µg/mL anti-IgE.
`Data shown are mean ± SEM inhibition of histamine release (HR)
`from 2 to 6 experiments; percent HR induced by anti-IgE in
`DMSO-treated cells was 36.1% ± 12.2%.
`
`incubated for 24 hours in the presence of increasing con-
`centrations (10–11 to 10–6 mol/L) of the indicated GC or
`DMSO diluent before challenge with 0.1 µg/mL anti-IgE.
`Treatment with GC induced a concentration-dependent
`inhibition of anti-IgE-induced histamine release, with
`maximum inhibition ranging from 59.7%, achieved by HC
`at 10–6 mol/L, to 81%, induced by BDP at 10–7 mol/L.
`Because a maximum effect was not reached in every
`experiment, for each GC we arbitrarily chose a defined net
`effect—50% inhibition of histamine release—calculated
`on the mean ± SEM of all experiments to express the con-
`centration at which each GC produced IC50. These values
`are indicated in Table I, showing a rank order of potency
`of FP > MF > BUD > BDP ≅ TAA >> HC.
`We next compared the ability of the various GCs to
`affect IL-5–sustained eosinophil survival by assessing
`eosinophil viability by erythrosin B dye exclusion after
`culture in the presence of GC. In a series of preliminary
`experiments, to identify a concentration of IL-5 able to
`prolong eosinophil survival without overriding the
`inhibitory activity of GC, we cultured eosinophils for 3
`days with a broad spectrum of IL-5 concentrations
`(0.002-5 ng/mL) in medium only, DMSO diluent, or 10–7
`mol/L BUD. A suboptimal concentration of IL-5 (0.01
`ng/mL) was chosen, at which both eosinophil viability
`and the inhibitory effect of the steroid were optimal (Fig
`2). The presence of DMSO did not affect eosinophil via-
`bility (Fig 2). Subsequently, we performed a series of
`experiments assessing the effect of 3 days of treatment
`with increasing concentrations (10–13 to 10–6 mol/L) of
`the different GC on eosinophil viability (n = 6). As was
`the case for basophil HR, a concentration-dependent
`inhibition of eosinophil survival was observed (Fig 3).
`Because of the relationship between IL-5 and steroid, the
`drugs did not reduce viability by 100%. Because the
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`626 Stellato et al
`
`J ALLERGY CLIN IMMUNOL
`SEPTEMBER 1999
`
`FIG 2. Titration of IL-5 concentration used to prolong eosinophil sur-
`vival (representative of n = 7 for IL-5 titration). Eosinophils were
`incubated with the indicated concentrations of IL-5 for 3 days in the
`presence of medium alone, DMSO diluent, or BUD (10–7 mol/L).
`Eosinophil viability was assessed by erythrosin B dye exclusion.
`Viability of cells is expressed as percent of total cells counted.
`
`FIG 4. Correlation among potencies of GCs in inhibiting basophil
`HR and eosinophil viability.
`
`FIG 3. Inhibition of eosinophil survival by GC. Eosinophils were
`cultured for 3 days in medium containing 0.01 ng/mL rhu-IL-5 in
`presence of indicated concentrations of GC, DMSO diluent, or
`medium alone. Viability in DMSO-treated cells was 62% ± 10%.
`Results shown are mean ± SEM of n = 6.
`
`steroids had a maximal effect of approximately 50%
`reduction of viability, a group maximum inhibition was
`determined and the concentration of each steroid that
`inhibited survival by half of this value was interpolated.
`The rank order of potency among the GCs in this assay
`differed slightly from the basophil assay, notably for tri-
`amcinolone and beclomethasone. The rank order of
`potency observed was FP > MF > BUD > TAA ≅ BDP >
`HC, as shown by the IC50 values displayed in Table I.
`
`FIG 5. Inhibition of VCAM-1 expression on BEAS-2B epithelial
`cells. BEAS-2B cells were preincubated with various concentra-
`tions of GC for 24 hours before stimulation with 10 ng/mL of TNF-
`α for another 24 hours. Expression of VCAM-1 was measured by
`flow cytometry. Results shown are means ± SEM of n = 3.
`(Reprinted from Atsuta J, Plitt J, Bochner BS, Schleimer RP. Inhi-
`bition of VCAM-1 expression in human bronchial epithelial cells
`by glucocorticoids. Am J Respir Cell Mol Biol 1999;20:643-50.
`Used with permission.)
`
`Nevertheless, for the series of drugs, a positive correla-
`tion existed (rs = 0.94, P < .005) between the potency
`observed in inhibiting basophil histamine release and
`eosinophil viability (Fig 4). In spite of the preservation of
`the relative rank order of potency, the basophil HR assay
`was more sensitive to all GCs than the eosinophil viabil-
`ity assay was, with a shift of sensitivity of 2- to 5-fold
`(Table I). Data in Fig 5 show previously published results
`from an assessment of the potency of the GC prepara-
`tions as inhibitors of VCAM-1 expression induced by
`TNF-α in cultured BEAS-2B airway epithelial cells.13
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`
`Stellato et al 627
`
`Previous studies have shown that stimulation of BEAS-
`2B cells with TNF-α and other cytokines induces expres-
`sion of the adhesion molecule VCAM-1.13 BEAS-2B
`cells were preincubated with various concentrations of
`GCs for 24 hours before stimulation with 10 ng/mL of
`TNF-α for another 24 hours. VCAM-1 expression was
`measured by flow cytometry. Data in Fig 5 show that all
`GCs tested inhibited VCAM-1 expression induced with
`10 ng/mL TNF-α in a concentration-dependent manner.
`β-E, used as a control steroid, did not change TNF-
`α–induced VCAM-1 expression. The concentrations at
`which each GC produced IC50 are listed in Table I, show-
`ing a rank order of potency MF ≅ FP >> BUD ≅ TAA >
`HC ≅ BDP. The most potent of the GCs tested were MF
`and FP, both of which had IC50s less than 10–11 mol/L.
`BUD and TAA had intermediate potency, and HC, BDP,
`and the BDP metabolite 17-BMP (IC50 = 9.2 × 10–9
`mol/L) were the least potent of the steroids tested. GC
`treatment had no effect on expression of intercellular
`adhesion molecule-1 (data not shown). Comparison of
`the potencies in the 3 assays revealed that BEAS-2B cells
`were the most sensitive, eosinophils were the least sensi-
`tive, and basophils had intermediate sensitivity to
`inhibitory effects for the GC (Fig 6). A correlation was
`found between the BEAS-2B assay and the basophil
`assay, but it did not reach statistical significance (rs =
`0.77, P = .072). A correlation was found between the
`potencies of the steroids in the BEAS-2B assay and the
`eosinophil assay (rs = 0.88, P < .05).
`
`DISCUSSION
`
`The purpose of the current study was to further char-
`acterize commonly used inhaled GCs for their relative
`potency in several in vitro assays of anti-inflammatory
`action. It is now well established that GCs achieve their
`potent anti-inflammatory effect by modulating the func-
`tion of multiple cell targets relevant in the establishment
`of allergic inflammation. Peripheral blood eosinophils
`and basophils are the effector cells selectively recruited
`within the tissue site of a chronic allergic reaction and
`during the late-phase response in experimental challenge
`models.19 Release of cytotoxic and proinflammatory
`mediators from these cells is believed to contribute to the
`structural damage of the airway mucosa seen in patients
`with respiratory allergies. Structural cells, such as airway
`epithelial cells, are also important sources of proinflam-
`matory mediators, cytokines, and eosinophil-specific
`chemoattractants,20 and they interact with inflammatory
`cells through several adhesion pathways.12 Epithelial
`cells, as well as eosinophils and basophils, are GC sensi-
`tive.21 Although one important effect of GC is the inhibi-
`tion of cytokine and chemokine production,11 GCs also
`have direct effects on inflammatory cells, including inhi-
`bition of basophil histamine release, decrease in
`eosinophil survival, and suppression of epithelial cell
`activation,
`independent of their ability to suppress
`cytokine synthesis. We have compared inhaled steroids
`commonly used for the treatment of asthma for their abil-
`
`FIG 6. Comparison of IC50 values obtained for various GCs for
`inhibition of basophil histamine release, eosinophil survival, and
`expression of VCAM-1 on bronchial epithelial cell line BEAS-2B.
`Asterisk, P < .05 compared with HC; pound sign, P < .05 compared
`with BDP; two asterisks, P < .05 compared with TAA.
`
`ity to inhibit basophil histamine release, eosinophil sur-
`vival, and epithelial activation for VCAM-1 expression.
`In a recent study, Spahn et al1 determined the potencies
`of several topical GCs in inhibiting phytohemagglutinin-
`induced proliferation of PBMCs from asthmatic patients.
`The rank order of potency found by these investigators in
`the inhibition of lymphocyte activation (BUD > TAA >
`BDP > HC) is almost identical to that found in this study
`in all 3 assays. Similarly, Umland et al22 report a very
`similar rank order of potency among topical GCs in their
`ability to suppress IL-4 and IL-5 production from periph-
`eral blood lymphocytes (MF = FP > BUD > BDP ≅
`TAA). The use of the standard test for determining rela-
`tive topical anti-inflammatory potency, the MacKenzie
`topical skin blanching test, as reported in the Second
`Expert Panel Report of the National Asthma Education
`and Prevention Program from the National Institues of
`Health, established a rank order of potency (FP > BUD >
`BDP > TAA)23 very similar to that reported in our study.
`The potency of several of the tested GCs, which
`included MF, FP, BDP, BUD, TAA, and HC, to inhibit
`basophil histamine release was severalfold greater than
`their potency in inhibiting eosinophil survival but lower
`than their actions on epithelial cells. The molecular
`explanation for this clear difference in potency in the 3
`assays is not obvious from the presented data. One pos-
`sible explanation for the relative resistance of the
`eosinophil assay is the presence of added cytokine,
`namely IL-5. Cytokines have been known for some time
`to be capable of antagonizing the action of GC in vitro.
`One of the first observations of this effect was the find-
`ing that the addition of IL-2 to lymphocyte proliferation
`assays could reverse the inhibitory effects of GCs on
`mitogenesis.15,24 In our early studies with human
`basophils we found that supernatants from activated T
`cells contained a factor that could antagonize the action
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`628 Stellato et al
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`SEPTEMBER 1999
`
`of GCs. We later found that this factor was the cytokine
`IL-3, which is a potent potentiator of basophil release of
`histamine and LT mediators.25 In the presence of high
`concentrations of IL-3, the GC effect was lost entirely.
`Similarly, in eosinophil assays we found that high con-
`centrations of GM-CSF could completely abrogate the
`inhibitory effects of GCs on eosinophil survival.26,27 This
`was confirmed by other laboratories studying eosinophil
`survival induced by IL-3 and IL-5 but not by IFN-γ.28,29
`The possible molecular mechanism that underlies this
`may be extrapolated from studies on the molecular action
`of GC on transcription factor systems. These studies have
`shown that the GC receptor complex can physically
`interact with several transcription factors, including acti-
`vator protein (AP)-1, and thereby block the activation of
`specific response elements for the AP-1 transcription fac-
`tor (the so-called TPA-response element).30 Conversely,
`however, the AP-1 transcription factor can, by virtue of
`the same physical interaction, prevent the GC receptor
`complex from interacting with its own receptor sites,
`including the glucocorticoid response element as well as
`a variety of transcription factors. Thus a strong elevation
`of transcription factors such as AP-1 could theoretically
`neutralize the GC receptor complexes in a given cell, ren-
`dering them incapable of suppressing the function of the
`cell. This type of acquired GC resistance at a cellular
`level has been described by Sher et al31 in lymphocyte
`systems where the combination of IL-2 and IL-4 can pro-
`duce a cellular phenotype reminiscent of that observed in
`GC-resistant patients. The 3- to 5-fold difference in
`potency of GCs in inhibiting eosinophil survival com-
`pared with basophil histamine release may be related to
`such a mechanism.
`The GCs studied were more potent at inhibiting epithe-
`lial cell activation and basophil histamine release than
`eosinophil survival. Nonetheless, the steroids with the
`highest binding affinity for the GC receptor32 were the
`most potent in inhibiting epithelial cell activation, basophil
`histamine release, and eosinophil survival. FP, MF, and
`BUD were all found to have particularly high potency,
`with IC50s below the nanomolar range in the basophil
`assays. In the lung BDP is hydrolized to 17-BMP, which
`displays a GC receptor affinity 25 times higher than that of
`BDP.33 The weak inhibition of VCAM expression
`obtained in our study with 17-BMP was therefore surpris-
`ing. We are currently evaluating whether this was the result
`of further degradation of 17-BMP into inactive metabo-
`lites. The large differences in potency among inhaled glu-
`cocorticoids in vitro in suppressing VCAM-1 expression,
`eosinophil survival, or basophil histamine release is not
`reflected in vivo when the potency of these drugs in revers-
`ing symptomatic endpoints (eg, FEV1, eosinophil number,
`or bronchial reactivity) is compared. It can be hypothe-
`sized that inhalation of any of these drugs will lead to tran-
`sient saturation or near saturation of glucocorticoid recep-
`tors in epithelial cells and other airway cells. In such a
`case, other parameters, such as the residence time of the
`drug in the airways and the dissociation rate of the steroid
`from receptors, may determine clinical potency.
`
`We thank Dr Donald MacGlashan, Jr, and Linda Friedhoff for
`helpful assistance in statistical analysis. We thank Dr Gunther
`Hochhaus for helpful discussion on data and Ms Bonnie Hebden for
`excellent secretarial assistance.
`
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