| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Section of Pulmonary and Critical Care Medicine, Depts of Medicine, Neurobiology Pharmacology and Physiology, Pediatrics, Anesthesia and Critical Care, and Committees on 2 Clinical Pharmacology and Pharmacogenomics, 3 Cell Physiology and Molecular Medicine, Division of the Biological Sciences, The University of Chicago, Chicago, USA
CORRESPONDENCE: A.R. Leff, Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC6076, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA. Fax: 1 7737029181. E-mail: aleff@medicine.bsd.uchicago.edu
Keywords: Adhesion Molecules, ß2-agonists, eosinophils, fluticasone propionate, glucocorticoids, salmeterol
Received: June 11, 2003
Accepted November 27, 2003
This work was supported by National Heart, Lung, and Blood Institute (NHLBI) Grant HL-46368, NHLBI SCOR Grant HL-56399 (A.R. Leff) and AI-52109 (X. Zhu), and GlaxoSmithKline, Research Triangle Park, NC, USA.
 |
Abstract |
|---|
 TOP Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Human eosinophils were isolated by negative magnetic selection. ß2-integrin-mediated eosinophil adhesion was measured by residual eosinophil peroxidase activity. Eosinophils were pretreated for 12 h to 24 h with FP and with or without SM for 30 min.
Both SM alone and FP alone inhibited eosinophil adhesion in concentration- and time-dependent manner. SM alone modestly (
30%) inhibited interleukin (IL)-5-induced eosinophil adhesion. Blockade of IL-5-induced eosinophil adhesion caused by 10–7 M FP at 24 h was augmented by 10–7 M SM from 41.5% to 72.5%. Similar blockade was also observed for eotaxin-induced eosinophil adhesion. Neither SM, FP, nor FP+SM blocked either: 1) upregulation of CD11b surface expression; or 2) phosphorylation of cPLA2.
Blockade of ß2-integrin-mediated eosinophil adhesion by fluticasone propionate is augmented by salmeterol. Decreased adhesion results from augmented blockade of nuclear translocation of cytosolic phospholipase A2 caused by addition of salmeterol to fluticasone.
Eosinophilic infiltration of tissues from the blood vessels involves multiple steps including directed chemotaxis, adhesion, and diapedesis 1. Eosinophil adhesion by the ß2-integrin, CD11b/CD18 (Mac-1), to immunoglobulin supergene, intercellular adhesion molecule I (ICAM-1), is an essential early step for cell migration in allergic inflammation as occurs in asthma 2–4. Blockade of the CD11b subunits inhibits human eosinophil adhesion 5–7 in vitro and causes the inhibition of eosinophil infiltration into airways in vivo 8–10.
Recent investigation has suggested that salmeterol (SM), a long-acting ß2-selective adrenoceptor agonist might also have an independent inhibitory effect on the inflammatory response in asthma 11. SM has been reported to reduce adherence of eosinophils in mucosal blood vessels in rat airways 12 and eosinophil adhesion to fibronectin induced by both interleukin (IL)-5 and platelet activating factor in vitro 13, 14. Other ß2 adrenoceptor agonists (fenoterol, salbutamol and procaterol) have been reported to inhibit CD11b upregulation in eosinophils caused by platelet activating factor 15, but another long-acting ß2-adrenergic receptor agonist, formoterol, did not inhibit the surface upregulation of CD11b 16.
Inhaled glucocorticoids also have been reported to diminish eosinophil infiltration in asthma; however, the mechanism by which glucocorticoids attenuate eosinophil adhesion is not established 17–19. The current authors have reported previously that cytosolic phospholipase A2 (cPLA2) activation is essential for ß1- and ß2-integrin-dependent adhesion of eosinophils 20, 21. Blockade of cPLA2 in vivo also blocked airway infiltration of eosinophils and attenuated airway responsiveness to methacholine 22. An essential regulatory step in eosinophil adhesion is phosphorylation of extracellular signal regulated kinase (ERK) 1/2. ERK 1/2 then phosphorylates cPLA2 at the serine505 position. Phosphorylation at this site converts the enzyme into its active hydrolytic form. Hydrolysis of membrane lipid into lysophospholipids that mediate eosinophil adhesion further requires the translocation of cPLA2 to the nuclear membrane 23.
This investigation was undertaken to determine the potential role of ß2-adrenoceptor stimulation and treatment with corticosteroid in blocking ß2-integrin-mediated eosinophil adhesion caused by IL-5 and eotaxin-1 in human eosinophils in vitro. Studies were conducted to determine the mechanism by which eosinophil adhesion caused by either IL-5 or eotaxin is blocked individually and additively by SM and fluticasone propionate (FP). The current authors’ data demonstrate that SM and FP, even in combination, do not block IL-5- or eotaxin-mediated upregulation of Mac-1 and do not block cPLA2 phosphorylation. Instead it was found that ß2-adrenoceptor stimulation after prior incubation with the corticosteroid, FP, blocks the translocation of activated cPLA2 to the perinuclear membrane, thereby blocking integrin-mediated adhesion caused by IL-5 and eotaxin.
|
Materials and Methods |
|---|
|
TOPAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Human peripheral blood eosinophils were isolated by a method modified from Hansel et al. 24. The method is based on Percoll centrifugation (density 1.089 g·mL–1) to isolate granulocytes, hypotonic lysis of red blood cells, and, finally, immunomagnetic depletion of neutrophils by the magnetic cell separation system using anti-CD16-coated MACS particles (Miltenyi Biotec, Sunnyvale, CA, USA). Eosinophil purity of 98% was routinely obtained, as assessed by Wright-Giemsa staining.
Treatment with fluticasone propionate and salmeterol
Eosinophils were resuspended in Roswell Park Memorial Institute (RPMI) buffer (10% foetal bovine serum, 250 U·mL–1 of penicillin and 250 µg·mL–1 streptomycin, 10 pg·mL–1 IL-5). For the time-dependent effect of SM on eosinophil adhesion, aliquots of 104 eosinophils were pre-incubated with buffer or 10–6 M SM for 5–60 min before adhesion assay. For the concentration-dependent effect of SM on eosinophil adhesion, eosinophils were pre-incubated with 10–10–10–6 M SM for 30 min. For the combinatorial effect of FP and SM, aliquots of eosinophils were incubated at 37°C with buffer or 10–10–10–6 M FP for 12–24 h, and then were incubated for an additional 30 min with buffer or SM at 10–10 and 10–7 M. Duplicated samples were prepared for adhesion assay. Recombinant human IL-5 (10 pg·mL–1) was added to maintain eosinophil viability during the experiment period, but had no effect on cellular adhesion 20, 25.
Eosinophil adhesion assay
Eosinophil adhesion was assessed as residual eosinophil peroxidase (EPO) activity of adherent cells 20, 25. Briefly, 96-well microplates were coated with 100 µl of bovine serum albumin (BSA, 10 µg·mL–1) dissolved in Hank’s balanced salt solution (HBSS) and incubated at 4°C overnight and blocked with 200 µL/well of heat-inactivated foetal bovine serum for 60 min at 37°C. Eosinophils (1x104/100 µl) were pre-incubated with different concentrations of FP or SM (GlaxoSmithKline, Uxbridge, UK) for indicated times at 37°C. Cells then were added to BSA-coated wells with or without stimulators and allowed to settle for 10 min on ice. Plates were rapidly warmed to 37° C and incubated for 30 min. After 3xwashing with HBSS, 100 µl of RPMI buffer was added to the reaction wells, and serial dilutions of original cell suspensions were added to the empty wells to generate a standard curve. 100 µl EPO substrate (1mM hydrogen peroxide, 1 mM O-phenylenediamine, and 0.1% triton X-100 in Tris buffer, pH 8.0) then was added to the wells. After 30 min incubation at room temperature, 50 µl of 4 M H2SO4 was added to stop the reaction. Absorbance was measured at 490 nM in a microplate reader (Thermomax; Molecular Devices, Menlo Park, CA). All assays were performed in duplicate. Stimulated eosinophil adhesion to plated BSA was ß2-integrin dependent, as anti-CD11b or anti-CD18 blocking antibody suppressed this adhesion substantially 25.
Immunofluorescence microscopy
After stimulation of cells, 5x105 cells were prepared from cytospin in a Shandon Cytospin Model 3 (Shandon, Pittsburgh, PA, USA) at 200xg for 2 min. Slides were then foil-wrapped and stored at 4°C until further use. For analysis, slides of eosinophils were fixed for 20 min in 2% paraformaldehyde in PBS and treated with 0.4% p-benzoquinone (Sigma, St Louis, MO, USA) in PBS for 10 min. Slides then were permeabilised using 0.1% saponin in PBS for 15 min. Following permeabilisation, slides were blocked using 2% human immunoglobulin (Ig)G (Reagent Grade I-4506; Sigma) for 60 min at room temperature. After a washing step, slides were incubated with monoclonal antibody (mAb) directed against cPLA2 (Santa Cruz Biotech, Santa Cruz, CA, USA) for 60 min at room temperature. Binding of mAb to cPLA2 was detected by incubating slides with 20 µg·mL–1 BODIPY FL-conjugated goat anti-mouse secondary antibody (Molecular Probes, Eugene, OR, USA) for 60 min at room temperature. After the final wash, 10 µL of anti-bleaching agent (0.4% n-propyl gallate (Sigma)) in 3:1 glycerol:TBS was used before coverslip application. Negative controls were carried out by replacing the primary antibody with PBS or mouse isotype control. Slides were viewed by fluorescence microscopy on a Zeiss Axoplan microscope (magnification x1000; Carl Zeiss, Thornwood, NY, USA).
Western blot analysis of cytosolic phospholipase A2 expression and phosphorylation
Eosinophils (2x106·group–1) were pre-incubated in the presence or absence of FP for 24 h and SM for 30 min. Then cells were stimulated with either 10 ng·mL–1 IL-5 or 100 ng·mL–1 eotaxin (R&D system, Minneapolis, MN, USA) for 30 min, and the reaction was stopped by brief centrifugation. The pellets were lysed in 80 µl of boiling buffer (50 mM Tris-HCl, 2 mM EDTA, 2 mM EGTA, 2 mM Na3VO4, 50 mM NaF, 2.5 mM DFP, 1% SDS, proteinase inhibitor cocktail), sonicated, and boiled for 5 min. Afterward, 14 µl of 6xloading buffer was added, and the mixture was boiled for 5 min and then saved at –80°C. Samples were subjected to SDS-PAGE, using 7.5% acrylamide gels under reducing condition (15 mA·gel–1). Electrotransfer of proteins from the gels to polyvinylidene fluoride membrane was achieved using a semi-dry system (400 mA, 60 min). The membrane was blocked with 2% BSA for 60 min, then incubated with 1:1000 anti-phosphorylated cPLA2 (Cell Signaling Technology, Beverly, MA, USA) or 1:400 anti-cPLA2 antibody diluted in Tris-buffered saline plus Tween-20 (TBS-T) for 60 min. The membranes then were washed three times for 20 min with TBS-T. Goat anti-rabbit Ig conjugated with horseradish peroxidase was diluted 1:3000 in TBS-T and incubated with polyvinylidene fluoride membrane for 60 min. The membranes were again washed three times with TBS-T and assayed by an enhanced chemiluminescence system (Amersham, Arlington Hts, IL, USA).
Analysis of surface CD11b expression by immunofluorescence flow cytometry
Eosinophils were pre-incubated with 10–7 M FP for 24 h and 10–7 M SM for 30 min, then stimulated with or without 10 ng·mL–1 IL-5 or 100 ng·mL–1 eotaxin, for 30 min. Thereafter, eosinophils were centrifuged at 400xg for 10 min, and the pellets were resuspended in PBS/2% BSA. Aliquots of 5x105 eosinophils were incubated with 10 µL of mAb CD11b (Bear 1) or isotype-matched control antibody for 30 min at 4°C. After 2 washes, the cells were incubated with an excess of fluorescein isothiocyanate-conjugated goat antimouse immunoglobulin for 30 min at 4°C. The cells were washed twice, resuspended in 1% paraformaldehyde and kept at 4°C until analysed. Flow cytometry was performed on a FACScan (Becton Dickinson, Mountain View, CA, USA). Fluorescence intensity was determined on at least 5,000 cells from each sample.
Determination of eosinophil viability after incubation with fluticasone propionate
To determine if FP affected eosinophil viability, trypan blue exclusion was assessed in eosinophils incubated with various concentration of FP. Aliquots of 104 eosinophils were incubated for 12–24 h at 37°C with 10–10–10–6 M FP in the presence of 10 pg·mL–1 of IL-5. Eosinophils then were centrifuged at 400xg and pellets were resuspended in 10 µl of HBSS. An equal volume of 0.01% trypan blue was added, and viable eosinophils were counted in a haemacytometer.
Statistical analysis
All values are expressed as the mean±sem. Differences between groups was assessed by paired t-test. Where more than two groups were compared, differences among groups were assessed by one-way analysis of variance (ANOVA). Where differences were found, comparisons among groups were made by Fisher’s least-protected difference test. Eosinophil adhesion was normalised as a per cent of the response in stimulated control in the same subjects using the following equation: adhesion (%)=100x(test sample data–negative control data)/(positive control data–negative control data). The combinatorial effect shown by SM and FP were designated as: synergistic when inhibition with two drugs combined > inhibition by FP alone + inhibition by SM alone. Additive inhibition was defined when the combined effect of FP and SM together > inhibition by either FP alone or SM alone.
|
Results |
|---|
|
TOPAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
). Accordingly a 30 min incubation time for SM was chosen for subsequent experiments. Concentration-responses curves were also generated after pre-incubation with SM (10–10 M–10–6 M) for 30 min. Statistically significant blockade of eosinophil adhesion was achieved with SM alone only at concentrations 
10–7 M. IL-5-induced eosinophil adhesion was 29% blocked by 10–7 M SM to 71.1±10.3% (p<0.05) of stimulated control and to 67.5±4.6% (p<0.01) of stimulated control in cells activated by eotaxin (fig. 1b).
|
). The combinatorial inhibition of eosinophil adhesion caused by IL-5 for eosinophils treated with SM (10–10 M or 10–7 M) + FP (10–10–10–6 M for 12 h) was additive (fig. 2a). After 24 h pretreatment with FP, SM and FP also produced additive inhibition. However, inhibition was synergistic for 10–10–10–8 M FP+10–7 M SM (fig. 2b).
|
). Progessive augmentation of inhibition of adhesion was elicited over full concentration range of FP with 10–10 SM and substantially augmented for the same concentrations of FP by co-treatment with 10–7 M SM (fig. 2c, d). As for IL-5, the effects observed at 12 h were substantially amplified at each concentration of FP after 24 h incubation (fig. 2c, d). In all circumstances, the blockade of adhesion by co-treatment with FP+SM was either synergistic (10–10–10–9 M FP at 24 h with 10–7 M SM) or additive (all other combinations).
At the end of each experiment, the effect of incubation with FP on cell viability was assessed. FP had no effect on eosinophil viability at 12 or 24 h as measured by trypan blue exclusion or propidium iodide. Viability at the beginning and end of each experiment was 98%. Accordingly, blockade of adhesion by FP was not related to diminished viability of the cells during treatment.
Effects of fluticasone propionate and salmeterol on CD11b surface expression
Studies were conducted to determine if blockade of Mac-1 (CD11b/CD18) upregulation accounted for the blockade of eosinophil adhesion caused by FP, SM or the additive combined effects of FP+SM. For these studies, large concentrations of both SM (10–7 M) and FP (10–7 M) were used to examine the effect of CD11b surface expression as measured by FACScan. Neither FP nor SM alone blocked the upregulation of surface CD11b caused by IL-5 (fig. 3a) or eotaxin (fig. 3b). The combination of FP+SM even at large concentrations also did not inhibit the upregulation of CD11b surface expression caused by IL-5 or eotaxin (fig. 3).
|
) also did not inhibit cPLA2 phosphorylation (fig. 4).
|
). Negative controls displayed minimal fluorescence background (fig. 5k). cPLA2 was predominantly localised to the cytoplasm in unstimulated cells (fig. 5l). After stimulation with either 2 µM A23187
[GenBank]
(as a positive control), 10 ng·mL–1 IL-5 or 100 ng·mL–1 eotaxin for 30 min, the immunofluorescent staining for cPLA2 was localised to the perinuclear region of the cells (fig. 5a, f, m). Pretreatment with 10–7 M FP for 24 h completely inhibited cPLA2 translocation to the nucleus caused by activation with both IL-5 and eotaxin (fig. 5b, g). Pretreatment with either 10–7 M SM for 30 min or 10–9 M FP for 24 h did not substantially block cPLA2 translocation (fig. 5c, d, h, i). However, the combination of 10–9 M FP +10–7 M SM inhibited completely the translocation of cPLA2 to the nuclear envelope (fig. 5e, j).
|
|
Discussion |
|---|
|
TOPAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
The current data indicate that both SM and FP inhibited IL-5- or eotaxin-induced eosinophil adhesion in a concentration- and a time-dependent manner, although neither compound was exceptionally effective alone in concentrations likely to be achieved in human airways during inhalation (GlaxoSmithKline, Uxbridge, UK). However, inhibition of IL-5- or eotaxin-induced eosinophil adhesion caused by 10–9 M FP was substantially enhanced by 10–7 M SM (fig. 2b, d), which caused a >2 log shift in the efficacy of FP at lower concentrations. The combination of SM and FP was synergistic (FP 
10–9 M at 24 h with 10–7 M SM for eotaxin and FP 10–8 M at 24 h with 10–7 M SM for IL-5) or additive (with all other combinations tested) in the blockade of the ß2-integrin adhesion of human eosinophils (fig. 2).
To determine the mechanism of additive and/or synergistic augmentation of adhesion between FP and SM, studies were performed to examine the effect of these compounds individually and in combination on 1) ß2-integrin upregulation on the eosinophil surface; 2) cPLA2-phosphorylation and 3) translocation of phosphorylated cPLA2 to the perinuclear membrane 23. Upregulation of ß2-integrin, cPLA2 phosphorylation and translocation of cPLA2 all are required for IL-5-stimulated eosinophil adhesion 20. Blockade of any step of these processes also causes blockade of adhesion 20, 26, 27. It was found that concentration of SM + FP that caused substantial blockade of eosinophil adhesion at 24 h had no effect either on Mac-1 upregulation (fig. 3) or cPLA2 phosphorylation (fig. 4) caused by IL-5 or eotaxin. By contrast, very small concentrations of SM+FP caused substantial blockade of cPLA2 translocation (fig. 5). The current authors have previously shown that FP blocks eosinophil secretion of LTC4 by preventing translocation of cPLA2 to the perinuclear membrane 28. The mechanism by which this occurs has not been further defined; however, inhibition required 48 h incubation, suggesting that FP-induced transcription is essential for the blockade of cPLA2 migration. This study found substantial augmentation of FP-induced blockade of adhesion at 24 h after treatment with SM. SM also greatly augmented the effect of FP in blocking translocation (fig 5). These data suggest that ß2-adrenoceptor stimulation, which has been shown to augment translocation of the cytosolic corticosteroid receptor to the nucleus, likely results in temporal and quantitative augmentation of transcriptional events that block integrin adhesion. The precise mechanism by which cPLA2 translocation is blocked, however, still remains to be elucidated.
In the experiments, the present authors ensured that eosinophil viability was maintained during culture. Zhang et al. 29 reported that FP induces eosinophil apoptosis and addition of IL-5 attenuates the toxic effect of FP on cell viability 29, 30. Accordingly, 10 pg·mL–1 IL-5 was added to the culture medium for all experiments in the current study. Viability of eosinophils after 24 h incubation with FP was >98%. However, IL-5 in concentrations used to maintain viability had no effect on eosinophil adhesion in these studies.
The current results contrast with some studies that found no effect of glucocorticoids on eosinophil adhesion to endothelial cells 18, 19. The mechanism underlying these differences is not certain but may be related to a difference in technique for measurement of adhesion. The current study used single purified ligands in concentrations established previously 20, 25 rather than cultured endothelial cells, which express multiple counter ligands such as ICAM-1, vascular cell adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 and have potential to secrete pro-inflammatory mediators. FP also has significantly greater potency than dexamethasone. Finally, in preliminary experiments for this study, the authors found little efficacy of FP in concentrations comparable to those achieved in airways after inhalation. The addition of a moderate concentration SM to FP was essential to achieve substantial blockade of inhibition, and this blockade was achieved within a relatively short time course (12–24 h) in comparison with prior studies using corticosteroids alone 28.
It is important to consider some important limitations of the current findings. While the concentrations of FP and SM used were those achieved in human airways (data provided by GlaxoSmithKline, Uxbridge, UK), these studies were nonetheless conducted in vitro. IL-5 was introduced to insure eosinophil survival, thus insuring that blockade of adhesion was not related to cell death. Freshly isolated human eosinophils were also used in these studies. Nonetheless, these data cannot be directly extrapolated to the human asthmatic state or to other cell types that may be involved in asthmatic airway inflammation. Accordingly, these data do not affirm or disprove a role for the eosinophil in human asthma. Rather, the current study shows, in an inflammatory cell that is highly prevalent in human asthma, that SM + FP act additively and synergistically to prevent translocation of cPLA2, which is an essential enzyme in all inflammatory cell adhesion and first step synthesis of prostaglandins and leukotrienes. Accordingly, the data suggest one mechanism by which the addition of a ß2-adrenoceptor agonist may augment at the cellular level the anti-inflammatory effects of corticosteroids.
To conclude, neither salmeterol nor fluticasone propionate alone cause substantial blockade of ß2-integrin adhesion caused by interleukin-5 or eotaxin. However, in combination, there is a significant additive effect, which, at low concentrations, is synergistic in blocking integrin adhesion. Augmented blockade of in vitro adhesion is caused neither by blockade of Mac-1 upregulation nor by blockade of cytosolic phospholipase A2 phosphorylation but rather by augmentation of corticosteroid-induced inhibition of cytosolic phospholipase A2 translocation to the perinuclear envelope.
|
Footnotes |
|---|
S. Myo and X. Zhu contributed equally to this work.
|
References |
|---|
|
TOPAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  A. Y. Meliton, N. M. Munoz, X. Zhu, and A. R. Leff Attenuated translocation of group IVa phospholipase A2 and up-regulated annexin-1 synthesis by glucocorticoid blocks {beta}2-integrin adhesion in neutrophils J. Leukoc. Biol., February 1, 2008; 83(2): 344 - 351. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. R. Leff and N. M. Munoz Future Treatment to Lessen Exacerbations of Chronic Obstructive Pulmonary Disease Proceedings of the ATS, December 1, 2007; 4(8): 659 - 666. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Y. Meliton, N. M. Munoz, A. Lambertino, E. Boetticher, J. Learoyd, X. Zhu, and A. R. Leff Phosphodiesterase 4 inhibition of {beta}2-integrin adhesion caused by leukotriene B4 and TNF-{alpha} in human neutrophils Eur. Respir. J., November 1, 2006; 28(5): 920 - 928. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. ten Hove, L. A. Houben, J. A. M. Raaijmakers, L. Koenderman, and M. Bracke Rapid Selective Priming of Fc{alpha}R on Eosinophils by Corticosteroids J. Immunol., November 1, 2006; 177(9): 6108 - 6114. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
R. Buhl and S. G. Farmer Future Directions in the Pharmacologic Therapy of Chronic Obstructive Pulmonary Disease Proceedings of the ATS, April 1, 2005; 2(1): 83 - 93. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S.P. Hart Additive blockade of {beta}2-integrin adhesion of eosinophils by salmeterol and fluticasone propionate Eur. Respir. J., August 1, 2004; 24(2): 333 - 333. [Full Text] [PDF]
|
|
|
|
|
|
A.R. Leff From the Authors Eur. Respir. J., August 1, 2004; 24(2): 334 - 334. [Full Text] [PDF]
|
|
|
|
|
|
A. Papi Investigating the steroids and long-acting {beta}2-agonists combination: why do we need more? Eur. Respir. J., April 1, 2004; 23(4): 501 - 502. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
). b) Concentration-dependent inhibition of SM on stimulated adhesion of eosinophils to plated BSA. Eosinophils were pre-incubated with SM for 30 min, then stimulated by either IL-5 (•) or eotaxin (
: 10–7 M SM; 
: The effect of 10–10 SM alone; 
: the effect of 10–7 SM alone. *: p<0.05 and **: p<0.01 compared with the sum of inhibition by each drug alone, and denotes the synergistic effect of FP+SM.
: 10–7 M FP-treated cells; – - – : 10–7 M SM-treated cells; – - - –: combination of 10–7 M FP and 10–7 M SM-treated cells.
