Overnight incubation of human eosinophils (Eøs)with the glucocorticoid hormone dexamethasone(DEX; 0.1 mM) resulted in lower expression of theCD11b, but not CD49d, antigen on their plasmamembrane, as assessed by � ow cytometry. DEXproduced a consistent inhibitory effect (rangingfrom 16% to 20%) when tested at a concentrationof 0.1 mM. Eø stimulation with 100 ng/ml eotaxinproduced an increase in CD11b (+26%), but notCD11c, levels and concomitantly a reduction( - 25%) on CD62L expression. The inhibitionexerted by DEX upon CD11b levels was alsoevident following eotax in upregulation, with adegree of inhibition similar to that seen on basallevels. These data highlight a novel mechanism ofaction by which glucocorticoid hormones may beeffective in reducing Eø accumulation duringallergic in� ammation in man.
In� ltration of eosinophils (Eøs) into sites ofallergic in�ammation is a characteristic featureof diseases such as asthma.1 The products ofactivated Eøs have been shown to play a role inmanifesting the symptoms of asthma.2 Althoughmany in�ammatory mediators (including lipidmediators, cytokines and chemokines) are po-tent activators of Eøs, very few act speci� callyon this cell type. Eotaxin, which is a member ofthe CC family of chemokines, was isolated fromthe bronchoalveolar lavage after allergen chal-lenge in sensitized guinea pigs.3 It is a potent,and more importantly speci� c, activator of Eøs.
Activation of leukocytes by in� ammatorymediators results in shedding of the selectinadhesion molecule CD62L from the cell surfaceand upregulation of the b2 integrin CD11b/CD18.4,5 This complex plays an important rolein adhesive and activation processes of Eøs6 - 8
and eotaxin increases levels of the CD11bsubunit (Mac-1) on the cell surface of humanEøs.9
Glucocorticoid hormones are used routinelyto treat allergic disorders, and indeed treatmentwith natural or synthetic glucocorticoids iseffective in reducing the symptoms of thesediseases, including asthma.10 Although little isknown about their mechanism of action, itappears that part of their anti-in� ammatoryactions may be mediated indirectly by reducing
the number of circulating Eøs thereby attenuat-ing Eø in� ltration into allergic tissues.11,12 Morerecently glucocorticoid hormones, in particulardexamathasone (DEX), have been shown toreduce adhesion molecule expression on se-lected target cells. For example, DEX was activeon endothelial cells where levels of CD62E andCD54 were increased with cytokines or lipo-polysaccharide.13,14 Such an effect of DEX onthe activated endothelium could contribute tothe inhibition exerted by this drug on leukocyteaccumulation during the host in�ammatoryresponse.13 Much less is known about the effectof DEX upon adhesion molecules expressed bycirculating leukocytes. A study with bovineneutrophils reported that administration of DEXin vivo reduced basal expression of CD62Land CD18 levels on this polymorphonuclearleukocyte.15
To date, no studies have investigated an effectof glucocorticoids on the adhesion moleculesexpressed on Eø plasma membrane. In thepresent study we have tested whether DEXaffected either basal or eotaxin-upregulatedCD11b levels on human Eøs.
Material and Methods
Isolation of human blood Eù s
Blood (80 ml) was obtained, out of the hay-fever season, from individuals who suffered
Mediators of In¯ ammation ´ Vol 6 ´ 1997 363
from allergic rhinitis, and it was collected intosodium citrate. After dextran sedimentation(�nal concentration 1.2% dextran; Pharmacia,Uppsala, Sweden) the buffy coat was layeredonto histopaque gradients (density 1.077;Sigma, Poole, UK). After centrifugation at400 3 g for 25 min the leukocyte pellet wascollected and all procedures were carried outat 48 C from this point. The remaining redblood cells in the granulocyte pellet werelysed hypotonically. Eøs were separated fromneutrophils by incubation with anti-CD16MACS microbeads (Miltenyi Biotec GmbH,Germany) at a concentration of 40 ml micro-beads per 1 3 108 granulocytes followed byelution of the Eøs on a CS-type MACS columnand magnetic separator (Milltenyi BiotecGmbH, Germany). The Eøs obtained weregreater than 96% pure with contaminatingcells constituting of mononuclear cells. Cellswere either incubated overnight with DEX orstimulated with eotaxin immediately (see be-low).
Overnight incubation of Eù s withDEX
Cells were resuspended at a concentration of5 3 106 Eøs per ml in RPMI 1640 mediumcontaining 10% heat-inactivated fetal bovineserum, 1% penicillin-streptomycin, 0.5% genta-mycin and 2 mM L-glutamine (all obtained fromSigma, Poole, UK). One hundred ml of the cellsuspension was added to �at-bottomed 96-wellplates (in duplicate for each treatment). Either100 ml buffer or different concentrations of DEX(1±0.001 mM �nal concentration; David BallLaboratories, Warwick, UK) was added to thewells and the cells incubated at 378 C with 5%CO2 in air for 18 h. After the incubation, thecells were washed and either (i) resuspended inphosphate buffered saline (PBS) containing0.2%bovine serum albumin (BSA) for the meas-urement of cell surface adhesion molecules or,(ii) resuspended in RPMI 1640 containing 0.1%BSA for stimulation with mediators.
Stimulation with mediators
Either fresh cells or cells which had beenincubated overnight with DEX or buffer wereincubated in a shaking water bath at 378 C for30 min in the presence of buffer, human recom-binant (hr) eotaxin (100 ng/ml �nal concentra-tion; PeproTech EC Ltd, London, UK) or 0.1 mM�nal concentration platelet activating factor(PAF; C16 form, Sigma, Poole, UK). The reactionwas stopped by placing the samples on ice for
5 min. The cells were then pelleted and resus-pended in PBS containing 0.2% BSA for themeasurement of cell surface adhesion mole-cules.
Measurement of cell surfaceadhesion molecules
The presence of CD11b, CD11c, CD62L andCD49d (a4 subunit of the b1 heterodimerCD49d/CD29) were measured on the cell sur-face using FACS analysis as previouslydescribed.16 For this purpose, the followingmonoclonal antibodies (mAb) were used:mouse-anti human CD11b (clone 44; Serotec,Oxford, UK); mouse-anti human CD11c (clone3.9; Serotec); mouse anti-human CD62L (cloneFMC46; Serotec), and mouse anti-human CD49d(MAX68P; kindly gifted by Dr M. Robinson,Celltech Therapeutics, Slough, UK). MAX68P isa well characterized monoclonal antibodywhich has been shown to bind to Chinesehamster ovary cells transfected with CD49d(personal communication, Dr M. Robinson,Celltech Therapeutics). Brie�y, cells wereplaced in �at-bottomed 96-well plates (2.5 3105 Eøs per well, 20 ml). Non-speci� c bindingsites were blocked with 20 ml human IgG(15 mg/ml) before the addition of 20 ml of theappropriate mAb (giving a �nal concentrationof 10±20 mg/ml). Control wells were incubatedwith similar amounts of mouse IgG (Sigma).Plates were incubated at 48 C for 1 h prior towashing and a further incubation with 40 ml ofFITC-conjugated sheep anti-mouse IgG antibody(1:50 dilution of stock; Sigma, Poole, UK) for30 min at 48 C. The plates were washed againand FACS analysis performed using a FACScan IIanalyser (Becton Dickinson, CA, USA) with air-cooled 100 mW argon ion laser tuned to488 nm and Consort 32 computer running LysisII software. The Eø population could be clearlyidenti�ed because of the side and forwardscatter characteristics (con� rming in this waythe high degree of purity) and the value of themean �uorescence intensity (MFI) associatedwith it was measured at 548 nm with a bandwidth of 10 nm (FL-1 channel). Results arereported as the MFI units for each sample andhave been corrected for the control (mouseIgG) value.
Statistics
Data are mean SEM of n experiments per-formed in duplicate. Statistical analysis wasperformed on raw data using the Student’spaired t-test.
364 Mediators of In¯ ammation ´ Vol 6 ´ 1997
A. M. Das et al.
Results
Effect of eotaxin on cell surfaceadhesion molecule levels
When human Eøs were incubated with eotaxinfor 30 min, there was a signi� cant increase(21%) in the levels of CD11b molecules on thecell surface with a concomitant 32% reductionin the CD62L levels (Fig. 1). In comparison, wefound no effect on the levels of CD11c. We alsoexamined the effect of another mediator, PAF,on the levels of CD11b and CD62L. From atypical experiment where the basal levels ofCD11b was 220 MFI units, after eotaxin or PAFexposure this increased to 277 (26% increase)and 303 (38% increase), respectively. Similarly,basal levels of CD62L was 131 MFI, and thiswas reduced to 117 and 74 MFI units aftereotaxin and PAF stimulation, respectively.
Effect of DEX on basal andstimulated CD11b levels
Next, we investigated the effect of DEX on thelevels of CD11b. For this series of experiments,Eøs were incubated for 18 h with or withoutthe glucocorticoid hormone after which either(i) cell surface CD11b levels were measured or,(ii) a 30 min stimulation with eotaxin wasperformed prior to quanti�cation of cell surfacelevels of CD11b. Incubation of Eøs overnight (inthe absence of DEX) did not affect CD11b
expression when compared to levels of theintegrin on freshly isolated Eøs. Further, over-night incubation did not alter the extent ofeotaxin-stimulated CD11b increase when com-pared to fresh Eøs (data not shown).
Following incubation with DEX there was asigni�cant reduction on basal CD11b expressionon Eø plasma membrane (Fig. 2). A similarreduction was also measured after Eø stimula-tion with eotaxin. The effect of DEX on basalCD11b levels was investigated further, �nding asimilar inhibition at all doses tested (a typicalexperiment is shown in the insert of Fig. 2). We�nally investigated whether this effect of DEXwas the result of a non-speci� c suppression ofEø genomic activity, or rather, was speci� c forCD11b. The data shown in Table 1 illustrates
control
stimulated
(4)
(4)*(2) (2)
(4)
(4)*
CD11b CD11c CD62L0
100
200
300
400
500
MF
I uni
ts
FIG. 1. The effect of eotaxin on Eù cell surface CD11b,CD11c and CD62L levels. Freshly isolated Eù s were incu-bated with either buffer (control) or 100 ng/ ml hr eotaxinfor 30 min at 378 C. Cell surface levels of the adhesionmolecules were measured using FACS analysis. Data aremean SEM of (n) experiments performed in duplicate.
P , 0.05 when compared with appropriate control.
*
*
control DEX
BASAL
control DEX
EOTAXIN-STIMULATED
DEX (mM)0 0.001.01 .1 1
150
250
350
MFI
uni
ts
70
85
100
115
130C
D11
b le
vels
(% o
f bas
al M
FI u
nits
)
FIG. 2. The effect of DEX on basal or eotaxin-stimulatedCD11b levels on Eù s. Eù s were incubated for 18 h witheither buffer (control) or 0.1 mM DEX. Cell surface CD11blevels were measured either immediately (basal) or afterthe cells were incubated with either buffer or 100 ng/ml hreotaxin for a further 30 min at 378 C. Basal value (100%) was347 MFI units. Data are mean SEM from three to fourexperiments performed in duplicate. The insert shows atypical dose response to DEX on basal CD11b levels.
P , 0.05 when compared with control (calculated onoriginal values).
Table 1. Effect of DEX on basal CD11b and CD49d levels onhuman Eù s
Adhesion molecule Control DEX(MFI units) (MFI units)
Eù s were incubated for 18 h with either buffer (control) or 0.1 mMDEX after which the cell surface levels of CD11b and CD49d weremeasured. Data are mean SEM of (n) experiments performed induplicate. P , 0.05 when compared with control values.
Mediators of In¯ ammation ´ Vol 6 ´ 1997 365
Dex ameth asone and eos inophil CD11b
the lack of effect of DEX incubation on basalCD49d expression on the plasma membrane ofEøs.
Discussion
In the present study we have investigated theeffect of DEX on the expression of adhesionmolecules on the surface of human Eøs. Weexamined the effect of DEX on basal, as well aseotaxin-stimulated, CD11b levels. We haveshown, to our knowledge for the � rst time, thatDEX is able to reduce the expression of CD11bon human Eøs.
We used the CC chemokine eotaxin tostimulate CD11b upregulation on Eøs. Theconcentration of eotaxin used was shown to bemaximal for upregulating CD11b on Eøs inprevious studies.9,17 Different levels of CD11bupregulation has been achieved between stud-ies. One reason for the difference could be thesource of Eøs; a previous study used Eøs fromnon-atopic donors9 whereas we have used Eøsfrom atopic subjects. Therefore, the Eøs in thepresent study might be desensitized to certainmediators through exposure in vivo . It alsoappears that the cells were more responsive toPAF than eotaxin which is in contrast toprevious �ndings.9 Again, this may be due tothe desensitization status of the cells.
Together with cytokines and chemokines,adhesion molecules play a pivotal role in theprocess of leukocyte recruitment. Recentlyadhesion molecules have been shown to be atarget (although not too sensitive) for glucocor-ticoid hormone action.13 Whereas DEX effecton CD62E or CD54 upregulation on endothelialcells, �broblasts and epithelial cells is wellaccepted, much less is known of the potentialeffect of this steroid on leukocyte adhesionmolecules. Few studies have reported an abilityof glucocorticoid hormones to reduce basalexpression of selected adhesion molecules,though some species speci� city has also beenfound. Prolonged (. 3 days) exposure to DEXreduced basal levels of CD62L and CD18 onbovine neutrophils;15 CD62L and CD11a, butnot CD11b, on rat neutrophils;18 and CD54, butnot CD11b, on rat monocytes.16 In man, treat-ment with methyl prednisolone reduced upreg-ulated, but not basal, CD11b expression onneutrophils after cardiopulmonary bypass, withno effect upon CD11a and CD11c.19 No studieshave addressed this question on Eøs.
In the present study DEX was found toreduce both basal CD11b expression on Eø cellsurface, and this reduction explained the lowerlevels measured after cell activation with eotax-
in. Therefore, the glucocorticoid affected thebasal level of the antigen expressed on the Eøplasma membrane but not the cellular respon-siveness to eotaxin. DEX effect was not due toan alteration of Eø viability. Further, it was notsecondary to a non-speci� c suppression of Eøgenomic activity as levels of a different integrin(CD49d) were not modi�ed with Eø incubationwith DEX. However, increased avidity, withoutalterations in total expression, of CD49d hasbeen reported following exposure to chemo-kines.20 It would therefore be of interest toinvestigate whether DEX exerted an effect onb1 avidity states.
From these initial data we cannot identify themolecular mechanism by which DEX reducesCD11b expression on human Eøs. It could bethe result of reduced gene transcription (whichmay be the case for CD54 as its promoterregion contains glucocorticoid response ele-ments (GRE)21 or, an effect on mRNA or proteinstability. The former hypothesis appears to beunlikely since GRE in the promoter region ofthe human CD11b gene has not been de-scribed.22 However, an indirect mechanism ofDEX through transcription factors or on CD11bvesicular turnover cannot be excluded.
Eø recruitment in experimental models ofallergic in�ammation relies on CD11b functionas demonstrated in vitro on Eø transendothelialmigration,6 and in vivo on eotaxin-induced Eøaccumulation.23 Based on the data presentedhere, the possibility that DEX may affect Eøaccumulation via, at least in part, an effect onCD11b should be taken into account, especiallywhen long-lasting dose regimens are used. Thiscan be particularly true for human diseases,where glucocorticoid hormones are given forprolonged periods for the treatment of asth-matic and allergic rhinitis.10
In conclusion, we believe the results fromthis study are important in understanding theanti-in�ammatory mechanism(s) of actions ex-erted by glucocorticoid hormones in diseases ofallergic in�ammation.
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ACKNOWLEDGEMENTS. This work was supported by the Wellcome Trust;R.J.F. is a principal fellow of the Wellcome Trust and M.P. is a postdoctoralfellow of the Arthritis and Rheumatism Council. The authors would like tothank Drs M. Robinson and A. Shock for helpful discussions.
Received 4 August 1997;accepted in revised form 29 August 1997
