(Circulation. 1997;96:295-301.)
© 1997 American Heart Association, Inc.
Articles |
Glucocorticoid Receptor Regulates Expression of L-Selectin and CD11/CD18 on Human Neutrophils
From the Research Center, Maisonneuve-Rosemont Hospital, Department of Medicine, University of Montréal, Québec, Canada.
Correspondence to János G. Filep, MD, Research Center, Maisonneuve-Rosemont Hospital, Department of Medicine, University of Montréal, 5415 Boulevard de l'Assomption, Montréal, Québec, Canada H1T 2M4.
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Abstract |
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Background Recent studies have raised the hypothesis that glucocorticoids could diminish the ability of endothelial cells to direct leukocyte traffic into inflamed tissues by inhibiting expression of the adhesion molecules endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1. The aim of the present study was to investigate whether glucocorticoids also regulate the expression of L-selectin and CD11/CD18 integrins on human neutrophil granulocytes.
Methods and Results Incubation of human whole blood with platelet-activating factor (PAF, 1 µmol/L) evoked downregulation of L-selectin and upregulation of CD11/CD18 adhesion receptors on neutrophils as measured by flow cytometry. While dexamethasone (0.1 nmol/L to 100 µmol/L) did not affect expression of adhesion molecules on resting neutrophils, it attenuated the PAF-induced changes in L-selectin and CD18 expression in a time- and concentration-dependent fashion with IC50 values of 31 and 13 nmol/L, respectively. These effects of dexamethasone were completely aborted by RU-486 (10 µmol/L), which blocks transcriptional activation of the glucocorticoid receptor, and by the protein synthesis inhibitor cycloheximide (35.5 µmol/L). Dexamethasone, up to a concentration of 1 µmol/L, neither affected significantly the release of granule enzymes nor interfered with PAF binding to its membrane receptors.
Conclusions Our results show that glucocorticoids at clinically relevant concentrations exert specific actions on expression of adhesion molecules on activated neutrophils, which are mediated through ligation of glucocorticoid receptors and induction of protein synthesis, and suggest a novel mechanism by which anti-inflammatory corticosteroids may inhibit leukocyte accumulation.
Key Words: leukocytes • receptors • proteins • L-selectin • CD11/CD18
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Introduction |
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Extravasation and activation of neutrophil granulocytes are essential in the inflammatory response. In some diseases such as autoimmune arthritides and vasculitis, the connection between neutrophil accumulation and exacerbation of the disease has been well documented.1 2 Leukocyte extravasation into inflamed areas involves complex interaction of leukocytes with endothelium through regulated expression of surface adhesion molecules. Among them, L-selectin (CD62L) appears to play a key role in the initial attachment of circulating leukocytes to endothelium.3 4 5 This adhesion molecule is constitutively expressed by most leukocytes and recognizes carbohydrate determinants found in several endothelial cell ligands, including GlyCAM-1,6 CD34,7 and P- (GMP-140) and E-selectins (endothelial-leukocyte adhesion molecule-1, ELAM-1).8 L-Selectin is rapidly shed after neutrophil activation with a concomitant upregulation of Mac-1(CD11b/CD18).9 10 The CD18 integrins Mac-1 and LFA-1(CD11a/CD18) are believed to be largely responsible for subsequent tightening of adhesion and transendothelial migration of neutrophils through interactions with their endothelial counterreceptors intercellular adhesion molecule (ICAM)-1 and ICAM-2.3
Inhibition of leukocyte accumulation in inflamed tissues and leukocytosis are well-known effects of anti-inflammatory corticosteroids in humans. Although glucocorticoids are widely used for the control of inflammatory human diseases, the mechanisms by which they reduce leukocyte influx and inflammation are poorly understood. Among other hypotheses, inhibition of phospholipases,11 12 inhibition of the transcription of various cytokines,13 14 15 and stabilization of lysosomal and other cell membranes16 have been proposed as major mechanisms of anti-inflammatory action of glucocorticoids. However, none of these hypotheses is sufficient to account for their effects on leukocyte traffic into inflamed tissues. Recent studies have demonstrated that dexamethasone and cortisol markedly attenuated adhesion of neutrophils to endotoxin-activated human endothelial cells through inhibition of the expression of ELAM-1 and ICAM-1.17 These studies, however, did not examine the possibility that glucocorticoids could also affect expression of adhesion molecules on human neutrophils.
In the present experiments, we studied the effects and the mechanisms of action of dexamethasone on the expression of L-selectin and CD11/CD18 integrins on human neutrophils challenged with platelet-activating factor (PAF). PAF was chosen as a stimulus because enhanced PAF production is one of the earliest events in microvascular injury18 19 and because membrane-associated PAF on activated endothelial cells may serve as a signal for neutrophils to bind to the endothelium.20 21
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Methods |
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Whole-Blood Incubation
Venous blood (10 mL, anticoagulated with sodium heparin 50 U/mL blood) was obtained by antecubital venipuncture from nonsmoking healthy volunteers (men and women, 24 to 56 years of age) who had not taken any drugs for at least 10 days before the experiments. Informed consent was obtained from all volunteers before participation in the study, and the protocol was approved by the Clinical Research Committee. White blood cell counts were between 5000 and 9000 cells/µL. Whole-blood aliquots (100 µL) were transferred to polypropylene centrifuge tubes, placed on a rotator, and incubated for 10 or 180 minutes at 37°C, 95% air/5% CO2 in the presence of dexamethasone (dexamethasone 21-phosphate disodium salt, 0.1 nmol/L to 100 µmol/L, Sigma Chemical Co), RU-486 (10 µmol/L, a gift from Dr E.E. Baulieu), and cycloheximide (35.5 µmol/L [10 µg/mL], Sigma) as indicated. Then, the cells were challenged with PAF (1 µmol/L, 1-O-hexadecyl-2-acetyl-sn-glycero-3-phosphocholine, Calbiochem) for 30 minutes at 37°C. Preliminary experiments showed that maximal changes in the expression of adhesion molecules can be achieved with 1 µmol/L PAF after 30 minutes of incubation. Although this incubation time was longer than that required to observe maximal responses of isolated neutrophils to PAF (see below), we have studied adhesion molecule expression on whole blood rather than isolated neutrophils to maintain the physiological environment and to avoid upregulation of CD11b/CD18 and a loss of L-selectin from the neutrophil surface during the isolation and purification procedures.22 Our preliminary experiments also showed that 10 µmol/L RU-486 was required to block completely the actions of dexamethasone in whole blood (ie, in the presence of albumin, to which the drug binds extensively23 ). This concentration is similar to that used in previous in vitro studies17 and lower than those detected in the plasma (50 to 200 µmol/L) of healthy volunteers after single oral administration of the drug.23
Flow Cytometry Analysis
At the end of the incubation period, saturating
concentrations of FITC-conjugated mouse anti-human L-selectin
monoclonal antibody (clone, DREG-56, Pharmingen) and
R-phycoerythrin–conjugated mouse monoclonal antibody directed against
the common ß-subunit (CD18) of human leukocyte ß2
integrins (clone, MEM-48, Monosan) were added to the pretreated
whole-blood samples. After incubation for 30 minutes at 22°C,
erythrocytes were lysed and leukocytes were fixed with 2 mL of a lysing
medium (FACS Lysing Solution, Becton Dickinson). The samples were then
centrifuged, washed, and resuspended in PBS containing 0.1%
sodium azide and stored at 4°C in the dark until fluorocytometric
analysis. Negative controls were obtained by omitting
monoclonal antibodies. Class-matched irrelevant FITC-conjugated mouse
IgG1 (Pharmingen) and R-phycoerythrin–conjugated mouse
IgG1 (clone, MOPC-21, Monosan) were used to evaluate
nonspecific antibody binding. Double-color
immunofluorescence staining was analyzed by
a cytofluorometer (FACScan, Becton Dickinson Immunocytometry System)
with Lysis II software. Data from 10 000 events per sample were
acquired. Antibody binding was determined as mean fluorescence
intensity after gating for neutrophils by their characteristic forward
and side scatter properties. The results are presented as
relative fluorescence units (RFU):
RFU=(FUexperimental-FUisotype)x100/(FUcontrol-FUisotype),
where FUexperimental and FUcontrol are the CD18
or L-selectin fluorescence intensity of treated cells and cells
cultured in medium only, respectively, and FUisotype is the
fluorescence intensity of class-matched irrelevant
antibody.
Isolation of Neutrophil Granulocytes
Neutrophil granulocytes were isolated from
peripheral blood by centrifugation through
Ficoll/Hypaque gradients (Pharmacia), sedimentation through dextran
(6% wt/vol), and hypotonic lysis of erythrocytes. The resultant cell
preparation contained >97% neutrophils with few contaminating
erythrocytes and platelets. Neutrophils were suspended in a
modified Hanks' balanced salt solution consisting of 145 mmol/L
NaCl, 10 mmol/L K2HPO4, 1.4 mmol/L
CaCl2, 1.2 mmol/L MgCl2, 10 mmol/L
glucose, and 250 µg/mL human serum albumin, pH 7.4. Cell
viability, estimated at the end of the experiments by trypan blue
exclusion, was >98%.
Enzymatic Activity in Supernatants From
Dexamethasone-Treated Neutrophils
Neutrophils (5x106 cells/mL) preincubated with
dexamethasone or its vehicle were treated with 10.4
µmol/L (5 µg/mL) cytochalasin B for 10 minutes and challenged with
PAF (1 µmol/L) for 10 minutes at 37°C. Then, cells were
pelleted by centrifugation, and supernatants were
assayed for ß-glucuronidase, lysozyme, and lactate dehydrogenase as
described.24 Results are reported as net enzyme release,
ie, the percentage of total cellular enzyme released by challenged
minus that released by identically treated but unchallenged cells.
Measurement of Prostaglandin E2 and
Leukotriene B4 Production
After activation of neutrophils with PAF for 10 minutes,
two volumes of ice-cold methanol was added to the tubes. After
centrifugation, the supernatants were collected and
evaporated under a stream of nitrogen and redissolved in assay buffer.
Concentrations of prostaglandin E2
(PGE2) and leukotriene B4
(LTB4) were measured by specific radioimmunoassays (IZINTA
and Amersham International, respectively). The PGE2
antiserum has 14% cross-reactivity with prostaglandin
E1 and <0.9% cross-reactivity with other prostanoids. The
LTB4 antiserum has <0.03% cross-reactivity with other
eicosanoids and arachidonic acid. The intra-assay
coefficients of variation were 5% and 8%, respectively.
Quantification of PAF binding
After preincubation with dexamethasone at 37°C for
indicated times, cell suspensions were cooled rapidly to 4°C. Binding
of [3H]-PAF (New England Nuclear) to neutrophils was
assayed as described previously.22 Nonspecific binding was
defined as the binding that was not inhibited by a 500-fold molar
excess of PAF over labeled PAF. Previous results indicated that under
the present experimental conditions, neutrophils do not metabolize
[3H]-PAF.24
Statistical Analysis
Results are expressed as mean±SEM. Binding data were
analyzed with the LIGAND software.25 Statistical
comparisons were made by ANOVA with ranks (Kruskal-Wallis test)
followed by Dunn's multiple contrast hypothesis test to identify
differences between various treatments or by the Mann-Whitney test for
unpaired observations. Values of P<.05 were considered
significant for all tests.
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Results |
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Dexamethasone Modulates Expression of L-Selectin and CD18 Integrins on PAF-Stimulated Neutrophils
Incubation of whole blood with PAF produced a significant decrease in L-selectin and a marked upregulation of CD18 adhesion receptors on neutrophils. Pretreatment of whole blood with dexamethasone for 10 minutes did not significantly affect the shedding of L-selectin and the upregulation of CD18 in response to 1 µmol/L PAF (Fig 1A
). On the other hand, 180 minutes of preincubation
with dexamethasone resulted in a concentration-dependent
attenuation of the PAF (1 µmol/L)–induced downregulation of
L-selectin and increased expression of CD18 with IC50
values of 31 and 13 nmol/L, respectively (Fig 1B). Fig 2
shows a representative experiment of the effect of
dexamethasone (1 µmol/L) on PAF-induced changes in
the expression of L-selectin and CD18. Dexamethasone, even
at a concentration as high as 100 µmol/L, failed to alter
significantly basal expression of these adhesion molecules after 210
minutes of incubation (mean fluorescence intensity for
L-selectin: dexamethasone, 215±17 versus medium only,
217±21; CD18: dexamethasone, 38±4 versus medium only,
41±3; n=6; both P>.1) or binding of isotype-matched
irrelevant antibodies (data not shown).
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To determine whether the modulation of expression of adhesion molecules
by dexamethasone was receptor mediated, we studied the
effects of RU-486, a noncompetitive antagonist of
glucocorticoid receptors. RU-486 at 10 µmol/L almost completely
reversed the effects of dexamethasone (1 µmol/L) on
the PAF-induced shedding of L-selectin and upregulation of CD18 (Fig 3). Furthermore, the protein synthesis
inhibitor cycloheximide (35.5 µmol/L) also reversed
the inhibitory action of dexamethasone (Fig 3).
Neither RU-486 nor cycloheximide had any effect on basal expression of
adhesion molecules (data not shown) and on the PAF-induced changes in
the expression of L-selectin and CD18 (Fig 3).
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Effects of Dexamethasone on Neutrophil
Activation
Release of ß-glucuronidase and lysozyme was used as a marker of
the azurophil and specific plus azurophil granule contents,
respectively. At lower concentrations, dexamethasone had no
significant effect on PAF-stimulated ß-glucuronidase and lysozyme
release, whereas a small, statistically significant inhibition was
detected at 100 µmol/L (see the Table). Lactate
dehydrogenase release never exceeded 2% of total cell content and was
similar in the absence and presence of dexamethasone (data
not shown), indicating that dexamethasone did not affect
cellular integrity.
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Dexamethasone inhibited the PAF (1
µmol/L)–stimulated release of PGE2 and LTB4
from neutrophils in a dose-dependent fashion with IC50
values of 8 and 11 nmol/L, respectively (Fig 4). The
inhibitory action of dexamethasone (1
µmol/L) was reversed by both RU-486 (10 µmol/L) and
cycloheximide (35.5 µmol/L). Dexamethasone (1
µmol/L) decreased PAF-induced PGE2 release from 4.6±0.5
to 1.5±0.3 ng/107 cells over 10 minutes (n=4,
P<.05), whereas neutrophils treated with RU-486 plus
dexamethasone and cycloheximide plus
dexamethasone released 4.4±0.5 and 4.3±0.4 ng
PGE2/107 cells over 10 minutes in response to
PAF, respectively (both P>.1 versus PAF-challenged cells).
LTB4 release was 90±16, 18±6, 88±12, and 86±10
pg/107 cells over 10 minutes (n=4) in response to PAF from
untreated, dexamethasone-treated, RU-486 plus
dexamethasone–treated, and cycloheximide plus
dexamethasone–treated neutrophils, respectively.
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Effect of Dexamethasone on Binding of PAF to
Neutrophils
To examine the possibility that dexamethasone could
interfere with binding of PAF to its receptors, the ability of
dexamethasone to compete with [3H]-PAF
binding to neutrophils was studied. Dexamethasone up to a
concentration of 1 µmol/L had no significant effect on the
specific binding of PAF to neutrophils, whereas at 10 to 100
µmol/L, a concentration-dependent inhibition was observed (Fig 5A). The degree of inhibition was similar when
neutrophils were preincubated with dexamethasone for 10 or
180 minutes. Nonspecific binding was barely inhibited even by the
highest concentrations of dexamethasone. Scatchard
analysis of PAF binding indicated that
dexamethasone 100 µmol/L decreased the affinity of
PAF for its high-affinity receptors (Kd,
1.3±0.5x10-10 and
5.5±1.2x10-10 mol/L in the absence and
presence of dexamethasone, respectively; n=4;
P<.05) without affecting the number of these receptors
(Bmax, 5.3±2.2x10-12 and
5.1±2.3x10-12 mol/L, respectively; Fig 5B).
The number of low-affinity binding sites for PAF (Bmax,
1.0±0.6x10-9 versus
0.9±0.4x10-9 mol/L) and binding affinity
(Kd, 2.6±1.4x10-8
versus 2.8±1.3x10-8 mol/L) were unaffected
by dexamethasone (Fig 5B).
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The principal observation of this study is that although ligation of glucocorticoid receptors has no detectable effect on expression of adhesion molecules on resting neutrophils, it attenuates downregulation of L-selectin and upregulation of CD11/CD18 adhesion molecules on human neutrophils in response to activation.
Incubation of whole blood with dexamethasone up to 100 µmol/L for up to 3 hours under nonstatic conditions did not affect the expression of L-selectin and CD11/CD18 on resting neutrophils. These observations are consistent with previous studies demonstrating unchanged expression of L-selectin on isolated human neutrophils after preincubation with 0.39 mmol/L (0.2 mg/mL) dexamethasone for 15 minutes.26 On the other hand, our results appear to differ from those of Burton et al,27 who found that in vivo glucocorticoid treatment induced significant downregulation of both L-selectin and CD18 expression on resting bovine neutrophils. However, these effects of glucocorticoids became detectable only 8 to 16 hours and 2 to 3 days after treatment, respectively. The delayed response, combined with the fact that neutrophils reside in the peripheral blood for only a few hours, would suggest that in cows glucocorticoids affected neutrophil precursors in bone marrow rather than circulating neutrophils.
The present results show that dexamethasone attenuated PAF-induced downregulation of L-selectin and upregulation of CD11/CD18 expression in a time- and concentration-dependent manner with a maximum inhibition of about 50% and 60%, respectively. Because prolonged (3-hour) incubation of whole blood with dexamethasone was required to detect an inhibitory action, one may assume that the inhibition observed might be attributed to induction of de novo synthesis of secondary regulatory elements (see below). The IC50 values for these actions of dexamethasone are lower than those required to inhibit cytokine production by human lung parenchyma (IC50, 100 to 200 nmol/L)14 15 but are one order of magnitude higher than those reported to cause 50% inhibition of endotoxin-induced expression of ELAM-1 and ICAM-1 on endothelial cells.17 These differences might be attributed to different mechanisms underlying the effects of glucocorticoids on genes, which contain or lack (eg, the gene for ELAM-1) glucocorticoid-responsive elements. In our study, dexamethasone was effective in modulating L-selectin and CD11/CD18 expression at nanomolar concentrations that can be detected in the plasma of patients undergoing long-term treatment with glucocorticoids.28
In regard to the mechanisms by which dexamethasone attenuated changes in L-selectin and CD18 expression in response to PAF, the following should be considered. Although at concentrations >1 µmol/L dexamethasone significantly inhibited specific binding of [3H]-PAF to its neutrophil receptors, competition with PAF binding cannot be accounted for the reversal of expression of adhesion molecules, for dexamethasone at concentrations that caused near-maximum attenuation of PAF-induced L-selectin downregulation and CD11/CD18 upregulation did not significantly affect PAF binding. Downregulation of PAF binding by dexamethasone may contribute to the acute effects of high-dose corticosteroid treatment. Furthermore, it is unlikely that dexamethasone stabilized neutrophil lysosomal and other cell membranes.16 Dexamethasone up to a concentration of 10 µmol/L had no significant effect on the release of ß-glucuronidase and lysozyme from neutrophils challenged with PAF compared with its effect at nanomolar concentrations on expression of adhesion molecules. Because the most readily mobilizable store of CD11b/CD18 is in a granule distinct from the classic azurophil and secondary granules,29 significant upregulation of CD18 can occur without degranulation of azurophil and secondary granules. Therefore, it is possible that this pool of CD11b/CD18 was responsible for upregulation of CD18 that can be inhibited by dexamethasone. The demonstration that RU-486 reverses the effects of dexamethasone on the expression of adhesion molecules and eicosanoid production is consistent with the hypothesis that the effects of glucocorticoids on neutrophils are mediated through glucocorticoid receptors. RU-486 blocks transcriptional activation of the glucocorticoid receptor and thereby inhibits transcription of genes containing glucocorticoid-responsive elements.30 31 Like RU-486, cycloheximide also partially prevented the effects of dexamethasone on the expression of L-selectin and CD11/CD18, suggesting a role for endogenous protein(s) in mediating these actions. Among these proteins, lipocortin-1, a member of the annexin superfamily, may be of particular interest. Glucocorticoids induce the formation and expression of lipocortin-1 in humans.32 Recombinant human lipocortin-1 has powerful anti-inflammatory effects in experimental animals such as the reduction of eicosanoid release33 and inhibition of neutrophil migration.34 Because dexamethasone inhibited PAF-induced eicosanoid release and upregulation of CD11/CD18 expression with similar IC50 values and these effects can be prevented by cycloheximide, one may assume that these actions of dexamethasone might be mediated by the same mechanism. However, it remains to be investigated whether lipocortin-1 could mimic the effects of glucocorticoids on the expression of adhesion molecules. Because the shedding of L-selectin in activated neutrophils takes place through a proteolytic cleavage,9 it is tempting to speculate that glucocorticoids induce the synthesis of a protein that could inhibit this proteolytic enzyme. The findings that neither RU-486 nor cycloheximide affected expression of L-selectin and CD11/CD18 on resting and PAF-stimulated neutrophils would argue against a nonspecific action of these agents on adhesion molecules.
Although shedding of L-selectin and upregulation of ß2 integrins are considered consecutive steps in neutrophil fixation and extravasation, recent studies with L-, E- or P-selectin–deficient mice raised the possibilities that the functions of adhesion molecules overlap and that they do not act sequentially but rather simultaneously.35 36 37 Furthermore, neutrophils can infiltrate certain tissues through a CD18-independent mechanism.38 39 40 41 Accordingly, adhesion molecule requirements for neutrophil accumulation may, to some extent, be organ specific. Therefore, the reversal of both L-selectin and CD11/CD18 expression by dexamethasone is particularly remarkable. The importance of these actions is underlain by the observation that treatment of healthy volunteers with glucocorticoids for 2 hours resulted in a marked inhibition of the adhesiveness of ex vivo neutrophils to nylon fibers.42 It is conceivable that the action of glucocorticoids on L-selectin and CD11/CD18 expression on activated neutrophils in combination with their effects on the expression of ELAM-1 and ICAM-1 on endothelial cells17 might account, at least in part, for the inhibition of adhesion of neutrophils to the endothelium and consequently for neutrophil migration into inflamed tissues. This may explain the beneficial effects of glucocorticoids in conditions such as autoimmune vasculitis, in which exacerbation of the disease is closely associated with neutrophil accumulation.1 2
In summary, our results demonstrate that dexamethasone at clinically relevant concentrations could partially reverse PAF-induced downregulation of L-selectin and upregulation of CD11/CD18 expression on human neutrophils and indicate that these actions of dexamethasone are mediated through ligation of glucocorticoid receptors and induction of protein synthesis. These observations represent a novel mechanism by which glucocorticoids could affect leukocyte adhesion to endothelial cells and their migration into inflamed tissues.
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Acknowledgments |
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This work was supported by grants from the Medical Research Council of Canada (MT-12573) and the Foundation of the Maisonneuve-Rosemont Hospital. Dr Filep is a Senior Scholar of the Fonds de la Recherche en Santé du Québec. We thank Dr E.E. Beaulieu (Institut National de la Santé et de la Recherche Médicale, Paris, France) for supplying us with RU-486.
Received August 26, 1996; revision received December 16, 1996; accepted January 20, 1997.
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