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Research Center, Maisonneuve-Rosemont Hospital and Department of Medicine, University of Montréal, Montréal, Québec, Canada H1T 2M4
1Correspondence: Research Center, Maisonneuve-Rosemont Hospital, 5415 blvd. de l’Assomption, Montréal, Québec, Canada H1T 2M4. E-mail: filepj{at}ere.umontreal.ca
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: lipocortin-1-derived peptides • L-selectin • integrin • neutrophil recruitment • inflammation
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and
neutrophil granulocytes (PMNs) (2)
. Glucocorticoid
injection in humans induces synthesis and surface expression of
lipocortin-1 on PMNs and monocytic cells (3
, 4)
.
Lipocortin-1 has been suggested to act as the ‘second messenger’ for
the glucocorticoid inhibition of leukocyte migration (5)
.
Recent evidence indicates that several regions of the lipocortin-1
molecule may be associated with biological activity.
Antiflammins, discovered as inhibitors of phospholipase
A2, are synthetic nonapeptides corresponding to a
region of high amino acid sequence similarity between lipocortin-1 and
uteroglobin (6)
. Antiflammin-1 (MQMKKVLDS) is equivalent
to the carboxyl-terminal part of 
-helix three in uteroglobin
(7)
, whereas antiflammin-2 (HDMNKVLDL) corresponds to
residues 246–254 of lipocortin-1 (8)
. Antiflammins show
potent anti-inflammatory effects. Although an apparent controversy
exists whether antiflammins can or cannot affect phospholipase
A2 activity (6
, 9
10
11)
, in
vitro, they inhibit synthesis of platelet-activating factor (PAF)
(12)
, PMN and mononuclear cell chemotaxis and aggregation
(12
, 13)
, and platelet aggregation (14)
.
Antiflammins also reduce the increase in vascular permeability and
leukocyte infiltration induced in rats by an Arthus reaction or by
intradermal injection of C5a (12)
, and inhibit murine ear
edema (15)
and endotoxin-induced uveitis in rats
(16)
. These studies raised the possibility that
antiflammins might be potent regulators of leukocyte trafficking, but
provided little insight into the underlying mechanisms.
Leukocyte extravasation into inflamed areas is a multistep process that
is regulated by several adhesion molecules (17
, 18)
. The
initial capture and tethering of circulating PMNs to endothelium is
mediated by L-selectin (CD62L) constitutively expressed by most
leukocytes (19
, 20)
and by P- and E-selectins expressed by
activated endothelium (17
, 18)
. L-selectin is rapidly shed
after cell activation with a concomitant up-regulation of Mac-1
(CD11b/CD18) (21)
. The ß2
integrins Mac-1 and LFA-1 (CD11a/CD18) are largely responsible for
subsequent tightening of the adhesion and transendothelial migration of
PMNs via interactions with their endothelial counterreceptors,
intercellular adhesion molecule-1 (ICAM-1) and ICAM-2 (17
, 18)
. PAF and interleukin-8 (IL-8) are thought to serve as
signals for PMNs to promote activation of ß2
integrins, leading to firm adhesion (22
, 23)
.
In the present experiments, we studied the effect and the cellular mechanisms of action of antiflammins on expression of adhesion molecules on human leukocytes and human coronary artery endothelial cells (HCAEC) and on binding of PMNs to HCAEC.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; anti-E-selectin mAb ENA-2
(IgG1, purified F(ab')2
fragments, Monosan) at 10 µg/ml (25)
; and anti-CD18 mAb
L130 (IgG1, Becton-Dickinson Immunocytometry
Systems, Mountain View, Calif.) at 10 µg/ml (26)
. The
irrelevant mAb MOPC-21 (IgG1, PharMingen) at 20
µg/ml was used as a negative control.
Synthetic antiflammin-1 and antiflammin-2 were obtained from Bachem
Bioscience (King of Prussia, Pa.). A scrambled sequence of
antiflammin-2 (MLNHKLDVD, synthesized by Biosynthesis Inc., Lewisville,
Tex.) and the unrelated synthetic peptide VPVEAVNPM corresponding to
residues 24–32 of the rat cholecystokinin prepro-sequence (Bachem)
were used as controls. Purity of the peptides, as analyzed by the
manufacturer, was >98%, amino acid composition, and molecular mass
were confirmed by mass spectrometry. Lipopolysaccharide (LPS,
Escherichia coli O111:B4) and dexamethasone 21-phosphate
were obtained from Sigma Chemical Co. (St. Louis, Mo.), PAF was from
Calbiochem (La Jolla, Calif.); human recombinant IL-8 was purchased
from R&D Systems (Minneapolis, Minn.). Recombinant human lipocortin-1
(8)
was a gift from Dr. R. B. Pepinsky (Biogen,
Cambridge, Mass.).
Whole blood incubation
Venous blood (anticoagulated with sodium heparin 50 U/ml) was
obtained from nonsmoking healthy volunteers (male and female, 24–45
years of age) who had not taken any drugs for at least 10 days before
the experiments. Informed consent was obtained from each volunteer, and
the protocol was approved by the Clinical Research Committee. White
blood cell counts were between 4500 and 9000 cells/µl. Whole blood
aliquots were incubated with various concentrations of antiflammin-1,
antiflammin-2, or lipocortin-1 for 30 min at 37°C, 95% air/5%
CO2 and then challenged with PAF (1 µmol/l) or
IL-8 (10 nmol/l) for 30 min.
Analysis of surface antigen expression
Direct immunofluorescence labeling of resting and treated
leukocytes in whole blood was performed as described (2
, 26)
. Leukocytes were stained with saturating concentration of
fluorescence dye-conjugated anti-human L-selectin or anti-human CD18
mAb. Nonspecific binding was evaluated by using appropriately labeled
mouse IgG1. Double- or single-color
immunofluorescence staining was analyzed by a cytofluorometer (FACScan,
Becton Dickinson) with Lysis II software. Antibody binding was
determined as mean fluorescence intensity after gating for PMNs,
monocytes, and lymphocytes by their characteristic forward and side
scatter properties.
Culture of endothelial cells
Normal HCAEC obtained from Clonetics Corp. (San Diego, Calif.)
were cultured as described (26)
. HCAEC (passages 3 to 6)
seeded into 24-well or 96-well microplates and grown to confluence were
used in the experiments. Two days before the experiments, the cells
were cultured in RPMI 1640 medium supplemented with 10% fetal bovine
serum only.
Expression of E-selectin and ICAM-1
After incubation for 4 h at 37°C in a 5%
CO2 atmosphere with LPS (1 µg/ml) in the
absence or presence of antiflammin-1, antiflammin-2 (100 µmol/l), or
dexamethasone (100 nmol/l), HCAEC were removed from the 24-well
microplates by exposure to EDTA (0.01%) in phosphate-buffered saline
(PBS) for 10 min at 37°C, followed by gentle trituration. Cells were
resuspended in ice-cold saline containing sodium azide (0.02%),
incubated with saturating concentration of fluorescein dye-conjugated
anti-E-selectin or anti-ICAM-1 mAb for 30 min at 4°C, washed, fixed
in formaldehyde (3.7% in PBS), and immunofluorescence was analyzed
with a cytofluorometer. Nonspecific binding was evaluated by using
appropriately labeled class-matched, irrelevant mouse
IgG1.
Neutrophil-endothelial cell adhesion assay
The adhesion assay was performed as described previously
(26)
. In brief, monolayers of HCAEC in 96-well microplates
were stimulated with LPS (1 µg/ml) with or without antiflammin-1,
antiflammin-2 (100 µmol/l), or dexamethasone (100 nmol/l) for 6 h at 37°C in a 5% CO2 atmosphere. The cells
were then washed three times and 2 x 105
51Cr-labeled PMNs in 100 µl were added. In some
experiments, PMNs were preincubated with antiflammin-1, antiflammin-2
for 30 min or with dexamethasone (100 nmol/l) for 120 min before
addition to HCAEC. In another set of experiments, LPS-activated HCAEC
were incubated for 15 min with ENA-2 or MOPC-21 mAb before addition of
PMNs. Radiolabeled PMNs were incubated with DREG-56, L130, or MOPC-21
mAb for 15 min before addition to HCAEC. After incubation of HCAEC with
PMNs for 30 min at 37°C on an orbital shaker at 90 rpm, loosely
adherent or unattached leukocytes were washed three times, and the
endothelial monolayer plus the adherent PMNs were lysed in 200 µl of
0.1% Triton X-100. The number of adhered PMNs in each experiment was
estimated from the radioactivity of a control sample. Treatment of
HCAEC with antiflammins did not affect the integrity of viable
endothelial monolayers.
Statistical analysis
Results are expressed as means ± SE.
Statistical comparisons were made by analysis of variance using ranks
(Kruskal-Wallis test), followed by Dunn’s multiple contrast hypothesis
test to identify differences between various treatments or by the
Mann-Whitney U test for unpaired observations. P
values <0.05 were considered significant for all tests.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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reports representative results illustrating the effects of
antiflammin-2 on PAF-activated neutrophils. Addition of ‘scrambled’
antiflammin-2 (MLNHKLDVD) (Fig. 1)
or the nonapeptide corresponding to
residues 24–32 of the rat prepro-cholecystokinin (data not shown) had
no effect on PAF-induced changes. Preincubation of blood with
antiflammin-1 or antiflammin-2 inhibited PAF-induced down-regulation of
L-selectin and up-regulation of CD18 expression on PMNs, monocytes, and
lymphocytes in a concentration-dependent fashion (Fig. 2
). The apparent IC50 values for antiflammin-1 and
antiflammin-2 were similar for PMNs (6.3 and 4.7 µmol/l,
respectively), monocytes (9.5 and 4.8 µmol/l, respectively), and
lymphocytes (5.2 and 7.8 µmol/l, respectively). Addition of
lipocortin-1 to whole blood gave a concentration-dependent attenuation
of PAF-induced changes in leukocyte adhesion molecule expression
(Fig. 3
). The apparent maximum inhibition that can be achieved with
antiflammins was similar to that detected with lipocortin-1 (Fig. 3)
and to that we had previously found with dexamethasone
(2)
. Denaturation of lipocortin-1 resulted in a complete
loss of its inhibitory actions (data not shown). As with PAF, both
antiflammin-1 and antiflammin-2 partially blocked IL-8-induced changes
in L-selectin and CD18 expression by PMNs with apparent
IC50 values of 9 and 18 µmol/l, respectively
(Fig. 4
). Immunostaining of leukocytes with an anti-CD11b mAb revealed changes
similar to those observed with the anti-CD18 mAb (data not shown).
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Effect of antiflammins on expression of E-selectin and ICAM-1 on
LPS-stimulated endothelial cells
After stimulation by LPS, HCAEC increased on average 25.8-fold and
2.2-fold the expression of E-selectin and ICAM-1, respectively
(n=3, both P<0.05) (Fig. 5
). Neither antiflammin-1 nor antiflammin-2 affected expression of these
adhesion molecules on unstimulated HCAEC (data not shown). Furthermore,
antiflammin-2 produced only a slight inhibition of LPS-induced changes
(Fig. 5)
. The maximum inhibition did not exceed 7%. Similar results
were obtained with antiflammin-1 (data not shown). Treatment of HCAEC
with dexamethasone (100 nmol/l) inhibited ~60% of the LPS-stimulated
expression of E-selectin and ICAM-1 (Fig. 5)
without altering basal
expression of these molecules (data not shown).
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Antiflammins inhibit PMN adhesion to endothelial cells
Activation of HCAEC with LPS resulted in a 3.7-fold increase in
the number of adherent PMNs (Fig. 6A
). Culture of HCAEC with LPS in the presence of
antiflammin-1 or antiflammin-2 produced only slight decreases in
adhesion, whereas culture of HCAEC with LPS in the presence of
dexamethasone (100 nmol/l) resulted in (on average) a 40% decrease in
the number of adherent PMNs (Fig. 6A
). Pretreatment of PMNs
with either antiflammin-1 or antiflammin-2 before addition to
LPS-activated HCAEC attenuated their attachment to endothelial cells in
a concentration-dependent fashion, with apparent
IC50 values of 1.2 and 3.0 µmol/l, respectively
(Fig. 6B
). At 100 µmol/l, antiflammin-1 and antiflammin-2
inhibited PMN adhesion by 54 ± 4 and 43 ± 4%, respectively
(n=3, both P<0.05) (Fig. 6B
). No
adhesion experiments were done when PAF or IL-8-activated neutrophils
were added to HCAEC stimulated in the presence of antiflammins, since
both PAF and IL-8 provoke neutrophil aggregation, making interpretation
of the results difficult. ‘Scrambled’ antiflammin-2 or the rat
prepro-cholecystokinin nonapeptide (1–100 µmol/l) had no detectable
effect on the number of adherent neutrophils (data not shown).
|
Since PMN adhesion to LPS-stimulated HCAEC is mediated by multiple
adhesion receptors (26)
, we assayed the contribution of
L-selectin, E-selectin, and CD18 to the binding interaction. A
significant proportion of PMN-HCAEC attachment was blocked by mAbs
binding to L-selectin (24±4%, n=3), CD18 (27±4%), or
E-selectin (35±5%) (Fig. 7
). The combination of these mAbs inhibited PMN adhesion by ~90%.
Treatment of PMNs with antiflammin-1 and anti-CD18 mAb resulted in only
a slightly greater inhibition of adhesion than observed with PMNs
treated with either antiflammin-1 or anti-CD18 mAb (Fig. 7)
. The
combination of antiflammin-1 with either anti-L-selectin mAb or
anti-E-selectin mAb resulted in additive inhibition, and the degree of
inhibition was similar to that observed when anti-L-selectin mAb or
anti-E-selectin mAb was combined with anti-CD18 mAb, respectively (Fig. 7)
. Combining antiflammin-1, anti-L-selectin mAb, and anti-E-selectin
mAb blocked ~82% of adhesion. Similar results were obtained with
antiflammin-2 (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The glucocorticoid-inducible protein lipocortin-1 appears to be an
important negative effector of leukocyte emigration in various
experimental models (27
28
29
30)
. Three regions of the
lipocortin-1 molecule—a 188 amino acid
NH2-terminal fragment (5)
, peptide
N-acetyl-2–26 (5)
, and peptide 246–254 (termed
antiflammin-2) (6)
—have been reported to inhibit
leukocyte accumulation. In our experiments, antiflammin-2 and
antiflammin-1 (the carboxyl-terminal part of -helix three in
uteroglobin) appeared to be equally potent inhibitors. The first two
residues (which differ in antiflammin-1 and antiflammin-2) can be
replaced, but not deleted, without loss of activity, indicating that
the length of antiflammins is critical for the biological activity
(6)
. Oxidation of the Met residue in position 1 in
antiflammin-1 has been suggested to account for the rapid (within 5
min) decline in the inhibitory action of antiflammin-1 incubated with
human isolated PMNs (12)
. However, this Met residue is not
a prerequisite for activity, since it is substituted with His in
antiflammin-2 (6)
. We could not detect any significant
loss of inhibitory action of antiflammin-1 in our experiments. Besides
differences in the experimental conditions (e.g., presence of serum in
our experiments), the reasons for this apparent discrepancy are not
known at present. The inhibitory action of antiflammins observed in
this study are specific for these peptide sequences, because no
inhibitory effects were detected with the peptide MLNHKLDVD (a
scrambled sequence of antiflammin-2) or the unrelated nonapeptide
VPVEAVNPH in the assays used.
Our study documents that although antiflammins had no effect on
expression of adhesion molecules on human resting leukocytes in whole
blood in vitro, they markedly attenuated changes in
L-selectin and CD11/CD18 expression evoked by PAF or IL-8. Thus,
antiflammins can fully duplicate the actions of lipocortin-1 on
leukocyte adhesion molecule expression. Assuming that no lipocortin-1
molecule has been denaturated during the experiments (8)
,
on a molar concentration basis, 30-fold more antiflammins than native
lipocortin-1 was needed to achieve a similar degree of inhibition. It
is likely that higher concentrations of antiflammins may be needed to
occupy the putative lipocortin-1 receptor. Nevertheless, it is
impressive that these nonapeptides are active in the whole blood and
overcame interactions with blood components to specifically regulate
leukocytes. Human PMNs and monocytic cells possess specific and
saturable binding sites for lipocortin-1 (30)
that mediate
the action of lipocortin-1 on these cells. Our results demonstrate that
in addition to consolidating binding to the target cells
(31)
, the antiflammin-2 sequence (residues 246–254 in
lipocortin-1) exerts actions similar to those of the parent protein.
These findings are also consistent with our previous study, which
showed that dexamethasone attenuates by ~60% the PAF and
formyl-Met-Leu-Phe-induced changes in L-selectin and CD11b/CD18
expression by human neutrophils through induction of protein synthesis
(2)
, most likely lipocortin-1 (5)
.
Within minutes of activation with PAF or IL-8, leukocytes release
L-selectin from their surface by a proteolytic enzyme. Inasmuch as this
enzyme appears to be constitutively active, formation of an appropriate
3-dimensional structure of L-selectin near the membrane is thought to
regulate this proteolytic process (32
, 33)
. Although the
nature of conformational changes required for the cleavage is not
understood at present, our study suggests that this can be partially
prevented by antiflammins. Phosphorylation of serine in the
intracellular tail of L-selectin (34)
and calmodulin
inhibitors (35)
were reported to induce proteolytic
shedding of L-selectin. Whether the action of antiflammins involves
activation of calmodulin or interference with a phosphorylation step
remains to be investigated. Leukocyte integrins change their
conformation during cell activation with characteristics of the active
molecule (i.e., increase in the binding avidity or affinity), depending
on the method of stimulation (36
, 37)
. Activation of
leukocytes with stimuli that increase intracellular
Ca2+ concentration, such as PAF or IL-8, induce
clustering of ß2 integrins, thereby increasing
the overall strength of binding without affecting affinity
(37)
. However, it seems unlikely that antiflammins might
interfere with Ca2+ signaling, because recent
results suggest that antiflammin-2 does not inhibit calcium-dependent
mobilization of arachidonic acid (13)
. Activation of
leukocytes from the outside of the cell with Mg2+
in the presence of EGTA results in the formation of a higher affinity
form of the integrins by either unmasking the ligand binding site or by
tertiary changes within the ligand binding domain (36
, 38)
. Although both mechanisms may operate in integrins, it is
not known how these mechanisms are activated.
Despite inhibition of L-selectin shedding from PMNs, which would be
expected to promote their adhesion to HCAEC, antiflammins markedly
reduced the number of adherent neutrophils. This inhibition can be
attributed primarily to their effect on PMNs rather than on HCAEC,
because antiflammins had little effect on LPS-stimulated expression of
E-selectin and ICAM-1 on HCAEC. The antiflammins or a function-blocking
anti-CD18 mAb resulted in similar decreases in PMN adhesion to HCAEC.
Furthermore, the actions of antiflammin-1 or antiflammin-2 and
anti-CD18 mAb were not additive, indicating that inhibition of
PMN-HCAEC adherence by antiflammins is predominantly attributable to
attenuation of up-regulation of CD11/CD18 expression on PMNs. Since the
inhibition with antiflammins was additive with anti-E-selectin and
anti-L-selectin mAbs, it is unlikely that antiflammins interfered with
E-selectin or L-selectin function or ligands. Inhibition of neutrophil
adhesion by antiflammins resembles that of peptide N-acetyl-2–26 of
lipocortin-1 (5)
. This peptide can also inhibit PMN
adhesion when using a leukocyte stimulus, but not when using
endothelial cell stimulation, although the underlying mechanisms of
action are not known at present.
This and earlier studies (1
, 2)
point to similarities and
striking differences between the actions of antiflammins and
glucocorticoids. Antiflammins exert similar inhibitory actions as
glucocorticoids on activation-induced changes in adhesion molecule
expression by PMNs, resulting in attenuation of PMN adhesion to HCAEC.
By contrast, dexamethasone, but not antiflammins, inhibits LPS-induced
expression of ICAM-1 and E-selectin on human umbilical vein endothelial
cells (1)
as well as on HCAEC (present study), leading to
decreases in PMN adherence. While most of the actions of
glucocorticoids are mediated by translocation of the
glucocorticoid–glucocorticoid receptor complex to the nucleus and its
binding to genes containing glucocorticoid-responsive elements
(1
, 39)
, the absence of a glucocorticoid-responsive
element in the gene for E-selectin (40)
suggests that
glucocorticoids either interfere directly with a transcriptional
regulator or induce the synthesis of a secondary regulatory element
(1)
. The lack of effect of antiflammins on E-selectin and
ICAM-1 expression on HCAEC would suggest that lipocortin-1 may not be
this regulatory element.
Our results may have relevance to inhibition of excessive trafficking
of leukocytes both in acute and chronic inflammation. By inhibiting
up-regulation of CD11/CD18 expression, antiflammins attenuate firm
adhesion of PMNs to the endothelium and consequently their
transendothelial migration, key events for leukocyte accumulation in
tissues. Therefore, antiflammins may be useful therapeutic agents to
prevent and/or attenuate the neutrophil-mediated tissue injury that
accompanies myocardial reperfusion injury (41
, 42)
, as
well as other chronic disease states such as rheumatoid arthritis
(43)
. The mechanisms of action of antiflammins differ from
those of nonsteroid anti-inflammatory drugs or the acute-phase reactant
C-reactive protein, which inhibit PMN binding to endothelial cells by
inducing shedding of L-selectin from the leukocyte surface without
affecting CD11/CD18 expression (26
, 44)
.
In conclusion, this study demonstrates that antiflammins mimic the actions of glucocorticoids on adhesion molecule expression on human leukocytes, but not on endothelial cells, and attenuate PMN adhesion to HCAEC via inhibition of cell activation-induced changes in CD11/CD18 expression. Therefore, antiflammins may represent a novel therapeutic approach in blocking leukocyte trafficking in host defense and inflammation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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regulates the expression of the adhesion molecule ELAM-1 and IL-6 production by human endothelial cells in vitro. J. Immunol. 145,2110-2114[Abstract]
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