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Blood, Vol. 94 No. 12 (December 15), 1999:
pp. 4132-4142
Anti-Inflammatory Actions of Lipoxin A4 Stable Analogs Are
Demonstrable in Human Whole Blood: Modulation of Leukocyte Adhesion
Molecules and Inhibition of Neutrophil-Endothelial Interactions
By
János G. Filep,
Christine Zouki,
Nicos A. Petasis,
Mohamed Hachicha, and
Charles N. Serhan
From the Research Center, Maisonneuve-Rosemont Hospital, Department
of Medicine, University of Montréal, Montréal,
Québec, Canada; the Department of Chemistry, University of
Southern California, Los Angeles, CA; and the Center for Experimental
Therapeutics and Reperfusion Injury, Department of Anesthesia, Brigham
and Women's Hospital and Harvard Medical School, Boston, MA.
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ABSTRACT |
We have examined in whole blood the actions of 2 lipoxin
A4 (LXA4) stable analogs,
15-R/S-methyl-LXA4 and 16-phenoxy-LXA4, for
their impact on the expression of adhesion molecules on human leukocytes and coronary artery endothelial cells (HCAEC) and on neutrophil adhesion to HCAEC in vitro. Both LXA4 analogs in
nanomolar to micromolar concentrations prevented shedding of L-selectin and downregulated CD11/CD18 expression on resting neutrophils, monocytes, and lymphocytes. Changes in CD11/CD18 expression were blocked by the mitogen-activated protein kinase kinase inhibitor PD98059. The LXA4 analogs also attenuated changes in
L-selectin and CD11/CD18 expression evoked by platelet-activating
factor (PAF), interleukin-8, or C-reactive protein-derived peptide
201-206 with IC50 values of 0.2 to 1.9 µmol/L, whereas
they did not affect lipopolysaccharide (LPS)- or tumor necrosis
factor- -stimulated expression of E-selectin and intercellular
adhesion molecule-1 on HCAEC. These LXA4
analogs markedly diminished adhesion of neutrophils to LPS-activated
HCAEC. Inhibition of adhesion was additive with function blocking
anti-E-selectin and anti-L-selectin antibodies, but was not additive
with anti-CD18 antibody. Combining LXA4 analogs with
dexamethasone (100 nmol/L) almost completely inhibited PAF-induced changes in adhesion molecule expression on leukocytes and gave additive
inhibition of neutrophil adhesion to HCAEC. Culture of HCAEC with
dexamethasone, but not with LXA4 analogs, also decreased neutrophil attachment. Together, these results indicate that
LXA4 stable analogs modulate expression of both L-selectin
and CD11/CD18 on resting and immunostimulated leukocytes and
inhibit neutrophil adhesion to HCAEC by attenuating CD11/CD18
expression. These actions are additive with those of glucocorticoids
and may represent a novel and potent regulatory mechanism by which
LXA4 and aspirin-triggered 15-epi-LXA4 modulate
leukocyte trafficking.
© 1999 by The American Society of Hematology.
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INTRODUCTION |
LIPOXINS (trihydroxytetraene-containing
eicosanoids) formed by leukocytes during cell-cell interactions
represent a unique class of lipid mediators with potent
anti-inflammatory actions.1 In particular, lipoxin
A4 (LXA4) was found to inhibit neutrophil and
eosinophil chemotaxis in vitro,2,3 to block transmigration
of neutrophil granulocytes (PMNL) across epithelial cells4
and endothelial monolayers,5 and to reduce PMNL entry into
inflamed renal tissues.6 Recent results showed that stable analogs of LXA4 resist rapid inactivation and retain
biological activities of native LXA4, including inhibition
of PMNL adhesion and transmigration across epithelial and endothelial
monolayers,7 inhibition of interleukin-8 (IL-8) production
with consequent impairment of the ability of bacteria-infected
epithelia to direct PMNL movement,8 and attenuation of
P-selectin-dependent PMNL adhesion and rolling on the mesenteric
microvasculature.9 These studies described novel actions of
LXA4 and aspirin-triggered LXA4 (15-epimer of
LXA4) on isolated cells, intestinal epithelial cells, or
isolated vessels after their topical application, yet the question of
whether these lipid mediators are active within the microenvironment of
human whole blood that possess a wide array of components that can bind
lipophilic compounds as well as the issue of the cellular mechanisms
that account for their novel inhibitory actions in leukocyte
trafficking have not been addressed.
Leukocyte extravasation into inflamed areas involves a multistep
interaction of leukocytes and endothelial cells via regulated expression of surface adhesion molecules.10,11 The initial capture and tethering of circulating neutrophils to endothelium is
mediated by L-selectin. L-selectin is constitutively expressed by most
leukocytes and is rapidly shed after cell activation with a concomitant
upregulation of Mac-1 (CD11b/CD18).12 The CD18 integrins,
Mac-1 and LFA-1 (CD11a/CD18), are largely responsible for subsequent
tightening of the adhesion and transendothelial migration of
neutrophils via interactions with their endothelial counterreceptors,
intercellular adhesion molecule-1 (ICAM-1) and ICAM-2.10,11
In the present experiments, we studied the impact of stable
LXA4 analogs in human whole blood and addressed their
cellular mechanisms of action with isolated cells. We examined the
expression of adhesion molecules on human leukocytes and human coronary
artery endothelial cells (HCAEC) and on adhesion of PMNL to HCAEC. We used 16-phenoxy-LXA4, an analog of the native
LXA4,7 and 15-R/S-methyl-LXA4, an
analog of aspirin-triggered 15-epi-LXA4.13
Platelet-activating factor (PAF) and IL-8 were chosen to activate
leukocytes, because these mediators can serve as signals for
neutrophils to bind tightly to the endothelium.14,15
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MATERIALS AND METHODS |
Antibodies and reagents.
In these studies, the monoclonal antibodies (MoAbs) used included
fluorescein isothiocyanate (FITC)-conjugated mouse antihuman L-selectin
MoAb DREG-56 (PharMingen, San Diego, CA), R-phycoerythrin-conjugated mouse antihuman CD18 MoAb MEM-48 (Monosan, Uden, The Netherlands), FITC-labeled mouse antihuman CD11a MoAb G-25.2 (Becton Dickinson Immunocytometry Systems, Mountain View, CA), FITC-labeled mouse antihuman E-selectin MoAb 1.2B6 (Serotec, Kidlington, UK), and R-phycoerythrin-conjugated mouse antihuman ICAM-1 MoAb HA58
(PharMingen). Appropriately labeled, class-matched irrelevant mouse
IgG1 was used as a negative control for each staining. The
following murine MoAbs were used in neutrophil-endothelial cell
adhesion assays: anti-L-selectin MoAb DREG-56 (IgG1;
PharMingen) at 20 µg/mL16; anti-E-selectin MoAb ENA-2
[IgG1, purified F(ab')2 fragments; Monosan] at 10 µg/mL17; and anti-CD18 MoAb L130
(IgG1; Becton Dickinson) at 10 µg/mL.18 The
irrelevant MoAb MOPC-21 (IgG1; PharMingen) at 20 µg/mL
was used as a negative control.
The LXA4 analogs, 15-R/S-methyl-LXA4,
16-phenoxy-LXA4 and 15-deoxy-LXA4, were
prepared by total organic synthesis7 and stored in ethanol
at  70°C. An aliquot was removed and diluted in assay medium
immediately before use. The highest concentration of ethanol (0.1%)
had no detectable effects in any of the assays used.
Lipopolysaccharide (LPS; Escherichia coli O111:B4), C-reactive
protein (CRP)-derived peptide 201-206 (Lys-Pro-Gln-Leu-Trp-Pro), dexamethasone 21-phosphate, wortmannin, and genistein were obtained from Sigma Chemical Co (St Louis, MO); PD98059, herbimycin A, and PAF
were from Calbiochem (San Diego, CA); and human recombinant IL-8 and
tumor necrosis factor- (TNF-) were purchased from R&D Systems
(Minneapolis, MN).
Whole blood incubation.
Venous blood (anticoagulated with 50 U/mL sodium heparin) was obtained
from nonsmoking healthy volunteers (male and female, 24 to 55 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 4,500 and 9,500 cells/µL. Whole blood
aliquots were incubated with one of the LXA4 analogs for 30 minutes at 37°C, 95% air/5% CO2. Preliminary experiments showed that the maximum effects of LXA4 analogs
could be achieved after 30 minutes of preincubation. Some blood samples were preincubated for 30 minutes with LXA4 analogs or
treated for 120 minutes with dexamethasone (100 nmol/L) with or without LXA4 analogs and then challenged with PAF (1 µmol/L),
IL-8 (10 nmol/L), or CRP peptide 201-206 (100 µg/mL), which
downregulates L-selectin expression without inducing cell
activation.18 In some experiments, blood samples were
challenged with Mg2+ (1 mmol/L) and EGTA (2 mmol/L) to
induce a higher affinity form of  2
integrins.19
Analysis of surface antigen expression.
Direct immunofluorescence labeling of resting and treated leukocytes in
whole blood was performed as described.18,20 Leukocytes were stained with saturating concentration of fluorescence
dye-conjugated antihuman L-selectin or antihuman CD18 MoAb. 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 fluoresence
intensity after gating for neutrophils, monocytes, and lymphocytes by
their characteristic forward and side scatter properties. The results are presented as relative fluorescence units (RFU): RFU = (Fuexperimental Fuisotype) × 100/(FUcontrol Fuisotype),
where FUexperimental and FUcontrol are
the L-selectin and CD18 fluorescence intensity of treated cells and
cells cultured in medium only, respectively, and FUisotype
is the fluorescence intensity of class-matched irrelevant antibody.
Isolation and treatment of neutrophils.
PMNL were isolated from peripheral blood by centrifugation through
Ficoll-Hypaque (Pharmacia Diagnostics, Uppsala, Sweden), sedimentation through dextran (3%, wt/vol), and hypotonic lysis of
erythrocytes. Neutrophils (5 × 106 cells/mL; purity,
>97%) were suspended in a modified Hanks' balanced salt solution;
incubated with the MAPK kinase (MEK) inhibitor PD98059, the
phosphatidylinositol 3-kinase inhibitor wortmannin, or the protein
tyrosine kinase inhibitors genistein or herbimycin A for 30 minutes at
37°C; and then challenged with 15-R/S-methyl-LXA4 for
30 minutes. Surface expression of L-selectin and CD18 was analyzed as
just described.
Culture of endothelial cells.
Normal HCAEC obtained from Clonetics Corp (San Diego, CA) were cultured
as described.18 HCAEC (passages 3 to 6) seeded into 24-well
or 96-well microplates and grown to confluence were used in the experiments.
Neutrophil-endothelial cell adhesion assay.
The adhesion assay was performed as in Zouki et al.18 In
brief, monolayers of HCAEC in 96-well microplates were stimulated with
LPS (1 µg/mL) with or without LXA4 analogs or
dexamethasone (100 nmol/L) for 6 hours at 37°C in a 5%
CO2 atmosphere. The cells were then washed 3 times, and 2 × 105 51Cr-labeled neutrophils in 100 µL were added. In some experiments, neutrophils were preincubated
with one of the LXA4 analogs for 30 minutes or with
dexamethasone (100 nmol/L) for 120 minutes with or without
LXA4 analogs for 30 minutes before the addition to HCAEC.
In another set of experiments, LPS-activated HCAEC were incubated for
15 minutes with ENA-2 or MOPC-21 MoAb before the addition of
neutrophils. Radiolabeled neutrophils were incubated with DREG-56,
L130, or MOPC-21 MoAb for 15 minutes before the addition to HCAEC.
After incubation of HCAEC with neutrophils for 30 minutes at 37°C
on an orbital shaker at 90 rpm, loosely adherent or unattached
neutrophils were washed 3 times and the endothelial monolayer plus the
adherent neutrophils were lysed in 200 µL of 0.1% Triton X-100. The
number of adhered neutrophils in each experiment was estimated from the
radioactivity of a control sample. Treatment of HCAEC with any of the
LXA4 analogs did not affect the integrity of viable
endothelial monolayers.
Expression of E-selectin and ICAM-1.
After incubation for 4 hours at 37°C in a 5% CO2
atmosphere with LPS (1 µg/mL) or TNF- (2.5 ng/mL) in the absence
or presence of various concentrations of
15-R/S-methyl-LXA4, HCAEC were removed from the 24-well
microplates by exposure to EDTA (0.01%) in phosphate-buffered saline
(PBS) for 10 minutes at 37°C, followed by gentle trituration. Cells
were resuspended in ice-cold saline containing sodium azide (0.02%),
incubated with a saturating concentration of fluorescein dye-conjugated
anti-E-selectin or anti-ICAM-1 MoAb for 30 minutes at 4°C,
washed, and fixed in formaldehyde (3.9% in PBS). Nonspecific binding
was evaluated by using appropriately labeled mouse IgG1. Immunofluorescence of HCAEC was then analyzed with a FACScan.
Data analysis.
Results are expressed as the means ± SEM. Statistical comparisons
were made by ANOVA using ranks (Kruskal-Wallis test), followed by
Dunn's multiple contrast hypothesis test to identify differences between various treatments, or by the Wilcoxon signed rank test for
paired observations. P values less than .05 were considered significant for all tests.
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RESULTS |
LXA4 analogs in whole blood modulate expression of
L-selectin and CD11/CD18 on resting and immunostimulated leukocytes.
Incubation of heparinized whole blood with either
15-R/S-methyl-LXA4 or 16-phenoxy-LXA4 resulted
in higher L-selectin expression on treated than control (untreated)
cells and gave concentration-dependent downregulation of CD18
expression on PMNL. Figure 1 reports
representative results illustrating the impact of
15-R/S-methyl-LXA4 added to whole blood. Incubation of
blood samples for 30 minutes at 37°C with 5 µmol/L of
15-R/S-methyl-LXA4 or 16-phenoxy-LXA4 resulted in 31% ± 8% and 32% ± 8% higher levels of expression for
L-selectin than on cells from untreated whole blood and gave 26% ± 5% and 27% ± 5% decreases in CD18 expression, respectively
(Fig 2). Similar changes were observed with
monocytes and lymphocytes (Fig 2). Neither 15-deoxy-LXA4
(Fig 2) nor structurally degraded (by thermal degradation)
15-R/S-methyl-LXA4 or 16-phenoxy-LXA4 affected
expression of adhesion molecules (data not shown). When blood samples
were incubated at 4°C, none of the LXA4 analogs studied
affected L-selectin expression. For instance, mean fluorescence
intensity for L-selectin on PMNL was 129 ± 8 and 120 ± 11 (n = 3, P > .5) in control samples and in the
presence of 15-R/S-methyl-LXA4, respectively.
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| Fig 1.
Whole blood actions of aspirin-triggered LXA4
analog (15-R/S-methyl-LXA4) on cell surface expression of
L-selectin and CD18 by human neutrophils. Whole blood was incubated
with 15-R/S-methyl-LXA4 (5 µmol/L) for 10 minutes at
37°C. In each histogram is also displayed the negative control of
immunostaining with class-matched irrelevant antibodies (C). Shown is a
representative experiment of 5 experiments.
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| Fig 2.
In whole blood, LXA4 analogs modulate surface
expression of L-selectin and CD11/CD18 on resting leukocytes. Blood
aliquots were incubated with LXA4 analogs for 30 minutes at
37°C. Adhesion molecule expression is presented as the percentage
of control (unchallenged cells). Control mean fluorescence intensity
for L-selectin: PMNL, 108 ± 7; monocytes, 30 ± 2; lymphocytes, 55 ± 3; for CD18: PMNL, 52 ± 4; monocytes, 113 ± 11; lymphocytes, 20 ± 1; n = 8. The results are the mean ± SEM of 4 to 8 experiments with different donor cell preparations. *P < .05 v control.
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The addition of PAF (1 µmol/L) to whole blood gave a significant
decrease in L-selectin and a marked upregulation of CD11/CD18 on
leukocytes (Figs 3 and
4). Figure 3 shows a representative experiment on the effect of 15-R/S-methyl-LXA4 on
PAF-induced changes in the expression of these adhesion molecules.
Preincubation of blood with either 15-R/S-methyl-LXA4 or
16-phenoxy-LXA4 attenuated PAF-induced upregulation of CD18
expression on PMNL, monocytes, and lymphocytes in a
concentration-dependent fashion, with IC50 values of
approximately 1.5 µmol/L (Fig 4). Essentially complete inhibition was
achieved with 5 µmol/L of the LXA4 analogs. PAF-induced decreases in L-selectin expression on PMNL and monocytes were also
markedly, although not completely, inhibited by these LXA4 analogs (Fig 4). Incubation of blood with IL-8 (10 nmol/L)
downregulated L-selectin and upregulated CD18 expression on PMNL
(Fig 5). As with PAF, both
15-R/S-methyl-LXA4 and 16-phenoxy-LXA4 at 5 µmol/L completely inhibited IL-8-induced upregulation of CD18,
whereas they partially inhibited changes in L-selectin expression (Fig 5). Immunostaining of leukocytes with an anti-CD11b MoAb showed similar
changes as those observed with the anti-CD18 MoAb (data not shown).
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| Fig 3.
Aspirin-triggered LXA4 analog
(15-R/S-methyl-LXA4) inhibits PAF-induced changes in cell
surface expression of L-selectin and CD18 by human neutrophils. Whole
blood was incubated in medium only (unstimulated) or with
15-R/S-methyl-LXA4 (5 µmol/L) for 10 minutes and then
with PAF (1 µmol/L) for 30 minutes at 37°C. In each histogram is
also displayed the negative control of immunostaining with
class-matched irrelevant antibodies (C). Shown is a representative
experiment of 5 experiments.
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| Fig 4.
Inhibition of PAF-induced changes in L-selectin and CD18
expression on leukocytes. Whole blood aliquots were incubated with
LXA4 analogs for 30 minutes and then challenged with 1 µmol/L PAF for 30 minutes at 37°C. Adhesion molecule expression
is presented as the percentage of control (unchallenged cells). Mean
fluorescence intensity for L-selectin: PMNL, control, 103 ± 9; PAF,
43 ± 5; monocytes, control, 30 ± 3; PAF, 20 ± 2; lymphocytes,
control, 53 ± 3; PAF, 50 ± 2, n = 5, all P < .05. Mean
fluorescence intensity for CD18: PMNL, control, 48 ± 3; PAF, 61 ± 6; P < .05; monocytes, control, 107 ± 12; PAF, 123 ± 13;
P < .05; lymphocytes, control, 19 ± 1; PAF, 21 ± 1. The
results are the mean ± SEM of 4 to 5 experiments with
different donor cell preparations. *P < .05 v
PAF-stimulated cells.
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| Fig 5.
Inhibition of IL-8-induced changes in L-selectin and
CD18 expression on human PMNL. Whole blood was preincubated with
LXA4 analogs for 30 minutes and then challenged with 10 nmol/L IL-8 for 30 minutes at 37°C. Adhesion molecule expression is
presented as the percentage of control (unchallenged cells). Mean
fluorescence intensity for L-selectin: control, 115 ± 8; IL-8, 61 ± 5; CD18: control, 45 ± 4; IL-8, 59 ± 9; n = 3; both
P < .05. Values are the means ± SEM of 3 experiments with
different donor cell preparations. *P < .05 v
IL-8-stimulated cells.
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We also investigated whether LXA4 analogs are able to
modify the affinity of 2 integrins. Formation of a
higher affinity form of LFA-1 and Mac-1 is thought to involve
conformational changes and association of the I domain with the
-propeller domain.21,22 The MoAb G-25.2 recognizes this
latter domain in LFA-1.22 Incubation of whole blood with
PAF or IL-8 in the absence or presence of 15-R/S-methyl-LXA4 was not associated with a detectable
increase in expression of MoAb G-25.2 (Fig
6A and B). On the other hand, activation of cells with Mg2+
and EGTA, which is known to induce the formation of a higher affinity
form of LFA-1 without inducing clustering,19 resulted in
increases in MoAb G-25.2 expression (Fig 6C) that was not affected by
15-R/S-methyl-LXA4 (Fig 6C and D) or
16-phenoxy-LXA4 (data not shown).
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| Fig 6.
Effect of 15-R/S-methyl-LXA4 on the
expression of LFA-1 epitope expressed by the -propeller domain of
LFA-1 on neutrophils. Mean fluorescence of MoAb G-25.2 on PMNL in whole
blood challenged with PAF (1 µmol/L; A) or IL-8 (10 nmol/L; B) for 30 minutes at 37°C in the presence of 15-R/S-methyl-LXA4
(5 µmol/L). The dotted line represents MoAb G-25.2 binding at
4°C. Shown is a representative of 3 experiments. (C) The expression
of MoAb G-25.2 on PMNL in whole blood challenged with
Mg2+ (1 mmol/L) and EGTA (2 mmol/L) for 30 minutes at
4°C (dotted line) and at 37°C in the absence or presence of
15-R/S-methyl-LXA4 (5 µmol/L). Shown is a representative
of 3 experiments. (D) Mean fluorescence of MoAb G-25.2 on neutrophils
challenged with Mg2+ and EGTA in the presence of
15-R/S-methyl-LXA4. Values are the means ± SEM of 3 experiments.
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Next, we examined whether LXA4 analogs when added to whole
blood could also affect the cell activation-independent downregulation of L-selectin expression. Incubation of blood with CRP peptide 201-206 (100 µg/mL) resulted in, on average, 49%, 42%, and 17% decreases
in L-selectin expression on PMNL, monocytes, and lymphocytes, respectively (Fig 7). These changes were
inhibited by 15-R/S-methyl-LXA4 in a
concentration-dependent manner, with apparent IC50 values of approximately 250 nmol/L (Fig 6). Similar inhibition was observed with 16-phenoxy-LXA4 (Fig 7).
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| Fig 7.
LXA4 analogs prevent cell
activation-independent downregulation of L-selectin expression on human
PMNL, monocytes, and lymphocytes. Whole blood aliquots were incubated
with LXA4 analogs for 30 minutes at 37°C and then
challenged with 100 µg/mL CRP peptide 201-206. Results are presented
as the percentage of control (unchallenged cells). Mean fluorescence
intensity for L-selectin: PMNL, control, 97 ± 6; peptide 201-206, 50 ± 14; monocytes, control, 35 ± 4; peptide 201-206, 22 ± 7;
lymphocytes, control, 61 ± 4; peptide 201-206, 51 ± 4; n = 3; all
P < .05. Values represent the mean ± SEM of 3 independent
experiments. *P < .05 v peptide 201-206-stimulated
cells.
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Inhibition of MAPK kinase reverses LXA4-induced changes
in CD11/CD18 expression on neutrophils.
Treatment of neutrophils with the MAPK kinase inhibitor PD98059
increased by approximately 27% L-selectin expression without altering
CD11/CD18 expression (Fig 8). PD98059
prevented 15-R/S-methyl-LXA4-induced downregulation of
CD11/CD18 expression in a concentration-dependent fashion, whereas
changes in L-selectin expression were not affected (Fig 8). Wortmannin
at 0.2 µmol/L had no effect, whereas at 2 µmol/L, it prevented
15-R/S-methyl-LXA4-induced downregulation of CD18 (Fig 8).
Furthermore, wortmannin (2 µmol/L) alone downregulated L-selectin
expression and inhibited the action of 15-R/S-methyl-LXA4 (Fig 8). 15-R/S-methyl-LXA4 induction was not affected by
the tyrosine kinase inhibitor genistein (Fig 8) or herbimycin-A (data not shown).
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| Fig 8.
Effects of MAPK kinase (MEK) and tyrosine kinase
inhibitors on neutrophil adhesion molecules. Isolated neutrophils
(107 cells/mL) were preincubated with PD98059, wortmannin,
or genistein for 30 minutes at 37°C and then challenged with 5 µmol/L 15-R/S-methyl-LXA4 for 30 minutes. Adhesion
molecule expression is presented as the percentage of control. Mean
fluorescence intensity for control samples, L-selectin: 35 ± 1; CD18:
82 ± 14; n = 4. Values are the mean ± SEM of 4 independent experiments.
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LXA4 analogs do not affect E-selectin or ICAM-1
expression on LPS- or TNF--stimulated endothelial cells.
After stimulation by LPS, HCAEC increased 27- and 2.6-fold the
expression of E-selectin and ICAM-1, respectively (n = 4, both P < .05; Table 1).
15-R/S-methyl-LXA4 did not affect basal expression of these
adhesion molecules (data not shown) and produced only a slight
inhibition of LPS-induced changes (Table 1). The maximum inhibition did
not exceed 10%. Similarly, 15-R/S-methyl-LXA4 failed to
significantly inhibit TNF--induced E-selectin and ICAM-1 expression (Table 1).
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Table 1.
15-R/S-methyl-LXA4 Does Not Alter E-Selectin
and ICAM-1 Expression on Human Coronary Artery Endothelial Cells
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LXA4 analogs inhibit neutrophil adhesion to endothelial
cells.
Activation of HCAEC with LPS resulted in a 4.1-fold increase in the
number of adherent neutrophils (Fig 9).
When HCAEC were challenged with LPS in the presence of one of the
stable LXA4 analogs, only slight decreases in adhesion
could be detected (Fig 9A). By contrast, addition of neutrophils
preincubated with either 15-R/S-methyl-LXA4 or
16-phenoxy-LXA4 to LPS-activated HCAEC attenuated neutrophil attachment in a concentration-dependent fashion (Fig 9A).
Significant inhibition of adhesion was detected with
15-R/S-methyl-LXA4 at a concentration as low as 50 nmol/L.
At 5 µmol/L, 15-R/S-methyl-LXA4 and
16-phenoxy-LXA4 inhibited neutrophil adhesion by 38% ± 4% and 36% ± 3%, respectively (n = 5, both P < .05;
Fig 9A). Neither 15-deoxy-LXA4 nor decomposed
15-R/S-methyl-LXA4 or 16-phenoxy-LXA4 affected
neutrophil adhesion (data not shown).
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| Fig 9.
Inhibition of neutrophil binding to endothelial cells by
LXA4 analogs (A) or by anti-E-selectin, anti-L-selectin,
and anti-CD18 MoAbs and 15-R/S-methyl-LXA4 (B). Confluent
HCAEC monolayers were cultured with 1 µg/mL LPS with or without
LXA4 analogs for 6 hours at 37°C. (A) Neutrophils were
preincubated for 30 minutes with LXA4 analogs or medium as
indicated before addition to activated HCAEC. Values are the means ± SEM of 3 to 6 experiments using neutrophils from different donors.
*P < .05 v LPS-treated HCAEC. (B) Neutrophils were
treated with 15-R/S-methyl-LXA4 (5 µmol/L) or the
indicated MoAbs before and during the assay. Neutrophil adhesion to
unstimulated HCAEC was 0.41 ± 0.03 × 104 cells per
well. The irrelevant MoAb MOPC-21 (IgG1) was used as a
negative control. The results represent the mean ± SEM of 5 experiments using neutrophils from different donors.
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Because multiple receptors are involved in neutrophil adhesion to
LPS-stimulated HCAEC18 and LXA4 analogs
affected expression of both L-selectin and CD11/CD18 on PMNL, we
assayed the contribution of L-selectin, E-selectin, and CD18 to the
binding interaction. A significant proportion of neutrophil-HCAEC
attachment was blocked by MoAbs binding to L-selectin (44% ± 4%,
n = 5), CD18 (31% ± 4%), or E-selectin (38% ± 5%; Fig 9B).
The combination of these MoAbs inhibited neutrophil adhesion by 87% to
98%. Treatment of neutrophils with 15-R/S-methyl-LXA4 and
anti-CD18 MoAb resulted in only a slightly greater inhibition of
adhesion than those observed with neutrophils treated with either
15-R/S-methyl-LXA4 or anti-CD18 MoAb (Fig 9B). The
combination of 15-R/S-methyl-LXA4 with either anti-L-selectin MoAb or anti-E-selectin MoAb resulted in additive inhibition, and the degree of inhibition was similar to those observed
when anti-L-selectin MoAb or anti-E-selectin MoAb was combined with
anti-CD18 MoAb, respectively (Fig 9B). Combining 15-R/S-methyl-LXA4, anti-L-selectin MoAb, and
anti-E-selectin MoAb blocked approximately 80% of adhesion. Similar
results were obtained with 16-phenoxy-LXA4 (data not shown).
Additive inhibition by LXA4 analogs and dexamethasone of
PAF-induced changes in neutrophil adhesion molecule expression and
binding to endothelial cells.
Dexamethasone was used at a concentration of 100 nmol/L, because our
previous results showed that the maximum inhibitory effect of
dexamethasone on neutrophil adhesion molecule expression can be
achieved with this concentration.20 As expected,
dexamethasone added alone attenuated by 35% to 45% PAF-induced
changes in L-selectin and CD11/CD18 expression on PMNL
(Fig 10A). Combining dexamethasone with
15-R/S-methyl-LXA4 resulted in an almost complete
inhibition (Fig 10A). Similar inhibition was observed with both
monocytes and lymphocytes (data not shown). Culture of HCAEC with
dexamethasone diminished the number of adherent neutrophils (Fig 10B).
However, no further decrements in neutrophil adhesion were detected in the presence of LXA4 analogs. On the other hand, the
inhibitory actions of dexamethasone and LXA4 analogs were
additive when neutrophils were preincubated with dexamethasone and
LXA4 analogs before exposure to LPS-activated HCAEC (Fig
10B).
View larger version (32K):
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| Fig 10.
Cooperative actions of dexamethasone and
LXA4 analogs. (A) Human whole blood aliquots were incubated
with 100 nmol/L dexamethasone for 120 minutes in the presence of
15-R/S-methyl-LXA4 (5 µmol/L) for the last 30 minutes of
incubation, as indicated, and then challenged with 1 µmol/L PAF for
30 minutes at 37°C. Adhesion molecule expression is presented as
the percentage of control. Mean fluorescence intensity for L-selectin:
control, 37 ± 3; PAF, 26 ± 4; CD18: control, 61 ± 4; PAF, 73 ± 7; n = 5; both P < .05. *P < .05; **P < .01 v control (untreated). L-selectin and CD18 expression on
dexamethasone plus 15-R/S-methyl-LXA4-treated cells did
not differ significantly from those of control. (B) Confluent HCAEC
monolayers were cultured for 6 hours with LPS (1 µg/mL) in the
presence of dexamethasone (DEX; 100 nmol/L),
15-R/S-methyl-LXA4, or 16-phenoxy-LXA4 (5 µmol/L). Neutrophils pretreated with dexa- methasone
and/or LXA4 analogs as described for whole blood were added
to activated HCAEC and then challenged with PAF (1 µmol/L). The
results are expressed as the mean ± SEM of 5 experiments using
neutrophils from different donors. *P < .05 v
LPS-treated.
|
|
| |
DISCUSSION |
In this report, we describe a novel mechanism(s) by which stable
LXA4 analogs can affect the inflammatory response, namely via the modulation of surface expression of adhesion molecules on
resting and immunostimulated leukocytes, and inhibition of neutrophil
adhesion to the activated endothelium. This inhibition is predominantly
mediated via actions of LXA4 analogs on leukocytes and is
distinct from those of glucocorticoids. Moreover and of particular
interest, these actions of stable LXA4 analogs were monitored for the first time within the whole blood environment, where
they clearly remain bioactive.
Because dehydrogenation of native lipoxins by human leukocytes results
in their rapid inactivation,23 we used LXA4
stable analogs that carry substituents at the carbon 15 through  20 end of the native LXA4 structure, resist
conversion,7,24 and retain the biological activities of
native LXA4 in leukocyte adhesion and migration
assays.5,24 15-R/S-methyl-LXA4 is an analog of
aspirin-triggered 15-epi-LXA4 and
16-phenoxy-LXA4 is an analog of native
LXA4.7,13 The effects of
15-R/S-methyl-LXA4 and 16-phenoxy-LXA4 observed
in this study are attributed to the molecules themselves, because
neither degraded LXA4 analogs nor 15-deoxy-LXA4 caused detectable changes in the assays used.
Our study documents the complex nature of actions of stable
LXA4 analogs on expression of leukocyte adhesion molecules.
These analogs upregulated L-selectin and downregulated CD11/CD18
expression on human resting neutrophils, monocytes, and, to a lesser
extent, lymphocytes in whole blood and markedly attenuated changes
evoked by immunostimulation. These results are consistent with earlier reports, which showed that native LXA4 inhibits
formyl-Met-Leu-Phe-stimulated upregulation of CD11b/CD18 on human
isolated neutrophils.25 The present results indicated that
this action is also demonstrable within whole blood with nanomolar to
micromolar concentrations of LXA4 analogs. As expected,
higher concentrations were required in whole blood to inhibit the
upregulation of CD18 expression than in isolated cells. This might
reflect interactions of LXA4 analogs with serum components
such as albumin. Nevertheless, it is impressive that these lipophilic
compounds are active in the microenvironment of whole blood and
overcome interactions with blood components to specifically regulate
leukocytes. Downregulation of CD11/CD18 expression on resting
neutrophils by LXA4 analogs does not involve
phosphatidylinositol 3-kinase or tyrosine kinases; rather, it appears
to be mediated via activation of MAPK kinase, as suggested by the
experiments with PD98059 and wortmannin, which, at a concentration of 2 µmol/L, also inhibits MAPK kinase.26 The LXA4
analogs did not initiate detectable changes in tyrosin phosphorylation
of either Erk-2 or p38 MAPK in resting human neutrophils as evaluated
by immunoblotting using specific antibodies to the phosphorylated forms
of these enzymes (Hachicha et al, unpublished observations). Recent results suggest that intracellular
arachidonic acid induces integrin-dependent homotypic adhesion of
neutrophils via the Raf-1/MAPK kinase/Erk pathway.27 With
respect to LXA4 receptor activation, it is possible that
other Erk or p38 MAPK-independent pathways may be involved in signaling
neutrophil adhesion molecule expression.
Physiological stimuli regulate adhesion by either altering the affinity
of the individual integrin molecule or by inducing clustering of
2 integrins, thereby increasing the overall strength of
binding.19,28,29 Increasing intracellular Ca2+
concentration does not induce detectable changes in the affinity of
LFA-1.22 Because leukocyte activation evoked by PAF or IL-8 is mediated through increases in intracellular Ca2+
concentration, these agonists would not increase integrin affinity. Indeed, neither PAF nor IL-8 affected MoAb G-25.2 expression. Activation of leukocytes with Mg2+ and EGTA, which results
in the formation of a higher affinity form of LFA-1,19
increased the expression of MoAb G-25.2, which did not appear to be
affected by the LXA4 analogs studied. These results suggest
that LXA4 analogs do not alter the affinity of either LFA-1
and probably of Mac-1, but rather they interfere with
activation-induced clustering of 2 integrins. However,
our results do not preclude the possibility that, in the presence of
integrin ligands, LXA4 analogs might affect a
ligand-induced affinity increase secondary to integrin clustering.
Comparison of L-selectin expression at 4°C and at 37°C
indicated that, within whole blood, these LXA4 analogs
prevented temperature-induced L-selectin shedding on unstimulated
leukocytes. For minutes of activation by either PAF or IL-8 or
challenge with CRP peptide 201-206, which does not activate cells,
leukocytes release L-selectin from their surface by a proteolytic
enzyme. Because 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.30,31 Whereas it remains to be established whether
the conformational changes induced by cell activation dependent or
independent stimuli are identical, our results demonstrate that these
events are effectively blocked by LXA4 analogs. A recent
study has reported that the intracellular tail of L-selectin is
phosphorylated on serine upon activation with
chemoattractants.32 It is of interest that calmodulin
inhibitors directly induce proteolytic shedding of
L-selectin.33 Whether this action of LXA4
involves calmodulin or interference with phosphorylation step(s)
remains a key area of inquiry.
Despite inhibition of L-selectin shedding from neutrophils within whole
blood, which is thought to promote neutrophil-HCAEC attachment,
LXA4 analogs actually inhibit isolated neutrophil adhesion
to HCAEC. This inhibition can primarily be attributed to their actions
on neutrophils rather than on HCAEC in this interaction, because only
slight decreases in the number of adherent neutrophils were observed
after culture of HCAEC with LPS in the presence of LXA4
analogs. Consistently, LXA4 analogs have little effect on
LPS- or TNF--stimulated expression of E-selectin and ICAM-1 on
HCAEC. Our results indicate that inhibition of neutrophil-HCAEC adherence by LXA4 analogs is predominantly attributable to
inhibition of CD11/CD18 expression on neutrophils. The LXA4
analogs or a function-blocking anti-CD18 MoAb resulted in similar
decreases in neutrophil adhesion to HCAEC. The action of
LXA4 analogs and anti-CD18 MoAb was not additive, whereas
the inhibition with LXA4 analogs was additive with
anti-E-selectin and anti-L-selectin MoAbs. These results suggest that
LXA4 analogs had little, if any, effect on E-selectin or
L-selectin function and did not interfere with E-selectin ligand. Taken
together, our data indicate that, in addition to inhibition of
P-selectin expression on rat intestinal venular endothelial
cells,8 lipoxins are potent regulators of both leukocyte
L-selectin and CD11/CD18 expression. Whereas inhibition of
P-selectin-dependent capture of neutrophils may be a key mechanism by
which LXA4 analogs inhibit neutrophil-endothelial interactions in the mesenteric circulation,8 functions of
adhesion molecules may overlap, and even neutrophil recruitment into
other vascular beds may not rely on a P-selectin-dependent
adhesion.34
The present study and previous studies20,35 indicate that
the mechanisms of action of LXA4 analogs differ from those
of glucocorticoids. Adhesion molecule expression on resting neutrophils can be modulated by LXA4 analogs, but not by
dexamethasone.20 Both dexamethasone and LXA4
analogs alone inhibited by approximately 20% to 45% PAF-induced
changes in L-selectin and CD11/CD18 expression on neutrophils,
representing the near maximum inhibition that can be achieved with
these compounds (Filep et al20 and the present study).
Interestingly, the inhibitory actions of dexamethasone and
LXA4 analogs were additive, resulting in almost complete
inhibition of PAF-stimulated changes. The inhibitory actions of
dexamethasone on PMNL requires de novo protein synthesis.20
Preincubation of neutrophils with either dexamethasone or
LXA4 analogs before addition to LPS-activated HCAEC
resulted in partial inhibition of adhesion, and the inhibitory actions
of dexamethasone and LXA4 analogs were additive. Culture of
HCAEC with LPS and dexamethasone, but not with LXA4
analogs, resulted in marked decreases in the number of adherent
neutrophils. These findings are consistent with the glucocorticoid
inhibition of LPS-induced expression of ICAM-1 and E-selectin on
endothelial cells.35 By contrast, LXA4 analogs
had little, if any, effect on expression of these adhesion molecules.
Interestingly, the mechanism by which LXA4 analogs inhibit
neutrophil-endothelial adhesion also differs from that of several
nonsteroidal anti-inflammatory drugs, which induce shedding of
L-selectin from neutrophils and inhibit L-selectin-mediated attachment.36 The actions of LXA4 analogs on
2 integrins appear to be similar to those reported for
piroxicam,37 tepoxalin, a dual cyclooxygenase/lipoxygenase
inhibitor,38 and the anti-inflammatory agent leumedin NPC
15669.39 Piroxicam prevents expression of a CD11b
activation neo-epitope,37 whereas tepoxalin and leumedin NPC 15669 inhibit upregulation of Mac-1.38,39 However,
unlike the LXA4 analogs, none of these agents affected
2 integrin expression on resting leukocytes.
In conclusion, this study demonstrates that stable LXA4
analogs modulate adhesion molecule expression on leukocytes monitored within the microenvironment of human whole blood and inhibit neutrophil adhesion to HCAEC via downregulation of CD11/CD18 expression. These
effects are distinct from those of either glucocorticoids or
nonsteroidal anti-inflammatory drugs. Therefore, stable analogs of
native LXA4 and aspirin-triggered 15-epi-LXA4
may represent a novel therapeutic approach for selective regulation of
leukocyte trafficking in host defense, inflammation, and reperfusion injury.
| |
ACKNOWLEDGMENT |
The authors thank Valery V. Fokin for expert assistance in the
synthesis of the lipoxin analogs.
| |
FOOTNOTES |
Submitted March 24, 1999; accepted August 10, 1999.
Supported by grants from the Medical Research Council of Canada
(MT-12573 to J.G.F.) and from the National Institutes of Health (GM-38765 and DK50305 to C.N.S.).
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Address reprint requests to János G. Filep, MD, Research Center,
Maisonneuve-Rosemont Hospital, 5415 boulevard de l'Assomption,
Montréal, Québec, Canada H1T 2M4; e-mail:
filepj{at}ere.umontreal.ca.
| |
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B. McMahon and C. Godson
Lipoxins: endogenous regulators of inflammation
Am J Physiol Renal Physiol,
February 1, 2004;
286(2):
F189 - F201.
[Abstract]
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P. Maderna, D. C. Cottell, G. Berlasconi, N. A. Petasis, H. R. Brady, and C. Godson
Lipoxins Induce Actin Reorganization in Monocytes and Macrophages But Not in Neutrophils : Differential Involvement of Rho GTPases
Am. J. Pathol.,
June 1, 2002;
160(6):
2275 - 2283.
[Abstract]
[Full Text]
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J. Goh, A. W. Baird, C. O'Keane, R. W. G. Watson, D. Cottell, G. Bernasconi, N. A. Petasis, C. Godson, H. R. Brady, and P. MacMathuna
Lipoxin A4 and Aspirin-Triggered 15-Epi-Lipoxin A4 Antagonize TNF-{alpha}-Stimulated Neutrophil-Enterocyte Interactions In Vitro and Attenuate TNF-{alpha}-Induced Chemokine Release and Colonocyte Apoptosis in Human Intestinal Mucosa Ex Vivo
J. Immunol.,
September 1, 2001;
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[Abstract]
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S. M. Oliani, M. J. Paul-Clark, H. C. Christian, R. J. Flower, and M. Perretti
Neutrophil Interaction with Inflamed Postcapillary Venule Endothelium Alters Annexin 1 Expression
Am. J. Pathol.,
February 1, 2001;
158(2):
603 - 615.
[Abstract]
[Full Text]
[PDF]
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M. V. Gomez-Gaviro, C. Dominguez-Jimenez, J. M. Carretero, P. Sabando, I. Gonzalez-Alvaro, F. Sanchez-Madrid, and F. Diaz-Gonzalez
Down-regulation of L-selectin expression in neutrophils by nonsteroidal anti-inflammatory drugs: role of intracellular ATP concentration
Blood,
November 15, 2000;
96(10):
3592 - 3600.
[Abstract]
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ABSTRACT
INTRODUCTION