INSERM U352, Laboratoire de Biochimie et Pharmacologie, Institut National des Sciences Appliquées de Lyon, Villeurbanne, France; and
Cátedra de Patología General y Fisiopatología, Instituto de Medicina Experimental, Facultad de Medicina, Universidad Central de Venezuela, Caracas
Correspondence: Annie-France Prigent, INSERM U352, Laboratoire de Biochimie et Pharmacologie, Bâtiment 406, Institut National des Sciences Appliquées de Lyon, 20 avenue Albert Einstein, 69621 Villeurbanne Cedex, France. E-mail: prigent{at}insa.insa-lyon.fr
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Key Words: lymphocyte-endothelial cell interactions • prostaglandin H and prostaglandin I2 synthases • atherothrombogenesis
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
HUVEC isolation and culture
Human umbilical cords were collected soon after birth and
processed within 24 h. Cells were isolated from umbilical cord
veins by digestion with collagenase IA as described by Jaffe et al.
[23
]. Endothelial cell cultures were grown to
subconfluence on T-25 Flasks (Falcon) coated with 2% gelatin, in a
humid atmosphere containing 5% CO2 at 37°C. Culture
medium consisted of M-199 medium containing 20% heat-inactivated
newborn calf serum, 100 µg/mL streptomycin, 100 U/mL penicillin, 100
µg/mL gentamicin, and 1% endothelial cell growth factor. The
identity of the endothelial cells was checked by their cobblestone
appearance under phase-contrast microscopy. After trypsin-EDTA
treatment, endothelial cells were subcultured in 24-well gelatin-coated
plates (Corning), allowed to grow to confluence (105
cells/well) under the conditions described above, and used at this
first passage.
Preparation of human peripheral blood lymphocytes (PBL)
Mononuclear cells were isolated from peripheral venous blood of
healthy subjects who had not taken any medication for 2 weeks before
blood donation (ETS, Lyon, France). Venous blood was drawn into
citrate-phosphate-dextrose anticoagulant, and mononuclear cells were
isolated by dextran sedimentation at 37°C for 30 min and
Ficoll-Hypaque density gradient centrifugation for 20 min at 600
g. Depletion of monocytes was performed by adhesion onto
polystyrene flasks as follows: cells were adjusted to 1 x
106 cells/mL in RPMI 1640 supplemented with 10% fetal calf
serum, antibiotics (100 µg/mL streptomycin, 100 U/mL penicillin), and
2 mmol/L L-glutamine, and then incubated for 2 x
1 h in a 75-cm2 tissue culture flask (Falcon) in
standard conditions, with a flask change between the incubations.
Nonadherent PBL were collected by gentle aspiration and then incubated
for 72 h in the absence or presence of 1 µg/mL
phytohemagglutinin (PHA). At the end of the incubation period, PBL were
recovered by centrifugation on a Ficoll-Hypaque density gradient,
washed three times with RPMI 1640, resuspended in serum-free medium
(RPMI 1640 supplemented with 100 U/mL penicillin, 100 µg/mL
streptomycin, and 2 mmol/L L-glutamine), and used for
interaction with HUVEC. The purity of these lymphocyte-enriched
preparations was assessed by flow cytometry analysis using CD14 mAb
(Leu M3, Becton Dickinson). CD14-positive cells (monocytes) were less
than 1% in the PHA-activated cells and less than 2.5% in the
nonactivated cells. Viability was greater than 95% according to the
trypan blue exclusion test.
PGI2 assay in supernatants from HUVEC coincubated with
lymphocytes
Confluent HUVEC cultured in 24-well gelatin-coated plates were
washed twice with RPMI 1640. Thereafter, monolayers were incubated at
37°C under standard conditions, with either serum-free medium
(control) or various concentrations of resting or PHA-activated
lymphocytes, for the indicated times, in a final volume of 0.5 mL. At
the end of the incubation period, supernatants were harvested,
centrifuged at 400 g to remove lymphocytes, and stored at
-20°C until assayed for PGI2 content. In experiments
designed to investigate whether PGI2 production was
affected by direct contact between lymphocytes and HUVEC or by soluble
products released in the medium, lymphocytes were incubated in an
insert (0.4-µm Costar filter). The final volumes of the luminal and
abluminal compartments were 1 and 0.4 mL, respectively. In some
experiments, lymphocytes (6 x 106 cells/mL) and
confluent HUVEC were pretreated for 30 min with 500 µg/mL of the
PGI2 synthase inhibitor tranylcypromine [24
,
25
], or with 25 µmol/L of the cPLA2
inhibitor, MAFP [26
], washed three times with serum-free
medium to remove any residual inhibitor before interaction for 20 h (tranylcypromine) or 4 h (MAFP) at 37°C. In some experiments,
lymphocytes (6 x 106 cells/mL) were pretreated with
10 µmol/L of the diacylglycerol (DAG) lipase inhibitor RHC80267
[27
] before interaction with confluent HUVEC for 4 h at 37°C. In other experiments confluent HUVEC were pretreated for
45 min with 100 µmol/L of the intracellular calcium chelator BAPTA/AM
before coincubation with lymphocytes, either in the presence or absence
of 5 mmol/L EGTA, for 4 h at 37°C. For all these experiments,
PGI2 released in the supernatant was quantified by EIA as
its stable breakdown product, 6-oxo-prostaglandin F1
(6-oxo-PGF1). Cross-reactivity with PGE2 was
<1%. In experiments designed to investigate whether arachidonic acid
used for the PGI2-stimulated synthesis was of lymphocyte or
endothelial origin, resting lymphocytes were labeled with 0.5 µCi/mL
(10 µmol/L) [14C]arachidonic acid for 1 h, and
confluent HUVEC were labeled overnight in the presence of 5% fetal
calf serum with the same amount of [14C]arachidonic acid.
Cells were washed three times before coincubation experiments in
gelatin-coated Petri dishes (28 cm2). At the end of the
coincubation (from 30 min to 8 h), culture media were acidified to
pH 3, and extracted twice with 3 vol ethylacetate. Dried lipid extracts
were spotted onto Silica gel G plates (Merck-Lipha, Darmstadt,
Germany), and the plates were developed in ethylacetate/isoctane/acetic
acid/H2O (55:25:10:50) as described by Xu et al.
[28
] and exposed to MP Hyperfilm for 14–30 days. Spots
corresponding to 6-oxo-PGF1 were scraped off, mixed with
picofluor (Packard), and the radioactivity determined by liquid
scintillation counting. Standard 6-oxo-PGF1,
PGE2, and arachidonic acid were chromatographed on the same
plate and visualized by iodine vapor staining for comparison.
Western blot of PGH and PGI2 synthases
In experiments where PGH synthases were analyzed, 1.5 x
106 HUVEC were cultured in T-25 flasks with 7.5 x
106 lymphocytes (lymphocyte/HUVEC ratio = 5) for
increasing periods of time from 1 up to 20 h. Then, HUVEC layers
were thoroughly washed, trypsinized, and lysed in 20 mmol/L Tris
buffer, pH 8.0, containing 1% Triton X-100, 150 mmol/L NaCl, 2 mmol/L
EDTA, 10% glycerol and protease inhibitors (1 mmol/L PMSF, 200 U/mL
aprotinin, 10 µg/mL leupeptin). For PGI2 synthase
immunodetection, 8 x 106 HUVEC and 1.5 x
108 PBL were lysed as above. In some experiments,
PGI2 synthase from lymphocyte lysates (corresponding to
108 cells) was immunoprecipitated by an
anti-PGI2 synthase monoclonal antibody complexed to protein
G-Sepharose according to published procedures [29
,
30
]. Proteins from lymphocyte immunoprecipitates or from
endothelial cells and lymphocyte lysates (50 µg) were separated by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)
and electrotransferred onto Immobilon-P membranes (Millipore).
Immunoreactive bands were detected using either 5 µg/mL PGHS-1
monoclonal antibody, 1:2000 dilution PGHS-2 polyclonal, or 1:1000
dilution PGI2 synthase polyclonal antibody and
HRP-conjugated anti-mouse or anti-rabbit IgG, and visualized by ECL.
Lymphocyte-HUVEC adhesion assay
PBL adhesion to HUVEC was assessed using
51Cr-labeled PBL as described elsewhere
[31
]. PBL (4 x 106/mL in PBS) were
incubated with 0.1 mCi/mL Na251CrO4
for 60 min at 37°C. After three washes, the chromium-labeled cells
were resuspended at 4 x 106 cells/mL in RPMI 1640
supplemented with 10% fetal calf serum and immediately used. Cell
viability, as measured by the trypan blue exclusion test, was higher
than 95%. Subcultured confluent HUVEC monolayers in 24-well plates
were used for the adhesion assays. The medium was removed and aliquots
of definite number of 51Cr-labeled PBL, activated or not,
were added to each well. After 1-h incubation at 37°C in 5%
CO2 humidified air, the wells were washed three times with
0.3 mL of PBS containing 5% fetal calf serum to remove nonadhering
lymphocytes. Adhering lymphocytes (plus endothelial cells) were lysed
in 0.1 mol/L NaOH (0.5 mL), at room temperature for 90 min, and the
radioactivity was counted in a gamma counter.
The percentage of PBL adhesion was calculated as follows: % adhesion = (cpm in 0.5 mL of lysate/cpm in the added PBL suspension) x 100.
Statistical analysis
Values are presented as means ± SE of
n independent experiments. All data were compared by
analysis of variance (Statview II for Macintosh) followed by protected
t test. P values of 0.05 or less were considered
statistically significant.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
(around 250 pg/105 HUVEC).
Resting lymphocytes (L) or lymphocytes previously activated with PHA
for 3 days and washed (PHA-L), incubated in the same conditions, did
not produce any detectable amount of PGI2. The coincubation
of confluent HUVEC (105 cells/well) with resting or
PHA-activated lymphocytes markedly increased PGI2
production (Fig. 1
). This lymphocyte-mediated PGI2 synthesis was directly
dependent on the number of lymphocytes added to the HUVEC monolayer. It
was already significant at a ratio of one lymphocyte for one
endothelial cell and then increased up to sixfold at the highest ratio
used. Whatever the ratio, no significant difference between resting
or PHA-activated lymphocytes was observed (Fig. 1)
. As shown in
Figure 2
, for a lymphocyte-to-endothelial cell ratio of 9, the stimulating
effect of resting lymphocytes on PGI2 synthesis was already
significant (threefold increase) after 20 min of coincubation and
nearly maximum (fivefold increase) after 4 h. A slight increase
was observed thereafter up to 20 h interaction (5.2-fold). Similar
time-courses in PGI2 production were observed when
PHA-activated lymphocytes were incubated with HUVEC (not shown).
Furthermore, when lymphocytes were disposed on a microporous insert
that allows the passage of secreted products but prevents lymphocyte to
endothelial cell contact, no enhancement of PGI2 synthesis
above control was observed (Fig. 3
).
 View larger version (33K): [in a new window] |
Figure 1. Effect of the lymphocyte-to-endothelial cell ratio on PGI2
production. Endothelial cells (105/well) were incubated
alone (control) or in the presence of an increasing number of resting
(HUVEC + L) or PHA-activated lymphocytes (HUVEC + PHA-L), in
a serum-free medium for 20 h at 37°C. 6-oxo-PGF1
was measured by EIA in supernatants. Results are expressed as picograms
6-oxo-PGF1 per well and are means ± SE
of 6–17 separate experiments performed in duplicate. Data were
analyzed by ANOVA and the means compared by Scheffé’s test;
*significantly different from control, P < 0.05.
|
 View larger version (13K): [in a new window] |
Figure 2. Time-course of the lymphocyte-mediated PGI2 production.
Endothelial cells (HUVEC) at confluence were coincubated with resting
lymphocytes (HUVEC + L) in serum-free medium as described in
Materials and Methods. 6-oxo-PGF1 was measured by EIA in
supernatants collected after 20 min and up to 20 h of
coincubation. Results are expressed as picograms
6-oxo-PGF1 per well and are means ± SE
of four determinations.
|
 View larger version (28K): [in a new window] |
Figure 3. Effect of cell-to-cell contact on PGI2 synthesis. Resting
(L) or PHA-activated lymphocytes (PHA-L; 9 x 105
cells) were added to endothelial cells (HUVEC; 105
cells/well) directly (cell-to-cell contact) or separated from them by
means of an insert well (separated), and incubated for 20 h at
37°C as described in Materials and Methods. 6-oxo-PGF1
was measured by EIA in supernatants. Results are expressed as picograms
6-oxo-PGF1 per well and are means ± SE
of three separate experiments performed in duplicate. Data were
analyzed by ANOVA and the means compared by Scheffé’s test;
*significantly different from HUVEC incubated alone,
P < 0.05.
|
Lack of endothelial PGH synthase up-regulation upon lymphocyte
adhesion
Arachidonic acid oxygenation by PGH synthases (PGHS) is the first
step of PGI2 synthesis. Whereas PGHS-1 is a constitutive
enzyme and as such not prone to transcriptional regulation, PGHS-2 is
known to be inducible by cytokines and mitogens in numerous cell types,
including endothelial cells [32
, 33
]. To
check for a possible influence of lymphocyte adhesion on PGHS
expression, HUVEC monolayers, which have been incubated with
lymphocytes for increasing periods of time (1–20 h), were lysed after
extensive washing to remove nonadherent lymphocytes, and cell lysates
were submitted to Western blot analysis. As a positive control, HUVEC
monolayers were also incubated with 1 µg/mL lipopolysaccharide (LPS)
for 4 or 20 h. As expected, an immunoreactive band with the same
electrophoretic mobility as PGHS-2 was already detectable after 4 h incubation of HUVEC with LPS, and approximately twofold increased at
20 h, whereas no PGHS-2 could be detected when HUVEC were
coincubated with lymphocytes up to 20 h (Fig. 4A
). Control HUVEC incubated without lymphocytes did not express
PGHS-2. In the same time, the expression of PGHS-1 was not modified in
HUVEC coincubated with lymphocytes as compared to HUVEC incubated alone
(Fig. 4B) . Consistent with the lack of endothelial PGH synthase
up-regulation, the pretreatment of HUVEC with 5 µg/mL cycloheximide
for 30 min before the coincubation did not inhibit but rather increased
the lymphocyte-induced PGI2 output (not shown), probably
due to an increased cPLA2 mRNA expression as reported by
Higaki et al. [34
].
 View larger version (49K): [in a new window] |
Figure 4. Western blot analysis of PGH synthases-1 and -2 from HUVEC coincubated
with resting lymphocytes. Confluent HUVEC were incubated in the absence
or presence of resting lymphocytes (lymphocyte/HUVEC ratio = 9)
for the indicated periods of time. Monolayers were then thoroughly
washed, cells were lysed, and proteins were resolved by
SDS-polyacrylamide gel electrophoresis (PAGE). PGHS-2 (A) and PGHS-1
(B) (20 µg protein/lane) were identified by Western blot analysis in
proteins from HUVEC and adherent lymphocytes because the latter are
assumed to possess very few of enzymes if any. (A) Lanes 1 and 2,
lysates from HUVEC incubated alone for 4 and 20 h, respectively;
lanes 3–6, lysates from HUVEC coincubated with lymphocytes for 1, 4,
8, and 20 h, respectively; lane 7 and 8, lysates from HUVEC
incubated with 1 µg/mL LPS for 4 and 20 h, respectively. (B)
Lanes 1 and 2, lysates from HUVEC incubated alone for 4 and 20 h,
respectively; lanes 3–6, lysates from HUVEC coincubated with
lymphocytes for 1, 4, 8, and 20 h, respectively. The figure
represents one of two separate experiments giving similar results.
|
 View larger version (20K): [in a new window] |
Figure 5. Western blot analysis of PGI2 synthase from lymphocytes and
HUVEC. Fifty micorgrams of proteins from platelet (lane 1), HUVEC (lane
2), and lymphocyte (lane 3) lysates, or lymphocyte proteins
immunoprecipitated with a PGI2 synthase monoclonal antibody
(lane 4), were separated by SDS-PAGE and immunodetected using a
PGI2 synthase polyclonal antibody as described in Materials
and Methods. The figure represents one of two separate experiments
giving similar results.
|
|
View this table: [in a new window] |
Table 1. Influence of Tranylcypromine Pretreatment on Lymphocyte-Mediated
PGI2 Synthesis
|
was measured by EIA in an aliquot fraction of
culture supernatant and 6-oxo-[14C]PGF1
was determined by thin-layer chromatography analysis of supernatant
lipid extracts and autoradiography as described in Materials and
Methods. As shown in Figure 6A
, radiolabeled HUVEC incubated alone produced low but detectable
amounts of 6-oxo-[14C]PGF1 increasing with
time from 148 dpm after 1 h incubation up to 600 dpm at 8 h
(Fig. 6E , open squares), the highest time point used in these
experiments. The coincubation of radiolabeled HUVEC with unlabeled
lymphocytes markedly stimulated
6-oxo-[14C]PGF1 synthesis (Fig. 6B)
with a
time-course similar to that of the total 6-oxo-PGF1 mass
(Fig. 6D and 6E
, filled diamonds). In marked contrast, no labeled
6-oxo-PGF1 could be detected when
[14C]arachidonate-labeled lymphocytes were incubated with
unlabeled HUVEC (Fig. 6C)
, whereas the increase in
6-oxo-PGF1 mass was in the expected range (HUVEC alone:
800 pg 6-oxo-PGF1/mL vs. HUVEC + L: 4000 pg
6-oxo-PGF1/mL). These results clearly show that the
direct contact between lymphocytes and HUVEC does not initiate
lymphocyte activation and arachidonic acid release from lymphocyte
phospholipids. In activated lymphocytes the main pathway of
arachidonate liberation involves the sequential action of a
phosphoinositide-specific PLC and diacylglycerol plus monoacylglycerol
lipases [39
]. To confirm that arachidonic acid used for
PGI2 synthesis during lymphocytes-HUVEC interactions was
not of lymphocyte origin, lymphocytes were pretreated with the
diglyceride lipase inhibitor RHC 80267 [27
,
39
] before coincubation experiments. As shown in
Table 2
, the pretreatment of lymphocytes with RHC 80267 has no significant
effect on the lymphocyte-mediated PGI2 synthesis.
 View larger version (51K): [in a new window] |
Figure 6. Arachidonic acid used for the lymphocyte-induced PGI2
synthesis originates from HUVEC. Endothelial cells grown in
gelatin-coated Petri dishes (28 cm2) were labeled overnight
with 0.5 µCi/mL [14C]arachidonic acid in the presence
of 5% fetal calf serum. Cells were then extensively washed to remove
non-incorporated radioactive material and used for coincubation
experiments. (A) Labeled HUVEC were incubated alone for increasing
periods of time from 1 to 8 h (lanes 1–3). In panel A, lane 4,
unlabeled HUVEC were incubated for 30 min with 10 µmol/L
[14C]arachidonic acid in a serum-free medium. Lane 5
shows the labeling medium before the coincubations. (B) Labeled HUVEC
were incubated with unlabeled resting lymphocytes at a
lymphocyte/endothelial cell ratio of 9 for increasing periods of time
from 30 min to 8 h (lanes 6–9). (C) Lymphocytes labeled for
1 h with 0.5 µCi/mL [14C]arachidonic acid in a
serum-free medium were incubated either alone (lane 10) or with
unlabeled HUVEC (lymphocyte/endothelial cell ratio = 9) for 4 h (lane 11). At the end of the incubations, culture supernatants were
extracted and lipids separated on TLC as described in Materials and
Methods. Plates were further exposed to MP Hyperfilm for 14 (A, B) or
30 days (C). Spots co-migrating with 6-oxo-PGF1 standard
were scraped off and the radioactivity determined by liquid
scintillation counting (E). Aliquots of each supernatant were also used
for total PGI2 measurement by EIA (D). Results are from one
experiment representative of two.
|
|
View this table: [in a new window] |
Table 2. Influence of Lymphocyte Pretreatment with RHC 80267 on the
Lymphocyte-Mediated PGI2 Synthesis
|
 View larger version (37K): [in a new window] |
Figure 7. Influence of MAFP pretreatment on the lymphocyte-mediated
PGI2 synthesis. Endothelial cells (105
cells/well) either pretreated for 30 min with 25 µmol/L MAFP or
untreated were incubated alone or in the presence of MAFP-pretreated
(25 µmol/L, 30 min) or untreated resting lymphocytes, in a serum-free
medium for 4 h at 37°C. 6-oxo-PGF1 was measured
by EIA in supernatants. Results are expressed as picograms
6-oxo-PGF1 per well and are means ± SE
of five separate experiments performed in duplicate. Data were analyzed
by ANOVA and the means compared by Fisher’s PLSD test.
*Significantly different from untreated HUVEC incubated
alone, P < 0.05; significantly different
from untreated HUVEC + untreated lymphocytes, P <
0.05.
|
 View larger version (31K): [in a new window] |
Figure 8. Influence of calcium chelators on the lymphocyte-induced
PGI2 synthesis. Endothelial cells (105
cells/well) either untreated (control, EGTA) or pretreated for 45 min
with 100 µmol/L BAPTA/AM (BAPTA/AM, EGTA + BAPTA/AM) were
incubated alone (open bars) or in the presence of resting lymphocytes
(hatched bars) in the absence (control, BAPTA/AM) or in the presence of
5 mmol/L EGTA, in a serum-free medium for 4 h at 37°C.
6-oxo-PGF1 was measured by EIA in supernatants. Results
are expressed as picograms 6-oxo-PGF1 per well and are
means ± SE of six separate experiments performed in
duplicate. Data were analyzed by ANOVA and the means compared by
Scheffé’s test. *Significantly different from
untreated HUVEC incubated alone, P < 0.05,
significantly different from untreated HUVEC +
untreated lymphocytes (control), P < 0.05.
|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Although it is generally accepted that lymphocytes are not able to oxygenate arachidonic acid [35 ], Iniguez et al. have recently reported an induction of PGHS-2 upon T lymphocyte activation by anti-CD3 plus anti-CD28 antibodies [43 ]. We cannot exclude that PGHS-2 could be present in adhered lymphocytes but it was undetectable given the low number of lymphocytes with respect to endothelial cells. However, Iniguez et al. [43 ] have shown that in activated T cells, PGHS-2 metabolites were produced at the nuclear level, where they activated gene transcription but were not released extracellularly. Thus, it is very unlikely that a hypothetical PGHS-2 present in lymphocytes could contribute significantly to the observed lymphocyte-induced PGI2 production. The present results suggest that the coincubation with HUVEC did not trigger lymphocyte activation and cytokine secretion. Thus, we hypothesize that the direct lymphocyte to HUVEC contact triggers a signaling pathway in endothelial cells, leading to increased arachidonic acid availability to PGH synthases. Although lymphocyte/HUVEC interactions have been extensively studied in the context of inflammation, tissue injury, or wound healing, relatively few reports have dealt with lymphocyte-mediated signaling in endothelial cells. It has been demonstrated that natural killer cell adhesion to cytokine-treated HUVEC elicited calcium oscillations associated with inositol phosphate generation [44 ]. Similar results have been reported for monoclonal antibodies directed against E-selectin and vascular cell adhesion molecule-1 (VCAM-1), whereas no calcium change was observed with antibodies directed against intercellular adhesion molecule-1 (ICAM-1) and platelet/endothelial cell adhesion molecule-1 (PECAM-1) [45 ]. This is at variance with results from Gurubhagavatula et al. [46 ] showing that engagement of PECAM-1 on HUVEC increased intracellular calcium concentration and stimulated PGI2 release. However, ELAM, VCAM-1, and ICAM-1 are clearly not involved in our experimental conditions because PGI2 synthesis was not suppressed when co-incubation experiments were performed in the presence of blocking antibodies directed against these adhesion molecules [Dominguez et al., unpublished results]. Because PECAM-1 is also expressed on some T cell subsets, homophilic interactions between lymphocyte and endothelial PECAM-1 could be envisaged [47 ].
Although the adhesion molecules involved in HUVEC-lymphocyte contact under static conditions remain to be identified, this cell-cell contact triggers an outside-in signaling in endothelial cells leading to arachidonic acid release through PLA2 activation. The strong inhibition of PGI2 synthesis observed with MAFP-pretreated HUVEC (Fig. 7) suggests that cytosolic but not secreted PLA2 were involved. This PLA2 showed a marked dependency on calcium because it was totally suppressed by the combination of the intracellular and extracellular calcium chelators, EGTA and BAPTA. These results rule out a possible role for the calcium-independent iPLA2 and support further the hypothesis of the cytosolic 85-kDA cPLA2 activation through direct lymphocyte/HUVEC contacts. Furthermore, the strong inhibition of PGI2 synthesis induced by EGTA alone (87%) indicates that calcium entry from the external space is required for cPLA2 activation. These results are in good agreement with those of Millanvoye-Van Brussel et al. [48 ] showing that in HUVEC, arachidonic acid release is directly related to calcium influx rather than to calcium mobilization from internal stores. The biochemical mechanisms responsible for endothelial cPLA2 activation upon lymphocyte addition to HUVEC are presently under investigation.
Tranylcypromine experiments (Fig. 6)
suggest that part of the
endothelial PGH2 could be metabolized by the lymphocyte
PGI2 synthase through transcellular exchange in a manner
reminiscent of what has been described by Wu et al. for interactions
between lymphocytes and platelets [36
]. Because
tranylcypromine has also been described as an inhibitor of arachidonic
acid release [25
], a passage of arachidonic acid from
lymphocytes to HUVEC could also be envisaged to explain the
lymphocyte-induced PGI2 synthesis. However, this latter
hypothesis seems to be unlikely for the following reasons. First, when
[14C]arachidonate-labeled lymphocytes were coincubated
with unlabeled HUVEC, no 14C-radiolabeled
6-oxo-PGF1 could be detected by TLC analysis and
autoradiography of the plates, whereas coincubations of
[14C]arachidonate-labeled HUVEC with unlabeled
lymphocytes produced a time-dependent synthesis of radiolabeled
6-oxo-PGF1. These results strongly suggest that
arachidonic acid used for the lymphocyte-mediated PGI2
synthesis does not originate from lymphocytes. Second, the
lymphocyte-induced PGI2 synthesis was not affected when
lymphocytes were pretreated with the DAG lipase inhibitor RHC 80267
(Table 1)
or the PLA2 inhibitor MAFP (Fig. 8)
. In human
lymphocytes, DAG lipase is the main pathway for arachidonic acid
release [39
]. Collectively, the present results
demonstrate that the direct contact of lymphocytes to HUVEC triggers a
signaling pathway in endothelial cells leading to increased arachidonic
acid release and synthesis of the endoperoxide PGH2, which
is further metabolized to PGI2 by both cell types. It can
be noticed on autoradiographies shown in Figure 6
that labeled HUVEC
incubated alone produced marked amounts of radioactive
PGE2. However, PGE2 synthesis was only modestly
stimulated by lymphocyte contact (4950 dpm for HUVEC alone vs. 8750 dpm
for HUVEC coincubated with lymphocytes, at 8 h), whereas
PGI2 synthesis was more than fourfold increased. Thus, the
stimulation of HUVEC either by a short incubation with radiolabeled
arachidonic acid (Fig. 6 , lane 4) or by lymphocytes preferentially
directed PGH2 metabolism to PGI2 synthesis.
The production of PGI2 by vascular endothelial cells is essential for the physiology of hemostasis. The capacity of lymphocytes to stimulate PGI2 synthesis provides the endothelium with a biochemical potential to regulate the vascular tone and to limit the extension of thrombotic events, which may accompany atherosclerosis. This enhancement of PGI2 synthesis may be viewed as a beneficial effect of lymphocytes together with their capacity to inhibit smooth muscle cell proliferation [8 ] and to induce endothelial nitric oxide synthesis [49 ], which may counteract some of their negative effects such as the induction of procoagulant activity through stimulation of tissue factor expression [42 , 50 ].
Received March 19, 2000; revised August 6, 2000; accepted August 10, 2000.
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
This article has been cited by other articles:
 |
|
 |
|
  M. D. Salvado, A. Alfranca, A. Escolano, J. Z. Haeggstrom, and J. M. Redondo COX-2 Limits Prostanoid Production in Activated HUVECs and Is a Source of PGH2 for Transcellular Metabolism to PGE2 by Tumor Cells Arterioscler. Thromb. Vasc. Biol., July 1, 2009; 29(7): 1131 - 1137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Nagaoka, T. W. Hein, A. Yoshida, and L. Kuo Simvastatin Elicits Dilation of Isolated Porcine Retinal Arterioles: Role of Nitric Oxide and Mevalonate-Rho Kinase Pathways Invest. Ophthalmol. Vis. Sci., February 1, 2007; 48(2): 825 - 832. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 G. Folco and R. C. Murphy Eicosanoid Transcellular Biosynthesis: From Cell-Cell Interactions to in Vivo Tissue Responses Pharmacol. Rev., September 1, 2006; 58(3): 375 - 388. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. F. Lamour and C. E. Chalfant Ceramide-1-phosphate: The "missing" link in eicosanoid biosynthesis and inflammation Mol. Interv., December 1, 2005; 5(6): 358 - 367. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 W. E. Ackerman IV, J. M. Robinson, and D. A. Kniss Despite Transcriptional and Functional Coordination, Cyclooxygenase-2 and Microsomal Prostaglandin E Synthase-1 Largely Reside in Distinct Lipid Microdomains in WISH Epithelial Cells J. Histochem. Cytochem., November 1, 2005; 53(11): 1391 - 1401. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 P.-E. Neagoe, C. Lemieux, and M. G. Sirois Vascular Endothelial Growth Factor (VEGF)-A165-induced Prostacyclin Synthesis Requires the Activation of VEGF Receptor-1 and -2 Heterodimer J. Biol. Chem., March 18, 2005; 280(11): 9904 - 9912. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Monaco, E. Andreakos, S. Young, M. Feldmann, and E. Paleolog T cell-mediated signaling to vascular endothelium: induction of cytokines, chemokines, and tissue factor J. Leukoc. Biol., April 1, 2002; 71(4): 659 - 668. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
