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(Journal of Leukocyte Biology. 2000;68:881-889.)
© 2000 by Society for Leukocyte Biology

Human lymphocytes stimulate prostacyclin synthesis in human umbilical vein endothelial cells. Involvement of endothelial cPLA2

Faten Merhi-Soussi, Zury Dominguez{dagger}, Olga Macovschi, Madeleine Dubois, Alain Savany, Michel Lagarde and Annie-France Prigent

INSERM U352, Laboratoire de Biochimie et Pharmacologie, Institut National des Sciences Appliquées de Lyon, Villeurbanne, France; and
{dagger} 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Prostacyclin (PGI2) contributes to the maintenance of a nonadhesive luminal surface in blood vessels due to its anti-platelet and vasodilatory properties. Here, we sought to determine whether peripheral blood lymphocytes (PBL) may regulate the PGI2 production of human umbilical vein endothelial cells (HUVEC). Cell-cell contact between HUVEC and lymphocytes markedly enhanced PGI2 synthesis as a function of the number of lymphocytes added. This stimulated synthesis was totally suppressed when lymphocytes and HUVEC were separated by a microporous insert. It was not due to prostaglandin H synthase up-regulation. The pretreatment of lymphocytes with the PGI2 synthase inhibitor tranylcypromine partially inhibited PGI2 synthesis (47%), suggesting a transcellular metabolism of the endothelial prostaglandin endoperoxide PGH2 by the lymphocyte PGI2 synthase. Experiments using [14C]arachidonate-labeled lymphocytes coincubated with unlabeled HUVEC, and [14C]arachidonate-labeled HUVEC coincubated with unlabeled lymphocytes showed that the arachidonic acid used for PGI2 synthesis was totally of endothelial origin. Furthermore, the PGI2 synthesis was strongly inhibited by the cytosolic phospholipase A2 inhibitor, MAFP and totally suppressed by the combination of the calcium chelators, BAPTA and EGTA. Collectively, these results suggest that lymphocytes trigger an outside-in signaling in endothelial cells involving cPLA2 activation. Overall, the switch-on for PGI2 synthesis induced by lymphocytes might serve as a protection against atherothrombogenesis.

Key Words: lymphocyte-endothelial cell interactions • prostaglandin H and prostaglandin I2 synthases • atherothrombogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Increased adherence of leukocytes to the endothelium and their transmigration into the arterial wall is one of the earliest events in atherogenesis [1 2 3 ]. Immunohistological investigations have shown that in addition to monocytes/macrophages, T lymphocytes also accumulate in early atherosclerotic lesions both in humans [4 ] and in experimental models [5 ]. Plaque lymphocytes usually account for up to 20% of the mononuclear cells [2 ]. In some experimental models such as rats fed an atherogenic diet, this percentage can even reach 60% in the initial phase of lesion formation [5 ]. The functional role of T lymphocytes in atherosclerotic lesions and their contribution to atherogenesis remains unclear at this time. Several in vivo studies have suggested that T cells might initiate or accelerate lesion formation. Thus, a marked reduction in the size of aortic lesions has been reported in CD4-T cell-depleted and nude C57BL/6 mice fed a high-fat atherogenic diet, compared with control mice fed the same regimen [6 ]. Similarly, athymic nude rats fed an atherogenic diet developed reduced aortic lesions almost devoid of T cells [7 ]. On the other hand, Hansson et al. [8 ] have shown that athymic rnu/rnu rats that lack T lymphocytes develop larger carotid arterial lesions after balloon-catheter injury than rnu/+ littermates with normal T cell levels [8 ]. Thus T lymphocytes may provide either negative or positive signals depending on the type of initial injury [9 ]. An initial event in the transmigration process is the binding of leukocytes to endothelial cells. These interactions through specific adhesion molecules are part of the normal functioning of vascular endothelium [10 ]. They trigger the synthesis of a large variety of bioactive substances that allow the endothelium to maintain a nonadhesive luminal surface in all blood vessels and to adjust blood flow [11 , 12 ]. The well-known endothelium-derived antiplatelet and vasodilatory factor is prostacyclin (PGI2), which is the main prostanoid synthesized by these cells [13 , 14 ]. Biosynthesis of PGI2 involves in succession phospholipase A2, which releases arachidonic acid from the sn-2 position of membrane phospholipids, prostaglandin H synthase (PGHS), which converts arachidonic acid to prostaglandin endoperoxide PGH2, and finally PGI2 synthase, which transforms PGH2 into PGI2 [15 ]. PGI2 inhibits the growth of human vascular smooth muscle cells in vivo [16 ], platelet aggregation and adhesion [17 ], leukocyte adhesion [18 ], foam cell formation, and cholesterol accumulation in the vascular wall [19 ]. It also stimulates fibrinolytic activity in atherosclerotic patients [20 ]. Among the various cytokines present in conditioned medium from stimulated blood mononuclear cells, interleukin-1 (IL-1) has been shown to be the most efficient to stimulate PGI2 synthesis in vascular cells [11 , 12 , 21 ]. It has also been demonstrated that direct cell-cell contact between purified monocytes and endothelial cells more strongly increased PGI2 synthesis than IL-1 [22 ], thus highlighting the crucial role of direct cell-cell contact in this process. Lymphocyte-derived cytokines have also been described to stimulate PGI2 synthesis, although contradictory results have been reported [11 , 21 ]. However, the influence of lymphocyte-endothelial cell contact on PGI2 synthesis has never been investigated with human cells. In this study, we investigated whether human lymphocytes may regulate the PGI2 production of HUVEC, a well-characterized in vitro model of human vascular endothelium [23 ].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and chemicals
RPMI 1640 medium with 25 mmol/L HEPES and bicarbonate and M-199 medium (containing 25 mmol/L HEPES buffer, Earle’s salts, L-glutamine, and L-amino acids), heat-inactivated newborn calf serum, L-glutamine, penicillin-streptomycin, gentamicin, endothelial cell growth factor, collagenase Type IA, trypsin-EDTA solution, gelatin type B, tranylcypromine, NaCl, Dextran, Histopaque-1077, trypan blue, phenylmethylsulfonyl fluoride (PMSF), leupeptin, aprotinin, Triton X-100, and tris(hydroxymethyl)aminomethane, EGTA, and BAPTA/AM were all purchased from Sigma-Aldrich (L’Isle d’Abeau, France). Glycerol was from SDS (Peypin, France). Fetal bovine serum was purchased from Biomedia (La Verpillère, France). PGI2 enzyme immunoassay (EIA) kits, PGI2 synthase monoclonal and polyclonal antibodies, PGHS-1 monoclonal antibody, and PGHS-2 polyclonal antibody, methyl arachidonoyl fluorophosphonate (MAFP; Cayman) were from SPI-Bio (Massy, France). Na251CrO4 was from NEN-Life Science Products (Le Blanc Mesnil, France). Protein G-Sepharose, MP Hyperfilm, enhanced chemiluminescence (ECL), horseradish peroxidase (HRP)-conjugated anti-mouse or anti-rabbit IgG were from Amersham-Pharmacia Biotech (Orsay, France). RHC80267 was from TEBU (Le Perray-en-Yvelines, France).

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{alpha} (6-oxo-PGF1{alpha}). 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{alpha} were scraped off, mixed with picofluor (Packard), and the radioactivity determined by liquid scintillation counting. Standard 6-oxo-PGF1{alpha}, 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Lymphocyte-induced stimulation of PGI2 synthesis in HUVEC
Endothelial cell monolayers incubated in serum-free medium for up to 20 h produced only small amounts of PGI2 measured as 6-oxo-PGF1{alpha} (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 ).



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  		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{alpha} was measured by EIA in supernatants. Results are expressed as picograms 6-oxo-PGF1{alpha} 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.

 


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  		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{alpha} 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{alpha} per well and are means ± SE of four determinations.

 


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  		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{alpha} was measured by EIA in supernatants. Results are expressed as picograms 6-oxo-PGF1{alpha} 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.

 
These transwell experiments clearly indicate that the physical association between lymphocytes and HUVEC is necessary to observe the lymphocyte-mediated stimulation of PGI2 synthesis. In parallel experiments using Na251CrO4-labeled resting or PHA-activated lymphocytes, it has been shown that the number of lymphocytes adhering to HUVEC increased linearly as a function of the number of lymphocytes added to HUVEC monolayers (not shown). As a consequence, the percentage of adhering lymphocytes remained constant whatever the lymphocyte-to-HUVEC ratio, resting lymphocytes adhering slightly less than PHA-activated ones (15.6 ± 2.8 vs. 21.7 ± 2.3%, n = 4).

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 ].



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  		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.

 
Contribution of lymphocytes to PGI2 synthesis in lymphocyte-HUVEC coincubations
Lymphocytes are known to have only a weak, if any, capability to oxygenate arachidonic acid [35 ]. However, results from Wu et al. [36 ], showing that lymphocytes coincubated with activated platelets devoid of PGI2 synthase could produce PGI2, suggested that human lymphocytes might express PGI2 synthase. To test this hypothesis, lymphocyte extracts were analyzed by Western blotting using a polyclonal anti-PGI2 synthase antibody. HUVEC and platelets were used as positive and negative controls, respectively [37 , 38 ]. The major immunoreactive band detected in lymphocyte lysates (lane 3, Fig. 5 ) has the same electrophoretic mobility as endothelial PGI2 synthase (lane 2, Fig. 5 ). To further confirm that this band corresponds to PGI2 synthase, lymphocyte lysates were immunoprecipitated with a PGI2 synthase monoclonal antibody, and immunoprecipitated proteins were analyzed by Western blotting. Only the 57-kDa band, with the same electrophoretic mobility as human PGI2 synthase, was recognized by the polyclonal PGI2 synthase antibody (lane 4, Fig. 5 ). Human platelet lysates, used as a negative control, did not show any immunoreactive band at 57 kDa (lane 1, Fig. 5 ). To evaluate the relative contribution of lymphocyte PGI2 synthase to the total PGI2 produced by HUVEC plus lymphocyte coincubations, either HUVEC or resting lymphocytes were pretreated with tranylcypromine, a well-known inhibitor of PGI2 synthase [24 , 25 ], before the coincubation. As shown in Table 1 , the pretreatment of either HUVEC or lymphocytes partially inhibited the lymphocyte-induced PGI2 synthesis (56 and 47%, respectively), whereas a total inhibition was observed when tranylcypromine was present in the culture medium during the overall coincubation. Similar results were obtained with lymphocytes previously activated by PHA for 3 days (not shown). These results suggest that in HUVEC-lymphocyte coincubations, part of endothelial PGH2 is metabolized by lymphocyte PGI2 synthase.



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  		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.

 

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  		Table 1. Influence of Tranylcypromine Pretreatment on Lymphocyte-Mediated PGI2 Synthesis

 
Lymphocytes fail to provide arachidonic acid for PGI2 synthesis in lymphocyte-HUVEC coincubations
To examine whether arachidonic acid used for the stimulated PGI2 synthesis originate partly from lymphocytes or exclusively from endothelial cells, either [14C]arachidonate-labeled lymphocytes were coincubated with unlabeled HUVEC or unlabeled lymphocytes were coincubated with [14C]arachidonate-labeled HUVEC (lymphocyte/HUVEC ratio = 9). At the end of coincubation, total 6-oxo-PGF1{alpha} was measured by EIA in an aliquot fraction of culture supernatant and 6-oxo-[14C]PGF1{alpha} 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{alpha} 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{alpha} synthesis (Fig. 6B) with a time-course similar to that of the total 6-oxo-PGF1{alpha} mass (Fig. 6D and 6E , filled diamonds). In marked contrast, no labeled 6-oxo-PGF1{alpha} could be detected when [14C]arachidonate-labeled lymphocytes were incubated with unlabeled HUVEC (Fig. 6C) , whereas the increase in 6-oxo-PGF1{alpha} mass was in the expected range (HUVEC alone: 800 pg 6-oxo-PGF1{alpha}/mL vs. HUVEC + L: 4000 pg 6-oxo-PGF1{alpha}/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.



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  		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{alpha} 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.

 

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  		Table 2. Influence of Lymphocyte Pretreatment with RHC 80267 on the Lymphocyte-Mediated PGI2 Synthesis

 
Lymphocytes stimulate PGI2 synthesis through activation of endothelial cPLA2
Because lymphocytes did not up-regulate PGH synthase expression, we hypothesize that they could act upstream, at the level of arachidonic acid release. To examine whether endothelial cPLA2 is involved in the lymphocyte-induced PGI2 synthesis, HUVEC were pretreated with the cPLA2 inhibitor MAFP at the concentration of 25 µM, known to inhibit both calcium-dependent and independent cytosolic PLA2 [26 ]. As a control, lymphocytes were also pretreated with MAFP before incubation with untreated HUVEC. As shown in Figure 7 , the inhibition of endothelial cytosolic PLA2 markedly decreased (-80%) the PGI2 output induced by the direct contact between lymphocytes and endothelium, whereas the pretreatment of lymphocytes with MAFP has no significant effect on the PGI2 response. These results strongly suggest that the direct contact of lymphocytes with HUVEC is sufficient to initiate a signaling cascade that involves cytosolic PLA2 activation and results in PGI2 synthesis. To determine whether the lymphocyte-induced PGI2 synthesis involved the calcium-dependent (cPLA2) or -independent (iPLA2) cytosolic enzyme, coincubation experiments were performed in the presence of the extracellular calcium chelator EGTA or HUVEC were pretreated with BAPTA/AM, a cell-permeant precursor of the calcium chelator BAPTA that buffers intracellular calcium movements, before the incubation with lymphocytes either in the presence or absence of EGTA in the culture medium. As shown in Figure 8 , the PLA2 involved in PGI2 output is very likely to be the calcium-dependent enzyme because PGI2 synthesis was drastically inhibited by each chelator and almost totally suppressed when BAPTA-loaded HUVEC were coincubated with lymphocytes in the presence of 5 mmol/L EGTA.



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  		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{alpha} was measured by EIA in supernatants. Results are expressed as picograms 6-oxo-PGF1{alpha} 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; {dagger}significantly different from untreated HUVEC + untreated lymphocytes, P < 0.05.

 


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  		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{alpha} was measured by EIA in supernatants. Results are expressed as picograms 6-oxo-PGF1{alpha} 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, {dagger}significantly different from untreated HUVEC + untreated lymphocytes (control), P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Results of this study demonstrate that human lymphocytes are able to stimulate the production of PGI2 by HUVEC in coincubation experiments performed in serum-free medium under static conditions, and thus, to regulate physiological processes occurring at the blood cell/vessel wall interface. PGI2 production increased as a function of the number of lymphocytes added, resting lymphocytes being as efficient as PHA-lymphoblasts. This result indicates that cell-surface molecules involved in the PGI2-induced synthesis were already present on resting lymphocytes and that the expression of very late activation antigen was not a prerequisite, although it increased adherence to endothelial cells. The lymphocyte-mediated PGI2 production by HUVEC was a rapid process, about 70% of the maximal response being already observed after 30 min (3.75-fold increase over basal level at 30 min vs. 5.2-fold at 20 h). This time-course is thus at variance with that described for human monocytes [22 ] where a 4-h coincubation was required to detect a significant enhancement of PGI2 synthesis, the maximal response being observed after 24-h coincubation. These findings strongly suggest that the mechanisms involved in lymphocyte- and monocyte-mediated PGI2 synthesis are quite different. Although the contact between endothelial cells and lymphocytes may provide T cell costimulation signals and induce cytokine synthesis [40 , 41 ], several lines of evidence suggest that the lymphocyte-mediated PGI2 production reported here does not involve the synthesis of soluble cytokines. First, PGI2 level was already significantly increased after 20–30 min of coincubation, whereas cytokine synthesis requires several hours. Second, no induction of PGI2 synthesis could be detected when the physical contact between lymphocytes and HUVEC was prevented by a microporous filter, which allowed the free passage of soluble molecules. In good agreement with the lack of cytokine involvement is the fact that no immunoreactive PGHS-2 known to be induced by cytokines in endothelial cells [11 , 12 , 21 ] could be detected in lysate of HUVEC plus adhered lymphocytes. However, we cannot exclude the presence of cytokines tightly bound to the cell surface of lymphocytes. Indeed, it has been demonstrated that human lymphocytes express a transmembrane form of tumor necrosis factor (mTNF) able to induce tissue factor expression in HUVEC through interactions with the 75-kDa form of TNF-R [42 ]. It is noteworthy that tissue factor synthesis in HUVEC also requires direct contact between lymphocytes and endothelial cells but displays slower kinetic patterns than we observed for PGI2 synthesis.

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{alpha} 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{alpha}. 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 ].


   ACKNOWLEDGEMENTS
 
This work was supported by INSERM and the Région Rhône-Alpes. F. M. S. was a recipient of a fellowship from the Association Sanofi-Thrombose pour la Recherche. Z. D., Assistant Professor on sabbatical leave from Venezuela to France, was supported by the Consejo de Desarrollo Cientifico y Humanistico, CDCH-Universidad Central de Venezuela. We thank the midwives from Tonkin Maternity for kindly providing us with fresh umbilical cords.

Received March 19, 2000; revised August 6, 2000; accepted August 10, 2000.


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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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