Mechanisms involved in the stimulation of prostacyclin synthesis by human lymphocytes in human umbilical vein endothelial cells

Abstract

1 Endothelial cells play an important role in the modulation of vascular tone because of their ability to produce vasoactive substances such as prostacyclin (PGI(2)). Cell-cell contact between human umbilical vein endothelial cells (HUVEC) and peripheral blood lymphocytes has been shown to stimulate endothelial PGI(2) synthesis by increasing free arachidonic acid availability through endothelial cytosolic phospholipase A2 (cPLA(2)) activation. In this study, we sought to determine whether phospholipase C (PLC) and D (PLD) activation also contributes, besides cPLA(2), to the lymphocyte-induced PGI(2) synthesis in HUVEC, and to delineate further the potential mechanisms of cPLA(2) activation triggered by the interaction of HUVEC with lymphocytes. 2 Pretreatment of endothelial cells with the PI-PLC inhibitor U-73122 before the coincubation with lymphocytes markedly inhibited the PGI(2) output whereas the diacylglycerol (DAG) lipase inhibitor RHC 80267 and ethanol had no effect. These results suggest that PLC may be involved through inositol trisphosphate generation and calcium mobilization, and that neither DAG nor phosphatidic acid (PtdOH) was used as sources of arachidonic acid. 3 The stimulated PGI(2) synthesis was protein kinase C (PKC)-independent but strongly inhibited by the mitogen-activated protein kinase kinase (MEK) inhibitors PD98059 and U-0126 and by the Src kinase inhibitor PP1. 4 Immunoblot experiments showed an increased phosphorylation of the extracellular signal-regulated kinases 1/2 (ERK1/2) upon lymphocyte addition till 4 h coincubation. Phosphorylation was markedly inhibited by U-0126 and PP1 addition. 5 Collectively, these results suggest that the signaling cascade triggered by lymphocytes in endothelial cells involves an Src kinase/ERK1/2 pathway leading to endothelial cPLA(2) activation.

Figure 1

Contribution of the PLC and PLD pathways to the lymphocyte-induced PGI₂ synthesis. Confluent HUVEC (10⁵ cells per well) either untreated (control) or pretreated with (a) 5 μM U-73122 (+U-73122) for 15 min, (b) 10 μM RHC 80267 (+RHC 80267) for 30 min or (c) 1% ethanol (+1% ethanol) for 1 h were incubated alone or in the presence of lymphocytes (lymphocyte to HUVEC ratio=15) in a serum-free medium for 4 h at 37°C. In (c), ethanol when present was maintained during the 4 h coincubation. 6-oxo-PGF_1α was measured by EIA in supernatants. Results are expressed as picograms 6-oxo-PGF_1α per well and are means ±s.e. of four (b, c) to five (a) separate experiments performed in triplicate. Data were analyzed by ANOVA and the means compared by Scheffé's test. ^*Significantly different from control HUVEC incubated alone; †Significantly different from untreated HUVEC coincubated with lymphocytes.

Figure 2

The lymphocyte-induced PGI₂ synthesis is independent of PKC. Confluent HUVEC (10⁵ cells per well) either untreated (control) or pretreated with (a) 1 μM BIM (+ BIM) for 30 min or (b) 100 nM PMA for 18 h (+ PMA 18 h) were incubated alone or in the presence of lymphocytes (lymphocyte to HUVEC ratio=15) in a serum-free medium for 4 h at 37°C. 6-oxo-PGF_1α was measured by EIA in supernatants. Results are expressed as picograms 6-oxo- PGF_1α per well and are means ± s.e. of five (a) or three (b) separate experiments performed in triplicate. Data were analyzed by ANOVA and the means compared by Scheffé's test. ^*Significantly different from control HUVEC incubated alone.

Figure 3

Effect of MEK inhibitors PD98054 and U-0126 on lymphocyte-induced PGI₂ synthesis. Confluent HUVEC (10⁵ cells per well) either untreated or pretreated with 25 μM PD98059 or 10 μM U-0126 for 30 min were incubated alone (HUVEC) or with lymphocytes (HUVEC+L) (lymphocyte to HUVEC ratio=15) for 4 h at 37°C. 6-oxo-PGF_1α was measured by EIA in supernatants. Results are expressed as picograms 6-oxo-PGF_1α per well and are means±s.e. of five separate experiments performed in triplicate. Data were analyzed by ANOVA and the means compared by Scheffé's test. ^*Significantly different from control HUVEC incubated alone; †Significantly different from HUVEC coincubated with lymphocytes in the absence of inhibitor.

Figure 4

Immunoblot analysis of phospho-ERK1/2 and ERK1/2 from HUVEC coincubated with resting lymphocytes. Confluent HUVEC were incubated in the absence (HUVEC) or presence of resting lymphocytes (HUVEC+L) (lymphocyte to HUVEC ratio=9) for the indicated periods of time. Monolayers were then thoroughly washed to remove lymphocytes; cells were lysed and proteins (30 μg protein per lane) were resolved by 10% SDS–polyacrylamide gel electrophoresis as described in Methods. The blots represent one of three separate experiments giving similar results. The upper panel shows the relative intensity of p-ERK1/2 bands normalized to their corresponding ERK1/2 controls. Values are means±s.e. of three separate experiments. ^*Significantly different from control HUVEC incubated alone.

Figure 5

Effects of the tyrosine kinase inhibitor genistein and the Src kinase inhibitor PP1 on lymphocyte-induced PGI₂ synthesis. Confluent HUVEC (10⁵ cells per well), either untreated or pretreated with 100 μM genistein or 50 μM PP1 for 30 min, were incubated alone (HUVEC) or with lymphocytes (HUVEC+L) (lymphocyte to HUVEC ratio=15) for 4 h at 37°C. 6-oxo-PGF_1α was measured by EIA in supernatants. Results are expressed as picograms 6-oxo-PGF_1α per well and are means±s.e. of three separate experiments performed in triplicate. Data were analyzed by ANOVA and the means compared by Scheffé's test. ^*Significantly different from control HUVEC incubated alone; †Significantly different from HUVEC coincubated with lymphocytes in the absence of inhibitor.

Figure 6

Inhibition of ERK phosphorylation by U-0126 and PP1. Confluent HUVEC (3 × 10⁵ cells per well) either untreated or pretreated with 10 μM U-0126 or 50 μM PP1 for 30 min were incubated alone (H) or with lymphocytes (H+L) (lymphocyte to HUVEC ratio=10) for 4 h at 37°C. Monolayers were then thoroughly washed to remove lymphocytes; cells were lysed and proteins (20 μg protein per lane) were resolved by 10% SDS–polyacrylamide gel electrophoresis as described in Methods. The blots represent one of four separate experiments giving similar results. The upper panel shows the relative intensity of p-ERK1/2 bands normalized to their corresponding ERK1/2 controls. Values are means±s.e. of four separate experiments. ^*Significantly different from control HUVEC incubated alone; †Significantly different from control HUVEC incubated with lymphocytes in the absence of inhibitor.

Figure 7

Proposed model for endothelial PGI₂ synthesis induced by lymphocyte contact. See the discussion for further explanation.?, unidentified receptor or adhesion molecule; AA, arachidonic acid.