Extracellular signal-regulated kinase plays an essential role in endothelin-1-induced homotypic adhesion of human neutrophil granulocytes

Abstract

1. Endothelin-1 (ET-1) stimulates integrin-dependent adhesion of neutrophil granulocytes to endothelial cells, one of the early key events in acute inflammation. However, the signalling pathway(s) of ET-1-stimulated neutrophil adhesive responses has not been elucidated. Previous studies indicated that extracellular signal-regulated kinase (ERK) activation could mediate rapid responses of neutrophil granulocytes to various stimuli. In this study, we investigated the role of ERK signalling in human neutrophil granulocytes challenged with ET-1. 2. ET-1 rapidly down-regulated the expression of L-selectin and up-regulated the expression of CD11b/CD18 on the neutrophil surface. Concomitantly, ET-1 induced homotypic adhesion (aggregation) of neutrophils, that was blocked by a monoclonal antibody to CD18. 3. ET-1, through ET(A) receptors, evoked activation of Ras and subsequent phosphorylation of Raf-1, mitogen-activated protein kinase kinase (MAPK/ERK kinase) and ERK 1/2. ERK activation by ET-1 was rapid, concordant with the kinetics of ET-1-stimulated neutrophil aggregation. 4. Neutrophil responses to ET-1 were markedly attenuated by the MAPK/ERK kinase inhibitor PD98059, whereas inhibitors of p38 MAPK, tyrosine kinases and phosphatidylinositol 3-kinase had no detectable effects. We have observed a tight correlation between neutrophil ERK activation and homotypic adhesion. 5. These data indicate an essential role for ERK in mediating ET-1-stimulated adhesive responses of human neutrophil granulocytes.

Figure 1

Effects of ET-1 on surface expression of L-selectin and CD11b on human neutrophils. Isolated neutrophils were challenged with ET-1 for 30 min at 37°C. (a) Representative immunostaining of neutrophils challenged with 30 nM ET-1. In each histogram is also displayed the negative control of immunostaining with class-matched irrelevant antibodies (C). Shown is a representative of six experiments using neutrophils from different blood donors. (b) Concentration-dependent effects of ET-1. Fluorescence intensity is presented as percentage of control, i.e. mean fluorescence intensity of neutrophils incubated in medium only. Results are the mean±s.e.mean. for six experiments using neutrophils from different donors. ^*P<0.05; ^**P<0.01; ^***P<0.001 compared with control.

Figure 2

Effect of ET-1 on the expression of CD11a/CD18 (LFA-1) epitope expressed by the β-propeller domain and associated with higher binding affinity of CD11a/CD18 on neutrophils. Mean fluorescence of mAb G-25.2 on neutrophils challenged with (a) 100 nM ET-1 or (b) Mg²⁺ (1 mM) and EGTA (2 mM) for 30 min at 37°C. The dotted lines represent control mAb G-25.2 binding at 4°C. Shown is a representative of three experiments.

Figure 3

Effects of protein kinase inhibitors on neutrophil expression of L-selectin and CD11b. Neutrophils were preincubated with the MAPK kinase inhibitor PD98059, the phosphatidylinositol 3-kinase inhibitor wortmannin, the tyrosine kinase inhibitor genistein or the p38 MAPK inhibitor SB 203580 for 20 min at 37°C, and then challenged with ET-1 (100 nM) for 30 min. Adhesion molecule expression is presented as the percentage of control. Mean fluorescence intensity for control samples, L-selectin: 78±9; CD11b: 580±38. Values are the mean±s.e.mean of four independent experiments. ^*P<0.05 compared with ET-1 alone (hatched columns).

Figure 4

ET-1 induces time-, and concentration-dependent phosphorylation of MEK and ERK in human neutrophils. Neutrophils were challenged with ET-1 (100 nM) for the indicated time periods (a) or with various concentrations of ET-1 for 2 min (b). The effects of PAF (1 μM) are shown for comparisons. Results are representative of four experiments.

Figure 5

ET-1 stimulates neutrophil ERK activity. (a) Neutrophils were challenged with ET-1 (100 nM), then lysed at the indicated times, and ERK activity was assayed (thin line) or assayed on-line for homotypic aggregation (thick line). (b) Concentration-dependent activation of ERK and induction of neutrophil aggregation. Neutrophils were challenged with ET-1 for 3 min at 37°C, then either lysed and analysed for ERK activity or homotypic aggregation was monitored on-line for 10 min. Results shown are the mean±s.e.mean or representative of four independent experiments. (c) ET-1 stimulation of human neutrophil granulocytes is MEK dependent. Neutrophils were incubated in the absence or presence of PD98059 (100 μM) for 10 min, challenged for 2 min with ET-1 (100 nM), lyzed and analysed for ERK activity or aggregation. Results shown are the mean±s.e.mean of 4 – 5 experiments.

Figure 6

ET-1 induces activation of Ras and Raf-1 kinase. (a) Activation of Raf-1. Neutrophils were challenged with ET-1 for 2 min at 37°C, lysed, Raf-1 was immunoprecipitated, and Raf-1 kinase activity was measured as described in Methods. Results shown are the mean±s.e.mean of four experiments. ^*P<0.05; ^**P<0.01 compared with unchallenged. Neutrophils were challenged (b) with various concentrations of ET-1 for 3 min or (c) with 100 nM ET-1 for the indicated times. GTP-bound active Ras was isolated from neutrophils by affinity precipitation with a GST-Ras binding domain fusion protein followed by immunoblot analysis with an anti-Ras antibody. Shown is a representative result, the experiments were repeated four times.

Figure 7

ET-1-induced gelatinase release is MEK dependent. Neutrophils were preincubated with PD 98059 (100 μM) for 20 min at 37°C, and then challenged with ET-1 for 30 min. Values are expressed as percentage of total cellular gelatinase activity released by neutrophils into the culture medium, and are means±s.e.mean of four independent experiments. ^*P<0.05; ^**P<0.01 compared with control (unstimulated).