Neutrophil adhesion to histamine stimulated cultured endothelial cells is primarily mediated via activation of phospholipase C and nitric oxide synthase isozymes

Abstract.

Objective and Design: In order to understand the underlying mechanism of histamine stimulated inflammatory responses, histamine receptor subtypes and signal transduction pathways by which histamine mediates the stimulation of neutrophil adhesion to endothelial cells has been studied in vitro.¶Material: Human neutrophils and human umbilical vein endothelial cells.¶Treatment: Confluent human endothelial cell layer were incubated with histamine (1 mM), H₁, H₂ or H₃ receptor agonists: fluorophenylhistamine (10 μM), amthamine (10 μm), methylhistamine (10 μM), respectively. Ten minutes prior to histamine (1 mM) stimulation H₁, H₂ or H₃ receptor antagonists (dimethindene, 100 μM; famotidine, 100 μM, thioperamid 100 μM, respectively) were added. Histamine stimulated signal transduction pathways were inhibited by adding phospholipase C inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamat (200 μM), adenylate cyclase inhibitor 9-(2 tetrahydrofuryl)adenine (80 μM), nitric oxide synthase isozymes inhibitor S-ethylisothiourea (1 μM) or guanylate cyclase inhibitor (LY 83583; 10 μM). Neutrophil adhesion was monitored at 30, 60, 90, 120, 150, 180 and 210 min.¶Methods: Neutrophil adhesion to endothelial cells was quantified by analysing alkaline phosphatase activity.¶Results: Histamine stimulation of endothelial cells resulted in a biphasic time and concentration dependent pattern of neutrophil adhesion. This pattern of neutrophil adhesion was mimicked by stimulation of endothelial cells with H₁ or H₂ agonists. Stimulation of endothelial cells with an H₃ agonist had no effect on neutrophil binding. Inhibition of phospholipase C (PLC), nitric oxide synthase isozymes (NOS) or guanylate cyclase (GC) resulted in a significant decrease of neutrophil binding to histamine or agonist stimulated endothelial cells. An increase of neutrophil binding to unstimulated or to agonist stimulated endothelial cells was observed during inhibition of adenylate cyclase.¶Conclusions: Our results suggest that histamine stimulated neutrophil adhesion is due to H₁ and H₂ receptor mediated activation of PLC, NOS and GC. Increase of cAMP concentration seems to mediate an inhibitory effect on PMN adhesion to endothelial cells