NEUTROPHIL ADHESION TO HISTAMINE-STIMULATED CULTURED ENDOTHELIAL-CELLS IS PRIMARILY MEDIATED VIA ACTIVATION OF PHOSPHOLIPASE-C AND NITRIC-OXIDE SYNTHASE ISOZYMES

Objective and Design: In order to understand the underlying mechanism of histamine stimulated inflammatory responses, histamine receptor sub types and signal transduction pathways by which histamine mediates the stimulation of neutrophil adhesion to endothelial cells has been stud ied in vitro. Material: Human neutrophils and human umbilical vein end othelial cells. Treatment: Confluent human endothelial cell. layer wer e incubated with histamine (1mM), H-1, H-2 or H-3 receptor agonists: f luorophenylhistamine (10 mu m), amthamine (10 mu m), methylhistamine ( 10 mu M), respectively. Ten minutes prior to histamine (1 mM) stimulat ion H-1, H-2 or H-3 receptor antagonists (dimethindene, 100 mu M famot idine, 100 mu M thioperamid 100 mu M, respectively) were added. Histam ine stimulated signal transduction pathways were inhibited by adding p hospholipase C inhibitor 2-nitro-4-carboxyphenyl N,N-diphenylcarbamat (200 mu M), adenylate cyclase inhibitor 9-(2 tetrahydrofuryl)adenine ( 80 mu M), nitric oxide synthase isozymes inhibitor S-ethylisothiourea (1 mu M) or guanylate cyclase inhibitor (LY 83583; 10 mu M). Neutrophi l adhesion was monitored at 30, 60, 90, 120, 150, 180 and 210 min. Met hods: Neutrophil adhesion to endothelial cells was quantified by analy sing alkaline phosphatase activity. Results: Histamine stimulation of endothelial cells resulted in a biphasic time and concentration depend ent pattern of neutrophil adhesion. This pattern of neutrophil adhesio n was mimicked by stimulation of endothelial cells with H-1 or H-2 ago nists. Stimulation of endothelial cells with an H-3 agonist had no eff ect 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 st imulated endothelial cells. An increase of neutrophil binding to unsti mulated or to agonist stimulated endothelial cells was observed during inhibition of adenylate cyclase. Conclusions: Our results suggest tha t histamine stimulated neutrophil adhesion is due to H-1 and H-2 recep tor mediated activation of PLC, NOS and GC. Increase of cAMP concentra tion seems to mediate an inhibitory effect on PMN adhesion to endothel ial cells.