EFFECTS OF ENDOGENOUS ENDOTHELIAL INTERLEUKIN-8 ON NEUTROPHIL MIGRATION ACROSS AN ENDOTHELIAL MONOLAYER

Objective: Recent studies suggest that interleukin-8 (IL-8) is involve d in the neutrophil infiltration of subendothelial myocardial tissue i n the ischaemia/reperfusion injury associated with acute myocardial in farction. The aim of this study was to investigate the effects of IL-8 on transendothelial neutrophil migration using an in vitro three dime nsional double chamber migration assay system. Methods: Human neutroph ils were incubated with human endothelial cell monolayers for 1 h, and adherent and migrated neutrophils were then counted. Expression of IL -8 mRNA and secretion of its protein by endothelial cells were analyse d respectively by northern blotting and ELISA. Results: Recombinant hu man (rh) IL-8 (50 ng . ml(-1)) placed in the lower compartment signifi cantly increased neutrophil adhesion 1.7-fold and transmigration 2.3-f old, compared with control conditions using medium alone in both compa rtments. In contrast, rh IL-8 (50 ng . ml(-1)) in the upper compartmen t significantly inhibited neutrophil adhesion and transmigration by 53 % and 61% respectively compared with controls. Neutrophil adhesion and transmigration was dependent on the IL-8 concentration gradient betwe en upper and lower compartments. Unstimulated endothelial cells showed no IL-8 expression, but endothelial cells pretreated with IL-1 beta ( 25 U . ml(-1)) markedly induced endogenous IL-8 mRNA and protein accum ulation. When endothelial cells were cocultured with neutrophils, enha nced endogenous IL-8 production was observed. Pretreatment of endothel ial cells with IL-1 beta for 4 and 24 h increased neutrophil transmigr ation 2.8-fold and 3.0-fold respectively, compared with unstimulated e ndothelial cells. The addition of anti-IL-8 monoclonal antibody (12.5 mu g . ml(-1)) to the upper compartment with IL-1 beta-pretreated endo thelial cells further enhanced transmigration from 2.8- to 3.3-fold an d from 3.0- to 4.3-fold respectively. Conclusions: Endogenous endothel ial IL-8, secreted from activated endothelial cells into the apical si de of endothelial cell monolayers, has an inhibitory effect on transen dothelial migration of neutrophils, suggesting that IL-8 may prevent e xcessive neutrophil infiltration of myocardial tissue from circulating blood in the reperfusion injury associated with acute myocardial infa rction.