Effects of endogenous endothelial interleukin-8 on neutrophil migration across an endothelial monolayer.
OBJECTIVE
Recent studies suggest that interleukin-8 (IL-8) is involved in the neutrophil infiltration of subendothelial myocardial tissue in the ischaemia/reperfusion injury associated with acute myocardial infarction. The aim of this study was to investigate the effects of IL-8 on transendothelial neutrophil migration using an in vitro three dimensional double chamber migration assay system.


METHODS
Human neutrophils 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 analysed respectively by northern blotting and ELISA.


RESULTS
Recombinant human (rh) IL-8 (50 ng.ml-1) placed in the lower compartment significantly increased neutrophil adhesion 1.7-fold and transmigration 2.3-fold, compared with control conditions using medium alone in both compartments. In contrast, rh IL-8 (50 ng.ml-1) in the upper compartment 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 between 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 accumulation. When endothelial cells were cocultured with neutrophils, enhanced endogenous IL-8 production was observed. Pretreatment of endothelial cells with IL-1 beta for 4 and 24 h increased neutrophil transmigration 2.8-fold and 3.0-fold respectively, compared with unstimulated endothelial cells. The addition of anti-IL-8 monoclonal antibody (12.5 micrograms.ml-1) to the upper compartment with IL-1 beta-pretreated endothelial cells further enhanced transmigration from 2.8- to 3.3-fold and from 3.0- to 4.3-fold respectively.


CONCLUSIONS
Endogenous endothelial IL-8, secreted from activated endothelial cells into the apical side of endothelial cell monolayers, has an inhibitory effect on transendothelial migration of neutrophils, suggesting that IL-8 may prevent excessive neutrophil infiltration of myocardial tissue from circulating blood in the reperfusion injury associated with acute myocardial infarction.
Takahashi
M
M
Jichi Medical School, Tochigi, Japan.
Masuyama
J
J
Ikeda
U
U
Kitagawa
S
S
Kasahara
T
T
Saito
M
M
Kano
S
S
Shimada
K
K
D015152:Blotting, Northern; D002448:Cell Adhesion / Q000187:drug effects; D002465:Cell Movement / Q000187:drug effects / Q000276:immunology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000276:immunology* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D016209:Interleukin-8 / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007958:Leukocyte Count; D009504:Neutrophils / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism
D007375:Interleukin-1; D016209:Interleukin-8; D012333:RNA, Messenger