LEUKOTRIENE-B(4) STIMULATES HUMAN POLYMORPHONUCLEAR LEUKOCYTES TO SYNTHESIZE AND RELEASE INTERLEUKIN-8 IN-VITRO

Interleukin-8 (IL-8) is a potent chemotactic protein for polymorphonuc lear leukocytes (PMN). Here we examine whether PMN synthesize and rele ase IL-8 in response to stimulation by leukotriene B4 (LTB4). PMN isol ated from normal heparinized peripheral human blood were incubated in RPMI culture medium at 37-degrees-C in 5 % CO2, with and without LTB4. The culture supernatants were tested for IL-8 bioactivity through che motactic activity measurements with and without neutralizing anti-IL-8 serum. Immunoreactive IL-8 was quantified by ELISA, and de novo IL-8 synthesis was evaluated by metabolic labeling with [S-35]cysteine foll owed by immunoprecipitation. LTB4 stimulated PMN to produce IL-8 in a dose- and time-dependent manner. The IL-8 concentrations reached maxim al levels after 16 h of incubation with LTB4. Significant increases in IL-8 production occurred with LTB4 doses of 10 to 1,000 nM/ml. Immuno precipitation of labeled IL-8 documented new synthesis of IL-8 by LTB4 -treated PMN. Northern blot analysis of total RNA from PMN using a 30 mer oligonucleotide for IL-8 demonstrated increased mRNA expression in LTB4-stimulated PMN compared with untreated PMN. These data show that peripheral blood PMN can be stimulated by LTB4 to synthesize and secr ete biologically active IL-8. PMN and other cells capable of producing LTB4 may induce IL-8 protein production by inflammatory PMN and there by amplify or perpetuate the acute inflammatory response by recruiting additional PMN into an inflammatory site.