Granulocyte/Macrophage Colony-stimulating Factor Stimulates Human Polymorphonuclear Leukocytes to Produce Interleukin-8 In Vitro

Interleukin-8 (IL-8) is a potent chemotactic factor for polymorphonuclear leukocytes (PMN). Here we examine whether PMN synthesize and release IL-8 in response to stimulation by selected inflammatory cytokines. PMN isolated from normal heparinized peripheral human blood were incubated in RPMI culture medium at 37 degrees C in 5% CO2, with and without granulocyte/macrophage colony-stimulating factor (GM-CSF). The culture supernatants were tested for chemotactic activity using a modified Boyden chamber. Immunoreactive IL-8 protein was measured by ELISA with a monoclonal antibody specific for IL-8. GM-CSF (0.01 to 50 ng/ml) stimulated PMN to produce chemotactic activity in a dose- and time-dependent manner. The amount of chemotactic activity reached maximal levels after 3 h of incubation with GM-CSF. Treatment of culture media supernatants with rabbit antiserum against IL-8 blocked the GM-CSF-induced chemotactic activity. IL-8 protein concentrations detected by ELISA closely paralleled the chemotactic bioactivity in both the dose-response and kinetic studies. Northern blot analysis of total RNA from PMN using a 30 mer oligonucleotide complementary to mRNA for IL-8 yielded a single 1.6-kb band. Its intensity increased 4-fold 2 h after treatment of PMN with GM-CSF. These data suggest that peripheral blood PMN can be stimulated by GM-CSF to synthesize and secrete bioactive IL-8. Since both IL-8 and GM-CSF accumulate in sites of acute inflammation, PMN may induce IL-8 gene expression in response to GM-CSF and thereby amplify the acute inflammatory response by recruiting additional PMN into inflammatory sites.