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Granulocyte/macrophage colony-stimulating factor stimulates human polymorphonuclear leukocytes to produce
R W McCain1, E N Dessypris, J W Christman
1Department of Veterans Affairs, Nashville, Tennessee.
Abstract:
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.
Insights
Granulocyte/macrophage colony-stimulating factor (GM-CSF) stimulates neutrophils (PMN) to produce and release interleukin-8 (IL-8). This suggests PMN can amplify inflammation by recruiting more neutrophils to infection sites.
Area of Science:
- Immunology
- Cell Biology
Background:
- Interleukin-8 (IL-8) is a key mediator in inflammatory responses, primarily attracting neutrophils (PMN).
- The capacity of PMN to synthesize and release IL-8 upon stimulation by inflammatory cytokines remains an area of investigation.
Purpose of the Study:
- To investigate whether peripheral blood PMN synthesize and release IL-8 in response to granulocyte/macrophage colony-stimulating factor (GM-CSF).
- To elucidate the role of PMN-derived IL-8 in amplifying acute inflammatory responses.
Main Methods:
- PMN were isolated and cultured with or without GM-CSF.
- Chemotactic activity of culture supernatants was assessed using a modified Boyden chamber.
- IL-8 protein levels were quantified via ELISA, and IL-8 mRNA expression was analyzed by Northern blot.
Main Results:
- GM-CSF dose-dependently stimulated PMN to produce chemotactic activity and release IL-8 protein.
- Maximal chemotactic activity and IL-8 production occurred within 3 hours of GM-CSF stimulation.
- GM-CSF treatment led to a significant increase in IL-8 mRNA levels in PMN.
Conclusions:
- Peripheral blood PMN can be induced by GM-CSF to synthesize and secrete bioactive IL-8.
- PMN-derived IL-8 may amplify acute inflammation by recruiting additional PMN to inflammatory sites.
- This autocrine or paracrine signaling loop involving GM-CSF and IL-8 highlights a mechanism for sustained neutrophil recruitment.