Interleukin (IL)-8 (CXCL8) induces cytokine expression and superoxide formation by guinea pig neutrophils infected with Mycobacterium tuberculosis

Setting: Interleukin (IL)-8, a neutrophil attracting chemokine, is known to be made by a variety of leukocyte populations following stimulation by Mycobacterium tuberculosis. Objective: The effect of recombinant guinea pig IL-8 on the ability of neutrophils to generate a cytokine response after infection with M. tuberculosis H37Ra was examined. Design: Recombinant gpIL-8 was produced by subcloning the gene into Escherichia coli and purification over a nickel column. The identity of the rgpIL-8 was confirmed by sequencing. Neutrophils were harvested from the blood of non-vaccinated or M. bovis BCG-vaccinated guinea pigs and tested for their ability to migrate toward media alone, 10μg/ml PPD, f-Met-Leu-Phe (f-MLP), or rgpIL-8 in 96-well chemotactic chambers. Neutrophils were also pre-stimulated with rgpIL-8 then restimulated with LPS (10μg/ml) or infected in vitro with M. tuberculosis H37Ra (MOI 1:1). Results: Recombinant gpIL-8 and f-MLP induced significant chemotaxis in neutrophils from both non-vaccinated and BCG-vaccinated guinea pigs, with the best chemotaxis occuring at a concentration of 10^-7M. Real-time PCR analysis revealed that pre-treatment of neutrophils induced elevated levels of IL-8 and TNF-α mRNA and protein as well as superoxide, but not mRNA for MCP-1, IFN-γ, or TGF-β when compared to neutrophils pre-stimulated with media alone. Conclusions: The presence of IL-8 early in the host response to M. tuberculosis infection may be an important contributor to a successful immune response. How essential a role IL-8 plays remains unknown and merits further study.