Interleukin-8 induces motile behavior and loss of focal adhesions in primary fibroblasts

Abstract

Interleukin-8 (IL-8) is a proinflammatory cytokine that promotes neutrophil migration. Although fibroblasts are known to secrete IL-8, the actions of this cytokine on fibroblasts have not been previously reported. We have found that in subconfluent populations of cultured primary fibroblasts, IL-8 causes an increase in the percentage of cells lacking focal adhesions. Most of the IL-8-stimulated cells not only exhibit a lack of focal adhesions but also have a migratory phenotype that includes a protrusive leading edge and trailing tail. In addition, IL-8 was found to promote primary fibroblast chemotaxis in modified Boyden chambers as well as chemokinesis on serum-coated coverslips. Human primary fibroblasts were also found to specifically bind to IL-8 with high affinity. We have previously shown that a lack of focal structures in primary fibroblasts can be used as an index of chemokinetic locomotion and have fully characterized this system using newborn rat heart conditioned medium. The main stimulus in heart conditioned medium that is responsible for the lack of focal adhesions in the majority of cells can be immunoprecipitated using a polyclonal antibody against recombinant human IL-8. Additionally, video microscopy assays using heart conditioned medium depleted with the IL-8 antibody show an increase in the percentage of stationary cells, a consequent decrease in the percentage of migrating cells, and a twofold increase in the mitotic rate. Interleukin-1 alpha and tumor necrosis factor-alpha, which are early inflammatory cytokines, have been previously shown to stimulate IL-8 production in macrophages, fibroblasts, endothelial and epithelial cells. Our findings indicate that these two cytokines also cause an increase in the percentage of fibroblasts without focal adhesions. Additionally, this increase in cells lacking focal structures can be largely attributed to the production and subsequent autocrine action of a factor immunoprecipitated with an IL-8 antibody. Conversely, GRO-alpha, which has a high homology with IL-8, does not cause a similar increase in the percentage of cells lacking focal adhesions, but was not antagonistic to the effects of IL-8.