INTERLEUKIN-8 INDUCES MOTILE BEHAVIOR AND LOSS OF FOCAL ADHESIONS IN PRIMARY FIBROBLASTS

Interleukin-8 (IL-8) is a proinflammatory cytokine that promotes neutr ophil migration. Although fibroblasts are known to secrete IL-8, the a ctions of this cytokine on fibroblasts have not been previously report ed. We have found that in subconfluent populations of cultured primary fibroblasts, IL-8 causes an increase in the percentage of cells lacki ng focal adhesions. Most of the IL-8-stimulated cells not only exhibit a lack of focal adhesions but also have a migratory phenotype that in cludes 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 cover-slips. Human pr imary fibroblasts were also found to specifically bind to IL-8 with hi gh affinity. We have previously shown that a lack of focal structures in primary fibroblasts can be used as an index of chemokinetic locomot ion and have fully characterized this system using newborn rat heart c onditioned medium. The main stimulus in heart conditioned medium that is responsible for the lack of focal adhesions in the majority of cell s can be immunoprecipitated using a polyclonal antibody against recomb inant human IL-8. Additionally, video microscopy assays using heart co nditioned medium depleted with the IL-8 antibody show an increase in t he percentage of stationary cells, a consequent decrease in the percen tage of migrating cells, and a twofold increase in the mitotic rate. I nterleukin-1 alpha and tumor necrosis factor-alpha, which are early in flammatory cytokines, have been previously shown to stimulate IL-8 pro duction 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. Additionall y, this increase in cells lacking focal structures can be largely attr ibuted to the production and subsequent autocrine action of a factor i mmunoprecipitated with an IL-8 antibody. Conversely, GRO-alpha, which has a high homology with IL-8, does not cause a similar increase in th e percentage of cells lacking focal adhesions, but was not antagonisti c to the effects of IL-8.