Neutrophil chemokines in cultured nasal fibroblasts.

Abstract

To gain insight into the mechanisms responsible for tissue neutrophil immigration in sinusitis, primary nasal fibroblasts are analyzed for synthesizing and delivering neutrophil chemokines. Primary nasal fibroblast cell culture was treated with tumor necrosis factor (TNF)-alpha concentrations of 20 and 200 ng/ml for 2, 8, 24 and 72 h. Chemokine concentrations in supernatants were determined by enzyme-linked immunoassay (ELISA) and chemokine mRNA expression in fibroblasts was measured by reverse transcriptase polymerase chain reaction (RT-PCR). Biological chemotactic activity was identified by three-step high-performance liquid chromatography (HPLC) and by bioassay measuring neutrophil chemotaxis in a single Boyden chamber system. Interleukin (IL)-8 and growth-related oncogene (GRO)-alpha were induced in nasal fibroblast culture by proinflammatory stimulus. After 24 h of stimulation neutrophil chemotactic activity only was detected for IL-8. Granulocyte chemotactic protein (GCP)-2 mRNA was already significantly up-regulated after 2 h of stimulation. Induction of IL-8 protein dominates chemokine synthesis 24 and 72 h after stimulation, whereas induction of GCP-2 mRNA seems to have a role in the early phase after 2 h of exposition with TNF-alpha.