IN-VITRO MODEL SYSTEM FOR THE IDENTIFICATION AND CHARACTERIZATION OF PROTEINS INVOLVED IN INFLAMMATORY PROCESSES

An in vitro model featuring important inflammatory cellular states was established, based on the murine monocyte/macrophage cell line RAW 26 4.7. Macrophages are key players in chronic inflammation, and major pa rts of the biochemical reactions taking place in vivo, e.g., the produ ction of proinflammatory cytokines, can be triggered in vitro by stimu lation of the cells with bacterial lipopolysaccharide (LPS). A masterg el, representing a synthetic image of the expressed basic set of cellu lar proteins, was designed by a computer-assisted overlay of a statist ically significant number of two-dimensional electrophoresis (2-DE) ge ls of unstimulated RAW 264.7 cells. This image served as a reference f or qualitative and quantitative changes in the protein pattern induced by stimulation of the macrophages with LPS. The optimal conditions fo r LPS stimulation were evaluated by monitoring the expression and secr etion of the proinflammatory cytokine tumor necrosis factor-alpha (TNF -alpha). The comparison of the mastergel with the 2-DE gels of LPS-sti mulated cells revealed several changes in the protein pattern. In orde r to prove the relevance of the presented model system, we focused on two low molecular weight proteins, which showed significant changes in the apparent concentration in a 2-DE pattern. These proteins were fur ther characterized by microsequencing of internal peptides. A comparis on of the obtained sequences with protein databases identified them as cofilin and keratinocyte lipid-binding protein.