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 264.7. Macrophages are key players in chronic inflammation, and major parts of the biochemical reactions taking place in vivo, e.g., the production of proinflammatory cytokines, can be triggered in vitro by stimulation of the cells with bacterial lipopolysaccharide (LPS). A mastergel, representing a synthetic image of the expressed basic set of cellular proteins, was designed by a computer-assisted overlay of a statistically significant number of two-dimensional electrophoresis (2-DE) gels of unstimulated RAW 264.7 cells. This image served as a reference for qualitative and quantitative changes in the protein pattern induced by stimulation of the macrophages with LPS. The optimal conditions for LPS stimulation were evaluated by monitoring the expression and secretion of the proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha). The comparison of the mastergel with the 2-DE gels of LPS-stimulated cells revealed several changes in the protein pattern. In order 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 further characterized by microsequencing of internal peptides. A comparison of the obtained sequences with protein databases identified them as cofilin and keratinocyte lipid-binding protein.