Mechanisms of Bacterial Endotoxin-induced Cell Activation by a Serum-independent Pathway.

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The present study was done to elucidate the biological properties of LPS receptor (s) and its signal transduction system, and to identify some molecules of them. A cell line, ST2, derived from murine bone marrow stroma, was mainly used in the present study. We examined the following points ; a) responsiveness to LPS antagonists and LPS agonists, b) detection of LPS-binding proteins on the ST2 cells and production of polyclonal antisera against these proteins, c) detection of tyrosine-phosphorylated proteins on the ST2 cells. We identified some molecules which is related to LPS-induced activation of ST2 cells.1) Adaptation of CD14-negative marrow stromal ST2 cells in serum-free medium and LPS-responsiveness of ST2 cellsTo exclude completely the effects of CD14 and serum components on LPS responsiveness, we established a subclone of CD14-negative marrow stromal ST2 cells growing under serum-free conditions.2) Detection of LPS-binding proteins on ST2 sells growing under serum-free conditions.A ligand-botting method and a cross-linker method using radiolabelled LPS demonstrated a number of LPS-binding proteins on ST2 cells.3) Production of polyclonal antisera against membrane fraction containing LPS-binding proteins on ST2 cells.Rabbits were immunized with partial purified LPS-binding proteins on ST2 cells and polyclonal antisera were collected. The biological immunological properties were determined.4) Detection of tyrosine-phosphorylated proteins on ST2 sells growing under serum-free conditions.A immunoprecipitation and western blotting methods revealed tyrosine-phosphorylated proteins on ST2 sells stimulated with LPS.5) Production of monoclonal antibodies against LPS-binding proteins and their associated proteins on ST2 cells.On based our previous finding, we produced monoclonal antibodies against LPS-binding proteins and their associated proteins on ST2 cells.