Objective: We have previously reported the characterization and regulation of interleukin-1 (IL-1) receptors utilizing [125I]IL-1 binding assay in male C57BL/6 mice and the mouse AtT-20 pituitary tumor cells. In the present study, we examine IL-1 receptors using an immunoblotting method to further characterize the mechanisms regulating the interactions of IL-1 receptors with endotoxin, lipopolysaccharide (LPS). Methods: We established Western blotting for IL-1 receptors using AtT-20 mouse pituitary tumor cells. Results: Several bands were seen; however, only the 105-kD band was neutralized with a 5-fold excess of IL-1 receptor- blocking peptides, suggesting that this band is specific for IL-1 receptors. Next, we investigated the effect of LPS and IL-1β on IL-1 receptors. Treatment of AtT-20 cells with 0.01 µg/ml of LPS did not affect IL-1 receptors. In contrast, 1 µg/ml of LPS significantly increased IL-1 receptors in AtT-20 cells compared with the control group. In addition, [125I]IL-1β binding was markedly increased followed by 1 µg/ml of LPS. In contrast, 1 nM recombinant human IL-1β significantly decreased IL-1 receptors in AtT-20 cells compared with the control group although treatment of AtT-20 cells with 0.01 nM IL-1β did not affect IL-1 receptors. LPS (0.1 and 1 µg/ml) did not affect IL-1β concentrations in the medium of AtT-20 cell culture. IL-1β concentrations in the homogenates from AtT-20 cells were significantly decreased after 1 µg/ml of LPS treatment but not after 0.01 µg/ml LPS. Conclusions: These data demonstrate that LPS and IL-1β differentially modulate IL-1 receptors in AtT-20 cells and LPS-induced modulation of IL-1 receptors may provide a novel mechanism for the actions of LPS to alter pituitary function during endotoxemia. Additional in vivo studies are necessary to determine the physiological relevance of this in vitro phenomenon.

Keywords:
AtT-20 cells, Interleukin-1 receptor, Lipopolysaccharide, Western blotting
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© 2003 S. Karger AG, Basel
2003
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