TITLE:
Characterization of macrophage mutants established by their resistance to LPS and cycloheximide-induced apopotic cell death
AUTHORS:
Fumio Amano, Shoko Tsukabe, Reiko Teshima, Keiko Waku, Kiyoko Kohama
KEYWORDS:
Macrophage; J774.1 Cell Line; LPS; Apoptosis; CD14
JOURNAL NAME:
Advances in Bioscience and Biotechnology,
Vol.3 No.6A,
October
30,
2012
ABSTRACT: Macrophages are activated by bacterial lipopolysaccharide (LPS) to produce inflammatory cytokines such as TNF-α or reactive oxygen species such as nitric oxide or superoxide anion. However, in the presence of an inhibitor of protein synthesis, cyclohex-imide (CHX), at 10 μg/mL, LPS at 100 ng/mL induced macrophage apoptosis rapidly without producing phenotypes of activated macrophages. In order to understand the mechanism underlying LPS-induced cytotoxicity toward macrophages, we isolated mutant cells from a macrophage-like cell line, J774.1, as clones resistant against the cytotoxic effects of LPS + CHX by using a somatic cell genetics protocol. All of the mutant clones, designated as LCR mutants, showed resistance to the cell death induced by LPS + CHX as well as to that induced by higher doses of LPS alone, as did the LPS1916 mutant cell line, which had been previously established by its resistance to 100 μg/mL LPS. Characterization of the activated macrophage phenotypes revealed that these mutants showed reduced production of TNF-α and nitric oxide in response to LPS. Further analysis showed a much reduced amount of [125I]LPS-binding and lower CD14 expression on the cell surface, in spite of an adequate intracellular expression of CD14 molecules. Besides, the molecular weight of CD14 on these mutants was around 40-48 kDa, smaller than that of the wild-type JA-4 cells (around 50-55 kDa), suggesting impaired CD14 maturation in these mutants. However, expression of Toll-like receptor 4 (TLR4) and Myd 88 on the cell surface was not different between the wild type and the mutant cells. These results suggest that LCR mutants have common phenotypes of mal-expression of CD14 molecules on the macrophage cell surface, leading to not only reduced responses to LPS-mediated macrophage activation but acquisition of resistance to LPS-induced apoptotic cell death in the presence of CHX.