Characterization of macrophage mutants established by their resistance to LPS and cycloheximide-induced apopotic cell death

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.

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Amano, F. , Tsukabe, S. , Teshima, R. , Waku, K. and Kohama, K. (2012) Characterization of macrophage mutants established by their resistance to LPS and cycloheximide-induced apopotic cell death. Advances in Bioscience and Biotechnology, 3, 770-781. doi: 10.4236/abb.2012.326097.

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1] P?lsson-McDermott, E.M. and O’Neill, LA. (2004) Signal transduction by the lipopolysaccharide receptor, Toll- like receptor-4. Immunology, 113, 153-162. doi:10.1111/j.1365-2567.2004.01976.x
[2] Nishijima, M., Amano, F., Akamatsu, Y., Akagawa, K., Tokunaga, T. and Raetz, C.R.H. (1985) Macrophage activation by monosaccharide precursors of Escherichia coli lipid A. Proceedings of the National Academy of Sciences of USA, 82, 282-286. doi:10.1073/pnas.82.2.282
[3] Johnston Jr., R.B., Godzik, C.A. and Cohn, Z.A. (1978) Increased superoxide anion production by immunologi- cally acti-vated and chemically elicited macrophages. Journal of Experimental Medicine, 148, 115-127. doi:10.1084/jem.148.1.115
[4] Alexander, P. and Evans, R. (1971) Endotoxin and double stranded RNA render macrophage cytotoxic. Nature New Biology, 232, 76-78.
[5] Jeyaseelan, S., Chu, H.W., Young, S.K., Freeman, M.W. and Worthen, G.S. (2005) Distinct roles of pattern recog- nition receptors CD14 and Toll-like re-ceptor 4 in acute lung injury. Infection and Immunity, 73, 1754-1763. doi:10.1128/IAI.73.3.1754-1763.2005
[6] Marino, M.W., Dunn, A., Grail, D., Inglese, M., Noguchi, Y., Ri-chards, E., Jungbluth, A., Wada, H., Moore, M., Wil-liamson, B., Basu, S. and Old, L.J. (1997) Charac- teriza-tion of tumor necrosis factor-deficient mice. Pro- ceedings of the National Academy of Sciences of USA, 94, 8093-8098. doi:10.1073/pnas.94.15.8093
[7] Szabó, C., Zingarelli, B., O’Connor, M. and Salzman, A.L. (1996) DNA strand breakage, activation of poly (ADP-ribose) synthetase, and cellular energy depletion are involved in the cytotoxicity of macrophages and smooth muscle cells exposed to peroxynitrite. Proceedings of the National Academy of Sciences of USA, 93, 1753-1758. doi:10.1073/pnas.93.5.1753
[8] Akagawa, K., Kamo-shita, K., Tomita, T., Yasuda, T. and Tokunaga, T. (1990) Regulatory mechanism of expres- sion of LPS binding site(s) and signaling events by LPS in macrophages. En-dotoxin, 256, 467-480.
[9] Amano, F. and Akamatsu, Y. (1991) A lipopolysaccha- ride (LPS)-resistant mutant isolated from a macrophagelike cell line, J774.1, exhibits an altered activated- macrophage phenotype in response to LPS. Infection and Immunity, 59, 2166-2174.
[10] Hara-Kuge, S., Amano, F., Nishijima, M. and Akamatsu, Y. (1990) Isolation of an LPS-resistant mutant, with defective LPS binding, of cultured macro-phage-like cells. Journal of Biological Chemistry, 265, 6606-6610.
[11] Noda, T. and Amano, F. (1998) Defect in induction of nitric oxide synthase by lipopolysaccharide (LPS) in an LPS-resistant mutant of a murine ma-crophage-like cell line, J774.1. Biological and Pharma-ceutical Bulletin, 21, 673-677. doi:10.1248/bpb.21.673
[12] Amano, F. and Karahashi, H. (1996) A cytotoxic effect of lipopolysaccharide (LPS) on a macrophage-like cell line, J774.1, in the presence of cycloheximide. Journal of Endotoxin Research, 3, 415-423.
[13] Karahashi, H., Nagata, K., Ishii, K. and Amano, F. (2000) A selective inhibitor of p38 MAP ki-nase, SB202190, induced apoptotic cell death of a lipo-polysaccharide (LPS)- treated macrophage-like cell line, J774.1. Biochimica et Biophysica Acta, 1502, 207-223. doi:10.1016/S0925-4439(00)00045-4
[14] Karahashi, H. and Amano, F. (1998) Apoptotic changes preceding ne-crosis in lipopolysaccharide-treated macro- phages in the presence of cycloheximide. Experimental Cell Research, 241, 373-383. doi:10.1006/excr.1998.4062
[15] Karahashi, H. and Amano, F. (1999) LPS-induced signals in activation of a caspase-3-like protease, a key enzyme regulating apoptotic cell damage to a macrophage-like cell line, J774.1, in the presence of cycloheximide. Jour- nal of Leukocyte Biology, 66, 689-696.
[16] Karahashi, H. and Amano, F. (2000) Changes of caspase activities involved in apoptosis of a macrophage-like cell line, J774.1/JA-4 treated with LPS and cycloheximide. Biological and Pharmaceutical Bulletin, 23, 140-144. doi:10.1248/bpb.23.140
[17] Karahashi, H. and Amano, F. (1998) Structure-activity relationships of lipopolysac-charide (LPS) in tumor necrosis factor-α (TNF-α) pro-duction and induction of macrophage cell death in the presence of cycloheximide (CHX) in a murine macro-phage-like cell line, J774.1. Biological and Pharmaceutical Bulletin, 21, 1102-1105.
[18] Amano, F. and Noda, T. (1995) Improved detection of nitric oxide radical (NO?) production in an activated macrophage culture with a radical scavenger, carboxy PTIO, and Griess reagent. FEBS Letters, 368, 425-428. doi:10.1016/0014-5793(95)00700-J
[19] Karahashi, H. and Amano, F. (2003) Endotoxin-tolerance to the cyto-toxicity toward a macrophage-like cell line, J774.1, in-duced by lipopolysaccharide and cycloheximide: Role of p38 MAPK in induction of the cytotoxicity. Biological and Pharmaceutical Bulletin, 26, 1249-1259. doi:10.1248/bpb.26.1249
[20] Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680-685. doi:10.1038/227680a0
[21] Ulevitch, R.J. (1978) The preparation and characterization of a radio-iodinated bac-terial lipopolysaccharide. Immunochemistry, 15, 157-164. doi:10.1016/0161-5890(78)90144-X
[22] Frey, T. and De Maio, A. (2009) The antifungal agent itraconazole induces the accumulation of high mannose glycoproteins in macrophages. Journal of Biological Chemistry, 284, 16882-16890. doi:10.1074/jbc.M109.007609
[23] Miller, S.I., Ernst, R.K. and Bader, M.W. (2005) LPS, TLR4 and infectious disease diversity. Nature Reviews Microbiology, 3, 36-46. doi:10.1038/nrmicro1068
[24] Schumann, R.R., Rietschel, E.T. and Loppnow, H. (1994) The role of CD14 and lipopolysaccharide-binding protein (LBP) in the activation of different cell types by endotoxin. Medical Microbiology and Immunology, 183, 279-297. doi:10.1007/BF00196679
[25] Kitchens, R.L. and Mun-ford, R.S. (1998) CD14-depen- dent internalization of bacterial lipopolysaccharide (LPS) is strongly influenced by LPS aggregation but not by cel- lular responses to LPS. Journal of Immunology, 160, 1920-1928.
[26] Triantafilou, K., Triantafilou, M. and Dedrick, R.L. (2001) Interactions of bacterial lipopoly-saccharide and peptidoglycan with a 70 kDa and an 80 kDa protein on the cell surface of CD14+ and CD14- cells. Human Immunology, 62, 50-63. doi:10.1016/S0198-8859(00)00222-6

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