Abstract
It is well known that natural killer (NK) cells are involved in defense against viruses and some tumors. NK cells kill target cells by the directed release of cytolytic granules that contain perforin, granzymes, and granulysin. It is increasingly important to evaluate NK cell function in immunotoxicity testing. NK cell function can be evaluated by determining cytolytic activity against target tumor cells by the 51Cr-release assay and also by determining the number of NK cells in peripheral blood in humans and in the spleen in animals using flow cytometry. Recently, the intracellular levels of perforin, granzymes, and granulysin determined by flow cytometry have also been used in the evaluation of NK cell function. This chapter will describe the methods for NK cell assays in immunotoxicity testing.
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References
Li Q, Kawada T (2006) The new mechanism of organophosphorus pesticides-induced inhibition of cytolytic activity of killer cells. Cell Mol Immunol 3:171–178
Li Q (2007) New mechanism of organophosphorus pesticide-induced immunotoxicity. J Nippon Med Sch 74:92–105
Li Q, Nagahara N, Takahashi H, Takeda K, Okumura K, Minami M (2002) Organophosphorus pesticides markedly inhibit the activities of natural killer, cytotoxic T lymphocyte and lymphokine-activated killer: a proposed inhibiting mechanism via granzyme inhibition. Toxicology 172:181–190
Hirata Y, Inagaki H, Shimizu T, Li Q, Nagahara N, Minami M et al (2006) Expression of enzymatically active human granzyme 3 in Escherichia coli for analysis of its substrate specificity. Arch Biochem Biophys 446:35–43
Li Q, Nakadai A, Ishizaki M, Morimoto K, Ueda A, Krensky AM et al (2005) Dimethyl 2, 2-dichlorovinyl phosphate (DDVP) markedly decreases the expression of perforin, granzyme A and granulysin in human NK-92CI cell line. Toxicology 213:107–116
Li Q, Kobayashi M, Kawada T (2008) DDVP markedly decreases the expression of granzyme B and granzyme 3/K in human NK cells. Toxicology 243:294–302
Okada S, Li Q, Whitin JC, Clayberger C, Krensky AM (2003) Intracellular mediators of granulysin-induced cell death. J Immunol 171:2556–2562
Li Q, Nakadai A, Takeda K, Kawada T (2004) Dimethyl 2, 2-dichlorovinyl phosphate (DDVP) markedly inhibits activities of natural killer cells, cytotoxic T lymphocytes and lymphokine-activated killer cells via the Fas-ligand/Fas pathway in perforin-knockout (PKO) mice. Toxicology 204:41–50
Kiessling R, Klein E, Wigzell H (1975) “Natural” killer cells in the mouse. I. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Specificity and distribution according to genotype. Eur J Immunol 5:112–117
Kiessling R, Klein E, Pross H, Wigzell H (1975) “Natural” killer cells in the mouse. II. Cytotoxic cells with specificity for mouse Moloney leukemia cells. Characteristics of the killer cell. Eur J Immunol 5:117–121
Li Q, Minami M, Inagaki H (1998) Acute and subchronic immunotoxicity of p-chloronitrobenzene in mice: I. Effect on natural killer, cytotoxic T lymphocyte activities and mitogen-stimulated lymphocyte proliferation. Toxicology 127:223–232
Li Q, Hirata Y, Piao S, Minami M (2000) Immunotoxicity of N, N-diethylaniline in mice: Effect on natural killer activity, cytotoxic T lymphocyte activity, lymphocyte proliferation response and cellular components of the spleen. Toxicology 150:181–191
Li Q, Hirata Y, Piao S, Minami M (2000) The by-products generated during sarin synthesis in the Tokyo sarin disaster induced inhibition of natural killer and cytotoxic T lymphocyte activity. Toxicology 146:209–220
Li Q, Nakadai A, Matsushima H, Miyazaki Y, Krensky AM, Kawada T et al (2006) Phytoncides (wood essential oils) induce human natural killer cell activity. Immunopharmacol Immunotoxicol 28:319–333
Li Q, Kobayashi M, Kawada T (2007) Organophosphorus pesticides induce apoptosis in human NK cells. Toxicology 239:89–95
Li Q, Morimoto K, Nakadai A, Inagaki H, Katsumata M, Shimizu T et al (2007) Forest bathing enhances human natural killer activity and expression of anti-cancer proteins. Int J Immunopathol Pharmacol 20:3–8
Li Q, Morimoto K, Kobayashi M, Inagaki H, Katsumata M, Hirata Y et al (2008) Visiting a forest, but not a city, increases human natural killer activity and expression of anti-cancer proteins. Int J Immunopathol Pharmacol 21:117–128
Li Q, Morimoto K, Kobayashi M, Inagaki H, Katsumata M, Hirata Y et al (2008) A forest bathing trip increases human natural killer activity and expression of anti-cancer proteins in female subjects. J Biol Regul Homeost Agents 22:45–55
Li Q, Minami M, Hanaoka T, Yamamura Y (1999) Acute immunotoxicity of p-chloronitrobenzene in mice: II. Effect of p-chloronitrobenzene on the immunophenotype of murine splenocytes determined by flow cytometry. Toxicology 137:35–45
Li Q, Morimoto K, Nakadai A, Qu T, Matsushima H, Katsumata M et al (2007) Healthy lifestyles are associated with higher levels of perforin, granulysin and granzymes A/B-expressing cells in peripheral blood lymphocytes. Prev Med 44:117–123
Hanson DA, Kaspar AA, Poulain FR, Krensky AM (1999) Biosynthesis of granulysin, a novel cytolytic molecule. Mol Immunol 36:413–422
Ito M, Watanabe M, Kamiya H, Sakurai M (1996) Non-radioactive assay of natural killer cell-mediated cytotoxicity against cytomegalovirus-infected fibroblasts by DNA fragmentation ELISA. J Virol Methods 56:77–84
Nagao F, Yabe T, Xu M, Yokoyama K, Saito K, Okumura K (1996) Application of non-radioactive europium (Eu3+) release assay to a measurement of human natural killer activity of healthy and patient populations. Immunol Invest 25:507–518
Kim GG, Donnenberg VS, Donnenberg AD, Gooding W, Whiteside TL (2007) A novel multiparametric flow cytometry-based cytotoxicity assay simultaneously immunophenotypes effector cells: comparisons to a 4 h 51Cr-release assay. J Immunol Methods 325:51–66
Acknowledgments
The parts of experimental data shown in this chapter and performed by author were supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (No. 09877077, No. 10770178, No. 12770206, No. 15590523, and No. 19590602). The author would like to thank Dr. Tomoyuki Kawada (Professor and Chief) at the Department of Hygiene and Public Health, Nippon Medical School, for his advice.
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Li, Q. (2010). NK Cell Assays in Immunotoxicity Testing. In: Dietert, R. (eds) Immunotoxicity Testing. Methods in Molecular Biology™, vol 598. Humana Press. https://doi.org/10.1007/978-1-60761-401-2_15
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DOI: https://doi.org/10.1007/978-1-60761-401-2_15
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