Abstract
Lymphocyte activation and fine tuning of downstream signaling circuits for the regulation of cytokine expression are critical for a successful immune response. Hence, technical protocols permitting simultaneous testing of these attributes in peripheral blood lymphocytes are of paramount importance. Phospho-specific flow cytometry is a novel methodology that detects phosphorylation of signaling effectors in multiple, rare cellular populations within peripheral blood. In addition, it allows the quantification of phosphorylation levels for signaling proteins within each single cell, and therefore is superior compared to traditional biochemical approaches, such as Western blotting. One such important signaling pathway within immune cells is the p38 MAPK pathway involved in the regulation of cytokine expression, cell proliferation and apoptosis. In this chapter, we provide technical instructions for culturing human peripheral blood lymphocytes for simultaneous monitoring of p38 MAPK phosphorylation and associated cytokine expression, especially in rare cell populations, such as NK and NKT.
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References
Loza, M. J., Faust, J. S., and Perussia, B. (2003) Multiple color immunofluorescence for cytokine detection at the single-cell level. Mol Biotechnol 23, 245–58.
Cheng, L. E., Ohlen, C., Nelson, B. H., and Greenberg, P. D. (2002) Enhanced signaling through the IL-2 receptor in CD8+ T cells regulated by antigen recognition results in preferential proliferation and expansion of responding CD8+ T cells rather than promotion of cell death. Proc Natl Acad Sci USA 99, 3001–6.
Bessoles, S., Fouret, F., Dudal, S., Besra, G. S., Sanchez, F., and Lafont, V. (2008) IL-2 triggers specific signaling pathways in human NKT cells leading to the production of pro- and anti-inflammatory cytokines. J Leukoc Biol 84, 224–33.
Wang, K. S., Ritz, J., and Frank, D. A. (1999) IL-2 induces STAT4 activation in primary NK cells and NK cell lines, but not in T cells. J Immunol 162, 299–304.
Saklatvala, J. (2004) The p38 MAP kinase pathway as a therapeutic target in inflammatory disease. Curr Opin Pharmacol 4, 372–7.
Clark, A. R., Dean, J. L., and Saklatvala, J. (2003) Post-transcriptional regulation of gene expression by mitogen-activated protein kinase p38. FEBS Lett 546, 37–44.
Dodeller, F., and Schulze-Koops, H. (2006) The p38 mitogen-activated protein kinase signaling cascade in CD4 T cells. Arthritis Res Ther 8, 205.
Perfettini, J. L., Castedo, M., Nardacci, R., Ciccosanti, F., Boya, P., Roumier, T., Larochette, N., Piacentini, M., and Kroemer, G. (2005) Essential role of p53 phosphorylation by p38 MAPK in apoptosis induction by the HIV-1 envelope. J Exp Med 201, 279–89.
Mavropoulos, A., Sully, G., Cope, A. P., and Clark, A. R. (2005) Stabilization of IFN-gamma mRNA by MAPK p38 in IL-12- and IL-18-stimulated human NK cells. Blood 105, 282–8.
Vivier, E., Nunes, J. A., and Vely, F. (2004) Natural killer cell signaling pathways. Science 306, 1517–9.
Far, D. F., and Rossi, B. (2000) Measurement of protein tyrosine phosphorylation in T-cell subsets by flow cytometry. Methods Mol Biol 134, 301–6.
Montag, D. T., and Lotze, M. T. (2006) Rapid flow cytometric measurement of cytokine-induced phosphorylation pathways [CIPP] in human peripheral blood leukocytes. Clin Immunol 121, 215–26.
Perez, O. D., and Nolan, G. P. (2002) Simulta-neous measurement of multiple active kinase states using polychromatic flow cytometry. Nat Biotechnol 20, 155–62.
Krutzik, P. O., Irish, J. M., Nolan, G. P., and Perez, O. D. (2004) Analysis of protein phosphorylation and cellular signaling events by flow cytometry: techniques and clinical applications. Clin Immunol 110, 206–21.
Grammer, A. C., Fischer, R., Lee, O., Zhang, X., and Lipsky, P. E. (2004) Flow cytometric assessment of the signaling status of human B lymphocytes from normal and autoimmune individuals. Arthritis Res Ther 6, 28–38.
Koprak, S., Staruch, M. J., and Dumont, F. J. (1999) A specific inhibitor of the p38 mitogen activated protein kinase affects differentially the production of various cytokines by activated human T cells: dependence on CD28 signaling and preferential inhibition of IL-10 production. Cell Immunol 192, 87–95.
Perez, O. D., Krutzik, P. O., and Nolan, G. P. (2004) Flow cytometric analysis of kinase signaling cascades. Methods Mol Biol 263, 67–94.
Irish, J. M., Hovland, R., Krutzik, P. O., Perez, O. D., Bruserud, O., Gjertsen, B. T., and Nolan, G. P. (2004) Single cell profiling of potentiated phospho-protein networks in cancer cells. Cell 118, 217–28.
Haas, A., Weckbecker, G., and Welzenbach, K. (2008) Intracellular Phospho-Flow cytometry reveals novel insights into TCR proximal signaling events. A comparison with Western blot. Cytometry A 73, 799–807.
Aerts, N. E., Ebo, D. G., Bridts, C. H., Stevens, W. J., and De Clerck, L. S. (2009) Flow cytometric analysis of phospho-p38 mitogen-activated kinase (MAPK): p38 MAPK does not mediate the effect of adalimumab on peripheral T cell cytokine production in rheumatoid arthritis. Cytokine 47, 178–84.
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Mavropoulos, A., Smyk, D., Rigopoulou, E.I., Bogdanos, D.P. (2012). Human Peripheral Blood Mononuclear Cell Culture for Flow Cytometric Analysis of Phosphorylated Mitogen-Activated Protein Kinases. In: Mitry, R., Hughes, R. (eds) Human Cell Culture Protocols. Methods in Molecular Biology, vol 806. Humana Press. https://doi.org/10.1007/978-1-61779-367-7_19
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DOI: https://doi.org/10.1007/978-1-61779-367-7_19
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