Abstract
To determine the function of a particular cell type, it is necessary either to have a large number of similar (ideally identical) cells or to use extremely sensitive methods to detect the activity of a single cell. Lymphocytes present special difficulties, because they have very precise antigen (Ag) recognition requirements, and, under physiological conditions, they will only be activated if they are exposed to their particular Ag. Polyclonal mitogens, such as phytohemagglutinin (PHA) or anti-CD3, will activate most T-cells, but may not elicit a truly physiological response in terms of cytokine production, and so on. Moreover, the biological readout (release of cytokines into culture supernatant) will represent the net balance of the integrated response of all the activated cells, minus any consumption of cytokines by the cultured cells.
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References
Ying, S., Durham, S. R., Corrigan, C. J., Hamid, Q., and Kay, A. B. (1995) Phenotype of cells expressing mRNA for TH2 type (IL-4 and IL-5) and TH1 type (IL-2 and IFN-Ī³) cytokines in bronchoalveolar lavage and bronchial biopsies from asthmatic and normal control subjects. Am. J. Respir. Cell Mol. Biol. 12, 477ā487.
Bradding, P., Roberts, J. A., Britten, K. M., Montefort, S., Djukanovic, R., Mueller, R., et al. (1994) Interleukins-4,-5,-6 and TNF-alpha in normal and asthmatic airways. Evidence for the human mast cell as an important source of these cytokines. Am. J. Respir. Cell Mol. Biol. 10, 471ā480.
Powers, G. D., Abbas, A. K., and Muller, R. A. (1988) Frequencies of IL-2 and IL-4 secreting, T., cells in naive and antigen-stimulated lymphocyte populations. J. Immunol. 140, 3352ā3357.
Ledever, J. A., Lion, J. S., Todd, M. D., Glimcher, L. H., and Lichtman, A. H. (1994) Regulation of cytokine gene expression in, T., helper cell subsets. J. Immunol. 152, 77ā86.
Leung, J. C. K., Lai, C. K. W., Chui, Y. L., Ho, R. T. H., Chan, C. H. S., and Lai, K. N. (1992) Characterisation of cytokine gene expression in CD4+and CD8+ T cells after activation with phorbol myristate acetate and phytohaemagglutinin. Clin. Exp. Immunol. 90, 147ā153.
Jung, T., Schauer, U., Heusser, C., Reumann, C., and Rieger, C. (1993) Detection of intracellular cytokines by flow cytometry. J. Immunol. Methods 159, 197.
Ferrick, D. A., Schrenzel, M. D., Mulvania, T., Hsieh, B., Ferlin, W. G., and Lepper, H. (1995) Differential production of interferon-Ī³ and interleukin-4 in response to Th1 and Th2-stimulating pathogens by Ī³Ī“ T-cells in vivo. Nature 373, 255.
Openshaw, P. J. M., Murphey, E. E., Hoskan, N. A., Maino, V., Davis, K., Murphey, K., and OāGara, A. (1995) Heterogeneity of intracellular cytokine synthesis at the single cell level in polarised T helper 1 and T helper 2 populations. J. Exp. Med. 182, 1357ā1367. 209
Bjƶrk, L., Fehniger, T. E., Andersson, U., and Andersson, J. (1995) Computerised assessment of production of multiple human cytokines at the single cell level using image analysis. J. Leuk. Biol. 59, 287ā295.
Manz, R., Assenmacher, M., PflĆ¼ger, E., Miltenyi, S., and Radbruch, A. (1995) Analysis and sorting of live cells according to secreted molecules relocated to a cell-surface affinity matrix. Proc. Natl. Acad. Sci. USA 92, 1921ā1925.
Krug, N., Madden, J., Redington, A. E., Lackie, P., Schauer, U., Holgate, S. T., Frew, A. J., and Howarth, P. H. (1996) T-cell cytokine profile evaluated at the single cell level in BAL and blood in allergic asthma. Am. J. Respir. Crit. Care Med. 14, 319ā326.
Madden, J., Krishna, M. T., Redington, A. E., Holgate, S. T., Frew, A. J., and Howarth, P. H. (1999) Time-course and stimulus-dependence of BAL T-cell cytokine production evaluated at the single cell level. Cytokine 11, 456.
Robinson, D. S., Bentley, A. M., Hartnell, A., Kay, A. B., and Durham, S. R. (1993) Activated memory T helper cells in bronchoalveolar lavage fluid from patients with atopic asthma:relation to asthma symptoms, lung function and bronchial responsiveness. Thorax 48, 26ā32.
Madden, J., Frew, A. J., Krishna, M. T., Redington, A. E., Holgate, S. T., and Howarth, P. H. Flow cytometric assessment of the relevance of T-cell subset and activation markers to cytokine protein production capacity in asthma. Am. J. Respir. Crit. Care. Med., in preparation.
Schall, T. J., OāHehir, R. E., Goeddel, D. V., and Lamb, J. R. (1992) Uncoupling of cytokine mRNA expression and protein secretion during the induction phase of T-cell anergy. J. Immunol. 148, 381ā387.
Hol, B. E. A., Hintzen, R. Q., van Lier, R. A. W., Alberts, C., Out, T. A., and Jansen, H. M. (1993) Soluble and cellular markers of T-cell activation in patients with pulmonary sarcoidosis. Am. Rev. Respir. Dis. 148, 643ā649.
Dasmahapatra, J., Hodges, E., Smith, J. L., Lanham, S., Krishna, M. T., Holgate, S. T., and Frew, A. J. (1998) T-cell receptor V-beta gene usage in bronchoalveolar lavage and peripheral blood T-cells from asthmatic and normal subjects. Clin. Exp. Immunol 112, 363ā374.
Jung, T., Wijdenes, J., Neumann, C., de Vries, J. E., and Yssel, H. (1996) Interleukin-13 is produced by activated human CD45RA+and CD45RO+T-cells: modulation by IL-4 and IL-12. Eur. J. Immunol. 26, 571ā577.
Dasmahapatra, J., Smith, J. L., and Frew, A. J. (1997) Effect of local allergen challenge on TcR VĪ² usage in BAL T-cells from human airways. Clin. Exp. Allergy 27, 1359.
Elson, L. H., Nutman, T. B., Metcalf, D. D., and Prussin, C. (1995) Flow cytometric analysis for cytokine production identifies Th1, Th2 and Th0 cells within the human CD4+CD27-lymphocyte subpopulation. J. Immunol. 154, 4294ā4301.
Jung, T., Schauer, U., Rieger, C., Wagner, K., Einsle, K., Neumann, C., and Heusser, C. (1995) Interleukin-4 and interleukin-5 are rarely co-expressed by human T-cells. Eur. J. Immunol. 25, 2413ā2416. 210
Zieg, G., Lack, G., Harbeck, R. J., Gelfand, E. W., and Leung, D. Y. M. (1994) In vivo effects of glucocorticoids on IgE production. J. Allergy Clin. Immunol. 94, 222ā230.
Jung, T., Lack, G., Schauer, U., Uberuck, W., Renz, H., Gelfand, E. W., and Rieger, C. H. L. (1995) Decreased frequency of interferon-gamma and IL-2-producing cells in patients with atopic diseases measured at the single cell level. J. Allergy Clin. Immunol. 96, 515ā527.
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Frew, A.J., Madden, J., Bakakos, P. (2000). Intracellular Cytokine Staining for Analysis by Flow Cytometry. In: Fan Chung, K., Adcock, I. (eds) Asthma. Methods in Molecular Medicineā¢, vol 44. Humana Press. https://doi.org/10.1385/1-59259-072-1:197
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DOI: https://doi.org/10.1385/1-59259-072-1:197
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