Summary
Systemic autoimmune diseases are characterized by extensive alterations in immune system function, with cytokines and autoantibodies contributing to impaired immunoregulation and tissue damage. Characterization of the expression and function of cytokines is important for elucidation of pathogenic mechanisms and for identification of therapeutic targets and strategies. We reviewed the utility of assays that reflect individual variability in cytokine gene sequence, expression of messenger ribonucleic acid (mRNA) or protein, as well as measurement of expression of target genes regulated by cytokines. Real-time reverse transcriptase polymerase chain reaction and intracellular staining for cytokine expression are two sensitive and quantitative approaches for analysis of cytokine mRNA and protein, respectively. Detailed methods are provided for these assays.
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References
Ronnblom, L. and Alm, G.V. (2001) A pivotal role for the natural interferon Ī±Ī±-producing cells (plasmacytoid dendritic cells) in the pathogenesis of lupus. J. Exp. Med. 194, 59ā63.
Chernysheva, A. D., Kirou, K. A., and Crow, M. K. (2002) T cell proliferation induced by autologous non-T cells is a response to apoptotic cells processed by dendritic cells. J. Immunol. 169, 1241ā1250.
Crow, M. K. (2003) Studies of autologous T cell activation in the Kunkel laboratory. Lupus 12, 163ā169.
Kirou, K. A., and Crow, M. K. (1999) New pieces to the SLE cytokine puzzle. Clin. Immunol. 91, 1ā5.
Llorente, L., Richaud-Patin, Y., Fior, R., Alcocer-Varela, J., Wijdenes, J., Fourrier, B. M., et al. (1994) In vivo production of interleukin-10 by non-T cells in rheumatoid arthritis, Sjƶrenās syndrome, and systemic lupus erythematosus. Arthritis Rheum. 11, 1647ā1655.
Hagiwara, E., Gourley, M. F., Lee, S., and Klinman, D. M. (1996) Disease severity in patients with systemic lupus erythematosus correlates with an increased ratio of interleukin-10:interferon-Ī³-secreting cells in the peripheral blood. Arthritis Rheum. 39, 379ā385.
Akira, S., Taga, T., and Kishimoto, T. (1993) IL-6 in biology and medicine. Adv. Immunol. 54, 1ā78.
Itoh, K. and Hirohata, S. (1995) The role of IL-10 in human B cell activation, proliferation, and differentiation. J. Immunol. 154, 4341ā4350.
Linker-Israeli, M., Deans, R. J., Wallace, D. J., Prehn, J., Ozeri-Chen, T., and Klinenberg, J. R. (1991) Elevated levels of endogenous IL-6 in systemic lupus erythematosus. A putative role in pathogenesis. J. Immunol. 147, 117ā123.
Al-Janadi, M., al-Balla, S., al-Dalaan, A., and Raziuddin, S. (1993) Cytokine profile in systemic lupus erythematosus, rheumatoid arthritis, and other rheumatic diseases. J. Clin. Immunol. 13, 58ā67.
Richaud-Patin, Y., Alcocer-Varela, J., and Llorente, L. (1995) High levels of TH2 cytokine gene expression in systemic lupus erythematosus. Rev. Invest. Clin. 47, 267ā272.
Swaak, A. J., van den Brink, H. G., and Aarden, L. A. (1996) Cytokine production (IL-6 and TNF alpha) in whole blood cell cultures of patients with systemic lupus erythematosus. Scand. J. Rheumatol. 25, 233ā238.
Barcellini, W., Rizzardi, G. P., Borghi, M. O., Nicoletti, F., Fain, C., Del Papa, N., et al. (1996) In vitro type-1 and type-2 cytokine production in systemic lupus erythematosus: lack of relationship with clinical disease activity. Lupus 5, 139ā145.
Linker-Israeli, M., Honda, M., Nand, R., Mandyam, R., Mengesha, E., Wallace, D. J., et al. (1999) Exogenous IL-10 and IL-4 down-regulate IL-6 production by SLE-derived PBMC. Clin. Immunol. 91, 6ā16.
Cerutti, A., Zan, H., Schaffer, A., Bergsagel, L., Harindranath, N., Max, E. E., et al. (1998) CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM + IgD + B cell line. J. Immunol. 160, 2145ā2157.
Ji, J. D., Tassiulas, I., Park-Min, K. H., Aydin, A., Mecklenbrauker, I., Tarakhovsky, A., et al. (2003) Inhibition of interleukin 10 signaling after Fc receptor ligation and during rheumatoid arthritis. J. Exp. Med. 197, 1573ā1583.
Chen-Kiang, S. (1995) Regulation of terminal differentiation of human B-cells by IL-6. Curr. Top. Microbiol. Immunol. 194, 189ā198.
Biron, C. A. (2001) Interferons alpha and beta as immune regulatorsāa new look. Immunity 4, 661ā664.
Hooks, J. J., Moutsopoulos, H. M., Geis, S. A., Stahl, N. I., Decker, J. L., and Notkins, A. L. (1979) Immune interferon in the circulation of patients with autoimmune disease. N. Engl. J. Med. 301, 5ā8.
Preble, O. T., Black, R. J., Friedman, R. M., Klippel, J. H., and Vilcek, J. (1982) Systemic lupus erythematosus: presence in human serum of an unusual acidlabile leukocyte interferon. Science 216, 429ā431.
Yee, A. M., Yip, Y. K., Fischer, H. D., and Buyon, J. P. (1990) Serum activity that confers acid lability to alpha-interferon in systemic lupus erythematosus: its association with disease activity and its independence from circulating alpha-interferon. Arthritis Rheum. 33, 563ā568.
Blanco, P., Palucha, A. K., Gill, M., Pascual, V., and Banchereau, J. (2001) Induction of dendritic cell differentiation by IFN-alpha in systemic lupus erythematosus. Science 294, 1540ā1543.
Crow, M. K., George, S., Paget, S. A., Ly, N., Woodward, R., Fry, K., et al. (2002) Expression of an interferon-alpha gene program in SLE. Arthritis Rheum. 46, S281.
Baechler, E. C., Batliwalla, F. M., Karypis, G., Gaffney, P. M., Ortmann, W. A., Espe, K. J., et al. (2003) Interferon-inducible gene expression signature in peripheral blood cells of patients with severe lupus. Proc. Natl. Acad. Sci. USA 100, 2610ā2615.
Bennett, L., Palucka, A. K., Arce, E., Cantrell, V., Borvak, J., Banchereau, J., et al. (2003) Interferon and granulopoiesis signatures in systemic lupus erythematosus blood. J. Exp. Med. 197, 711ā723.
Han, G.-M., Chen, S.-L., Shen, N., Ye, S., Bao, C.-D., and Gu, Y.-Y. (2003) Analysis of gene expression profiles in human systemic lupus erythematosus using oligonucleotide microarray. Genes Immun. 4, 177ā186.
Siegel, F. P., Kadowaki, N., Shodell, M., Fitzgerald-Bocarsly, P. A., Shah, K., Ho, S., et al. (1999) The nature of the principal type I interferon-producing cells in human blood. Science 284, 1835ā1837.
Svensson, H., Johannisson, A., Nikkila, T., Alm, G. V., and Cederblad, B. (1996) The cell surface phenotype of human natural interferon-Ī±Ī± producing cells as determined by flow cytometry. Scand. J. Immunol. 44, 164ā172.
Vallin, H., Blomberg, S., Alm, G. V., Cederblad, B., and Ronnblom, L. (1999) Patients with systemic lupus erythematosus (SLE) have a circulating inducer of interferon-alpha (IFN-Ī±Ī±) production acting on leukocytes resembling immature dendritic cells. Clin. Exp. Immunol. 115, 196ā202.
Bave, U., Vallin, H., Alm, G. V., and Ronnblom, L. (2001) Activation of natural interferon-alpha producing cells by apoptotic U937 cells combined with lupus IgG and its regulation by cytokines. J. Autoimmun. 17, 71ā80.
Magnusson, M., Magnusson, S., Vallin, H., Ronnblom, L., and Alm, G. V. (2001) Importance of CpG dinucleotides in activation of natural IFN-alpha-producing cells by a lupus-related oligodeoxynucleotide. Scand. J. Immunol. 54, 543ā550.
Ronnblom, L. E., Alm, G. V., and Oberg, K. E. (1990) Possible induction of systemic lupus erythematosus by interferon-Ī±Ī± treatment in a patient with a malignant carcinoid tumor. J. Intern. Med. 227, 207ā210.
Crow, M. K. and Kirou, K.A. (2001) Regulation of CD40 ligand expression in systemic lupus erythematosus. Curr. Opin. Rheumatol. 13, 361ā369.
Santiago-Raber, M. L., Baccala, R., Haraldsson, K. M., Choubey, D., Stewart, T. A., Kono, D. H., et al. (2003) Type-I interferon receptor deficiency reduces lupuslike disease in NZB mice. J. Exp. Med. 197, 777ā788.
Peng, S. L., Moslehi, J., and Craft, J. (1997) Roles of interferon-gamma and interleukin-4 in murine lupus. J. Clin. Invest. 99, 1936ā1946.
Balomenos, D., Rumold, R., and Theophilopoulos, A. N. (1998) Interferongamma is required for lupus-like disease and lymphoaccumulation in MRL-lpr mice. J. Clin. Invest. 101, 364ā371.
Haas, C., Ryffel, B., and Le Hir, M. (1998) IFN-gamma receptor deletion prevents autoantibody production and glomerulonephritis in lupus-prone (NZB x NZW)F1 mice. J. Immunol. 160, 3713ā3718.
Pachman, L. M., Liotta-Davis, M. R., Hong, D. K., Kinsella, T. R., Mendez, E. P., Kinder, J. M., et al. (2000) TNFĪ±-308A allele in juvenile dermatomyositis: association with increased production of tumor necrosis factor Ī±, disease duration, and pathologic calcifications. Arthritis Rheum. 43, 2368ā2377.
Wilson, A. G., DiGiovine, F. S., Blakemore, A. I. F., and Duff, G. W. (1992) Single base polymorphism in the human tumor necrosis factor (TNFĪ±). Hum. Mol. Genet. 1, 353.
Linker-Israeli, M., Wallace, D. J., Prehn, J., Michael, D., Honda, M., Taylor, K. D., et al. (1999) Association of IL-6 gene alleles with systemic lupus erythematosus (SLE) and with elevated IL-6 expression. Genes Immun. 1, 45ā52.
Ansari, A. A. and Mayne, A. E. (2002) Cytokine analysis by intracellular staining. Methods Mol. Med. 72, 423ā435.
Loza, M. J., Faust, J. S., and Perussia, G. (2003) Multiple color immunofluorescence for cytokine detection at the single-cell level. Mol. Biotechnol. 23, 245ā258.
Kalyuzhny, A. and Stark, S. (2001) A simple method to reduce the background and improve well-to-well reproducibility of staining in ELISPOT assays. J. Immunol. Methods 257, 93ā97.
Mashishi, T. and Gray, C. M. (2002) The ELISPOT assay: an easily transferable method for measuring cellular responses and identifying T cell epitopes. Clin. Chem. Lab. Med. 40, 903ā910.
Van Besouw, N. M., Vaessen, L. M., Zuijderwijk, J. M., van Vliet, M., Ijermans, J. N., van Der Meide, P. H., et al. (2003) The frequency of interferon-Ī³ producing cells reflects alloreactivity against minor histocompatibility antigens. Transplantation 75, 1400ā1404.
Salmon, J. E., Millard, S., Schachter, L. A., Arnett, F. C., Ginzler, E. M., Gourley, M. F., et al. (1996) Fc gamma RIIA alleles are heritable risk factors for lupus nephritis in African Americans. J. Clin. Invest. 97, 1348ā1354.
Gabriel, S. B., Schaffner, S. F., Nguyen, H., Moore, J. M., Roy, J., Blumenstiel, B., et al. (2002) The structure of haplotype blocks in the human genome. Science 296, 2225ā2229.
Von Wolff, M. and Tabibzadeh, S. (1999) Multiprobe Rnase protection assay with internally labeled radioactive probes, generated by RT-PCR and nested PCR. Front. Biosci. 4, C1āC3.
Ceol, M., Forino, M., Gambaro, G., Sauer, U., Schleicher, E. D., DāAngelo, A., et al. (2001) Quantitation of TGF-beta1 mRNA in porcine mesangial cells by comparative kinetic RT/PCR: comparison with ribonuclease protection assay and in situ hybridization. J. Clin. Lab. Anal. 15, 215ā222.
Glue, C., Hansen, J. B., Schjerling, P., Jinquan, T., and Poulsen, L. K. (2002) LPS-induced cytokine production in the monocytic cell line THP-1 determined by multiple quantitative competitive PCR (QC-PCR). Scand. J. Clin. Lab. Invest. 62, 405ā412.
Bustin, S. A. (2002) Quantification of mRNA using real-time reverse transcription PCR (RT-PCR): trends and problems. J. Mol. Endocrinol. 29, 23ā39.
Ramos-Payan, R., Aguilar-Medina, M., Estrada-Parra, S., Gonzalez-Y-Merchand, J. A., Favila-Castillo, L., Monroy-Ostria, A., et al. (2003) Quantification of cytokine gene expression using an economical real-time polymerase chain reaction method based on SYBR Green I. Scand. J. Immunol. 57, 439ā445.
Zuker, M. (2003) Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res. 31, 3406ā3415.
Ririe, K. M., Rasmussen, R. P., and Wittwer, C. T. (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal. Biochem. 245, 154ā160.
Pfaffl, M. W. (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29, E45āE45.
Murakawa, Y., Takada, S., Ueda, Y., Suzuki, N., Hoshino, T., and Sakane, T. (1985) Characterization of T lymphocyte subpopulations responsible for deficient interleukin 2 activity in patients with systemic lupus erythematosus. J. Immunol. 134, 187ā195.
Solomou, E. E., Juang, Y. T., Gourley, M. F., Kammer, G. M., and Tsokos, G. C. (2001) Molecular basis of deficient IL-2 production in T cells from patients with systemic lupus erythematosus. J. Immunol. 166, 4216ā4222.
Schmittgen, T. D. and Zakrajsek, B. A. (2000) Effect of experimental treatment on housekeeping gene expression: validation by real-time, quantitative RT-PCR. J. Biochem. Biophys. Methods 46, 69ā81.
OāNeil-Andersen, N. J. and Lawrence, D. A. (2002) Differential modulation of surface and intracellular protein expression by T cells after stimulation in the presence of monensin or brefeldin A. Clin. Diagn. Lab. Immunol. 9, 243ā250.
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Kirou, K.A., Lee, C., Crow, M.K. (2004). Measurement of Cytokines in Autoimmune Disease. In: Perl, A. (eds) Autoimmunity. Methods in Molecular Medicineā¢, vol 102. Humana Press. https://doi.org/10.1385/1-59259-805-6:129
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DOI: https://doi.org/10.1385/1-59259-805-6:129
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