Abstract
Polymorphonuclear neutrophils, traditionally viewed as short-lived effector cells, are nowadays regarded as important components of effector and regulatory circuits in the innate and adaptive immune systems. Most of the physiological functions of neutrophils as crucial players in the host immune response, able not only to act in the early phases of acute inflammation but also to condition the progression of the inflammatory reaction and the subsequent initiation of the specific immune response, relies on their capacity to produce and release a number of proinflammatory and immunoregulatory cytokines. This fact has reevaluated the importance, the role, and the physiological and pathological significance of neutrophils in the pathogenesis of inflammatory, infectious, autoimmune, and neoplastic diseases and has identified neutrophils as an important potential target for selective pharmacological intervention to both promote and restrain inflammation. In this context, understanding the mechanisms of modulation of neutrophil-derived cytokines and chemokines represents a critical step toward a better understanding of how neutrophils may influence pathophysiological processes in vivo. Herein, we describe and discuss an updated version of the methods that we have developed to rapidly and precisely characterize the pattern of cytokine expression in in vitro-activated human neutrophils. The validation of the reverse transcription quantitative real-time PCR assay as a suitable strategy for an accurate, sensitive, reliable, and bona fide analysis of cytokine gene expression in human neutrophils overcomes several problems strictly specific to neutrophils and offers an important tool, in the neutrophil research area, to test many experimental conditions for gene expression analysis.
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References
Ley K, Hoffman HM, Kubes P et al (2018) Neutrophils: new insights and open questions. Sci Immunol 3:4579
Tamassia N, Bianchetto-Aguilera F, Arruda-Silva F et al (2018) Cytokine production by human neutrophils: revisiting the “dark side of the moon”. Eur J Clin Investig 48(Suppl 2):e12952
Wang T, Brown MJ (1999) mRNA quantification by real time TaqMan polymerase chain reaction: validation and comparison with RNase protection. Anal Biochem 269:198–201
Malinen E, Kassinen A, Rinttila T et al (2003) Comparison of real-time PCR with SYBR green I or 5′-nuclease assays and dot-blot hybridization with rDNA-targeted oligonucleotide probes in quantification of selected faecal bacteria. Microbiology 149:269–277
Palmer S, Wiegand AP, Maldarelli F et al (2003) New real-time reverse transcriptase-initiated PCR assay with single-copy sensitivity for human immunodeficiency virus type 1 RNA in plasma. J Clin Microbiol 41:4531–4536
Arikawa E, Sun Y, Wang J et al (2008) Cross-platform comparison of SYBR green real-time PCR with TaqMan PCR, microarrays and other gene expression measurement technologies evaluated in the MicroArray quality control (MAQC) study. BMC Genomics 9:328
Nagalakshmi U, Wang Z, Waern K et al (2008) The transcriptional landscape of the yeast genome defined by RNA sequencing. Science 320:1344–1349
Pelletier M, Maggi L, Micheletti A et al (2010) Evidence for a cross-talk between human neutrophils and Th17 cells. Blood 115:335–343
Bazzoni F, Cassatella MA, Laudanna C et al (1991) Phagocytosis of opsonized yeast induces tumor necrosis factor-alpha mRNA accumulation and protein release by human polymorphonuclear leukocytes. J Leukoc Biol 50:223–228
Tamassia N, Le Moigne V, Calzetti F et al (2007) The MyD88-independent pathway is not mobilized in human neutrophils stimulated via TLR4. J Immunol 178:7344–7356
Davey MS, Tamassia N, Rossato M et al (2011) Failure to detect production of IL-10 by activated human neutrophils. Nat Immunol 12:1017–1018; author reply 1018-1020
Wang P, Wu P, Anthes JC et al (1994) Interleukin-10 inhibits interleukin-8 production in human neutrophils. Blood 83:2678–2683
Reglier H, Arce-Vicioso M, Fay M et al (1998) Lack of IL-10 and IL-13 production by human polymorphonuclear neutrophils. Cytokine 10:192–198
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(−Delta Delta C(T)) method. Methods 25:402–408
Ruijter JM, Ramakers C, Hoogaars WM et al (2009) Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res 37:e45
Muller PY, Janovjak H, Miserez AR et al (2002) Processing of gene expression data generated by quantitative real-time RT-PCR. Biotechniques 32:1372–1374. 1376, 1378–1379
Lefever S, Vandesompele J, Speleman F et al (2009) RTPrimerDB: the portal for real-time PCR primers and probes. Nucleic Acids Res 37:D942–D945
Marshall OJ (2004) PerlPrimer: cross-platform, graphical primer design for standard, bisulphite and real-time PCR. Bioinformatics 20:2471–2472
Ye J, Coulouris G, Zaretskaya I et al (2012) Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction. BMC Bioinformatics 13:134
Love MI, Huber W, Anders S (2014) Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol 15:550
Tamassia N, Cassatella MA, Bazzoni F (2014) Fast and accurate quantitative analysis of cytokine gene expression in human neutrophils. Methods Mol Biol 1124:451–467
Acknowledgments
This work was supported by grants from Associazione Italiana per la Ricerca sul Cancro, (IG-20339); Ministero dell’Istruzione, dell’Università e della Ricerca and (2015YYKPNN), and Fondazione CARIPLO (2015-0584) to M.A.C.
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Tamassia, N., Cassatella, M.A., Bazzoni, F. (2020). Fast and Accurate Quantitative Analysis of Cytokine Gene Expression in Human Neutrophils by Reverse Transcription Real-Time PCR. In: Quinn, M., DeLeo, F. (eds) Neutrophil. Methods in Molecular Biology, vol 2087. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0154-9_19
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DOI: https://doi.org/10.1007/978-1-0716-0154-9_19
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