Abstract
The pro-inflammatory cytokine interleukin (IL)-1β is an important mediator of the inflammatory response. In order to perform its role in the inflammatory cascade, IL-1β must be secreted from the cell, yet it lacks a signal peptide that is required for conventional secretion, and the exact mechanism of release remains undefined. Conventional biochemical methods have limited the investigation into the processes involved in IL-1β secretion to population dynamics, yet heterogeneity between cells has been observed at a single-cell level. Here, greater sensitivity is achieved with the use of a newly developed vector that codes for a fluorescently labelled version of IL-1β. Combining this with real-time single-cell confocal microscopy using the methods described here, we have developed an effective protocol for investigating the mechanisms of IL-1β secretion and the testing of the hypothesis that IL-1β secretion requires membrane permeabilisation.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Lopez-Castejon G, Brough D (2011) Understanding the mechanism of IL-1beta secretion. Cytokine Growth Factor Rev 22:189–195
Auron PE, Webb AC, Rosenwasser LJ, Mucci SF, Rich A, Wolff SM, Dinarello CA (1984) Nucleotide sequence of human monocyte interleukin 1 precursor cDNA. Proc Natl Acad Sci U S A 81:7907–7911
Rubartelli A, Cozzolino F, Talio M, Sitia R (1990) A novel secretory pathway for interleukin-1 beta, a protein lacking a signal sequence. EMBO J 9:1503–1510
Ma C, Fan R, Ahmad H, Shi Q, Comin-Anduix B, Chodon T, Koya RC, Liu CC, Kwong GA, Radu CG, Ribas A, Heath JR (2011) A clinical microchip for evaluation of single immune cells reveals high functional heterogeneity in phenotypically similar T cells. Nat Med 17:738–743
Shirasaki Y, Yamagishi M, Suzuki N, Izawa K, Nakahara A, Mizuno J, Shoji S, Heike T, Harada Y, Nishikomori R, Ohara O (2014) Real-time single-cell imaging of protein secretion. Sci Rep 4:4736
Liu T, Yamaguchi Y, Shirasaki Y, Shikada K, Yamagishi M, Hoshino K, Kaisho T, Takemoto K, Suzuki T, Kuranaga E, Ohara O, Miura M (2014) Single-cell imaging of caspase-1 dynamics reveals an all-or-none inflammasome signaling response. Cell Rep 8:974–982
Perregaux D, Gabel CA (1994) Interleukin-1 beta maturation and release in response to ATP and nigericin. Evidence that potassium depletion mediated by these agents is a necessary and common feature of their activity. J Biol Chem 269:15195–15203
Ferrari D, Chiozzi P, Falzoni S, Dal Susino M, Melchiorri L, Baricordi OR, Di Virgilio F (1997) Extracellular ATP triggers IL-1 beta release by activating the purinergic P2Z receptor of human macrophages. J Immunol 159:1451–1458
Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, Roose-Girma M, Lee WP, Weinrauch Y, Monack DM, Dixit VM (2006) Cryopyrin activates the inflammasome in response to toxins and ATP. Nature 440:228–232
Shen H, Nelson G, Nelson DE, Kennedy S, Spiller DG, Griffiths T, Paton N, Oliver SG, White MR, Kell DB (2006) Automated tracking of gene expression in individual cells and cell compartments. J R Soc Interface 3:787–794
Du CJ, Marcello M, Spiller DG, White MR, Bretschneider T (2010) Interactive segmentation of clustered cells via geodesic commute distance and constrained density weighted Nystrom method. Cytometry A 77:1137–1147
Katzen F (2007) Gateway((R)) recombinational cloning: a biological operating system. Expert Opin Drug Discov 2:571–589
Bagnall J (2011) Single-cell imaging and mathematical modelling of the hypoxia-inducible factor signalling network. PhD thesis, University of Liverpool
Acknowledgements
The authors are grateful to Prof Claire Bryant from the University of Cambridge for providing the immortalised BMDMs. P.P. holds a BBSRC David Phillips Research Fellowship (BB/I017976/1). This work was also supported by M.R.C. (MR/K015885/1) and BBSRC (BB/K003097/1). The work leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2012-2017) under grant agreement n° 305564.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Diamond, C. et al. (2016). Investigating IL-1β Secretion Using Real-Time Single-Cell Imaging. In: Di Virgilio, F., Pelegrín, P. (eds) NLR Proteins. Methods in Molecular Biology, vol 1417. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3566-6_4
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3566-6_4
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3564-2
Online ISBN: 978-1-4939-3566-6
eBook Packages: Springer Protocols