Skip to main content
SpringerLink
Log in

Models for the Study of the Cross Talk Between Inflammation and Cell Cycle

  • Protocol
Cyclin-Dependent Kinase (CDK) Inhibitors

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1336))

Abstract

Cyclin-dependent kinases (CDKs) have been traditionally associated with the cell cycle. However, it is now known that CDK7 and CDK9 regulate transcriptional activity via phosphorylation of RNA polymerase II and subsequent synthesis of, for example, inflammatory mediators and factors that influence the apoptotic process; including apoptosis of granulocytes such as neutrophils and eosinophils. Successful resolution of inflammation and restoration of normal tissue homeostasis requires apoptosis of these inflammatory cells and subsequent clearance of apoptotic bodies by phagocytes such as macrophages. It is believed that CDK7 and CDK9 influence resolution of inflammation since they are involved in the transcription of anti-apoptotic proteins such as Mcl-1 which is especially important in granulocyte survival.

This chapter describes various in vitro and in vivo models used to investigate CDKs and their inhibitors in granulocytes and particularly the role of CDKs in the apoptosis pathway. This can be performed in vitro by isolation and use of primary granulocytes and in vivo using animal models of inflammatory disease in rodents and zebrafish. Some of the methods described here to assess the role of CDKs in inflammation and apoptosis include flow cytometry and western blotting, together with imaging and quantification of apoptosis in fixed tissue, as well as in vivo models of inflammation.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 89.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 119.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Leitch AE, Duffin R, Haslett C, Rossi AG (2008) Relevance of granulocyte apoptosis to resolution of inflammation at the respiratory mucosa. Mucosal Immunol 1(5):350–363

    Article  CAS  PubMed  Google Scholar 

  2. Fox S, Leitch AE, Duffin R, Haslett C, Rossi AG (2010) Neutrophil apoptosis: relevance to the innate immune response and inflammatory disease. J Innate Immun 2:216–227

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  3. Leitch AE, Haslett C, Rossi AG (2009) Review: cyclin-dependent kinase inhibitor drugs as potential novel anti-inflammatory and pro-resolution agents. Br J Pharmacol 158:1004–1016

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  4. Leitch AE, Lucas CD, Marwick JA, Duffin R, Haslett C, Rossi AG (2012) Cyclin-dependent kinases 7 and 9 specifically regulate neutrophil transcription and their inhibition drives apoptosis to promote resolution of inflammation. Cell Death Differ 1–12

    Google Scholar 

  5. Rossi AG, Sawatzky DA, Walker A, Ward C, Sheldrake TA, Riley NA, Caldicott A, Martinez-Losa M, Walker TR, Duffin R, Gray M, Crescenzi E, Martin MC, Brady HJ, Savill JS, Dransfield I, Haslett C (2006) Cyclin-dependent kinase inhibitors enhance the resolution of inflammation by promoting inflammatory cell apoptosis. Nat Med 12(9):1056–1064

    Article  CAS  PubMed  Google Scholar 

  6. Lucas CD, Dorward DA, Tait MA, Fox S, Marwick JA, Allen KC, Robb CT, Hirani N, Haslett C, Duffin R, Rossi AG (2013) Downregulation of Mcl-1 has anti-inflammatory pro-resolution effects and enhances bacterial clearance from the lung. Mucosal Immunol 10

    Google Scholar 

  7. Leitch AE, Riley NA, Sheldrake TA, Festa M, Fox S, Duffin R, Haslett C, Rossi AG (2010) The cyclin-dependent kinase inhibitor R-Roscovitine down-regulates Mcl-1 to override pro-inflammatory signalling and drive neutrophil apoptosis. Eur J Immunol 40:1127–1138

    Article  CAS  PubMed  Google Scholar 

  8. Alessandri AL, Duffin R, Leitch AE, Lucas CD, Sheldrake TA, Dorward DA, Hiriani N, Pinho V, de Sousa LP, Teixeira MM, Lyons JF, Haslett C, Rossi AG (2011) Induction of eosinophil apoptosis by the cyclin-dependent kinase inhibitor AT7519 promotes the resolution of eosinophil-dominant allergic inflammation. PLoS One 6(9):1–10

    Article  Google Scholar 

  9. Smallie T, Ricchetti G, Horwood NJ, Feldmann M, Clark AR, Williams LM (2010) IL-10 inhibits transcription elongation of the human TNF gene in primary macrophages. J Exp Med 207(10):2081–2088

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Wang S, Fischer PM (2008) Cyclin-dependent kinase 9: a key transcriptional regulator and potential drug target in oncology, virology and cardiology. Trends Pharmacol Sci 29(9):302–313

    Article  PubMed  Google Scholar 

  11. Haslett C, Guthrie LA, Kopaniak MM, Johnston RB, Henson PM (1985) Modulation of multiple neutrophil functions by preparative methods or trace concentrations of bacterial lipopolysaccharide. Am J Pathol 119:101–110

    CAS  PubMed Central  PubMed  Google Scholar 

  12. Youssef PP, Mantzioris BX, Roberts-Thomson PJ, Ahern MJ, Smith MD (1995) Effects of ex vivo manipulation on the expression of cell adhesion molecules on neutrophils. J Immunol Methods 186:217–224

    Article  CAS  PubMed  Google Scholar 

  13. Macey MG, McCarthy DA, Vordermeier S, Newland AC, Brown KA (1995) Effects of cell purification methods on CD11b and L-selectin expression as well as the adherence and activation of leucocytes. J Immunol Methods 181:211–219

    Article  CAS  PubMed  Google Scholar 

  14. Hu Y (2012) Isolation of human and mouse neutrophils ex vivo and in vitro. Methods Mol Biol 844:101–113

    Article  CAS  PubMed  Google Scholar 

  15. Dorward DA, Lucas CD, Alessandri AL, Marwick JA, Rossi F, Dransfield I, Haslett C, Dhaliwal K, Rossi AG (2013) Technical advance: autofluorescence-based sorting: rapid and nonperturbing isolation of ultrapure neutrophils to determine cytokine production. J Leukoc Biol 94(1):193–202

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  16. Renshaw SA, Loynes CA, Trushell DMI, Elworthy S, Ingham PW, Whyte MKB (2006) A transgenic zebrafish model of neutrophilic inflammation. Blood 108(13):3976–3978

    Article  CAS  PubMed  Google Scholar 

  17. Gray C, Loynes CA, Whyte MKB, Crossman DC, Renshaw SA, Chico TJA (2010) Simultaneous intravital imaging of macrophage and neutrophil behaviour during inflammation using a novel transgenic zebrafish. Thromb Haemost 105(5):811–819

    Article  Google Scholar 

  18. Loynes CA, Martin JS, Robertson A, Trushell DMI, Ingham PW, Whyte MKB, Renshaw SA (2010) Pivotal advance: pharmacological manipulation of inflammation resolution during spontaneously resolving tissue neutrophilia in the zebrafish. J Leukoc Biol 87:203–212

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  19. Lucas CD, Allen KC, Dorward DA, Hoodless LJ, Melrose LA, Marwick JA, Tucker CS, Haslett C, Duffin R, Rossi AG (2013) Flavones induce neutrophil apoptosis by down-regulation of Mcl-1 via a proteasomal-dependent pathway. FASEB J 27(3):1084–1094

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Michlewska S, Dransfield I, Megson IL, Rossi AG (2009) Macrophage phagocytosis of apoptotic neutrophils is critically regulated by the opposing actions of pro-inflammatory and anti-inflammatory agents: key role for TNF-α. FASEB J 23:844–854

    Article  CAS  PubMed  Google Scholar 

  21. Chomarat P, Banchereau J, Davoust J, Palucka AK (2000) IL-6 switches the differentiation of monocytes from dendritic cells to macrophages. Nat Immunol 1(6):510–514

    Article  CAS  PubMed  Google Scholar 

  22. Walker A, Ward C, Dransfield I, Haslett C, Rossi AG (2003) Regulation of granulocyte apoptosis by hemopoietic growth factors, cytokines and drugs: potential relevance to allergic inflammation. Curr Drug Targets Inflamm Allergy 2(4):339–347

    Article  CAS  PubMed  Google Scholar 

  23. Felton JM, Lucas CD, Rossi AG, Dransfield I (2014) Eosinophils in the lung – modulating apoptosis and efferocytosis in airway inflammation. Front Immunol 5(302):1–11

    CAS  Google Scholar 

  24. Yamaguchi Y, Hyashi YI, Sugama Y, Miura Y, Kasahara T, Kitamura S, Torisuj M, Mita S, Tominaga A, Takatsu K, Suda T (1988) Highly purified murine interleukin 5 (IL-5) stimulates eosinophil function and prolongs in vitro survival. J Exp Med 167:1737–1742

    Article  CAS  PubMed  Google Scholar 

  25. Farahi N, Uller L, Juss JK, Langton AJ, Cowburn AS, Gibson A, Foster MR, Farrow SN, Marco-Casanova P, Sobolewski A, Condliffe AM, Chilvers ER (2011) Effects of the cyclin-dependent kinase inhibitor R-Roscovitine on eosinophil survival and clearance. Clin Exp Allergy 41(5):673–687

    Article  CAS  PubMed  Google Scholar 

  26. Newman SL, Henson JE, Henson PM (1982) Phagocytosis of senescent neutrophils by human monocyte-derived macrophages and rabbit inflammatory macrophages. J Exp Med 156(2):430–442

    Article  CAS  PubMed  Google Scholar 

  27. Nicoletti I, Migliorati G, Pagliacci MC, Grignani F, Riccardi C (1991) A rapid and simple method for measuring thymocyte apoptosis by propidium iodide staining and flow cytometry. J Immunol Methods 139(2):271–279

    Article  CAS  PubMed  Google Scholar 

  28. Coutinho P (2005) Models of human genetic diseases. BMC Bioinformatics 2005:6 (Suppl 4:P7)

    Google Scholar 

  29. Kettleborough RN, Busch-Nentwich EM, Harvey SA, Dooley CM, de Bruijn E, van Eeden F, Sealy I, White RJ, Herd C, Nijman IJ, Fényes F, Mehroke S, Scahill C, Gibbons R, Wali N, Carruthers S, Hall A, Yen J, Cuppen E, Stemple DL (2013) A systematic genome-wide analysis of zebrafish protein-coding gene function. Nature 496(7446):494–947

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  30. Howe K, Clark MD, Torroja CF, Torrance J, Berthelot C, Muffato M, Collins JE, Humphray S, McLaren K, Matthews L, McLaren S, Sealy I, Caccamo M, Churcher C, Scott C, Barrett JC, Koch R, Rauch GJ, White S, Chow W, Kilian B, Quintais LT, Guerra-Assunção JA, Zhou Y, Gu Y, Yen J, Vogel JH, Eyre T, Redmond S, Banerjee R, Chi J, Fu B, Langley E, Maguire SF, Laird GK, Lloyd D, Kenyon E, Donaldson S, Sehra H, Almeida-King J, Loveland J, Trevanion S, Jones M, Quail M, Willey D, Hunt A, Burton J, Sims S, McLay K, Plumb B, Davis J, Clee C, Oliver K, Clark R, Riddle C, Elliot D, Threadgold G, Harden G, Ware D, Begum S, Mortimore B, Kerry G, Heath P, Phillimore B, Tracey A, Corby N, Dunn M, Johnson C, Wood J, Clark S, Pelan S, Griffiths G, Smith M, Glithero R, Howden P, Barker N, Lloyd C, Stevens C, Harley J, Holt K, Panagiotidis G, Lovell J, Beasley H, Henderson C, Gordon D, Auger K, Wright D, Collins J, Raisen C, Dyer L, Leung K, Robertson L, Ambridge K, Leongamornlert D, McGuire S, Gilderthorp R, Griffiths C, Manthravadi D, Nichol S, Barker G, Whitehead S, Kay M, Brown J, Murnane C, Gray E, Humphries M, Sycamore N, Barker D, Saunders D, Wallis J, Babbage A, Hammond S, Mashreghi-Mohammadi M, Barr L, Martin S, Wray P, Ellington A, Matthews N, Ellwood M, Woodmansey R, Clark G, Cooper J, Tromans A, Grafham D, Skuce C, Pandian R, Andrews R, Harrison E, Kimberley A, Garnett J, Fosker N, Hall R, Garner P, Kelly D, Bird C, Palmer S, Gehring I, Berger A, Dooley CM, Ersan-Ürün Z, Eser C, Geiger H, Geisler M, Karotki L, Kirn A, Konantz J, Konantz M, Oberländer M, Rudolph-Geiger S, Teucke M, Lanz C, Raddatz G, Osoegawa K, Zhu B, Rapp A, Widaa S, Langford C, Yang F, Schuster SC, Carter NP, Harrow J, Ning Z, Herrero J, Searle SM, Enright A, Geisler R, Plasterk RH, Lee C, Westerfield M, de Jong PJ, Zon LI, Postlethwait JH, Nüsslein-Volhard C, Hubbard TJ, Roest, Crollius H, Rogers J, DL. S (2013) The zebrafish reference genome sequence and its relationship to the human genome. Nature 496(7446):498–503

    Google Scholar 

  31. Lieschke GJ, Currie PD (2007) Animal models of human disease: zebrafish swim into view. Nat Rev Genet 8:353–367

    Article  CAS  PubMed  Google Scholar 

  32. Kwan KM, Fujimoto E, Grabher C, Mangum BD, Hardy ME, Campbell DS, Parant JM, Yost HJ, Kanki JP, Chien CB (2007) The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs. Dev Dyn 236(11):3088–3099

    Article  CAS  PubMed  Google Scholar 

  33. Ellett F, Pase L, Hayman JW, Andrianopoulos A, Lieschke GJ (2010) mpeg1 promoter transgenes direct macrophage-lineage expression in zebrafish. Blood 117(4):e49–e56

    Article  PubMed  Google Scholar 

  34. Hall C, Flores MV, Storm T, Crosier K, Crosier P (2007) The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish. BMC Dev Biol 7(42)

    Google Scholar 

  35. Henry KM, Loynes CA, Whyte MKB, Renshaw SA (2013) Zebrafish as a model for the study of neutrophil biology. J Leukoc Biol 94:1–10

    Article  Google Scholar 

  36. Renshaw SA, Loynes CA, Elworthy S, Ingham PW, Whyte MKB (2007) Modeling inflammation in the zebrafish: how a fish can help us understand lung disease. Exp Lung Res 33:549–554

    Article  CAS  PubMed  Google Scholar 

  37. Sieger D, Moritz C, Ziegenhals T, Prykhozhij S, Peri F (2012) Long-range Ca2+ waves transmit brain-damage signals to microglia. Dev Cell 22(6)

    Google Scholar 

  38. Jagadeeswaran P, Carrillo M, Radhakrishnan UP, Rajpurohit SK, Kim S (2011) Laser-induced thrombosis in zebrafish. Methods Cell Biol 101:197–203

    Article  PubMed  Google Scholar 

  39. Prajsnar TK, Cunliffe VT, Foster SJ, Renshaw SA (2008) A novel vertebrate model of Staphylococcus aureus infection reveals phagocyte-dependent resistance of zebrafish to non-host specialized pathogens. Cell Microbiol 10(11):2312–2325

    Article  CAS  PubMed  Google Scholar 

  40. Brown SB, Tucker CS, Ford C, Lee Y, Dunbar DR, Mullins JJ (2007) Class III antiarrhythmic methanesulfonanilides inhibit leukocyte recruitment in zebrafish. J Leukoc Biol 82(1):79–84

    Article  CAS  PubMed  Google Scholar 

  41. Enyedi B, Kala S, Nikolich-Zugich T, Niethammer P (2013) Tissue damage detection by osmotic surveillance. Nat Cell Biol 15:1123–1130

    Article  CAS  PubMed  Google Scholar 

  42. Auer TO, Del Bene F (2014) CRISPR/Cas9 and TALEN-mediated knock-in approaches in zebrafish. Methods 14

    Google Scholar 

  43. Hruscha A, Krawitz P, Rechenberg A, Heinrich V, Hecht J, Haass C, Schmid B (2013) Efficient CRISPR/Cas9 genome editing with low off-target effects in zebrafish. Development 10

    Google Scholar 

  44. Moulton JD, Yan YL (2008) Using morpholinos to control gene expression. Curr Protoc Mol Biol 26(28):21–29

    Google Scholar 

  45. Milan DJ, Peterson TA, Ruskin JN, Peterson RT, MacRae CA (2003) Drugs that induce repolarization abnormalities cause Bradycardia in zebrafish. Circulation 107:1355–1358

    Article  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. MRC Centre for Inflammation Research, The Queen’s Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK

    Laura J. Hoodless, Calum T. Robb, Jennifer M. Felton & Adriano G. Rossi

  2. BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, The University of Edinburgh, 47 Little France Crescent, Edinburgh, EH16 4TJ, UK

    Carl S. Tucker

Authors
  1. Laura J. Hoodless
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Calum T. Robb
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jennifer M. Felton
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Carl S. Tucker
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Adriano G. Rossi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Adriano G. Rossi .

Editor information

Editors and Affiliations

  1. Medicinal Chemistry, Centro de Investigación Príncipe Felipe, Valencia, Spain

    Mar Orzáez

  2. Medicinal Chemistry, Centro de Investigación Príncipe Felipe, Valencia, Spain

    Mónica Sancho Medina

  3. IBV-CSIC and Centro de Investigación Príncipe Felipe, Valencia, Spain

    Enrique Pérez-Payá

Rights and permissions

Reprints and permissions

Copyright information

© 2016 Springer Science+Business Media, LLC

About this protocol

Cite this protocol

Hoodless, L.J., Robb, C.T., Felton, J.M., Tucker, C.S., Rossi, A.G. (2016). Models for the Study of the Cross Talk Between Inflammation and Cell Cycle. In: Orzáez, M., Sancho Medina, M., Pérez-Payá, E. (eds) Cyclin-Dependent Kinase (CDK) Inhibitors. Methods in Molecular Biology, vol 1336. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2926-9_15

Download citation

  • DOI: https://doi.org/10.1007/978-1-4939-2926-9_15

  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-2925-2

  • Online ISBN: 978-1-4939-2926-9

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation