SpringerProtocols: Abstract: The Generation of Highly Purified Primary Human Neutrophils and Assessment of Apoptosis in Response to Toll-like Receptor Ligands

12. The Generation of Highly Purified Primary Human Neutrophils and Assessment of Apoptosis in Response to Toll-like Receptor Ligands

By: Lisa C. Parker³, Lynne R. Prince³, David J. Buttle³, Ian Sabroe³

Abstract

Full Text

Download PDF (321K)

Neutrophils are crucial components of our defence against microbial assault. They are short-lived cells, with regulation of their lifespan being a primary mechanism involved in the regulation of their function. Delay of apoptosis facilitates their clearance of pathogens, whilst appropriate induction of cell death facilitates wound healing. A variety of methods are available to study neutrophil function: purification of human neutrophils and analysis of their lifespan are described here.

Affiliation(s): (3) School of Medicine and Biomedical Sciences, University of Sheffield, L Floor, Royal Hallamshire Hospital, Sheffield S10 2JF, UK

Book Title: Toll-Like Receptors: Methods and Protocols

Series: Methods in Molecular Biology | Volume: 517 | Pub. Date: Jun-01-2009 | Page Range: 1-14 | DOI: 10.1007/978-1-59745-541-1_12

Subject: Immunology

Key Words: Granulocytes - Phoshotidylserine - Annexin V - To-Pro-3 - Caspase - Flow cytometry

References

Download References

1.	Bianchi, S. M., Dockrell, D. H., Renshaw, S. A., Sabroe, I., and Whyte, M. K. (2006) Granulocyte apoptosis in the pathogenesis and resolution of lung diseaseClin Sci (Lond). 110, 293–304.

2.	Colotta, F., Re, F., Polentarutti, N., Sozzani, S.,and Mantovani, A. (1992) Modulation of granulocyte survival and programmed cell death by cytokines and bacterial productsBlood 80, 2012–2020.

3.	Lee, A., Whyte, M. K., and Haslett, C. (1993) Inhibition of apoptosis and prolongation of neutrophil functional longevity by inflammatory mediatorsJ. Leukoc Biol. 54, 283–288.

4.	Kobayashi, S. D., Braughton, K. R., Whitney, A. R., Voyich, J. M., Schwan, T. G., Musser, J. M., and DeLeo, F. R. (2003) Bacterial pathogens modulate an apoptosis differentiation program in human neutrophilsProc Natl Acad Sci U.S.A. 100, 10948–10953.

5.	Sabroe, I., Jones, E. C., Usher, L. R., Whyte, M. K., and Dower, S. K. (2002) Toll-like receptor (TLR)2 and TLR4 in human peripheral blood granulocytes: a critical role for monocytes in leukocyte lipopolysaccharide responsesJ. Immunol. 168, 4701–4710.

6.	Hayashi, F., Means, T. K., and Luster, A. D. (2003) Toll-like receptors stimulate human neutrophil functionBlood 102, 2660–2669.

7.	Kurt-Jones, E. A., Mandell, L., Whitney, C., Padgett, A., Gosselin, K., Newburger, P. E., and Finberg, R. W. (2002) Role of toll-like receptor 2 (TLR2) in neutrophil activation: GM-CSF enhances TLR2 expression and TLR2-mediated interleukin 8 responses in neutrophilsBlood 100, 1860–1868.

8.	Parker, L. C., Whyte, M. K., Dower, S. K., and Sabroe, I. (2005) The expression and roles of Toll-like receptors in the biology of the human neutrophilJ. Leukoc Biol. 77, 886–892.

9.	Sabroe, I., Prince, L. R., Jones, E. C., Horsburgh, M. J., Foster, S. J., Vogel, S. N., Dower, S. K., and Whyte, M. K. (2003) Selective roles for Toll-like receptor (TLR)2 and TLR4 in the regulation of neutrophil activation and life spanJImmunol. 170, 5268–5275.

10.	Radsak, M. P., Salih, H. R., Rammensee, H. G., and Schild, H. (2004) Triggering receptor expressed on myeloid cells-1 in neutrophil inflammatory responses: differential regulation of activation and survivalJ. Immunol. 172, 4956–4963.

11.	Lotz, S., Aga, E., Wilde, I., van Zandbergen, G., Hartung, T., Solbach, W., and Laskay, T. (2004) Highly purified lipoteichoic acid activates neutrophil granulocytes and delays their spontaneous apoptosis via CD14 and TLR2J. Leukoc Biol. 75, 467–477.

12.	Jozsef, L., Khreiss, T., and Filep, J. G. (2004) CpG motifs in bacterial DNA delay apoptosis of neutrophil granulocytesFASEB J. 18, 1776–1778.

13.	Savill, J. S., Wyllie, A. H., Henson, J. E., Walport, M. J., Henson, P. M., and Haslett, C. (1989) Macrophage phagocytosis of aging neutrophils in inflammation. Programmed cell death in the neutrophil leads to its recognition by macrophagesJ. Clin Invest. 83, 865–875.

14.	Fadok, V. A., Voelker, D. R., Campbell, P. A., Cohen, J. J., Bratton, D. L., and Henson, P. M. (1992) Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophagesJ Immunol. 148, 2207–2216.

15.	Chang, H. Y., and Yang, X. (2000) Proteases for cell suicide: functions and regulation of caspasesMicrobiol Mol Biol Rev. 64, 821–846.

16.	Homburg, C. H., de Haas, M., von dem Borne, A. E., Verhoeven, A. J., Reutelingsperger, C. P., and Roos, D. (1995) Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitroBlood 85, 532–540.

17.	Dransfield, I., Buckle, A. M., Savill, J. S., McDowall, A., Haslett, C., and Hogg, N. (1994) Neutrophil apoptosis is associated with a reduction in CD16 (Fc gamma RIII) expressionJImmunol. 153, 1254–1263.

18.	Miller, D. K. (1997) The role of the caspase family of cysteine proteases in apoptosisSemin Immunol. 9, 35–49.

19.	Los, M., Walczak, H., Schulze-Osthoff, K., and Reed, J. C. (2000) Fluorogenic substrates as detectors of caspase activity during natural killer cell-induced apoptosisMethods Mol Biol. 121, 155–162.

20.	Niwa, M., Hara, A., Kanamori, Y., Matsuno, H.,Kozawa, O., Yoshimi, N., Mori, H., and Uematsu, T. (1999) Inhibition of tumor necrosis factor-alpha induced neutrophil apoptosis by cyclic AMP: involvement of caspase cascadeEur JPharmacol. 371, 59–67.

21.	Wilkie, R. P., Vissers, M. C., Dragunow, M., and Hampton, M. B. (2007) A functional NADPH oxidase prevents caspase involvement in the clearance of phagocytic neutrophilsInfect Immun. 75, 3256–3263.

22.	Stennicke, H. R., and Salvesen, G. S. (2000) Caspase assaysMethods Enzymol. 322, 91–100.

23.	Rawlings, N. D., and Barrett, A. J. (1990) FLUSYS: a software package for the collection and analysis of kinetic and scanning data from Perkin-Elmer fluorimetersComput Appl Biosci. 6, 118–119.

24.	Sabroe, I., Prince, L. R., Dower, S. K., Walmsley, S. R., Chilvers, E. R., and Whyte, M. K. (2004) What can we learn from highly purified neutrophils? Biochem Soc Trans. 32, 468–469.

Comments (Loading...)

Post comment/View all comments