Abstract
Neutrophil polarization and directed migration (chemotaxis) are critical for the inflammatory response. Neutrophil chemotaxis is achieved by the sensing of narrow gradients of chemoattractant and the subsequent polarization and directed migration toward the chemotactic source. Despite recent progress, the signaling mechanisms that regulate neutrophil polarization during chemotaxis have not been clearly defined. Here, we describe methods to analyze neutrophil polarization and asymmetric redistribution of signaling components induced by chemoattractant using immunofluorescence. Further, methods are described to dissect the role of specific signaling pathways during chemotaxis by the use of murine neutrophils from transgenic mouse models. Finally, methods for time-lapse microscopy and transwell assay for the analysis of neutrophil chemotaxis will also be discussed.
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References
Nathan, C. (2006) Neutrophils and immunity: challenges and opportunities. Nat. Rev. Immunol. 6, 173–182.
Mollinedo, F., Borregaard, N., and Boxer, L. A. (1999) Novel trends in neutrophil structure, function and development. Immunol. Today 20, 535–537.
Van Haastert, P. J. and Devreotes, P. N. (2004) Chemotaxis: signalling the way forward. Nat. Rev. Mol. Cell Biol. 5, 626–634.
Postma, M., Bosgraaf, L., Loovers, H. M., and Van Haastert, P. J. (2004) Chemotaxis: signalling modules join hands at front and tail. EMBO Rep. 5, 35–40.
Iijima, M., Huang, Y. E., and Devreotes, P. (2002) Temporal and spatial regulation of chemotaxis. Dev. Cell 3, 469–478.
Chung, C. Y., Funamoto, S., and Firtel, R. A. (2001) Signaling pathways controlling cell polarity and chemotaxis. Trends Biochem. Sci. 26, 557–566.
Meili, R., Sasaki, A. T., and Firtel, R. A. (2005) Rho Rocks PTEN. Nat. Cell Biol. 7, 334–335.
Meili, R. and Firtel, R. A. (2003) Two poles and a compass. Cell 114, 153–156.
Weiner, O. D., Rentel, M. C., Ott, A. et al. (2006) Hem-1 complexes are essential for Rac activation, actin polymerization, and myosin regulation during neutrophil chemotaxis. PLoS. Biol. 4, e38.
Dekker, L. V. and Segal, A. W. (2000) Perspectives: signal transduction. Signals to move cells. Science 287, 982–3,985.
Weiner, O. D., Servant, G., Welch, M. D., Mitchison, T. J., Sedat, J. W., and Bourne, H. R. (1999) Spatial control of actin polymerization during neutrophil chemotaxis. Nat. Cell Biol. 1, 75–81.
Heit, B., Tavener, S., Raharjo, E., and Kubes, P. (2002) An intracellular signaling hierarchy determines direction of migration in opposing chemotactic gradients. J. Cell Biol. 159, 91–102.
Foxman, E. F., Kunkel, E. J., and Butcher, E. C. (1999) Integrating conflicting chemotactic signals. The role of memory in leukocyte navigation. J. Cell Biol. 147, 577–588.
Foxman, E. F., Campbell, J. J., and Butcher, E. C. (1997) Multistep navigation and the combinatorial control of leukocyte chemotaxis. J. Cell Biol. 139, 1349–1360.
Hogg, N., Henderson, R., Leitinger, B., McDowall, A., Porter, J., and Stanley, P. (2002) Mechanisms contributing to the activity of integrins on leukocytes. Immunol. Rev. 186, 164–171.
Pinkerton, P. H., Robinson, J. B., and Senn, J. S. (1978) Lazy leucocyte syndrome—disorder of the granulocyte membrane? J. Clin. Pathol. 31, 300–308.
Miller, M. E., Oski, F. A., and Harris, M. B. (1971) Lazy-leucocyte syndrome. A new disorder of neutrophil function. Lancet 1, 665–669.
Lokuta, M. A., Cooper, K. M., Aksentijevich, I., Kastner, D. L., and Huttenlocher, A. (2005) Neutrophil chemotaxis in a patient with neonatal-onset multisystem inflammatory disease and Muckle-Wells syndrome. Ann. Allergy Asthma Immunol. 95, 394–399.
Eddy, R. J., Pierini, L. M., and Maxfield, F. R. (2002) Microtubule asymmetry during neutrophil polarization and migration. Mol. Biol. Cell 13, 4470–4483.
Ding, M., Robinson, J. M., Behrens, B. C., and Vandre, D. D. (1995) The microtubule cytoskeleton in human phagocytic leukocytes is a highly dynamic structure. Eur. J. Cell Biol. 66, 234–245.
Kindzelskii, A. L., Sitrin, R. G., and Petty, H. R. (2004) Cutting edge: optical microspectrophotometry supports the existence of gel phase lipid rafts at the lamellipodium of neutrophils: apparent role in calcium signaling. J. Immunol. 172, 4681–4685.
Nuzzi, P. A., Lokuta, M. A., and Huttenlocher, A. (2006) Analysis of neutrophil chemotaxis. Methods Mol. Biol. 370, 23–35.
Ruoslahti, E., Hayman, E. G., Pierschbacher, M., and Engvall, E. (1982) Fibronectin: purification, immunochemical properties, and biological activities. Methods Enzymol. 82 Pt A, 803–831.
Hauert, A. B., Martinelli, S., Marone, C., and Niggli, V. (2002) Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis. Int. J. Biochem. Cell Biol. 34, 838–854.
Hoff, J. (2000) Methods of blood collection in the mouse. Lab Animal 29, 47–53.
Miner, N. A., Koehler, J., and Greenaway, L. (1969) Intraperitoneal injection of mice. Appl. Microbiol. 17, 250–251.
Eddy, R. J., Pierini, L. M., and Maxfield, F. R. (2002) Microtubule asymmetry during neutrophil polarization and migration. Mol. Biol. Cell 13, 4470–4483.
Kindzelskii, A. L., Sitrin, R. G., and Petty, H. R. (2004) Cutting edge: optical microspectrophotometry supports the existence of gel phase lipid rafts at the lamellipodium of neutrophils: apparent role in calcium signaling. J. Immunol. 172, 4681–4685.
Ding, M., Robinson, J. M., Behrens, B. C., and Vandre, D. D. (1995) The microtubule cytoskeleton in human phagocytic leukocytes is a highly dynamic structure. Eur. J. Cell Biol. 66, 234–245.
Heit, B. and Kubes, P. (2003) Measuring chemotaxis and chemokinesis: the underagarose cell migration assay. Sci. STKE. 2003, L5.
Foxman, E. F., Kunkel, E. J., and Butcher, E. C. (1999) Integrating conflicting chemotactic signals. The role of memory in leukocyte navigation. J. Cell Biol. 147, 577–588.
Wells, C. M. and Ridley, A. J. (2005) Analysis of cell migration using the Dunn chemotaxis chamber and time-lapse microscopy. Methods Mol. Biol. 294, 31–41.
Rhoads, D. S., Nadkarni, S. M., Song, L., Voeltz, C., Bodenschatz, E., and Guan, J. L. (2005) Using microfluidic channel networks to generate gradients for studying cell migration. Methods Mol. Biol. 294, 347–357.
Abhyankar, V. V., Lokuta, M. A., Huttenlocher, A., and Beebe, D. J. (2006) Characterization of a membrane-based gradient generator for use in cell-signaling studies. Lab. Chip. 6, 389–393.
Nuzzi, P. A., Lokuta, M. A., and Huttenlocher, A. (2006) Analysis of neutrophil chemotaxis. Methods Mol. Biol. 96, In press.
Zigmond, S. H. (1977) Ability of polymorphonuclear leukocytes to orient in gradients of chemotactic factors. J. Cell Biol. 75, 606–616.
Zigmond, S. H. and Hirsch, J. G. (1973) Leukocyte locomotion and chemotaxis. New methods for evaluation, and demonstration of a cell-derived chemotactic factor. J. Exp. Med. 137, 387–410.
Lokuta, M. A., Nuzzi, P. A., and Huttenlocher, A. (2003) Calpain regulates neutrophil chemotaxis. Proc. Natl. Acad. Sci. USA 100, 4006–4011.
Popesko, P., Rajtova, V., and Horak, J. (2003) A Colour Atlas of Anatomy of Small Laboratory Animals. Elsevier.
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Lokuta, M.A., Nuzzi, P.A., Huttenlocher, A. (2007). Analysis of Neutrophil Polarization and Chemotaxis. In: Quinn, M.T., DeLeo, F.R., Bokoch, G.M. (eds) Neutrophil Methods and Protocols. Methods in Molecular Biology™, vol 412. Humana Press. https://doi.org/10.1007/978-1-59745-467-4_14
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DOI: https://doi.org/10.1007/978-1-59745-467-4_14
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