Abstract
Polymorphonuclear leukocyte (PMN) transmigration into tissues is a highly regulated process and plays a central role in host defense. In inflammatory human diseases such as ulcerative colitis and Crohn’s disease, the infiltration of intestinal mucosa by large numbers of PMNs contributes to epithelial pathophysiology. The sequence of events that fine-tune PMN migration across epithelial cells is not well-understood. In this chapter, we describe a method to study PMN transmigration across intestinal epithelial T84 monolayers using a modified Boyden chamber system. This in vitro model system consists of three main components: the epithelium, purified PMN, and a chemoattractant gradient. Intestinal epithelial cells are cultured as inverted monolayers on permeable filter supports to facilitate the study of PMN transmigration in the physiologically relevant basolateral-to-apical direction. PMNs are isolated from human blood using dextran sedimentation followed by Ficoll density gradient centrifugation. PMN transmigration is elicited using N-formyl-methionyl-leucyl-phenylalanine gradients and is quantified by assaying for myeloperoxidase activity. The advantages of this model are its reductionist approach and the fact that the system can be easily manipulated. Studies using this model system will shed more light on the mechanisms regulating PMN responses in acute inflammatory diseases.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Weiland, J. E., Davis, W. B., Holter, J. F., Mohammed, J. R., Dorinsky, P. M., and Gadel, J. E. (1986). Lung neutrophils in the adult respiratory distress syndrome. Clinical and pathophysiologic significance. Am. Rev. Respir. Dis. 133, 218–225.
Hawker, P. C., McKay, J. S., and Turnberg, L. A. (1980) Electrolyte transport across colonic mucosa from patients with inflammatory bowel disease. Gastroenterology 79, 508–511.
Nusrat, A., Parkos, C. A., Liang, T. W., Carnes, D. K., and Madara, J. L. (1997) Neutrophil migration across model intestinal epithelia: monolayer disruption and subsequent events in epithelial repair. Gastroenterology 113, 1489–1500.
Koyama, S., Rennard, S. I., Leikauf, G. D., et al. (1991) Endotoxin stimulates bronchial epithelial cells to release chemotactic factors for neutrophils. A potential mechanism for neutrophil recruitment, cytotoxicity, and inhibition of proliferation in bronchial inflammation. J. Immunol. 147, 4293–4301.
Winn, R. K., Vedder, N. B., Sharar, S. R., and Harlan, J. M. (1997) Leukocyteendothelial cell interactions in ischemia-reperfusion injury. Ann. NY Acad. Sci. 832, 311–321.
Kakkar, A. K. and Lefer, D. J. (2004) Leukocyte and endothelial adhesion molecule studies in knockout mice. Curr. Opin. Pharmacol. 4, 154–158.
Jordan, J. E., Zhao, Z.-Q., and Vinten-Johansen, J. (1999) The role of neutrophils in myocardial ischemia-reperfusion injury. Cardiovasc. Res. 43, 860–878.
Frangogiannis, N. G., Smith, C. W., and Entman, M. L. (2002) The inflammatory response in myocardial infarction. Cardiovasc. Res. 53, 31–47.
Liu, Y., Shaw, S. K., Ma, S., Yang, L., Luscinskas, F. W., and Parkos, C. A. (2004) Regulation of leukocyte transmigration: cell surface interactions and signaling events. J. Immunol. 172, 7–13.
Bianchi, E., Bender, J. R., Blasi, F., and Pardi, R. (1997) Through and beyond the wall: late steps in leukocyte transendothelial migration. Immunol. Today 18, 586–591.
Feng, D., Nagy, J. A., Pyne, K., Dvorak, H. F., and Dvorak, A. M. (1998) Neutrophils emigrate from venules by a transendothelial cell pathway in response to FMLP. J. Exp. Med. 187, 903–915.
Carman, C. V. and Springer, T. A. (2004) A transmigratory cup in leukocyte diapedesis both through individual vascular endothelial cells and between them. J. Cell Biol. 167, 377–388.
McCormick, B., Hofman, P., Kim, J., Carnes, D., Miller, S., and Madara, J. (1995) Surface attachment of Salmonella typhimurium to intestinal epithelia imprints the subepithelial matrix with gradients chemotactic for neutrophils. J. Cell Biol. 131, 1599–1608.
Loike, J. D., el Khoury, J. Cao, L., et al. (1995) Fibrin regulates neutrophil migration in response to interleukin 8, leukotriene B4, tumor necrosis factor, and formylmethionyl-leucyl-phenylalanine. J. Exp. Med. 181, 1763–1772.
Loike, J. D., Cao, L., Budhu, S., et al. (1999) Differential regulation of beta 1 integrins by chemoattractants regulates neutrophil migration through fibrin. J. Cell Biol. 144, 1047–1056.
Loike, J. D., Cao, L., Budhu, S., Hoffman, S., and Silverstein, S. C. (2001) Blockade of a5β1 integrins reverses the inhibitory effect of tenascin on chemotaxis of human monocytes and polymorphonuclear leukocytes through three-dimensional gels of extracellular matrix proteins. J. Immunol. 166, 7534–7542.
Parkos, C. A., Delp, C., Arnaout, M. A., and Madara, J. L. (1991) Neutrophil migration across a cultured intestinal epithelium. Dependence on a CD11b/CD18-mediated event and enhanced efficiency in physiological direction. J. Clin. Invest. 88, 1605–1612.
Tosi, M., Hamedani, A., Brosovich, J., and Alpert, S. (1994) ICAM-1-independent, CD18-dependent adhesion between neutrophils and human airway epithelial cells exposed in vitro to ozone. J. Immunol. 152, 1935–1942.
Agace, W., Patarrayo, M., Svensson, M., Carlemalm, E., and Svanborg, C. (1995) Escherichia coli induces transuroepithelial neutrophil migration by an intercellular adhesion molecule-1-dependent mechanism. Infect. Immun. 63, 4054–4062.
Zen, K., Babbin, B. A., Liu, Y., Whelan, J. B., Nusrat, A., and Parkos, C. A. (2004) JAM-C is a component of desmosomes and a ligand for CD11b/CD18-mediated neutrophil transepithelial migration. Mol. Biol. Cell 15, 3926–3937.
Liu, Y., Buhring, H.-J., Zen, K., et al. (2002) Signal regulatory protein (SIRPalpha), a cellular ligand for CD47, regulates neutrophil transmigration. J. Biol. Chem. 277, 10,028–10,036.
Liu, Y., Merlin, D., Burst, S. L., Pochet, M., Madara, J. L., and Parkos, C. A. (2001) The Role of CD47 in neutrophil transmigration. Increased rate of migration correlates with increased cell surface expression of CD47. J. Biol. Chem. 276, 40,156–40,166.
Parkos, C., Colgan, S., Liang, T., et al. (1996) CD47 mediates post-adhesive events required for neutrophil migration across polarized intestinal epithelia. J. Cell Biol. 132, 437–450.
Colgan, S., Parkos, C., Delp, C., Aranaout, M., and Madara, J. (1993) Neutrophil migration across cultured intestinal epithelial monolayers is modulated by epithelial exposure to IFN-gamma in a highly polarized fashion. J. Cell Biol. 120, 785–798.
Cereijido, M., Robbins, E., Dolan, W., Rotunno, C., and Sabatini, D. (1978) Polarized monolayers formed by epithelial cells on a permeable and translucent support. J. Cell Biol. 77, 853–880.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2006 Humana Press Inc.
About this protocol
Cite this protocol
Lee, W.Y., Chin, A.C., Voss, S., Parkos, C.A. (2006). In Vitro Neutrophil Transepithelial Migration. In: Colgan, S.P. (eds) Cell-Cell Interactions. Methods in Molecular Biology™, vol 341. Humana Press. https://doi.org/10.1385/1-59745-113-4:205
Download citation
DOI: https://doi.org/10.1385/1-59745-113-4:205
Publisher Name: Humana Press
Print ISBN: 978-1-58829-523-1
Online ISBN: 978-1-59745-113-0
eBook Packages: Springer Protocols