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Qualitative and quantitative analysis of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction

  • Cell and Tissue Models
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Summary

A novel method for qualitative and quantitative analysis of monocyte transendothelial migration is described. By labeling monocytes and endothelial cells with different fluorophores, and utilizing confocal microscopy and three-dimensional image reconstruction, transmigrating monocytes were resolved and quantified within a subendothelial collagen gel. Comparison of monocyte migration across endothelial monolayers derived from human brain microvessels versus umbilical veins revealed diapedesis across brain endothelium to be significantly delayed. Inclusion of astrocytes within the subendothelial collagen gel resulted in the formation of an array of astrocytic processes that simulated the glia limitans surrounding brain microvessels in situ, thus yielding a more physiologic paradigm of the blood-brain barrier. By virtue of its unique capacity to provide information on the total number of migrating cells, this analytic approach overcomes significant caveats associated with sampling only aspects of the migration process. The potential adaptability of this method to computer-assisted analysis further enhances its prospective use in high-throughput screening.

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References

  • Abbott, N. J.; Revest, P. A.; Romero, I. A.. Astrocyte-endothelial interaction: physiology and pathology. Neuropathol. Appl. Neurobiol. 18:424–433; 1992.

    PubMed  CAS  Google Scholar 

  • Abbruscato, T. J.; Davis, T. P. Protein expression of brain endothelial cell E-cadherin after hypoxia/aglycemia: influence of astrocyte contact. Brain Res. 842:277–286; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Andersson, P. B.; Perry, V. H.; Gordon, S. The acute inflammatory response to lipopolysaccharide in CNS parenchyma differs from that in other body tissues. Neuroscience 48:169–186; 1992a.

    Article  PubMed  CAS  Google Scholar 

  • Andersson, P. B.; Perry, V. H.; Gordon, S. Intracerebral injection of proin-flammatory cytokines or leukocyte chemotaxins induces minimal myelomonocytic cell recruitment to the parenchyma of the central nervous system J. Exp. Med. 176:255–259; 1992b.

    Article  PubMed  CAS  Google Scholar 

  • Andjelkovic, A. V.; Kerkovich, D.; Pachter, J. S. Monocyte: astrocyte interactions regulate MCP-1 expression in both cell types. J. Leukoc. Biol. 68:545–552; 2000.

    PubMed  CAS  Google Scholar 

  • Andjelkovic, A. V.; Kerkovich, D.; Shanley, J.; Pulliam, L.; Pachter, J. S. Expression of chemokine binding sites on human astrocytes. Glia 28:225–235; 1999a.

    Article  PubMed  CAS  Google Scholar 

  • Andjelkovic, A. V.; Spencer, D. D.; Pachter, J. S. Visualization of chemokine binding sites on human brain microvessels. J. Cell Biol. 145:403–412 1999b.

    Article  PubMed  CAS  Google Scholar 

  • Bell, M. D.; Taub, D. D.; Perry, V. H. Overriding the brain's intrinsic resistance to leukocyte recruitment with intraparenchymal injection of recombinant chemokines. Neuroscience 74:283–292; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Biegel, D.; Pachter, J. S. Growth of brain microvessel endothelial cells on collagen gels: applications to the study of blood-brain barrier physiology and CNS inflammation. In Vitro Cell. Dev. Biol. 30A.:581–588; 1994.

    Google Scholar 

  • Biegel, D.; Spencer, D. D.; Pachter, J. S. Isolation and culture of human brain microvessel endothelial cells for the study of blood-brain barrier properties in vitro. Brain Res. 692:183–189; 1995.

    Article  PubMed  CAS  Google Scholar 

  • Brightman, M. W.; Tao-Chang, J. H. Tight junctions of brain endothelium and epithelium. In: Pardridge W. M., ed. The blood-brain barrier. New York: Raven Press; 1993:107–125.

    Google Scholar 

  • Christenson, L. K.; Stouffer, R. L. Isolation and culture of microvascular endothelial cells from the primate corpus luteum. Biol. Repord. 55:1397–1404; 1997.

    Article  Google Scholar 

  • Coggeshall, R. E.; Lekan, H. A. Methods for determing numbers of cells and synapses: a case for more uniform standards of review. J. Comp. Neurol. 364:6–15;1996.

    Article  PubMed  CAS  Google Scholar 

  • Crone, C.; Oleson, S. P. Electrical resistance of brain microvessel endothelium. Brain Res. 241:49–55; 1982.

    Article  PubMed  CAS  Google Scholar 

  • Deli, M. A.; Joo, F. Cultured vascular endothelial cells of the brain. Keio. J. Med. 445:183–198; 1997.

    Google Scholar 

  • Denholm, E. M.; Wolber, F. M. A simple method for the purification of human peripheral blood monocytes. A substitute for Sepracell-MN. J. Immunol. Methods 144:247–251; 1991.

    Article  PubMed  CAS  Google Scholar 

  • Fabbri, M.; Bianchi, E.; Fumagalli, L.; Pardi, R. Regulation of lymphocyte traffic by adhesion molecules. Inflammation Res. 48:239–246; 1999.

    Article  CAS  Google Scholar 

  • Furie, M. B.; Naprstek, B. L.; Silverstein, S. C. Migration of neutrophils across monolayers of cultured microvascular endothelial cells. An in vitro model of leukocyte extravasation. J. Cell Sci. 88:161–175; 1987.

    PubMed  CAS  Google Scholar 

  • Gahmberg, C. G.; Valmu, L.; Kotovuori, A., et al. Biosci. Rep. 19:273–281; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Groger, M.; Matsumura, T.; Kohrgruber, N.; Maurer, D.; Wolff, K.; Petzelbauer, P. A standardized, computer-assisted in vitro assay for the assessment of neutrophil transmigration across endothelial monolayers. J. Immunol. Methods 222:101–109 1999.

    Article  PubMed  CAS  Google Scholar 

  • Gu, L.; Tseng,. S. C.; Rollins, B. J.; Monocyte chemoattractant protein-1. Chen. Immunol. 27–29, 1999.

  • Hoogewerf, A. J.; Kuschert, G. S.; Proudfoot, A. E., et al. Glycosaminoglycans mediate cell surface oligomerization of chemokines. Biochemistry 36:13570–13578; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Hurwitz, A. A.; Berman, J. W.; Rashbaum, W. K.; Lyman, W. D. Human fetal astrocytes induce the expression of blood-brain barrier specific proteins by autologous endothelial cells. Brain Res. 625:238–243; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Imhof, B. A.; Dunon, D. Basic mechanisms of leukocyte migration. Hormone Metabol. Res. 29:614–621; 1997.

    Article  CAS  Google Scholar 

  • Janzer, R. C. The blood-brain barrier: cellular basis. J. Inherit. Metab. Dis. 16:639–647; 1993.

    Article  PubMed  CAS  Google Scholar 

  • Jayne, D. G.; O'Leary, R.; Gill, A.; Guilou, P. J. A three-dimensional in-vitro model of peritoneal tumour metastasis. Clin. Exp. Metastasis 17:515–523; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Joo, F. Endothelial cells of the brain and other organ systems: some similarities and differences. Prog. Neurobiol. 48:255–273; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Joseph, J.; Lublin, F. D.; Knobler, R. L. Modulation of T-cell endothelial adhesion by astrocyte conditioned medium. Glia 21:408–412; 1997.

    Article  PubMed  CAS  Google Scholar 

  • Kuschert, G. S.; Coulin, F.; Power, C. A., et al. Glycosaminoglycans interact with chemokines and modulate receptor binding and cellular responses. Biochemistry 38:12959–12968; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Lassmann, H.; Zimprich, F.; Vass, K.; Hickey, W. F. Microglial cells are a component of the perivascular glia limitans. J. Neurosci. Res. 28:236–243; 1991.

    Article  PubMed  CAS  Google Scholar 

  • London, J. A.; Biegel, D.; Pachter, J. S. Neurocytopathic effects of betaamyloid-stimulated monocytes: a potential mechanism for central nervous system damage in Alzheimer disease. Proc. Natl. Acad. Sci. USA 93:4147–4152; 1996.

    Article  PubMed  CAS  Google Scholar 

  • Mackarel, A. J.; Cottell, D. C.; Fitzgerald, M. X.; O'Conner, C. M. Human endothelial cells on microporous filters used for leukocyte transmigration studies on both sides of the filter. In Vitro Cell. Dev. Biol. 35:346–351; 1999.

    CAS  Google Scholar 

  • Pawlowski, N. A.; Kaplan, G.; Abraham, E.; Cohn, Z. A. The selective binding and transmigration of monocytes through the junctional complexes of human endothelium. J. Exp. Med. 168:1865–1882; 1988.

    Article  PubMed  CAS  Google Scholar 

  • Perry, V. H.; Anthony, D. C.; Brown, H. C. The blood-brain barrier and the inflammatory response. Mol. Med. Today 3:337–341; 1997.

    Google Scholar 

  • Persidsky, Y.; Stins, M.; Way, D., et al. A model for monocyte migration through the blood-brain barrier during HIV-1 encephalitis J. Immunol. 158:3499–3510; 1997.

    PubMed  CAS  Google Scholar 

  • Rosenkranz, A. R.; Mayadas, T. N. Leukocyte-endothelial cell interactions—lessons from knockout mice. Exp. Nephrol 7:125–136; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Rutten, M. J.; Hoover, R. L.; Karnovksy M. J. Electrical resistance and macromolecular permeability of brain endothelial monolayer cultures. Brain Res. 425:301–310; 1987.

    Article  PubMed  CAS  Google Scholar 

  • Shasby, S. S. Endothelial cells grown on permeable membrane supports. J. Tissue Cult. Methods 14:247–252; 1992.

    Article  Google Scholar 

  • Tan, P.; Luscinskas, F. W.; Homer-Vanniasinkam, S. Cellular and molecular mechanisms of inflammation and thrombosis. Eur. J. Vasc. Endovasc. Surg. 17:373–389; 1999.

    Article  PubMed  CAS  Google Scholar 

  • Vestweber, D.; Blanks, J. E. Mechanisms that regulate the function of the selectins and their ligands. Physiol. Rev. 79:181–213; 1999.

    PubMed  CAS  Google Scholar 

  • Vorbrodt, A. W. Ultrastructural cytochemistry of blood-brain barrier endothelia. Prog. Histochem. Cytochem. 18:1–96; 1988.

    CAS  Google Scholar 

  • West, M. J.; Slominanka, L.; Gundersen, H. J. G. Unblased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat. Rec. 231:482–497; 1991.

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. Blood-Brain Barrier Laboratory, Department of Pharmacology, University of Connecticut Health Center, 263 Farmington Avenue, 06030, Connecticut, Farmington

    Anuska V. Andjelkovic & Joel S. Pachter

  2. Center for Biomedical Imaging, University of Connecticut Health Center, 263 Farmington Avenue, 06030, Farmington, Connecticut

    Frank Morgan

  3. Department of Molecular and Cellular Physiology, Yale University School of Medicine, 333 Cedar St., 06510, New Haven, Connecticut

    Michal R. Zochowski

Authors
  1. Anuska V. Andjelkovic
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  2. Michal R. Zochowski
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  3. Frank Morgan
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  4. Joel S. Pachter
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Correspondence to Joel S. Pachter.

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Andjelkovic, A.V., Zochowski, M.R., Morgan, F. et al. Qualitative and quantitative analysis of monocyte transendothelial migration by confocal microscopy and three-dimensional image reconstruction. In Vitro Cell.Dev.Biol.-Animal 37, 111–120 (2001). https://doi.org/10.1290/1071-2690(2001)037<0111:QAQAOM>2.0.CO;2

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  • DOI: https://doi.org/10.1290/1071-2690(2001)037<0111:QAQAOM>2.0.CO;2

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