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. 1992 Sep 1;176(3):819–828. doi: 10.1084/jem.176.3.819

Monocyte-selective transendothelial migration: dissection of the binding and transmigration phases by an in vitro assay

PMCID: PMC2119361  PMID: 1512545

Abstract

We describe a quantitative assay of transendothelial migration (TEM) that allows us to selectively study the interaction of monocytes with confluent human endothelial cell (HEC) monolayers. The HEC are grown on hydrated collagen gels; the monocytes need not be purified. 100% of monocytes transmigrated the monolayer within 1 h at 37 degrees C and accumulated in the subendothelial collagen; TEM of lymphocytes was not detected within this time. Migration of neutrophils from the same donor was much slower and incomplete, with only 14% of PMN transmigrating in 2 h. This rapid TEM occurs in the absence of exogenous chemoattractants, and HEC in this system do not express cytokine- inducible leukocyte adhesion molecules. A slight modification of the TEM assay allowed us to separate binding to the apical HEC surface from TEM. We found that tight apical surface binding was the rate-limiting step for TEM. Two-thirds of this binding and TEM could be blocked by a monoclonal antibody against the leukocyte beta 2 integrin chain CD18. This assay will allow us to dissect the mechanisms of both the binding and transmigration stages of diapedesis.

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Selected References

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  1. Albelda S. M., Buck C. A. Integrins and other cell adhesion molecules. FASEB J. 1990 Aug;4(11):2868–2880. [PubMed] [Google Scholar]
  2. Atherton A., Born G. V. Quantitative investigations of the adhesiveness of circulating polymorphonuclear leucocytes to blood vessel walls. J Physiol. 1972 Apr;222(2):447–474. doi: 10.1113/jphysiol.1972.sp009808. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bevilacqua M. P., Pober J. S., Mendrick D. L., Cotran R. S., Gimbrone M. A., Jr Identification of an inducible endothelial-leukocyte adhesion molecule. Proc Natl Acad Sci U S A. 1987 Dec;84(24):9238–9242. doi: 10.1073/pnas.84.24.9238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Collins T., Lapierre L. A., Fiers W., Strominger J. L., Pober J. S. Recombinant human tumor necrosis factor increases mRNA levels and surface expression of HLA-A,B antigens in vascular endothelial cells and dermal fibroblasts in vitro. Proc Natl Acad Sci U S A. 1986 Jan;83(2):446–450. doi: 10.1073/pnas.83.2.446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cushing S. D., Berliner J. A., Valente A. J., Territo M. C., Navab M., Parhami F., Gerrity R., Schwartz C. J., Fogelman A. M. Minimally modified low density lipoprotein induces monocyte chemotactic protein 1 in human endothelial cells and smooth muscle cells. Proc Natl Acad Sci U S A. 1990 Jul;87(13):5134–5138. doi: 10.1073/pnas.87.13.5134. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Deuel T. F., Senior R. M., Huang J. S., Griffin G. L. Chemotaxis of monocytes and neutrophils to platelet-derived growth factor. J Clin Invest. 1982 Apr;69(4):1046–1049. doi: 10.1172/JCI110509. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Furie M. B., Naprstek B. L., Silverstein S. C. Migration of neutrophils across monolayers of cultured microvascular endothelial cells. An in vitro model of leucocyte extravasation. J Cell Sci. 1987 Sep;88(Pt 2):161–175. doi: 10.1242/jcs.88.2.161. [DOI] [PubMed] [Google Scholar]
  8. Graham R. C., Jr, Karnovsky M. J. The early stages of absorption of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J Histochem Cytochem. 1966 Apr;14(4):291–302. doi: 10.1177/14.4.291. [DOI] [PubMed] [Google Scholar]
  9. Hakkert B. C., Rentenaar J. M., Van Aken W. G., Roos D., Van Mourik J. A. A three-dimensional model system to study the interactions between human leukocytes and endothelial cells. Eur J Immunol. 1990 Dec;20(12):2775–2781. doi: 10.1002/eji.1830201236. [DOI] [PubMed] [Google Scholar]
  10. Harlan J. M. Leukocyte-endothelial interactions. Blood. 1985 Mar;65(3):513–525. [PubMed] [Google Scholar]
  11. Lawrence M. B., Springer T. A. Leukocytes roll on a selectin at physiologic flow rates: distinction from and prerequisite for adhesion through integrins. Cell. 1991 May 31;65(5):859–873. doi: 10.1016/0092-8674(91)90393-d. [DOI] [PubMed] [Google Scholar]
  12. Lo S. K., Lee S., Ramos R. A., Lobb R., Rosa M., Chi-Rosso G., Wright S. D. Endothelial-leukocyte adhesion molecule 1 stimulates the adhesive activity of leukocyte integrin CR3 (CD11b/CD18, Mac-1, alpha m beta 2) on human neutrophils. J Exp Med. 1991 Jun 1;173(6):1493–1500. doi: 10.1084/jem.173.6.1493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Luscinskas F. W., Cybulsky M. I., Kiely J. M., Peckins C. S., Davis V. M., Gimbrone M. A., Jr Cytokine-activated human endothelial monolayers support enhanced neutrophil transmigration via a mechanism involving both endothelial-leukocyte adhesion molecule-1 and intercellular adhesion molecule-1. J Immunol. 1991 Mar 1;146(5):1617–1625. [PubMed] [Google Scholar]
  14. MARCHESI V. T., FLOREY H. W. Electron micrographic observations on the emigration of leucocytes. Q J Exp Physiol Cogn Med Sci. 1960 Oct;45:343–348. doi: 10.1113/expphysiol.1960.sp001489. [DOI] [PubMed] [Google Scholar]
  15. MARCHESI V. T. The site of leucocyte emigration during inflammation. Q J Exp Physiol Cogn Med Sci. 1961 Apr;46:115–118. doi: 10.1113/expphysiol.1961.sp001522. [DOI] [PubMed] [Google Scholar]
  16. Moser R., Schleiffenbaum B., Groscurth P., Fehr J. Interleukin 1 and tumor necrosis factor stimulate human vascular endothelial cells to promote transendothelial neutrophil passage. J Clin Invest. 1989 Feb;83(2):444–455. doi: 10.1172/JCI113903. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Muller W. A., Ratti C. M., McDonnell S. L., Cohn Z. A. A human endothelial cell-restricted, externally disposed plasmalemmal protein enriched in intercellular junctions. J Exp Med. 1989 Aug 1;170(2):399–414. doi: 10.1084/jem.170.2.399. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Navab M., Hough G. P., Stevenson L. W., Drinkwater D. C., Laks H., Fogelman A. M. Monocyte migration into the subendothelial space of a coculture of adult human aortic endothelial and smooth muscle cells. J Clin Invest. 1988 Dec;82(6):1853–1863. doi: 10.1172/JCI113802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Newman P. J., Berndt M. C., Gorski J., White G. C., 2nd, Lyman S., Paddock C., Muller W. A. PECAM-1 (CD31) cloning and relation to adhesion molecules of the immunoglobulin gene superfamily. Science. 1990 Mar 9;247(4947):1219–1222. doi: 10.1126/science.1690453. [DOI] [PubMed] [Google Scholar]
  20. Osborn L., Hession C., Tizard R., Vassallo C., Luhowskyj S., Chi-Rosso G., Lobb R. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell. 1989 Dec 22;59(6):1203–1211. doi: 10.1016/0092-8674(89)90775-7. [DOI] [PubMed] [Google Scholar]
  21. Pawlowski N. A., Abraham E. L., Pontier S., Scott W. A., Cohn Z. A. Human monocyte-endothelial cell interaction in vitro. Proc Natl Acad Sci U S A. 1985 Dec;82(23):8208–8212. doi: 10.1073/pnas.82.23.8208. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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. 1988 Nov 1;168(5):1865–1882. doi: 10.1084/jem.168.5.1865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Pober J. S., Gimbrone M. A., Jr, Lapierre L. A., Mendrick D. L., Fiers W., Rothlein R., Springer T. A. Overlapping patterns of activation of human endothelial cells by interleukin 1, tumor necrosis factor, and immune interferon. J Immunol. 1986 Sep 15;137(6):1893–1896. [PubMed] [Google Scholar]
  24. Smith C. W., Rothlein R., Hughes B. J., Mariscalco M. M., Rudloff H. E., Schmalstieg F. C., Anderson D. C. Recognition of an endothelial determinant for CD 18-dependent human neutrophil adherence and transendothelial migration. J Clin Invest. 1988 Nov;82(5):1746–1756. doi: 10.1172/JCI113788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Springer T. A. Adhesion receptors of the immune system. Nature. 1990 Aug 2;346(6283):425–434. doi: 10.1038/346425a0. [DOI] [PubMed] [Google Scholar]
  26. Wright S. D., Silverstein S. C. Tumor-promoting phorbol esters stimulate C3b and C3b' receptor-mediated phagocytosis in cultured human monocytes. J Exp Med. 1982 Oct 1;156(4):1149–1164. doi: 10.1084/jem.156.4.1149. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. von Andrian U. H., Chambers J. D., McEvoy L. M., Bargatze R. F., Arfors K. E., Butcher E. C. Two-step model of leukocyte-endothelial cell interaction in inflammation: distinct roles for LECAM-1 and the leukocyte beta 2 integrins in vivo. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7538–7542. doi: 10.1073/pnas.88.17.7538. [DOI] [PMC free article] [PubMed] [Google Scholar]

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