Galectins pp 215-229 | Cite as

Assessing the Roles of Galectins in Regulating Dendritic Cell Migration Through Extracellular Matrix and Across Lymphatic Endothelial Cells

  • Sandra Thiemann
  • Jeanette H. Man
  • Linda G. BaumEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1207)


Leukocyte migration from the bloodstream into tissues, and from tissues to lymph nodes, depends on expression of specific adhesion and signaling molecules by vascular endothelial cells and lymphatic endothelial cells. Tissue damage and microbial infection induce vascular endothelial cells to up-regulate expression of adhesion molecules to facilitate entry of several leukocyte populations from blood into tissues. Many of these cells then leave inflamed tissue and migrate to regional lymph nodes. A critical population that emigrates from inflamed tissue is dendritic cells. Dendritic cells in tissue have to migrate through extracellular matrix and across a layer of lymphatic endothelial cells to enter the lymphatic vasculature. Little is known about the adhesion molecules expressed by lymphatic endothelial cells or the processes required for the critical step of dendritic cell exit from tissues, specifically migration through the extracellular matrix and basal-to-apical migration across the lymphatic endothelial cell layer into lymphatic vasculature.

Members of the galectin family of carbohydrate binding proteins are expressed in both vascular and lymphatic endothelial cells. Dynamic changes in galectin expression during inflammation are known to regulate leukocyte tissue entry during inflammation. However, the roles of galectin family members expressed by lymphatic endothelial cells in leukocyte tissue exit remain to be explored.

Here, we describe an in vitro transmigration assay that mimics dendritic cell tissue exit in the presence and absence of galectin protein. Fluorescently labeled human dendritic cell migration through extracellular matrix and across human lymphatic endothelial cells is examined in the presence and absence of recombinant human galectin protein.

Key words

Leukocyte migration Human dendritic cell Human lymphatic endothelial cell Tissue exit Extracellular matrix Galectin Transmigration insert 


  1. 1.
    Luster AD, Alon R, von Andrian UH (2005) Immune cell migration in inflammation: present and future therapeutic targets. Nat Immunol 6(12):1182–1190PubMedCrossRefGoogle Scholar
  2. 2.
    Ley K, Laudanna C, Cybulsky MI, Nourshargh S (2007) Getting to the site of inflammation: the leukocyte adhesion cascade updated. Nat Rev Immunol 7(9):678–689PubMedCrossRefGoogle Scholar
  3. 3.
    Sorokin L (2010) The impact of the extracellular matrix on inflammation. Nat Rev Immunol 10(10):712–723. doi: 10.1038/nri2852 PubMedCrossRefGoogle Scholar
  4. 4.
    Elola MT, Wolfenstein-Todel C, Troncoso MF, Vasta GR, Rabinovich GA (2007) Galectins: matricellular glycan-binding proteins linking cell adhesion, migration, and survival. Cell Mol Life Sci 64(13):1679–1700PubMedCrossRefGoogle Scholar
  5. 5.
    Angeli V, Randolph GJ (2006) Inflammation, lymphatic function, and dendritic cell migration. Lymphat Res Biol 4(4):217–228PubMedCrossRefGoogle Scholar
  6. 6.
    von Andrian UH, Mempel TR (2003) Homing and cellular traffic in lymph nodes. Nat Rev Immunol 3(11):867–878. doi: 10.1038/nri1222 CrossRefGoogle Scholar
  7. 7.
    Randolph GJ, Angeli V, Swartz MA (2005) Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat Rev Immunol 5(8):617–628PubMedCrossRefGoogle Scholar
  8. 8.
    Macatonia SE, Knight SC, Edwards AJ, Griffiths S, Fryer P (1987) Localization of antigen on lymph node dendritic cells after exposure to the contact sensitizer fluorescein isothiocyanate. Functional and morphological studies. J Exp Med 166(6):1654–1667PubMedCrossRefGoogle Scholar
  9. 9.
    Fulcher JA, Chang MH, Wang S, Almazan T, Hashimi ST, Eriksson AU, Wen X, Pang M, Baum LG, Singh RR, Lee B (2009) Galectin-1 co-clusters CD43/CD45 on dendritic cells and induces cell activation and migration through Syk and protein kinase C signaling. J Biol Chem 284(39):26860–26870PubMedCrossRefPubMedCentralGoogle Scholar
  10. 10.
    Johnson LA, Clasper S, Holt AP, Lalor PF, Baban D, Jackson DG (2006) An inflammation-induced mechanism for leukocyte transmigration across lymphatic vessel endothelium. J Exp Med 203(12):2763–2777PubMedCrossRefPubMedCentralGoogle Scholar
  11. 11.
    Johnson LA, Jackson DG (2010) Inflammation-induced secretion of CCL21 in lymphatic endothelium is a key regulator of integrin-mediated dendritic cell transmigration. Int Immunol. doi: 10.1093/intimm/dxq435 PubMedGoogle Scholar
  12. 12.
    Lutz MB, Schuler G (2002) Immature, semi-mature and fully mature dendritic cells: which signals induce tolerance or immunity? Trends Immunol 23(9):445–449PubMedCrossRefGoogle Scholar
  13. 13.
    He J, Baum LG (2006) Endothelial cell expression of galectin-1 induced by prostate cancer cells inhibits T-cell transendothelial migration. Lab Invest 86(6):578–590PubMedGoogle Scholar
  14. 14.
    Roth SJ, Carr MW, Rose SS, Springer TA (1995) Characterization of transendothelial chemotaxis of T lymphocytes. J Immunol Methods 188(1):97–116PubMedCrossRefGoogle Scholar
  15. 15.
    Miteva DO, Rutkowski JM, Dixon JB, Kilarski W, Shields JD, Swartz MA (2010) Transmural flow modulates cell and fluid transport functions of lymphatic endothelium. Circ Res 106(5):920–931PubMedCrossRefGoogle Scholar
  16. 16.
    Podgrabinska S, Braun P, Velasco P, Kloos B, Pepper MS, Skobe M (2002) Molecular characterization of lymphatic endothelial cells. Proc Natl Acad Sci U S A 99(25):16069–16074PubMedCrossRefPubMedCentralGoogle Scholar
  17. 17.
    Ogunbiyi S, Chinien G, Field D, Humphries J, Burand K, Sawyer B, Jeffrey S, Mortimer P, Clasper S, Jackson D, Smith A (2011) Molecular characterization of dermal lymphatic endothelial cells from primary lymphedema skin. Lymphat Res Biol 9(1):19–30. doi: 10.1089/lrb.2010.0019 PubMedCrossRefGoogle Scholar
  18. 18.
    Sallusto F, Lanzavecchia A (1994) Efficient presentation of soluble antigen by cultured human dendritic cells is maintained by granulocyte/macrophage colony-stimulating factor plus interleukin 4 and downregulated by tumor necrosis factor alpha. J Exp Med 179(4):1109–1118PubMedCrossRefGoogle Scholar
  19. 19.
    Osugi Y, Vuckovic S, Hart DN (2002) Myeloid blood CD11c(+) dendritic cells and monocyte-derived dendritic cells differ in their ability to stimulate T lymphocytes. Blood 100(8):2858–2866. doi: 10.1182/blood.V100.8.2858 PubMedCrossRefGoogle Scholar
  20. 20.
    Granucci F, Vizzardelli C, Virzi E, Rescigno M, Ricciardi-Castagnoli P (2001) Transcriptional reprogramming of dendritic cells by differentiation stimuli. Eur J Immunol 31(9):2539–2546. doi: 10.1002/1521-4141(200109)31:9<2539::AID-IMMU2539>3.0.CO;2-9 PubMedCrossRefGoogle Scholar
  21. 21.
    Podgrabinska S, Kamalu O, Mayer L, Shimaoka M, Snoeck H, Randolph GJ, Skobe M (2009) Inflamed lymphatic endothelium suppresses dendritic cell maturation and function via Mac-1/ICAM-1-dependent mechanism. J Immunol 183(3):1767–1779PubMedCrossRefGoogle Scholar
  22. 22.
    Teijeira A, Palazon A, Garasa S, Marre D, Auba C, Rogel A, Murillo O, Martinez-Forero I, Lang F, Melero I, Rouzaut A (2012) CD137 on inflamed lymphatic endothelial cells enhances CCL21-guided migration of dendritic cells. FASEB J 26(8):3380–3392. doi: 10.1096/fj.11-201061 PubMedCrossRefGoogle Scholar
  23. 23.
    He J, Baum LG (2006) Galectin interactions with extracellular matrix and effects on cellular function. Methods Enzymol 417:247–256PubMedCrossRefGoogle Scholar
  24. 24.
    Fainaru O, Almog N, Yung CW, Nakai K, Montoya-Zavala M, Abdollahi A, D’Amato R, Ingber DE (2010) Tumor growth and angiogenesis are dependent on the presence of immature dendritic cells. FASEB J 24(5):1411–1418. doi: 10.1096/fj.09-147025 PubMedCrossRefPubMedCentralGoogle Scholar
  25. 25.
    Bi S, Hong PW, Lee B, Baum LG (2011) Galectin-9 binding to cell surface protein disulfide isomerase regulates the redox environment to enhance T-cell migration and HIV entry. Proc Natl Acad Sci U S A 108(26):10650–10655PubMedCrossRefPubMedCentralGoogle Scholar
  26. 26.
    Baum LG, Seilhamer JJ, Pang M, Levine WB, Beynon D, Berliner JA (1995) Synthesis of an endogeneous lectin, galectin-1, by human endothelial cells is up-regulated by endothelial cell activation. Glycoconj J 12(1):63–68PubMedCrossRefGoogle Scholar
  27. 27.
    Thijssen VL, Hulsmans S, Griffioen AW (2008) The galectin profile of the endothelium: altered expression and localization in activated and tumor endothelial cells. Am J Pathol 172(2):545–553PubMedCrossRefPubMedCentralGoogle Scholar
  28. 28.
    Thiemann S, Baum LG (2011) The road less traveled: regulation of leukocyte migration across vascular and lymphatic endothelium by galectins. J Clin Immunol 31(1):2–9PubMedCrossRefPubMedCentralGoogle Scholar
  29. 29.
    Cueni LN, Detmar M (2009) Galectin-8 interacts with podoplanin and modulates lymphatic endothelial cell functions. Exp Cell Res 315(10):1715–1723PubMedCrossRefPubMedCentralGoogle Scholar
  30. 30.
    Hsu DK, Chernyavsky AI, Chen HY, Yu L, Grando SA, Liu FT (2009) Endogenous galectin-3 is localized in membrane lipid rafts and regulates migration of dendritic cells. J Invest Dermatol 129(3):573–583PubMedCrossRefPubMedCentralGoogle Scholar
  31. 31.
    Brewer CF, Miceli MC, Baum LG (2002) Clusters, bundles, arrays and lattices: novel mechanisms for lectin-saccharide-mediated cellular interactions. Curr Opin Struct Biol 12(5):616–623PubMedCrossRefGoogle Scholar
  32. 32.
    Rabinovich GA, Liu FT, Hirashima M, Anderson A (2007) An emerging role for galectins in tuning the immune response: lessons from experimental models of inflammatory disease, autoimmunity and cancer. Scand J Immunol 66(2–3):143–158PubMedCrossRefGoogle Scholar
  33. 33.
    Stowell SR, Arthur CM, Mehta P, Slanina KA, Blixt O, Leffler H, Smith DF, Cummings RD (2008) Galectin-1, -2, and -3 exhibit differential recognition of sialylated glycans and blood group antigens. J Biol Chem 283(15):10109–10123PubMedCrossRefPubMedCentralGoogle Scholar
  34. 34.
    Earl LA, Baum LG (2008) CD45 glycosylation controls T-cell life and death. Immunol Cell Biol 86(7):608–615PubMedCrossRefGoogle Scholar
  35. 35.
    Song X, Xia B, Stowell SR, Lasanajak Y, Smith DF, Cummings RD (2009) Novel fluorescent glycan microarray strategy reveals ligands for galectins. Chem Biol 16(1):36–47PubMedCrossRefPubMedCentralGoogle Scholar
  36. 36.
    Nagae M, Nishi N, Murata T, Usui T, Nakamura T, Wakatsuki S, Kato R (2006) Crystal structure of the galectin-9 N-terminal carbohydrate recognition domain from Mus musculus reveals the basic mechanism of carbohydrate recognition. J Biol Chem 281(47):35884–35893PubMedCrossRefGoogle Scholar
  37. 37.
    Ahmad N, Gabius HJ, Andre S, Kaltner H, Sabesan S, Roy R, Liu B, Macaluso F, Brewer CF (2004) Galectin-3 precipitates as a pentamer with synthetic multivalent carbohydrates and forms heterogeneous cross-linked complexes. J Biol Chem 279(12):10841–10847PubMedCrossRefGoogle Scholar
  38. 38.
    Thijssen VL, Barkan B, Shoji H, Aries IM, Mathieu V, Deltour L, Hackeng TM, Kiss R, Kloog Y, Poirier F, Griffioen AW (2010) Tumor cells secrete galectin-1 to enhance endothelial cell activity. Cancer Res 70(15):6216–6224. doi: 10.1158/0008-5472.CAN-09-4150 PubMedCrossRefGoogle Scholar
  39. 39.
    Liu FT, Hsu DK, Zuberi RI, Kuwabara I, Chi EY, Henderson WR Jr (1995) Expression and function of galectin-3, a beta-galactoside-binding lectin, in human monocytes and macrophages. Am J Pathol 147(4):1016–1028PubMedPubMedCentralGoogle Scholar
  40. 40.
    Oomizu S, Arikawa T, Niki T, Kadowaki T, Ueno M, Nishi N, Yamauchi A, Hattori T, Masaki T, Hirashima M (2012) Cell surface galectin-9 expressing th cells regulate Th17 and Foxp3+ Treg development by galectin-9 secretion. PLoS One 7(11):e48574. doi: 10.1371/journal.pone.0048574 PubMedCrossRefPubMedCentralGoogle Scholar
  41. 41.
    Nair S, Archer GE, Tedder TF (2012) Isolation and generation of human dendritic cells. In: Coligan JE (ed) Current protocols in immunology, vol 99. Wiley, New York, pp 7.32.31–37.32.23Google Scholar
  42. 42.
    Humrich JY, Humrich JH, Averbeck M, Thumann P, Termeer C, Kampgen E, Schuler G, Jenne L (2006) Mature monocyte-derived dendritic cells respond more strongly to CCL19 than to CXCL12: consequences for directional migration. Immunology 117(2):238–247. doi: 10.1111/j.1365-2567.2005.02292.x PubMedCrossRefPubMedCentralGoogle Scholar
  43. 43.
    Makinen T, Norrmen C, Petrova TV (2007) Molecular mechanisms of lymphatic vascular development. Cell Mol Life Sci 64(15):1915–1929. doi: 10.1007/s00018-007-7040-z PubMedCrossRefGoogle Scholar
  44. 44.
    Fulcher JA, Hashimi ST, Levroney EL, Pang M, Gurney KB, Baum LG, Lee B (2006) Galectin-1-matured human monocyte-derived dendritic cells have enhanced migration through extracellular matrix. J Immunol 177(1):216–226PubMedCrossRefGoogle Scholar
  45. 45.
    Mobergslien A, Sioud M (2010) Optimized protocols for siRNA delivery into monocytes and dendritic cells. Methods Mol Biol 629:71–85. doi: 10.1007/978-1-60761-657-3_5 PubMedGoogle Scholar
  46. 46.
    Cambi A, Beeren I, Joosten B, Fransen JA, Figdor CG (2009) The C-type lectin DC-SIGN internalizes soluble antigens and HIV-1 virions via a clathrin-dependent mechanism. Eur J Immunol 39(7):1923–1928. doi: 10.1002/eji.200939351 PubMedCrossRefGoogle Scholar
  47. 47.
    Pace KE, Hahn HP, Baum LG (2003) Preparation of recombinant human galectin-1 and use in T-cell death assays. Methods Enzymol 363:499–518. doi: 10.1016/S0076-6879(03)01075-9 PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Sandra Thiemann
    • 1
  • Jeanette H. Man
    • 2
  • Linda G. Baum
    • 1
    Email author
  1. 1.Department of Pathology and Laboratory Medicine, UCLA School of MedicineUniversity of CaliforniaLos AngelesUSA
  2. 2.Department of Pathology and Laboratory Medicine, UCLA School of MedicineUniversity of California, Medical College of WisconsinMilwaukeeUSA

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