Skip to main content

Advertisement

SpringerLink
Log in

Generation and characterization of a mouse lymphatic endothelial cell line

  • Regular Article
  • Published:
Cell and Tissue Research Aims and scope Submit manuscript

Abstract

Lymphatic vessels, by channeling fluid and leukocytes from the periphery into lymph nodes, play a central role in the development of the immune response. Despite their importance in homeostasis and disease, the difficulties in enriching and culturing lymphatic endothelial cells limit studies of their biology. Here, we report the isolation, stabilization, and characterization of a mouse lymphatic endothelial cell line (MELC) and the generated clones thereof. Cells were isolated from benign lymphangiomas induced by intraperitoneal injections of incomplete Freund’s adjuvant. The MELC line expressed molecules typical of lymphatic endothelium, including VEGFR3/Flt-4, podoplanin, Prox-1, and D6, but not LYVE-1. It also expressed CD34, ICAM-1, VCAM, and JAM-A, but not CD31, VE-cadherin, E-selectin, or CX3CL1/fractalkine (both TNFα-induced), at variance with vascular endothelial cells tested in parallel. The inflammatory cytokines TNFα and IL-4 regulated production of selected adhesion molecules (VCAM), cytokines (IL-6), and chemokines (CCL2/JE). Whole genome transcriptional profiling identified a set of 150 known genes differentially expressed in MELC versus vascular endothelial cells. Thus, the MELC line may represent an invaluable source of lymphatic endothelium.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Alitalo K, Carmeliet P (2002) Molecular mechanisms of lymphangiogenesis in health and disease. Cancer Cell 1:219–227

    Article  PubMed  CAS  Google Scholar 

  • Breiteneder-Geleff S, Soleiman A, Kowalski H, Horvat R, Amann G, Kriehuber E, Diem K, Weninger W, Tschachler E, Alitalo K, Kerjaschki D (1999) Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am J Pathol 154:385–394

    PubMed  CAS  Google Scholar 

  • Brint EK, Xu D, Liu H, Dunne A, McKenzie AN, O’Neill LA, Liew FY (2004) ST2 is an inhibitor of interleukin 1 receptor and Toll-like receptor 4 signaling and maintains endotoxin tolerance. Nat Immunol 5:373–379

    Article  PubMed  CAS  Google Scholar 

  • Carlos TM, Harlan JM (1994) Leukocyte-endothelial adhesion molecules. Blood 84:2068–2101

    PubMed  CAS  Google Scholar 

  • Carreira C, Romero C, Rodriguez R, Martin de Francisco J, Urbasos M, Pinto J (2001) A cavernous haemangioma of breast in male: radiological-pathological correlation. Eur Radiol 11:292–294

    Article  PubMed  CAS  Google Scholar 

  • Corada M, Zanetta L, Orsenigo F, Breviario F, Lampugnani M G, Bernasconi S, Liao F, Hicklin D J, Bohlen P, Dejana E (2002) A monoclonal antibody to vascular endothelial-cadherin inhibits tumor angiogenesis without side effects on endothelial permeability. Blood 100:905–911

    Article  PubMed  CAS  Google Scholar 

  • Dong QG, Bernasconi S, Lostaglio S, De Calmanovici RW, Martin-Padura I, Breviario F, Garlanda C, Ramponi S, Mantovani A, Vecchi A (1997) A general strategy for isolation of endothelial cells from murine tissues. Characterization of two endothelial cell lines from the murine lung and subcutaneous sponge implants. Arterioscler Thromb Vasc Biol 17:1599–1604

    PubMed  CAS  Google Scholar 

  • Garlanda C, Parravicini C, Sironi M, De Rossi M, Wainstok de Calmanovici R, Carozzi F, Bussolino F, Colotta F, Mantovani A, Vecchi A (1994) Progressive growth in immunodeficient mice and host cell recruitment by mouse endothelial cells transformed by polyoma middle-sized T antigen: implications for the pathogenesis of opportunistic vascular tumors. Proc Natl Acad Sci USA 91:7291–7295

    Article  PubMed  CAS  Google Scholar 

  • Garlanda C, Berthier R, Garin J, Stoppacciaro A, Ruco L, Vittet D, Gulino D, Matteucci C, Mantovani A, Vecchi A, Dejana E (1997) Characterization of MEC 14.7, a new monoclonal antibody recognizing mouse CD34: a useful reagent for identifying and characterizing blood vessels and hematopoietic precursors. Eur J Cell Biol 73:368–377

    PubMed  CAS  Google Scholar 

  • Garlanda C, Riva F, Polentarutti N, Buracchi C, Sironi M, De Bortoli M, Muzio M, Bergottini R, Scanziani E, Vecchi A, Hirsch E, Mantovani A (2004) Intestinal inflammation in mice deficient in Tir8, an inhibitory member of the IL-1 receptor family. Proc Natl Acad Sci USA 101:3522–3526

    Article  PubMed  CAS  Google Scholar 

  • Gunn MD, Kyuwa S, Tam C, Kakiuchi T, Matsuzawa A, Williams LT, Nakano H (1999) Mice lacking expression of secondary lymphoid organ chemokine have defects in lymphocyte homing and dendritic cell localization. J Exp Med 189:451–460

    Article  PubMed  CAS  Google Scholar 

  • Hamrah P, Chen L, Zhang Q, Dana MR (2003) Novel expression of vascular endothelial growth factor receptor (VEGFR)-3 and VEGF-C on corneal dendritic cells. Am J Pathol 163:57–68

    PubMed  CAS  Google Scholar 

  • Irjala H, Alanen K, Grenman R, Heikkila P, Joensuu H, Jalkanen S (2003) Mannose receptor (MR) and common lymphatic endothelial and vascular endothelial receptor (CLEVER)-1 direct the binding of cancer cells to the lymph vessel endothelium. Cancer Res 63:4671–4676

    PubMed  CAS  Google Scholar 

  • Jackson DG (2003) The lymphatics revisited: new perspectives from the hyaluronan receptor LYVE-1. Trends Cardiovasc Med 13:1–7

    Article  PubMed  CAS  Google Scholar 

  • Jackson DG, Prevo R, Clasper S, Banerji S (2001) LYVE-1, the lymphatic system and tumor lymphangiogenesis. Trends Immunol 22:317–321

    Article  PubMed  CAS  Google Scholar 

  • Kaipainen A, Korhonen J, Mustonen T, Hinsbergh VW van, Fang G H, Dumont D, Breitman M, Alitalo K (1995) Expression of the fms-like tyrosine kinase 4 gene becomes restricted to lymphatic endothelium during development. Proc Natl Acad Sci USA 92:3566–3570

    Article  PubMed  CAS  Google Scholar 

  • Kriehuber E, Breiteneder-Geleff S, Groeger M, Soleiman A, Schoppmann S F, Stingl G, Kerjaschki D, Maurer D (2001) Isolation and characterization of dermal lymphatic and blood endothelial cells reveal stable and functionally specialized cell lineages. J Exp Med 194:797–808

    Article  PubMed  CAS  Google Scholar 

  • Makinen T, Veikkola T, Mustjoki S, Karpanen T, Catimel B, Nice E C, Wise L, Mercer A, Kowalski H, Kerjaschki D, Stacker SA, Achen M G, Alitalo K (2001) Isolated lymphatic endothelial cells transduce growth, survival and migratory signals via the VEGF-C/D receptor VEGFR-3. EMBO J 20:4762–4773

    Article  PubMed  CAS  Google Scholar 

  • Mancardi S, Stanta G, Dusetti N, Bestagno M, Jussila L, Zweyer M, Lunazzi G, Dumont D, Alitalo K, Burrone OR (1999) Lymphatic endothelial tumors induced by intraperitoneal injection of incomplete Freund’s adjuvant. Exp Cell Res 246:368–375

    Article  PubMed  CAS  Google Scholar 

  • Mancardi S, Vecile E, Dusetti N, Calvo E, Stanta G, Burrone O R, Dobrina A (2003) Evidence of CXC, CC and C chemokine production by lymphatic endothelial cells. Immunology 108:523–530

    Article  PubMed  CAS  Google Scholar 

  • Mantovani A, Bussolino F, Dejana E (1992) Cytokine regulation of endothelial cell function. FASEB J 6:2591–2599

    PubMed  CAS  Google Scholar 

  • Mantovani A, Locati M, Vecchi A, Sozzani S, Allavena P (2001) Decoy receptors: a strategy to regulate inflammatory cytokines and chemokines. Trends Immunol 22:328–336

    Article  PubMed  CAS  Google Scholar 

  • Martin-Padura I, Lostaglio S, Schneemann M, Williams L, Romano M, Fruscella P, Panzeri C, Stoppacciaro A, Ruco L, Villa A, Simmons D, Dejana E (1998) Junctional adhesion molecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctions and modulates monocyte transmigration. J Cell Biol 142:117–127

    Article  PubMed  CAS  Google Scholar 

  • Netten JP van, Cann SA, Maxwell CA,Finegan RP (1998) Lymphagenesis and cancer metastasis. Br J Cancer 78:277–278

    Article  PubMed  Google Scholar 

  • Nibbs RJ, Kriehuber E, Ponath PD, Parent D, Qin S, Campbell JD, Henderson A, Kerjaschki D, Maurer D, Graham G J, Rot A (2001) The beta-chemokine receptor D6 is expressed by lymphatic endothelium and a subset of vascular tumors. Am J Pathol 158:867–877

    PubMed  CAS  Google Scholar 

  • Partanen TA, Arola J, Saaristo A, Jussila L, Ora A, Miettinen M, Stacker SA, Achen MG, Alitalo K (2000) VEGF-C and VEGF-D expression in neuroendocrine cells and their receptor, VEGFR-3, in fenestrated blood vessels in human tissues. FASEB J 14:2087–2096

    Article  PubMed  CAS  Google Scholar 

  • Petrova TV, Makinen T, Makela TP, Saarela J, Virtanen I, Ferrell RE, Finegold DN, Kerjaschki D, Yla-Herttuala S, Alitalo K (2002) Lymphatic endothelial reprogramming of vascular endothelial cells by the Prox-1 homeobox transcription factor. EMBO J 21:4593–4599

    Article  PubMed  CAS  Google Scholar 

  • Podgrabinska S, Braun P, Velasco P, Kloos B, Pepper MS, Skobe M (2002) Molecular characterization of lymphatic endothelial cells. Proc Natl Acad Sci USA 99:16069–16074

    Article  PubMed  CAS  Google Scholar 

  • Rodriguez-Niedenfuhr M, Papoutsi M, Christ B, Nicolaides KH, von Kaisenberg CS, Tomarev SI, Wilting J (2001) Prox1 is a marker of ectodermal placodes, endodermal compartments, lymphatic endothelium and lymphangioblasts. Anat Embryol (Berl) 204:399–406

    Article  CAS  Google Scholar 

  • Saeki H, Moore A M, Brown M J, Hwang ST (1999) Cutting edge: secondary lymphoid-tissue chemokine (SLC) and CC chemokine receptor 7 (CCR7) participate in the emigration pathway of mature dendritic cells from the skin to regional lymph nodes. J Immunol 162:2472–2475

    PubMed  CAS  Google Scholar 

  • Schmelz M, Way DL, Borgs P, Peitsch WK, Schmidt H, Witte MH, Witte CL, Franke WW, Moll R (1998) A novel type of adhering junction in an epithelioid tumorigenic rat cell culture line. Cell Tissue Res 294:11–25

    Article  PubMed  CAS  Google Scholar 

  • Sinzelle E, Van Huyen JP, Breiteneder-Geleff S, Braunberger E, Deloche A, Kerjaschki D, Bruneval P (2000) Intrapericardial lymphangioma with podoplanin immunohistochemical characterization of lymphatic endothelial cells. Histopathology 37:93–94

    Article  PubMed  CAS  Google Scholar 

  • Sironi M, Sciacca F L, Matteucci C, Conni M, Vecchi A, Bernasconi S, Minty A, Caput D, Ferrara P, Colotta F, et al (1994) Regulation of endothelial and mesothelial cell function by interleukin-13: selective induction of vascular cell adhesion molecule-1 and amplification of interleukin-6 production. Blood 84:1913–1921

    PubMed  CAS  Google Scholar 

  • Vecchi A, Garlanda C, Lampugnani M G, Resnati M, Matteucci C, Stoppacciaro A, Schnurch H, Risau W, Ruco L, Mantovani A, et al (1994) Monoclonal antibodies specific for endothelial cells of mouse blood vessels. Their application in the identification of adult and embryonic endothelium. Eur J Cell Biol 63:247–254

    PubMed  CAS  Google Scholar 

  • Wald D, Qin J, Zhao Z, Qian Y, Naramura M, Tian L, Towne J, Sims JE, Stark GR, Li X (2003) SIGIRR, a negative regulator of Toll-like receptor-interleukin 1 receptor signaling. Nat Immunol 4:920–927

    Article  PubMed  CAS  Google Scholar 

  • Wang HW, Trotter M W, Lagos D, Bourboulia D, Henderson S, Makinen T, Elliman S, Flanagan A M, Alitalo K, Boshoff C (2004) Kaposi sarcoma herpesvirus-induced cellular reprogramming contributes to the lymphatic endothelial gene expression in Kaposi sarcoma. Nat Genet 36:687–693

    Article  PubMed  CAS  Google Scholar 

  • Wigle JT, Oliver G (1999) Prox1 function is required for the development of the murine lymphatic system. Cell 98:769–778

    Article  PubMed  CAS  Google Scholar 

  • Williams RL, Courtneidge SA, Wagner EF (1988) Embryonic lethalities and endothelial tumors in chimeric mice expressing polyoma virus middle T oncogene. Cell 52:121–131

    Article  PubMed  CAS  Google Scholar 

  • Witmer AN, Blijswijk BC van, Dai J, Hofman P, Partanen TA, Vrensen GF, Schlingemann RO (2001) VEGFR-3 in adult angiogenesis. J Pathol 195:490–497

    Article  PubMed  CAS  Google Scholar 

  • Witte MH, Bernas MJ, Martin CP, Witte CL (2001) Lymphangiogenesis and lymphangiodysplasia: from molecular to clinical lymphology. Microsc Res Tech 55:122–145

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. Marina Sironi, Alberto Mantovani & Annunciata Vecchi

    Present address: Research Laboratory in Immunology and Inflammation, Istituto Clinico Humanitas, Via Manzoni 56, 20089, Rozzano, MI, Italy

Authors and Affiliations

  1. Department of Immunology and Cell Biology, Mario Negri Institute for Pharmacological Research, Milan, Italy

    Marina Sironi, Sergio Bernasconi, Maida De Bortoli, Alberto Mantovani, Elisabetta Dejana & Annunciata Vecchi

  2. Italian Foundation for Cancer Research (FIRC), Institute of Molecular Oncology, Milan, Italy

    Annarita Conti & Elisabetta Dejana

  3. Department of Biotechnology, University of Brescia, Brescia, Italy

    Anna M. Fra

  4. Institute of General Pathology, Faculty of Medicine, University of Milan, Milan, Italy

    Fabio Pasqualini & Alberto Mantovani

  5. Pathology Unit, L. Sacco Institute of Medical Science, L. Sacco Hospital, University of Milan, Milan, Italy

    Manuela Nebuloni & Eleonora Lauri

  6. Department of Biomolecular and Biothecnological Sciences, Faculty of Sciences, University of Milan, Milan, Italy

    Elisabetta Dejana

Authors
  1. Marina Sironi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Annarita Conti
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sergio Bernasconi
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Anna M. Fra
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Fabio Pasqualini
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Manuela Nebuloni
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Eleonora Lauri
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Maida De Bortoli
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Alberto Mantovani
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Elisabetta Dejana
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Annunciata Vecchi
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Annunciata Vecchi.

Additional information

This work was supported by the Associazione Italiana per la Ricerca sul Cancro; the Italian Ministry of University and Scientific and Technological Research, Piano Nazionale Oncologia, tema 11; the AIDS Special Program of the Istituto Superiore di Sanità, Rome, contract 30D.83; the European Community (QLRT-2001-02059; Integrated Project Contract no. LSHG-CT-2004-503573; NoE MAIN 502935; and NoE EVGN 503254).

Rights and permissions

Reprints and permissions

About this article

Cite this article

Sironi, M., Conti, A., Bernasconi, S. et al. Generation and characterization of a mouse lymphatic endothelial cell line. Cell Tissue Res 325, 91–100 (2006). https://doi.org/10.1007/s00441-006-0171-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00441-006-0171-y

Keywords

Search

Navigation