, Volume 17, Issue 2, pp 395–406 | Cite as

The effects of inflammatory cytokines on lymphatic endothelial barrier function

  • Walter E. Cromer
  • Scott D. Zawieja
  • Binu Tharakan
  • Ed W. Childs
  • M. Karen Newell
  • David C. Zawieja
Original Paper


Proper lymphatic function is necessary for the transport of fluids, macromolecules, antigens and immune cells out of the interstitium. The lymphatic endothelium plays important roles in the modulation of lymphatic contractile activity and lymph transport, but it’s role as a barrier between the lymph and interstitial compartments is less well understood. Alterations in lymphatic function have long been associated with edema and inflammation although the integrity of the lymphatic endothelial barrier during inflammation is not well-defined. In this paper we evaluated the integrity of the lymphatic barrier in response to inflammatory stimuli commonly associated with increased blood endothelial permeability. We utilized in vitro assays of lymphatic endothelial cell (LEC) monolayer barrier function after treatment with different inflammatory cytokines and signaling molecules including TNF-α, IL-6, IL-1β, IFN-γ and LPS. Moderate increases in an index of monolayer barrier dysfunction were noted with all treatments (20–60 % increase) except IFN-γ which caused a greater than 2.5-fold increase. Cytokine-induced barrier dysfunction was blocked or reduced by the addition of LNAME, except for IL-1β and LPS treatments, suggesting a regulatory role for nitric oxide. The decreased LEC barrier was associated with modulation of both intercellular adhesion and intracellular cytoskeletal activation. Cytokine treatments reduced the expression of VE-cadherin and increased scavenging of β-catenin in the LECs and this was partially reversed by LNAME. Likewise the phosphorylation of myosin light chain 20 at the regulatory serine 19 site, which accompanied the elevated monolayer barrier dysfunction in response to cytokine treatment, was also blunted by LNAME application. This suggests that the lymphatic barrier is regulated during inflammation and that certain inflammatory signals may induce large increases in permeability.


Lymphatic Endothelial Permeability Cytokines Barrier 

Supplementary material

10456_2013_9393_MOESM1_ESM.tif (23 mb)
Supplementary Fig 1 Dose response curves of cytokines tested on RLEC monolayer permeability. Each graph is a single experiment with n = 3. These data along with the literature was used to determine which doses to use for further experiments. (TIFF 23578 kb)
10456_2013_9393_MOESM2_ESM.tif (12.3 mb)
Supplementary Fig 2 Western blots representative data for β-catenin, VE-cadherin and pMLC20/MLC20 ratios from TNF-α, IL-6, IL-1β, LPS and IFN-γ treated RLECs at 1 hour. (TIFF 12587 kb)


  1. 1.
    Casley-Smith JR (1968) How the lymphatic system works. Lymphology 1(3):77–80PubMedGoogle Scholar
  2. 2.
    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):920–931. doi:10.1161/CIRCRESAHA.109.207274 PubMedCrossRefGoogle Scholar
  3. 3.
    Angeli V, Ginhoux F, Llodra J, Quemeneur L, Frenette PS, Skobe M, Jessberger R, Merad M, Randolph GJ (2006) B cell-driven lymphangiogenesis in inflamed lymph nodes enhances dendritic cell mobilization. Immunity 24(2):203–215. doi:10.1016/j.immuni.2006.01.003 PubMedCrossRefGoogle Scholar
  4. 4.
    Angeli V, Randolph GJ (2006) Inflammation, lymphatic function, and dendritic cell migration. Lymphat Res Biol 4(4):217–228. doi:10.1089/lrb 2006.4406PubMedCrossRefGoogle Scholar
  5. 5.
    Jakubzick C, Bogunovic M, Bonito AJ, Kuan EL, Merad M, Randolph GJ (2008) Lymph-migrating, tissue-derived dendritic cells are minor constituents within steady-state lymph nodes. J Exp Med 205(12):2839–2850. doi:10.1084/jem.20081430 PubMedCentralPubMedCrossRefGoogle Scholar
  6. 6.
    Maruyama K, Ii M, Cursiefen C, Jackson DG, Keino H, Tomita M, Van Rooijen N, Takenaka H, D’Amore PA, Stein-Streilein J, Losordo DW, Streilein JW (2005) Inflammation-induced lymphangiogenesis in the cornea arises from CD11b-positive macrophages. J Clin Invest 115(9):2363–2372PubMedCentralPubMedCrossRefGoogle Scholar
  7. 7.
    Muller WA, Randolph GJ (1999) Migration of leukocytes across endothelium and beyond: molecules involved in the transmigration and fate of monocytes. J Leukoc Biol 66(5):698–704PubMedGoogle Scholar
  8. 8.
    Randolph GJ, Angeli V, Swartz MA (2005) Dendritic-cell trafficking to lymph nodes through lymphatic vessels. Nat Rev Immunol 5(8):617–628. doi:10.1038/nri1670 PubMedCrossRefGoogle Scholar
  9. 9.
    Skobe M, Detmar M (2000) Structure, function, and molecular control of the skin lymphatic system. J Investig Dermatol Symp Proc 5(1):14–19. doi:10.1046/j.1087-0024.2000.00001.x PubMedCrossRefGoogle Scholar
  10. 10.
    Zawieja SD, Wang W, Wu X, Nepiyushchikh ZV, Zawieja DC, Muthuchamy M (2012) Impairments in the intrinsic contractility of mesenteric collecting lymphatics in a rat model of metabolic syndrome. Am J Physiol Heart Circ Physiol 302(3):H643–H653. doi:10.1152/ajpheart.00606.2011 PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Sessa WC (2009) Molecular control of blood flow and angiogenesis: role of nitric oxide. J Thromb Haemost 7(Suppl 1):35–37PubMedCrossRefGoogle Scholar
  12. 12.
    Spyridopoulos I, Luedemann C, Chen D, Kearney M, Chen D, Murohara T, Principe N, Isner JM, Losordo DW (2002) Divergence of angiogenic and vascular permeability signaling by VEGF: inhibition of protein kinase C suppresses VEGF-induced angiogenesis, but promotes VEGF-induced, NO-dependent vascular permeability. Arterioscler Thromb Vasc Biol 22(6):901–906PubMedCrossRefGoogle Scholar
  13. 13.
    Moncada S, Higgs EA (1991) Endogenous nitric oxide: physiology, pathology and clinical relevance. Eur J Clin Invest 21(4):361–374PubMedCrossRefGoogle Scholar
  14. 14.
    Davenpeck KL, Gauthier TW, Lefer AM (1994) Inhibition of endothelial-derived nitric oxide promotes P-selectin expression and actions in the rat microcirculation. Gastroenterology 107(4):1050–1058PubMedGoogle Scholar
  15. 15.
    Krieglstein CF, Anthoni C, Cerwinka WH, Stokes KY, Russell J, Grisham MB, Granger DN (2007) Role of blood- and tissue-associated inducible nitric-oxide synthase in colonic inflammation. Am J Pathol 170(2):490–496PubMedCentralPubMedCrossRefGoogle Scholar
  16. 16.
    Gasheva OY, Zawieja DC, Gashev AA (2006) Contraction-initiated NO-dependent lymphatic relaxation: a self-regulatory mechanism in rat thoracic duct. J Physiol 575(Pt 3):821–832. doi:10.1113/jphysiol.2006.115212 PubMedCentralPubMedCrossRefGoogle Scholar
  17. 17.
    Schmid-Schonbein GW (2012) Nitric oxide (NO) side of lymphatic flow and immune surveillance. Proc Natl Acad Sci U S A 109(1):3–4. doi:10.1073/pnas.1117710109 PubMedCentralPubMedCrossRefGoogle Scholar
  18. 18.
    Wu TF, Carati CJ, Macnaughton WK, von der Weid PY (2006) Contractile activity of lymphatic vessels is altered in the TNBS model of guinea pig ileitis. Am J Physiol Gastrointest Liver Physiol 291(4):G566–G574. doi:10.1152/ajpgi.0 0058.2006PubMedCrossRefGoogle Scholar
  19. 19.
    von der Weid PY, Muthuchamy M (2010) Regulatory mechanisms in lymphatic vessel contraction under normal and inflammatory conditions. Pathophysiology 17(4):263–276. doi: 10.1016 Google Scholar
  20. 20.
    Hayes H, Kossmann E, Wilson E, Meininger C, Zawieja D (2003) Development and characterization of endothelial cells from rat microlymphatics. Lymphat Res Biol 1(2):101–119PubMedCrossRefGoogle Scholar
  21. 21.
    Chakravortty D, Koide N, Kato Y, Sugiyama T, Kawai M, Fukada M, Yoshida T, Yokochi T (2000) Cytoskeletal alterations in lipopolysaccharide-induced bovine vascular endothelial cell injury and its prevention by sodium arsenite. Clin Diagn Lab Immunol 7(2):218–225PubMedCentralPubMedGoogle Scholar
  22. 22.
    Dudek SM, Munoz NM, Desai A, Osan CM, Meliton AY, Leff AR (2011) Group V phospholipase A2 mediates barrier disruption of human pulmonary endothelial cells caused by LPS in vitro. Am J Respir Cell Mol Biol 44(3):361–368. doi:10.1165/rcmb.2009-0446OC PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Chaitanya GV, Franks SE, Cromer W, Wells SR, Bienkowska M, Jennings MH, Ruddell A, Ando T, Wang Y, Gu Y, Sapp M, Mathis JM, Jordan PA, Minagar A, Alexander JS (2010) Differential cytokine responses in human and mouse lymphatic endothelial cells to cytokines in vitro. Lymphat Res Biol 8(3):155–164. doi:10.1089/lrb2010.0004 PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Puhlmann M, Weinreich DM, Farma JM, Carroll NM, Turner EM, Alexander HR Jr (2005) Interleukin-1beta induced vascular permeability is dependent on induction of endothelial tissue factor (TF) activity. J Transl Med 3:37. doi:10.1186/1479-5876-3-37 PubMedCentralPubMedCrossRefGoogle Scholar
  25. 25.
    Bove K, Neumann P, Gertzberg N, Johnson A (2001) Role of ecNOS-derived NO in mediating TNF-induced endothelial barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 280(5):L914–L922PubMedGoogle Scholar
  26. 26.
    Breslin JW, Yuan SY, Wu MH (2007) VEGF-C alters barrier function of cultured lymphatic endothelial cells through a VEGFR-3-dependent mechanism. Lymphat Res Biol 5(2):105–113. doi:10.1089/lrb2007.1004 PubMedCentralPubMedCrossRefGoogle Scholar
  27. 27.
    Baluk P, Fuxe J, Hashizume H, Romano T, Lashnits E, Butz S, Vestweber D, Corada M, Molendini C, Dejana E, McDonald DM (2007) Functionally specialized junctions between endothelial cells of lymphatic vessels. J Exp Med 204(10):2349–2362. doi:10.1084/jem.20062596 PubMedCentralPubMedCrossRefGoogle Scholar
  28. 28.
    Leak LV, Burke JF (1968) Electron microscopic study of lymphatic capillaries in the removal of connective tissue fluids and particulate substances. Lymphology 1(2):39–52PubMedGoogle Scholar
  29. 29.
    Chaitanya GV, Cromer W, Wells S, Jennings M, Mathis JM, Minagar A, Alexander JS (2012) Metabolic modulation of cytokine-induced brain endothelial adhesion molecule expression. Microcirculation 19(2):155–165. doi:10.1111/j.1549-8719.2011.00141.x PubMedCrossRefGoogle Scholar
  30. 30.
    Barbieri SS, Weksler BB (2007) Tobacco smoke cooperates with interleukin-1beta to alter beta-catenin trafficking in vascular endothelium resulting in increased permeability and induction of cyclooxygenase-2 expression in vitro and in vivo. Faseb J 21(8):1831–1843. doi:10.1096/fj.06-7557com PubMedCrossRefGoogle Scholar
  31. 31.
    Sola-Villa D, Camacho M, Sola R, Soler M, Diaz JM, Vila L (2006) IL-1beta induces VEGF, independently of PGE2 induction, mainly through the PI3-K/mTOR pathway in renal mesangial cells. Kidney Int 70(11):1935–1941PubMedGoogle Scholar
  32. 32.
    Alexander JS, Chaitanya GV, Grisham MB, Boktor M (2010) Emerging roles of lymphatics in inflammatory bowel disease. Ann N Y Acad Sci 1207(Suppl 1):E75–E85. doi:10.1111/j.1749-6632.2010.05757.x PubMedCrossRefGoogle Scholar
  33. 33.
    Breslin JW, Gaudreault N, Watson KD, Reynoso R, Yuan SY, Wu MH (2007) Vascular endothelial growth factor-C stimulates the lymphatic pump by a VEGF receptor-3-dependent mechanism. Am J Physiol Heart Circ Physiol 293(1):H709–H718. doi:10.1152/ajpheart.00102.2007 PubMedCrossRefGoogle Scholar
  34. 34.
    Chatterjee V, Gashev AA (2012) Aging-associated shifts in functional status of mast cells located by adult and aged mesenteric lymphatic vessels. Am J Physiol Heart Circ Physiol 303(6):H693–H702. doi:10.1152/ajpheart.00378.2012 PubMedCentralPubMedCrossRefGoogle Scholar
  35. 35.
    Breslin JW (2011) ROCK and cAMP promote lymphatic endothelial cell barrier integrity and modulate histamine and thrombin-induced barrier dysfunction. Lymphat Res Biol 9(1):3–11. doi:10.1089/lrb2010.0016 PubMedCentralPubMedCrossRefGoogle Scholar
  36. 36.
    Nooteboom A, Van Der Linden CJ, Hendriks T (2002) Tumor necrosis factor-alpha and interleukin-1beta mediate endothelial permeability induced by lipopolysaccharide-stimulated whole blood. Crit Care Med 30(9):2063–2068. doi:10.1097/01.CCM.0000021522.67956.E6 PubMedCrossRefGoogle Scholar
  37. 37.
    Hunziker T, Brand CU, Kapp A, Waelti ER, Braathen LR (1992) Increased levels of inflammatory cytokines in human skin lymph derived from sodium lauryl sulphate-induced contact dermatitis. Br J Dermatol 127(3):254–257PubMedCrossRefGoogle Scholar
  38. 38.
    Olszewski WL, Pazdur J, Kubasiewicz E, Zaleska M, Cooke CJ, Miller NE (2001) Lymph draining from foot joints in rheumatoid arthritis provides insight into local cytokine and chemokine production and transport to lymph nodes. Arthritis Rheum 44(3):541–549. doi:10.1002/1529-0131(200103)44:3<541:AID-ANR102>3.0.CO;2-6 PubMedCrossRefGoogle Scholar
  39. 39.
    Glass CA, Harper SJ, Bates DO (2006) The anti-angiogenic VEGF isoform VEGF165b transiently increases hydraulic conductivity, probably through VEGF receptor 1 in vivo. J Physiol 572(Pt 1):243–257PubMedCentralPubMedGoogle Scholar
  40. 40.
    Cromer W, Jennings MH, Odaka Y, Mathis JM, Alexander JS (2010) Murine rVEGF164b, an inhibitory VEGF reduces VEGF-A-dependent endothelial proliferation and barrier dysfunction. Microcirculation 17(7):536–547 Google Scholar
  41. 41.
    Wu F, Han M, Wilson JX (2009) Tripterine prevents endothelial barrier dysfunction by inhibiting endogenous peroxynitrite formation. Br J Pharmacol 157(6):1014–1023. doi:10.1111/j.1476-5381.2009.00292.x PubMedCentralPubMedCrossRefGoogle Scholar
  42. 42.
    Sawa Y, Ueki T, Hata M, Iwasawa K, Tsuruga E, Kojima H, Ishikawa H, Yoshida S (2008) LPS-induced IL-6, IL-8, VCAM-1, and ICAM-1 expression in human lymphatic endothelium. J Histochem Cytochem 56(2):97–109. doi:10.1369/jhc.7A 7299.2007PubMedCentralPubMedCrossRefGoogle Scholar
  43. 43.
    Ghoshal S, Witta J, Zhong J, de Villiers W, Eckhardt E (2009) Chylomicrons promote intestinal absorption of lipopolysaccharides. J Lipid Res 50(1):90–97. doi:10.1194/jlr.M800156-JLR200 PubMedCrossRefGoogle Scholar
  44. 44.
    Tzima E, Irani-Tehrani M, Kiosses WB, Dejana E, Schultz DA, Engelhardt B, Cao G, DeLisser H, Schwartz MA (2005) A mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Nature 437(7057):426–431. doi:10.1038/nature03952 PubMedCrossRefGoogle Scholar
  45. 45.
    Tharakan B, Hellman J, Sawant DA, Tinsley JH, Parrish AR, Hunter FA, Smythe WR, Childs EW (2011) beta-Catenin Dynamics in the Regulation of Microvascular Endothelial Cell Hyperpermeability. Shock. doi:10.1097/SHK.0b013e318240b564 Google Scholar
  46. 46.
    Ding H, Keller KC, Martinez IK, Geransar RM, zur Nieden KO, Nishikawa SG, Rancourt DE, zur Nieden NI (2012) NO-beta-catenin crosstalk modulates primitive streak formation prior to embryonic stem cell osteogenic differentiation. J Cell Sci 125(Pt 22):5564–5577. doi:10.1242/jcs.081703 PubMedCrossRefGoogle Scholar
  47. 47.
    Kang DE, Soriano S, Frosch MP, Collins T, Naruse S, Sisodia SS, Leibowitz G, Levine F, Koo EH (1999) Presenilin 1 facilitates the constitutive turnover of beta-catenin: differential activity of Alzheimer’s disease-linked PS1 mutants in the beta-catenin-signaling pathway. J Neurosci 19(11):4229–4237PubMedGoogle Scholar
  48. 48.
    Soriano S, Kang DE, Fu M, Pestell R, Chevallier N, Zheng H, Koo EH (2001) Presenilin 1 negatively regulates beta-catenin/T cell factor/lymphoid enhancer factor-1 signaling independently of beta-amyloid precursor protein and notch processing. J Cell Biol 152(4):785–794PubMedCentralPubMedCrossRefGoogle Scholar
  49. 49.
    Strawitz JG, Eto K, Mitsuoka H, Olney C, Pairent FW, Howard JM (1968) Molecular weight dependence of lymphatic permeability: the concept of regional cancer chemotheraphy by lymphatic perfusion. Microvasc Res 1(1):58–67CrossRefGoogle Scholar
  50. 50.
    Rigor RR, Shen Q, Pivetti CD, Wu MH, Yuan SY (2012) Myosin Light Chain Kinase Signaling in Endothelial Barrier Dysfunction. Med Res Rev. doi:10.1002/med.21270 PubMedCentralPubMedGoogle Scholar
  51. 51.
    Yoshikawa H, Takada K, Muranishi S (1984) Molecular weight dependence of permselectivity to rat small intestinal blood-lymph barrier for exogenous macromolecules absorbed from lumen. J Pharmacobiodyn 7(1):1–6PubMedCrossRefGoogle Scholar
  52. 52.
    Scallan JP, Huxley VH (2010) In vivo determination of collecting lymphatic vessel permeability to albumin: a role for lymphatics in exchange. J Physiol 588(Pt 1):243–254. doi:10.1113/jphysiol.2009.179622 PubMedCentralPubMedCrossRefGoogle Scholar
  53. 53.
    Dunworth WP, Fritz-Six KL, Caron KM (2008) Adrenomedullin stabilizes the lymphatic endothelial barrier in vitro and in vivo. Peptides 29(12):2243–2249. doi:10.1016/j.peptides.2008.09.009 PubMedCentralPubMedCrossRefGoogle Scholar
  54. 54.
    Price GM, Chrobak KM, Tien J (2008) Effect of cyclic AMP on barrier function of human lymphatic microvascular tubes. Microvasc Res 76(1):46–51. doi:10.1016/j.mvr.2008.02.003 PubMedCentralPubMedCrossRefGoogle Scholar
  55. 55.
    Hou WH, Liu IH, Tsai CC, Johnson FE, Huang SS, Huang JS (2011) CRSBP-1/LYVE-1 ligands disrupt lymphatic intercellular adhesion by inducing tyrosine phosphorylation and internalization of VE-cadherin. J Cell Sci 124(Pt 8):1231–1244. doi:10.1242/jcs.078154 PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Walter E. Cromer
    • 1
  • Scott D. Zawieja
    • 1
  • Binu Tharakan
    • 2
  • Ed W. Childs
    • 3
  • M. Karen Newell
    • 2
  • David C. Zawieja
    • 1
  1. 1.Department of Systems Biology and Translational MedicineTexas A&M HSCCollege StationUSA
  2. 2.Department of Surgery, Scott and WhiteTexas A&M HSCTempleUSA
  3. 3.Department of SurgeryMorehouse School of MedicineAtlantaGeorgia

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