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The effects of inflammatory cytokines on lymphatic endothelial barrier function

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Abstract

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.

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Acknowledgments

Supported by NIH grants HL070308, DK099221, HL094269, CA140732 and the Scott & White Wigley award.

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Authors and Affiliations

  1. Department of Systems Biology and Translational Medicine, Texas A&M HSC, College Station, TX, USA

    Walter E. Cromer, Scott D. Zawieja & David C. Zawieja

  2. Department of Surgery, Scott and White, Texas A&M HSC, Temple, TX, USA

    Binu Tharakan & M. Karen Newell

  3. Department of Surgery, Morehouse School of Medicine, Atlanta, Georgia

    Ed W. Childs

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  1. Walter E. Cromer
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Correspondence to Walter E. Cromer.

Electronic supplementary material

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10456_2013_9393_MOESM1_ESM.tif

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

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)

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Cromer, W.E., Zawieja, S.D., Tharakan, B. et al. The effects of inflammatory cytokines on lymphatic endothelial barrier function. Angiogenesis 17, 395–406 (2014). https://doi.org/10.1007/s10456-013-9393-2

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