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Cited by 116Lymphatic System and DiseasesLymphatic Disorders and TreatmentsParasitic Diseases Research and Treatment

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Th2 Cytokines Inhibit Lymphangiogenesis

Open access
Savetsky, Ira L., Ghanta, Swapna, Gardenier, Jason C., Torrisi, Jeremy S., García Nores, Gabriela D., Hespe, Geoffrey E., Nitti, Matthew D., Kataru, Raghu P., Mehrara, Babak J.·2015·PLOS ONE
DOI 10.1371/journal.pone.0126908PMC PMC4454507
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Abstract

Lymphangiogenesis is the process by which new lymphatic vessels grow in response to pathologic stimuli such as wound healing, inflammation, and tumor metastasis. It is well-recognized that growth factors and cytokines regulate lymphangiogenesis by promoting or inhibiting lymphatic endothelial cell (LEC) proliferation, migration and differentiation. Our group has shown that the expression of T-helper 2 (Th2) cytokines is markedly increased in lymphedema, and that these cytokines inhibit lymphatic function by increasing fibrosis and promoting changes in the extracellular matrix. However, while the evidence supporting a role for T cells and Th2 cytokines as negative regulators of lymphatic function is clear, the direct effects of Th2 cytokines on isolated LECs remains poorly understood. Using in vitro and in vivo studies, we show that physiologic doses of interleukin-4 (IL-4) and interleukin-13 (IL-13) have profound anti-lymphangiogenic effects and potently impair LEC survival, proliferation, migration, and tubule formation. Inhibition of these cytokines with targeted monoclonal antibodies in the cornea suture model specifically increases inflammatory lymphangiogenesis without concomitant changes in angiogenesis. These findings suggest that manipulation of anti-lymphangiogenic pathways may represent a novel and potent means of improving lymphangiogenesis.

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Figures

Fig 1 hLECs express IL-4Rα and respond to rhIL-4/IL-13 with increased apoptosis. A. Representative immunofluorescent images of IL-4Rα and Prox-1 staining from cultured hLECs. Scale bar = 20μm. B. Flow cytometry analysis for Annexin V and propidium iodide of cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Representative contour plots. Quantification of apoptotic hLECs. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS).
Fig 1 hLECs express IL-4Rα and respond to rhIL-4/IL-13 with increased apoptosis. A. Representative immunofluorescent images of IL-4Rα and Prox-1 staining from cultured hLECs. Scale bar = 20μm. B. Flow cytometry analysis for Annexin V and propidium iodide of cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Representative contour plots. Quantification of apoptotic hLECs. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS).CC BY · PubMed Central
Fig 2 rhIL-4 and rhIL-13 decrease LEC proliferation and downregulate expression of Prox-1. A. Flow cytometry analysis for incorporated EdU by cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Representative histograms. Quantification of EdU positive hLECs. Control vs. rhIL-4 (n = 5 in each; *p<0.01); control vs. rhIL-13 (n = 5 in each; *p<0.01); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS). B. Quantification of hLEC proliferation using a WST-1 cell assay kit. Control vs. rhIL-4 (n = 10 in each; *p<0.01 for both concentrations); 50 ng/ml rhIL-4 vs. 100 ng/ml rhIL-4 (n = 10 in each; *p<0.05); control vs. rhIL-13 (n = 10 in each; *p<0.01 for both concentrations); 50 ng/ml rhIL-13 vs. 100 ng/ml rhIL-13 (n = 10 in each; p = NS). C. Representative immunofluorescent staining for Ki67 and VEGF-R3 in cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Scale bar = 100μm. Quantification of Ki67+ cells per high-power field. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS). D. Representative immunofluorescent staining for Prox-1 in cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Scale bar = 100μm. Quantification of Prox-1 signal intensity using MetaMorph analysis per high-power field. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS).
Fig 2 rhIL-4 and rhIL-13 decrease LEC proliferation and downregulate expression of Prox-1. A. Flow cytometry analysis for incorporated EdU by cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Representative histograms. Quantification of EdU positive hLECs. Control vs. rhIL-4 (n = 5 in each; *p<0.01); control vs. rhIL-13 (n = 5 in each; *p<0.01); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS). B. Quantification of hLEC proliferation using a WST-1 cell assay kit. Control vs. rhIL-4 (n = 10 in each; *p<0.01 for both concentrations); 50 ng/ml rhIL-4 vs. 100 ng/ml rhIL-4 (n = 10 in each; *p<0.05); control vs. rhIL-13 (n = 10 in each; *p<0.01 for both concentrations); 50 ng/ml rhIL-13 vs. 100 ng/ml rhIL-13 (n = 10 in each; p = NS). C. Representative immunofluorescent staining for Ki67 and VEGF-R3 in cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Scale bar = 100μm. Quantification of Ki67+ cells per high-power field. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS). D. Representative immunofluorescent staining for Prox-1 in cultured hLECs after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 24 hours. Scale bar = 100μm. Quantification of Prox-1 signal intensity using MetaMorph analysis per high-power field. Control vs. rhIL-4 (n = 5 in each; *p<0.001); control vs. rhIL-13 (n = 5 in each; *p<0.001); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS).CC BY · PubMed Central
Fig 3 rhIL-4 and rhIL-13 inhibit hLEC tubule formation. A. Representative images of cultured hLECs tubule formation on matrigel after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 12 hours. B. Quantification of tubule formation per high-power field. Control vs. rhIL-4 (n = 5–8 in each; *p<0.01); control vs. rhIL-13 (n = 5–8 in each; *p<0.01); rhIL-4 vs. rhIL-13 (n = 5–8 in each; p = NS).
Fig 3 rhIL-4 and rhIL-13 inhibit hLEC tubule formation. A. Representative images of cultured hLECs tubule formation on matrigel after treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) for 12 hours. B. Quantification of tubule formation per high-power field. Control vs. rhIL-4 (n = 5–8 in each; *p<0.01); control vs. rhIL-13 (n = 5–8 in each; *p<0.01); rhIL-4 vs. rhIL-13 (n = 5–8 in each; p = NS).CC BY · PubMed Central
Fig 4 rhIL-4 and rhIL-13 inhibit hLEC migration. A. Representative images of cultured hLECs after scratch wound and treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) at 0, 12 and 24 hours. White dotted lines represent LEC migration borders. Black solid lines represent initial gap created by scratch. B. Quantification of hLEC migration at 12 and 24 hours. Control vs. rhIL-4 (n = 5 in each; *p<0.01 for both time points); control vs. rhIL-13 (n = 5 in each; *p<0.01 for both time points); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS for both time points).
Fig 4 rhIL-4 and rhIL-13 inhibit hLEC migration. A. Representative images of cultured hLECs after scratch wound and treatment with 50 ng/ml rhIL-4 or rhIL-13 protein, or media alone (control) at 0, 12 and 24 hours. White dotted lines represent LEC migration borders. Black solid lines represent initial gap created by scratch. B. Quantification of hLEC migration at 12 and 24 hours. Control vs. rhIL-4 (n = 5 in each; *p<0.01 for both time points); control vs. rhIL-13 (n = 5 in each; *p<0.01 for both time points); rhIL-4 vs. rhIL-13 (n = 5 in each; p = NS for both time points).CC BY · PubMed Central
Fig 5 IL-4 or IL-13 blockade increases inflammatory lymphangiogenesis. A. Representative gross images of corneas 14 days after suture placement. B. Representative immunofluorescent cornea whole mount images for LYVE-1. Scale bar = 100μm. C. Quantification of lymphatic vessel area per 0.25mm2. Control vs. IL-4mAb (n = 8 in each; *p<0.001); control vs. IL-13mAb (n = 8 in each; *p<0.001); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS). D. Quantification of lymphatic vessel volume per 0.25mm2. Control vs. IL-4mAb (n = 8 in each; *p<0.001); control vs. IL-13mAb (n = 8 in each; *p<0.001); IL-4mAb vs. IL-13mAb (n = 8 in each; *p<0.05).
Fig 5 IL-4 or IL-13 blockade increases inflammatory lymphangiogenesis. A. Representative gross images of corneas 14 days after suture placement. B. Representative immunofluorescent cornea whole mount images for LYVE-1. Scale bar = 100μm. C. Quantification of lymphatic vessel area per 0.25mm2. Control vs. IL-4mAb (n = 8 in each; *p<0.001); control vs. IL-13mAb (n = 8 in each; *p<0.001); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS). D. Quantification of lymphatic vessel volume per 0.25mm2. Control vs. IL-4mAb (n = 8 in each; *p<0.001); control vs. IL-13mAb (n = 8 in each; *p<0.001); IL-4mAb vs. IL-13mAb (n = 8 in each; *p<0.05).CC BY · PubMed Central
Fig 6 IL-4 and IL-13 blockade increases LEC proliferation in vivo. A. Representative immunofluorescent cornea whole mount images stained for Ki67 and LYVE-1. Scale bar = 100μm. B. Quantification of Ki67+/LYVE-1+ cells (n/0.25mm2). Control vs. IL-4mAb (n = 5 in each; *p<0.01); control vs. IL-13mAb (n = 5 in each; *p<0.01); IL-4mAb vs. IL-13mAb (n = 5 in each; *p<0.01).
Fig 6 IL-4 and IL-13 blockade increases LEC proliferation in vivo. A. Representative immunofluorescent cornea whole mount images stained for Ki67 and LYVE-1. Scale bar = 100μm. B. Quantification of Ki67+/LYVE-1+ cells (n/0.25mm2). Control vs. IL-4mAb (n = 5 in each; *p<0.01); control vs. IL-13mAb (n = 5 in each; *p<0.01); IL-4mAb vs. IL-13mAb (n = 5 in each; *p<0.01).CC BY · PubMed Central
Fig 7 IL-4 and IL-13 blockade does not increase corneal angiogenesis. A. Representative immunofluorescent cornea whole mount images stained for CD31 and LYVE-1. Scale bar = 100μm. B. Quantification of blood vessel area per 0.25mm2. Control vs. IL-4mAb (n = 8 in each; p = NS); control vs. IL-13mAb (n = 8 in each; p = NS); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS). C. Quantification of blood vessel volume per 0.25mm2. Control vs. IL-4mAb (n = 8 in each; p = NS); control vs. IL-13mAb (n = 8 in each; p = NS); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS).
Fig 7 IL-4 and IL-13 blockade does not increase corneal angiogenesis. A. Representative immunofluorescent cornea whole mount images stained for CD31 and LYVE-1. Scale bar = 100μm. B. Quantification of blood vessel area per 0.25mm2. Control vs. IL-4mAb (n = 8 in each; p = NS); control vs. IL-13mAb (n = 8 in each; p = NS); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS). C. Quantification of blood vessel volume per 0.25mm2. Control vs. IL-4mAb (n = 8 in each; p = NS); control vs. IL-13mAb (n = 8 in each; p = NS); IL-4mAb vs. IL-13mAb (n = 8 in each; p = NS).CC BY · PubMed Central

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Metadata

Authors
Savetsky, I. L., Ghanta, S., Gardenier, J. C., Torrisi, J. S., García Nores, G. D., Hespe, G. E., Nitti, M. D., Kataru, R. P., & Mehrara, B. J.
Affiliations
Memorial Sloan Kettering Cancer Center (US)
Corresponding author
Mehrara, Babak J.
ORCID
Venue
PLOS ONE
Publisher
Public Library of Science (PLoS)
Published
2015-06-03
Volume
10
Issue
6
Pages
e0126908
ISSN
1932-6203 (ISSN-L)
DOI
10.1371/journal.pone.0126908
PMC
PMC4454507
OpenAlex
W560736474
Cited by
114
References
23
Citations by year
2026: 8 · 2025: 11 · 2024: 11 · 2023: 6 · 2022: 18 · 2021: 10 · 2020: 11 · 2019: 8 · 2018: 11 · 2017: 12 · 2016: 6 · 2015: 2
FWCI
4.51
Type
journal article
Language
en
Open access
Licence
Locations
  • PLoS ONE (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Memorial Sloan-Kettering Cancer Center (CA008748, P30 CA008748)
  • National Heart, Lung, and Blood Institute (R01HL111130)
  • National Cancer Institute (T32CA009685, P30CA008748, T32CA009501)
Topics
  • Health Sciences › Medicine › Oncology › Lymphatic System and Diseases
  • Health Sciences › Medicine › Surgery › Lymphatic Disorders and Treatments
  • Health Sciences › Medicine › Infectious Diseases › Parasitic Diseases Research and Treatment
Keywords
lymphangiogenesis, Th2 cytokines, IL-4, IL-13, lymphatic endothelial cells, lymphedema, anti-lymphangiogenic therapy, lymphatic fibrosis, extracellular matrix, monoclonal antibodies
MeSH terms
Animals, Cell Movement, Cell Survival, Male, Interleukin-4, Th2 Cells, Interleukin-13, Lymphangiogenesis, Endothelial Cells, Cell Proliferation, Mice

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