IL-17 promotes lymphangiogenesis by inducing VEGF-D secretion, and proliferation of and tube formation by LECs. (A) Pellets containing 100 ng of IL-1β, IL-17, or IL-17 along with systemic blockade of IL-1β or VEGFR3, were placed in corneal micropockets (n = 6 mice/group) to induce angiogenesis.¹⁷  After 7 days, corneas were evaluated biomicroscopically and then harvested for immunostaining with CD31 (green) and Lyve1 (red). Digital micrographs using epifluorescence microscopy were captured, and ImageJ 1.34s software was used to quantify the growth of (B) blood (CD31^hiLyve1⁻) and (C) lymphatic (CD31^loLyve1⁺) vessels. (D) Primary human corneal epithelial cells were cultured with 10 ng/mL concentration of IL-1β, IL-17, and IL-17 with IL-1β–blocking antibodies for 24 hours, and expression levels of VEGF-A, VEGF-C, and VEGF-D mRNA in cells, and protein in culture supernatants, were measured by real-time PCR and ELISA, respectively. (E) Primary human LECs were cultured with 10 ng/mL concentrations of IL-1β, IL-17, and IL-17 with blockade of IL-1β or VEGFR-3 for 24 hours, and then proliferation was measured using BrdU incorporation assay. (F) In a transwell Matrigel assay, in vitro polarized 2 × 10⁵ Th17 cells (in transwell) were cocultured with 5 × 10⁴ LECs (on Matrigel) in basal MEM with reduced serum (2% FBS). Positive controls consisted of LEC cultures on Matrigel in MEM supplemented with growth factors (5% FBS, VEGF, FGF, EGF). Negative controls consisted of LEC cultures on Matrigel in basal MEM only with reduced serum (2% FBS). In some wells of Th17-LEC coculture, soluble-IL-17receptor-Fc or soluble-VEGFR3-Fc was added in media. After 8- and 24-hour incubation at 37°C, wells were visualized under bright-field inverted microscope to study the LEC tube formations on Matrigel, and digital micrographs were then captured for quantitative analysis of tube length. Data are mean ± SEM values of 3 independent experiments. *P < .05, as determined by Student t test. n.s. indicates not significant.