IL-18 Regulates Melanoma VLA-4 Integrin Activation through a Hierarchized Sequence of Inflammatory Factors  María Valcárcel, Teresa Carrascal, Olatz Crende, Fernando Vidal-Vanaclocha  Journal of Investigative Dermatology  Volume 134, Issue 2, Pages 470-480 (February 2014) DOI: 10.1038/jid.2013.342 Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Concentration course and time course-dependent proinflammatory factor mediated B16 melanoma cell adhesion to HSE in vitro. (a–f) B16 melanoma cells received increasing concentrations of rmIL-18 and rmTNFα for 6 hours, rmVEGF, and rhIL-1β for 4 hours; rmPGE2 and H2O2 for 2 hours. (g–l) B16 melanoma cells received 10 ng ml−1 rmIL-18 and rmTNFα, 100 ng ml−1 rmVEGF and rmPGE2, 1 ng ml−1 rhIL-1β and 10 μM H2O2. Three separate experiments were done, each in six replicates (n=18). *Statistically significant (P<0.01) as compared with untreated B16 cells. H2O2, hydrogen peroxide; HSE, hepatic sinusoidal endothelium; PGE2, prostaglandin E2; rh, recombinant human; rm, recombinant murine; TNFα, tumor necrosis factor-α; VEGF, vascular endothelial growth factor. Journal of Investigative Dermatology 2014 134, 470-480DOI: (10.1038/jid.2013.342) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Integrin activation accounts for inflammation induced endothelial adhesion. (a) Effect of anti-VLA-4 antibodies on proinflammatory factor-induced B16 melanoma cell adhesion to HSE. B16 cells received 10 ng ml−1 rmIL-18 and rmTNFα (6 hours); 100 ng ml−1 rmVEGF and 1 ng ml−1 rhIL-1β (4 hours); and 100 ng ml−1 rmPGE2 and 10 μM H2O2 (2 hours). B16 cells received 10 μg ml−1 anti-murine VLA-4 antibody or nonspecific IgG 30 minutes before the adhesion assay. Three separate experiments were done, each in six replicates (n=18). Statistically significant (P<0.01) as compared with untreated cells (*) and to inflammatory factor-treated cells (**). (b) Flow cytometry on VLA-4 expression by B16 melanoma cells. Cells were labeled with rat anti-mouse VLA-4 antibody (30 minutes; 4 °C; 10 μg/106 cells) followed by FITC-IgG anti-rat. (c) Proinflammatory factor-treated B16 melanoma cell adhesion assay to immobilize recombinant human VCAM-1. B16 cells received 1 ng ml−1 rmIL-18 (6 hours), 100 ng ml−1 rmVEGF (4 hours), 1 ng ml−1 IL-1β (4 hours), and 10 μM H2O2 (2 hours). Three separate experiments were done, each in eight replicates (n=18). (*) Statistically significant (P<0.01) as compared with untreated B16 cells. (d) Detection of active conformation VLA-4-expressing A375 melanoma cells by flow cytometry. Permeabilized A375 melanoma cells were labeled with mouse anti-human active conformation β1 integrin antibody (5 μg/106 cells; 30 minutes; 4 °C) followed by conjugated FITC-IgG anti-mouse. H2O2, hydrogen peroxide; HSE, hepatic sinusoidal endothelium; PGE2, prostaglandin E2; rh, recombinant human; rm, recombinant murine; TNFα, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor; VLA-4, very late antigen-4. Journal of Investigative Dermatology 2014 134, 470-480DOI: (10.1038/jid.2013.342) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Effect of sequential inflammatory factor blockade on single inflammatory factor (a–f) and H2O2-induced B16 cell adhesion (g). B16 cells received 1 μg ml−1 anti-TNFα antibody, 1 μg ml−1 anti-VEGF antibody, 100 ng ml−1 IL-1ra, 1 μg ml−1 celecoxib and 1,000 unit ml−1 catalase 30 minutes before inflammatory factors: 10 ng ml−1 rmIL-18, 6 hours (a), 10 ng ml−1 rmTNFα, 6 hours (b), 100 ng ml−1 rmPGE2, 2 hours (c), 100 ng ml−1 rmVEGF, 4 hours (d), 1 ng ml−1 rhIL-1β, 4 hours (e), basal medium (f), and 10 μM H2O2 (2 hours) (g). Three separate experiments were done, each in six replicates (n=18). Statistically significant (P<0.01) as compared with untreated B16 cells (*), and to inflammatory factor-treated B16 cells (**). H2O2, hydrogen peroxide; IL-1ra, IL-1 receptor antagonist; PGE2, prostaglandin E2; rh, recombinant human; rm, recombinant murine; TNFα, tumor necrosis factor-α; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor. Journal of Investigative Dermatology 2014 134, 470-480DOI: (10.1038/jid.2013.342) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 (a) Effect of anti-IL-18, anti-TNFα, anti-VEGF, IL-1ra, and celecoxib on H2O2 production from inflammatory factor-induced B16 melanoma. B16 cells received anti-inflammatory and inflammatory molecules as in Figure 3a–f. Next, they were incubated with DCFH-DA and H2O2 production was determined by plate scanner microfluorimetry. Three separate experiments were done, each in six replicates (n=18). Statistically significant (P<0.01) as compared with untreated cells (*), and to inflammatory factor-treated cells. (b) Model on melanoma VLA-4-activation through a hierarchized sequence of IL-18-induced inflammatory factors. DCFH-DA, 2′7′-dichlorofluorescein-diacetate; H2O2, hydrogen peroxide; IL-1ra, IL-1 receptor antagonist; TNFα, tumor necrosis factor-α; VEGF, vascular endothelial growth factor; VLA-4, very late antigen-4. Journal of Investigative Dermatology 2014 134, 470-480DOI: (10.1038/jid.2013.342) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 (a) RT-PCR and (b) flow cytometry on IL-18R, TNFR1, and VEGFR2 expression by IL-18-treated B16 melanoma. IL-18 (10 ng ml−1) was added for 1 hour (a) or 6 hours (b) before analyses. Mean values ±SD of three separate experiments were statistically significant (P<0.01) as compared with IL-18R (*), TNFR1 (**) and VEGFR2 (***) expression by untreated cells. (c) ELISA on TNFα and VEGF concentration in the supernatant of IL-18-treated B16 melanoma. Three separate experiments were done, each in sextuplicate. Statistically significant (P<0.01) as compared with TNFα (*) and VEGF secretion (**) by untreated cells. (d–h) Functional comparison of B16 melanoma cells with active and inactive VLA-4: flow cytometry on IL-18R, TNFR1, and VEGFR2 expression (d); quantitative melanoma cell adhesion assay to immobilized VCAM-1 (e); ELISA on VEGF concentration in the supernatant of cultured melanoma cells (f); quantitative assay for hepatic endothelial cell migration-stimulating activity measurement in melanoma cell supernatants (g); and hepatic metastasis growth of intrasplenically injected B16 cells in syngeneic mice (h). Data represent the mean ±SD of three separate experiments. Statistically significant (*) P<0.01 as compared with active VLA-4-expressing B16 cells. H2O2, hydrogen peroxide; RT–PCR, Reverse transcriptase–PCR; TNF, tumor necrosis factor; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor; VLA-4, very late antigen-4 Journal of Investigative Dermatology 2014 134, 470-480DOI: (10.1038/jid.2013.342) Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions