Endocytosis of the ManR ligand mannan increased after 3-hour LSEC/C26 coculture, but no increase in endocytosis of the stabilin-2 ligands CSPG or FSA22 was observed. Control experiments omitting the presence of C26 cells gave no increased endocytosis via the two receptors (Fig. 1A-C). No change in ManR-mediated endocytosis was detected in

LSECs that had been either cocultured with C26 cells in separate compartments or treated with C26 CM for 6 hours, suggesting a cell-to-cell contact-mediated mechanism in the activation of ManR-mediated endocytosis (Fig. 1D). C26 cells cultured alone did not take up any of investigated ligands, neither under basal conditions nor under LSEC/CM BMN 673 concentration treatment conditions. We previously reported that LSECs secrete IL-1 in response to tumor-derived factors.23 Herein, IL-1 also increased (P < 0.05) in the supernatant of C26/LSEC cocultures, but not in those obtained from LSECs coincubated with C26 cells in different compartments, or in the presence of C26/CM (Fig. 1E). C26 cell supernatant had nondetectable levels

of IL-1 in the same conditions as above. Addition of anti-murine IL-1RI antibodies to LSECs prior to their coculture with C26 cells for 6 hours abolished tumor-induced ManR-mediated PLX4032 purchase endocytosis (Fig. 1F). Inhibition of IL-1–converting enzyme (ICE) with an irreversible inhibitor given to LSECs prior to C26 cell addition also abrogated tumor-induced endocytosis, whereas the addition of IL-1 to ICE inhibitor-treated 上海皓元医药股份有限公司 LSECs restored endocytosis up-regulation. ICE mediates production of both IL-1 and IL-18.24 However, addition of IL-18–neutralizing antibodies to coincubated LSEC/C26 cells did not alter tumor-induced ManR-mediated endocytosis (Fig. 1F). Hepatic uptake of fluorescently labeled ManR ligand FITC-OVA also increased in mice bearing hepatic C26 tumors, compared with the uptake of FITC-OVA by C26 cell-free control

mice. FITC-OVA uptake increased by 50% on the 36th hour after C26 cell injection, when a majority of cancer cells had reached the liver. In vivo blockade of IL-1 with IL-1Ra (single intraperitoneal injection, 5 mg/kg, 2 hours prior to C26 cell injection) abrogated tumor-dependent increased hepatic FITC-OVA uptake augmentation. FITC-OVA uptake was not significantly affected by IL-1Ra–treated C26 cell-free mice. Calculated clearance constants (k = FITC-OVA flow rate/maximum uptake) for each treatment were as follows: 1.78 min−1, after C26 injection, 2.59 min−1, after saline injection, 3.18 min−1, after C26 injection in IL-1Ra–treated mice, and 2.42 min−1 in saline-injected mice given IL-1Ra (Fig. 2A). An ELISA study confirmed the increase (P < 0.05, n = 20) of IL-1 concentration in the hepatic blood on the 36th hour after injection of C26 cells in mice (41.8 ± 8 pg/mL) as compared with saline-injected mice (23.2 ± 11 pg/mL).