INTERLEUKIN-1-MEDIATED H2O2 PRODUCTION BY HEPATIC SINUSOIDAL ENDOTHELIUM IN RESPONSE TO B16 MELANOMA CELL-ADHESION

We have examined H2O2 production by in vitro enriched hepatic sinusoid al endothelium (HSE) during interleukin-1 beta (IL-1 beta) stimulation and B16 melanoma cell adhesion. Production of H2O2 was quantified by flow cytometry and multiwell plate-scanning fluorimetry of intracellul ar 2', 7'-dichlorofluorescein (DCFH) oxidation in HSE. Under IL-1 beta treatment there was a 6-fold increase in endothelial cells producing H2O2 (67%) and a 4-fold augmentation in the Kupffer cell population (8 6%). The average H,O, content per cell size unit significantly (P < 0. 01) increased in endothelial cells (2.6-fold) and Kupffer cells (1.7-f old). In contrast to the homogeneity of Kupffer cells, H2O2 production intensity was largely heterogeneous in IL-1 beta-activated HSE. Enhan cement of H2O2 production by IL-P-treated HSE started at the 4th h and peaked 2-3 h later. The addition of increasing concentrations of IL-1 beta to HSE for 4 h caused the progressive activation of H2O2 product ion by treated cells. The addition of 80 M excess of IL-1 receptor ant agonist (IL-1 Ra) 10 min before IL-1 beta treatment abrogated IL-1 bet a-mediated enhancement of H2O2. From the 2nd h of B16 melanoma adhesio n to HSE there was a significant (P < 0.05) enhancement of H2O2 conten t in HSE. This activation increased 2.25-fold by the 3rd h of cocultur e and had reduced again by the 5th h. IL-1Ra (80 ng/ml) given to HSE 1 0 min before melanoma cells abrogated the HSE response to melanoma cel ls. The addition of 1% paraformaldehyde (PFA)-fixed B16 melanoma cells to HSE did not affect H2O2 production response, indicating that HSE-a ctivating agents were on the melanoma cell) surface. Preincubation of B16 melanoma cells in the presence of 5 mu g/ml anti-mouse IL-1 beta n eutralizing antibody reduced the melanoma cell-induced HSE production of H2O2 by 80%. On the contrary, B16 melanoma cell-conditioned medium did not vary HSE production of H2O2 compared to control HSE. Western b lot analysis of cytosolic and membrane sediments from B16 melanoma cel ls confirmed the presence of IL-1 beta (17.4 kDa) in both cell compart ments. Thus, HSE responded to melanoma cell contact with a rapid produ ction of H2O2. HSE activation was IL-1-dependent. This cytokine was di rectly provided to HSE by the cell surface of adhered melanoma cells. (C) 1996 Wiley-Liss, Inc.