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Interleukin‐1‐mediated H 2 O 2 production by hepatic sinusoidal endothelium in response to B16 melanoma cell adhesion
Author(s) -
Anasagasti M.J.,
Alvarez A.,
Avivi C.,
VidalVanaclocha F.
Publication year - 1996
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/(sici)1097-4652(199605)167:2<314::aid-jcp16>3.0.co;2-7
Subject(s) - flow cytometry , chemistry , microbiology and biotechnology , dichlorofluorescein , population , endothelium , cell culture , intracellular , biology , medicine , biochemistry , endocrinology , environmental health , genetics
We have examined H 2 O 2 production by in vitro enriched hepatic sinusoidal endothelium (HSE) during interleukin‐1β (IL‐1β) stimulation and B16 melanoma cell adhesion. Production of H 2 O 2 was quantified by flow cytometry and multiwell plate‐scanning fluorimetry of intracellular 2′, 7′‐dichlorofluorescein (DCFH) oxidation in HSE. Under IL‐1β treatment there was a 6‐fold increase in endothelial cells producing H 2 O 2 (67%) and a 4‐fold augmentation in the Kupffer cell population (86%). The average H 2 O 2 content per cell size unit significantly ( P < 0.01) increased in endothelial cells (2.6‐fold) and Kupffer cells (1.7‐fold). In contrast to the homogeneity of Kupffer cells, H 2 O 2 production intensity was largely heterogeneous in IL‐1β‐activated HSE. Enhancement of H 2 O 2 production by IL‐β‐treated HSE started at the 4th h and peaked 2–3 h later. The addition of increasing concentrations of IL‐1β to HSE for 4 h caused the progressive activation of H 2 O 2 production by treated cells. The addition of 80 M excess of IL‐1 receptor antagonist (IL‐1Ra) 10 min before IL‐1β treatment abrogated IL‐1β‐mediated enhancement of H 2 O 2 . From the 2nd h of B16 melanoma adhesion to HSE there was a significant ( P < 0.05) enhancement of H 2 O 2 content in HSE. This activation increased 2.25‐fold by the 3rd h of coculture and had reduced again by the 5th h. IL‐1Ra (80 ng/ml) given to HSE 10 min before melanoma cells abrogated the HSE response to melanoma cells. The addition of 1% paraformaldehyde (PFA)‐fixed B16 melanoma cells to HSE did not affect H 2 O 2 production response, indicating that HSE‐activating agents were on the melanoma cell surface. Preincubation of B16 melanoma cells in the presence of 5 μg/ml anti‐mouse IL‐1β neutralizing antibody reduced the melanoma cell‐induced HSE production of H 2 O 2 by 80%. On the contrary, B16 melanoma cell‐conditioned medium did not vary HSE production of H 2 O 2 compared to control HSE. Western blot analysis of cytosolic and membrane sediments from B16 melanoma cells confirmed the presence of IL‐1β (17.4 kDa) in both cell compartments. Thus, HSE responded to melanoma cell contact with a rapid production of H 2 O 2 . HSE activation was IL‐1‐dependent. This cytokine was directly provided to HSE by the cell surface of adhered melanoma cells. © 1996 Wiley‐Liss, Inc.