Prevention of endotoxin-induced mortality is associated with blockade of both PGE2 synthesis and calcium flux in Kupffer cells

Abstract

Hepatic Kupffer cells (KC) comprise a major macrophage population responsible for synthesis of the mediators, TNF-a and PGE 2, implicated in septic mortality. The rare earth metal, Gadolinium chloride (Gd), when internalized by KC, inhibits KC phagocytosis and is known to alter Ca ++ flux in other cell types. We examined the effect of Gd on a lethal LPS challenge and the associated effects on PGE 2, [Ca++] i, and TNF-a production by KC. Methods: Rats were injected with either Gd (7mg/kg i.v.) or vehicle for the two days prior to a lethal LPS dose (30mg/kg i.v.) or prior to isolation of KC by collagenase digestion and plastic adherence. Supematants from cultured LPS-stimulated KC were assayed for TNF-a using the L929 bioassay and for PGE 2 by RIA. To determine Ca ++ flux in fura-2-AM-loaded KC, fluorescence changes were measured [using a spectrofluorimeter] after exposure to CaC12, PAF or innomycin in the presence or absence of Gd (1.0 mM). Ca ++dependent (innomycin) or Ca ++ independent (TPA) PGE 2 synthesis by KC were stimulated in the presence or absence of Gd (10 mM).Results: Two doses of Gd in vivo completely protected against the lethal dose of LPS (0% vs. 100% mortality for vehicle control, p<0.001). Gd-treated KC released PGE 2 in response to in vitro LPS (0.025 mg/ml) and exhibited impaired Ca ++ flux when exposed to innomycin. Passive and receptor-mediated influx of Ca ++ after PAF stimulation into Ca++-depleted KC were inhibited by in vitro Gd. Gd treatment reduced the iunomycinmediated, Ca++-dependent pathway of PGE 2 synthesis but did not affect the TPA-mediated, Ca++-independent pathway. In vivo GD treatment significantly (p<0.05) increased TNF-a response (vs decreased PGE 2) to in vitro LPS when compared to control KC. The associated increase of TNF-a by Gd reflects the decrease of autocoid inhibition of TNF-a by PGE 2 and suggests that TNF-a is not directly responsible for septic death.