Abstracts
1998 Digestive Disease Week

#1076

PREVENTION OF ENDOTOXIN-INDUCED MORTALITY IS ASSOC-IATED WITH BLOCKADE OF BOTH PGE2 SYNTHESIS AND CALCIUM FLUX IN KUPFFER CELLS. CR Roland, B Naziruddin, Y Nakafusa, T Mohanakumar, MW Flye, Washington University School of Medicine. St. Louis, MO.

Hepatic Kupffer cells (KC) comprise a major macrophage population responsible for synthesis of the mediators, TNF-_ and PGE2, 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 PGE2, [Ca++]i, and TNF-_ 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. Supernatants from cultured LPS-stimulated KC were assayed for TNF-_ using the L929 bioassay and for PGE2 by RIA. To determine Ca++ flux in fura-2-AM-loaded KC, fluorescence changes were measured [using a spectrofluorimeter] after exposure to CaCl2, PAF or ionomycin in the presence or absence of Gd (1.0 mM). Ca++-dependent (ionomycin) or Ca++ independent (TPA) PGE2 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 PGE2 in response to in vitro LPS (0.025 mg/ml) and exhibited impaired Ca++ flux when exposed to ionomycin. Passive and receptor-mediated influx of Ca++ after PAF stimulation into Ca++-depleted KC were inhibited by in vitro Gd. Gd treatment reduced the ionomycinmediated, Ca++-dependent pathway of PGE2 synthesis but did not affect the TPA-mediated, Ca++-independent pathway. In vivo GD treatment significantly (p<0.05) increased TNF-_ response (vs decreased PGE2) to in vitro LPS when compared to control KC. The associated increase of TNF-_ by Gd reflects the decrease of autocoid inhibition of TNF-_ by PGE2 and suggests that TNF-_ is not directly responsible for septic death.

Conclusions: While death from sepsis is multifactorial, PGE2-mediated immunosuppression and [Ca++]i dysregulation both occur during sepsis. However, the relative contribution of each to septic mortality remains illdefined. The same in vivo dose of Gd which completely prevented LPS lethality was shown to also impair PGE2 release and Ca++ flux, thus supporting the hypothesis that elevated [Ca++]i and immunosuppression contribute to septic mortality.

Copyright 1996 - 1998, SSAT, Inc. Revised 29 June 1998.