Production of inflammatory mediators by human macrophages obtained from ascites

Ascites is a readily available source of human macrophages (Mø), which can be used to study Mø functions in vitro. We characterized the mediators of inflammation produced by human peritoneal Mø (hp-Mø) obtained from patients with portal hypertension and ascites. The production of the cytokines interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) was found to be lipopolysaccharide (LPS) concentration dependent (0–10 μg/ml) with a maximal production at 10 μg/ml and also dependent on the time of exposure to the stimulus (0–36 h). IL-1β, IL-6 and TNF-α production after LPS administration reached a plateau at 24 h. In vitro stimulation for 24 h with LPS does not influence the eicosanoid production from endogenous arachidonate. 13 min of exposure of the cells to the calcium ionophore A23187 gives a significant increase in eicosanoid production from both exogenous and endogenous arachidonate. The main eicosanoids produced are the 5-lipoxgenase products LTB4 and 5-hydroxyeicosatetraenoic acid (HETE). The increase in production of the other eicosanoids is not significant. The eicosanoid production depends on the stimulus concentration. The optimal A23187 concentration is 1 μM. Oxygen radical production was measured in the Mø by a flowcytometric method. The fluorescence intensity of phorbol 12-myristate 13-acetate stimulated and dihydro-rhodamine 123 loaded. hp-Mø increases significantly after 15 min. We conclude that LPS stimulation of hp-Mø from liver disease results in similar production of IL-1β, IL-6 and TNF-α, but that the profile of the eicosanoid production of these Mø stimulated with LPS and A23187 differs from Mø of other origin and species.

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