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Production of inflammatory mediators by human macrophages obtained from ascites
W M Pruimboom1, A P van Dijk, C J Tak
1Department of Internal Medicine II, University Hospital Dijkzigt Rotterdam, The Netherlands.
Abstract:
Ascites is a readily available source of human macrophages (M phi), which can be used to study M phi functions in vitro. We characterized the mediators of inflammation produced by human peritoneal M phi (hp-M phi) obtained from patients with portal hypertension and ascites. The production of the cytokines interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) was found to be lipopolysaccharide (LPS) concentration dependent (0-10 micrograms/ml) with a maximal production at 10 micrograms/ml and also dependent on the time of exposure to the stimulus (0-36 h). IL-1 beta, IL-6 and TNF-alpha 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 microM. Oxygen radical production was measured in the M phi by a flowcytometric method. The fluorescence intensity of phorbol 12-myristate 13-acetate stimulated and dihydro-rhodamine 123 loaded hp-M phi increases significantly after 15 min. We conclude that LPS stimulation of hp-M phi from liver disease results in similar production of IL-1 beta, IL-6 and TNF-alpha, but that the profile of the eicosanoid production of these M phi stimulated with LPS and A23187 differs from M phi of other origin and species.
Insights
Human peritoneal macrophages (hp-M phi) from ascites patients produce inflammatory cytokines IL-1 beta, IL-6, and TNF-alpha in response to lipopolysaccharide (LPS). Eicosanoid production differs from other macrophage types.
Area of Science:
- Immunology
- Cell Biology
- Gastroenterology
Background:
- Ascites provides a source of human peritoneal macrophages (hp-M phi) for in vitro studies.
- Understanding macrophage inflammatory mediator production is crucial for liver disease research.
Purpose of the Study:
- To characterize inflammatory mediators produced by hp-M phi from patients with portal hypertension and ascites.
- To investigate the effects of lipopolysaccharide (LPS) and calcium ionophore A23187 on hp-M phi.
Main Methods:
- Human peritoneal macrophages (hp-M phi) were isolated from ascites fluid.
- Cytokine production (IL-1 beta, IL-6, TNF-alpha) was measured following LPS stimulation.
- Eicosanoid production was assessed after stimulation with LPS and calcium ionophore A23187.
- Oxygen radical production was quantified using flow cytometry.
Main Results:
- LPS-induced production of IL-1 beta, IL-6, and TNF-alpha was dose- and time-dependent, plateauing at 24 hours.
- LPS stimulation did not affect endogenous eicosanoid production.
- Calcium ionophore A23187 significantly increased eicosanoid production, primarily LTB4 and 5-HETE.
- Optimal A23187 concentration was 1 microM.
- Oxygen radical production increased significantly after 15 minutes of stimulation.
Conclusions:
- LPS stimulation of hp-M phi from liver disease patients yields consistent IL-1 beta, IL-6, and TNF-alpha production.
- The eicosanoid production profile of these hp-M phi differs from macrophages of other origins/species when stimulated with LPS and A23187.