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3R-Project 63-97

In vitro study of pathogenesis of sepsis in a conditionally immortalized Kupffer cell line

Regine Landmann
Division Infectious Deseases, Department Research, University Hospital Basel, 4031 Basel, Switzerland

Keywords: mice; liver; inflammation; septic shock models; cell cultures: organ-specific; cell cultures: transgenic; reduction

Duration: 2 years Project Completion: 2000

Background and Aim
Hepatic macrophages (Kupffer cells) represent the major first-line defense in sepsis. Unfortunately, freshly isolated Kupffer cells survive and maintain their phenotype only for a short time in vitro. The goal of this project is to develop a Kupffer cell line suitable for culturing in vitro, so that interactions between these cells and bacteria or bacterial products in vitro may be studied.

Method and Results
1) Conditionally immortalized Kupffer cell lines from the H-2Kb-tsA58 transgenic mouse (Jat PS, PNAS, 1991) have been generated.
2) The inflammatory reaction and clearing function of these cells in response to gram negative bacterial products will be studied. Conditions under which there is an optimal balance between clearing and inflammatory response are searched. The modulation of cytokine liberation and clearing by in-vitro transfection of genes involved in both effector functions will be assessed.
Kupffer cells have been isolated from transgenic mice carrying a thermolabile SV40 large tumor antigen under the H2Kb promoter (kind gift of D. Kioussis, NIMR, London). The cells grow with Interferon-gamma at 33oC, at which temperature the promoter is turned on and the SV40T Ag is active. They differentiate at 39oC. These cells are now being characterised: cytokine and NO liberation is stimulated, surface receptors are assessed at the mRNA and protein level, and phagocytosis and uptake of bacterial components are being measured. Results will be compared with the functional characteristics of primary Kupffer cells isolated from normal mice (see also 3R project 73-00)

Conclusions and Relevance for 3R
If the Kupffer cells derived from the transgenic mice prove functionally normal, they will replace studies with primary Kupffer cells. Accordingly, animals will no longer be required for cell isolation.