Semester

Spring

Date of Graduation

2000

Document Type

Dissertation

Degree Type

PhD

College

School of Pharmacy

Department

Pharmaceutical Sciences

Committee Chair

Yon Rojanasakul.

Abstract

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays an important regulatory role in inflammatory diseases. The overall goal of this research is to investigate the potential therapeutic effect of IL-10 gene therapy for the treatment of pulmonary inflammation and to develop appropriate gene delivery and expression systems for efficient gene transfer to the lung. We evaluated various liposomal and non-liposomal agents including LipofectAMINE RTM, LipofectinRTM, DOTAP, DEAE-dextran, and the DNA condensing agent protamine sulfate for their ability to promote gene transfection in macrophages. We have developed an efficient transfection protocol which is at least 20--25 fold superior to frequently used protocols for macrophage transfection. However, gene transfection in macrophages is still low. Since macrophages are known to be highly susceptible to endotoxin stimulation and endotoxin is a major contaminant of plasmid DNA preparations used in most transfection studies, we investigated the effect of endotoxin on gene transfer and the role of reactive oxygen species (ROS) in the process. Our results indicate that this decreased transfection was dependent on ROS-mediated cellular toxicity induced by endotoxin and that ·OH radicals play a major role in this process. The mechanism of anti-inflammatory action of IL-10 is incompletely understood. The present study also investigates the possible role of ROS in NF-kappaB activation and IkappaBalpha degradation and their inhibition by IL-10 in macrophages. Addition of IL-10 inhibited both IkappaBalpha degradation and generation of ·OH radicals in response to LPS stimulation. These results provide, for the first time, direct evidence for the role of IL-10 in ROS-dependent NF-kappaB activation. The next step was to develop a protocol for in vivo gene transfer. Cationic liposomes represent a class of nonviral vectors that hold great promise as gene delivery vehicles. However, recent studies have shown that they may be associated with some toxicity. We tested the hypothesis that the positive charge of liposomes is a key determinant of toxicity. ROS play a key role in cationic lipid-mediated toxicity. Finally, we established an efficient gene transfer protocol that results in clinically relevant concentrations of IL-10 protein that can downregulate mouse lung inflammation in response to LPS stimulation.

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