Date of Graduation

2003

Document Type

Thesis

Degree Type

MS

Committee Chair

Yongyut Rojanasakul

Abstract

The first section of this work addressed the relationship between apoptotic and necrotic cell death and their role in inflammatory lung injury induced by endotoxin. Pulmonary administration of lipopolysaccharide (LPS) into the lung of mice caused a time-dependent increase in the levels of pro-inflammatory cytokine TNF-α and inflammatory cell influx in the bronchoalveolar lavage (BAL) fluids. Control mice showed only resident alveolar macrophages with no apoptosis as determined by terminal deoxyribonucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) and histone-bound DNA fragmentation assays. In contrast, LPS-treated mice showed clear apoptosis of BAL cells. Microscopic studies confirmed the presence of apoptotic neutrophils and macrophages ingesting apoptotic bodies. The number of apoptotic neutrophils increased concomitantly with the increase in neutrophil influx which peaked 1 day after the treatment. However, necrosis was not detected at this early time point, whereas apoptosis continued to increase and peaked at day 2 post-treatment. Necrosis gradually increased by day 2 and reached a maximal level by day 3 where it subsequently declined. The levels of necrosis and apoptosis were both elevated

and prolonged at high LPS doses. Inhibition of macrophage phagocytosis of apoptotic cells by phosphatidylserine (PS)-containing liposomes increased the level of necrosis caused by LPS, whereas control PS-lacking liposomes had no effect. Gathered data suggests that necrosis occurs secondary to apoptosis and that this development, in contrast to the general belief, is not the result of direct insult by LPS treatment but rather a consequence of inefficient phagocytic clearance of apoptotic cells. Because necrosis is associated with cellular damage and release of cytotoxic contents, this development is likely to play a role in determining the severity and duration of inflammatory lung injury following endotoxin exposure. In the second section, the focus shifted to the role of ROS in FasL-induced cell death. Given that Fas/FasL pathway has been implicated in the pathogenesis of numerous inflammatory disorders, potential therapeutic utility of antioxidants was investigated in an in vitro model. ESR studies of RAW 264.7 cell response to Fas ligation showed clear production of hydroxyl radicals. The response was visible as early as 5 min and peaked at approximately 30 min following the

treatment. Morphological analysis of total death response (apoptosis vs. necrosis) showed dose and time dependency with the peak response time at 12 h post-treatment. Apoptosis levels were significantly elevated as early as 6 h while necrosis remained at a baseline level. Only at a 35-fold increase in apoptosis did the necrosis levels become significantly elevated. Catalase and deferoxamine, ROS scavengers, inhibited the total death response as well as the ESR signal, while SOD and sodium formate showed no effect. This established a role for ROS, specifically H2O2 and . OH, as important participants in the Fas death pathway. Further, morphological analysis during inhibition studies showed apoptosis dominance over necrosis suggesting that apoptosis was the primary mode of death in RAW 264.7 cells and that necrosis came secondary. Because Fas pathway is implicated in toxicities associated with inflammatory conditions, utilization of antioxidants as potential therapeutic agents may be advantageous.

Based on the findings presented within this work, excessive apoptosis and consequent inability of the tissues’s phagocytes to promptly clear apoptotic bodies results in secondary necrosis and amplification of the overall tissue damage via release of the noxious cellular contents into the environment. Such relationship illustrates the apoptosis-necrosis paradox and suggests that strategies directed towards apoptosis inhibition and/or stimulation of the phagocyte system may show therapeutic utility in the treatment of inflammatory disorders.

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