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Lipopolysaccharide induced apoptosis of a bovine pulmonary endothelial cell line

University of British Columbia

Haemophilus influenzae type b (Hib) was found to induce gross morphological changes and cell death in both bovine brain endothelial cells (BBEC) and a bovine pulmonary endothelial cell line (CPAE). The causative agent was identified as lipopolysaccharide (LPS), an integral component of the outer membrane of gram-negative bacteria. The cytotoxic effect of LPS was demonstrated by the MTT and LDH release assays and was dose and time dependent. Similar cytotoxicity was seen using LPS from Escherichia coli and Salmonella typhimurium. Other cell lines tested, including human and mouse endothelial cells, were resistant to LPS. The presence of serum was essential for LPS mediated cell death. Antibodies against CD 14, a macrophage/monocyte LPS receptor, blocked LPS-induced endothelial cell death. While CD14 is not found on endothelial cells, a soluble form is found in serum, sCD14. The involvement of sCD14 in endothelial cell death was confirmed by immunodepleting sCD14 from serum. The mode of cell death was investigated to distinguish between necrosis and apoptosis, two processes that are biochemically and morphologically distinct. DNA fragmentation characteristic of apoptosis was observed, and the occurance of apoptosis was confirmed by transmission electron microscopy. Potential LPS-induced signal transduction events were investigated for their role in apoptosis of CPAE cells. Calcium, nitric oxide and inositol phosphates did not appear to be involved in cell death, and inhibitors blocking protein synthesis, kinases, vacuolar acidification, microtubule rearrangements, and GTP proteins had no effect. In contrast, inhibitors of tyrosine phosphatases (vanadate/H202) and microfilament assembly (cytochalasin D) blocked cell death. Other studies were initiated to determine whether LPS acts by mimicking ceramide, as LPS and ceramide share structural similarity. Ceramide is a second messenger in the sphingomyelin pathway, and has been implicated in the process of apoptosis. Ceramide induced apoptosis in CPAE cells, but the mechanism appeared to be morphologically and kinetically different to that of LPS-induced apoptosis.

Thesis/Dissertation

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2009-04-03

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http://hdl.handle.net/2429/6778

Microbiology and Immunology

University of British Columbia

UBCV

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