DOI
https://doi.org/10.25772/J9TD-3218
Defense Date
2006
Document Type
Thesis
Degree Name
Master of Science
Department
Biochemistry
First Advisor
Dr. Suzanne E. Barbour
Abstract
Localized Aggressive Periodontitis (LAgP) is an aggressive, early onset form of periodontitis characterized by a unique myeloid cell phenotype. In addition to its bacterial origin, the unique phenotype of the myeloid cell contributes to disease pathogenesis and progression through mechanisms mediating host inflammatory and immune responses. LAgP monocytes synthesize increased levels of the potent proinflammatory lipid mediator, Prostaglandin E2 (PGE2), preferentially differentiate into dendritic cells, and lead to increased IgG2 production. In addition, levels of Platelet Activating Factor (PAF) have shown to be elevated in the gingival tissue and gingival crevicular fluid of subjects with periodontitis. The aim of this study was to further characterize the unique phenotype of the myeloid cell by investigating its role in the increased levels of PAF in periodontitis subjects, examining differences in gene expression of the immune response gene, STATl which is involved in IFN-γ signaling, and by examining the differential expression and function of the scavenger receptor CD36. LAgP monocytes have exhibited decreased activity of the PAF-acetylhydrolase (PAFAH), the catalytic enzyme that breaks down PAF. Since PAF levels are regulated by synthesis and degradation, we hypothesized that synthesis by myeloid cells, monocytes or PMN, also contribute to the increased PAF levels in LAgP. We also hypothesized, based on initial microarray data that myeloid cells have decreased gene expression of STATl and downstream IFNy related genes in LAgP. In addition, based on the initial microarray results, we hypothesized that LAgP monocytes have increased CD36 expression with increased capacity for the binding and uptake of chemically modified versions of LDL. Monocytes were isolated from the peripheral blood of LAgP and NP control subjects over a Ficoll gradient. A radiolabeled PAF assay was used to quantify total PAF synthesis in both resting monocytes and PMN, and in monocytes and PMN stimulated with calcium ionophore A23 187. Quantitative RT-PCR was used to quantify STATl and CD36 gene expression from RNA isolated from adherent monocytes, and CD36 expression and AcLDL (acetylated LDL) uptake was quantified using flow cytometry. Our results indicate that PAF synthesis is increased in LAgP PMN but not in monocytes. LAgP monocytes synthesize less PAF compared to NP control, and their response to calcium ionophore A23 187 (IoA), expressed as fold increase, was blunted. LAgP and NP monocytes did not differ in STATl gene expression as determined by quantitative RT-PCR, and CD36 experiments suggest the possibility that dendritic cells express increased scavenger receptor CD36 than macrophage cells. In conclusion, LAgP myeloid cells are unique in their response to A23 187, and LAgP PMN contribute to increased PAF primarily through synthesis, whereas the LAgP monocytes contributes to elevated PAF through decreased catabolism. STAT1 gene expression did not differ between LAgP and NP monocytes, however this does not rule out the possibility of differential STATl signaling in LAgP monocytes though inhibitory proteins or differential phosphorylation of STATl. Finally, CD36 expression appears from preliminary data to be increased in dendritic cells. These findings add to the current understanding of the unique phenotype of the LAgP monoctye and further experiments will continue to expand our understanding of how unique biology of myeloid cells and their ability to facilitate crosstalk between the innate and adaptive immune system, and the host inflammatory system.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
June 2008