Browsing by Author "Tentke, Annika"

This project was about the molecular mechanisms involved in the generation of eicosanoids in human mast cells with particular emphasis on lipid bodies as a source and/or site of lipid mediator biogenesis. The cells to be used are isolated from human peripheral blood provided by Finnish Red Cross Blood Transfusion Service and collected from healthy donors. Human mast cells are found in connective tissue. They contain granules filled with histamine, heparine and proteases. Human mast cells are potent effector cells in host-defense mechanisms of innate immunity, including inflammatory diseases such as atherosclerosis. Activation of mast cells by different stimuli triggers the release of a huge range of mediators, including de-novo synthesized eicosanoids, which are highly biologically active lipid mediators. The major eicosanoid released by activated mast cells is prostanoid prostaglandin D2 (PGD2). The aim of this project was to find out whether mast cell lipid bodies are the cellular compartments of PGD2 synthesis, what are the enzymes involved in AA liberation from TGs, and whether TG-derived AA is a source for PGD2 production. The enzymes of special interest were hormone sensitive lipase (HSL) and adipose triglyceride lipase (ATGL). We were also interested about hematopoietic prostaglandin D synthase (HPGDS), the key enzyme in the production of D and J series of prostanoids. Methods used in this pro gradu work include siRNA transfections, RNA isolation, cDNA synthesis, qPCR, immunoblotting, ELISA and conventional fluorescence microscopy. Immediate increase in the amount of PGD2 released from mast cells sensitized with human IgE (1 µg/ml) and activated by polyclonal rabbit anti-human IgE (1 µg/ml) was observed. The increase was most prominent after one hour of activation, and slowly decreased to basal levels at 48 h post-activation. siRNA transfection affected the amount of enzyme DNA in mast cells and the amount of PGD2 released. HSL, ATGL and HSL+ATGL double knockdowns all reduced the amount of PGD2 released in acute (5 to 30 minutes) term activation compared to control cells. However, no significant changes were observed in the mRNA expression levels of ATGL, HSL, CGI-58, HPGDS or COX-1 under mast cell activation. The only significant changes in mRNA expression levels were observed with COX-2. However, the relative expression of HPGDS increased in IgE treated mast cells compared to control treated cells and the expression was even greater in mast cells treated with αIgE also. Both ATGL and HPGDS were recognized throughout the cytosolic area in the non-activated Ctrl cells. Although HPGDS located also in the circumference of mast cells, no clear localization of HPGDS was observed in the circumference of mast cell lipid droplets. The experiments carried out at the Wihuri Research Institute, including those presented here, have established that, in addition to phospholipids, the triglycerides present in mast cell lipid droplet core are also an important source of eicosanoids, and that also ATGL and HSL, not just cPLA, can release arachidonic acid for eicosanoid production. The ramifications of this study include the possibility that arachidonic acid release from triglycerides for the formation of eicosanoids could take an indirect or a direct route to supply precursors for cellular eicosanoid biosynthesis. The key is the pathway of AA release. In the direct pathway, AA is released from LD TGs by ATGL or HSL and this free AA is used for the generation of PGs by either COX-1 or COX-2, depending on the status of the cell. In the indirect pathway, AA is liberated from LD TGs by ATGL or HSL and then further re-esterified into phospholipids from where AA is then finally released by cPLA2 for the generation of eicosanoids.