Mechanisms of Lipid Mediator-Generation in Human Mast Cells

The inflammatory process in emerging atherosclerotic lesions is characterized by the presence of three different types of inflammatory cells, including macrophages, T cells, and mast cells (MCs), which represent powerful activatable effectors in the arterial wall. The activation of MCs is characterized by exocytosis of cytoplasmic secretory granules in a process called degranulation. The exocytosed granules affect extracellular matrix remodeling and may lead to various metabolic changes in neighbouring cells through potent biologically active mediators, which include de novo-synthesized lipid mediators, e.g., metabolites of arachidonic acid such as prostaglanding (PG) D2, the leukotrienes (LTs) LTB4, and LTC4, the parent molecule of cysteinyl-LTs. To date, only few insights into the origin and biosynthesis of lipid mediators in MCs exist. Particularly, our knowledge about the mechanisms governing the generation of lipid mediators derived from lipids stored in the form of cytoplasmic granules or "lipid bodies" is very limited. Prompted by these shortcomings, the current proposal is directed towards the identification of key components and the elucidation of regulatory processes orchestrating lipid mediator generation in MCs. The approaches to shed light on these aspect have three major goals: 1) to analyze the spectrum of lipolytic and lipogenic enzymes in human MCs, and to identify which of these enzymes are implicated in acute eiconsanoid production upon activation of the MCs; 2) to determine which members of the low-density lipoprotein receptor (LDLDR) family in addition to the LDLR are expressed by human MCs and may function in the delivery of lipoprotein-associated arachidonic acid or precursors; and 3) to analyze the previously described "lipid bodies" of MCs for their content of proteins characteristic of important organelles known as lipid droplets, which are found to be metabolically highly active compartments in a wide variety of cell types. The research program at the host Institute on MC biology has thus far focused on extracellular mechanisms which clearly link them to atherosclerotic plaque formation and plaque rupture, the event that triggers most heart attacks. The proposed novel investigations of intracellular molecular mechanisms ultimately leading to the release of potent lipid mediators which determine the proinflammatory phenotype of human MCs should help to (re)define their role in atherosclerotic pathology. It is anticipated that uncovering novel lipid regulatory pathways in human MCs may also identify new pharmacologic targets that regulate the inflammatory activity of these potentially harmful cells.

Mast cells are a certain type of immune cell with very powerful characteristics in host-defense mechanisms, including various different inflammatory diseases such as asthma, rheumatoid arthritis, and atherosclerosis. Mast cells possess a huge range of different molecules on their cell surface, through which they can receive specific signals from outside the cell. Depending on the stimulus, the incoming signal triggers different cellular events inside the mast cell and determines how the cell can respond to its environment. Under these conditions, the mast cell turns into an activated mode and reacts via the release of special mediators, which are highly biologically active molecules that are able to initiate, enhance or dampen inflammatory responses. Among these molecules, there is one class of mediators, called eicosanoids. These are lipid signalling molecules which are made from a common precursor omega-6 fatty acid, termed arachidonic acid, which can be usually found in a certain membrane lipid class, called phospholipids. Human lung mast cells have been demonstrated to contain a huge amount of arachidonic acid also in triglycerides, which belong to the class of neutral lipids and are stored in lipid droplets. Lipid droplets are specific cellular organelles made out of a neutral lipid core that is surrounded by a phospholipid monolayer containing various different proteins. Here, we investigated the possibility, whether mast cells can use arachidonic acid from triglycerides located in the neutral lipid core of lipid droplets for the production of eicosanoids. For our studies, we generated human mast cells isolated from peripheral blood-derived progenitors. Based on our experimental observations, these mast cells contain lipid droplets and are able to incorporate huge amounts of arachidonic acid in triglycerides. Mast cells release a certain eicosanoid, called prostaglandin D2, when the cells are getting immunologically activated by a specific stimulus. Therefore, we used a biochemical approach to silence the expression of the enzyme adipose triglyceride lipase (ATGL), which mediates the release of fatty acids from triglycerides. Indeed, ATGL silencing in human mast cells significantly reduced the formation of PGD2 upon cellular activation. In addition, silencing of ATGL resulted also in the accumulation of neutral lipids in the lipid droplets, thus reflecting an impaired triglyceride hydrolysis. This novel finding supports our hypothesis, that ATGL plays an essential function for providing arachidonic acid for eicosanoid formation in human mast cells.