Targeting Phospholipase D Genetically and Pharmacologically for Studying Leukocyte Function.

Phospholipase D (PLD), is a protein that breaks down phospholipids, maintaining structural integrity and remodeling of cellular or intracellular membranes, as well as mediating protein trafficking and cytoskeletal dynamics during cell motility. One of the reaction products of PLD action is phosphatidic acid (PA). PA is a mitogen involved in a large variety of physiological cellular functions, such as cell growth, cell cycle progression, and cell motility. We have chosen as cell models the leukocyte polymorphonuclear neutrophil and the macrophage as examples of cell motility. We provide a three-part method for targeting PLD genetically and pharmacologically to study its role in cell migration. In the first part, we begin with genetically deficient mice PLD1-KO and PLD2-KO. We describe bone marrow neutrophil (BMN) isolation; BMN is labeled fluorescently and can be used for studying tissue-damaging neutrophilia in ischemia-reperfusion injury (IRI). In the second part, we begin also with PLD1-KO and PLD2-KO and prepare bone marrow-derived macrophages (BMDM), first from monocytes and then inducing macrophage differentiation in culture with continuous incubation of cytokines. We use BMDM to find experimentally if PLD woul play a role in cholesterol phagocytosis, which is the first step in atherosclerosis progression. In the third part, we study PLD function in BMN and BMDM with PLD enzyme pharmacological inhibitors instead of genetically deficient mice, to ascertain the particular contributions of isoforms PLD1 and PLD2 on leukocyte function. By using the three-step thorough approach, we could understand the molecular underpinning of PLD in the pathological conditions indicated above, IRI-neutrophilia and atherosclerosis.