Exploration of Platelet and Mast Cell Communication: A Study of Membrane Lipids, Asthma, and Inflammation

Abstract

This work examines how environmental factors such as lipid membrane concentration, opioid agonist exposure, and inflammatory diseases impact cell communication. It explores the use of different cell models, specifically platelets and mast cells, to understand how disease states can impact cellular function. Throughout the thesis, a variety of analytical techniques including electrochemistry, mass spectrometry, dark field imaging, and microfluidics, are used to understand exocytosis, lipid concentration, manufactured inflammatory mediators, adhesion, and shape change in platelets and mast cells. Platelets are cell-like bodies that travel through the bloodstream and are known for their role in hemostasis and diseases like stroke and myocardial infarction. They have also been implicated in inflammatory diseases such as asthma. In addition, their anucleate nature and easy isolation make them an ideal model for studying variations in cell communication upon the modification of their lipid content. Platelets communicate through the exocytosis of their three distinct granule types (δ, α, and lysosome). These granules contain molecules that assist in the transmigration of immune cells to the site of activation and help with additional platelet aggregation and adhesion. In contrast, mast cells are found throughout the body in connective tissue and are one of the immune system’s first lines of defense. They are primarily known for their role in allergies and asthma. Upon detection of antigens that they are sensitized to, the mast cell secretes manufactured chemokines and pre-formed granule mediators, including histamine and serotonin, calling other inflammatory cells to the site of infection. Chapter One reviews single cell analysis techniques with a particular emphasis on the techniques used in this thesis, including electrochemistry and mass spectrometry. Chapter Two through four are focused on understanding how variations in membrane lipids and structure affect platelet function and exocytosis in general. Chapter Two focuses on understanding the variations that the fusion pore undergoes when granules are being exocytosed. Traditionally, a granule release event, monitored using carbon-fiber microelectrode amperometry, has a quick rise in current (spike) and gradual decay. The variations to this spike are classified as different forms of pre- and post-spike features and non-traditional granule secretion events. The role of cholesterol in changing the frequency and duration of these features is also discussed. Chapter Three discusses the role of phosphatidylserine (PS) in cellular communication using a platelet model. In this chapter, we explore how the stereochemistry of the head group and concentration of PS affects various platelet functions including granular content secretion, manufactured lipid release, and adhesion. The cholesterol level change upon addition of PS is also monitored. Finally, Chapter Four aims to understand how natural lipid variations affect cell function by comparing platelets from different species. This chapter highlights the importance of understanding your cell model relative to the actual cells involved in the disease or function being studied. Chapter Five and Six progresses from lipid function into developing a better understanding of how platelets respond to their environment, particularly in the context of inflammatory diseases. Chapter Five’s focus is on platelet response to opioids like those that are used in the treatment of pain due to inflammatory diseases, cancer, or surgery. Specifically, the effects on cell exocytosis as well as the presence of and role that opioid receptors play in platelets are characterized. Chapter Six focuses on studying how platelets respond to allergic asthma, including response to allergens and the chemoattractants (CXCL10 and CCL5) released during an asthma attack. Using bulk and single cell methods in conjunction allows us to obtain in-depth information on both the overall response and the granule fusion pore during exocytosis. Chapter Seven and Eight focus on mouse peritoneal mast cell (MPMC) function in the context of inflammatory diseases including allergic asthma and neurogenic inflammation, respectively. Chapter Seven aims to state the importance of understanding the cell line you are using since variations in response to allergens are noted between commonly used mast cell models (rat basophilic leukocytes cell line and primary culture MPMC). In addition, MPMC response to CXCL10 and CCL5 was monitored. Finally, Chapter Eight explores the role of MPMC in neurogenic inflammation, a process wherein neurons release the neuropeptides substance P and calcitonin gene-related peptide. Mast cell response to these neuropeptides has been highly disputed, and this chapter focuses on the impact of IgE on MPMC bulk granular content secretion. It also aims to understand how these neuropeptides affect the fusion pore opening and closing during exocytosis.