Dr Amanda Mackenzie  

Profile

My research areas range from inflammation, molecular physiology, biophysics to stem cell physiology. Specific projects available in the lab include:

1. The functional role of ATP-gated ion channels expressed in inflammatory macrophages (live cell imaging, electrophysiology and protein biochemistry).
2. The interaction between endothelial cells and monocytes during inflammation (live cell imaging and protein biochemistry).
3. The regulation and molecular characterization of ion channels in stem cell megakaryocytes (live cell imaging and electrophysiology).

ATP-gated P2X receptors

Extracellular adenosine 5'-triphosphate (ATP) is an established intercellular signaling molecule in the immune system acting at two classes of receptor: metabotropic G protein coupled P2Y receptors and ionotropic P2X receptors. P2X receptors (P2X1-P2X7) can function as homomeric or heteromeric subunit assemblies forming a non-selective cation channel. Each P2X receptor subtype can be distinguished by distinct pharmacological or biophysical properties with a range of permeabilities to calcium. P2X7 receptors are predominately expressed by cells of the immune system, including monocytes, neutrophils and lymphocytes, and exocrine secretory glands. The P2X7 receptor is uniquely characterized by a decreased sensitivity to extracellular ATP compared with other P2X receptors and rapid coupling to a membrane 'pore' that transports large molecular weight dyes.

Cell biology of immune cells

My research laboratory is interested in the functional role of ion channels, particularly P2X receptors, in the innate immune response to pathogen-derived molecules. In particular, we are interested in the molecular signaling pathways underlying the production of the inflammatory cytokine interleukin 1 (IL-1Beta). The production of bioactive IL-1Beta is a tightly controlled multi-step process: first IL-1Beta is produced as an inactivate precursor 31 kD pro-IL-1Beta , this is cleaved to produce bioactive 17 kD IL-1Beta and then finally the bioactive IL-1Beta is rapidly secreted from the macrophage. Processing and cleavage of the IL-1Beta precursor is achieved by a protease called caspase-1 that is activated within a multi-protein complex termed the inflammasome. Activation of Toll like receptors triggers the transcription and translation of pro-IL-1Beta. Stimulation of an ATP-gated ion channel, the P2X7 receptor, leads to the rapid activation of caspase-1 and secretion of bioactive IL-1Beta . The cytokine IL-1Beta lacks a signal peptide sequence and is secreted via a non-classical pathway in microvesicles. We are interested in the molecular mechanism underlying the secretion of pro-inflammatory cytokines in microvesicles.

Moreover, activation of P2X7 receptors couples to gross changes in cellular morphology typically associated with cell death [MacKenzie et al, 2005, 2001]. These events include dynamic membrane bleb formation, microvesicle formation, phosphatidylserine exposure on the outer leaflet of the plasma membrane and calcium dependent swelling of mitochondria. However, if receptor activation is brief, these events can occur in the absence of cell death. We are interested in signal transduction pathways underlying these dramatic events and the downstream physiological/pathophysiological role.

Inflammation and Vascular Endothelial cells

The vascular endothelium plays an essential role in orchestrating the inflammatory response. Through the expression of cell adhesion molecules on the endothelial cell surface, circulating immune cells may bind to and cross the endothelium and enter regions of inflammation.

How do endothelial cells interact with blood monocytes? This is a complex process where close interaction between the endothelium and immune cells is vital. We are interested in the interaction of immune cells and the endothelium in the disease sepsis, a systemic inflammatory response.>

Currently, we are studying the involvement of bacterial peptides in inducing the release of inflammatory signalling molecules that are involved in communication between the endothelium and immune cells. In particular we are interested the role of P2 receptors, and the consequential effects on pro-inflammatory cytokine release.

Functional role of ion channels in megakaryocytes

Can ion channels signal to the nucleus to regulate stem cell function? Megakaryocytes are stem cells resident in the bone marrow; cytokine stimulation triggers the differentiation of megakaryocytes that culminates in the production of blood platelets.

We are using molecular and biophysicalapproaches to identify the ion channels expressed by megakaryocytes and the role these proteins play in the process of differentiation and the production of blood platelets. Understanding the molecular mechanisms underlying megakaryocyte differentiation and platelet production will be potentially important to identify drug targets for the treatment of clinical or inherited thrombocytopenia.

Our research is funded by the British Heart Foundation, Heart Research UK, the University of Bath and the Royal Society.

Publications

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