The Role of Phosphatidylinositol 3-Kinase in P2X7R Gated Signalling in T Lymphocytes

John G Foster

Research output: ThesisDoctoral Thesis

Abstract

Background: The role of P2X7R in the immune response has been investigated and this receptor clearly has important roles in inflammation. However, the mechanisms which integrate P2X7R activation with biochemical changes in T lymphocytes such as: proliferation, migration and regulation of adhesion molecule expression are less well understood. Many of these processes are controlled by the PI3K pathway, which is an important signalling cascade involved in development and immunity that it is frequently altered in disease. This study sought to investigate if PI3K was responsible for integrating P2X7R dependent signalling in primary human T lymphocytes, with an emphasis on regulation of the adhesion molecule CD62L.
Results: Whole cell patch clamp electrophysiology is an important technique for characterising ion channel expression. This technique was optimised for the first time in primary human naïve CD4+ T lymphocytes and used to show P2X7R expression in this study. The pharmacology of ATP in the process of CD62L down-regulation in these cells was explored using new P2X7R antagonists with improved selectivity over previous compounds. Remarkably, PI3K/mTOR, MAPK and PKC signalling was shown to be dispensable for this down-regulation of cell surface CD62L expression. However, while investigating novel mechanisms for ATP induced CD62L down-regulation, it was revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R dependent CD62L down-regulation by increasing ATP potency. The mechanism for this was further explored and this effect may arise from enhanced superoxide generation in the mitochondria of rotenone and antimycin A treated cells. Crucially, although ATP alone did not cause apoptosis of cell, perturbation of the mitochondria of cell with these compounds followed by ATP treatment, revealed P2X7R exposure of phosphatidyl serine.
Discussion: This major new finding may have implications for the clearance of naïve CD4+ T lymphocytes which have undergone mitochondrial damage. A novel protective mechanism for the potential removal of cells with damaged mitochondria is presented, whereby, P2X7R dependent PS exposure occurs only when cells have enhanced mitochondrial ROS generation. Given the potential role of P2X7R in a number of diseases with a mitochondrial element, the findings of this thesis are of great importance for the targeting of P2X7R in inflammation.
LanguageEnglish
QualificationPh.D.
Awarding Institution
  • University of Bath
Supervisors/Advisors
  • Ward, Stephen, Supervisor
  • Mackenzie, Amanda, Supervisor
Award date31 Jul 2012
StatusPublished - 2012

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Phosphatidylinositol 3-Kinase
T-Lymphocytes
Adenosine Triphosphate
Down-Regulation
Phosphatidylinositol 3-Kinases
Mitochondria
Pharmacology
Antimycin A
Inflammation
Rotenone
NADPH Oxidase
Electrophysiology
Phosphatidylserines
Ion Channels
Superoxides
Immunity
Apoptosis

Cite this

The Role of Phosphatidylinositol 3-Kinase in P2X7R Gated Signalling in T Lymphocytes. / Foster, John G.

2012. 261 p.

Research output: ThesisDoctoral Thesis

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title = "The Role of Phosphatidylinositol 3-Kinase in P2X7R Gated Signalling in T Lymphocytes",
abstract = "Background: The role of P2X7R in the immune response has been investigated and this receptor clearly has important roles in inflammation. However, the mechanisms which integrate P2X7R activation with biochemical changes in T lymphocytes such as: proliferation, migration and regulation of adhesion molecule expression are less well understood. Many of these processes are controlled by the PI3K pathway, which is an important signalling cascade involved in development and immunity that it is frequently altered in disease. This study sought to investigate if PI3K was responsible for integrating P2X7R dependent signalling in primary human T lymphocytes, with an emphasis on regulation of the adhesion molecule CD62L.Results: Whole cell patch clamp electrophysiology is an important technique for characterising ion channel expression. This technique was optimised for the first time in primary human na{\"i}ve CD4+ T lymphocytes and used to show P2X7R expression in this study. The pharmacology of ATP in the process of CD62L down-regulation in these cells was explored using new P2X7R antagonists with improved selectivity over previous compounds. Remarkably, PI3K/mTOR, MAPK and PKC signalling was shown to be dispensable for this down-regulation of cell surface CD62L expression. However, while investigating novel mechanisms for ATP induced CD62L down-regulation, it was revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R dependent CD62L down-regulation by increasing ATP potency. The mechanism for this was further explored and this effect may arise from enhanced superoxide generation in the mitochondria of rotenone and antimycin A treated cells. Crucially, although ATP alone did not cause apoptosis of cell, perturbation of the mitochondria of cell with these compounds followed by ATP treatment, revealed P2X7R exposure of phosphatidyl serine.Discussion: This major new finding may have implications for the clearance of na{\"i}ve CD4+ T lymphocytes which have undergone mitochondrial damage. A novel protective mechanism for the potential removal of cells with damaged mitochondria is presented, whereby, P2X7R dependent PS exposure occurs only when cells have enhanced mitochondrial ROS generation. Given the potential role of P2X7R in a number of diseases with a mitochondrial element, the findings of this thesis are of great importance for the targeting of P2X7R in inflammation.",
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T1 - The Role of Phosphatidylinositol 3-Kinase in P2X7R Gated Signalling in T Lymphocytes

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PY - 2012

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N2 - Background: The role of P2X7R in the immune response has been investigated and this receptor clearly has important roles in inflammation. However, the mechanisms which integrate P2X7R activation with biochemical changes in T lymphocytes such as: proliferation, migration and regulation of adhesion molecule expression are less well understood. Many of these processes are controlled by the PI3K pathway, which is an important signalling cascade involved in development and immunity that it is frequently altered in disease. This study sought to investigate if PI3K was responsible for integrating P2X7R dependent signalling in primary human T lymphocytes, with an emphasis on regulation of the adhesion molecule CD62L.Results: Whole cell patch clamp electrophysiology is an important technique for characterising ion channel expression. This technique was optimised for the first time in primary human naïve CD4+ T lymphocytes and used to show P2X7R expression in this study. The pharmacology of ATP in the process of CD62L down-regulation in these cells was explored using new P2X7R antagonists with improved selectivity over previous compounds. Remarkably, PI3K/mTOR, MAPK and PKC signalling was shown to be dispensable for this down-regulation of cell surface CD62L expression. However, while investigating novel mechanisms for ATP induced CD62L down-regulation, it was revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R dependent CD62L down-regulation by increasing ATP potency. The mechanism for this was further explored and this effect may arise from enhanced superoxide generation in the mitochondria of rotenone and antimycin A treated cells. Crucially, although ATP alone did not cause apoptosis of cell, perturbation of the mitochondria of cell with these compounds followed by ATP treatment, revealed P2X7R exposure of phosphatidyl serine.Discussion: This major new finding may have implications for the clearance of naïve CD4+ T lymphocytes which have undergone mitochondrial damage. A novel protective mechanism for the potential removal of cells with damaged mitochondria is presented, whereby, P2X7R dependent PS exposure occurs only when cells have enhanced mitochondrial ROS generation. Given the potential role of P2X7R in a number of diseases with a mitochondrial element, the findings of this thesis are of great importance for the targeting of P2X7R in inflammation.

AB - Background: The role of P2X7R in the immune response has been investigated and this receptor clearly has important roles in inflammation. However, the mechanisms which integrate P2X7R activation with biochemical changes in T lymphocytes such as: proliferation, migration and regulation of adhesion molecule expression are less well understood. Many of these processes are controlled by the PI3K pathway, which is an important signalling cascade involved in development and immunity that it is frequently altered in disease. This study sought to investigate if PI3K was responsible for integrating P2X7R dependent signalling in primary human T lymphocytes, with an emphasis on regulation of the adhesion molecule CD62L.Results: Whole cell patch clamp electrophysiology is an important technique for characterising ion channel expression. This technique was optimised for the first time in primary human naïve CD4+ T lymphocytes and used to show P2X7R expression in this study. The pharmacology of ATP in the process of CD62L down-regulation in these cells was explored using new P2X7R antagonists with improved selectivity over previous compounds. Remarkably, PI3K/mTOR, MAPK and PKC signalling was shown to be dispensable for this down-regulation of cell surface CD62L expression. However, while investigating novel mechanisms for ATP induced CD62L down-regulation, it was revealed that pharmacological modulation of mitochondrial complex I or III, but not inhibition of NADPH oxidase, enhanced P2X7R dependent CD62L down-regulation by increasing ATP potency. The mechanism for this was further explored and this effect may arise from enhanced superoxide generation in the mitochondria of rotenone and antimycin A treated cells. Crucially, although ATP alone did not cause apoptosis of cell, perturbation of the mitochondria of cell with these compounds followed by ATP treatment, revealed P2X7R exposure of phosphatidyl serine.Discussion: This major new finding may have implications for the clearance of naïve CD4+ T lymphocytes which have undergone mitochondrial damage. A novel protective mechanism for the potential removal of cells with damaged mitochondria is presented, whereby, P2X7R dependent PS exposure occurs only when cells have enhanced mitochondrial ROS generation. Given the potential role of P2X7R in a number of diseases with a mitochondrial element, the findings of this thesis are of great importance for the targeting of P2X7R in inflammation.

M3 - Doctoral Thesis

ER -