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P2X7 Activation of Non-Primed Myeloid Cells Promotes the Shedding of Stimulatory Materials Within Microvesicles

Thomas, Louis Michael (2011) P2X7 Activation of Non-Primed Myeloid Cells Promotes the Shedding of Stimulatory Materials Within Microvesicles. Doctoral Dissertation, University of Pittsburgh.

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    Abstract

    There is an increasing need to understand how inflammation is initiated by endogenous factors in the absence of infection. Various diseases such as atherosclerosis and arthritis are shaped by endogenous mediators. In situations such as transplant or trauma where there is extensive amount of tissue damage, endogenous factors can be released to influence inflammation. The modes of activation in which immune cells liberate endogenous factors for incurring immune responses remain elusive.Adenosine triphosphate (ATP) activation of the puringeric receptor P2X7 has been implicated in several immune responses. P2X7 promotes the shedding of microvesicles (MV) and the secretion of inflammatory mediators. I hypothesized that P2X7-induced MV containing some of these inflammatory mediators would promote the activation of innate immune cells such as macrophages.Using murine bone marrow derived macrophages as a model for macrophage function, I describe that harvested P2X7-induced MV from myeloid cells promote macrophage activation including pro-inflammatory cytokine secretion and co-stimulatory ligand upregulation. Phospholipids from P2X7-induced MV are partially responsible for the observed macrophage activation. Isolated phospholipids from P2X7-induced MV activate TLR4. Secondly, I describe mature cathepsin D release into P2X7-induced MV from myeloid cells.P2X7-induced MV from myeloid cells contain both intermediate and mature forms of cathepsin D. Furthermore, P2X7 stimulation of myeloid cells promotes the peripheral displacement of cathepsin D and dynamin. Dynasore, a selective and potent dynamin inhibitor, significantly reduced the secretion of mature but not intermediate cathepsin D.Lastly, I describe a novel morphological alteration following P2X7 activation of myeloid cells. ATP stimulates de novo filopodia production. These filopodia are the result of actin polymerization, Rho kinases, and phospholipases. Furthermore, P2X7 promotes the re-localization of lipids and actin-based machinery to the periphery of ATP treated cells.Collectively, these results demonstrate that P2X7-induced MV possess stimulatory cargo including phospholipids that can activate macrophages and cathepsins that are potentially capable of degrading extracellular matrix components. This data would suggest a provocative role for P2X7-induced MV and actin-based processes in promoting sterile disease.


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    Item Type: University of Pittsburgh ETD
    ETD Committee:
    ETD Committee TypeCommittee MemberEmail
    Committee ChairSalter, Russell D.rds@pitt.edu
    Committee MemberKane, Lawrence P.lkane@pitt.edu
    Committee MemberWeisz, Ora A.weisz@pitt.edu
    Committee MemberBinder, Robert J.rjb42@pitt.edu
    Committee MemberWatkins, Simon C.swatkins@pitt.edu
    Title: P2X7 Activation of Non-Primed Myeloid Cells Promotes the Shedding of Stimulatory Materials Within Microvesicles
    Status: Unpublished
    Abstract: There is an increasing need to understand how inflammation is initiated by endogenous factors in the absence of infection. Various diseases such as atherosclerosis and arthritis are shaped by endogenous mediators. In situations such as transplant or trauma where there is extensive amount of tissue damage, endogenous factors can be released to influence inflammation. The modes of activation in which immune cells liberate endogenous factors for incurring immune responses remain elusive.Adenosine triphosphate (ATP) activation of the puringeric receptor P2X7 has been implicated in several immune responses. P2X7 promotes the shedding of microvesicles (MV) and the secretion of inflammatory mediators. I hypothesized that P2X7-induced MV containing some of these inflammatory mediators would promote the activation of innate immune cells such as macrophages.Using murine bone marrow derived macrophages as a model for macrophage function, I describe that harvested P2X7-induced MV from myeloid cells promote macrophage activation including pro-inflammatory cytokine secretion and co-stimulatory ligand upregulation. Phospholipids from P2X7-induced MV are partially responsible for the observed macrophage activation. Isolated phospholipids from P2X7-induced MV activate TLR4. Secondly, I describe mature cathepsin D release into P2X7-induced MV from myeloid cells.P2X7-induced MV from myeloid cells contain both intermediate and mature forms of cathepsin D. Furthermore, P2X7 stimulation of myeloid cells promotes the peripheral displacement of cathepsin D and dynamin. Dynasore, a selective and potent dynamin inhibitor, significantly reduced the secretion of mature but not intermediate cathepsin D.Lastly, I describe a novel morphological alteration following P2X7 activation of myeloid cells. ATP stimulates de novo filopodia production. These filopodia are the result of actin polymerization, Rho kinases, and phospholipases. Furthermore, P2X7 promotes the re-localization of lipids and actin-based machinery to the periphery of ATP treated cells.Collectively, these results demonstrate that P2X7-induced MV possess stimulatory cargo including phospholipids that can activate macrophages and cathepsins that are potentially capable of degrading extracellular matrix components. This data would suggest a provocative role for P2X7-induced MV and actin-based processes in promoting sterile disease.
    Date: 17 February 2011
    Date Type: Completion
    Defense Date: 02 February 2011
    Approval Date: 17 February 2011
    Submission Date: 13 February 2011
    Access Restriction: No restriction; Release the ETD for access worldwide immediately.
    Patent pending: No
    Institution: University of Pittsburgh
    Thesis Type: Doctoral Dissertation
    Refereed: Yes
    Degree: PhD - Doctor of Philosophy
    URN: etd-02132011-224435
    Uncontrolled Keywords: damage associated molecular patterns; endogenous danger signals; exosome; innate immunity; lysosome; microvesicle; phospholipase D
    Schools and Programs: School of Medicine > Immunology
    Date Deposited: 10 Nov 2011 14:31
    Last Modified: 17 Feb 2012 14:10
    Other ID: http://etd.library.pitt.edu/ETD/available/etd-02132011-224435/, etd-02132011-224435

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