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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. (Unpublished)

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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
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Thomas, Louis Michaellmt27@pitt.eduLMT27
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairSalter, Russell D.rds@pitt.eduRDS
Committee MemberKane, Lawrence P.lkane@pitt.eduLKANE
Committee MemberWeisz, Ora A.weisz@pitt.eduWEISZ
Committee MemberBinder, Robert J.rjb42@pitt.eduRJB42
Committee MemberWatkins, Simon C.swatkins@pitt.eduSWATKINS
Date: 17 February 2011
Date Type: Completion
Defense Date: 2 February 2011
Approval Date: 17 February 2011
Submission Date: 13 February 2011
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: School of Medicine > Immunology
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: damage associated molecular patterns; endogenous danger signals; exosome; innate immunity; lysosome; microvesicle; phospholipase D
Other ID:, etd-02132011-224435
Date Deposited: 10 Nov 2011 19:31
Last Modified: 15 Nov 2016 13:36


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