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Yi's dissertation Final (modified).pdf (2.99 MB)

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Sequential Priming of Neutrophils

Yao, Yi
http://rave.ohiolink.edu/etdc/view?acc_num=mco1389612809

Abstract Details

2014, Doctor of Philosophy (PhD), University of Toledo, College of Medicine.
Upon recruitment to inflammatory sites, neutrophils markedly elevate phagocyte functionality as measured by oxidative burst and bactericidal activity. The purpose of this study is to characterize this process known as neutrophil “priming.” GeneChip analyses unveiled a cluster of signature genes including CD54, dectin-2, and interleukin-1ß (IL-1ß) that are markedly (>30-fold) elevated in Ly6G+ neutrophils recovered from acute peritonitis lesions compared to those in bone marrow (BM). These observations were confirmed at mRNA and protein levels by real-time PCR and FACS, respectively. Thus, we reasoned that neutrophils undergo priming upon extravasation and/or exposure to granulocyte-macrophage colony-stimulating factor (GM-CSF) present at high levels in inflammatory sites. In fact, BM neutrophils that had migrated toward two C-X-C chemokine receptor ligands, CXCL1 and CXCL2, added to the lower chamber in a standard 2 h chemotaxis assay exhibited markedly elevated production of reactive oxygen species (ROS) and bacterial uptake compared to those that had remained in the upper chamber. Moreover, mRNA and protein expression of the above signature genes occurred preferentially in the “migrated” neutrophil population during subsequent exposure to GM-CSF. Maximal surface expression of CD54 and dectin-2 was observed 16-24 h after exposure to GM-CSF. We next sought to visualize the process of neutrophil priming using our recently constructed transgenic mice expressing DsRed reporter gene under the control of IL-1ß promoter (pIL1-DsRed). In these mice, only small fractions of Ly6G+ neutrophils expressed DsRed fluorescence signals in BM and peripheral blood, whereas robust DsRed signals became readily detectable in virtually all (~95%) neutrophils recovered from inflamed sites of both thioglycollate-induced peritonitis and allergen-induced lung inflammation. DsRed signals also became detectable in a majority of the neutrophils that had migrated toward CXCL1 and CXCL2 in the chemotaxis assay. Furthermore, surface expression of CD54 and dectin-2 occurred almost exclusively within the DsRed+ neutrophil population during culture with GM-CSF, indicating that DsRed expression (i.e., IL-1ß promoter activation) serves as a marker of primed neutrophils. Notably, by testing >60 cytokines and chemokines, GM-CSF was found to be the most potent to induce DsRed expression in neutrophils. To test in vivo relevance of these observations, we performed intravital confocal imaging experiments. Few, if any, DsRed+ cells were found in the skin of DsRed reporter mice under the steady state. Topical application of oxazolone, a contact sensitizer, triggered profound emergence of DsRed+ cells in the extravascular space of inflamed skin. Those DsRed+ cells showed amoeba-like motile behavior and expressed typical neutrophil markers including Ly6G, 7/4 and CD11b, indicating that they were neutrophils. Interestingly, although both DsRed+ and DsRed– neutrophil populations migrated in random directions, the former exhibited significantly higher motility than the latter. Thus, we conclude that neutrophil priming occurs in a sequential manner with rapid acquisition of phagocyte function (ROS production and bacterial uptake), followed by surface expression of CD54 and dectin-2 at the late phase. Importantly, this process of neutrophil priming can now be visualized in reporter mice by monitoring acquisition of DsRed signals. These findings provide a new conceptual framework for our understanding of the process of neutrophil priming.
Akira Takashima (Advisor)
Kevin Pan (Committee Chair)
Fan Dong (Committee Member)
Stanislaw Stepkowski (Committee Member)
Mark Wooten (Committee Member)
120 p.
Immunology

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Citations

  • Yao, Y. (2014). Sequential Priming of Neutrophils [Doctoral dissertation, University of Toledo]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=mco1389612809

    APA Style (7th edition)

  • Yao, Yi. Sequential Priming of Neutrophils. 2014. University of Toledo, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=mco1389612809.

    MLA Style (8th edition)

  • Yao, Yi. "Sequential Priming of Neutrophils." Doctoral dissertation, University of Toledo, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=mco1389612809

    Chicago Manual of Style (17th edition)

mco1389612809
491
© 2014, all rights reserved.
This open access ETD is published by University of Toledo Health Science Campus and OhioLINK.