Date Approved

2014

Document Type

Dissertation

Degree Name

PhD in Cell and Molecular Biology

Department

Cell Biology

College

Graduate School of Biomedical Sciences

First Advisor

Kingsley Yin, PhD

Committee Member 1

John Pastorino, PhD

Committee Member 2

Bernd Spur, PhD

Committee Member 3

Robert Nagele, PhD

Committee Member 4

T. Peter Stein, PhD

Subject(s)

Lipoxins, Neutrophils, Phagocytes, Sepsis, CD 64 Antigens, IgG Receptors

Disciplines

Biological Phenomena, Cell Phenomena, and Immunity | Cell Biology | Laboratory and Basic Science Research | Medicine and Health Sciences | Molecular Biology | Pathogenic Microbiology | Pathological Conditions, Signs and Symptoms

Abstract

During sepsis, neutrophils are inappropriately activated and have prolonged lifespans, thus becoming dysfunctional. Excessive neutrophil activation can lead to tissue injury while neutrophil dysfunction can lead to decreased free radical production and reduced phagocytosis, preventing the host from clearing preexisting infections. Lipoxin A4 (LXA4) is a specialized pro-resolution mediator which reduces neutrophil migration and expression of proinflammatory mediators. Intact neutrophil functions are critical for the host to efficiently clear invading pathogens. The effects of LXA4 on neutrophil function in sepsis have not been established. Using the cecal ligation and puncture (CLP) model of sepsis, LXA4 administered 1 h after sepsis induction reduced blood bacterial load, as well as peritoneal neutrophils iNOS and TNF-α expression. Furthermore, LXA4 increased blood neutrophil apoptosis, decreased free radical (ROS) production and markedly enhanced phagocytic index. When LXA4 was given directly to blood leukocytes ex vivo, apoptosis and ROS generation were not altered. However, LXA4 given ex vivo directly augmented the phagocytic index of blood neutrophils with no effect on peritoneal neutrophils, suggesting that the effects of LXA4 were compartment specific, where it directly enhanced blood neutrophil phagocytic ability while the augmentation of peritoneal neutrophil phagocytic ability observed in vivo is through an indirect mechanism. Immunophenotyping showed that administration of LXA4 in vivo led to increased expression of the Fcγ RI receptor, CD64. Blockade of CD64 led to a dramatic decrease in the phagocytic index of LXA4 treated animals. LXA4 treatment increased the percentage of blood neutrophils with activated caspase 8, both before and after ex vivo phagocytosis of E. coli, suggesting LXA4 promotes neutrophil apoptosis by enhancing phagocytosis induced cell death (PICD). Taken together, the results suggest that LXA4 increased bacterial clearance in blood by enhancing neutrophil phagocytosis through increased surface CD64 expression, resulting in PICD. The resultant neutrophil phenotype is more efficient at clearing pathogens while indirectly triggering cell death and thus reverses sepsis-induced neutrophil dysfunction.

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