Date of Award

12-12-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Pharmacology, Physiology and Therapeutics

First Advisor

James E. Porter

Abstract

The adrenergic receptor (AR) family is a group of G protein-coupled receptors activated by the endogenous catcholamines epinephrine (Epi) and norepinephrine (NE). There are nine different AR subtypes that are differentially expressed throughout the body including on immune cells. Levels of Epi and NE are known to influence immune responses however, the AR subtype expression and function are not well characterized. The purpose of this study was to: 1) characterize the AR subtype expression profile on monocytes and macrophages; 2) elucidate how AR activation regulates cytokine/chemokine production; and 3) determine the mechanism(s) by which individual AR subtypes modulate cytokine production. Traditional immunoblot methods and radioligand binding assays with subtype-selective antagonists were used to characterize AR profiles on monocyte and macrophages. Human monocytes expressed a homogenous population of α1B-ARs that switched to a heterogenous population of α1A- and α 1D-ARs upon differentiation into macrophages. Murine macrophages also expressed a homogenous α1B-AR population, while radioligand binding demonstrated no α2-AR expression on either human monocytes or macrophages. A heterogenous β1- and β 2-AR population was demonstrated to be expressed on human monocytes. Commercially available inflammatory mediator antibody arrays were used to characterize global changes in cytokine/chemokine production from cells stimulated with a selective AR agonist in combination with lipopolysaccharide (LPS), an activator of Toll-like receptor (TLR)4. LPS-mediated IL-1β production was synergistically increased with both α1B- and β 1-AR stimulation therefore, signaling mechanisms associated with this change were examined. Immunoblots examining mitogen-activated protein kinase (MAPK) activation with α1-AR and TLR4 stimulation demonstrated increased p38 MAPK phosphorylation, which was necessary for increases in IL-1β. RNA interference showed β-arrestin (β-ARR)2 scaffolding to MEK3/6 was required for p38 MAPK activation by α1-ARs and TLR4 however, β-ARR1 was necessary for enhanced IL-1β production. Luciferase and electrophoretic gel mobility shift assays (EMSA) demonstrated α1-ARs and TLR4 stimulation inhibited NF-κB while activating the transcription factor AP-1, which was necessary for IL-1β responses. Schild analysis linked β1-AR activation to cAMP generation, which was required for synergistic IL-1β generation with β-AR stimulation. Immunoblots demonstrated PKA activated JNK and p38 MAPK pathways with β-AR stimulation, which were necessary for synergistic increases in IL-1β production.

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