Adrenergic Receptor Gene Expression in Bovine Leukocytes and Effects of Adrenergic Agonists on Neutrophil and Eosinophil Function
Mammalian responses to stressors that alter homeostasis are mediated by the hypothalamic-pituitary-adrenal (HPA) and sympathetic-adrenal-medullary (SAM) axes. The SAM axis releases catecholamines, which include the neurotransmitters epinephrine (E) and norepinephrine (NE). The α- and β-adrenergic receptors (ARs) are encoded by 6 α- and 3 β-AR genes and these ARs mediate interactions between the stress hormones E and NE and immune cells. However, AR gene expression and function have been studied to a limited extent in bovine immune cells. Furthermore, a thorough survey has never been published in any species to determine which of the 9 AR genes are expressed in blood leukocytes and if there are significant differences in AR gene expression when comparing among leukocyte lineages. In this study, AR gene expression was quantified in bovine leukocytes isolated from whole blood, peripheral blood mononuclear cells (PBMCs), polymorphonuclear cells (PMNs), and purified T cells, B cells, monocytes, innate lymphoid cells, neutrophils and eosinophils. While transcript abundance for the β2-, α2A-, and α1A-AR genes tended to be greatest in leukocytes isolated from bovine whole blood, marked differences in AR gene expression were observed when leukocytes were separated into individual lineages. Variation in AR gene expression among leukocyte lineages provided the first evidence that individual bovine leukocyte lineages may also differ in their responses to E and NE. Adrenergic receptor gene expression was also quantified in whole blood leukocytes following maternal separation (weaning) and transportation of suckling beef calves. These stressors were associated with significant increases in transcript abundance for the β1-, β2, β3-, and α2A-AR genes in blood leukocytes at different time points throughout the 28-day post-weaning and transportation period. Thus, the capacity of the immune system to respond to E and NE may increase significantly when animals respond to stressful changes in their environment. Following confirmation that AR genes were expressed in bovine leukocytes, PMNs were chosen for further analysis of AR gene expression and function. I developed and validated an appropriate flow cytometric method to specifically identify neutrophils and eosinophils and then flow cytometry was used to analyze the response of resting and activated PMN to adrenergic agonists. Eosinophils were identified as autofluorescence high and CD44 high cells, while neutrophils were characterized as autofluorescence low and CD44 low. PMNs are frequently assumed to be primarily neutrophils with a small proportion of eosinophils. However, I observed, depending on the individual animal and time of year, eosinophils comprised 1.8 - 29.4% of isolated PMN populations. Subsequent analysis of neutrophils and eosinophils confirmed each population responded differently to co-stimulation with opsonized zymosan and IFN and displayed significantly different responses to adrenergic agonists. Short-term treatment with E, NE, phenylephrine (an α1-AR agonist), dexmedetomidine (an α2-AR agonist), and isoproterenol (a β-AR agonist) modified neutrophil and eosinophil expression of intracellular reactive oxygen species, CD11b, L-selectin, CD16 and CD44. Analysis of these markers revealed significant differences in the response of these two PMN subpopulations to these adrenergic agonists. Resting neutrophils demonstrated an L-selectin “shedding” response to E and NE, consistent with a phenotype which may lead to decreased neutrophil exit from, and increased marginated neutrophil entry, into circulation. At the same time, resting neutrophils treated with E and NE increased CD11b and intracellular reactive oxygen species (iROS), consistent with cell activation. This short-term state could be representative of increased neutrophil surveillance in response to stress, leading to an increased inflammatory response to tissue pathogens, as in the case of bovine respiratory disease. This response is consistent with the short-term neutrophilia and leukocyte activation that has been observed in response to multiple stressors such as weaning and transportation in cattle. Similar responses were observed in activated neutrophils in response to NE. My research also demonstrated a potential role for eosinophils during a stress response, as eosinophils responded to NE with an activation phenotype (L-selectin high, CD11b high, CD44 high, CD16 high). This eosinophil activation may augment short-term tissue invasion by eosinophils following stimulation with agonists such as NE. Interestingly, synthetic agonists targeting individual AR families (phenylephrine, dexmedetomidine, and isoproterenol) induced some responses that were similar to those observed with E and NE. However, these agonists consistently decreased iROS in both neutrophils and eosinophils. Further research is required to determine why physiological and synthetic AR agonists had different effects on iROS. Collectively, my analyses of AR gene expression provided evidence that the sympathetic-adrenal-medullary (SAM) axis may interact with all the leukocyte subpopulations examined. Further work is required to determine how this interaction may alter the function of individual leukocyte lineages or subpopulations. My analysis of AR function in PMNs provided evidence both neutrophils and eosinophils express functional 1-, 2- and -AR, but these two PMN subpopulations differ significantly in their response to adrenergic agonists. However, adrenergic agonists consistently increased iROS and altered expression of surface adhesion molecules by resting neutrophils and eosinophils. Therefore, an acute stress response with increased release of catecholamines could rapidly increase the capacity of neutrophils and eosinophils to respond to infection or tissue damage.
adrenergic receptors, polymorphonuclear cells
Master of Science (M.Sc.)
School of Public Health
Vaccinology and Immunotherapeutics