Molecular Regulation of Inducible Nitric Oxide Synthase Gene in Chicken Macrophages

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Title: Molecular Regulation of Inducible Nitric Oxide Synthase Gene in Chicken Macrophages
Author: Dil, Nyla
Advisors: J. B. Allen, Committee Member
P. B. Carter, Committee Member
F. W. Edens, Committee Member
M. A. Qureshi, Committee Chair
Abstract: Inducible nitric oxide synthase serves as an important mediator of macrophage immunobiology by regulating the biosynthesis of nitric oxide that in turn mediates several immunological and physiological functions. Previous studies have demonstrated that macrophages from Cornell K-strain chickens (B15B15) were hyper-responsive to Escherichia coli LPS whereas macrophages from GB1 (B13B13) and GB2 (B6B6) chickens were hypo-responsive for inducible nitric oxide synthase (iNOS) expression and activity. Furthermore, this differential iNOS induction in response to LPS was transcriptionally regulated. The current study was conducted to explore the molecular basis of strain-based differential iNOS induction in chicken macrophages. In the first study, macrophages from K-strain, GB1, GB2 chickens and MQ-NCSU macrophage cell line were exposed to LPS from three sources other than E.coli and both iNOS activity and expression were studied. iNOS mRNA and nitrite data showed that these strain-based differences are intrinsic in nature and not limited to the bacterial source of LPS. In addition, when surface expression of LPS-related receptors was studied via flow cytometry, macrophages from the K-strain exhibited higher numbers of LPS signal transducing molecule TLR4 constitutively in comparison to GB2 macrophages. In the second study, inducible expression of LPS related receptors as well as NFkappaB activation and the involvement of these proteins in iNOS induction was studied. LPS exposure induced significantly higher numbers of inducible TLR4 and CD14 molecules on K-strain macrophages and a significantly higher corresponding increase in NFkappaB activation as compared with GB2 macrophages. Furthermore, blocking studies revealed that CD14, TLR4 and NFkappaB activation were all involved in LPS mediated iNOS induction in chicken macrophages. The objective of the third study was to examine the expression of IL-1beta and to explore if LPS-mediated induction of IL-1beta modulates iNOS induction in these genetic lines of chickens. Initially GB2 and K strain macrophages produced comparable IL-1beta mRNA soon after LPS stimulation. However, IL-1beta mRNA persisted up to 9 h only on GB2 macrophages and LPS-inducible IL-1 surface receptor expression was also found to be higher in GB2 than on K-strain macrophages. In addition, blocking of IL-1 receptor by the anti-IL-1 receptor antibody inhibited the LPS mediated iNOS induction by 50% both in K and GB2 chickens indicating that IL-1beta does not contribute to differential iNOS induction in these chicken lines. Overall these studies demonstrate that strain based differences in iNOS expression in chickens are mediated via enhanced expression of LPS related receptors.
Date: 2002-08-19
Degree: PhD
Discipline: Immunology

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