The expression and regulation of inducible nitric oxide synthase gene differ in macrophages from chickens of different genetic background

Abstract

It has been previously reported that lipopolysaccharide (LPS)-stimulated macrophages from Cornell K-Strain chickens (B 15B 15) and a transformed cell line, MQ-NCSU, (broiler origin) produced significantly higher levels of inducible nitric oxide synthase (iNOS) mRNA than macrophages isolated from GB1 (B 13B 13) and GB2 (B 6B 6) chickens. The purpose of this study was to determine the basis of such differential iNOS gene expression and to study the relationship of high or low expression of iNOS mRNA with iNOS enzyme activity in macrophages from GB2 (low iNOS mRNA expresser), K-strain and MQ-NCSU (high iNOS mRNA expressers). The enzyme activity in lysates from LPS-stimulated macrophages was lower in GB2 (range: 23 to 41 μM, P<0.05) as compared with the K-strain and MQ-NCSU macrophages that exhibited intermediate (range: 27 to 59 μM) and the highest (range: 144 to 217 μM) activity, respectively. Total RNA collected from LPS-treated macrophages at various time-points post-actinomycin D treatment revealed comparable iNOS mRNA levels in MQ-NCSU, GB2, and K-strain macrophages, suggesting that post-transcriptional regulation mechanism(s) do not account for the difference in iNOS mRNA expression. To determine if differences in the transcription rate are the basis of the differential iNOS gene expression, macrophages were stimulated with or without LPS and nuclei-isolated. Inducible NOS mRNA probes were generated and hybridized with immobilized iNOS cDNA (reverse Northern blot). The resulting lumigraph yielded enhanced transcriptional activity from K-strain and MQ-NCSU macrophages whereas this activity was lower in GB2 macrophages. Therefore, these studies suggest that the previously reported genetically-based difference in iNOS mRNA expression further translates into differences in iNOS enzyme activity, and that the iNOS gene in chickens is transcriptionally regulated.