Involvement of lipopolysaccharide related receptors and nuclear factor κB in differential expression of inducible nitric oxide synthase in chicken macrophages from different genetic backgrounds

Macrophages from Cornell K-strain chickens (B15B15) are hyper and from GB2 chickens (B6B6) are hypo-responders to LPS-mediated inducible NOS (iNOS) expression and activity. The molecular mechanism(s) responsible for this differential expression is not yet fully understood. We have previously reported that macrophages from K (iNOS hyper-responder) and GB2 (iNOS hypo-responder) chickens differ in constitutive expression of TLR4 but not in CD14 molecules. The objectives of the current study was to determine if the iNOS differences between K and GB2 macrophages are possibly due to differential expression of LPS-induced TLR4, CD14 and/or nuclear factor κB (NFκB). The results showed that Sephadex-elicited, adherence purified K macrophages expressed more inducible TLR4 and CD14 receptors (P<0.05) at 6 and 12 h post-LPS stimulation than GB2 macrophages as measured by flow cytometry. In addition, pre-incubation of macrophages from a transformed chicken macrophage cell line, MQ-NCSU, with 50 μg/ml anti-CD14 and anti-TLR4 antibodies significantly reduced where as pre-incubation with 100 μg/ml completely blocked LPS-mediated iNOS activity as measured by nitrite levels. Furthermore, the amount of nuclear bound NFκB was found to be significantly greater in K than in GB2 macrophages at 30 min post-LPS stimulation. This nuclear localization of NFκB as well as iNOS activity was completely inhibited by pretreatment of macrophages with 50 μM MG132, a proteosome inhibitor, both in K and GB2 macrophages. Taken together, these findings suggest that a differential and perhaps more stronger LPS-mediated signaling via CD14, TLR4 and NFκB is responsible for the heightened iNOS gene induction in K-strain (hyper-responder) macrophages than in GB2 (hypo-responder) chickens.