Submitted to: American Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: March 31, 2004
Publication Date: April 1, 2004
Citation: Li, C., Carbaugh, D., Kahl, S., Elsasser, T.H. 2004. Concomitant dual wavelength fluorescence evaluation of inducible nitric oxide synthase and cytokine responses to endotoxin stimulation of peripheral blood mononuclear cells [abstract]. Journal of Animal Science 82(Suppl.1):180. Technical Abstract: The free radical nitric oxide (NO) or a NO-releasing product is synthesized within mammalian immune, cardiovascular, and neural systems, where it functions as a signaling or cytotoxic molecular. Cytokines exert autocrine and paracrine effects on the immune response of mammals and some of which may be mediated by inducible nitric oxide synthase (i-NOS). On the turn, enhanced formation of NO, especially by inducible nitric oxide synthase (i-NOS) is largely stimulated by tumor necrosis factor (TNF). Since cytokines are rapidly released from producing cells upon stimulation, it is difficult to evaluate the cellular level of cytokine, as well as its correlation to the i-NOS activity. In the present work, we analyzed the effects of LPS and cytokines on i-NOS activity and the correlation between intracellular cytokines and i-NOS in bovine peripheral blood mononuclear cells (PBMC) and possible signaling pathway that regulates the i-NOS activity. After in vivo and in vitro activation in the presence of protein transport inhibitor to facilitate the accumulation of intracellular accumulation of cytokine, bovine peripheral blood mononuclear cells were analyzed by immunofluorescent staining and flow cytometry and Western blot. Our data indicate that upon activation, the intracellular levels of TNF and i-NOS increases significantly (P< 0.01) and the levels of TNF and i-NOS are correlated in monocytes. We also investigated the interaction between TNF and i-NOS and the possible role of specific phosphorylations in the regulation of the function of i-NOS. By understanding the functional inportance of i-NOS and cytokines in the animal immune responses, we then shall be able to develop strategies to maintain animal health.