Nitric oxide synthase activity and mRNA expression in chicken macrophages

Abstract

The activity of inducible nitric oxide synthase (iNOS) enzyme was quantified in chicken macrophages. Macrophages from Cornell K-strain (B15B15), GB1 (B13B13), and GB2 (B6B6) chickens and a transformed cell line (MQ-NCSU) were incubated with or without varying concentrations of bacterial lipopolysaccharide (LPS). The culture supernatants were tested for the presence of nitrite. Macrophages from either source produced minimal nitrite (< 4.4 microM/1 x 10(6) cells) levels without LPS stimulation. However, nitrite levels produced by K-strain (42 microM) and MQ-NCSU (41 microM) macrophages were higher (P < 0.05) than those produced by the GB1 (14 microM) and GB2 (14 microM) per 1 x 10(6) macrophages with optimum LPS concentration range of 50 ng to 1 microgram/mL. The addition of an L-arginine analog, NGMMLA, at a concentration of 200 microM completely abolished nitrite production. The addition of 10% vol/vol lymphokines exhibited an additive effect on nitrite production in conjunction with LPS. The increased nitrite production by the K-strain and MQ-NCSU macrophages corresponded to an increased expression of iNOS mRNA as compared to the mRNA produced by GB1 and GB2 macrophages. The iNOS mRNA kinetics study revealed that mRNA levels peaked between 6 to 12 h. The cells from avian lymphoid lineage failed to produce any detectable iNOS activity. These studies showed that macrophages from varying sources differ in NOS activity and implied that genetic background may dictate the extent of arginine-mediated contribution in various biological and immunological functions.