Nitric oxide production and expression of inducible nitric oxide synthase by bovine alveolar macrophages

Alveolar macrophages play a central role in host defense in the lower respiratory tract. Production of the reactive intermediate nitric oxide (NO), via expression of inducible nitric oxide synthase (iNOS) is an important microbicidal effector mechanism possessed by macrophages. In this study, cytokine regulation of NO production by bovine alveolar macrophages (bAM) was evaluated. Bovine alveolar macrophages were exposed to one or more of the following: recombinant human (rh) and recombinant bovine (rb) IFN gamma, rh- and rbIL-1 beta, rbGM-CSF, rhTNF alpha, rhIL-4 endotoxin (LPS), fetal bovine serum (FBS), mitogen-stimulated bovine splenic supernatant (SS), and purified human TGF beta-1. LPS alone, or in combination with SS, rbIFN gamma, or rbIL-1 beta stimulated production of NO in a time and dose dependent fashion. Recombinant bovine IFN gamma, rbIL-1 beta, and rhTNF alpha in combination produced maximal stimulation which was not further enhanced by LPS. Recombinant human IFN gamma, rhIL-1 beta, and rbGM-CSF had minimal effect either as single stimuli, or in combination with LPS, rbIFN gamma, rbIL-1 beta, or rhTNF alpha. Nitric oxide production was inhibited by rhIL-4, and the L-arginine analogue antagonists of iNOS, N-(G)-monomethyl-L-arginine (N(G)MMA) and aminoguanidine (AG). Purified human TGF beta-1 did not inhibit NO production. Messenger RNA for iNOS was maximally expressed by 8 h and remained detectable for at least 48 h. Expression of iNOS mRNA induced by cytokines and LPS varied with strength of the stimulus as determined by nitrite production in culture supernatant.