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

Abstract

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) IFNγ, rh- and rbIL-1β, rbGM-CSF, rhTNFα, rhIL-4, endotoxin (LPS), fetal bovine serum (FBS), mitogen-stimulated bovine splenic supernatant (SS), and purified human TGFβ-1. LPS alone, or in combination with SS, rbIFNγ, or rbIL-1β stimulated production of NO in a time and dose dependent fashion. Recombinant bovine IFNγ, rbIL-1β, and rhTNFα in combination produced maximal stimulation which was not further enhanced by LPS. Recombinant human IFNγ, rhIL-1β, and rbGM-CSF had minimal effect either as single stimuli, or in combination with LPS, rbIFNγ, rbIL-1β, or rhTNFα. Nitric oxide production was inhibited by rhIL-4, and the L-arginine analogue antagonists of iNOS, N- G-monomethyl-L-arginine (N GMMA) and aminoguanidine (AG). Purified human TGFβ-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.