L-Arginine-Dependent Production of a Reactive Nitrogen Intermediate by Macrophages of a Uricotelic Species

Abstract

L-arginine-dependent production of reactive nitrogen intermediates (RNIs: nitric oxide, nitrite, and nitrate) by mammalian macrophages has been proposed to occur via an L-arginine oxidative deimination pathway and is known to be responsible for certain antineoplastic and antimicrobial effector functions. The present study represents the first examination of this pathway in a non-mammalian vertebrate. Because chickens, unlike mammals, lack a urea cycle and are incapable of de novo synthesis of L-arginine, the possible existence of an avian macrophage pathway for production of RNIs is questionable. We have defined conditions under which chicken macrophages are able to produce nitrite. Sephadex-elicited chicken peritoneal macrophages required a bacterial lipopolysaccharide (LPS from Escherichia coli) signal to produce nitrite during 24 hour cultures in the presence of L-arginine. As little as 5 ng/ml LPS resulted in significant nitrite production in culture. The relationship of nitrite production to both LPS and L-arginine levels was dose-dependent. D-arginine was unable to substitute for L-arginine but also produced no inhibitory effect. In contrast, L-NG-monomethyl arginine showed a significant inhibitory effect on nitrite production. A virus-transformed chicken macrophage cell line, HD11, also produced nitrite in a dose-dependent manner relative to both LPS and L-arginine concentration. Concentrations as low as 5 ng/ml LPS and 0.1 mM L-arginine resulted in significant nitrite production, while maximum levels of nitrite production were obtained using greater than or equal to 0.5 micrograms/ml LPS and greater than or equal to 0.4 mM L-arginine. These results indicate that chicken macrophages can produce RNIs. This production is dependent upon activation and is influenced by local L-arginine concentration. Moreover, because the chicken does not possess the ability to synthesize arginine and has an absolute nutritional requirement for this amino acid, the chicken represents a highly controllable system to examine the in vivo effects of L-arginine on macrophage-related immune functions.

Avian Reovirus Major μ-Class Outer Capsid Protein Influences Efficiency of Productive Macrophage Infection in a Virus Strain-Specific Manner

“…Incubation of HD-11 cells in the presence of LPS stimulated iNOS activity, as indicated by the high levels of sodium nitrite detected in culture supernatants at 12 h posttreatment (Fig. 3) and as previously reported (33). Neither virus was capable of inhibiting NO production by activated macrophages (Fig.…”

“…The HD-11 myelomonocytic cell line is a chicken bone marrow-derived cell line obtained by transformation with the replication-defective avian retrovirus MC29 (1). This cell line displays many macrophage-like properties, including high phagocytic capability and expression of Fc receptors, secretion of interleukin-1B, macrophage inflammatory protein 1␣, interleukin-8, and tumor necrosis factor alpha (TNF-␣), and iNOS activity following stimulation with TNF-␣ and/or LPS (1,31,33,39,40). We used the HD-11 cell line as a model system to assess ARV-176 and ARV-138 macrophage interactions using a focus-forming assay.…”

Campylobacter jejuni-Induced Cytokine Responses in Avian Cells

“…Nitric oxide production was measured by assaying cell culture supernatant fluid for the presence of nitrite, using the standard Griess assay (9,40). The absorbance was read at 550 nm, using an Anthos Lab Systems microtiter plate reader (Labtech, Ringmer, United Kingdom).…”

The effects of nitric oxide on the immune response during giardiasis

“…This is confi rmed by the wide occurrence of the enzyme responsible for NO production: NO-synthase (NOS) responses in several species, ranging from invertebrates such as Limulus polyphemus, 5 insects, 6,7 to mammals and non-mammal vertebrates. [8][9][10] The NO signaling mechanisms would evolve only later for signaling. In mammals, the NO response is raised in response to infection by a wide variety of intracellular pathogen unicellular organisms such as bacteria, yeast and parasites (e.g., Leishmania spp.…”