Mammalian nitrate biosynthesis: mouse macrophages produce nitrite and nitrate in response to Escherichia coli lipopolysaccharide.

Abstract

Escherichia coli lipopolysaccharide (LPS)-induced nitrate biosynthesis was studied in LPS-sensitive C3H/He and LPS-resistant C3H/HeJ mice. Intraperitoneal injection of 15 ,ug ofLPS led to a temporary 5-to 6-fold increase in blood nitrate concentration in the C3H/He strain. Levels of nitrate excreted in the urine were also increased. In contrast, no increase was observed in the C3H/HeJ strain with LPS injections up to 175 ,ug. Furthermore, thioglycolate-elicited peritoneal macrophages from C3H/He, but not from C3H/HeJ mice, produced nitrite (60%) and nitrate (40%) when cultured with LPS (10 jig/ml). T-lymphocyte addition/depletion experiments showed the presence of T cells enhanced this response. However, LPS did not cause nitrite or nitrate production in cultures of spleen lymphocytes from either strain. LPS-induced nitrate synthesis was also observed with nude mice and CBA/N mice, indicating that neither functional T lymphocytes nor LPS-responsive B lymphocytes were required for the response in vivo. This was consistent with the in vitro results showing macrophages alone were competent. Mycobacterium bovis infection of C3H/He and C3H/HeJ mice resulted in a large increase in nitrate production over the course of the infection for both strains, suggesting T-lymphocyte-mediated activation of macrophages as a potent stimulus for nitrate biosynthesis.The synthesis of nitrite is significant in that it can directly participate in the endogenous formation of nitrosamines and may also be involved in some aspect of the chemistry of cytotoxicity.A number of studies have now established that mammals on low nitrate diets excrete more nitrate than they ingest (1-3). At first it was thought that this excess urinary nitrate was the product of intestinal microbial metabolism (1), but subsequent studies with germ-free and conventional rats showed that the process of nitrate biosynthesis was by a mammalian pathway (2). The concern for this process stems from the demonstrated carcinogenicity of nitrosamines (4), the association between exposure to high levels of nitrate (NO-) and stomach cancer (5-8), and the potential for the endogenous formation of nitrosamines by reaction of nitrite derived from nitrate by bacterial reduction (9-11) with endogenous or exogenous amines.Tannenbaum and colleagues (12) Frederick, MD). All mice used were 9-12 weeks old except for the BALB/c and CBA mice, which were 7 and 5 weeks old, respectively.Housing and Urine Collection. Mice were housed three to a cage in Nalgene metabolic cages (Thomas) and were given a nutritionally complete low-nitrate diet (12) and distilled drinking water. The urine from three mice was automatically pooled and collected every 24 hr. The cages were cleaned daily with germicidal soap. In all cases, the amount of nitrate in the urine after a 24-hr collection period for three mice was divided by three and reported as the mean daily nitrate production per mouse. As an additional precaution against infection, nude mice were housed in an isolated cubicle, given sterile...

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