Mice lacking inducible nitric oxide synthase are not resistant to lipopolysaccharide-induced death.

Abstract

Nitric oxide produced by cytokine-inducible nitric oxide synthase (iNOS) is thought to be important in the pathogenesis of septic shock To further our understanding of the role of iNOS in normal biology and in a variety of inflammatory disorders, including septic shock, we have used gene targeting to generate a mouse strain that lacks iNOS. Mice lacking iNOS were indistinguishable from wild-type mice in appearance and histology. Upon treatment with lipopolysaccharide and interferon y, peritoneal macrophages from the mutant mice did not produce nitric oxide measured as nitrite in the culture medium. In addition, lysates of these cells did not contain iNOS protein by immunoblot analysis or iNOS enzyme activity. In a Northern analysis of total RNA, no iNOS transcript of the correct size was detected. No increases in serum nitrite plus nitrate levels were observed in homozygous mutant mice treated with a lethal dose of lipopolysaccharide, but the mutant mice exhibited no significant survival advantage over wild-type mice. These results show that lack of iNOS activity does not prevent mortality in this murine model for septic shockIn biological systems, nitric oxide (NO) is produced via the oxidation of L-arginine by enzymes known as nitric oxide synthases (NOSs). Three NOS isozymes have been described (for recent reviews see refs. 1 and 2). These include constitutively expressed neuronal (3, 4) and endothelial (5, 6) enzymes and a cytokine-induced enzyme found in macrophages (7), hepatocytes (8), and a variety of other cells (9, 10). Although the biological functions of these enzymes are not completely understood, neuronal NOS is thought to play a role in neurotransmission (11), endothelial NOS is involved in regulation ofvascular tone (12, 13), and inducible NOS (iNOS) is involved in immune defense (14,15). The constitutively synthesized neuronal and endothelial enzymes produce small amounts of NO in response to increases in intracellular calcium levels. In contrast, iNOS is synthesized de novo in response to a variety of inflammatory stimuli and produces large amounts of NO over prolonged periods of time (16). NO produced by iNOS has been shown to be beneficial through its antitumor (17, 18) and antimicrobial (15) activities, but it is also thought to cause tissue damage and contribute to pathology in a variety of inflammatory conditions including rheumatoid arthritis (19,20), inflammatory bowel disease (21), and septic shock (22,23).Septic shock is usually the result of a systemic Gram-negative bacterial infection and is characterized by hypotension and the failure of a number of organ systems, especially the liver, kidney, and heart (24). The bacterial membrane component, lipopolysaccharide (LPS), induces the production of host inflammatory mediators such as tumor necrosis factor a, interferon y (IFN-,y), and interleukin 1,B, which in turn cause an increase in the expression of iNOS. The large amount of NO produced by iNOS has been hypothesized to contribute to LPS-induced hypotension and mortality.To be...

Prevention of neural tube defects by loss of function of inducible nitric oxide synthase in fetuses of a mouse model of streptozotocin-induced diabetes

“…The genetic background of iNos −/− mice is C57BL/6J. The iNos −/− line of mice has been previously established [24,25]; although they are deficient in iNOS expression, they are nevertheless fertile and no breeding problems have been reported [26]. The mice were bred under specific pathogen-free conditions at the Animal Facility of Nagoya University.…”

Characterization of the non‐adrenergic/non‐cholinergic response to perivascular nerve stimulation in the double‐perfused mesenteric bed of the mouse

“…Breeding couples of homozygous iNOS KO (Laubach et al, 1995) or eNOS KO (Shesely et al, 1996) mice, with the same C57BL/6J genetic background, were obtained from Jackson Laboratory (Bar Harbour, ME, USA). All animals were kept at constant room temperature (723 1C) and humidity (B78%) under a controlled light-dark cycle (0600-1800 hours) with free access to food and water.…”

Cutting Edge: Systemic Inhibition of Angiogenesis Underlies Resistance to Tumors During Acute Toxoplasmosis

“…Macrophages destroy tumor cells using two principal mediators, NO produced by the enzyme inducible NO synthase and TNF-␣ (15,16). Because B16 melanomas cells are sensitive to NO but not TNF-␣ (17), mice lacking inducible NO synthase (18) were chosen for further studies. In addition, neoplastic cells could be destroyed by CTLs and NK cells; thus we also used scid-beige mice where CTLs are absent and NK cells are not cytotoxic, and perforin (Pfp) knockout mice where cytolytic functions are essentially absent (19).…”