Search

We tested the hypotheses that 1) systemic IL-10, after adenoviral gene transfer, protects arteries from impaired relaxation produced by LPS; 2) local expression of IL-10 within the arterial wall protects against vasomotor dysfunction after LPS; and 3) IL-10 protects against vascular dysfunction mediated by inducible NO synthase (iNOS) after LPS. In IL-10-deficient (IL-10-/-) and wild-type (WT, IL-10+/+) mice, LPS in vivo impaired relaxation of arteries to acetylcholine and gene transfer of IL-10 improved responses to acetylcholine. Superoxide levels were elevated in arteries after LPS, and increased levels of superoxide were prevented by gene transfer of IL-10. In arteries incubated with a low concentration of LPS in vitro to eliminate systemic effects of LPS and IL-10 from nonvascular sources, responses to acetylcholine were impaired in IL-10-deficient mice and impairment was largely prevented by gene transfer in vitro of IL-10. In arteries from WT mice in vitro, the low concentration of LPS did not impair responses to acetylcholine. Thus IL-10 within the vessel wall protects against LPS-induced dysfunction. In IL-10-deficient mice, aminoguanidine, which inhibits iNOS, protected against vasomotor dysfunction after LPS. In arteries from iNOS-deficient mice, LPS did not impair responses to acetylcholine. These findings suggest that both systemic and local effects of IL-10 provide important protection of arteries against an inflammatory stimulus and that IL-10 decreases iNOS-mediated impairment of vasorelaxation after LPS.

The injection of repeated doses of lipopolysaccharide (LPS) results in attenuation of the febrile response, which is called endotoxin tolerance. We tested the hypothesis that nitric oxide (NO) arising from inducible NO synthase (iNOS) plays a role in endotoxin tolerance, using not only pharmacological trials but also genetically engineered mice. Body core temperature was measured by biotelemetry in mice treated with NG-monomethyl-L-arginine (L-NMMA, 40 mg/kg; a nonselective NO synthase inhibitor) or aminoguanidine (AG, 10 mg/kg; a selective iNOS inhibitor) and in mice deficient in the iNOS gene (iNOS KO) mice. Tolerance to LPS was induced by means of three consecutive LPS (100 microg/kg) intraperitoneal injections at 24-h intervals. In wild-type mice, we observed a significant reduction of the febrile response to repeated administration of LPS. Injection of L-NMMA and AG markedly enhanced the febrile response to LPS in tolerant animals. Conversely, iNOS-KO mice repeatedly injected with LPS did not become tolerant to the pyrogenic effect of LPS. These data are consistent with the notion that NO modulates LPS tolerance in mice and that iNOS isoform is involved in NO synthesis during LPS tolerance.

The aim of this study was to evaluate the impact of the lack of inducible NO synthase (iNOS) on body weight and adipose tissue mass as well as on plasma leptin and adiponectin in basal conditions and 6 h after lipopolysaccharide (LPS) administration in mice. Body weight was not different among male, six-week-old wild-type (WT) and iNOS-/- animals. However, the amount of epididymal white adipose tissue (EWAT) in iNOS-/- mice was significantly reduced (P<0.05). Circulating leptin and leptin mRNA in EWAT were decreased in iNOS-/- mice (P<0.05 and P<0.01, respectively). Plasma adiponectin and adiponectin mRNA were unchanged. LPS administration increased plasma leptin in both genotypes (P<0.05). Neither genotype nor treatment changed plasma adiponectin. In summary, iNOS-/- mice exhibited normal body weight but reduced adipose mass accompanied by hypoleptinemia. Leptin responsiveness to LPS in iNOS-/- mutants is preserved, showing that the LPS-induced rise in leptin is independent of the presence of functional iNOS. In addition, iNOS deficiency or LPS does not influence expression and circulating levels of adiponectin.

Acute respiratory distress syndrome (ARDS) is a life-threatening ailment characterized by severe lung injury involving inflammatory cell recruitment to the lung, cytokine production, surfactant dysfunction, and up-regulation of nitric oxide synthase 2 (NOS2) resulting in nitric oxide (NO) production. We hypothesized that NO production from NOS2 expressed in lung parenchymal cells in a murine model of ARDS would correlate with abnormal surfactant function and reduced surfactant protein-B (SP-B) expression. Pulmonary responses to nebulized endotoxin (lipopolysaccharide, LPS) were evaluated in wild-type (WT) mice, NOS2 null (-/-) mice, and NOS2-chimeric animals derived from bone marrow transplantation. NOS2-/- animals exhibited significantly less physiologic lung dysfunction and loss of SP-B expression than did WT animals. However, lung neutrophil recruitment and bronchoalveolar lavage cytokine levels did not significantly differ between NOS2-/- and WT animals. Chimeric animals for NOS2 exhibited the phenotype of the recipient and therefore demonstrated that parenchymal production of NOS2 is critical for the development of LPS-induced lung injury. Furthermore, administration of NO donors, independent of cytokine stimulation, decreased SP-B promoter activity and mRNA expression in mouse lung epithelial cells. This study demonstrates that expression of NOS2 in lung epithelial cells is critical for the development of lung injury and mediates surfactant dysfunction independent of NOS2 inflammatory cell expression and cytokine production.

Asthma results from an intrapulmonary allergen-driven Th2 response and is characterized by intermittent airway obstruction, airway hyperreactivity, and airway inflammation. An inverse association between allergic asthma and microbial infections has been observed. Microbial infections could prevent allergic responses by inducing the secretion of the type 1 cytokines, IL-12 and IFN-gamma. In this study, we examined whether administration of bacterial LPS, a prototypic bacterial product that activates innate immune cells via the Toll-like receptor 4 (TLR4) could suppress early and late allergic responses in a murine model of asthma. We report that LPS administration suppresses the IgE-mediated and mast cell-dependent passive cutaneous anaphylaxis, pulmonary inflammation, airway eosinophilia, mucus production, and airway hyperactivity. The suppression of asthma-like responses was not due to Th1 shift as it persisted in IL-12(-/-) or IFN-gamma(-/-) mice. However, the suppressive effect of LPS was not observed in TLR4- or NO synthase 2-deficient mice. Our findings demonstrate, for the first time, that LPS suppresses Th2 responses in vivo via the TLR4-dependent pathway that triggers NO synthase 2 activity.

Although platelets have been implicated in the pathogenesis of vascular diseases, little is known about factors that regulate interactions between platelets and the vessel wall under physiological conditions. The objectives of this study were to 1) define the contribution of nitric oxide (NO) to endotoxin (lipopolysaccharide, LPS)-induced platelet-endothelial cell (P/E) adhesion in murine intestinal venules and 2) determine whether the antiadhesive action of NO is mediated by soluble guanylate cyclase (sGC). Adhesive interactions between platelets and endothelial cells were monitored by intravital microscopy. LPS administration into control wild-type mice (WT) resulted in a >15-fold increase in P/E adhesion. Similar responses were observed using endothelial NO synthase (eNOS)-deficient platelets. However, treatment with the NO donor diethylenetriamine-nitric oxide (DETA-NO) attenuated the P/E adhesion response to LPS, whereas the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester or eNOS deficiency resulted in an exacerbation. P/E adhesion response did not differ between LPS-treated WT and inducible NOS-deficient mice. Inhibition of sGC abolished the attenuating effects of DETA-NO, whereas the sGC activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole (YC-1) reduced LPS-induced P/E adhesion. These findings indicate that 1) eNOS-derived NO attenuates endotoxin-induced P/E adhesion and 2) sGC is responsible for the antiadhesive action of NO.

Lipopolysaccharide (LPS, a Gram-negative bacterium cell wall component) is a potent macrophage activator that inhibits macrophage proliferation and stimulates production of nitric oxide (NO) via NO synthase II (NOSII). We investigated whether NO mediates the LPS-stimulated cell cycle arrest in mouse bone marrow-derived macrophages (BMM). The addition of the NO donor DETA NONOate (200 microM) inhibited BMM proliferation by approx. 80%. However, despite NO being an antimitogen, LPS was as potent at inhibiting proliferation in BMM derived from NOSII-/- mice as from wild-type mice. Consistent with these findings, LPS-induced cell cycle arrest in normal BMM was not reversed by the addition of the NOSII inhibitor S-methylisothiourea. Moreover, in both normal and NOSII-/- BMM, LPS inhibited the expression of cyclin D1, a protein that is essential for proliferation in many cell types. Despite inhibiting proliferation DETA NONOate had no effect on cyclin D1 expression. Our data indicate that while both LPS and NO inhibit BMM proliferation, LPS inhibition of BMM proliferation can occur independently of NOSII induction.

Although bacterial endotoxins [lipopolysaccharide (LPS)] can confer tissue resistance to subsequent inflammatory insults, the mechanisms that underlie this LPS-preconditioning (LPS-PC) response remain poorly defined. The dual-radiolabeled monoclonal antibody technique was used to examine whether LPS-PC alters the upregulation (protein) of E- and P-selectins after subsequent LPS challenge. In the gut of wild-type (C57BL/6J) mice, LPS-PC was associated with a reduction in E- (66%) and P-selectin (33%) expression. A similar reduction in E-selectin expression was observed in mutant mice that were genetically deficient in either the endothelial or inducible isoform of nitric oxide synthase or that overexpressed the human gene for Cu/Zn superoxide dismutase. Severe combined immunodeficient mice, genetically devoid of lymphocytes, did exhibit partial inhibition of the LPS-PC response. We conclude that 1) LPS-PC can be demonstrated for E- and P-selectins in some vascular beds (e.g., gut), 2) the mechanism(s) underlying this blunted selectin response does not include a major role for either nitric oxide and superoxide, and 3) circulating lymphocytes may contribute to the LPS-PC response.

The deleterious effects of lipopolysaccharide (LPS) during endotoxic shock are associated with the secretion of tumor necrosis factor (TNF) and the production of nitric oxide (NO), both predominantly released by tissue macrophages. We analyzed the mechanism by which LPS induces apoptosis in bone marrow-derived macrophages (BMDM). LPS-induced apoptosis reached a plateau at about 6 hours of stimulation, whereas the production of NO by the inducible NO-synthase (iNOS) required between 12 and 24 hours. Furthermore, LPS-induced early apoptosis was only moderately reduced in the presence of an inhibitor of iNOS or when using macrophages from iNOS -/-mice. In contrast, early apoptosis was paralleled by the rapid secretion of TNF and was almost absent in macrophages from mice deficient for one (p55) or both (p55 and p75) TNF-receptors. During the late phase of apoptosis (12-24 hours) NO significantly contributed to the death of macrophages even in the absence of TNF-receptor signaling. NO-mediated cell death, but not apoptosis induced by TNF, correlated with the induction of p53 and Bax genes. Thus, LPS-induced apoptosis results from 2 independent mechanisms: first and predominantly, through the autocrine secretion of TNF-alpha (early apoptotic events), and second, through the production of NO (late phase of apoptosis). (Blood. 2000;95:3823-3831)

Background: In animal models, endotoxin (lipopolysaccharide) challenge impairs the pulmonary vasodilator response to inhaled nitric oxide (NO). This impairment is prevented by treatment with inhibitors of NO synthase 2 (NOS2), including glucocorticoids and L-arginine analogs. However, because these inhibitors are not specific for NOS2, the role of this enzyme in the impairment of NO responsiveness by lipopolysaccharide remains incompletely defined., Methods: To investigate the role of NOS2 in the development of lipopolysaccharide-induced impairment of NO responsiveness, the authors measured the vasodilator response to inhalation of 0.4, 4, and 40 ppm NO in isolated, perfused, and ventilated lungs obtained from lipopolysaccharide-pretreated (50 mg/kg intraperitoneally 16 h before lung perfusion) and untreated wild-type and NOS2-deficient mice. The authors also evaluated the effects of breathing NO for 16 h on pulmonary vascular responsiveness during subsequent ventilation with NO., Results: In wild-type mice, lipopolysaccharide challenge impaired the pulmonary vasodilator response to 0.4 and 4 ppm NO (reduced 79% and 45%, respectively, P < 0.001), but not to 40 ppm. In contrast, lipopolysaccharide administration did not impair the vasodilator response to inhaled NO in NOS2-deficient mice. Breathing 20 ppm NO for 16 h decreased the vasodilator response to subsequent ventilation with NO in lipopolysaccharide-pretreated NOS2-deficient mice, but not in lipopolysaccharide-pretreated wild-type, untreated NOS2-deficient or untreated wild-type mice., Conclusions: In response to endotoxin challenge, NO, either endogenously produced by NOS2 in wild-type mice or added to the air inhaled by NOS2-deficient mice, is necessary to impair vascular responsiveness to inhaled NO. Prolonged NO breathing, without endotoxin, does not impair vasodilation in response to subsequent NO inhalation. These results suggest that NO, plus other lipopolysaccharide-induced products, are necessary to impair responsiveness to inhaled NO in a murine sepsis model.

The goal of this study was to evaluate the importance of the inducible nitric oxide synthase (iNOS) in lipopolysaccharide (LPS)-induced diaphragmatic contractile dysfunction. Many investigators have proposed that iNOS induction in the ventilatory and limb muscles of animals injected with Escherichia coli LPS leads to impaired muscle contractility and increased fatigability. We tested this proposal by examining wild-type mice and iNOS-deficient (iNOS knockout) mice. Both types of mice were injected with either saline (control) or E. coli LPS and killed after 12 h. Diaphragm nitric oxide synthase (NOS) activity, NOS expression, and muscle contractility were assessed with L-citrulline assay, immunoblotting, and in vitro bath preparation, respectively. LPS injection in wild-type mice induced iNOS protein expression and augmented total diaphragmatic NOS activity, which coincided with impaired muscle force generated at frequencies higher than 30 Hz. In iNOS knockout mice, injection of LPS augmented constitutive muscle NOS activity, upregulated the expression of the neuronal NOS (nNOS), but elicited a significantly greater decline in force generated in response to high frequency of stimulation compared with wild-type animals. We conclude that iNOS may play a protective role in attenuating the inhibitory influence of LPS on muscle contractility.

The inducible isoform of nitric oxide synthase (iNOS) is expressed after systemic administration of lipopolysaccharide (LPS). The importance of expression of iNOS in blood vessels is poorly defined. Because nitric oxide from iNOS may alter vasomotor function, we examined effects of LPS on vasomotor function in carotid arteries from iNOS-deficient mice. We studied contraction of the carotid artery from wild-type and iNOS-deficient mice in vitro 12 h after injection of LPS (20 mg/kg ip). Contractile responses to PGF2alpha (3-30 microM) and thromboxane A2 analog (U-46619; 3-100 nM) were evaluated using vascular rings from mice treated with vehicle or LPS. Maximum force of contraction generated by rings in response to PGF2alpha was 0.39 +/- 0.02 and 0.25 +/- 0.01 (SE) g (n = 14) in vehicle and LPS-treated wild-type mice, respectively (P < 0.001 vs. vehicle). Thus LPS reduced constrictor responses in wild-type mice. Thiocitrulline and aminoguanidine (inhibitors of iNOS) improved contractile responses from LPS-treated wild-type vessels. Indomethacin also improved constrictor responses in arteries from wild-type mice injected with LPS. In contrast, contraction of the carotid arteries in response to PGF2alpha and U-46619 was not impaired in LPS-treated iNOS-deficient mice, and contraction was not altered by inhibitors of iNOS. Expression of iNOS mRNA was confirmed using RT-PCR in carotid arteries from wild-type mice after injection of LPS but not vehicle. PCR products for iNOS were not observed in iNOS-deficient mice. These findings provide the first direct evidence that iNOS mediates impairment of vascular contraction after treatment with LPS.

Purpose: To investigate the roles of tumor necrosis factor-alpha (TNF-alpha), interleukin-4 (IL-4), and inducible nitric oxide synthase (iNOS) in endotoxin-induced uveitis (EIU) using gene knock-out mice., Methods: Mice (C57BL/6 x 129) either of normal phenotype or deficient in the genes encoding one or both tumor necrosis factor receptors (TNFR p55 and TNFR p75), IL-4, or iNOS were given footpad injections of 400 micrograms Escherichia coli lipopolysaccharide. Animals were killed 24 hours later, and infiltrating cells were counted on 5-micron ocular cross-sections through the optic nerve., Results: All abnormal mouse phenotypes were susceptible to EIU. Yet, TNFR p55 and IL-4 gene knock-out mice experienced less ocular inflammation than control animals (P = 0.021 and 0.007, respectively), whereas disease was not reduced for iNOS-deficient mice. Mice deficient in TNFR p55 and TNFR p75 experienced milder EIU than mice lacking TNFR p75 alone (P = 0.046)., Conclusions: Mice deficient in TNFR p55 and TNFR p75, IL-4, or iNOS retain the susceptibility to EIU, but TNF-alpha and IL-4 influence the influx of inflammatory cells to the eye during this disease.

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 gamma, 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 shock.