Adenovirus infection increases iNOS and peroxynitrite production in the lung

Host inflammatory and immune responses limit viral gene expression after administration of replication-deficient adenoviruses to the lung. The current study asks whether inducible nitric oxide synthase (iNOS) expression and peroxynitrite generation accompanied the inflammatory response following intratracheal administration of adenovirus. Pulmonary iNOS mRNA and protein were increased 2, 7, and 14 days following administration of 2 x 10(9) plaque-forming units of recombinant adenovirus (Av1Luc1) to BALB/c mice. Adenovirus infection was associated with a marked increase in nitrotyrosine staining. Intense nitrotyrosine staining was observed in alveolar macrophages, respiratory epithelial cells, conducting airways, and alveolar spaces 2 days postinfection. Two weeks after exposure to adenovirus, nitrotyrosine staining was detected within alveolar macrophages, suggesting adenovirus enhanced the nitration of proteins that were subsequently taken up by alveolar macrophages. Western blot analysis using anti-nitrotyrosine antibody did not demonstrate accumulation of nitrated surfactant protein A (SP-A), although a small fraction of aggregated SP-A comigrated with a nitrotyrosine-positive protein. iNOS expression, peroxynitrite, and nitrotyrosine generation accompany and may contribute to inflammatory responses to adenovirus in the lung.     

Association between the expression of inducible nitric oxide synthase by chondrocytes and its nitric oxide-generating activity in adjuvant arthritis in rats.

Inducible nitric oxide synthase (iNOS) is one of the clinical targets in rheumatoid arthritis. Synoviocytes, macrophages, and chondrocytes in the joints of patients with rheumatoid arthritis appear to express iNOS, but the contribution of iNOS molecules to rheumatoid arthritis is not yet clear. This study used adjuvant-induced arthritis in rats as a model to examine the association between the iNOS expression and its activity in rheumatoid arthritis. In adjuvant-injected rats, arthritic changes in the paw were first observed between days 10 and 12. NO-generation activity was precisely determined by combining an electron spin resonance/nitric oxide (NO)-trapping method with the method of standard addition using an NO generator, and we found that the activity in the joint samples was extremely high on day 10. The administration of S-(2-aminoethyl)isothiourea, a selective iNOS inhibitor, from day 0 to day 10, effectively reduced the paw swelling. Immunohistological studies showed that chondrocytes expressed iNOS on days 7-14 and that nitrotyrosine residues, a footprint of NO generation, were produced on day 10. This indicates that NO generation by iNOS induced in chondrocytes is a key event in the induction of adjuvant arthritis.     

Expression of Inducible Nitric Oxide Synthase and Fas/Fas Ligand Correlates with the Incidence of Apoptotic Cell Death in Atheromatous Plaques of Human Coronary Arteries

It was recently reported that inducible nitric oxide synthase was expressed in advanced atheromatous plaques. So we investigated the effect of NO or peroxynitrite reactive product of NO or O(2)(-) released by iNOS induced in macrophages or T lymphocytes on inflammatory cells in atheromatous plaques of human coronary arteries by immunohistochemistry. We found that iNOS was expressed in T lymphocytes and macrophages in T lymphocytes and macrophages coexisted advanced atheromatous areas. Most of the smooth muscle cells are not coexisted with T lymphocytes. We could not find iNOS in those smooth muscle cells. Only a small number of iNOS-positive smooth muscle cells were found close to T lymphocytes and macrophages. Markers for apoptotic cells induced in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) showed that many apoptotic T lymphocytes and macrophages existed near iNOS induced cells. Fas and Fas ligand were expressed in almost same areas that iNOS was expressed. By double-label immunostaining, Fas was expressed in T lymphocytes but Fas ligand was expressed in macrophages and in some T lymphocytes. These results suggest that NO from iNOS induces Fas and Fas ligand-mediated apoptosis and associates with regression of atherosclerosis. On the other hand, nitrotyrosine was detected wider areas than iNOS. So peroxynitrite from iNOS damages cells and tissues widely and may associate with progression of atherosclerosis. These results suggest an important role of iNOS in mediating both regressive changes and progressive change in atheromatous plaques.     

Expression and potential role of inducible nitric oxide synthase in the central nervous system of Theiler's murine encephalomyelitis virus-induced demyelinating disease.

Intracerebral inoculation of susceptible strains of mice with Theiler's murine encephalomyelitis virus (TMEV) results in immune-mediated demyelinating disease. We examined the pathogenic roles of nitric oxide (NO) and inducible NO synthase (iNOS) in TMEV-induced demyelinating disease (TMEV-IDD). The presence of iNOS was confirmed in the spinal cords of TMEV-infected mice using immunohistochemical staining with anti-iNOS antibody on day 0 (control) and days 15, 30, 60, and 120. Aminoguanidine (AG), a specific inhibitor of iNOS, was injected intraperitoneally (ip) on 1, 3, 5, 8, 10, and 12 days post-TMEV inoculation as induction phase or 15, 17, 19, 22, 24, and 26 days as effector phase. Control animals in each experiment received phosphate-buffered saline (PBS) ip at similar time intervals. Few iNOS-positive cells were observed in the spinal cords of naive SJL/J mice. In the early phase (day 15) of TMEV-IDD, an increase of iNOS-positive cells was detected in the leptomeninges and perivascular space of the spinal cords. The number of iNOS-positive cells was increased and reached its peak on day 60, when histology of the animals showed peak infiltration with inflammatory cells. The clinical course of TMEV-IDD on each day postintracerebral infection was significantly reduced in mice treated with AG in the effector phase, and there was no significant difference between mice treated with AG in induction phase versus those administered PBS. Thus, NO production via iNOS appears to be a pathogenic factor in the effector phase of TMEV-IDD.     

An effect of parasite-encoded arginase on the outcome of murine cutaneous leishmaniasis

Classical activation of macrophages infected with Leishmania species results in expression and activation of inducible NO synthase (iNOS) leading to intracellular parasite killing. Macrophages can contrastingly undergo alternative activation with increased arginase activity, metabolism of arginine along the polyamine pathway, and consequent parasite survival. An active role for parasite-encoded arginase in host microbicidal responses has not previously been documented. To test the hypothesis that parasite-encoded arginase can influence macrophage responses to intracellular Leishmania, a comparative genetic approach featuring arginase-deficient mutants of L. mexicana lacking both alleles of the gene encoding arginase (Deltaarg), as well as wild-type and complemented Deltaarg controls (Deltaarg[pArg]), was implemented. The studies showed: 1) the absence of parasite arginase resulted in a significantly attenuated infection of mice (p<0.05); 2) poorer survival of Deltaarg in mouse macrophages than controls correlated with greater NO generation; 3) the difference between Deltaarg or control intracellular survival was abrogated in iNOS-deficient macrophages, suggesting iNOS activity was responsible for increased Deltaarg killing; 4) consistently, immunohistochemistry showed enhanced nitrotyrosine modifications in tissues of mice infected with Deltaarg compared with control parasites. Furthermore, 5) in the face of decreased parasite survival, lymph node cells draining cutaneous lesions of Deltaarg parasites produced more IFN-gamma and less IL-4 and IL-10 than controls. These data intimate that parasite-encoded arginase of Leishmania mexicana subverts macrophage microbicidal activity by diverting arginine away from iNOS.     

Nitric Oxide Protects against Infection-Induced Neuroinflammation by Preserving the Stability of the Blood-Brain Barrier

Nitric oxide (NO) generated by inducible NO synthase (iNOS) is critical for defense against intracellular pathogens but may mediate inflammatory tissue damage. To elucidate the role of iNOS in neuroinflammation, infections with encephalitogenic Trypanosoma brucei parasites were compared in inos ^-/- and wild-type mice. Inos ^-/- mice showed enhanced brain invasion by parasites and T cells, and elevated protein permeability of cerebral vessels, but similar parasitemia levels. Trypanosome infection stimulated T cell- and TNF-mediated iNOS expression in perivascular macrophages. NO nitrosylated and inactivated pro-inflammatory molecules such as NF-κΒp65, and reduced TNF expression and signalling. iNOS-derived NO hampered both TNF- and T cell-mediated parasite brain invasion. In inos ^-/- mice, TNF stimulated MMP, including MMP9 activity that increased cerebral vessel permeability. Thus, iNOS-generated NO by perivascular macrophages, strategically located at sites of leukocyte brain penetration, can serve as a negative feed-back regulator that prevents unlimited influx of inflammatory cells by restoring the integrity of the blood-brain barrier.     

Effects of oxidant stress on inflammation and survival of iNOS knockout mice after marrow transplantation

In a model of idiopathic pneumonia syndrome after bone marrow transplantation (BMT), injection of allogeneic T cells induces nitric oxide (.NO), and the addition of cyclophosphamide (Cy) generates superoxide (O.) and a tissue-damaging nitrating oxidant. We hypothesized that.NO and O. balance are major determinants of post-BMT survival and inflammation. Inducible nitric oxide synthase (iNOS) deletional mutant mice (-/-) given donor bone marrow and spleen T cells (BMS) exhibited improved survival compared with matched BMS controls. Bronchoalveolar lavage fluids obtained on day 7 post-BMT from iNOS(-/-) BMS mice contained less tumor necrosis factor-alpha and interferon-gamma, indicating that.NO stimulated the production of proinflammatory cytokines. However, despite suppressed inflammation and decreased nitrotyrosine staining, iNOS(-/-) mice given both donor T cells and Cy (BMS + Cy) died earlier than iNOS-sufficient BMS + Cy mice. Alveolar macrophages from iNOS(-/-) BMS + Cy mice did not produce.NO but persisted to generate strong oxidants as assessed by the oxidation of the intracellular fluorescent probe 2',7'-dichlorofluorescin. We concluded that.NO amplifies T cell-dependent inflammation and addition of Cy exacerbates.NO-dependent mortality. However, the lack of.NO during Cy-induced oxidant stress decreases survival of T cell-recipient mice, most likely by generation of.NO-independent toxic oxidants.     

Posttraumatic Stress Disorder Disturbs Coronary Tone and Its Regulatory Mechanisms

Posttraumatic stress disorder (PTSD) is associated with myocardial injury, but changes in coronary regulatory mechanisms in PTSD have not been investigated. This study evaluated the effect of PTSD-inducing stress on coronary tone and its regulation by nitric oxide (NO) and voltage-gated K⁺ channels. PTSD was induced by exposing rats to predator stress, 15 min daily for 10 days, followed by 14 stress-free days. Presence of PTSD was confirmed by the elevated plus-maze test. Coronary tone was evaluated from changes in coronary perfusion pressure of Langendorff isolated hearts. Predator stress induced significant decreases in coronary tone of isolated hearts and in blood pressure of intact rats. L-NAME, a non-selective NO synthase (NOS) inhibitor, but not S-MT, a selective iNOS inhibitor, and increased coronary tone of control rats. In PTSD rats, both L-NAME and S-MT increased coronary tone. Therefore, the stress-induced coronary vasodilation resulted from NO overproduction by both iNOS and eNOS. NOS induction was apparently due to systemic inflammation as evidenced by increased serum interleukin-1β and C-reactive protein in PTSD rats. Decreased corticosterone in PTSD rats may have contributed to inflammation and its effect on coronary tone. PTSD was also associated with voltage-gated K⁺ channel dysfunction, which would have also reduced coronary tone.     

Severity of neurological signs and degree of inflammatory lesions in the brains of rats with Borna disease correlate with the induction of nitric oxide synthase.

The putative role of nitric oxide in the neuropathogenesis of Borna disease was investigated by determining changes in the expression of inducible nitric oxide synthase (iNOS) mRNA and constitutively expressed NOS (cNOS) mRNA in brains of Borna disease virus (BDV)-infected rats. iNOS mRNA was not detected in normal rat brain but was identified in BDV-infected brain at 14 days postinfection (p.i.), reaching maximum levels at 21 days p.i., when neurological signs and inflammatory reactions in the brain were also at a peak. cNOS mRNA was expressed in both normal brain and infected brain, increasing markedly at 17 days p.i. and reaching a peak at 21 days p.i. In situ hybridization analysis revealed iNOS mRNA in some, but not all, BDV-infected regions of the brain, particularly in the basolateral cortex and the hippocampus. iNOS-positive cells, as identified immunohistologically, were preferentially localized in perivascular areas of the hippocampus and in outer cortical layers. These iNOS-positive cells resembled monocytes/macrophages in morphology and distribution pattern but were significantly fewer. The correlation of iNOS and cNOS mRNA expression with the development of neurological disease, as well as the enhanced expression of iNOS within brain regions with inflammatory lesions, strongly suggests that NO may contribute to pathogenesis of Borna disease.     

Inducible nitric oxide synthase and guinea-pig ileitis induced by adjuvant

We sought to establish a model of inflammatory bowel disease by augmenting the activity of the local immune system with Freund's complete adjuvant, and to determine if inducible nitric oxide synthase (iNOS) expression and peroxynitrite formation accompanied the inflammatory condition. In anaesthetized guinea-pigs, a loop of distal ileum received intraluminal 50% ethanol followed by Freund's complete adjuvant. Control animals were sham operated. When the animals were killed 7 or 14 days later, loop lavage fluid was examined for nitrite and PGE(2) levels; mucosal levels of granulocyte and macrophages were estimated by myeloperoxidase (MPO) and N-acetyl-D-glucosaminidase (NAG) activity, respectively. Cellular localization if iNOS and peroxynitrite formation were determined by immunohistochemistry with polyclonal antibodies directed against peptide epitopes of mouse iNOS and nitrotyrosine, respectfully. Adjuvant administration resulted in a persistent ileitis, featuring gut thickening, crypt hyperplasia, villus tip swelling and disruption, and cellular infiltration. Lavage levels of PGE(2) and nitrite were markedly elevated by adjuvant treatment. Immunoreactive iNOS and nitrotyrosine bordered on detectability in normal animals but were markedly evident with adjuvant treatment at day 7 and particularly day 14. Immunohistochemistry suggested that enteric neurons and epithelia were major sites of iNOS activity and peroxynitrite formation. We conclude that local administration of adjuvant establishes a chronic ileitis. Inducible nitric oxide synthase may contribute to the inflammatory process.     

Inducible nitric oxide synthase and nitrotyrosine in the central nervous system of mice chronically infected with Trypanosoma brucei brucei

Human African trypanosomiasis, or sleeping sickness, evolves toward a meningoencephalitic stage, with a breakage in the blood-brain barrier, perivascular infiltrates, and astrocytosis. The involvement of nitric oxide (NO) has been evoked in the pathogenic development of the illness, since NO was found to be increased in the brain of animals infected with Trypanosoma brucei (T. b.) brucei. An excessive NO production can lead to alterations of neuronal signaling and to cell damage through the cytotoxicity of NO and its derivatives, especially peroxynitrites. In African trypanosomiasis, the sites of NO production and its role in the pathogenicity of lesions in the central nervous system (CNS) are unknown. In a chronic model of African trypanosomiasis (mice infected with T. b. brucei surviving with episodic suramin administration), NADPH-diaphorase staining of brain slides revealed that NO synthase (NOS) activity is located not only in endothelial cells, choroid plexus ependymal cells, and neurons as in control mice but also in mononuclear inflammatory cells located in perivascular and parenchyma infiltrates. An immunohistochemical study showed that the mononuclear inflammatory cells expressed an inducible NOS activity. Furthermore, the presence of nitrotyrosine in inflammatory lesions demonstrated an increased NO production and the intermediate formation of peroxynitrites. The detection of extensive formation of nitrotyrosine in the CNS parenchyma was observed in mice having shown neurological disorders, suggesting the role of peroxynitrites in the appearance of neurological troubles. In conclusion, this study confirmed the increased NO synthesis in the CNS of mice infected with T. b. brucei and suggests a deleterious role for NO, through the formation of peroxynitrites, in the pathogenesis of African CNS trypanosomiasis.     

Suppression of bleomycin-induced nitric oxide production in mice by taurine and niacin.

The effects of taurine (T) and niacin (N) on the influx of inflammatory cells and nitric oxide (NO) levels in bronchoalveolar lavage fluid (BALF) and expression of inducible NO synthase (iNOS) mRNA and iNOS protein in lungs were evaluated in the bleomycin (BL)-mouse model of lung fibrosis. Mice were placed into four groups: saline-instilled (SA) with a control diet (CD) (SA + CD); saline-instilled with TN (1% taurine in water + 2.5% (w/w) niacin in diet) (SA + TN); BL-instilled with CD (BL + CD); and BL-instilled with TN treatment (BL + TN). There was no difference in differential cell counts in BALF between the SA + CD and SA + TN control groups. Intratracheal instillation (IT) of BL (0.1 U/mouse) in mice stimulated an early influx of neutrophils followed by an increase in lymphocytes and macrophages in the BL + CD group. Taurine and niacin treatment significantly reduced the numbers of neutrophils, lymphocytes, and macrophages in the BL + TN group and caused significant reductions in BL-induced increases in the lung hydroxyproline content at 14 and 21 days in the BL + TN group. The mice in the SA + CD and SA + TN control groups had low levels of NO in BALF, whereas mice in the BL + CD group as compared to the SA + CD control group had elevated levels of NO from day 3 through day 21. Taurine and niacin treatment caused significant reductions in BL-induced increases in NO levels in BALF from mice in the BL + TN group at 7, 14, and 21 days as compared to the corresponding BL + CD group. The increases in NO levels in BALF from the BL + CD group were associated with elevated levels of iNOS gene expression and protein in the lungs. RT-PCR analysis of total RNA isolated from the lungs indicated that taurine and niacin treatment suppressed the BL-induced increases in iNOS message and iNOS protein. The ability of taurine and niacin to suppress the BL-induced increased production of NO secondary to decreases in iNOS mRNA and protein appears to be one of the mechanisms for their anti-inflammatory and antifibrotic effects.     

Immune evasion by Helicobacter pylori is mediated by induction of macrophage arginase II

Helicobacter pylori infection persists for the life of the host due to the failure of the immune response to eradicate the bacterium. Determining how H. pylori escapes the immune response in its gastric niche is clinically important. We have demonstrated in vitro that macrophage NO production can kill H. pylori, but induction of macrophage arginase II (Arg2) inhibits inducible NO synthase (iNOS) translation, causes apoptosis, and restricts bacterial killing. Using a chronic H. pylori infection model, we determined whether Arg2 impairs host defense in vivo. In C57BL/6 mice, expression of Arg2, but not arginase I, was abundant and localized to gastric macrophages. Arg2(-/-) mice had increased histologic gastritis and decreased bacterial colonization compared with wild-type (WT) mice. Increased gastritis scores correlated with decreased colonization in individual Arg2(-/-) mice but not in WT mice. When mice infected with H. pylori were compared, Arg2(-/-) mice had more gastric macrophages, more of these cells were iNOS(+), and these cells expressed higher levels of iNOS protein, as determined by flow cytometry and immunofluorescence microscopy. There was enhanced nitrotyrosine staining in infected Arg2(-/-) versus WT mice, indicating increased NO generation. Infected Arg2(-/-) mice exhibited decreased macrophage apoptosis, as well as enhanced IFN-γ, IL-17a, and IL-12p40 expression, and reduced IL-10 levels consistent with a more vigorous Th1/Th17 response. These studies demonstrate that Arg2 contributes to the immune evasion of H. pylori by limiting macrophage iNOS protein expression and NO production, mediating macrophage apoptosis, and restraining proinflammatory cytokine responses.     

Decreased alveolar macrophage apoptosis is associated with increased pulmonary inflammation in a murine model of pneumococcal pneumonia

Regulation of the inflammatory infiltrate is critical to the successful outcome of pneumonia. Alveolar macrophage apoptosis is a feature of pneumococcal infection and aids disease resolution. The host benefits of macrophage apoptosis during the innate response to bacterial infection are incompletely defined. Because NO is required for optimal macrophage apoptosis during pneumococcal infection, we have explored the role of macrophage apoptosis in regulating inflammatory responses during pneumococcal pneumonia, using inducible NO synthase (iNOS)-deficient mice. iNOS(-/-) mice demonstrated decreased numbers of apoptotic macrophages as compared with wild-type C57BL/6 mice following pneumococcal challenge, greater recruitment of neutrophils to the lung and enhanced expression of TNF-alpha. Pharmacologic inhibition of iNOS produced similar results. Greater pulmonary inflammation was associated with greater levels of early bacteremia, IL-6 production, lung inflammation, and mortality within the first 48 h in iNOS(-/-) mice. Labeled apoptotic alveolar macrophages were phagocytosed by resident macrophages in the lung and intratracheal instillation of exogenous apoptotic macrophages decreased neutrophil recruitment in iNOS(-/-) mice and decreased TNF-alpha mRNA in lungs and protein in bronchial alveolar lavage, as well as chemokines and cytokines including IL-6. These changes were associated with a lower probability of mice becoming bacteremic. This demonstrates the potential of apoptotic macrophages to down-regulate the inflammatory response and for the first time in vivo demonstrates that clearance of apoptotic macrophages decreases neutrophil recruitment and invasive bacterial disease during pneumonia.     

Dual role of inducible nitric oxide synthase in acute asbestos-induced lung injury

Reactive oxygen and nitrogen species have been implicated in the pathogenesis of asbestos fibers-associated pulmonary diseases. By comparing the responses of inducible nitric oxide synthase (iNOS) knockout and wild-type mice we investigated the consequences of iNOS expression for the development of the inflammatory response and tissue injury upon intratracheal instillation of asbestos fibers. Exposure to asbestos fibers resulted in an increased iNOS mRNA and protein expression in the lungs from wild-type mice. Moreover, iNOS knockout mice exhibited an exceeded pulmonary expression and production of TNF-alpha as well as a higher influx of neutrophils into the alveolar space than wild-type mice. In contrast, iNOS knockout animals displayed an attenuated oxidant-related tissue injury reflected in a decrease in protein leakage and LDH release into the alveolar space as well as weaker nitrotyrosine staining of lung tissue compared to wild-type mice. Data presented here indicate that iNOS-derived NO exerts a dichotomous role in acute asbestos-induced lung injury in that iNOS deficiency resulted in an exacerbated inflammatory response but improved oxidant-promoted lung tissue damage.     

Coronary artery remodeling in a model of left ventricular pressure overload is influenced by platelets and inflammatory cells

Left ventricular hypertrophy (LVH) is usually accompanied by intensive interstitial and perivascular fibrosis, which may contribute to arrhythmogenic sudden cardiac death. The mechanisms underlying the development of cardiac fibrosis are incompletely understood. To investigate the role of perivascular inflammation in coronary artery remodeling and cardiac fibrosis during hypertrophic ventricular remodeling, we used a well-established mouse model of LVH (transverse aortic constriction [TAC]). Three days after pressure overload, macrophages and T lymphocytes accumulated around and along left coronary arteries in association with luminal platelet deposition. Consistent with these histological findings, cardiac expression of IL-10 was upregulated and in the systemic circulation, platelet white blood cell aggregates tended to be higher in TAC animals compared to sham controls. Since platelets can dynamically modulate perivascular inflammation, we investigated the impact of thrombocytopenia on the response to TAC. Immunodepletion of platelets decreased early perivascular T lymphocytes' accumulation and altered subsequent coronary artery remodeling. The contribution of lymphocytes were examined in Rag1(-/-) mice, which displayed significantly more intimal hyperplasia and perivascular fibrosis compared to wild-type mice following TAC. Collectively, our studies support a role of early perivascular accumulation of platelets and T lymphocytes in pressure overload-induced inflammation.     

Oxidative and nitrosative mediators in hepatic injury caused by whole body hyperthermia in rats

The involvement of oxidative and nitrosative mediators in liver injury caused by heat stress remains unclear. This study aimed to elucidate the role of endothelial nitric oxide synthase (eNOS), and inducible NOS (iNOS)-derived NO and nitrotyrosine in the whole-body hyperthermia (WBH)-induced liver injury. Rats were anesthetized with intraperitoneal pentobarbital, and were exposed to a heating lamp for 60 min to raise the core temperature to 42.5 degrees C. The rats were maintained at the hyperthermic state for an additional 50 min. Blood urea nitrogen, creatinine, aspartate aminotransferase, alanine aminotransferase, lactic dehydrogenase, creatine phosphokinase, amylase, lipase, nitrate/nitrite, methyl guanidine, and proinflammatory cytokines (tumor necrosis factoralpha, interleukin-1beta and interleukin-10) were measured before and 14 h after hyperthermia. Immunohistochemical staining was employed to detect the eNOS, iNOS and nitrotyrosine levels. Western blotting was used to examine the expression of heatshock protein 70 (HSP 70). Histopathological examination of the liver tissue was performed. WBH caused liver injury accompanied with significant increases in biochemical factors, nitrate/nitrite, methyl guanidine, and proinflammatory cytokines. In addition, WBH enhanced the eNOS, iNOS, nitrotyrosine and HSP 70 levels. WBH caused hepatic injury. The pathogenetic mechanism is likely mediated through the NOS-derived NO, free radical, proinflammatory cytokines and nitrotyrosine. The enhanced expression of HSP 70 may play a protective role.     

Role of nitric oxide and superoxide in acute cardiac allograft rejection in rats

The role of NO and superoxide (O(2)(-)) in tissue injury during cardiac allograft rejection was investigated by using a rat ex vivo organ perfusion system. Excessive NO production and inducible NO synthase (iNOS) expression were observed in cardiac allografts at 5 days after cardiac transplantation, but not in cardiac isografts, as identified by electron spin resonance spectroscopy and Northern blotting. Cardiac isografts or allografts obtained on Day 5 after transplantation were perfused with Krebs bicarbonate buffer with or without various antidotes for NO or O(2)-, including N(omega)-monomethyl-L-arginine (L-NMMA; 1 mM), 2-phenyl-4,4,5, 5-tetramethylimidazoline-1-oxyl 3-oxide (PTIO; 100 microM), 4-amino-6-hydroxypyrazolo[3,4-d]pyrimidine (AHPP; a xanthine oxidase inhibitor; 100 microM), and superoxide dismutase (SOD; 100 units/ml). Treatment of the cardiac allografts with PTIO showed most remarkable improvement of the cardiac injury as revealed by significant reduction in aspartate transaminase, lactate dehydrogenase, and creatine phosphokinase concentrations in the perfusate. Similar but less potent protective effect on the allograft injury was observed by treatment with L-NMMA, AHPP, and SOD. Immunohistochemical analyses for iNOS and nitrotyrosine indicated that iNOS is mainly expressed by macrophages infiltrating the allograft tissues, and nitrotyrosine formation was demonstrated not only in macrophages but also in cardiac myocytes of the allografts, providing indirect evidence for the generation of peroxynitrite during allograft rejection. Our results suggest that tissue injury in rat cardiac allografts during acute rejection is mediated by both NO and O(2)(-), possibly through peroxynitrite formation.     

Reactive nitrogen species formation in eosinophils and imbalance in nitric oxide metabolism are involved in atopic dermatitis-like skin lesions in NC/Nga mice

Nitric oxide (NO) and reactive nitrogen species (RNS) have been implicated in the pathogenesis of inflammatory diseases. However, the involvement of NO and RNS in atopic dermatitis (AD), a pruritic inflammatory skin diseases, is not fully understood. In this study, we investigated the contribution of NO and RNS to the development of AD-like skin lesions in NC/Nga mice, an animal model for human AD. AD-like skin lesions were observed in NC/Nga mice kept under conventional conditions but not in specific pathogen-free conditions. The expression of inducible NO synthase (iNOS) and endothelial NOS (eNOS) proteins was upregulated in the dermal lesions, and that of neuronal NOS (nNOS) was downregulated in the epidermal lesions of the skin. Although the concentrations of NO2(-) and NO3(-) were lower, protein-bound nitrotyrosine content was significantly increased in the skin lesions. Immunohistochemical localization of nitrotyrosine was observed in almost all eosinophils. These results suggest that RNS formation in eosinophils and imbalance of NO metabolism are involved in the pathogenesis of AD-like skin lesions in NC/Nga mice.