FK 506 and aminoguanidine suppress iNOS induction in orthotopic corneal allografts and prolong graft survival in mice.

The aim of this study was to compare the effectiveness of immunosuppressant FK 506 and the specific inhibitor of inducible nitric oxide synthase (iNOS) aminoguanidine (AG) in prevention of corneal graft rejection and to investigate the iNOS expression in the rejection process. Orthotopic corneal allografting in mice was performed (C57BL/10; H-2(b) to BALB/c; H-2(d)). FK 506 (0.3 mg/kg per day) or AG (100 mg/kg per day) was injected intraperitoneally for 4 weeks. Grafted mice without therapy served as controls. Immunohistological evaluation of iNOS-positive cells and macrophage infiltration in grafts 27th day after grafting was performed. Within 4 weeks FK 506 prevented graft rejection in 71% and AG in 57% of animals compared to 29% of clear grafts in controls. A significant proportion of iNOS-positive cells was detected in the rejected grafts of the control and AG-treated groups. The treatment with FK 506 resulted in the inhibition of iNOS expression to a high degree in the rejected corneas. Non-rejected corneas of all groups and non-transplanted corneas exhibited no iNOS-positive cells. A massive infiltration of macrophages was detected in the rejected grafts, whereas non-rejected grafts exhibited only slight infiltration of macrophages. The presented data suggest that overexpression of iNOS and/or activation of iNOS is one of the several influential factors that contribute to the rejection process and that iNOS suppression delays corneal allograft rejection. FK 506 and AG are effective drugs in preventing corneal allograft rejection. Higher beneficial effect of FK 506 on graft survival could be explained by its well-known selective T-cell immunosuppression.     

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.     

Involvement of nitric oxide (NO) and TNF-alpha in the oxidative stress associated with anemia in experimental Trypanosoma cruzi infection

Trypanosoma cruzi infection in mice is associated with severe hematological changes, including anemia, which may contribute to mortality. TNF-alpha and nitric oxide (NO) play a critical role in establishing host resistance to this pathogen. We hypothesized that phagocyte-derived NO damages erythrocytes and contributes to the anemia observed during T. cruzi infection. To test this hypothesis, two strains of mice that differed in susceptibility and NO response to T. cruzi infection were used in these studies. We also blocked endogenous NO production by aminoguanidine (AG) treatment or blocked TNF-alpha with a neutralizing antibody and used mice that cannot produce phagocyte-derived NO (C57BL/6 iNOS(-/-)). Following infection with T. cruzi, resistant (C57BL/6) and susceptible (Swiss) mice displayed a parasitemia that peaked at the same time (i.e., day 9), yet parasitemia was 3-fold higher in Swiss mice (P < 0.05). All Swiss mice were dead by day 23 post-infection, while no C57BL/6 mice died during the study. At 14 days post-infection anemia in C57BL/6 mice was more severe than in Swiss mice. Treatment of both strains with the NO inhibitor, AG (50 mg/kg), and the use of iNOS(-/-) mice, revealed that the anemia in T. cruzi-infected mice is not caused by NO. However, the reticulocytosis that occurs during infection was significantly reduced after treatment with AG in both Swiss and C57BL/6 mice (P < 0.05). In addition, we showed that neutralization of TNF-alpha in vivo induced a significant increase in circulating reticulocytes in T. cruzi-infected C57BL/6 mice (P < 0.05), but did not modify other hematologic parameters in these mice. The evaluation of the oxidative stress after induction by t-butyl hydroperoxide (t-BHT) revealed that the treatment with AG completely protected against NO-mediated haemoglobin oxidation. Further, treatment with AG, but not with anti-TNF-alpha, protected against the infection-induced reduction of antioxidant capacity of erythrocytes as assessed by oxygen uptake and induction time. In summary, this is the first report showing the participation of NO and TNF-alpha in the oxidative stress to erythrocytes in acute T. cruzi infection. Further, our data suggest that NO does not play a direct role in development of the anemia. However, NO may contribute to other hematological changes noted during T. cruzi infection, such as the elevation of circulating reticulocytes and the reduction in circulating leukocytes and neutrophils.     

Effect of anti-B7-1 and anti-B7-2 mAb on Theiler's murine encephalomyelitis virus-induced demyelinating disease

We examined the role of B7-1 and B7-2, costimulatory molecules critical to full activation of T cells, in the development of Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD). Treatment with mAbs to B7-1 resulted in significant suppression of the development of this disease both clinically and histologically. In mice treated with these mAbs, the production of TNF-alpha and IFN-gamma in the spleen cells was decreased. The delayed-type hypersensitivity and T cell proliferative response specific for TMEV were decreased by this treatment. In contrast, treatment with Abs to B7-2, resulted in no effect on TMEV-IDD. These data suggest that B7-1 is critically involved in the pathogenesis of TMEV-IDD and that Abs to B7-1 could be a novel therapeutic approach in the clinical treatment of demyelinating diseases such as human multiple sclerosis.     

Induction of autoreactive CD8+ cytotoxic T cells during Theiler's murine encephalomyelitis virus infection: implications for autoimmunity

Theiler's murine encephalomyelitis virus (TMEV) belongs to the family Picornaviridae and causes demyelinating disease in the spinal cords of infected mice. Although immune responses have been shown to play an important role in demyelination, the precise effector mechanism(s) is unknown. Potentially autoreactive cytotoxic cells could contribute to the destruction. We tested whether an autoreactive cell induced by TMEV infection mediated cytotoxicity by using a 5-h (51)Cr release assay in SJL/J mice. Spleen cells from TMEV-infected mice were stimulated with irradiated TMEV antigen-presenting cells and used as effector cells. The effector cells differed from conventional cytotoxic T cells since these cells could kill both TMEV-infected and uninfected syngeneic or semisyngenic cell lines (PSJLSV and BxSF11gSV) but could not kill an allogeneic cell line (C57SV). The TMEV-induced autoreactive cells were also different from conventional natural killer (NK) cells or lymphokine-activated killer (LAK) cells, because they could kill neither NK cell-sensitive YAC-1 nor NK cell-resistant P815 and EL4 cells. Induction of autoreactive cells was not detected in vaccinia virus infection. The autoreactive killing required direct cell-to-cell contact and was mediated by a Fas-FasL pathway but not by a perforin pathway. The phenotype of the killer cells was CD3(+) CD4(-) CD8(+). Intracerebral inoculation of the effector cells into naive mice caused meningitis and perivascular cuffing not only in the brain parenchyma but also in the spinal cord, with no evidence of viral antigen-positive cells. This is the first report demonstrating that TMEV can induce autoreactive cytotoxic cells that induce central nervous system pathology.     

Role of nitric oxide in tolerance to lipopolysaccharide in mice.

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.     

Effects of iNOS inhibitor on IFN-gamma production and apoptosis of splenocytes in genetically different strains of mice infected with Toxoplasma gondii

To evaluate the role of nitric oxide (NO) in IFN-gamma production and apoptosis of splenocytes in genetically different strains of mice with toxoplasmosis, BALB/c (a toxoplasmosis resistant strain) and C57BL/6 (a toxoplasmosis susceptible strain) mice were infected with Toxoplasma gondii cysts orally and subsequently injected intraperitoneally with aminoguanidine, an iNOS inhibitor (AG; 35 mg/kg per mouse daily for 14 days). When BALB/c or C57BL/6 mice were infected with T. gondii without AG treatment, number of brain cysts, NO and IFN-gamma production by splenocytes, and percentages of apoptotic splenocytes were increased compared to uninfected control mice without AG treatment. AG treatment increased the number of brain cysts, and reduced NO and IFN-gamma production in T. gondii-infected C57BL/6 mice. In contrast, in T. gondii-infected BABL/c mice, the number of brain cysts, and NO and IFN-gamma production of splenocytes was not altered by treatment with AG. However, the percentages of apoptotic splenocytes in T. gondii-infected BALB/c or C57BL/6 mice were not affected by AG treatment. These results suggest that NO modulates IFN-gamma production in T. gondii-infected C57BL/6 mice, and that NO is involved in mediating a protective response in toxoplasmosis susceptible, but not resistant, mice strain during acute infection.     

The protective role of nitric oxide in a neurotoxicant-induced demyelinating model.

Demyelination is often associated with acute inflammatory events involving the recruitment-activation of microglia/macrophage, astrocytes, and leukocytes. The ultimate role of inflammatory products in demyelinating disease and in the survival of oligodendrocytes, the myelin forming cells, is unresolved. The current study examines the role of inducible NO synthase (iNOS)-derived NO in a neurotoxicant-induced model of demyelination. NO levels were greatly elevated in the midline corpus callosum during demyelination in genetically intact C57BL/6 mice, and this NO was due solely to the induction of iNOS, as the correlates of NO were not found in mice lacking iNOS. C57BL/6 mice lacking iNOS exhibited more demyelination, but did not display an increased overall cellularity in the corpus callosum, attributable to an unimpeded microglia/macrophage presence. An enhanced course of pathology was noted in mice lacking iNOS. This was associated with a greater depletion of mature oligodendrocytes, most likely due to apoptosis of oligodendrocytes. Microglia and astrocytes did not undergo apoptosis during treatment. Our results suggest a moderately protective role for NO during acute inflammation-association demyelination.     

Disassociation between the in vitro and in vivo effects of nitric oxide on a neurotropic murine coronavirus.

Intranasal inoculation of the neuroattenuated OBLV60 strain of mouse hepatitis virus results in infection of mitral neurons in the olfactory bulb, followed by spread along olfactory and limbic pathways to the brain. Immunocompetent BALB/c mice were able to clear virus by 11 days postinfection (p.i.). Gamma interferon (IFN-gamma) may play a role in clearance of OBLV60 from infected immunocompetent BALB/c mice through a nonlytic mechanism. Among the variety of immunomodulatory activities of IFN-gamma is the induction of expression of inducible nitric oxide synthase (iNOS), an enzyme responsible for the production of nitric oxide (NO). Studies were undertaken to investigate the role of IFN-gamma and NO in host defense and clearance of OBLV60 from the central nervous system (CNS). Exposure of OBLV60-infected OBL21a cells, a mouse neuronal cell line, to the NO-generating compound S-nitroso-L-acetyl penicillamine resulted in a significant decrease in viral replication, indicating that NO interfered with viral replication. Furthermore, infection of IFN-gamma knockout (GKO) mice and athymic nude mice with OBLV60 resulted in low-level expression of iNOS mRNA and protein in the brains compared to that of OBLV60-infected BALB/c mice. Nude mice were unable to clear virus and eventually died between days 11 and 14 p.i. (B. D. Pearce, M. V. Hobbs, T. S. McGraw, and M. J. Buchmeier, J. Virol. 68:5483-5495, 1994); however, GKO mice survived infection and cleared virus by day 18 p.i. These data suggest that IFN-gamma production in the olfactory bulb contributed to but may not be essential for clearance of OBLV60 from the brain. In addition, treatment of OBLV60-infected BALB/c mice with aminoguanidine, a selective inhibitor of iNOS activity, did not result in any increase in mortality, and the mice cleared the virus by 11 days p.i. These data suggest that although NO was able to block replication of virus in vitro, expression of iNOS with NO release in vivo did not appear to be the determinant factor in clearance of OBLV60 from CNS neurons.     

Endothelial nitric oxide synthase is protective in the initiation of caerulein-induced acute pancreatitis in mice

The effect of inhibiting nitric oxide (NO) synthase (NOS) or enhancing NO on the course of acute pancreatitis (AP) is controversial, in part because three NOS isoforms exist: neuronal (nNOS), endothelial (eNOS), and inducible (iNOS). We investigated whether inhibition or selective gene deletion of NOS isoforms modified the initiation phase of caerulein-induced AP in mice and explored whether this affected pancreatic microvascular blood flow (PMBF). We investigated the effects of nonspecific NOS inhibition with N(omega)-nitro-l-arginine (l-NNA; 10 mg/kg ip) or targeted deletion of eNOS, nNOS, or iNOS genes on the initiation phase of caerulein-induced AP in mice using in vivo and in vitro models. Western blot analysis was performed to assess eNOS phosphorylation status, an indicator of enzyme activity, and microsphere studies were used to measure PMBF. l-NNA and eNOS deletion, but not nNOS or iNOS deletion, increased pancreatic trypsin activity and serum lipase during the initiation phase of in vivo caerulein-induced AP. l-NNA and eNOS did not affect trypsin activity in caerulein-hyperstimulated isolated acini, suggesting that nonacinar events mediate the effect of NOS blockade in vivo. The initiation phase of AP in wild-type mice was associated with eNOS Thr(495) residue dephosphorylation, which accompanies eNOS activation, and a 178% increase in PMBF; these effects were absent in eNOS-deleted mice. Thus eNOS is the main isoform influencing the initiation of caerulein-induced AP. eNOS-derived NO exerts a protective effect through actions on nonacinar cell types, most likely endothelial cells, to produce greater PMBF.     

iNOS expression in dystrophinopathies can be reduced by somatic gene transfer of dystrophin or utrophin

BACKGROUND: Nitric oxide (NO) is an inorganic gas produced by a family of NO synthase (NOS) proteins. The presence and the distribution of inducible-NOS (NOS II or iNOS), and NADPH-diaphorase (NADPH-d), a marker for NOS catalytic activity, were determined in muscle sections from control, DMD, and BMD patients. MATERIALS AND METHODS: NADPH-d reactivity, iNOS- and nNOS (NOS I)-immunolocalization were studied in muscles from mdx mice before and after somatic gene transfer of dystrophin or utrophin. RESULTS: In control patients, few fibers (<2%) demonstrated focal accumulation of iNOS in sarcolemma. In DMD patients, a strong iNOS immunoreactivity was observed in some necrotic muscle fibers as well as in some mononuclear cells, and regenerating muscle fibers had diffusely positive iNOS immunoreactivity. In DMD patients, NADPH-d reactivity was increased and mainly localized in regenerating muscle fibers. In mdx mice quadriceps, iNOS expression was mainly observed in regenerating muscle fibers, but not prior to 4 weeks postnatal, and was still present 8 weeks after birth. The expression of dystrophin and the overexpression of utrophin using adenovirus-mediated constructs reduced the number of iNOS-positive fibers in mdx quadriceps muscles. The correction of some pathology in mdx by dystrophin expression or utrophin overexpression was independent of the presence of nNOS. CONCLUSIONS: These results suggest that iNOS could play a role in the physiopathology of DMD and that the abnormal expression of iNOS could be corrected by gene therapy.     

Nitric oxide participates in the recovery of normal jejunal epithelial ion transport following exposure to the superantigen, Staphylococcus aureus enterotoxin B.

Bacterial superantigens (SAgs) are potent T cell activators. Mice treated 4 h previously with the SAg, Staphylococcus aureus enterotoxin B (SEB), display reduced ion transport (assessed by short circuit current) responses to prosecretory stimuli, which normalize 24 h posttreatment. Here, mice were treated with SEB alone or in combination with an inhibitor of the inducible form of NO synthase (iNOS), l -NIL. Subsequently, jejunal iNOS expression was detected by immunohistochemistry, ion transport was evaluated in Ussing chambers, and serum levels of TNF-alpha and IFN-gamma were measured by ELISA. SEB-treated mice had increased epithelial iNOS immunoreactivity, and numerous iNOS-positive CD3+ T cells occurred in their mucosa and submucosa. Concomitant treatment with l -NIL did not affect the reduced short circuit current responsiveness to electrical nerve stimulation or the prosecretory agents, carbachol and forskolin, that occurred 4 h post-SEB (5 microgram) treatment. However, Isc responses in l -NIL- plus SEB-treated mice were still significantly reduced 24 h posttreatment, indicating a role for NO in the restoration of normal ion transport following exposure to SAgs. The prolongation of epithelial ion transport abnormalities correlated with elevated serum levels of TNF-alpha and IFN-gamma in mice treated 24 h previously with l -NIL plus SEB compared with those in controls and SEB-only-treated mice. Additionally, mice treated with l -NIL plus SEB and TNF-alpha- or IFN-gamma-neutralizing Abs displayed normal jejunal ion transport characteristics 24 h posttreatment. We conclude that NO mobilization is important in the homeostatic recovery response following immune stimulation by SAgs and that the beneficial effect of NO in this model system is probably via regulation of TNF-alpha and IFN-gamma production.     

脊髓水平iNOS在吗啡依赖大鼠戒断反应中的作用

目的:探讨脊髓水平诱导型一氧化氮合酶在吗啡依赖大鼠戒断反应中的作用。方法:健康雄性SD大鼠72只,体重200~250 g,吗啡剂量每次10 mg/kg,每日2次,隔日每次增加10 mg/kg,至第6天末次注射50 mg/kg,大鼠腹腔注射纳洛酮4 mg/kg建立吗啡依赖及戒断模型,在纳洛酮激发戒断前30 min鞘内注射iNOS特异性抑制剂氨基胍(AG)150μg。分为正常对照组、吗啡依赖组、吗啡戒断组、AG组。采用行为学(n=8)、免疫组织化学(n=6)和Western blot(n=4)方法观察鞘内应用iNOS特异性抑制剂氨基胍对吗啡依赖大鼠纳洛酮催促戒断反应和脊髓神经元iNOS表达的影响。结果:AG组戒断症状评分和戒断组促诱发痛评分均低于戒断组(P<0.05)。免疫组织化学和Western blot显示戒断组大鼠脊髓iNOS阳性神经元的数目和蛋白的表达增高,而AG组大鼠脊髓iNOS阳性神经元的数目和iNOS蛋白的表达低于戒断组(P<0.05)。结论:脊髓水平iNOS表达上调可能参与介导吗啡戒断反应。     

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.     

Effect of endogenously generated nitric oxide on the energy metabolism of peritoneal macrophages

To understand the role of nitric oxide (NO) in the regulation of cellular metabolism in peritoneal macrophages under physiological low oxygen tension, its effect on the respiration and energy metabolism was examined with casein-induced peritoneal macrophages from the rat. Intraperitoneal injection of casein transiently induced peritoneal infiltration of neutrophils (peaked on day 1) followed by the migration of macrophages that peaked on day 2. Western blotting analysis using antibodies against inducible type of NO synthase (iNOS) revealed that macrophages appeared in the peritoneal cavity during an early stage (approximately day 2) but not during the late stage (day 3 approximately) of inflammation expressed iNOS and generated substantial amounts of NO by a mechanism that was inhibited by N-iminoethyl-L-ornithine (NIO), a specific inhibitor of iNOS. Although NO reversibly but strongly inhibited the respiration of macrophages from both stages particularly under physiologically low oxygen tension, NIO markedly enhanced the respiration of macrophages obtained from the early period but not from the late period of inflammation. The ATP level in the macrophages from the late period but not from the early period was markedly decreased by NO. Biochemical analysis revealed that the glycolytic activity in the macrophages obtained from the early period was significantly higher than that from the late period of inflammation. These results indicate that significant fractions of cellular ATP in iNOS-positive peritoneal macrophages are synthesized by the increased activity of glycolysis particularly under physiological low oxygen tensions where the mitochondrial respiration is strongly inhibited by endogenously generated NO by macrophages and neutrophils.     

Immunoregulatory role of nitric oxide in Kilham rat virus-induced autoimmune diabetes in DR-BB rats

Macrophages play a critical role in the pathogenesis of Kilham rat virus (KRV)-induced autoimmune diabetes in diabetes-resistant BioBreeding (DR-BB) rats. This investigation was initiated to determine the role of macrophage-derived soluble mediators, particularly NO, in the pathogenesis of KRV-induced diabetes in DR-BB rats. We found that the expression of inducible NO synthase (iNOS), an enzyme responsible for NO production, was significantly increased during the early phase of KRV infection. Inhibition of iNOS by aminoguanidine (AG) treatment resulted in the prevention of diabetes in KRV-infected animals. The expression of IL-1beta, TNF-alpha, and IL-12 was significantly decreased in the spleen of AG-treated, KRV-infected DR-BB rats compared with PBS-treated, KRV-infected control rats. Subsequent experiments revealed that AG treatment exerted its preventive effect in KRV-infected rats by maintaining the finely tuned immune balance normally disrupted by KRV, evidenced by a significant decrease in the expression of IFN-gamma, but not IL-4, and a decrease in Th1-type chemokine receptors CCR5, CXCR3, and CXCR4. We also found that iNOS inhibition by AG decreased the KRV-induced expression of MHC class II molecules and IL-2R alpha-chain, resulting in the suppression of T cell activation, evidenced by the decreased cytolytic activity of CD8(+) T cells. We conclude that NO plays a critical immunoregulatory role by up-regulating macrophage-derived proinflammatory cytokines, up-regulating the Th1 immune response, and activating T cells, leading to type 1 diabetes after KRV infection, whereas suppression of NO production by AG treatment prevents KRV-induced autoimmune diabetes in DR-BB rats.     

Protection by pioglitazone in the MPTP model of Parkinson's disease correlates with I kappa B alpha induction and block of NF kappa B and iNOS activation

Inflammation has been implicated in the pathogenesis of Parkinson's disease (PD). In the chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of PD, inducible NO synthase (iNOS) derived nitric oxide (NO) is an important mediator of dopaminergic cell death. Ligands of the peroxisome proliferator-activated receptor (PPAR) exert anti-inflammatory effects. We here investigated whether pioglitazone, a PPARgamma agonist, protected mice from MPTP-induced dopaminergic cell loss, glial activation, and loss of catecholamines in the striatum. As shown by western blot, PPARgamma was expressed in the striatum and the substantia nigra of vehicle- and MPTP-treated mice. Oral administration of 20 mg/(kg day) of pioglitazone protected tyrosine hydroxylase (TH)-positive substantia nigra neurons from death induced by 5 x 30 mg/kg MPTP. However, the decrease of dopamine in the striatum was only partially prevented. In mice treated with pioglitazone, there were a reduced activation of microglia, reduced induction of iNOS-positive cells and less glial fibrillary acidic protein positive cells in both striatum and substantia nigra pars compacta. In addition, treatment with pioglitazone almost completely blocked staining of TH-positive neurons for nitrotyrosine, a marker of NO-mediated cell damage. Because an increase in inhibitory protein-kappa-Balpha (IkappaBalpha) expression and inhibition of translocation of the nuclear factor kappaB (NFkappaB) subunit p65 to the nucleus in dopaminergic neurons, glial cells and astrocytes correlated with the protective effects of pioglitazone, our results suggest that pioglitazone sequentially acts through PPARgamma activation, IkappaBalpha induction, block of NFkappaB activation, iNOS induction and NO-mediated toxicity. In conclusion, treatment with pioglitazone may offer a treatment opportunity in PD to slow the progression of disease that is mediated by inflammation.     

Epitope spreading initiates in the CNS in two mouse models of multiple sclerosis

Chronic progression of two T cell-mediated central nervous system (CNS) demyelinating models of multiple sclerosis, relapsing EAE (R-EAE) and Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) is dependent on the activation of T cells to endogenous myelin epitopes (epitope spreading). Using transfer of carboxyfluorescein succinyl ester (CFSE)-labeled T-cell receptor (TCR)-transgenic T cells and mixed bone marrow chimeras, we show that activation of naive proteolipid protein (PLP)139-151-specific T cells in SJL mice undergoing PLP178-191-induced R-EAE or TMEV-IDD occurs directly in the CNS and not in the cervical lymph nodes or other peripheral lymphoid organs. Examination of the antigen-presentation capacity of antigen-presenting cell (APC) populations purified from the CNS of mice with PLP178-191-induced R-EAE shows that only F4/80-CD11c+CD45hi dendritic cells (DCs) efficiently present endogenous antigen to activate naive PLP139-151-specific T cells in vitro. In contrast, DCs as well as F4/80+CD45hi macrophages and F4/80+CD45lo microglia activate a PLP139-151-specific helper T cell line. The data suggest that naive T cells enter the inflamed CNS and are activated by local APCs, possibly DCs, to initiate epitope spreading.     

Experimental autoimmune uveoretinitis initiated by non‐phagocytic destruction of inner segments of photoreceptor cells by Mac‐1+ mononuclear cells

EAU in mice is a model of human posterior uveitis. EAU is a Th1‐dependent disease that has been assumed to target the neural retina and related tissues; however, in situ effector cells and the target have not yet been clearly demonstrated. In the present study, we induced EAU in B10R mice by immunizing them with human interphotoreceptor retinoid‐binding protein peptide 161–180. Histological examinations revealed that EAU occurred approximately 11 days after the immunization and reached a peak on day 14. Retinae from normal or EAU mice were treated with proteases to obtain mono‐dispersed cells. The mono‐dispersed cells thus obtained were separated into three to four fractions by discontinuous Percoll density‐gradient (e.g. PBS/40/60) centrifugation. In normal mice, 94% of the total cells were recovered in two fractions (i.e. PBS/40 and pellet); and these fractions mainly contained inner and outer segments and cell bodies of photoreceptor cells and RPE cells, respectively. In EAU mice, additional cells (i.e. inflammatory cells) were obtained at the 40/60 interface. Electron microscopic examination showed that tissue damage during EAU was initiated by non‐phagocytic destruction of inner segments by Mac‐1⁺ mononuclear cells on day 11, followed by phagocytic activity of macrophages against outer segments and RPE cells on day 14. In vitro culturing of normal retinal cells with EAU infiltrates suggested the involvement of TNF‐α and NO in the tissue damage. These results indicate that EAU was initiated by non‐phagocytic destruction of inner segments of photoreceptor cells by Mac‐1⁺ mononuclear cells.